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WO1994007500A1 - 2,5-dihydro-2,5-dioxo-1h-azepines et 2,5-dihydro-2-oxo-1h-azepines et leurs utilisations en tant qu'antagonistes du recepteur de glycine et d'acides amines excitateurs - Google Patents

2,5-dihydro-2,5-dioxo-1h-azepines et 2,5-dihydro-2-oxo-1h-azepines et leurs utilisations en tant qu'antagonistes du recepteur de glycine et d'acides amines excitateurs Download PDF

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Publication number
WO1994007500A1
WO1994007500A1 PCT/US1993/009288 US9309288W WO9407500A1 WO 1994007500 A1 WO1994007500 A1 WO 1994007500A1 US 9309288 W US9309288 W US 9309288W WO 9407500 A1 WO9407500 A1 WO 9407500A1
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hydroxy
dihydro
benzazepine
dioxo
nitro
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PCT/US1993/009288
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English (en)
Inventor
Eckard Weber
John F. W. Keana
Kenton J. Swartz
Walter J. Koroshetz
Alun H. Rees
James E. Huettner
Original Assignee
Eckard Weber
Keana John F W
Swartz Kenton J
Koroshetz Walter J
Rees Alun H
Huettner James E
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Application filed by Eckard Weber, Keana John F W, Swartz Kenton J, Koroshetz Walter J, Rees Alun H, Huettner James E filed Critical Eckard Weber
Priority to AU53498/94A priority Critical patent/AU5349894A/en
Publication of WO1994007500A1 publication Critical patent/WO1994007500A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/45—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by at least one doubly—bound oxygen atom, not being part of a —CHO group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole

Definitions

  • the present invention was made with U.S. government support.
  • the present invention is in the field of medicinal chemistry.
  • the present invention relates to novel substituted 2,5-dihydro-2,5- dioxo-lH-azepines and 2,5-dihydro-2-oxo-lH-azepines and their use to treat or prevent neuronal degeneration associated with ischemia, pathophysiologic conditions associated with neuronal degeneration, convulsions, anxiety, chronic pain and to induce anesthesia.
  • Glutamate is thought to be the major excitatory neurotransmitter in the brain. There are three major subtypes of glutamate receptors in the CNS.
  • NMDA receptors are found in the membranes of virtually every neuron in the brain. NMDA receptors are ligand-gated cation channels that allow Na + , K + and Ca ++ to permeate when they are activated by glutamate or aspartate (non-selective, endogenous agonists) or by NMDA (a selective, synthetic agonist) (Wong and Kemp, Ann. Rev. Pharmacol. Toxicol. 37:401- 425 (1991)). Glutamate alone cannot activate the NMDA receptor.
  • the NMDA receptor channel In order to become activated by glutamate, the NMDA receptor channel must first bind glycine at a specific, high affinity glycine binding site which is separate from the glutamate/NMDA binding site on the receptor protein (Johnson and Ascher, Nature 325:329-331 (1987)). Glycine is therefore an obligatory co- agonist at the NMDA receptor/channel complex (Kemp, J.A., et al. , Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)).
  • the NMDA receptor carries a number of other functionally important binding sites. These include binding sites for Mg ++ , Zn ++ , polyamines, arachidonic acid and phencyclidine (PCP) (Reynolds and Miller, Adv. in Pharmacol. 27: 101-126 (1990); Miller, B., et al. , Nature 355:722-725 (1992)).
  • the PCP binding site now commonly referred to as the PCP receptor—is located inside the pore of the ionophore of the NMDA receptor/channel complex (Wong, E.H.F., et al , Proc. Natl. Acad. Sci. USA 83:7104-7108 (1986); Huettner and Bean, Proc.
  • PCP In order for PCP to gain access to the PCP receptor, the channel must first be opened by glutamate and glycine. In the absence of glutamate and glycine, PCP cannot bind to the PCP receptor although some studies have suggested that a small amount of PCP binding can occur even in the absence of glutamate and glycine (Sircar and Zukin, Brain Res. 556:280-284 (1991)). Once PCP binds to the PCP receptor, it blocks ion flux through the open channel. Therefore, PCP is an open channel blocker and a non-competitive glutamate antagonist at the NMDA receptor/channel complex.
  • MK-801 One of the most potent and selective drugs that bind to the PCP receptor is the anticonvulsant drug MK-801.
  • This drug has a K d of approximately 3nM at the PCP receptor (Wong, E.H.F., et al , Proc. Natl. Acad. Sci. USA 83:7104-7108 (1986)).
  • Both PCP and MK-801 as well as other PCP receptor ligands [e.g. dextromethorphan, ketamine and N,N,N'-trisubstituted guanidines] have neuroprotective efficacy both in vitro and in vivo (Gill, R. , et al. , J. Neurosci.
  • PCP receptor drugs as ischemia rescue agents in stroke has been severely hampered by the fact that these drugs have strong PCP-like behavioral side effects (psychotomimetic behavioral effects) which appear to be due to the interaction of these drugs with the PCP receptor (Tricklebank, M.D., et al. , Eur. J. Pharmacol. 767:127-135 (1989); Koek, W., et al, J. Pharmacol. Exp. Ther. 245:969 (1989); Willets and Balster, Neuropharmacology 27: 1249 (1988)).
  • PCP-like behavioral side effects appear to have caused the withdrawal of MK801 from clinical development as an ischemia rescue agent.
  • these PCP receptor ligands appear to have considerable abuse potential as demonstrated by the abuse liability of PCP itself.
  • PCP and related PCP receptor ligands cause a behavioral excitation (hyperlocomotion) in rodents (Tricklebank, M.D., et al. , Eur. J. Pharmacol. 767: 127-135 (1989)) and a characteristic katalepsy in pigeons (Koek, W., et al., J. Pharmacol. Exp. Ther.
  • Drugs acting as competitive antagonists at the glutamate binding site of the NMDA receptor such as CGS 19755 and LY274614 also have neuroprotective efficacy because these drugs-like the PCP receptor ligands- can prevent excessive Ca ++ flux through NMDA receptor/channels in ischemia (Boast, C.A., et al. , Brain Res. 442:345-348 (1988); Schoepp, D.D., et al. , J. Neural. Trans. 85: 131-143 (1991)).
  • NMDA receptor channel activation is by using antagonists at the glycine binding site of the NMDA receptor. Since glycine must bind to the glycine site in order for glutamate to effect channel opening (Johnson and Ascher, Nature 325: 329-331 (1987); Kemp, J.A., et al. , Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)), a glycine antagonist can completely prevent ion flux through the NMDA receptor channel— even in the presence of a large amount of glutamate.
  • glycine antagonists should be very powerful neuroprotective agents, because they can prevent the opening of NMDA channels by glutamate non- competitively and therefore—unlike competitive NMDA antagonists— do not have to overcome the large concentrations of endogenous glutamate that are released in the ischemic brain region.
  • glycine antagonists act at neither the glutamate/- NMDA nor the PCP binding sites to prevent NMDA channel opening, these drugs might not cause the PCP-like behavioral side effect seen with both PCP receptor ligands and competitive NMDA receptor antagonists (Tricklebank, M.D., et al , Eur. J. Pharmacol. 767: 127-135 (1989); Koek, W., et al, J. Pharmacol. Exp. Ther. 245:969 (1989); Willets and Balster, Neuropharma ⁇ cology 27: 1249 (1988); Tricklebank, M.D., et al., Eur. J. Pharmacol. 767: 127-135 (1989); Zukin, S.R., et al , Brain Res. 294: 174 (1984); Brady,
  • glycine antagonists as clinically useful neuroprotective agents: A. Most available glycine antagonists with relatively high receptor binding affinity in vitro such as 7-Cl-kynurenic acid (Kemp, J.A., et al. , Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)), 5,7-dichlorokynurenic acid (DCK) (McNamara, D., et al. , Neuroscience Lett. 120: 17-20 (1990)) and indole-2-carboxylic acid (Gray,
  • HA-966 (Fletcher and Lodge, Eur. J. Pharmacol. 757: 161-162 (1988))— is a partial agonist with micromolar affinity for the glycine binding site.
  • a neuroprotective efficacy for HA-966 in vivo has not been demonstrated nor has it been demonstrated for the other available glycine antagonists because they lack bioavailability in vivo.
  • a need continues to exist for potent and selective glycine/NMDA antagonists which can penetrate the blood/brain barrier and which: • lack the PCP-like behavioral side effects common to the PCP- like NMDA channel blockers such as MK801 or to the competitive NMDA receptor antagonists such as CGS 19755;
  • R' represents an alkyl group or halogen or hydrogen atom in position 7 and/or 8; n may be 1 or 2; and R represents an alkyl group or a hydrogen atom.
  • the benzazepines are useful as intermediates for the preparation of kynurenic acid and its analogs.
  • James et al. also disclose the preparation of 7-nitro-2,3,4,5-tetrahydro-2,4,5-trioxo-lH- benzazepine from 7-nitro-3,4-epoxy-2,3,4,5-tetrahydro-2,5-dioxo-lH- benzazepine by epoxide rearrangement with concentrated sulfuric acid.
