WO1994007500A1 - 2,5-dihydro-2,5-dioxo-1h-azepines and 2,5-dihydro-2-oxo-1h-azepines and the use thereof as excitatory amino acid and glycine receptor antagonists - Google Patents

2,5-dihydro-2,5-dioxo-1h-azepines and 2,5-dihydro-2-oxo-1h-azepines and the use thereof as excitatory amino acid and glycine receptor antagonists Download PDF

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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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French (fr)
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Eckard Weber
John F. W. Keana
Kenton J. Swartz
Walter J. Koroshetz
Alun H. Rees
James E. Huettner
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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/en

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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.

Abstract

Methods 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'ssyndrome, treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, as well as treating anxiety, chronic pain, convulsions and inducing anesthesia are disclosed by administering to an animal in need of such treatment a substituted benzazepine which has high binding to the glycine receptor.

Description

TITLE OF THE INVENTION
2,5-DIHYDRO-2,5-DIOXO-lH-AZEPINES AND 2,5-DIΗYDRO-2-OXO-lH-
AZEPINES AND THE USE THEREOF AS EXCITATORY AMINO ACID
AND GLYCINE RECEPTOR ANTAGONISTS
The present invention was made with U.S. government support.
Therefore, the U.S. Government has certain rights in the invention.
Cross Reference to Related Applications
The present application is a continuation-in part of U.S. Application Serial No. 07/953,689, filed September 30, 1992, the contents of which are fully incorporated by reference in their entirety.
Field of the Invention
The present invention is in the field of medicinal chemistry. In particular, 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.
Background of the Invention
Glutamate is thought to be the major excitatory neurotransmitter in the brain. There are three major subtypes of glutamate receptors in the CNS.
These are commonly referred to as ainate, AMPA and N-methyl-D-aspartate (NMDA) receptors (Watkins and Olverman, Trends in Neurosci. 7:265-272 (1987)). 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. 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)).
Besides the binding sites for glutamate/NMDA and glycine, 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.
Natl. Acad. Sci. USA 85: 1307-1311 (1988); MacDonald, J.F., et al. , Neurophysiol. 58:251-266 (1987)). 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.
One of the most potent and selective drugs that bind to the PCP receptor is the anticonvulsant drug MK-801. This drug has a Kd 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. 7:3343-3349 (1987); Keana, J.F.W. , et al. , Proc. Natl. Acad. Sci. USA 86:5631-5635 (1989); Steinberg, G.K., et al. , Neuroscience Lett. 89: 193-197 1988); Church, J., et al. , In: Sigma and Phencyclidine-Like Compounds as Molecular Probes in Biology, Domino and Kamenka, eds. , Ann Arbor: NPP
Books, pp. 747-756 (1988)). The well-characterized neuroprotective efficacy of these drugs is largely due to their capacity to block excessive Ca++ influx into neurons through NMDA receptor channels which become over activated by excessive glutamate release in conditions of brain ischemia (e.g. in stroke, cardiac arrest ischemia etc.) (Collins, R.C. , Metabol. Br. Dis. 7:231-240
(1986); Collins, R.C., et al. , Annals Int. Med. 770:992-1000 (1989)).
However, the therapeutic potential of these 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)). These PCP-like behavioral side effects appear to have caused the withdrawal of MK801 from clinical development as an ischemia rescue agent. Furthermore, these PCP receptor ligands appear to have considerable abuse potential as demonstrated by the abuse liability of PCP itself.
The PCP-like behavioral effects of the PCP receptor ligands can be demonstrated in animal models: 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. 245:969 (1989); Willets and Balster, Neuropharmacology 27: 1249 (1988)); in drug discrimination paradigms, there is a strong correlation between the PCP receptor affinity of these drugs and their potency to induce a PCP-appropriate response behavior (Zukin, S.R., et al., Brain Res. 294: 174 (1984); Brady, K.T., et al , Science 275: 178 (1982); Tricklebank, M.D., et al., Eur. J. Pharmacol. 141:497 (1987)).
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)). However, competitive NMDA receptor antagonists also have PCP-like behavioral side-effects in animal models (behavioral excitation, activity in PCP drug discrimination tests) although not as potently as MK-801 and PCP (Tricklebank, M.D., et al. , Eur. J. Pharmacol. 767:127-135 (1989)).
An alternate way of inhibiting 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. Recent in vivo microdialysis studies have demonstrated that in the rat focal ischemia model, there is a large increase in glutamate release in the ischemic brain region with no significant increase in glycine release (Globus, M.Y.T., et al., J. Neurochem. 57:470-478 (1991)). Thus, theoretically, 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.
Furthermore, because 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,
K.T., et al. , Science 275: 178 (1982); Tricklebank, M.D., et al. , Eur. J. Pharmacol. 141:497 (1987)). That glycine antagonists may indeed be devoid of PCP-like behavioral side effects has been suggested by recent studies in which available glycine antagonists were injected directly into the brains of rodents without resulting in PCP-like behaviors (Tricklebank, M.D., et al. ,
Eur. J. Pharmacol. 767: 127-135 (1989)).
However, there have been two major problems which have prevented the development of 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,
N.M., et al. , J. Med. Chem. 34:1283-1292 (1991)) cannot penetrate the blood/brain barrier and therefore have no utility as therapeutic agents;
B. The only widely available glycine antagonist that sufficiently penetrates the blood/brain barrier-the drug
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;
• show potent anti-ischemic efficacy because of the non- competitive nature of their glutamate antagonism at the NMDA receptor;
• have utility as novel anticonvulsants with fewer side-effects than the PCP-like NMDA channel blockers or the competitive NMDA antagonists;
• help in defining the functional significance of the glycine binding site of the NMDA receptor in vivo.
There have been a number of reports in the literature regarding the preparation of benzazepines. For example, U.K. Patent No. 1,340,334, discloses the preparation of benzazepines having the formula:
Figure imgf000008_0001
wherein 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. According to this patent, the benzazepines are useful as intermediates for the preparation of kynurenic acid and its analogs.
Birchall and Rees, Can. J. Chem. 52:610 (1974), discloses the preparation of substituted benzazepines including 2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 4-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 3-acetoxy-2,5-dihydro-2,5-dioxo-lH-benzazepine, 2,5-dihydro- 2,5-dioxo-3-methoxy-lH-benzazepine, 7,8-dimethyl-2,5-dihydro-2,5-dioxo-3- methoxy-lH-benzazepine, by the ring expansion of the corresponding 1,4- naphthoqui nones with hydrazoic acid. Rees, A.Η., J. Chem. Soc. 3111-3116 (1959), discloses the preparation of 7,8-dimethyl-2,3,4,5-tetrahydro-2,4,5-trioxo-lH-benzazepine from 7,8- dimethyl-2,3,4,5-tetrahydro-2,5-dioxo-lH-benzazepine by condensation with N,N-dimethyl-p-nitrosoaniline followed by acid hydrolysis. According to Rees, the product was alkali-soluble but prolonged reaction with alkali gave two products, 4-carboxy-6,7-dimethyl-2-hydroxyquinoline and 5,6- dimethylisatin. See also, Moriconi and Maniscalco, J. Org. Chem. 37(2):208- 215 (1972).
James, R.A. et al., J. Heterocycl. Chem. 26(3)793-5 (1989), discloses the preparation of 2,3,4,5-tetrahydro-2,4,5-trioxo-lH-benzazepine from 3,4- epoxy-2,3,4,5-tetrahydro-2,5-dioxo-lH-benzazepine by epoxide rearrangement with concentrated sulfuric acid. When 3,4-epoxy-2,3,4,5-tetrahydro-2,5- dioxo-lH-benzazepine was heated at 85°C in acetic acid, a low yield of 2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine was obtained. 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.
Summary of the Invention
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)
Figure imgf000010_0001
I
or a tautomer thereof; wherein:
Rj is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R, and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 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)
Figure imgf000010_0002
II or a tautomer thereof; wherein:
Rj is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and R7 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 (III)
Figure imgf000011_0001
III
or a tautomer thereof; wherein:
R, is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 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)
Figure imgf000012_0001
IV
or a tautomer thereof; wherein: R, is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R, and R5 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; Re is hydrogen; R7 is hydrogen, acyl or alkyl; and Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM 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)
Figure imgf000013_0001
V
or a tautomer thereof; wherein: Rj is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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 Re is hydrogen.
The present invention also relates to the novel substituted azepines disclosed herein, and pharmaceutical compositions thereof.
The present invention resulted from the initial discovery that substituted
2,5-dihydro-2,5-dioxo-lH-benzazepines have high binding to the glycine receptor. In addition, 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. In addition, 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. Thus, the compounds of the present invention are useful for treating pathophysiologic conditions, without significant side effects or toxicity.
Description of the Figures
Figure 1 depicts graphs showing the inhibition of membrane current by 100 μM 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine evoked by 100 μM kainate (A), or 20 μM NMDA plus 300 nM glycine (B), at holding potentials of +50 mV and -80 mV. With these concentrations, DDΗB 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). This difference is not significant at p < 0.05 (Student's t test). Applicationofl00μM2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepinealone had no effect at +50 or -80 mV (C).
Figure 2A depicts a graph showing the control currents activated by 10 μM to 10 mM kainate. Figure 2B depicts a graph showing the competitive antagonism of kainate currents by 50 μM 8-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine which were activated by 40 μM 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 μM 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (13 applications in five cells), 20 μM 8-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine (■) (eight applications in five cells), or 50 μM 8- methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (•) (seven applications in three cells). Points, mean ± standard error of the normalized currents (7//max). Smooth curves, best fit of eq. 2 to all of the data points for all four antagonist concentrations (0, 8, 20, and 50 μM), with agonist EC50 = 120 μM (111-131 μM, 95 % confidence interval), slope factor = 1.39 (1.29-1.47), and antagonist K = 6.4 μM (5.5-7.5 μM). Individual fits of eq. 1 were not significantly better, at the 5 % level, than the simultaneous fit achieved with eq. 2 (FJ)197 = 0.71).
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 μM glutamate alone or with 8, 20 or 50 μM 8-methyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine. For each application of L- glutamate that included 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, the cell was first equilibrated in control external solution containing 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine at the same concentration. MK-801 was included in all solutions at 1 μM to block
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 μM L-glutamate alone). Bars, mean + standard error for 8 μM (20 applications in eight cells), 20 μM (23 applications in nine cells), and 50 μM (23 applications in 10 cells) 8-Me-DDΗB. 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 μM to 1 mM glutamate (A). In a different cell, currents were evoked by 4 μM to 1 mM L-glutamate with 50 μM 8-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy- lH-benzazepine and a control response to glutamate alone (B). Holding potential, -70 mV. MK-801 was included in all solutions at 1 μM 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 μM 8- methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (όcells), 20 μM
8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (■) (6 cells), or 50 μM 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (•) (eight cells). Points, mean + standard error of the normalized currents (7/7max). Smooth curves, best fit of eq. 2 to all of the data points for all four antagonist concentrations (0, 8, 20, and 50 μM), with agonist EC50 = 17 μM (15-19 μM,
95 % confidence interval), slope factor = 1.64 (1.74-1.80), and antagonist KB = 9.6 μM (7.8-11.8 μM). Individual fits of eq. 1 were not significantly better, at the 5 % level, than the simultaneous fit achieved with eq. 2 (F5tll7 = 1.17). 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 (ECJ0' _+ 95 % confidence limits) determined from individual fits of eq. 1 (see examples), plotted as a function of antagonist KB plus antagonist concentration (KB + [8-
Me-DDΗB]), for kainate (A) and L-glutamate (B). Straight lines, relationship expected for simple competitive antagonism given by the equation EC50' = (EC50/7^B)-»(7<rB + [antagonist]). Values for ECJQ and KB were determined from the fit of eq. 2 (see examples), as in Figs. 3 and 7. For kainate, EC50 = 120 μM; KB for 8-Me-DDΗB versus kainate = 6.4 μM. For L-glutamate, ECJ0
= 17 μM; KB for 8-Me-DDHB versus glutamate = 9.6 μM.
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 μM 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, as well as a control response to NMDA plus 50 μM 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 μM 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (8 applications in 4 cells), 10 μM 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine (■) (9 applications in 3 cells), or 50 μM 8-methyl-2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine (•) (thirteen applications in 4 cells). Points, mean +_ standard error of the normalized currents (///„_„). Smooth curves, best fit of eq. 2 to all of the data points for all four antagonist concentrations (0, 2, 10, and 50 μM), with agonist EC50 = 770 nM (690-850 nM, 95 % confidence interval), slope factor = 1.29 (1.20-1.37), and antagonist KB = 470 nM (410-540 nM). Individual fits of eq. 1 were not significantly better, at the 5% level, than the simultaneous fit achieved with eq. 2 (F5 217 =
2.03).