  • the invention relates to a method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemiaand surgery, as well as treating neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, treating or preventing the adverse consequences of the overstimulation of the excitatory amino acids, as well as treating anxiety, convulsions, chronic pain and inducing anesthesia, comprising administering to an animal in need of such treatment a compound of the Formula (I)
  • Rj is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
  • R 2 , R 3 , R, and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carbonylamido or alkylthiol;
  • R e is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH 2 CONHAr, -NHCONHAr, -NHCOCH 2 Ar, -COCH 2 Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
  • R 7 is hydrogen, acyl or alkyl.
  • the invention also relates to a method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia and surgery, as well as treating neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, treating or preventing the adverse consequences of the overstimulation of the excitatory amino acids, as well as treating anxiety, convulsions, chronic pain and inducing anesthesia, comprising administering to an animal in need of such treatment a compound of the Formula (II)
  • Rj is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
  • R 2 , R 3 , R 4 and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carbonylamido or alkylthiol;
  • Rj is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH 2 CONHAr, -NHCONHAr, -NHCOCH 2 Ar, -COCH 2 Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or
  • the invention also relates to a method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia and surgery, as well as treating neurodegenerative diseases including
  • Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome treating or preventing the adverse consequences of the overstimulation of the excitatory amino acids, as well as treating anxiety, convulsions, chronic pain and inducing anesthesia, comprising administering to an animal in need of such treatment a compound of the Formula (III)
  • R is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
  • R 2 , R 3 , R 4 and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carbonylamido or alkylthiol;
  • Rg is hydrogen
  • R 7 is hydrogen, acyl or alkyl
  • X is -NHCO-Ar, NHCOCH 2 -Ar, NHCONH-Ar, -NHCONH 2 , -NHCONHR 8 or -NHCONR 8 R 9 , wherein R 8 and R 9 are C alkyl groups and Ar is an aryl group which may be substituted by a halo group.
  • the invention also relates to a method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia and surgery, as well as treating neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, treating or preventing the adverse consequences of the overstimulation of the excitatory amino acids, as well as treating anxiety, convulsions, chronic pain and inducing anesthesia, comprising administering to an animal in need of such treatment a compound of the Formula (IV)
  • R is H 2 , H(OH), H(acyloxy), or oxo
  • R 2 , R 3 , R, and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carboxyamido or alkylthiol;
  • R e is hydrogen;
  • R 7 is hydrogen, acyl or alkyl; and Y is alkyl, -NHCO-Ar, NHCOCH 2 -Ar, NHCONH-Ar, -NHCONH 2 , -NHCONHR 8 or -NHCONR 8 R 9 , wherein R 8 and R 9 are C M alkyl groups and Ar is an aryl group which may be substituted by a halo group.
  • the invention also relates to a method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia and surgery, as well as treating neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, treating or preventing the adverse consequences of the overstimulation of the excitatory amino acids, as well as treating anxiety, convulsions, chronic pain and inducing anesthesia, comprising administering to an animal in need of such treatment a compound of the Formula (V)
  • Rj is H 2 , H(OH), H(acyloxy), or oxo
  • R 2 , R 3 , R 4 and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carboxyamido or alkylthiol; and R e is hydrogen.
  • the present invention also relates to the novel substituted azepines disclosed herein, and pharmaceutical compositions thereof.
  • 2,5-dihydro-2,5-dioxo-lH-benzazepines have high binding to the glycine receptor.
  • certain of the compounds of the present invention easily cross the blood/brain barrier, thus making them highly suitable for treating central nervous system neurodegeneration.
  • the compounds of the present invention may not exhibit the PCP-like behavioral side effects common to the PCP-like NMDA channel blockers such as MK-801 and other NMDA antagonists such as CGS 19755.
  • the compounds of the present invention are useful for treating pathophysiologic conditions, without significant side effects or toxicity.
  • Figure 1 depicts graphs showing the inhibition of membrane current by 100 ⁇ M 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine evoked by 100 ⁇ M kainate (A), or 20 ⁇ M NMDA plus 300 nM glycine (B), at holding potentials of +50 mV and -80 mV. With these concentrations, DD ⁇ B completely blocked the response to NMDA plus glycine at both holding potentials. Inhibition of current evoked by kainate was to 59 ⁇ 3.6% of control at -80 mV (five experiments) and to 55 ⁇ 1.5 % of control at +50 mV (three experiments).
  • Figure 2A depicts a graph showing the control currents activated by 10 ⁇ M to 10 mM kainate.
  • Figure 2B depicts a graph showing the competitive antagonism of kainate currents by 50 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine which were activated by 40 ⁇ M to 10 mM kainate and a control response to 10 mM kainate alone. Holding potential, -70 mV.
  • Figure 3 depicts a graph showing the concentration-response relationship for kainate alone (o) (13 applications in four cells) or in the presence of 8 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (13 applications in five cells), 20 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine ( ⁇ ) (eight applications in five cells), or 50 ⁇ M 8- methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (•) (seven applications in three cells).
  • Figure 4 depicts graphs showing the inhibition by 8-methyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine of the initial transient current activated by L-glutamate (Glu). Currents were activated by the rapid application of 500 ⁇ M glutamate alone or with 8, 20 or 50 ⁇ M 8-methyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
  • NMDA receptor channels Holding potential was -70 mV.
  • Figure 5 depicts a bar graph showing the inhibition by 8-methyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine of the initial transient current activated by L-glutamate.
  • the values represent the peak inward current minus steady state current, expressed as a percentage of the control response (peak current minus steady state current for 100 ⁇ M L-glutamate alone). Bars, mean + standard error for 8 ⁇ M (20 applications in eight cells), 20 ⁇ M (23 applications in nine cells), and 50 ⁇ M (23 applications in 10 cells) 8-Me-DD ⁇ B.
  • Figure 6 depicts graphs showing the competitive antagonism by 8- methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine of the steady state current activated by L-glutamate.
  • Currents were activated by application of 1 ⁇ M to 1 mM glutamate (A).
  • A 1 ⁇ M glutamate
  • B 50 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy- lH-benzazepine and a control response to glutamate alone (B).
  • MK-801 was included in all solutions at 1 ⁇ M to block NMDA receptor channels.
  • Figure 7 depicts a graph showing the concentration-response relationship for glutamate alone (o) (10 cells) or in the presence of 8 ⁇ M 8- methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) ( ⁇ cells), 20 ⁇ M
  • Figure 8 depicts graphs showing the competitive antagonism by 8- methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine to activation of steady state current by kainate (A) and glutamate (B) at non-NMDA receptors. Points, apparent half-maximal concentration of agonist (EC J0 ' _+ 95 % confidence limits) determined from individual fits of eq. 1 (see examples), plotted as a function of antagonist K B plus antagonist concentration (K B + [8-
  • Figure 9 depicts graphs showing the competitive antagonism of glycine potentiation at the NMDA receptor by 8-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine.
  • Figure 9A shows the whole cell currents elicited by 1 mM NMDA and six concentrations of glycine. In a different cell, currents gated by NMDA and five concentrations of glycine were determined in the presence of 10 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, as well as a control response to NMDA plus 50 ⁇ M glycine without antagonist (B). Holding potential was -70 mV.
  • Figure 10 depicts a graph showing the concentration-response relationship for glycine alone (o) (15 applications in 5 cells) or in the presence of 2 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (8 applications in 4 cells), 10 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine ( ⁇ ) (9 applications in 3 cells), or 50 ⁇ M 8-methyl-2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine (•) (thirteen applications in 4 cells).
  • Figure 11 depicts a graph showing the competitive antagonism by 8- methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepinetoglycineactivation at the glycine allosteric site on the NMDA receptor.
  • Points apparent half- maximal concentration of agonist (EC 50 ; +_ 95 % confidence limits) determined from individual fits of eq. 1, plotted as a function of antagonist K B plus antagonist concentration (K B + [8-Me-DD ⁇ B]).
  • Values for EC 50 and K B were determined from the fit of eq. 2, as in Fig. 8.
  • the EC 50 for glycine 770 nM; K B for 8-Me-
  • Figure 12 depicts a graph showing that increasing concentrations of NMDA reduce the EC 50 for glycine at the allosteric potentiation site. The concentration-response relationship is shown for glycine in the presence of 25 ⁇ M NMDA (o) (9 applications in 5 cells) or in the presence of 1 mM NMDA
  • FIG. 13 depicts graphs showing the antagonism of the NMDA recognition site by 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
  • A Currents elicited by 1 ⁇ M to 1 mM NMDA, all with 1 mM D-serine added to saturate the glycine allosteric site.
  • B In a different cell, currents gated by 4 ⁇ M to ImM NMDA in the presence of 50 ⁇ M 8-Me-DD ⁇ B and a control response to 1 mM NMDA without antagonist (all containing ImM D-serine).
  • Figure 14 depicts a graph showing the concentration-response relations for NMDA plus 1 mM D-serine (O) (17 applications in seven cells) and in the presence of 50 ⁇ M 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (•) (nine applications in three cells).
  • Figure 15 depicts a graph showing the antagonism of kainate by 2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-bromo-2,5-dihydro-2,5-dioxo- 3-hydroxy-lH-benzazepine, and 7-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine.
  • Figure 16 depicts a graph showing the antagonism at the glycine allosteric site on NMDA receptors by 7-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 4-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, and 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine. All currents were activated by variable concentrations of glycine in the presence of 1 mM NMDA, to provide saturation of the NMDA recognition site.