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 (EC50 ; +_ 95 % confidence limits) determined from individual fits of eq. 1, plotted as a function of antagonist KB plus antagonist concentration (KB + [8-Me-DDΗB]). Straight line, relationship expected for simple competitive antagonism, given by the equation EC50' = (EC50/ΛTB)-»(7-vB + [antagonist]). Values for EC50 and KB were determined from the fit of eq. 2, as in Fig. 8. The EC50 for glycine = 770 nM; KB for 8-Me-
DDHB versus glycine = 470 nM.
Figure 12 depicts a graph showing that increasing concentrations of NMDA reduce the EC50 for glycine at the allosteric potentiation site. The concentration-response relationship is shown for glycine in the presence of 25 μM NMDA (o) (9 applications in 5 cells) or in the presence of 1 mM NMDA
(A) (15 applications in 5 cells). Points, mean +_ standard error of the normalized currents (7/7max). Smooth curves, best fits of eq. 1 to all of the data points for each concentration of NMDA. With 25 μM NMDA, the EC50 for glycine was 308 nM (279-339 nM, 95 % confidence interval; n = 1.4), compared with an EC50 of 770 nM (690-850 nM; n= 1.3) with ImM NMDA. Dotted lines, concentration-response relations for glycine predicted from computer simulations of scheme 2 of Beneveniste et al. , J. Physiol. (Lond.) 428:333-357 (1990). The simulations yielded an ECJ0 for glycine of 294 nM (n = 1.2) when 5 μM NMDA was used, an EC50 of 586 nM (n = 1.1) for 25 μM NMDA, and an EC50 of 853 nM (n = 1.2) with 1 mM NMDA. Figure 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 μM 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 μM to ImM NMDA in the presence of 50 μM 8-Me-DDΗB and a control response to 1 mM NMDA without antagonist (all containing ImM D-serine).
Holding potential, -70 mV.
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 μM 8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (•) (nine applications in three cells). Points, mean +_ standard error of the normalized currents (7/7.---J. Smooth curves, best fit of eq. 2 to all of the data points for 0 and 50 μM antagonist, with agonist EC^ = 13 μM (12-14 μM, 95 % confidence interval), slope factor = 1.33 (1.25 - 1.42), and antagonist K = 27 μM (23-32 μM). Individual fits of eq. 1 were not significantly better, at the 5 % level, than the simultaneous fit achieved with eq. 2 (F, 136 =
0.58).
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. Concentration-response relations for kainate alone (O) (15 applications in five cells) or in the presence of 100 μM 2,5-dihydro-2,5-dioxo- 3-hydroxy-lH-benzazepine (•) (six applications in two cells), 100 μM 4- bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (11 applications in two cells), or 100 μM 7-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine (■) (six applications in two cells). Points, mean +_ standard error of the normalized currents I/I^). Smooth curves, best fits of eq. 1 to each concentration-response relation, with the slope factor constrained to be the same for all four curves. From the optimal fit, n = 1.47. Individual fitting of eq. 1 without the constraint of equivalent slopes was not significantly superior at the 5 % level (F3 ι85 = 0.63). The KB for each antagonist was determined from the best fit of eq. 2 to the antagonist and control data, with the slope factor held constant at the optimal value for all four curves (n = 1.47). For kainate alone, ECJQ = 120 μM (112-129 μM, 95% confidence interval). With 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, the EC50' = 305 μM (279-333 μM) and KB = 65 μM (53-80 μM). With 4-bromo-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, the EC50' = 310 μM (288-335 μM) and KB = 63 μM (53-74 μM). With 7-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, the EC50' = 566 μM (511-627 μM) and KB = 27 μM (22-32 μM).
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 100μM 2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine (•) (nine applications in two cells), 100 μM 4-bromo-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (A) (10 applications in three cells), or 100 μM 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,-^). Smooth curves, best fits of eq. 1 to each concentration-response relation. For glycine alone, EC50 = 665 μM (634-697 μM, 95 % confidence interval) and n = 1.19. With 2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, the EC50' = 23 μM (22-24 μM) and n = 1.34. With 4-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, the ECJ0' = 3.3 μM (3.2-3.5 μM) and n = 1.46. With 7-methyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, the EC50' = 7.7 μM (7.4 - 8.4 μM) and n = 1.58. 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. All currents were elicited by variable concentrations of NMDA in the presence of 1 mM D-serine, to saturate the glycine allosteric site. Concentration-response relations for NMDA alone (O) (17 applications in seven cells) or in the presence of 100 μM 7-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine (•) (11 applications in four cells), 100 μM 4-bromo-2,5-dihydro-
2,5-dioxo-3-hydroxy-lH-benzazepine (A) (10 applications in three cells), or 100 μM 7-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (■) (10 applications in three cells). Points, mean +_ standard error of the normalized currents (I/I^. Smooth curves, best fits of eq. 1 to each concentration- response relation. For NMDA alone, EC50 = 13.2 μM (12.8 - 13.7 μM, 95 % confidence interval) and n = 1.31. With DDΗB, the EC50' = 95 μM (92=99 μM) and n = 1.59. With 4-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, the EC50' = 29 μM (28-30 μM) and n = 1.75. With 7-methyl- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, the EC50' = 25 μM (24-26 μM) and n = 1.33.
Description of the Preferred Embodiments
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):
Figure imgf000021_0001
I
or a tautomer thereof; wherein:
R, is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R, and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CΗ2CONΗAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and R7 is hydrogen, acyl or alkyl.
Other substituted 2,5-dihydro-2,5-dioxo-lH-benzazepines have the Formula (II):
Figure imgf000021_0002
II
or a tautomer thereof; wherein R,-R7 are as defined above. With respect to compounds having Formula II (here R7 is H), it has been reported that the compounds exist in the triketo form and are highly insoluble. See, Moriconi and Maniscalco, J. Org. Chem. 37(2):208-15 (1972); and Maniscalco, J.A., Diss. Abstr. Int. B 32(2):781 (1971). 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 Na2CO3, NaOH, KOH or choline hydroxide. However, prolonged exposure to alkali results in the formation of the corresponding 4-carboxy-2-hydroxyquinoline. See. Rees, A.H., J. Chem. Soc. 3111-3116 (1959). Thus, the compounds having Formula 77 should be dissolved in aqueous alkali immediately before administration to an animal.
Preferred compounds within the scope of Formula I are wherein R2 is halo or nitro, R3 is hydrogen or halo, R4 is halo or haloalkyl and R5 is hydrogen. Especially preferred compounds are wherein R3 is hydrogen. Other preferred compounds within the scope of Formula I are wherein one of
R2, R3, R4 (especially R4) is amino and R5 is hydrogen. Other preferred compounds are wherein R,, R2, R5, Re and R7 are hydrogen, R3 is alkyl, e.g. methyl, and R4 is halo, e.g. bromo or chloro.
Other preferred compounds having Formula I are wherein R2 is halo or nitro, R3 is alkyl, R4 is alkyl and R5 is hydrogen. A further preferred compound is where R,, R2, R3, R5, Re and R7 are H and R4 is alkyl or halo, especially bromo.
The invention also relates to compounds of the Formula (III)
Figure imgf000022_0001
III or a tautomer thereof; wherein:
Ri is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM 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)
Figure imgf000023_0001
or a tautomer thereof; wherein:
R, is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM 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)
Figure imgf000024_0001
V
or a tautomer thereof; wherein:
Ri is H2, H(OH), H(acyloxy), or oxo; R2, R3, R, and R5 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
Re is hydrogen; with the proviso that at least one of R1, R2, R3, and R4 is other than hydrogen.
It is expected that 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 Ce.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 CM alkyl groups mentioned above.
Typical alkylthio groups include sulphur substituted by one of the C,^ alkyl groups mentioned above.
Typical acylamino groups include any C1-6acyl 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, j8-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinyl groups.
Typical amino groups include -NΗ2, -NHR8, and -NR8R9, wherein R8 and R9 are Cw alkyl groups as defined above.
Typical carbonylamido groups are carbonyl groups substituted by -NH2, -NHR8, and -NR R9 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,
8-methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-methyl-2,5- dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6,8-dichloro-2,5-dihydro-2,5- dioxo-3-hydroxy-lH-benzazepine, 6,8-dichloro-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-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 7,8-dichloro-6-bromo-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 7,8-dichloro-6-bromo-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 7,8-dibromo-6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 7,8-dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 7,8-difluoro-6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 7,8-difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine,7-chloro-8-bromo-6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine,7-chloro-8-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 7-bromo-8-chloro-6-nitro-2,5-dihydro-2 ,5-dioxo-3- hydroxy-lH-benzazepine,7-bromo-8-chloro-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 7-fluoro-8-chloro-6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 7-fluoro-8-chloro-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 8-fluoro-7-chloro-6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 8-fluoro-7-chloro-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine,7-fluoro-8-bromo-6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 7-fluoro-8-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 8-fluoro-7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 8-fluoro-7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-3-hydroxy-lH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8- trifluoromethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 6-chloro-9- nitro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8- trichloro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6,7,8,9- tetrafluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8,9- tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-nitro-7,8- dibromo-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 6-nitro-7,8- dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8- trifluoromethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6-bromo-8- trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-bromo-8- fluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6-bromo-8-fluoro- 2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-chloro-8-methyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6-chloro-8-methyl-2,5-dihydro-
2,5-dioxo-4-hydroxy-lH-benzazepine,7-chloro-8-methyl-6-nitro-2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine,7-chloro-8-methyl-6-nitro-2,5-dihydro- 2,5-dioxo-4-hydroxy- IH-benzazepine, 7-chloro-8-methyl-6-bromo-2,5-dihydro- 2,5-dioxo-3-hydroxy- IH-benzazepine, 7-chloro-8-methyl-6-bromo-2,5-dihydro- 2,5-dioxo-4-hydroxy- IH-benzazepine, 7-bromo-8-methyl-6-nitro-2,5-dihydro-
2,5-dioxo-3-hydroxy-lH-benzazepine,7-bromo-8-methyl-6-nitro-2,5-dihydro- 2,5-dioxo-4-hydroxy-lH-benzazepine,7-fluoro-8-methyl-6-nitro-2,5-dihydro- 2 , 5 -dioxo-3 -hydroxy- 1 H-benzazepine , 7-fluoro-8-methyl-6-nitro-2 , 5 -dihydro- 2, 5-dioxo-4-hydroxy- IH-benzazepine, 8-methyl-6-trifluoromethyl-2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine, 8-methyl-6-trifluoromethyl-2,5-dihydro-
2,5-dioxo-4-hydroxy- IH-benzazepine, 8-methyl-7-nitro-6-trifluoromethyl-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 8-methyl-7-nitro-6- trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 8-methyl-9- nitro-6-trifluoromethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8- methyl-9-nitro-6-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy- IH- benzazepine, 8-methyl-6,7-dichloro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8-methyl-6,7-dichloro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 8-methyl-6,7,9-trifluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8-methyl-6,7,9-trifluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 8-methyl-6,7-dibromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8-methyl-6,7-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 8-methyl-7-bromo-6-trifluoromethyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 8-methyl-7-bromo-6-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 8-methyl-7-bromo-6-fluoro-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 8-methyl-7-bromo-6-fluoro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 7,8-dimethyl-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 7-methyl-8-chloro-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine, 7-methyl-8-chloro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 7-methyl-8-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 7-methyl-8-bromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 7, 8-trimethylene-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 7, 8-trimethylene-2,5-dihydro-2,5-dioxo-4-hydroxy- IH- benzazepine and 2,5-dihydro-2,5-dioxo-3,4-epoxy-lH-benzazepine.