  • Concentration-response relations for glycine alone (O) 23 applications in eight cells) or in the presence of 100 ⁇ M 2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine (•) (nine applications in two cells), 100 ⁇ M 4-bromo-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (10 applications in three cells), or 100 ⁇ M 7-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine ( ⁇ ) (10 applications in four cells). Points, mean +_ standard error of the normalized currents (7/7,- ⁇ ).
  • Figure 17 depicts a graph showing the antagonism at the agonist recognition site on NMDA receptors by 7-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 4-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, and 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
  • the present invention relates to novel substituted 2,5-dihydro-2,5- dioxo-lH-benzazepines which are highly selective, competitive antagonists of the glycine binding site of the NMDA receptor and of the excitatory amino acids.
  • the substituted 2,5-dihydro-2,5-dioxo-lH-benzazepines of the invention have the following Formula (I):
  • R is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
  • R 2 , R 3 , R, and R 5 are hydrogen, halo, haloalkyl, aryl, a fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxyl, carbonylamido or alkylthiol;
  • R e is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -C ⁇ 2 CON ⁇ Ar, -NHCONHAr, -NHCOCH 2 Ar, -COCH 2 Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and R 7 is hydrogen, acyl or alkyl.
  • R,-R 7 are as defined above.
  • R 7 is H
  • these compounds may be solubilized by raising the pH of the solution to about 8.5 to about 10 by the addition of an aqueous base such as Na 2 CO 3 , NaOH, KOH or choline hydroxide.
  • Preferred compounds within the scope of Formula I are wherein R 2 is halo or nitro, R 3 is hydrogen or halo, R 4 is halo or haloalkyl and R 5 is hydrogen. Especially preferred compounds are wherein R 3 is hydrogen.
  • Other preferred compounds within the scope of Formula I are wherein one of
  • R 2 , R 3 , R 4 (especially R 4 ) is amino and R 5 is hydrogen.
  • R, R 2 , R 5 , Re and R 7 are hydrogen
  • R 3 is alkyl, e.g. methyl
  • R 4 is halo, e.g. bromo or chloro.
  • R 2 is halo or nitro
  • R 3 is alkyl
  • R 4 is alkyl
  • R 5 is hydrogen
  • R, R 2 , R 3 , R 5 , R e and R 7 are H and R 4 is alkyl or halo, especially bromo.
  • the invention also relates to compounds of the Formula (III)
  • Ri is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
  • R 2 , R 3 , R 4 and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carbonylamido or alkylthiol;
  • R e is hydrogen
  • R 7 is hydrogen, acyl or alkyl
  • X is -NHCO-Ar, NHCOCH 2 -Ar, NHCONH-Ar, -NHCONH 2 , -NHCONHR 8 or -NHCONR 8 R 9 , wherein R 8 and R 9 are C M alkyl groups and Ar is an aryl group which may be substituted by a halo group.
  • the invention also relates to compounds of the Formula (IV)
  • R is H 2 , H(OH), H(acyloxy), or oxo;
  • R 2 , R 3 , R 4 and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carbonylamido or alkylthiol;
  • R e is hydrogen
  • R 7 is hydrogen, acyl or alkyl
  • Y is alkyl, -NHCO-Ar, NHCOCH 2 -Ar, NHCONH-Ar, -NHCONH 2 , -NHCONHR 8 or -NHCONR 8 R 9 , wherein R 8 and R 9 are C M alkyl groups and Ar is an aryl group which may be substituted by a halo group.
  • the invention also relates to compounds of the Formula (V)
  • Ri is H 2 , H(OH), H(acyloxy), or oxo;
  • R 2 , R 3 , R, and R 5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamido, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy, carboxyamido or alkylthiol; and
  • R e is hydrogen; with the proviso that at least one of R 1 , R 2 , R 3 , and R 4 is other than hydrogen.
  • compounds having Formula (V) will be especially lipophilic and, thus, will easily cross the blood-brain barrier.
  • Compounds having Formula (V) are expected to have high binding affinity to the glycine binding site and may serve as pro-drugs for the corresponding 2,5-dihydro-2,5-dioxo- 3-hydroxy-lH-benzazepines which may form by rearrangement.
  • Typical C e . 14 aryl groups include phenyl, naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups.
  • Typical fused aryl rings are benzo and naphtho groups fused to the 7,8- position of the azepine ring.
  • Typical halo groups include fluorine, chlorine, bromine and iodine.
  • Typical C, ⁇ alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and tert. -butyl groups. Also contemplated is a trimethylene group substituted on the 7 and 8 positions of any one of the compounds having Formulae I-V.
  • Typical haloalkyl groups include C, ⁇ alkyl groups substituted by one or more fluorine, chlorine, bromine or iodine atoms, e.g. fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1, 1-difluoroethyl and trichloromethyl groups.
  • Typical alkoxy groups include oxygen substituted by one of the C M alkyl groups mentioned above.
  • Typical alkylthio groups include sulphur substituted by one of the C, ⁇ alkyl groups mentioned above.
  • Typical acylamino groups include any C 1-6 acyl substituted nitrogen, e.g. acetamido, propionamido, butanoylamido, pentanoylamido, hexanoylamidoand the like.
  • Typical acyloxy groups include any C 6 acyloxy groups, e.g. acetoxy, propionoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy and the like.
  • Typical heterocyclic groups include tetrahydrofuranyl, pyranyl, piperidinyl, piperizinyl, pyrrolidinyl, imidazolindinyl, imidazolinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinyl and pyrazolinyl groups.
  • Typical heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl, naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, 5aH- carbozolyl, carbozolyl,
  • Typical amino groups include -N ⁇ 2 , -NHR 8 , and -NR 8 R 9 , wherein R 8 and R 9 are C w alkyl groups as defined above.
  • Typical carbonylamido groups are carbonyl groups substituted by -NH 2 , -NHR 8 , and -NR R 9 groups' as defined above.
  • Particularly preferred substituted 2,5-dihydro-2,5-dioxo-lH- benzazepines of the present invention include, but are not limited to 4-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 3-bromo-2,5-dihydro-2,5- dioxo-4-hydroxy-lH-benzazepine, 7-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine,7-methyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,
  • Certain of the compounds of the present invention are expected to be potent anticonvulsants in animal models and will prevent ischemia-induced nerve cell death in the gerbil global ischemia model after i.p. administration.
  • 6-Chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine 6-Chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8-dichloro-6-nitro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine 7,8- dichloro-5-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 8-bromo-
  • the compounds of the present invention are active in treating or preventing neuronal loss, neurodegenerative diseases, chronic pain, are active as anticonvulsants and inducing anesthesia. Certain of the compounds of the present invention are expected to exhibit little or no untoward side effects caused by non-selective binding with other receptors, particularly, the PCP and glutamate receptors associated with the NMDA receptor. In addition, certain of the compounds block kainate, AMPA and quisqualate receptors and are therefore useful as broad-spectrum excitatory amino acid receptor antagonists. Moreover, the compounds of the present invention are effective in treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, e.g. those which are involved in the NMDA receptor system, by blocking the glycine receptors and preventing the ligand-gated cation channels from opening and allowing excessive influx of Ca ++ into neurons, as occurs during ischemia.
  • Neurodegenerative diseases which may be treated with the compounds of the present invention include those selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome.
  • the compounds of the present invention find particular utility in the treatment or prevention of neuronal loss associated with multiple strokes which give rise to dementia. After a patient has been diagnosed as suffering from a stroke, the compounds of the present invention may be administered to ameliorate the immediate ischemia and prevent further neuronal damage that may occur from recurrent strokes.
  • the compounds of the present invention are able to cross the blood/brain barrier which makes them particularly useful for treating or preventing conditions involving the central nervous system.
  • the compounds of the invention find particular utility in treating or preventing the adverse neurological consequences of surgery.
  • coronary bypass surgery requires the use of heart-lung machines which tend to introduce air bubbles into the circulatory system which may lodge in the brain. The presence of such air bubbles robs neuronal tissue of oxygen, resulting in anoxia and ischemia.
  • Pre- or post- surgical administration of the compounds of the present invention will treat or prevent the resulting ischemia.
  • the compounds of the invention are administered to patients undergoing cardiopulmonary bypass surgery or carotid endarterectomy surgery.
  • the compounds of the present invention also find utility in treating or preventing chronic pain. Such chronic pain may be the result of surgery, trauma, headache, arthritis, or other degenerative disease.
  • the compounds of the present invention also find particular utility in the treatment of phantom pain that results from amputation of an extremity.
  • the compounds of the invention are also expected to be useful in inducing anesthesia, either general or local anesthesia, for example, during surgery.
  • novel glycine and excitatory amino acid antagonists may be tested for in vivo anticonvulsant activity after intraperitoneal injection using a number of anticonvulsant tests in mice (audiogenic seizure model in DBA-2 mice, pentylenetetrazol-induced seizures in mice, NMDA-induced death).
  • the compounds may also be tested in drug discrimination tests in rats trained to discriminate PCP from saline. It is expected that most of the compounds of the present invention will not generalize to PCP at any dose. In addition, it is also expected that none of the compounds will produce a behavioral excitation in locomotor activity tests in the mouse.
  • novel glycine, AMPA, kainate and quisqualate antagonists of the present invention do not show the PCP-like behavioral side effects that are common to NMDA channel blockers such as MK-801 and PCP or to competitive NMDA antagonists such as CGS 19755.
  • the novel glycine and excitatory amino acid antagonists are also expected to show potent activity in vivo after intraperitoneal injection suggesting that these compounds can penetrate the blood/brain barrier.
  • the compounds of the present invention may be tested for potential glycine antagonist activity by observing the inhibition of binding of l ⁇ M glycine-stimulated [ 3 H]-MK-801 in rat or guinea pig brain membrane homogenates.
  • the compounds of the present invention may be prepared by the general methods taught by Bichall and Rees, Can. J. Chem. 52:610 (1974), the contents of which are fully incorporated by reference herein.
  • the methods involve the preparation of an appropriately substituted 2-alkoxy- 1 ,4-naphthoquinone followed by reaction with hydrazoic acid to give the corresponding 2,3,4,5-tetrahydro-2,4,5-trioxo-lH-l-benzazepine (VI) (see Scheme I).
  • one may prepare 6-bromo-2-alkoxy-l,4- naphthoquinone according to Schaffner-Sabba, K. et al., J. Med. Chem. 27:990 (1984) and treat it with hydrazoic acid to give 8-bromo-2,3,4,5- tetrahydro-2 ,4,5-trioxo- 1H- 1 -benzazepine.
  • a further method involves the oxidation of the corresponding substituted 1- or 2-oxo-l ,2,3,4-tetrahydronaphthylene (XII) with molecular oxygen in the presence of potassium te/ ⁇ .-butoxide (see Scheme III) to give the corresponding 2-hydroxy-l,4-dioxo-l,4-dihydronaphthylene (XIII).
  • 1-Oxo-l, 2,3,4- tetrahydronaphthylene may be prepared by Friedel-Crafts reaction of benzene with butyrolactone. The product of these reactions (XIII) may be carried on to the corresponding 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-l-benzazepine according to Scheme I.
  • Naphthalene itself is known to be oxidized to 1 ,4-naphthoquinone in 35% yield (see Braude and Fawcett, Org. Synth. Coll. Vol. IV, p. 698).
  • chlorinated or nitrated naphthalenes oxidize them to the corresponding chlorinated and nitrated 1,4- naphthoqui nones, which may be carried on to the chlorinated and nitrated 2,5- dihydro-2,5-dioxo-3-hydroxy-lH-l-benzazepines as outlined in Scheme II.
  • the naphthoquinones may be prepared by a Diels-Alder condensation reaction between benzoquinone and a diene followed by oxidation.
  • Diels-Alder condensation reaction between benzoquinone and a diene followed by oxidation.
  • the condensation of 2-chloro-3-methylbutadiene with benzoquinone followed by oxidation gives 6-chloro-7-methyl-l ,4- benzoquinone.
  • the electronic withdrawing groups on the benzene ring may be added by preparation of the silyl derivative (XIV) with a halotrialkylsilane followed by an electrophilic substitution reaction. It is expected that reaction of the silyl ether (XIV) with N-chlorosuccinamide and removal of the silyl group (e.g. with fluoride anion) will give the 4-chloro (XV) or 7-chloro derivative (XVI). It is also expected that reaction of (XIV) with nitric acid and removal of the silyl group will give the nitro derivative (XVII) (see Scheme IV).
  • a reactive halide for example, deprotonation of 7,8- dichloro-6-nitro-2,5-dihydro-2,5-dioxo-3-t-butyldimethylsiloxy-lH-l- benzazepine (XIX) with a base such as lithium diisopropylamide will give the corresponding anion (XX).
  • Alkylation with an ⁇ -haloester such as methyl bromoacetate followed by acid hydrolysis will give the corresponding acid (XXI).
  • Condensation of the acid with an arylamine in the presence of a dehydrating agent such as DCC gives the arylamide (XVIII).
  • R e -NHCONHAr (XXII)
  • the compound may be prepared by reaction of the aminate anion XXIII with chloramine, mesitylenesulfonyl- oxyamine (Tamura, Y et al , Synthesis 1, 1977), or hydroxylamine-O-sulfonic acid (procedure of Wallace, R.G., Org. Prep. Proced. Int 14:269 (1982)) to give the N-amino-2,5-dihydro-2,5-dioxo-3-t-butyldimethylsiloxy-lH-l- benzazepine intermediate XXIV.
  • N-nitrosylation of the Nj amide nitrogen atom followed by reduction will give the N-amino 2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-l-benzazepines intermediate.
  • Acylation of the free amino group with, for example, phenylisocyanate will give XXII.
  • Rg is -N ⁇ COC ⁇ 2 Ar (XXV)
  • acylation of the intermediate XXIV with phenylacetyl chloride leads to XXV (see Scheme VI).
  • the corresponding isomeric 2,3,4,5-tetrahydro-2,4,5-trioxo-lH-l- benzazepines may be prepared according to Moore, ⁇ .W. et al., Tetr. Lett., 1243 (1960) and Rees, A. ⁇ ., J. Chem. Soc, 3111 (1959).
  • a substituted 2,3,4,5-tetrahydro-2,4,5-trioxo-lH-benzazepine may be prepared from the corresponding substituted 2,3,4,5-tetrahydro-2,5-dioxo-lH- benzazepine by condensation with N,N-dimethyl-p-nitrosoaniline followed by acid hydrolysis.
  • substituted 2,3,4,5-tetrahydro-2,4,5-trioxo-lH-benzazepine may be prepared from the corresponding 3,4-epoxy-2,3,4,5-tetrahydro-2,5-dioxo-lH-benzazepine by epoxide rearrangement with concentrated sulfuric acid. See, James, R.A. et al, J. Heterocycl. Chem. 26(3)793-5 (1989).
  • the base solubility of 9 was preliminarily investigated. It was found that 9 dissolved in 0.1 M sodium bicarbonate at a concentration of 1 mg/mL when a suspension was gently warmed to give a yellow solution. No precipitation was noted when the solution was allowed to cool to room temperature. The concentration could be raised to 2.5 mg/mL if the suspension was vigorously heated. TLC analysis of this solution showed some degradation had occurred. Also, a portion of the material precipitated out of solution upon cooling to room temperature. The binding affinity of compound 9 was quite unexpected. Whereas the affinity of 9 was expected to be higher than that of 7, the measurements showed the potency of 9 to be approximately 40 times less than that of 7 (Table 1).
  • the most down field proton (8.03) has been assigned to position 6 because it is ortho to one other proton and is ortho to the electron withdrawing 5-carbonyl. Therefore, the doublet at 7.46 must then be the signal for the proton at position 9.
  • the most up field triplet (7.26) has been assigned to the proton at position 7 since it is para to the electron donating amide nitrogen. A resonance structure may be drawn whereby electron density from the nitrogen may be placed at that position. That leaves the remaining triplet (7.61) for the proton at position 8.
  • An 8-substituted benzazepine skeleton may be prepared by the ring expansion reaction of a 7-substituted 2-methoxynaphtho-l,4-quinone (Birchall and Rees, Can. J. Chem. 52:610-615 (1974)), which is prepared by the treatment of the 2-hydroxy analog with diazomethane (Fieser, L.J., 7. Am. Chem. Soc. ⁇ 8:2928-2937 (1926)).
  • the 2-hydroxyquinones may be prepared by the base promoted oxidation of 7-substituted- 1-tetralones (Baillie and Thompson, J. Chem. Soc.
  • the present invention is directed to compounds having high binding to the glycine receptor and low binding to the kainate and AMPA sites.
  • Particular compounds of the invention have high antagonist potency at the kainate, AMPA and quisqualate receptors in addition to the glycine receptor.
  • those compounds having high binding to the glycine receptor exhibit a glycine binding affinity (Kj) of about 10 ⁇ M or less in a glycine binding assay (see the Examples).
  • Kj glycine binding affinity
  • the compounds of the present invention exhibit a K, of 1 ⁇ M or less.
  • the compounds of the present invention exhibit a Kj of 0.1 ⁇ M or less.
  • the compounds exhibit high binding to the kainate and AMPA sites if they exhibit a Kj of about 10 ⁇ M or less, especially, 1 ⁇ M or less in a kainate or AMPA binding assay.
  • the glycine antagonist potency in vitro may be determined using a l ⁇ M glycine- stimulated [ 3 ⁇ ]-MK801 binding assay.
  • This assay takes advantage of the fact that the binding of [ 3 H]-MK801 to the PCP receptor inside the pore of the NMDA channel is dependent on the presence of both glutamate and glycine.
  • [ 3 H]-MK801 cannot bind effectively to the PCP receptor, because the NMDA channel remains closed and access of [ 3 H]-MK801 to the PCP receptor inside the closed channel pore is severely restricted.
  • the assay is conducted using rat brain membrane homogenates which are enriched in NMDA receptors.
  • the membranes are prepared as follows. Frozen rat brains (obtained from Pel-Freez, Rogers, Arkansas) are homogenized in 15 volumes (w/v) of ice cold 0.32 M sucrose. The homogenate is spun at 1,000 x g for ten minutes. The supernatant is collected and spun for 20 minutes at 44,000 x g. The pellet is suspended in 15 volumes of water (relative to original brain weight). The homogenate is again spun at 44,000 x g for twenty minutes. The pellet is resuspended in 5 volumes of water and the suspension is freeze-thawed 2 times.
  • the suspension is brought to 15 volumes with water and spun at 44,000 x g for twenty minutes.
  • the pellet is resuspended in 5 volumes of ice-cold lOmM HEPES, and is titrated to pH 7.4 with KOH containing 0.04% Triton X-100.