Other compounds which may be used in the practice of the invention include 6-chloro-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 7-chloro- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-chloro-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 9-chloro-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine, 6, 7-dichloro-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 6, 8-dichloro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,9-dichloro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,7,8-dichloro- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8,9-dichloro-2,5-dihydro-
2, 5-dioxo-3-hydroxy- IH-benzazepine, 6,8,9-trichloro-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 7, 8,9-trichloro-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 6,7,9-trichloro-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8,9- tetrachloro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6-iodo-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7-iodo-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 8-iodo-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 9-iodo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7- diiodo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,8-diiodo-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,9-diiodo-2,5-dihydro-2,5- dioxo-3-hydroxy-lH-benzazepine, 7,8-diiodo-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine, 8,9-diiodo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,8,9-triiodo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7,8,9-triiodo- 2 ,5-dihydro-2 ,5-dioxo-3-hydroxy- 1 H-benzazepine ,6,7, 9-triiodo-2 , 5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine,6,7,8-triiodo-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 6,7,8,9-tetraiodo-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine,6-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7-bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-bromo-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 9-bromo-2,5-dihydro-2,5-dioxo- 3-hydroxy- IH-benzazepine, 6,7-dibromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,
6,9-dibromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,7,8-dibromo- 2,5-dihydro-2, 5-dioxo-3-hydroxy- IH-benzazepine, 8, 9-dibromo-2,5-dihydro- 2, 5-dioxo-3-hydroxy- IH-benzazepine, 6,8,9-tribromo-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 7,8,9-tribromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7 , 9-tribromo-2,5-dihydro-2 ,5-dioxo-3-hydroxy- IH-benzazepine,
6,7,8-tribromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8,9- tetrabromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6-fluoro-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 7-fluoro-2,5-dihydro-2,5-dioxo- 3-hydroxy- IH-benzazepine, 8-fluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 9-fluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7- difluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,8-difluoro-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,9-difluoro-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 7,8-difluoro-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 8,9-difluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,8,9-trifluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,
7,8,9-trifluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,9- trifluoro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8-trifluoro-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 6,7,8,9-tetrafluoro-2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine, 6-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 7-nitro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8-nitro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 9- nitro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7-dinitro-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,8-dinitro-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 6,9-dinitro-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 7,8-dinitro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8,9-dinitro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,
6,8,9-trinitro-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7,8,9-trinitro- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,6,7,9-trinitro-2,5-dihydro- 2, 5-dioxo-3-hydroxy- IH-benzazepine, 6,7,8-trinitro-2,5-dihydro-2,5-dioxo-3- hydroxy- 1 H-benzazepine , 6,7,8, 9-tetranitro-2 ,5-dihydro-2 ,5-dioxo-3-hydroxy- IH-benzazepine, 6-cyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,
7-cyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-cyano-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 9-cyano-2,5-dihydro-2,5-dioxo- 3-hydroxy- IH-benzazepine, 6,7-dicyano-2 ,5-dihydro-2 ,5-dioxo-3-hydroxy- IH- benzazepine, 6,8-dicyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,9-dicyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7,8-dicyano-
2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8,9-dicyano-2,5-dihydro- 2,5-dioxo-3-hydroxy- IH-benzazepine, 6,8,9-tricyano-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 7, 8,9-tricyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,9-tricyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8-tricyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8,9- tetracyano-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6-methyl-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 9-methyl-2,5-dihydro-2,5-dioxo- 3-hydroxy-lH-benzazepine, 6,7-dimethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine^, 8-dimethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 6,9-dimethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7,8-dimethyl-
2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,8,9-dimethyl-2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine, 6,8,9-trimethyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 7,8,9-trimethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine, 6, 7, 9-trimethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 6,7,8-trimethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,8,9-tetramethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6-ethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 7- ethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 8-ethyl-2,5-dihydro- 2, 5-dioxo-3-hydroxy- IH-benzazepine, 9-ethyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 6,7-diethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,8-diethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,
6,9-diethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7,8-diethyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8,9-diethyl-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 6,8,9-triethyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 7,8,9-triethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,9-triethyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine,
6,7,8-triethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6,7,8,9- tetraethyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 6-n-propyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7-n-propyl-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 8-n-propyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 9-n-propyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7-di-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6, 8-di-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 6, 9-di-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 7,8-di-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8,9-di-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,8,9-tri-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 7,8,9-tri-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,9-tri-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,8-tri-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,8,9-tetra-(n-propyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6-isopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7-isopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-isopropyl-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 9-isopropyl-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 6,7-diisopropyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 6,8-diisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 6, 9-diisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 7,8-diisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8, 9-diisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 6,8,9-triisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 7,8,9-triisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,9-triisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,8-triisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,8,9-tetraisopropyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6-t-?/ -butyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 7-t-?/ϊ-butyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-tt?/t-butyl-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 9-tert-butyl-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 6,7-di-(tert-butyl)-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 6, 8-di-(tι?/τ-butyl)-2 ,5-dihydro-2 ,5-dioxo-3-hydroxy- lH-benzazepine, 6,9-di-(t-?rf-butyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 7,8-di-(t-?rt-butyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8,9-di-(t-?rt-butyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,8,9-tri-(t-?rt-butyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 7,8,9-tri-(t-?rt-butyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,9-tri-(t-?rt-butyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 6,7,8-tri-(t-?rt-butyl)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 5-benzamido-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5-
(phenylacetamido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'- phenylureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-ureido-2,5- dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-methylureido)-2,5-dihydro-2- oxo-3-hydroxy- IH-benzazepine, 5-(N'-dimethylureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N'-trimethylureido)-2,5-dihydro-2-oxo-3-hydroxy- lH-benzazepine, 4-benzamido-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 4-(phenylacetamido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 4-(N'-phenylueido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 4-ureido-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4- (N'-methylureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'- dimethylureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'- trimethylureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,5-(N'-(2- chlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(3- chlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(4- chlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'- (2,3-dichlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-
(N'-(2,4-dichlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N '-(2, 5-dichlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH- benzazepine, 5-(N'-(2,6-dichlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- lH-benzazepine, 5-(N'-(3,4-dichlorophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N'-(3,5-dichlorophenyl)ureido)-2,5-dihydro-2- oxo-3-hydroxy-lH-benzazepine, 5-(N'-(2,3,4-trichlorophenyl)ureido)-2,5- dihydro-2-oxo-3-hyd roxy- l H-benzazepi ne , 5 - (N ' - (2 , 3 , 5 - trichlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine,5-(N'- (2,3,6-trichlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N ' -(2 ,4 , 6-trichlorophenyl)-ureido)-2 ,5-dihydro-2-oxo-3-hydroxy- IH- benzazepine, 5-(N'-(2,3,4,5-tetrachlorophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N'-(2,3,4,6-tetrachlorophenyl)ureido)-2,5- dihydro-2-oxo-3 -hydroxy- IH-benzazepine , 5-(N ' -(2 , 3 ,5 , 6- tetrachlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5-(N'- (2,3,4,5,6-pentachlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH- benzazepine, 5-(N'-(2-iodophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH- benzazepine, 5-(N'-(3-iodophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH- benzazepine, 5-(N'-(4-iodophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH- benzazepine, 5-(N'-(2,3-diiodophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5-(N'-(2,4-diiodophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N'-(2,5-diiodophenyl)ureido)-2,5-dihydro-2-oxo- 3-hydroxy- IH-benzazepine, 5-(N'-(2,6-diiodophenyl)ureido)-2,5-dihydro-2- oxo-3-hydroxy-lH-benzazepine5-(N'-(3,4-diiodophenyl)ureido)-2,5-dihydro- 2-oxo-3-hydroxy- IH-benzazepine, 5-(N'-(3,5-diiodophenyl)ureido)-2,5- dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(2,3,4-triiodophenyl)ureido)- 2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(2,3,5-triiodophenyl)- ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5-(N '-(2,3,6- triiodophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'- (2,4,6-triiodophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5- (N'-(2,3,4,5-tetraiodophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH- benzazepine, 5-(N'-(2,3,4,6-tetraiodophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N'-(2,3,5,6-tetraiodophenyl)ureido)-2,5-dihydro- 2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(2,3,4,5,6-pentaiodophenyl)ureido)- 2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine5-(N'-(2-fluorophenyl)ureido)- 2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine5-(N'-(3-fluorophenyl)ureido)- 2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine5-(N'-(4-fluorophenyl)ureido)-
2 , 5 -dihydro-2-oxo-3 -hydroxy- IH-benzazepine , 5-(N ' -(2 , 3- difluorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'- (2,4-difluorophenyl)-ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5- (N ' -(2 ,5-difluorophenyl)ureido)-2 ,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5-(N'-(2,6-difluorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH- benzazepine, 5-(N'-(3,4-difluorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- lH-benzazepine, 5-(N'-(3,5-difluorophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy-lH-benzazepine, 5-(N'-(2,3,4-trifluorophenyl)ureido)-2,5-dihydro-2- oxo-3-hydroxy- IH-benzazepine, 5-(N'-(2,3,5-trifluorophenyl)ureido)-2,5- d ihydro-2-oxo-3-hydroxy- l H-benzazep i ne , 5 -(N ' - (2 , 3 , 6- trifluorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine,5-(N'- (2,4,6-trifluorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(2,3,4,5-tetrafluorophenyl)-ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH- benzazepine, 5-(N'-(2,3,4,6-tetrafluorophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N ' -(2 , 3 ,5 , 6-tetrafluorophenyl)ureido)-2 ,5-dihydro-
2-oxo-3-hydroxy- IH-benzazepine, 5-(N'-(2,3,4,5,6-pentafluorophenyl)ureido)- 2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(2-bromophenyl)ureido)- 2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(3-bromophenyl)ureido)- 2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5-(N'-(4-bromophenyl)ureido)- 2 , 5 -dihydro-2-oxo-3 -hydroxy- IH-benzazepine , 5- (N ' -(2 , 3- dibromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine,5-(N'-
(2,4-dibromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 5- (N'-(2,5-dibromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5-(N'-(2,6-dibromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH- benzazepine, 5-(N'-(3,4-dibromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 5-(N'-(3,5-dibromophenyl)-ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N'-(2,3,4-tribromophenyl)ureido)-2,5-dihydro-2- oxo-3-hydroxy- IH-benzazepine, 5-(N'-(2,3,5-tribromophenyl)ureido)-2,5- d ihydro-2-oxo-3 -hydroxy- l H-benzazepine , 5 - (N ' - (2 , 3 , 6- tribromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine,5-(N'- (2,4,6-tribromophenyl)ureido)-2 ,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine,
5-(N'-(2,3,4,5-tetrabromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH- benzazepine, 5-(N'-(2,3,4,6-tetrabromophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-benzazepine, 5-(N'-(2,3,5,6-tetrabromophenyl)-ureido)-2,5- dihydro-2-oxo-3 -hydroxy- IH-benzazepine, 5-(N ' -(2 , 3 ,4 ,5 , 6- pentabromophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 4-
(N'-(2-chlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(3-chlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine4-(N'-(4-chlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine, 4-(N'-(2,3-dichlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine,4-(N'-(2,4-dichlorophenyl)ureido)-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2,5-dichlorophenyl)ureido)-2,5- dihydro-2 , 5 -dioxo-3-hydroxy- I H-benzazepine , 4-(N ' -(2 , 6- dichlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4- (N'-(3,4-dichlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 4-(N'-(3,5-dichlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 4-(N'-(2,3,4-trichlorophenyl)ureido)-2,5-dihydro- 2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N '-(2,3 ,5-trichlorophenyl)ureido)-2,5- di hydro-2 , 5 -dioxo-3 -hydroxy- IH-benzazepine, 4-(N ' -(2 ,3 , 6- trichlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4- (N'-(2,4,6-trichlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 4-(N'-(2,3,4,5-tetrachlorophenyl)ureido)-2,5-dihydro-2,5-oxo-3- hydroxy- IH-benzazepine, 4-(N'-(2,3,4,6-tetrachlorophenyl)ureido)-2,5- dihydro-2 ,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N '-(2 ,3 ,5 ,6- tetrachlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2,3,4,5,6-pentachlorophenyl)ureido)-2,5-dihydro-2,5-oxo-3-hydroxy-lH- benzazepine, 4-(N'-(2-iodophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy- lH-benzazepine, 4-(N'-(3-iodophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 4-(N'-(4-iodophenyl)ureido)-2,5-dihydro-2,5-dioxo- 3-hydroxy- IH-benzazepine, 4-(N'-(2,3-diiodophenyl)ureido)-2,5-dihydro-2,5- dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2,4-diiodophenyl)ureido)-2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2,5-diiodophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2,6-diiodophenyl)ureido)- 2 , 5-dihydro-2 ,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N '-(3 ,4- diiodophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N'- (3,5-diiodophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2,3,4-triiodophenyl)ureido)-2,5-dihydro-2,4-dioxo-3-hydroxy-lH- benzazepine, 4-(N'-(2,3,5-triiodophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 4-(N'-(2,3,6-triiodophenyl)ureido)-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2,4,6-triiodophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2,3,4,5- tetraiodophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,4-
(N'-(2,3,4,6-tetraiodophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine,4-(N '-(2,3,5, 6-tetraiodophenyl)ureido)-2 ,5-dihydro-2 , 5-dioxo-3- hydroxy- IH-benzazepine, 4-(N'-(2,3,4,5,6-pentaiodophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2-fluorophenyl)ureido)- 2 ,5-dihydro-2 ,5-dioxo-3-hydroxy- lH-benzazepine, 4-(N ' -(3- fluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'- (4-fluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,4- (N'-(2,3-difluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 4-(N'-(2,4-difluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 4-(N'-(2,5-difluorophenyl)-ureido)-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2,6-difluorophenyl)ureido)-2,5- d ihydro-2 , 5 -dioxo-3 -hydroxy- I H-benzazepine , 4-(N ' -(3 , 4- difluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4- (N'-(3,5-difluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine , 4-(N ' -(2 , 3 ,4-trifluorophenyl)ureido)-2 ,5-dihydro-2 , 5-dioxo-3- hydroxy- IH-benzazepine, 4-(N' -(2,3 ,5-trifluorophenyl)ureido)-2 ,5-dihydro-2 ,5- dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2,3,6-trifluorophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2,4,6-trifluorophenyl)- ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2, 3,4,5- tetrafluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(2,3,4,6-tetrafluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 4-(N'-(2,3,5,6-tetrafluorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 4-(N'-(2, 3,4,5, 6-pentafluorophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2-bromophenyl)ureido)- 2 , 5 -dihydro-2 , 5 -dioxo-3 -hydroxy- IH-benzazepine, 4-(N ' -(3- bromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N'-
(4-bromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4- (N'-(2,3-dibromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy- IH- benzazepine, 4-(N'-(2,4-dibromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 4-(N'-(2,5-dibromophenyl)ureido)-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2,6-dibromophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(3,4-dibromophenyl)- ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N'-(3,5- dibromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4- (N'-(2,3,4-tribromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 4-(N'-(2,3,5-tribromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 4-(N'-(2,3,6-tribromophenyl)ureido)-2,5-dihydro- 2 ,5-dioxo-3-hydroxy- 1 H-benzazepine , 4-(N '-(2 ,4 , 6-tribromophenyl)ureido)- 2, 5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine, 4-(N'-(2,3 ,4,5- tetrabromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 4-(N ' -(2 , 3 ,4 , 6-tetrabromophenyl)ureido)-2,5-dihydro-2 ,5-dioxo-3-hydroxy- 1H- benzazepine, 4-(N'-(2,3,5,6-tetrabromophenyl)ureido)-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 4-(N'-(2,3,4,5,6-pentabromophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-chloro-6-iodo-5-(N'- phenylureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine, 8-methyl-6-iodo- 5-(N'-phenylureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, 8-methyl-6- chloro-5-(N'-phenylureido)-2,5-dihydro-2-oxo-3-hydroxy-lH-benzazepine,8- methyl-7-chloro-6-iodo-5-(N'-phenylureido)-2,5-dihydro-2-oxo-3-hydroxy- IH- benzazepine, 8-ethyl-6-chloro-5-(N'-phenylureido)-2,5-dihydro-2-oxo-3- hydroxy-lH-benzazepine, 8-ethyl-7-chloro-6-iodo-5-(N'-phenylureido)-2,5- dihydro-2-oxo-3-hydroxy-lH-benzazepine, 8-nitro-7-cyano-2,5-dihydro-2,5- dioxo-3-hydroxy-lH-benzazepine, 8-nitro-7-cyano-5-(N'-phenylureido)-2,5- dihydro-2-oxo-3-hydroxy-lH-benzazepine, 10-nitro-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-naphtho[l,2-f]azepine}0-nitro-5-(N'-phenylureido)-2,5-dihydro- 2-oxo-3-hydroxy-lH-naphtho[l,2-f]azepine, 8-chloro-6-iodo-2,5-dihydro-2,5- dioxo-3-hydroxy- IH-benzazepine, 8-methyl-6-iodo-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine, 8-chloro-6-iodo-4-(N'-phenylureido)-2,5-dihydro-2- oxo-3-hydroxy- IH-benzazepine, 8-chloro-6-iodo-4-(N'-phenylureido)-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-methyl-6-iodo-4-(N'- phenylureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine,8-methyl-7- chloro-6-iodo-4-(N'-phenylureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8-methyl-6-chloro-4-(N'-phenylureido)-2,5-dihydro-2,5-dioxo-3- hydroxy-lH-benzazepine, 8-ethyl-7-chloro-6-iodo-4-(N'-phenylureido)-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-ethyl-6-chloro-4-(N'- phenylureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-chloro-6- iodo-4-(N'-(2,4-dichlorophenyl)ureido)-2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine, 8-methyl-7-chloro-6-iodo-4-(N'-(2,4-dichlorophenyl)ureido)-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine, 8-methyl-7-chloro-6-iodo-5-(N'- (2,4-dichlorophenyl)ureido)-2,5-dihydro-2-oxo-3-hydroxy- IH-benzazepine, and 8-chloro-6-iodo-5-(N'-(2,4-dichlorophenyl)ureido)-2,5-dihydro-2-oxo-3- hydroxy- IH-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-
2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine and 8-bromo-2,5-dihydro- 2, 5-dioxo-4-hydroxy- IH-benzazepine are expected to be especially potent.
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.
Moreover, 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. For example, 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. In a preferred embodiment, 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. In addition to treatment of pain, the compounds of the invention are also expected to be useful in inducing anesthesia, either general or local anesthesia, for example, during surgery.
The 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. It is expected that such results will suggest that the 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 lμM glycine-stimulated [3H]-MK-801 in rat or guinea pig brain membrane homogenates. The more potent the glycine antagonist, the less [3H]-MK-801 can bind since the [3H]-MK801 binding site (PCP receptor) is accessible only upon opening of the ion channel by glutamate and glycine (Fletcher, E.L., et al. , in Glycine Neurotransmission, Otterson, P., et al. (eds.), John Wiley and Sons (1990); Johnson, J.W., et al , Nature 325:529 (1987)).
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. In general, 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). For example, 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. Scheme I
Figure imgf000042_0001
R *= H R = Me
Alternatively, one may treat an appropriately substituted 1,4- naphthoquinone (VII) with acetic anhydride and boron trifluoride in ether to give the corresponding substituted 1,2,4-triacetoxynaphthylene (VIII). Alternatively, this transformation may be carried out in acetic anhydride and sulfuric acid. See Thiele, J. and Winter, E., Ann. Chem. 377:341-352 (1900). The product (VIII) which may be hydrolyzed in the presence of sodium methoxide and air in methanol to give the corresponding 2-hydroxy-l,4- naphthoquinone (IX). Alkylation of the hydroxy group with, for example, diazomethane gives the substituted 2-alkoxy-l,4-naphthoquinone (X). Treatment of this compound with hydrazoic acid then gives the corresponding 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-l-benzazepine (XI) according to Birchall and Rees, Can. J. Chem. 52:610-615 (1974) (See Scheme II). Scheme II
Figure imgf000043_0001
υ i i U I I I
Figure imgf000043_0002
I X
Figure imgf000043_0003
XI
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/τ.-butoxide (see Scheme III) to give the corresponding 2-hydroxy-l,4-dioxo-l,4-dihydronaphthylene (XIII). See, Baillie and Thomson, J. Chem. Soc. (Q:2184-2186 (1966). 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.
Figure imgf000044_0001
XII
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). Using this method, one may prepare chlorinated or nitrated naphthalenes and 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. Alternatively, the naphthoquinones may be prepared by a Diels-Alder condensation reaction between benzoquinone and a diene followed by oxidation. For example, 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). Scheme TV
Figure imgf000045_0001
xiu XU
Figure imgf000045_0002
XU I I
The compounds having Formula XVIII (Re = CH2CONHAr) may be prepared by N-alkylation of the corresponding hydroxy-protected anion with a reactive halide (see Scheme V). 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 α-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). Scheme V
Figure imgf000046_0001
XIX XX
Figure imgf000046_0002
XXI XUIII
Where Re = -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. Alternatively, 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. Alternatively, where Rg is -NΗCOCΗ2Ar (XXV), acylation of the intermediate XXIV with phenylacetyl chloride leads to XXV (see Scheme VI). Scheme VI
C l - NH-
Figure imgf000047_0001
XX I I I or xxiυ
Figure imgf000047_0002
XX I I xxυ
The corresponding isomeric 2,3,4,5-tetrahydro-2,4,5-trioxo-lH-l- benzazepines may be prepared according to Moore, Η.W. et al., Tetr. Lett., 1243 (1960) and Rees, A.Η., J. Chem. Soc, 3111 (1959). For example, 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. See, Rees, A.Η., supra. Alternatively, the 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 ring expansion reaction of 2-alkoxynaphtho-l,4-quinones in the presence of hydrazoic acid to yield 2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepines (7) was reported by Birchall and Rees (Can. J. Chem. 52:610- 615 (1974)). The work detailed the preparation of compounds 1, 4, 5 and 6 as shown in Scheme VII as well as 7,8-dimethyl-2,5-dihydro-2,5-dioxo-3- hydroxy- IH-benzazepine.
Scheme VII
Figure imgf000049_0001
Figure imgf000049_0003
Figure imgf000049_0002
Treatment of 2-methoxynaphtho-l,4-quinone (7), which is readily prepared by the reaction of the corresponding hydroxy compound with diazomethane in ether (Fieser, L.J., J. Amer. Chem. Soc. 48:2922-2937 (1926)), with sodium azide in sulfuric acid was reported to yield 7 in 60% yield. Isolation of compound 5 (3-methoxy derivative) was possible if care was taken (slow addition to crushed ice with sodium bicarbonate neutralization) during the hydrolysis of the acid reaction mixture. Compound 5 could also be prepared by allowing diazomethane to react with 7 in methanol. Treatment of 7 with bromine in acetic acid yielded the 4-substituted bromide 4, while reaction with acetic anhydride in pyridine yielded the acetate 6. Compound 7 was prepared in 78% yield. Treatment with sodium azide (1.05 eq) in sulfuric acid for 18 hours cleanly yielded 5 (53 %) with no special precautions being taken. No sign of 7 was noted in the crude reaction product by TLC analysis. In the original work, it is possible that the filter cake was not sufficiently washed and residual acid was present. This residual acid would lead to the hydrolysis of 5 during the crystallization procedure resulting in the formation of 7. Employing a large excess of sodium azide does not appear to be detrimental to the reaction. The use of 1.05 to 1.10 eq resulted in 7 still being present after 18 hours at room temperature, while the use of 2 eq resulted in no 7 being noted. No side product formation was noted with the larger excess if the reaction was run at room temperature but it was noted if the reaction was heated to 50°C. The hydrolysis of 5 to 7 (82% mass recovery, 52% crystallized yield) was readily performed in warm ethanol/water in the presence of sulfuric acid. Azepine derivatives substituted on the aromatic portion were then prepared. Our initial attempt was the electrophilic aromatic nitration of compound 5. The use of potassium nitrate in sulfuric acid is a convenient method for the nitration of quinoxalinediones (Cheeseman, G.W.H., J. Chem. Soc: 1170-1176 (1962)). Since compound 5 is prepared by the treatment of compound 7 with sodium azide in sulfuric acid, it was reasoned that the in situ preparation of 5 followed by the addition of potassium nitrate to the reaction mixture would be a simple way to prepare a nitrated derivative. Thus, the addition of 1.1 eq. of potassium nitrate to the ring expanded reaction mixture resulted in a new product as determined by TLC analysis (Scheme VIII). The formation of this new product was complete 2.5 hours after the addition of the potassium nitrate at room temperature. The product 8 was isolated in 40% yield as near colorless laths. Scheme VIII
Figure imgf000051_0001
The hydrolysis of 8 to 9 in warm acidic ethanol/water goes smoothly on a scale up to 250 mg in 60% yield. Conversely, a 500 mg reaction resulted in side product formation and no pure 9 could be isolated. On the smaller scale, the product precipitated out of the reaction mixture after a relatively short period of time. On the larger scale, precipitation was delayed, thus exposing 9 to the acidic hydrolysis conditions for an extended period, which led to side product formation. Purposely exposing 9 to these conditions led to the formation of the same side products.
The position of nitration was tentatively deduced by *Η NMR spectroscopy (DMSO-d6). The aromatic region of 9 exhibited three, single proton signals at 7.55 (d, 7=9.0 Hz), 8.39 (dd, 7=9.2, 2.7 Hz) and 8.70 (d, 7=2.7 Hz). The most down field aromatic proton did not display ortho coupling but did display meta. Conversely, the most up field proton exhibited ortho coupling but not meta. These data indicated that the protons associated with these signals occupy positions 6 and 9 with positions 7 and 8 being the possible sites of substitution. Electronic considerations indicated that the preferred site of substitution is position 7 since that position is meta to the meta directing 5-carbonyl group and para to the ortho/para directing amide nitrogen. This tentative assignment has since been confirmed by X-ray crystallographic data for compound 8. These data show the nitro to be in the 7-position, that the molecule is almost planar with slight puckering occurring in the 7-membered ring and that there is hydrogen bonding between the amide hydrogens and the 2-carbonyl groups of two adjacent molecules. Even though the *H NMR and HRMS data (see experimental section) conform with the structural assignment of 9, it was necessary to confirm that a rearrangement or ring contraction reaction had not occurred during hydrolysis. This was a possibility since the Η NMR and MS data for the above described side products could readily be confused with that of 9. This confirmation was obtained by allowing 9 to react with diazomethane in methanol/dichloromethane to yield 8 as demonstrated by TLC analysis. The reaction appears to go cleanly until 9 is consumed. However, the addition of excess diazomethane results in the formation of a spectrum of products. Treatment of authentic 8 with diazomethane in a similar manner gives this same product distribution.