  • Membranes are incubated with the Triton/HEPES buffer at 37 °C for 15 minutes. The volume is then brought to 15 with ice-cold 10 mM HEPES, pH 7.4, and spun/washed three times with spins of 44,000 x g between washes.
  • the final pellet is suspended in three volumes of 50 mM HEPES, pH 7.4 and the protein concentration is determined with a standard dye-binding protein assay (Bio-Rad, Richmond, CA). The suspension is stored at -80°C until used. Only HPLC grade water is used for all buffers and suspensions/washings. The extensive washings are necessary to remove as much endogenous glycine from the membrane preparation as possible.
  • Nonspecific binding is defined as the difference in binding that occurs in the absence or presence of PCP (final concentration: 100 ⁇ M).
  • PCP final concentration: 100 ⁇ M.
  • bound radioactivity in the presence of 10 ⁇ M glutamate alone (final concentration) is subtracted from the bound radioactivity in the presence of both 10 ⁇ M glutamate and 1 ⁇ M glycine (final concentration).
  • a 500 nM concentration (final) of 5,7- dichlorokynurenic (DCK) acid is added to all assay tubes. This concentration of the glycine antagonist DCK "buffers" most of the residual endogenous glycine that is not removed by the extensive washing steps that are carried out during the membrane preparation procedure. The 500 nM DCK does not interfere with the stimulation of [ 3 H]-MK801 binding that is effected by the addition of 1 ⁇ M exogenous glycine.
  • the assays are incubated for 120 minutes at room temperature after which time the membrane-bound radioactivity is isolated from the free radioactivity by vacuum filtration through Whatman glass fiber filters that had been pretreated with 0.3 % polyethyleneimine. Filtration is accomplished using a Brandel 48 well cell harvester. Filtered membranes are washed three times with 3 ml each of ice cold buffer. Filters are transferred to scintillation vials and 5 ml of scintillation cocktail is added. The vials are shaken overnight and the radioactivity is counted by liquid scintillation spectroscopy. The assays are done in triplicate and all experiments are conducted at least three times.
  • Inhibition dose response curves are constructed using increasing concentrations of glycine antagonists from 5 nM to 330 ⁇ M. IC 50 values are determined for compounds active in inhibiting 1 ⁇ M glycine-stimulated [ 3 H]- MK801 binding by computer-assisted plotting of the inhibition curves and interpolation. When compounds are found to inhibit glycine-stimulated [ 3 H]- MK801 binding, experiments are conducted to determine whether the inhibition of the glycine-stimulated [ 3 H]-MK801 binding is indeed mediated at the glycine binding site of the NMDA receptor.
  • Kj values for the glycine antagonists are calculated using the Cheng and Prusoff equation employing the experimentally determined IC 50 values, the known concentration of glycine in the assay (1 ⁇ M) and the known affinity of glycine for the glycine binding site of the NMDA receptor (100 nM).
  • the same rat brain membrane homogenates used for the 1 ⁇ M glycine- stimulated [ 3 H]-MK801 binding assay are used for the [ 3 H]-AMPA radioligand binding assay.
  • the frozen membranes (prepared as described above) are thawed and diluted with 30mM Tris/HCl buffer containing 2.5 mM CaCl 2 and 100 mM KSCN, pH 7.4, to yield a final membrane concentration of 1.25 mg/ml membrane protein.
  • 0.8ml of membrane homogenate is added to polypropylene tubes followed by 0.033 ml drug and 0.067 ml buffer (or for controls by 0.1 ml buffer alone) and 0.1 ml buffer containing 200,000 cpm of [ 3 H]-AMPA.
  • the assay is incubated for 30 minutes on ice. Bound radioactivity is separated from free radioactivity by filtration over Whatman glass fiber filters (pretreated with 0.3 % polyethyleneimine) using a Brandel 48 well cell harvester.
  • Filtered membranes are washed three times with 3 ml each of ice cold buffer.
  • the filters are transferred to scintillation vials and 5 ml of scintillation cocktail is added.
  • the vials are shaken overnight and radioactivity is counted by liquid scintillation spectroscopy.
  • Nonspecific binding is determined by the radioactivity that remains bound to the membranes in the presence 10 mM glutamate.
  • Inhibition dose response curves are constructed by adding increasing concentrations of drug from 10 nM to 100 ⁇ M.
  • the same membrane preparation as that used for the [3H]-AMPA binding assay may be used for the [ 3 H]-Kainate radioligand binding assay.
  • the frozen rat brain membranes are thawed and 5 mM Tris/HCl buffer, pH 7.4, is added to yield a final concentration of 0.5 mg/ml membrane protein.
  • 0.8 ml of membrane homogenate is added to polypropylene tubes followed by 0.033 ml drug and 0.067 ml buffer (or for controls by 0.1 ml buffer alone) and 0.1 ml buffer containing 200,000 cpm of [ 3 H]-kainate.
  • the assay is incubated for 2 hours on ice. Bound radioactivity is separated from free radioactivity by filtration over Whatman glass fiber filters (pretreated with 0.3% polyethyleneimine) using a Brandel 48 well cell harvester.
  • Filtered membranes are washed three times with 3 ml each of ice cold buffer.
  • the filters are transferred to scintillation vials and 5 ml of scintillation cocktail is added.
  • the vials are shaken overnight and radioactivity is counted by liquid scintillation spectroscopy. Nonspecific binding is determined by the radioactivity that remains bound to the membranes in the presence 10 mM glutamate.
  • Inhibition dose response curves are constructed by adding increasing concentrations of drug from 250 nM to 330 ⁇ M.
  • the anxiolytic activity of any particular compound of the present invention may be determined by use of any of the recognized animal models for anxiety.
  • a preferred model is described by Jones, B.J. etal., Br. J. Phar ⁇ macol. 93:985-993 (1988).
  • This model involves administering the compound in question to mice which have a high basal level of anxiety.
  • the test is based on the finding that such mice find it aversive when taken from a dark home environment in a dark testing room and placed in an area which is painted white and brightly lit.
  • the test box has two compartments, one white and brightly illuminated and one black and non-illuminated.
  • the mouse has access to both compartments via an opening at floor level in the divider between the two compartments.
  • mice are placed in the center of the brightly illuminated area. After locating the opening to the dark area, the mice are free to pass back and forth between the two compartments. Control mice tend to spend a larger proportion of time in the dark compartment. When given an anxiolytic agent, the mice spend more time exploring the more novel brightly lit compartment and exhibit a delayed latency to move to the dark compartment. Moreover, the mice treated with the anxiolytic agent exhibit more behavior in the white compartment, as measured by exploratory rearings and line crossings. Since the mice can habituate to the test situation, naive mice should always be used in the test.
  • the administration of the compounds of the present invention is expected to result in the mice spending more time in the larger, brightly lit area of the test chamber.
  • the anxiolytic activity of a putative agent can be identified by the increase of the numbers of line crossings and rears in the light compartment at the expense of the numbers of line crossings and rears in the dark compartment, in comparison with control mice.
  • a second preferred animal model is the rat social interaction test described by Jones, B.J. et al., supra, wherein the time that two mice spend in social interaction is quantified.
  • the anxiolytic activity of a putative agent can be identified by the increase in the time that pairs of male rats spend in active social interaction (90% of the behaviors are investigatory in nature). Both the familiarity and the light level of the test arena may be manipulated. Undrugged rats show the highest level of social interaction when the test arena is familiar and is lit by low light. Social interaction declines if the arena is unfamiliar to the rats or is lit by bright light. Anxiolytic agents prevent this decline. The overall level of motor activity may also be measured to allow detection of drug effects specific to social behaviors.
  • the efficacy of the glycine and excitatory amino acid antagonists to inhibit glutamate neurotoxicity in rat brain cortex neuron cell culture system may be determined as follows.
  • An excitotoxicity model modified after that developed by Choi Choi, D.W., J. Neuroscience 7:357 (1987) may be used to test anti-excitotoxic efficacy of the novel glycine and excitatory amino acid antagonists.
  • Fetuses from rat embryonic day 19 are removed from time-mated pregnant rats. The brains are removed from the fetuses and the cerebral cortex is dissected.
  • Cells from the dissected cortex are dissociated by a combination of mechanical agitation and enzymatic digestion according to the method of Landon and Robbins (Methods in Enz mology 124:412 (1986)).
  • the dissociated cells are passed through a 80 micron nitex screen and the viability of the cells are assessed by Trypan Blue.
  • the cells are plated on poly-D-lysine coated plates and incubated at 37°C in an atmosphere containing
  • LDH lactate dehydrogenase
  • the anticonvulsant activity of the glycine and excitatory amino acid antagonists may be assessed in the audiogenic seizure model in DBA-2 mice as follows. DBA-2 mice may be obtained from Jackson Laboratories, Bar Harbor, Maine.
  • mice at an age of ⁇ 27 days develop a tonic seizure within 5-10 seconds and die when they are exposed to a sound of 14 kHz (sinus wave) at 110 dB (Lonsdale, D. , Dev. Pharmacol. Ther. 4:28 (1982)).
  • Seizure protection is defined when animals injected with drug 30 minutes prior to sound exposure do not develop a seizure and do not die during a 1 minute exposure to the sound. 21 day old DBA-2 mice are used for all experiments. Compounds are given intraperitoneally in either saline, DMSO or polyethyleneglycol-400. Appropriate solvent controls are included in each experiment. Dose response curves are constructed by giving increasing doses of drug from 1 mg/kg to 100 mg/kg. Each dose group (or solvent control) consists of at least six animals.