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). This surprising result combined with the knowledge that compound 2, the 8-methyl derivative, showed increased affinity relative to compound 7 led to the preparation of a derivative incorporating an electron donating substituent. The most direct way to do this was to reduce the available nitro analogs to the corresponding amines. Table 1. Structure-activity relationships for 7-substituted benzazepines.
Figure imgf000053_0001
Cpd # R IC50 (μM) Ki (μM) Potency relative to DCK 1 H 7.5 (7.8) 0.68 (0.71) 35 % (32%) 9 N02 300 27.3 0.90% 15 AcNH 92.5 8.4 2.6 % 17 N3 inactive 21 Br inactive
The preparation of the amine analogs is outlined in Scheme IX. The synthesis started with compound 8 because of its ready availability and its superior solubility characteristics relative to compound 9. The initial attempts at reduction were with the reportedly mild reducing conditions utilizing stannous chloride in either ethyl acetate or ethanol (Bellamy and Ou, Tetrahedron Letts. 25 8j:839-842 (1984)). These conditions proved to be unsuitable for the reduction of 8. Compound 8 has very limited solubility in either solvent at room temperature. No detectable reaction was noted at room temperature when a suspension of 8 in either solvent was allowed to stir in the presence of the reducing agent, though some discoloration was noted. Heating the reaction mixture with either solvent resulted in decomposition. This
decomposition is not surprising considering the acidic nature of stannous chloride reagent and the sensitivity of these benzazepines to acidic conditions.
Scheme IX
DECOMPOSITION WHEN HEATED
NO REACTION AT ROOM TEMPERATURE
Figure imgf000054_0001
13
ι-> , Pcl/C
13 (Not synthetical ly ef fecti ye)
QUI NOLINE
The next attempts utilized the method of catalytic hydrogenation. As a baseline experiment, 8 was hydrogenated in a solution of 1: 1 methanol/dichloromethane in the presence of a palladium on carbon catalyst (10%) at 20 psi for one hour. TLC analysis showed the total consumption of 8 and the formation of one new spot. However, !H NMR analysis (DMSO-d6) showed the presence of the desired amine 10 and the over- reduced analog 77 in a ratio of about 4 to 6 respectively. Reducing the pressure to 5 psi and the reaction time to 15 minutes improved the ratio to about 8 to 2 but the presence of under-reduced material, presumed to be the hydroxyl amine 72, was noted by TLC analysis. At this point, it did not seem probable that simply reducing the reaction pressure and time would improve the ratio of 10 to 11 without obtaining an unacceptable percentage of 72. Therefore, the catalyst poison quinoline (Lindlar, H., Helv. Chim. Acta 35:446-450 (1952)) (20% by weight relative to the catalyst) was added to the reaction mixture. With the poison present and with a hydrogen pressure of 5 psi, it took 60 minutes for the reaction to achieve an acceptable point of completion. TLC analysis at this point showed the amine(s) to be the major spot with a detectable amount of 72 being present. !H NMR analysis showed 10, 11 and 72 to be present in a ratio of approximately 90:5:5. Precipitation from methanol/ethyl acetate yielded pure 10 as a orange powder in 39% yield. It may also be possible to reduce the amount of reduction of the 3,4-double bond by reducing the 3-ethoxy analog instead of the 3-methoxy compound 8. Hydrolysis of 10 under the conditions employed for 8 failed to yield
73. Initially, it was believed that the failure was due to the probable high solubility of protonated 73 in aqueous media which prevented its precipitation from the reaction medium. As noted above, prolonged exposure of these compounds to aqueous acid results in decomposition. Allowing the reaction to proceed at room temperature slowly converted 10 to 73 as shown by TLC analysis but degradation of the product occurred at rate competitive with its formation.
Direct catalytic reduction of 9 was also attempted. This was not effective under the conditions employed for the reduction of 8 because of the reduced solubility of 9. Employing DMF as the reaction solvent gave a mixture of 73 plus its over- and under-reduced analogs. Unfortunately, purification could not be readily effected and the attempt was abandoned.
Since 73 was not readily accessible, the preparation of acetamide 75, a potential intermediate for the preparation of the corresponding amine, was attempted (Scheme X). The reduction of 8 as described above yielded the crude mixture of 10, 11, and 72. After filtration to remove the catalyst, the resulting solution was treated with an excess of acetic anhydride. TLC analysis indicated complete conversion of the amine mixture to a slightly lower Rf spot after a few hours. Solvent removal yielded crude 14 as a yellow powder. An analytical sample was prepared by precipitation from ethanol (57% recovery). The hydrolysis of crude 8 to 9 in warm, acidic ethanol/water readily gave 75 in 46% yield after crystallization from ethanol. No trace of over- or under-reduced side products were noted by *H NMR analysis.
Scheme X
Figure imgf000056_0001
Again, the binding data were unexpected. Compound 75 had a lower affinity for the glycine site than did compound 7 (Table 1). Since both an electron withdrawing and donating substituent at position 7 reduced the binding affinity relative to compound 7, it was reasoned that the steric bulk of substituents at position 7 may not allow the molecule to readily fit into the receptor site. In support of this, the 7-methyl analog also exhibits decreased binding affinity relative to compound 7. To help confirm this hypothesis, we attempted the preparation of the 7-halo and the 7-azido analogs in order to determine if this positional inhibitory effect was a general phenomenon for this class of compounds.
The preparation of azide 77 from crude 10 is outlined in Scheme XI. The crude amine reaction mixture, derived from the catalytic reduction of 9, was converted to the corresponding hydrochloride by the acidification of an ethyl acetate solution with anhydrous HC1 at room temperature followed by solvent removal. Diazatization was accomplished in a cold (0 to 5°C) hydrochloric acid solution by the addition of sodium nitrite. The addition of sodium azide, followed by stirring at room temperature yielded a brown precipitate, which consisted of a mixture of 77, the methoxy analog (16) and side products. The mass recovery from this reaction was poor (30 to 40% based on three runs) and appeared to be independent of the concentration at which the reaction was run. The side products were removed at this point by a precipitation from ethanol. Treatment with warm, acidic ethanol/water cleanly converted the residual 76 to 77. The final yield of 77 based on starting amine hydrochloride was 18%. Compound 77 exhibited the characteristic strong absorbance for azides in its IR spectrum (2116 cm"1, KBr).
Scheme XI
Figure imgf000057_0001
16 R = CH;
17 R = H
The attempts made to prepare a 7-halo derivative are outlined in Scheme XII. Treatment of compound 5 with either NCS or NBS in DMF (Mitchell, R.H., et al. , J. Org. Chem. ^:4733-4735 (1979)) yielded the 4- chloro (78) and the 4-bromo (79) derivatives respectively. Interestingly, the reaction utilizing NCS was much cleaner than that utilizing NBS. Whereas the addition of one equivalent of NCS to 5 gave a fairly clean mixture of 5 and 78 after 24 hours at room temperature, as determined by TLC and !H NMR analysis, the addition of one equivalent of NBS gave a mixture of 5, 79 and substantial quantities of side products under the same conditions. In neither case was there evidence for aromatic substitution.
Figure imgf000058_0002
20
H2S04 , H20 , E tOH , Δ
Figure imgf000058_0001
A second attempt was made utilizing bromine and silver sulfate in sulfuric acid, which has been found to be an effective method for bromination of quinoxalinediones (Cheeseman, G.W.H., 7. Chem. Soc : 1170-1176 (1962)). As in the case with the nitration reaction, compound 5 was prepared in situ by the treatment of compound 7 with sodium azide in sulfuric acid, after which the bromination reagents (1 eq. each) were added. After 72 hours at room temperature, little if any reaction was noted. As a hunch, NBS was added at this point and the reaction was allowed to stir for an additional 24 hours at room temperature. TLC and *H NMR analysis indicated the possibility that aromatic substitution had occurred.
In order to insure that NBS was in and of itself responsible for the above observed reaction, a new run was performed in which compound 5 was prepared in situ, followed by the addition of NBS (1.1 eq). After 24 hours, !H NMR analysis of a hydrolyzed sample showed approximately 60% conversion of 5. The addition of more NBS (0.55 eq) and continued stirring
(96 hours) resulted in total consumption of 5. TLC analysis indicated that at least 4 products were formed. After an aqueous workup, the major product was readily isolated owing to its relatively poor solubility in organic solvents. The material isolated from the workup was boiled in a solution of 10% methanol, 90% ethyl acetate to yield a red solution and a yellow solid. The solid was near pure major product. Crystallization yielded the 3-methoxy derivative as gold plates (6%). Treatment with warm, acidic ethanol/water cleanly converted this 3-methoxy product to the 3-hydroxy analog (74%).
Since the analytical data of the crude reaction mixture indicated that there may be multiple sites of substitution, the position of bromine substitution cannot be made by analogy to the single isomer nitration reaction. However, these compounds have been tentatively assigned structures 20 and 27 (7- bromo) based on the Η NMR spectroscopic evidence. An examination of the spectrum of compound 1 (DMSO-rf6) shows four distinct aromatic proton signals. These are as follows δ 7.26 (t, J = 7.5 Hz, 1 H), 7.46 (d, J = 8.1
Hz, 1 H), 7.61 (t, J = 7.2 Hz, 1 H), 8.03 (d, J = 7.8 Hz, 1 H). 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.
Comparing these data with those of the aromatic region of 27 (DMSO- d6), δ 7.41 (d, J = 9.0 Hz, 1 H), 7.80 (dd, J = 8.7, 2.4 Hz, 1 H), 8.10 (d,
J = 2.1 Hz, 1H)) one can see that the signal associated with proton 7 in compound 1 is no longer present, while the other three signals are still present with only modest changes in their chemical shifts. It is also apparent that the doublet of doublets assigned to the proton at position 8 is ortho coupled to the doublet at 7.41 (assigned to the proton at position 9) and meta coupled to the doublet at 8.10 (assigned to the proton at position 6). This assignment has since been confirmed by an independent total synthesis of the 8-isomer (vide infra.)
The preparation of 8-substituted benzazepines was also attempted. Since our experience has shown that direct electrophilic substitution occurs preferably at position 7, the preparation of compounds with other substituent positions will require the preparation of the benzazepine with the substituent already in place. A retrosynthetic scheme for doing this at the 8 position is shown in Scheme XIII. Scheme XIII
Figure imgf000061_0001
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. (C):2184-2186 (1966)), which are obtained by the cyclization of a 4'-substituted-4-phenylbutyric acids (Evans and Smith, J. Chem. Soc : 798-800 (1954)). These may be obtained by the hydrogenation of corresponding 2- or 3-butenoic acids, which may be synthesized by the palladium catalyzed coupling of a 4-substituted phenyl tributyl tin reagent with methyl 4-bromocrotonoate (Owton and Brunavs, Synth. Commun. 21 (8&9): 981-987 (1991)) or via a Wittig reaction of a 4-substituted benzaldehyde with the triphenylphosphonium salt of 3-bromopropionic acid (Beugelmans, R., et al , J. Org. Chem. 50:4933-4938 (1985)).
For the sake of speed and efficiency, one would wish to start up as far on the retrosynthetic scheme as possible. Attempted nitrations of 2-hydroxy and 2-methoxynaphtho-l,4-quinones failed to yield aromatic substituted products. However, nitration of 1-tetralone (22, Scheme XIV) under the conditions described above (potassium nitrate in sulfuric acid) yielded both -(23) and 5-nitro-l-tetralone (24).
Catalytic hydrogenation of 23 (H2, Pd, 2-propanol) yielded the corresponding 7-amino analog 25 in 76% yield. Treatment of this amine with acetic anhydride in methanol yielded the corresponding 7-acetamide 26 in 89% yield. Also, the 7-azide 27was prepared in 90% yield by reacting 23 initially with sodium nitrite and then with sodium azide in aqueous hydrochloric acid.
Unfortunately, exposing 23, 25, and 26 to the conditions described for the oxidation of tetralones to 2-hydroxynaphtho-l ,4-quinones (0.3 M potassium t-butoxide in t-butanol in the presence of oxygen; Baillie and Thompson, 7.
Chem. Soc. C2184-2186 (1966)), failed to yield the desired oxidation products. Compound 27 yielded the desired quinone 28 in very poor yield.
Scheme XIV
Figure imgf000063_0001
Attempts have also been made to brominate 22 (Scheme XV). It is hoped that preparing a bromo substituted benzazepine by this route will confirm the assignment made for 27 and also yield a new bromo isomer. The initial attempt utilized NBS in DMF (Mitchell, R.H., et al. , J. Org. Chem. 44:4733-4735 (1979)). No reaction was noted by either TLC or Η NMR analysis. Based on the experience with the benzazepines, 22 was allowed to react with NBS in sulfuric acid. A mixture of products was obtained from which 7-bromo (29) and 5-bromo-l-tetralone (30) were isolated in poor yield (4% each, Scheme XV). Compound 29 was oxidized to 37 (34%), which was methylated to 32 (87%). The ring expansion reaction of 32 yielded the desired benzazepine (33) in poor yield (5%). An unknown side product was also isolated.