  • the anticonvulsant efficacy of the glycine receptor antagonists may be assessed in the pentylenetetrazol (PTZ)-induced seizure test as follows. Swiss/Webster mice, when injected with 50 mg/kg PTZ (i.p.) develop a minimal clonic seizure of approximately 5 seconds in length within 5-15 minutes after drug injection.
  • Anticonvulsant efficacy of a glycine/excitatory amino acid antagonist (or other) drug is defined as the absence of a seizure when a drug is given 30 minutes prior to PTZ application and a seizure does not develop for up to 45 minutes following PTZ administration.
  • Glycine/excitatory amino acid antagonist or other drugs are given intraperitoneally in either saline, DMSO or polyethyleneglycol-400. Appropriate solvent controls are included in each experiment. Dose response curves are constructed by giving increasing doses of drug from 1 mg/kg to 100 mg/kg. Each dose group (or solvent control) consists of at least six animals.
  • mice are injected with 200 mg/kg N-methyl-D-aspartate (NMDA) i.p., the animals will develop seizures followed by death within 5-10 minutes.
  • Glycine/excitatory amino acid antagonists are tested for their ability to prevent NMDA-induced death by giving the drugs i.p. 30 minutes prior to the NMDA application.
  • Glycine/excitatory amino acid antagonist or other drugs are given intraperitoneally in either saline, DMSO or polyethyleneglycol-400. Appropriate solvent controls are included in each experiment. Dose response curves are constructed by giving increasing doses of drug from 1 mg/kg to 100 mg/kg. Each dose group (or solvent control) consists of at least six animals.
  • the series of different evaluations may be conducted on doses of the glycine/excitatory amino acid antagonists of the invention to determine the biological activity of the compounds both in normal gerbils and in animals exposed to 5 minutes of bilateral carotid occlusion. See Scheme XVI.
  • naive gerbils are injected with either saline or differing doses of the antagonist.
  • the behavioral changes are assessed using a photobeam locomotor activity chamber which is a two foot circular diameter arena with photobeam detection. Animals are individually placed in the 2 foot diameter chambers. The chambers are housed in a cabinet which is closed and noise is abated using both a background white noise generator and a fan. Animals are placed in these chambers in the case of the initial pharmacological evaluation for a period of 6 hours and the total activity during each successive hour is accumulated using the computer control systems.
  • mice Following the initiation of reperfusion, animals are placed into the circular locomotor activity testing apparatus and the activity at the beginning of the first hour following reperfusion is monitored for the subsequent four hours.
  • Control animals not exposed to ischemia and given injections of saline prior to being placed in the locomotor activity chamber show a characteristic pattern of activity which in the first hour of locomotor activity is substantially higher than during all other hours and progressively declined over the four hours to a very low value.
  • control animals that are exposed to five minutes of cortical ischemia demonstrate a completely different pattern of locomotor activity.
  • gerbils are pretreated with the glycine/excitatory amino acid antagonists of the invention 30 minutes before the onset of carotid occlusion and then placed into the locomotor activity following one hour of reperfusion. It is expected that pretreatment of the gerbils with the glycine/- excitatory amino acid antagonists of the invention will prevent both the post- ischemic decrease and increase in activity. Post-ischemic decreases in activity are expected to be near zero during the first hour following reperfusion. Pretreatment with the glycine/excitatory amino acid antagonists of the invention is expected to reduce or prevent this early depression of behavior. In addition, the glycine/excitatory amino acid antagonists of the invention are expected to prevent the post-ischemic stimulation of behavior.
  • gerbils are also evaluated with multiple injections of the glycine/excitatory amino acid antagonists of the invention. Doses are administered I.P. at 6 hours, 4 hours, 2 hours and 30 minutes prior to the onset of 5 minutes of ischemia.
  • the effects of 5 minutes of bilateral carotid occlusion on neuronal cell death in the dorsal hippocampus may be evaluated in animals 7 days after ischemia reperfusion injury. Previous studies have demonstrated that neuronal degeneration begins to occur around 3 days following cerebral ischemia. By 7 days those neurons which have been affected and will undergo cytolysis and have either completed degeneration or are readily apparent as dark nuclei and displaced nuclei with eosinophilic cytoplasm with pycnotic nuclei. The lesion with 5 minutes of ischemia is essentially restricted within the hippocampus to the CAl region of the dorsal hippocampus.
  • the intermedial lateral zone of the horn is unaffected and the dentate gyrus and/or in CA3 do not show pathology.
  • Gerbils are anesthetized on day 7 following ischemia with 60 mg/kg of pentobarbital. Brains are perfused transcardiac with ice-cold saline followed by buffered paraformaldehyde (10%). Brains are removed, imbedded and sections made. Sections are stained with hematoxylin-eosin and neuronal cell counts are determined in terms of number of neuronal nuclei/ 100 micrometers. Normal control animals (not exposed to ischemia reperfusion injury) will not demonstrate any significant change in normal density nuclei within this region.
  • Exposure to five minutes of bilateral carotid occlusion results in a significant reduction in the number of nuclei present in the CAl region. In general, this lesion results in a patchy necrosis instead of a confluent necrosis which is seen if 10 minutes of ischemia is employed.
  • Pretreatment with the glycine receptor antagonists of the invention are expected to produce a significant protection of hippocampal neuronal degeneration.
  • Tissue injury such as that caused by injecting a small amount of formalin subcutaneously into the hindpaw of a test animal has been shown to produce an immediate increase of glutamate and aspartate in the spinal cord (Skilling,
  • NMDA receptor antagonists can block dorsal horn neuron response induced by subcutaneous formalin injection
  • NMDA receptor antagonists have potential for the treatment of chronic pain such as pain which is caused by surgery or by amputation (phantom pain) or by infliction of other wounds (wound pain) .
  • chronic pain such as pain which is caused by surgery or by amputation (phantom pain) or by infliction of other wounds (wound pain) .
  • conventional NMDA antagonists such as MK801 or CGS 19755, in preventing or treating chronic pain, is severely limited by the adverse PCP-like behavioral side effects that are caused by these drugs.
  • the glycine receptor antagonists that are the subject of this invention will be highly effective in preventing chronic pain in mice induced by injecting formalin subcutaneously into the hindpaw of the animals. Because the glycine/excitatory amino acid antagonists of this invention are expected to be free of PCP-like side effects, these drugs are highly useful in preventing or treating chronic pain without causing PCP-like adverse behavioral side effects.
  • mice Male Swiss/Webster mice weighing 25-35 grams are housed five to a cage with free access to food and water and are maintained on a 12 hour light cycle (light onset at 0800h).
  • the glycine receptor antagonist is dissolved in DMSO at a concentration of 1-40 and 5-40 mg/ml, respectively. DMSO is used as vehicle control. All drugs are injected intraperitoneally (l ⁇ l/g). The formalin test is performed as described (Dubuisson and Dennis, Pain 4:H161-174 (1977)). Mice are observed in a plexiglass cylinder, 25cm in diameter and 30cm in height.
  • the plantar surface of one hindpaw is injected subcutaneously with 20/il of 5 % formalin.
  • the degree of pain is determined by measuring the amount of time the animal spends licking the formalin-injected paw during the following time intervals: 0-5' (early phase); 5'-10', 10'-15' and 15'-50' (late phase).
  • vehicle (DMSO) or drugs dissolved in vehicle at doses of lmg/kg to 40mg/kg are injected intraperitoneally 30 minutes prior to the formalin injection. For each dose of drug or vehicle control at least six animals are used.
  • compositions within the scope of this invention include all composi ⁇ tions wherein the compounds of the present invention are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is with the skill of the art.
  • the compounds may be administered to mammals, e.g.
  • a suitable intramuscular dose would be about 0.0025 to about 15 mg/kg, and most preferably, from about 0.01 to about 10 mg/kg.
  • the pharmaceutical compositions of the invention may comprise the compounds of the present invention at a unit dose level of about 0.01 to about 50 mg/kg of body weight, or an equivalent amount of the pharmaceutically acceptable salt thereof, on a regimen of 1-4 times per day.
  • the compounds of the invention When used to treat chronic pain or to induce anesthesia, the compounds of the invention may be administered at a unit dosage level of from about 0.01 to about 50mg/kg of body weight, or an equivalent amount of the pharmaceutically acceptable salt thereof, on a regimen of 1-4 times per day.
  • a unit dosage level of from about 0.01 to about 50mg/kg of body weight, or an equivalent amount of the pharmaceutically acceptable salt thereof, on a regimen of 1-4 times per day.
  • the exact treatment level will depend upon the case history of the animal, e.g., human being, that is treated. The precise treatment level can be determined by one of ordinary skill in the art without undue experimentation.
  • the unit oral dose may comprise from about 0.01 to about 50 mg, preferably about 0.1 to about 10 mg of the compound.
  • the unit dose may be administered one or more times daily as one or more tablets each containing from about 0.1 to about 10, conveniently about 0.25 to 50 mg of the compound or its solvates.
  • the compounds of the invention may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 0.25 to 75 percent of active compound(s), together with the excipient.
  • non- toxic pharmaceutically acceptable salts of the compounds of the present invention are also included within the scope of the present invention.