Compound 33 was poorly soluble in all solvents including DMSO. Hydrolysis of 33 yielded 34 (57%) but with a purity of only 90% as determined by HPLC analysis. Since only 12 mg of material was available, it was tested for glycine antagonism without further purification. The K_ of 34 at the glycine receptor was 80 nM.
Scheme XV
Figure imgf000064_0001
2, 5-Dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine may also be prepared according to Moore et al. , Tet. Lett 16: 1243-1245 (1969). Azide 36 was prepared in 44% yielded by the treatment of corresponding sodium sulfonate 35 with sodium azide in water. Dissolving 35 in ice bath cold coned, sulfuric acid and allowing the resulting solution to slowly warm to room temperature gave 37 in 81 % yield.
Figure imgf000065_0001
Thus, 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. According to the present invention, those compounds having high binding to the glycine receptor exhibit a glycine binding affinity (Kj) of about 10 μM or less in a glycine binding assay (see the Examples). Preferably, the compounds of the present invention exhibit a K, of 1 μM or less. Most preferably, the compounds of the present invention exhibit a Kj of 0.1 μM or less. The compounds exhibit high binding to the kainate and AMPA sites if they exhibit a Kj of about 10 μM or less, especially, 1 μM or less in a kainate or AMPA binding assay.
The glycine antagonist potency in vitro may be determined using a lμM glycine- stimulated [3Η]-MK801 binding assay. This assay takes advantage of the fact that the binding of [3H]-MK801 to the PCP receptor inside the pore of the NMDA channel is dependent on the presence of both glutamate and glycine. In the absence of glycine but in the presence of glutamate, [3H]-MK801 cannot bind effectively to the PCP receptor, because the NMDA channel remains closed and access of [3H]-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. After the final thaw cycle, 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.
On the day of the assay, the previously prepared membranes are thawed and 5 mM Tris/HCl buffer, pH 7.4, is added to yield a final protein concentration of 0.156 mg/ml. For binding assays, 0.8 ml of membranes are pipetted into polypropylene tubes followed by 0.033 ml of 15.1 μM 5,7- dichlorokynurenic acid (DCK), 0.033 ml of 30.3 μM glycine in buffer (or buffer alone), 0.033 ml of 303 μM glutamate in buffer (or for controls, 0.1 ml ImM PCP instead of DCK/gly/glu), 0.033 ml glycine antagonist in buffer (or buffer alone) and 0.1 ml buffer containing 200,000 cpm [3H]-MK801. Nonspecific binding is defined as the difference in binding that occurs in the absence or presence of PCP (final concentration: 100 μM). To determine the effect of 1 μM glycine on the binding of [3H]-MK801, bound radioactivity in the presence of 10 μM glutamate alone (final concentration) is subtracted from the bound radioactivity in the presence of both 10 μM glutamate and 1 μM 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 [3H]-MK801 binding that is effected by the addition of 1 μM 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 μM. IC50 values are determined for compounds active in inhibiting 1 μM glycine-stimulated [3H]- MK801 binding by computer-assisted plotting of the inhibition curves and interpolation. When compounds are found to inhibit glycine-stimulated [3H]- MK801 binding, experiments are conducted to determine whether the inhibition of the glycine-stimulated [3H]-MK801 binding is indeed mediated at the glycine binding site of the NMDA receptor. In these experiments, a fixed concentration of antagonist sufficient to produce a > 95 % inhibition of the 1 μM glycine-stimulated [3H]-MK801 binding is incubated with the membranes without any additional glycine (above 1 μM) and in the presence of increasing concentrations of additional glycine (2 μM to 1 μM). If the inhibition of [3H]-
MK801 binding by the drug in the presence of 1 μM glycine is fully reversed by adding increasing concentrations of glycine, then the inhibition of [3H]- MK801 binding is mediated by the drug acting as an antagonist at the glycine binding site of the NMDA receptor. After constructing inhibition dose response curves and determination of glycine reversibility, Kj values for the glycine antagonists are calculated using the Cheng and Prusoff equation employing the experimentally determined IC50 values, the known concentration of glycine in the assay (1 μM) 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 μM glycine- stimulated [3H]-MK801 binding assay are used for the [3H]-AMPA radioligand binding assay. On the day of the assay the frozen membranes (prepared as described above) are thawed and diluted with 30mM Tris/HCl buffer containing 2.5 mM CaCl2 and 100 mM KSCN, pH 7.4, to yield a final membrane concentration of 1.25 mg/ml membrane protein. For the binding assay, 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 [3H]-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 μM. The same membrane preparation as that used for the [3H]-AMPA binding assay may be used for the [3H]-Kainate radioligand binding assay. On the day of the 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. For the binding assay, 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 [3H]-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 μM.
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. The 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. Five parameters may be measured: the latency to entry into the dark compartment, the time spent in each area, the number of transitions between compartments, the number of lines crossed in each compartment, and the number of rears in each compartment. 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.
In the light/dark exploration model, 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
91 % O2/9% CO2. Six days later, fluoro-d-uracil is added for two days to suppress non-neural cell growth. At culture day 12, the primary neuron cultures are exposed to 100 μM glutamate for 5 minutes with or without increasing doses of glycine and excitatory amino acid antagonist or other drugs. After 5 minutes the cultures are washed and incubated for 24 hours at
37 °C. Neuronal cell damage is quantitated by measuring lactate dehydrogenase (LDH) activity that is released into the culture medium. The LDH activity is measured according to the method of Decker et al. (Decker et al. , J. Immunol. Methods 75: 16 (1988)). 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. These 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.
The efficacy of glycine/excitatory amino acid antagonists to protect mice from NMDA-induced death may be assessed as follows. When 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.
Scheme XVI
Gerbil Ischemia Model
Figure imgf000074_0001
1. Surgery to Expose Carotid Arteries
2. Recovery for 48 h
Figure imgf000074_0002
These studies are conducted in animals who are conscious and have no other pharmacological agents administered to them. Gerbils are preinstrumented 48-hours prior to ischemia to allow for the complete elimination of the pentobarbital anesthetic which is employed. When tested with drugs, animals are given IP injections of the glycine/excitatory amino acid antagonist or vehicle. In the case of multiple injections, animals are given IP injections 2 hours apart and the final injection is given 30 minutes prior to the ischemic period or in the case of post treatment, the animals are given injections at 30 minutes, 2 hours, 4 hours and 6 hours post-ischemic reperfusion.
In order to assess the direct pharmacological activity or potential activity of the glycine/excitatory amino acid antagonists, 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.
Saline results in an initial high rate of activity, with the control animals showing a first hour activity level of about 1600 counts. This level of control activity is typical for the gerbil under these experimental conditions. As the session progressed, animals decrease their exploratory activity and at the terminal period the activity declines to about 250 counts per hour. It is expected that the glycine/excitatory amino acid antagonists of the present invention will have no significant effect on either the initial exploratory rate or the terminal rate of exploration. In a next phase of the evaluation of the glycine/excitatory amino acid antagonists, gerbils are pretreated with varying doses of the antagonists and then exposed to a five minute period of bilateral carotid occlusion. 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. In contrast to the progressive decline in activity over the four hour testing period, control animals that are exposed to five minutes of cortical ischemia demonstrate a completely different pattern of locomotor activity. During the first hour there is a significant decline in activity which is followed by a progressive increase in which the activity during the fourth hour is ten-fold higher than that demonstrated by animals not exposed to carotid occlusion. These results are typical and are a reliable result of the alterations caused by five minutes of bilateral carotid occlusion in the gerbil.
Separate groups of 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.
Subsequent to completion of the single dose pretreatment evaluations, 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.
At 24 hours all animals are evaluated for differences in patrolling behavior using a 8-arm radial maze. In this procedure, animals are placed into the center start chamber of the maze, the barrier removed and the amount of time and the number of times the animal makes an error recorded prior to completion of exploration in all 8 arms of the maze. An error is defined as the revisiting of an arm by entering to the extent of the entire body without including tail by the animal. If the animal perseveres or fails to leave the arm for longer than five minutes, the session is terminated. In the control population of the animals, the number of errors and exploration of the maze with no prior experience (naive) is approximately 6 errors. This is an average value for an N of 28 gerbils. Following 5 minutes of bilateral carotid occlusion and testing at 24 hours, gerbils make an average number of errors of 21. When animals are pretreated with the glycine/excitatory amino acid antagonists of the invention, there is expected to be a significant reduction in the number of errors made. There is also expected to be a significant sparing of the behavioral changes that are induced in the radial arm maze performance. It is also expected that post treatment the glycine/excitatory amino acid antagonists of the invention will reduce the short term memory impairment 24 hours post ischemic/reperfusion.
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.
It is known that NMDA receptors are critically involved in the development of persistent pain following nerve and tissue injury. 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,
S.R., et al, J. Neurosci. 70: 1309-1318 (1990)). Administration of NMDA receptor blockers reduces the response of spinal cord dorsal horn neurons following formalin injection (Dickenson and Aydar, Neuroscience Lett. 727:263-266 (1991); Haley, J.E., et al., Brain Res. 578:218-226 (1990)). These dorsal horn neurons are critical in carrying the pain signal from the spinal cord to the brain and a reduced response of these neurons is indicative of a reduction in pain perceived by the test animal to which pain has been inflicted by subcutaneous formalin injection.
Because of the observation that 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) . However, the use of 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. It is expected that 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.
The effects of the glycine receptor antagonists of the present invention on chronic pain may be evaluated as follows. 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 (lμl/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). To test whether the glycine/excitatory amino acid antagonists prevent chronic pain in the test animals, 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. Compared to vehicle control, it is expected that the intraperitoneal injection of the glycine receptor antagonists 30 minutes prior to formalin injection into the hindpaw will significantly inhibit formalin-induced chronic pain in a dose-dependent manner as determined by the reduction of the time spent licking by the mouse of the formalin injected hindpaw caused by increasing doses of glycine/excitatory amino acid antagonist. 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. Typically, the compounds may be administered to mammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated for anxiety disorders, e.g., generalized anxiety disorder, phobic disorders, obsessional compulsive disorder, panic disorder, and post traumatic stress disorders. Preferably, about 0.01 to about 10 mg/kg is orally administered to treat or prevent such disorders. For intramuscular injection, the dose is generally about one-half of the oral dose. For example, for treatment or prevention of anxiety, 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. In the method of treatment or prevention of neuronal loss in ischemia, brain and spinal cord trauma, hypoxia, hypoglycemia, and surgery, as well as for the treatment of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's Syndrome, or in a method of treating a disease in which the pathophysiology of the disorder involves hyperactivity of the excitatory amino acids or NMDA receptor-ion channel related neurotoxicity, 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. 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. Of course, it is understood that 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.
In addition to administering the compound as a raw chemical, 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. Preferably, 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.
Also included within the scope of the present invention are the non- toxic pharmaceutically acceptable salts of the compounds 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.
The pharmaceutical compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.
The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, 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.
In addition to the pharmacologically active compounds, 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. Preferably, 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.
The pharmaceutical preparations 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. Thus, 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. If desired, 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. For this purpose, 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. In order to produce coatings resistant to gastric juices, solutions of 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. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, 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. In addition, it is also possible to use 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. In addition, 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. Optionally, the suspension may also contain stabilizers.
The characterization of glycine binding sites in vitro has been difficult because of the lack of selective drug ligands. Thus, 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 3H, πC, 1 C, 15N, or 18F.
The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention. Examples
General. Reagents were used as received unless otherwise noted. Melting points were taken on a Mel-Temp melting point apparatus and are uncorrected. Analytical thin layer chromatography was performed on plastic- backed silica gel 60 F254 plates and visualization was effected with an ultraviolet lamp. *H NMR spectra were recorded on a 300 MHz General Electric QE-300; chemical shifts are reported in delta units referenced to residual proton signals of the deuterated solvents (CHC13, δ 7.26; CD3SOCD2H, δ 2.49). Infrared spectra were obtained on a Nicolet 5DXB FT- IR spectrophotometer. Absorptions are recorded in wave numbers (cm-1) and the intensity of the absorptions are indicated by the letters s (strong), m (medium), and w (weak). Mass Spectra were recorded on a VG ZAB-2-HF mass spectrometer with a VG- 11-250 data system, in the electron ionization mode (70 eV) unless otherwise indicated. X-Ray crystallographic data were obtained on a Enraf-Nonius CAD-4 diffractometer.