  • Acid addition salts are formed by mixing a solution of the particular azepine of the present invention with a solution of a pharmaceutically acceptable non-toxic acid such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, and the like.
  • Basic salts are formed by mixing a solution of the particular azepine of the present invention with a solution of a pharmaceutically acceptable non-toxic base such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate and the like.
  • compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention.
  • animals Foremost among such animals are humans, although the invention is not intended to be so limited.
  • compositions of the present invention may be administered by any means that achieve their intended purpose.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the new pharmaceutical preparations may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, are present at a concentration of from about 0.01 to 99 percent, together with the excipient.
  • compositions of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes.
  • pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose,
  • disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetyl- cellulose phthalate or hydroxypropymethyl-cellulose phthalate, are used.
  • Dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin.
  • stabilizers may be added.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active compounds with a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water- soluble salts and alkaline solutions.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400).
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the glycine ligands of the present invention may be used to characterize the glycine binding site.
  • Particularly preferred azepines of the present invention which may be used for this purpose are isotopically radiolabelled derivatives, e.g. where one or more of the atoms are replaced with 3 H, ⁇ C, 1 C, 15 N, or 18 F.
  • Diazomethane This reagent was prepared according to the method of Black (Black, T. H., Aldrichchimica Acta 16(1):3-10 (1983)). To a stirred solution of potassium hydroxide (25 g) in water/95 % ethanol (40 mL/50 mL) at 65°C, contained in a single unit reaction vessel/efficient water-cooled distillation condenser and equipped with a dry ice/acetone receiving flask, there was added in a dropwise manner a solution of Diazald R (25.0 g, 117 mmol) in diethyl ether (225 mL). The drip rate was so adjusted so that a steady stream of etherial diazomethane distilled into the collection flask.
  • the reaction was added in portions with swirling to 50 mL of crushed ice, with additional ice being added as needed. The final volume was 125 mL.
  • the resulting cream precipitate was collected by filtration and washed with ice water (4 x 15 mL). The precipitate was resuspended in water (100 mL) and the suspension was adjusted to pH 8 with solid sodium bicarbonate. A 250 mL portion of 30% methanol, 70% chloroform was added to dissolve the product. The layers were separated and the organic portion was washed with 30% methanol, 70% water (3 x 170 mL). The organic portion was filtered through a cotton plug and the solvent was removed in vacuo to yield a bone powder (2.6 g). Crystallization from 95 % ethanol
  • the catalyst was removed by filtration and was washed with fresh reaction solvent (50 mL). The solvent was removed in vacuo to yield an orange powder. A second portion of 8 was similarly treated. The combined crude reaction products were precipitated from methanol/ethyl acetate (1: 1, v/v, dissolved in 150 mL, concentrated to
  • the combined brown powder consisted of a mixture of 16, 17 and side products as determined by TLC analysis (57% 2-propanol, 20% dioxane, 11.5 % water, 11.5 % ammonium hydroxide).
  • the side products were removed by precipitation from 95 % ethanol (dissolve in 75 mL, concentrate to 25 mL) to yield 91 mg of a brown powder.
  • This mixture was dissolved in boiling 95% ethanol (100 mL). The resulting solution was allowed to stir at room temperature for one minute then 10% aqueous sulfuric acid (10 mL) was added. The reaction was allowed to stir at room temperature for 5 minutes and an additional portion of the acid solution (90 mL) was added. A precipitate formed after a few minutes.
  • the solid was suspended in 10% methanol, 90% ethyl acetate (30 mL) and heated to boiling to yield a solid suspended in an orange solution.
  • the solid was collected by filtration, washed with ethyl acetate (2 2 mL) and dried in vacuo to yield a yellow powder (120 mg).
  • TLC analysis ethyl acetate indicated the powder to be the major reaction product, while the solution contained a mixture of at least 4 components.
  • the faster eluting fraction was crystallized from hexanes (dissolved in 300 mL, concentrated to 200 mL) with charcoal decolorization (0.5 g) to yield 5-nitro-l-tetralone as pale yellow laths (3.9 g, 12%); mp 99-100°C (hexanes), lit. (Biggs et al., J. Med. Chem.
  • the reaction was allowed to stir at rt for 1.5 hr, after which no further gas evolution was noted (an orange oil was present).
  • the reaction mixture was extracted with EtOAc (3 x 100 mL).
  • the extract was washed with water (2 x 100 mL), dried over anhyd Mg 2 SO 4 and the solvent removed in vacuo to yield a brown oil (1.4 g).
  • the crude material was purified chromatographically (silica gel, 10 mL, 2% EtOAc, 98% hexanes).
  • the aqueous portion was decanted from a solid.
  • the solid was suspended in EtOAc (150 mL), collected by filtration and washed with a second portion of EtOAc (100 mL). The solid residue was discarded.
  • the above aqueous portion was extracted with EtOAc (2 x 100 mL). All EtOAc portions were combined and washed with brine (2 x 100 mL), saturated NaHCO 3 (200 mL) and brine (2 x 100 mL).
  • the organic portion was dried over anhydrous Na 2 SO 4 and the solvent removed in vacuo to yield a dark oil.
  • the oil was washed with hexanes (3 x 200 mL) and the remaining residue discarded.
  • the hexanes portion was allowed to stand for 4 d at rt to yield a brown solid.
  • the hexanes were decanted (vide infra), the solid collected, washed with hexanes (3 x 10 mL) and dried in vacuo (2.7 g).
  • a second portion was collected at an oven temperature of 90-95°C (7.1 g), which consisted of a volatile product mixture containing 2 major and multiple minor products.
  • This material was subjected to silica gel chromatography (Mallinkrodt, Grade 62, 60-200 mesh, 50 x 5.5 cm) with EtOAc, hexanes elution (5%, 95 %).
  • the two major products were isolated to yield a faster eluting material (oil, 1.9 g), a mixture (oil, 2.3 g), and a slower eluting material (solid, 2.5 g).
  • the faster eluting oil was dissolved in boiling petroleum ether (37-57°C, 8 mL) and the solution was allowed to cool to rt to yield an oil.
  • 4-Br-DDHB was prepared by bromination of DDHB, as described (Birchall, G.R. and Rees, A.H., Can. J. Chem. 52:610-615 (1974)).
  • Benzazepines were dissolved in the standard external solution by sonication at 40°. Lack of visible precipitate after centrifugation at 3000 rpm for 5 min was considered evidence for complete dissolution.
  • the maximal solubility of 8-Me-DDHB was approximately 50 ⁇ M, whereas that of the other benzazepines was slightly higher.
  • the parent molecule, DDHB has previously been described (Birchall, G.R. and Rees, A.H., Can. J. Chem.
  • Dizocilpine (MK-801; donated by Merck, Sharp & Dohme) was added to the external solution at 1 ⁇ M for experiments with L-glutamate, to block current through channels gated by the NMDA receptor.
  • Drug solutions were applied by local perfusion from a linear array of eight microcapillary tubes (2- ⁇ l Drummond microcaps, 64-mm length). Solution flow was driven by gravity in most cases. For rapid applications of L-glutamate, the solutions were driven by a peristaltic pump and flow to the microcapillary tubes was gated by a set of three-way valves (Vyticiany et al. , 7. Physiol.
  • Fig. 1 shows the inhibition of kainate- and NMDA-gated currents by DDHB at holding potentials of +50 mV and -80 mV.
  • DDHB blocked 40-45 % of the current evoked by 100 ⁇ M kainate and produced complete block of current activated by 20 ⁇ M NMDA plus 300 nM glycine. Both the onset of and recovery from block were complete within seconds.
  • Non-NMDA or kainate/ AMPA receptor-linked channels can be activated by L-glutamate, quisqualate, kainate, and AMPA (Mayer, M.L. et al., Prog. Neurobiol. 28: 197-276 (1987); Dingledine, R., et al., Crit. Rev. Neurobiol. 4: 1-96 (1988)).
  • concentration-response relation for kainate and L-glutamate in the presence of 0, 8, 20, and 50 ⁇ M antagonist.
  • 8-Me-DDHB produced a concentration-dependent blockade of the current elicited by kainate.
  • the inhibition produced by 8-Me- DDHB was completely overcome by increasing the concentration of kainate, a property expected for a competitive mechanism of antagonism.
  • Waud Waud, D.R., Methods Pharmacol. 3:471-506 (1975)
  • the model for simple competitive antagonism embodied in eq. 2 was fit simultaneously to all four concentration-response curves shown in Fig. 3.
  • Eq. 2 uses the logistic curve (eq. 1) to describe the shape of the concentration- response relationship. This method incorporates the essential features of simple competitive antagonism (Gaddum, J.H. , 7. Physiol. (Lond.) 61 : 141-150
  • kainate alone produced half-maximal activation at a concentration of 120 ⁇ M (111-131 ⁇ M, 95 % confidence interval for EC 50 from the fit of eq. 2).
  • 8-Me-DDHB antagonized the current gated by kainate with a K B of 6.4 ⁇ M (5.5-7.5 ⁇ M).
  • L-Glutamate elicits both a transient and a sustained current when applied rapidly enough to central neurons (onset, ⁇ 30-50 msec) (Kiskin, N.I., et al, Neurosci. Lett. 63:225-230 (1986)). As shown in Figs. 4 and 5, increasing concentrations of 8-Me-DDHB progressively blocked the fast transient current evoked by rapid application of 500 ⁇ M L-glutamate.