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 DiazaldR (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. After this addition was complete, additional ether (20 mL) was added as above in order to purge the system of residual diazomethane, as evidenced by the lack of the characteristic yellow color in the distillate. This procedure should yield approximately 80 mmol of the reagent. Example 1 Preparation of 2,5-dihydro-2,5-dioxo-3-hydroxy-lH- benzazepine (1).
2-methoxynaphtho-l,4-quinone (7) Compound 7 was prepared according to the method of Fieser (Fieser, L.J., J. Amer. Chem. Soc. 48: 2922-2937 (1926)). To a stirred, room temperature suspension of
2-hydroxynaphtho-l,4-quinone (13.0 g, 74.6 mmol, Aldrich, 97%) in diethyl ether (200 mL), there was added an etherial solution of diazomethane (approximately 80 mmol, 250 mL, -78°C) in one portion. Gas immediately evolved and the initial orange suspension transformed into a yellow suspension. The reaction was allowed to stir for one hour at room temperature. The resulting precipitate was collected by filtration, washed with ether (3 x 15 mL) and partially air dried. The resulting yellow powder was crystallized from boiling 95 % ethanol (dissolved in 600 mL, concentrated to 400 mL) to yield 7 as yellow needles (10.1 g, 72 %); mp 177-178°C (ethanol), lit. mp (Fieser, L.J., 7. Amer. Chem. Soc. 48: 2922-2937 (1926)) 183-185°C;
Η NMR (OMSO-d6) δ 3.85 (s, 3 H), 6.34 (s, 1 H), 7.82 (m, 2 H), 7.96 (m, 2 H).
2,5-dihydro-2,5-dioxo-3-methoxy-lH-benz zepine (5). To stirred, ice bath cold, cone, sulfuric acid (16.5 mL), there was added 7 (2.50 g, 13.3 mmol, mp 177-178°C) in portions. A deep red solution resulted. To this cold solution, sodium azide (907 mg, 13.9 mmol, Aldrich) was added in one portion. The reaction was allowed to slowly attain room temperature with stirring. Gas evolution was noted. After stirring 20 hours, TLC analysis of a hydrolyzed portion indicated near quantitative conversion of the quinone to the ring expanded product (10% methanol, 90% chloroform, Rfs 0.8 and 0.5 respectively). 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
(dissolved in 350 mL, concentrated to 200 mL) with charcoal decolorization (1 g) yielded 1.44 g (53 %) of pale yellow plates (concentration of the mother liquor failed to yield further pure product); mp 243-244°C, (ethanol), lit. (Birchall and Rees, Can J. Chem. 52:610-615 (1974)): 255°C (methanol) (crystallization from ethanol may have produced a small amount of the ethyl ether that depresses the melting point of the methyl ether); *H NMR (DMSO- d6) 3.78 (s, 3 H), 6.33 (s, 1 H), 7.22 (t, J= 7.8 Hz, 1 H), 7.38 (d, J = 8.1
Hz, 1 H), 7.59 (t, 7 = 7.8 Hz, 1 H), 7.90 (d, 1 =8.1 Hz), 11.31 (s, 1 H).
2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine (1). A solution of 5 (150 mg, 738 mol) in boiling 95 % ethanol (35 mL) was prepared. The warm solution was stirred for one minute without further heating and a solution of 10% aqueous sulfuric acid (4 mL) was added. The reaction was allowed to stir for 5 minutes, then an additional portion of the acid solution (31 mL) was added. Further stirring for 10 minutes yielded a near colorless precipitate. The ethanol was removed in vacuo. The precipitate was collected, washed with water (10 x 3 mL) and dried in vacuo to yield 115 mg (82%) of a near colorless solid, which was pure by TLC analysis (57% isopropanol, 20% dioxane, 11.5 % water, 11.5% ammonium hydroxide). Crystallization from 95 % ethanol (dissolved in 25mL, concentrated to 10 mL) yielded 69 mg (49%) of the product as near colorless micro-plates; mp 255 °C dec. (instantaneous melt, ethanol), lit. (Birchall and Rees, supra): 245-248°C (ethanol); Η NMR (OMSO-d6) δ 6.41 (s, 1 H), 7.26 (t, 7 = 7.5 Hz, 1 H), 7.46 (d, J = 8.1 Hz, 1 H), 7.61 (t, 7 = 7.2 Hz, 1 H), 8.03 (d, 7 = 7.8 Hz, 1 H), 10.65 (bs, 1 H), 11.60 (s, 1 H). Example 2 Preparation of 2,5-dihydro-2,5-dioxo-3-hydroxy-7-nitro-lH- benzazepine (9).
2, 5-dihydro-2, 5-dioxo-3-methoxy-7-nitro-lH-benzazepine (8). Compound 5 was prepared in situ as described above from 7 (10.0 g, 53.1 mmol, mp 177-178°C) in cone, sulfuric acid (66 mL) with sodium azide
(3.80 g, 58.4 mmol, Aldrich). The resulting reaction mixture was re-cooled in an ice bath and potassium nitrate (5.90 g, 58.4 mmol, Baker) was added in portions. Foaming was noted after this addition. The cooling bath was removed and the reaction was allowed to stir at room temperature for 3.5 hours. TLC analysis (1.2 % methanol, 98.8% chloroform) indicated complete conversion to the nitrated product (Rf 0.25). A minor side product was also noted (Rf 0.6). The reaction was slowly added to crushed ice (200 mL). Additional ice was added during the addition so that the final volume after addition was approximately 600 ml. The mustard precipitate was collected by filtration to yield a paste. This was resuspended in water (200 mL) and carefully neutralized with solid sodium bicarbonate. A solution of 30% methanol, 70% chloroform (1 L) was stirred in so as to dissolve the solid product. The phases were separated and the organic portion was washed with 30% methanol, 70% water (2 x 500 mL). The organic portion was filtered through a cotton plug and the solvent was removed in vacuo. The resulting wet, yellow solid was crystallized from 95% ethanol (dissolved in 1.5 L, concentrated to 800 mL), with charcoal decolorization (unnecessary with this work up procedure) to yield 5.2 g (40%) of the product as near colorless laths; mp 252-253°C (ethanol); Η NMR (DMSO-< 6) δ 3.83 (s, 3 H), 6.42 (s, 1 H), 7.55 (d, 7 = 9.0 Hz, 1 H), 8.39 (dd, 7 = 9.2, 2.7 Hz, 1 H), 8.70
(d, 7 = 2.7 Hz, 1 H), 11.84 (s, 1 H).
2,5-dihydro-2,5-dioxo-3-hydroxy-7-nitro-lH-benzazepine (9). A solution of 8 (225 mg, 914 μmol) in 80 mL boiling 95 % ethanol was prepared. The warm solution was stirred for one minute without further heating and a solution of 10% aqueous sulfuric acid (10 mL) was added. The reaction was allowed to stir for 5 minutes, then an additional portion of the acid solution (70 mL) was added. Further stirring for 10 minutes yielded a near colorless precipitate. The ethanol was removed in vacuo. The precipitate was collected and washed with water (5 x 5 mL). The damp filter cake was crystallized from 95 % ethanol (dissolved in 50 mL, concentrated to
35 mL) to yield 124 mg (58%) of the product as near colorless micro-blades; mp 250°C dec; Η NMR (DMSO-^) δ 6.45 (s, 1 H), 7.61 (d, 7 = 9.0 Hz, 1H), 8.39 (dd, 1 = 9.0, 2.7 Hz, 1 H), 8.79 (d, 7 = 2.7 Hz, 1 H), 11.14 (bs, 1 H), 12.04 (s, 1 H); HRMS Calcd. for C10H6N2O5: 234.0277; Found: 234.0279. Anal. Calcd. for C10H2N2O5- C, 51.29; H, 2.58; N„ 11.96.
Found: C, 51.44; H, 2.44; N, 11.97.
Example 3 Preparation of 2,5-dihydro-2,5-dioxo-3-methoxy-7-amino- IH-benzazepine (10).
2, 5-dihydro-2, 5-dioxo-3-methoxy-7-amino-lH-benzazepine (10). A solution of 8 (250 mg, 1.00 mmol) and quinoline (20 mg) in dichloromethane/methanol (1: 1, 250 mL) was prepared. A suspension of Pd/C (10%, 100 mg) in 5 mL of dichloromethane was added. The mixture was hydrogenated on a Parr hydrogenator at 5 psi for one hour at room temperature. TLC analysis (10% methanol, 90% chloroform) indicated near total conversion to the amine with a minor amount of under reduced material being present (Rfs 0.45 and 0.40 respectively). 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
75 mL), with charcoal decolorization to yield 10 (170 mg, 39%) as an orange powder; mp 247-249°C, dec. (methanol/ethyl acetate); Η NMR DMSO-d6) δ 3.76 (s, 3 H), 5.31 (s, 2 H), 6.28 (s, 1 H), 6.85 (dd, 7 = 8.7, 2.7 Hz, 1 H), 7.12 (m, 2 H), 11.01 (s, 1 H). Anal. Calcd. for CπH10N2O3: C, 60.54; H, 4.62; N, 12.84. Found: C, 60.33; H, 4.44; N, 12.75.
Example 4 Preparation of2,5-dihydro-2,5-dioxo-3-hydroxy-7-acetamido- IH-benzazepine (15).
2, 5-dihydro-2, 5-dioxo-3-methoxy-7-acetamido-lH-benzazepine (14).
Compound 8 (400 mg, 1.61 mmol) was hydrogenated as described above (250 mL of solvent, 100 mg of catalyst, 20 mg of quinoline) followed by filtration of the catalyst. To the resulting orange solution there was added acetic anhydride (3 mL). The reaction mixture was allowed to stir for one hour at room temperature to yield a cloudy, canary yellow solution. TLC analysis (10% methanol, 90% chloroform) indicated complete conversion of the amine to the amide (Rf of amide 0.40). The reaction was clarified by filtration. Solvent removal yielded a yellow powder (386 mg, 92%), which had a purity of approximately 90% by 'H NMR analysis (DMSO--76). This material was suitable for use in the next reaction without further purification.
An analytical sample was prepared by precipitation from 95 % ethanol. A 142 mg quantity of crude material (dissolved in 75 mL, concentrated to 30 mL) yielded 81 mg of a yellow powder; mp 304-306°C, dec. (ethanol), pre-heated block, 300°C; Η NMR (DMSO-rf6) δ 2.03 (s, 3 H), 3.79 (s, 3 H), 6.34 (s, 1 H), 7.35 (d, 1 = 8.7 Hz, 1 H), 7.80 (dd, 7 = 8.6, 2.0 Hz, 1 H), 8.14 (d,
7 = 1.8 Hz, 1 H), 10.13 (s, 1 H), 11.28 (s, 1 H). Anal. Calcd. for C13H12N2O4: C, 60.00; H, 4.65; N, 10.76. Found: C, 59.84; H, 4.59; N, 10.68.
2, 5-dihydro-2, 5-dioxo-3-hydroxy-7-acetamido-lH-benzazepine (15). A solution of 14 (125 mg, —432 μmol, 90% purity) was prepared by dissolving in boiling 95 % ethanol (75 mL). The solution was allowed to cool for 5 minutes and a solution of 10% aqueous sulfuric acid was added. The reaction was allowed to stir in a 40°C oil bath for one hour. A suspension was present. TLC analysis of the mixture (57% 2-propanol, 20% dioxane, 11.5 % water, 11.5 % ammonium hydroxide) indicated near total conversion of the starting material to the desired product (Rfs 0.9 and 0.7 respectively). The reaction was poured onto crushed ice (75 mL) and was allowed to stir until the ice melted. The resulting yellow solid was collected and washed to neutrality with water. The damp filter cake was crystallized from 95 % ethanol
(dissolved in 150 mL, concentrated to 100 mL) to yield 54 mg ( — 51 %) of yellow micro-blades; mp 310°C, dec. (ethanol), pre-heated block, 300°C; Η NMR (DMSO--76) δ 2.03 (s, 3 H), 6.41 (s, 1 H), 7.41 (d, 7 = 8.7 Hz, 1 H), 7.87 (dd, 7 = 8.7, 2.1 Hz, 1 H), 8.21 (d, J = 1.8 Hz, 1 H), 10.15 (s, 1 H), 10.60 (bs, 1 H), 11.59 (s, 1 H); HRMS Calcd. for C12H10N2O4:
246.0640; Found: 246.0659. Anal. Calcd. for C12H10N2O40.40 H2O: C, 56.87; H, 4.30; N, 11.05. Found: C, 56.85; H, 3.94; N, 10.83.
Example 5 Preparation of 2,5-dihydro-2,5-dioxo-3-hydroxy-7-azido- lH-benzazepine (17).
2, 5-dihydro-2, 5-dioxo-3-hydroxy-7-azido-lH-benzazepine (17).
Compound 8 (400 mg, 1.61 mmol) was hydrogenated as described above. After solvent removal, the resulting orange powder was dissolved in boiling ethyl acetate (700 ml), with any insoluble material being removed by filtration. The resulting clear orange solution was allowed to cool to room temperature. Acidification with anhydrous HCl yielded a near colorless cloudy solution.