  • the magnitudes of the shifts indicate a K B of 470 nM (410-540 nM) for 8-Me- DDHB at the glycine site.
  • glycine potentiated the response to NMDA, with an EC 50 of 770 nM (690-850 nM). This value is somewhat higher than previously reported EC 50 values, which range from 90 to 700 nM (Kleckner, N.W., et al, Science (Washington, D.C.) 241 : 835-837 (1988); Huettner, J.E., Science (Washington, D. C.) 243:1611-1613 (1989);
  • Physiol. (Lond.) 428:333-357 (1990)) have recently proposed a model for desensitization of NMDA receptors in which binding of NMDA to the transmitter recognition site reduces the affinity for glycine at the allosteric potentiation site. Therefore, we considered whether the anomalously low affinity for glycine obtained in Fig. 10 could be due to the high concentration of NMDA (1 mM) used in this experiment. As shown in Fig. 12, the EC 50 for potentiation of steady state current by glycine was sensitive to the concentration of NMDA.
  • Fig. 12 represent the concentration-response relations for glycine with 5 ⁇ M, 25 ⁇ M, and 1 mM NMDA predicted by scheme 2 of Benveniste et al. (Benveniste, M., et al, J. Physiol. (Lond.) 428:333-357 (1990)).
  • the glycine EC 50 of 853 nM predicted by their model falls just outside the 95 % confidence interval of our experimental EC 50 (690-850 nM). Consistent with previous reports (Huettner, J.E., Science (Washington, D.
  • antagonist potency at the NMDA recognition site is approximately 60-fold lower than at the glycine allosteric site.
  • Inhibition produced by 50 ⁇ M 8-Me-DDHB was completely overcome by increasing the concentration of NMDA.
  • the shift in the EC 50 for NMDA from 13 to 28 ⁇ M after the addition of 50 ⁇ M 8-Me-DDHB indicates a K B of 27 ⁇ M (23-32 ⁇ M).
  • the control EC 50 for NMDA (13 ⁇ M) is consistent with previous findings (Huettner, J.E. , Science (Washington, D. C.) 243: 1611-1613
  • NMDA indicates that there was no appreciable binding of 8-Me-DDHB to the glycine allosteric site in the presence of 1 mM D-serine.
  • Structural analogues of 8-Me-DDHB were each tested, at a concentration of 100 ⁇ M, against kainate, glycine, and NMDA. As shown in Fig. 15, the three compounds shifted the kainate dose-response relation toward higher concentrations. 7-Me-DDHB produced the largest displacement, indicating a K of 27 ⁇ M (22-32 ⁇ M), compared with 63 ⁇ M (53-74 ⁇ M) for 4-Br-DDHB and 65 ⁇ M (53-80 ⁇ M) for DDHB.
  • Fig. 16 shows the antagonism produced by 100 ⁇ M DDHB, 4-Br- DDHB, and 7-Me-DDHB at the glycine allosteric site on the NMDA receptor.
  • K B values were calculated for the three antagonists assuming a competitive mechanism, in order to compare their potencies with that of 8-Me-DDHB. From the shift in the EC 50 for glycine, the K B for DDHB is estimated at 3.0 ⁇ M, compared with 9.5 ⁇ M for 7-Me-DDHB and 25 ⁇ M for 4-Br-DDHB. These values are all significantly higher than the K B of 470 nM obtained for 8-Me-DDHB (Fig.
  • DDHB DDHB-phosphate-semiconductor
  • Fig. 17 shows the shifts produced by 100 ⁇ M DDHB, 4-Br-DDHB, and 7-Me-DDHB in the concentration-response relation for NMDA. All four curves were generated in the presence of 1 mM D-serine, to saturate the glycine potentiation site.
  • NMDA at 1 mM completely overcame the inhibition produced by each of the three antagonists.
  • the K B calculated for DDHB from the data in Fig. 17 was 16 ⁇ M, which is lower than the K B value of 27 ⁇ M obtained for 8-Me-DDHB (Fig. 14). 7-Me-DDHB displayed a K of 108 ⁇ M, whereas that for 4-Br-DDHB was 81 ⁇ M.
  • Table 3 presents some additional binding data for some 7- and 8-substituted benzazepines.
  • DDHB 27 ⁇ M versus NMDA.
  • Competitive antagonism Two lines of evidence suggest that benzazepines inhibit the activation of excitatory amino acid receptors by a competitive mechanism of antagonism. First, the inhibition produced by all of the derivatives could be completely overcome by increasing the agonist concentration.
  • a key feature of the simple competitive model is that a competitive inhibitor will exhibit the same K B regardless of which agonist is used to activate the receptor; this property provides one of the main pharmacological tools for defining receptor subtypes (see, Colquhoun, D., Handb. Exp. Pharmacol. 59:59-113 (1986); Colquhoun, D., "The Relation Between Classical and Cooperative Models For Drug Action,” in Drug Receptors (H.P. Rang, ed.), Macmillan, London (1973) 149-182).
  • Our results for steady state antagonism of kainate and glutamate are in fairly good agreement with recent work (Boulter, J., et al., Science (Washington, D. C.) 249: 1033-1037 (1990); Keinanen, K., et al, Science (Washington, D.C.) 249:556-560
  • DDHB and its derivatives share a number of structural features with these parent compounds, kynurenic acid, indole-2-carboxylic acid, and quinoxaline-2,3-dione. Although direct comparison of the potency of the four parent compounds is difficult, due to the different methods that have been used to assess antagonist affinity, the available data suggest that DDHB represents an attractive lead compound. DDHB acted at the glycine modulation site, the NMDA recognition site, and non-NMDA receptors, with apparent dissociation constants of 3, 16 and 65 ⁇ M, respectively (Table 1).
  • Indole-2- carboxylic acid binds to the glycine site with a K B of approximately 25 ⁇ M (Huettner, J.E. , Science (Washington, D. C.) 243: 1611-1613 (1989)), but it has very low affinity for the other two sites (K > 0.5-1 mM).
  • a compound such as 6,8-dichloro-DDHB might be particularly potent as a glycine site antagonist, by analogy to 5,7-dichlorokynurenic acid (Baron,
  • NMDA receptor antagonists may contribute separately to neuroprotection.
  • kynurenic acid indole-2-carboxylic acid, quinoxaline-2,3-dione and 2,5-dihydro-2,5-dioxo-3-hydroxy-7H- benzazepine (DD ⁇ B)
  • DD ⁇ B has the highest apparent affinity at the glycine allosteric site, at the NMDA recognition site, and at non-NMDA receptors.
  • DD ⁇ B and derivatives thereof are neuroprotective in both in vitro and in vivo assays.

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Abstract

L'invention concerne des procédés de traitement ou de prévention de la perte neuronal associée aux attaques, à l'ischémie, aux traumatismes du système nerveux central, à l'hypoglycémie et à la chirurgie, ainsi que de traitement de maladies neurodégénératives parmi lesquelles la maladie d'Alzheimer, la sclérose latérale amiotrophique, la maladie de Huntington et le syndrome de Down, de traitement ou de prévention des conséquences néfastes de l'hyperactivité des acides aminés excitateurs, ainsi que de traitement de l'anxiété, de la douleur chronique, des convulsions et induisant l'anesthésie par administration à un animal nécessitant un tel traitement d'une benzazépine substituée possédant un pouvoir puissant de fixation au récepteur de glycine.
PCT/US1993/009288 1992-09-30 1993-09-30 2,5-dihydro-2,5-dioxo-1h-azepines et 2,5-dihydro-2-oxo-1h-azepines et leurs utilisations en tant qu'antagonistes du recepteur de glycine et d'acides amines excitateurs WO1994007500A1 (fr)

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US5476933A (en) * 1994-11-16 1995-12-19 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon Azepine synthesis via a diels-alder reaction
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US5514680A (en) * 1992-06-22 1996-05-07 The State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University Glycine receptor antagonists and the use thereof
US5597922A (en) * 1994-07-29 1997-01-28 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon Glycine receptor antagonist pharmacophore
US5801168A (en) * 1994-06-09 1998-09-01 Zeneca Limited Substituted nitrogen heterocycles

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US5514680A (en) * 1992-06-22 1996-05-07 The State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University Glycine receptor antagonists and the use thereof
US5620979A (en) * 1992-06-22 1997-04-15 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon, Eugene Oregon Glycine receptor antagonists and the use thereof
US5622952A (en) * 1992-06-22 1997-04-22 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon, Eugene Oregon Glycine receptor antagonists and the use thereof
US5502048A (en) * 1993-06-10 1996-03-26 Zeneca Limited Substituted nitrogen heterocycles
US5656626A (en) * 1993-06-10 1997-08-12 Zeneca Limited Substituted nitrogen heterocycles
US5801168A (en) * 1994-06-09 1998-09-01 Zeneca Limited Substituted nitrogen heterocycles
US5597922A (en) * 1994-07-29 1997-01-28 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon Glycine receptor antagonist pharmacophore
US5476933A (en) * 1994-11-16 1995-12-19 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon Azepine synthesis via a diels-alder reaction
WO1996015112A1 (fr) * 1994-11-16 1996-05-23 Acea Pharmaceuticals, Inc. Synthese d'azepines par reaction de diels-alder
US5708168A (en) * 1994-11-16 1998-01-13 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon, Eugene Oregon Azepine compounds

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CA2098446A1 (fr) 1994-03-31

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