Solvent removal yielded 382 mg (93 %) of an orange/brown powder, which was utilized without further purification. The purity was judged to be 90% by *H NMR analysis (DMSO-d6). To a stirred, ice bath cooled, solution/suspension of the above prepared hydrochloride salt (125 mg, — 440 μmol) in water (19 mL), there was added cone. HCl (9 mL) so that the temperature of the mixture was maintained between 0 and 5°C. To this there was added a solution of sodium nitrite (37 mg, 530 μmol) in water (1 mL) in one portion. The reaction was allowed to stir between 0 and 5°C for one hour to yield a near homogeneous yellow solution. To this there was added sodium azide (35 mg, 530 mol) as a solid in one portion. The ice bath was removed and the reaction mixture was allowed to warm with stirring to 20°C over a period of 2 hours. Gas evolution was noted during this period. The resulting precipitate was collected by filtration, washed to neutrality with water (6 x 2 mL) and was dried in vacuo to yield a brown powder (50 mg). A second portion of amine hydrochloride (231 mg, — 840 /xmol, — 90%) was treated in a similar manner to yield an additional 80 mg. 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. After 12 minutes of total reaction time, the mixture was added to crushed ice (50 mL) and the ethanol was removed in vacuo (35-40 °C). The solid was collected by filtration and washed to neutrality with water (10 x 2 mL). The damp filter cake was crystallized from 95 % ethanol (dissolved in 150 mL, concentrated to 40 mL) to yield 55 mg (— 18%) as orange micro-blades; mp 190°C, dec. without melting (ethanol); Η NMR (OMSO-d6) δ 6.42 (s, 1 H), 7.39 (dd, J = 8.7, 2.7 Hz, 1 H), 7.51 (d, 7 = 8.7 Hz, 1 H), 7.66 (d, 7 = 2.7 Hz, 1 H), 10.78 (bs, 1 H), 11.70 (s 1 H); IR (KBr) 2116 (s); HRMS Cacld. for C10H6N4O3: 230.0440;
Found: 230.0443. Anal. Calcd. for C10H6N4O3: C, 52.18; H, 2.63; N, 24.34. Found: C, 52.35; H, 2.49; N, 24.20. Example 6 Preparation of 2,5-dihydro-2,5-dioxo-3-hydroxy-7-bromo- IH-benz zepine (21).
2, 5-dihydro-2, 5-dioxo-3-methoxy-7-bromo-lH-benzazepine (20),
Compound 5 was prepared in situ as described above from 7 (1.00 g, 5.31 mmol, mp 177-178°C) in cone sulfuric acid {6.6 mL) with sodium azide
(690 mg, 10.6 mmol, Aldrich). To the resulting reaction mixture, NBS (1.03 g, 5.80 mmol, Aldrich) was added in one portion and the reaction was allowed to stir at room temperature. After 48 hours, *H NMR analysis (DMSO-ti6) of a hydrolyzed portion indicated approximately 60% conversion of the starting material, as determined by the integration of the azepine nitrogen proton (δ =
11.32 and 11.45 for starting material and product respectively). A second portion of NBS (500 mg, 2.81 mmol) was added and the reaction was allowed to stir an additional 24 hours. The analysis now showed complete consumption of the starting material. The reaction was carefully added to 50 ml of crushed ice and a solution of 30% methanol, 70% chloroform (100 ml) was stirred . in. The layers were separated and the aqueous portion was extracted with 30% methanol, 70% chloroform (3 x 35 mL). The extract was washed with 30% methanol, 70% water (3 x 50 mL), filtered through a cotton plug, dried over anhydrous sodium sulfate and the solvent was removed in vacuo to yield a yellow solid (900 mg). 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 powder was crystallized from 95 % ethanol (dissolved in 100 mL, concentrated to 60 mL) to yield 94 mg (6%) of gold plates; mp 290- 291 °C (ethanol); Η NMR (DMSO-^) δ 3.80 (s, 3 H), 6.36 (s, 1 H), 7.35 (d, 7 = 8.7 Hz, 1 H), 7.78 (dd, 7 = 8.7, 2.1 Hz, 1 H), 7.99 (d, 7 = 2.1 Hz, 1 H), 11.45 (s, 1 H). Anal. Calcd. for CπH8BrNO3: C, 46.84; H, 2.86; N, 4.96. Found: C, 47.08; H, 2.70; N, 4.73.
2,5-dihydro-2,5-dioxo-3-hydroxy-7-bromo-lH-benzazepine (21). A solution of 20 (78 mg, 280 μmol) in 100 mL of boiling 95% ethanol was prepared. The hot solution was stirred without further heating for one minute and a solution of 10% aqueous sulfuric acid (10 mL) was added. The reaction was allowed to stir for 5 minutes, then an additional portion of the acid solution (90 mL) was added. A pale yellow precipitate formed a few minutes after the second addition. After 12 minutes of total reaction time, the mixture was added to crushed ice (50 mL). The ethanol was removed in vacuo at 35 to 40°C. The precipitate was collected and washed with water to neutrality (6 x 2 mL). The damp filter cake was crystallized from 95% ethanol (dissolved in 50 mL, concentrated to 25 mL) to yield 55 mg (74%) of the product as a gold micro-blades; mp 284-285°C dec. (preheat to 280°C); Η NMR (DMSO-ti6) δ 6.42 (s, 1 H), 7.41 (d, 7 = 9.0 Hz, 1 H), 7.80 (dd,
7 = 8.7, 2.4 Hz, 1 H), 8.10 (d, 7 = 2.1 Hz, 1 H), 10.86 (bs, 1 H), 11.71 (s, 1 H); HRMS Cacld. for C10H6BrNO3: 266.9532; Found: 266.9528. Anal. Calcd. for C10H6BrNO30.16 H2O: C, 44.33; H, 2.35; N, 5.17. Found: C, 44.62; H, 2.13; N, 4.77.
Example 7 Preparation of 5- and 7-nitro-l-tetralone.
To stirred, ice bath cold, coned H2SO4 (165 mL), 1-tetralone (25.00 g, 171 mmol, Aldrich, distilled prior to use) was added in portions over 5 min to yield a yellow/brown solution. To this, potassium nitrate (20.8 g, 205 mmol) was added in very small portions over 2.5 hr to yield an orange suspension. The reaction was allowed to stir for 20 min in an ice bath. TLC analysis of a hydrolyzed portion (50% EtOAc, 50% hexanes) showed a mixture of the nitro- 1-tetralones, and a side product (Rfs 0.4, 0.1 and 0.01 respectively). The reaction was carefully added to crushed ice (500 ml) and diluted with water (500 ml) to yield an orange precipitate. The precipitate was collected and washed to neutrality with water (10 x 150 mL). The filter cake was air dried to give an orange powder which was extracted with boiling hexanes (2 x 500 mL and 2 x 250 mL) leaving a brown residue (side product). A yellow solid formed from the hexanes solution upon cooling. The mixture was re-heated to boiling to give a yellow solution, which was decanted from some residual water, decolorized with activated charcoal (4 g), hot filtered and allowed to re-cool to room temperature. The resulting solid proved to be a mixture of isomers. The solvent was removed from the hexanes portion and all hexanes soluble materials were combined to yield a yellow solid (approximately 10 g). This material was subjected to silica gel chromatography (Mallinkrodt, Grade 12, 28-200 mesh, 40 x 3.5 cm) with EtOAc, hexanes elution (initial 10%, 90%, increased EtOAc 1 % every 250 mL until all the initial isomer eluted then washed column with 75%, 25 %). Two major fractions were collected. 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. 79:472-475 (1976)) 98-102°C; Η NMR (CDC13) 52.17 (p, j=6.3 Hz, 2 H), 2.72 (t, J=6.6 Hz, 2 H), 3.22 (t, J=6.0 Hz, 2 H), 7.48 (t, J = 8.1 Hz, 1 H); 8.08 (d J=8.1 Hz, 1 H), 8.34 (d, J=8.1 Hz, 1H). The slower eluting fraction was crystallized from hexanes (dissolved in 900 mL, concentrated to 600 mL) with charcoal decolorization (1 g) to yield 7-nitro-l-tetralone as a fluffy colorless solid (5.2 g, 16%); mp 104-105°C (hexanes), lit. (Biggs et al, J. Med. Chem 79:472-475 (1976)) 105-106°C; Η NMR (CDC13) δ 2.20 (p, 7=6.6 Hz, 2 H), 2.73 (t, 7=6.6 Hz, 2 H), 3.08 (t, 7=6.3 Hz, 2 H), 7.45 (d,
7=8.7 Hz, 1 H); 8.29 (dd 7=8.4, 2.4 Hz, 1 H), 8.45 (d, 7=2.1 Hz, 1H).
Example 8 Preparation of 7-amino-l-tetraloήe.
A solution of 7-nitro-l-tetralone (2.00 g, 10.4 mmol.) in 2-propanol (250 mL, prepared with warming) was hydrogenated over a Pd/C catalyst (10%, 200 mg, Aldrich) at 0-5 psi for 20 min. TLC analysis 50% EtOAc, 50% hexanes) indicated total consumption of the starting material. The catalyst was removed by filtration (Celite) and the solvent removed in vacuo to yield a yellow solid (1.8g). The solid was crystallized from EtOAc/hexanes (dissolve in EtOAc (35 mL) then added hexanes (90 mL) while maintaining boil) to yield yellow needles (1.27 g, 76%); mp 134-135°C (EtOAc/hexanes), lit. (Biggs, et al., J. Med. Chem 79:472-475 (1976)) 135-137°C; Η NMR (CDC13) δ 2.09 (p, 7=6.3 Hz, 2 H), 2.61 (t, 7=6.6 Hz, 2 H), 2.85 (t, 7=6.0 Hz, 2 H), 3.69 (bs, 2 H), 6.83 (dd 7=8.1, 2.7 Hz, 1 H), 7.05 (d, 7=8.1 Hz, 1H), 7.32 (d, 7=2.4 Hz, 1H).
Example 9 Preparation of 7-acetamido-l-tetralone.
To a stirred solution of 7-amino-l-tetralone (100 mg, 620 mmol) there was added acetic anhydride (0.5 mL). The yellow solution paled to near colorless. After 5 min, TLC analysis (50% EtOAc, 50% hexanes) indicated that the reaction was complete. The solvent was removed in vacuo, the residue washed with water (3 x 2 mL) and dried in vacuo to yield a colorless solid (112 mg, 89%); mp 161-162°C, lit (Biggs, et al., 7. Med. Chem 19:472- 475 (1976)) 162-164°C; Η NMR (CDC13) δ 2.13 (p, 7=6.3 Hz, 2 H), 2.21 (s, 3 H), 2.65 (t, 7=6.6 Hz, 2 H), 2.93 (t, 7=6.0 Hz, 2H), 7.24 (d, 7=8.7 Hz, 1 H), 7.82 (d 7=2.1, Hz, 1 H), 8.07 (bs, 1 H), 8.16 (dd, 7=8.1, 2.4 Hz,
1 H).
Example 10 Preparation of 7-azido-l-tetralone.
To a stirred solution of 7-amino-l-tetralone (1.25 g, 7.75 mmol) in aqueous HCl (62.5 mL coned HCl, 25 mL H2O) cooled to 0-5 °C in an ice bath, there was added a solution of NaNO2 (588 mg, 8.53 mmol, Baker) in water (3 mL) over a 5 min period. The ice bath cooled reaction mixture was allowed to stir for 1 hr (yellow solution to orange solution). The reaction mixture was added in portions to a rt solution of NaN3 (5 g, 77.5 mmol) in water (150 mL) over 2 min. 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 Mg2SO4 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). Solvent removal yielded a near colorless oil, which crystallized upon standing (1.3 g, 90%); mp 39-39.5°C; Η NMR (CDC13) δ 2.13 (p, 7=6.3 Hz, 2 H), 2.60 (t, 7=6.6 Hz, 2 H), 2.94 (t, 7=6.0 Hz, 2 H), 7.09
(dd, 7=8.1, 2.4 Hz, 1 H), 7.25 (d 7=7.8 Hz, 1 H), 7.72 (d, 7= 2.4 Hz, l H); IR (neat film) 2109 s (N3).
Example 11 Preparation of Bromo-1-tetralones.
To stirred, ice bath cold, coned H2SO4 (165 mL), there was added 1- tetralone (25.0 g, 171 mmol, Aldrich, distilled prior to use) in portions over a 10 min period to yield an orange/brown solution. NBS (30.4 g, 171 mmol, Aldrich, used as received) was added to the cold reaction mixture over a 45 min period. The reaction was allowed to stir at rt for 48 h and a second portion of NBS was added in portions at rt. The reaction was allowed to stir an additional 5 d at rt. The reaction was carefully added in portions to enough crushed ice so that the mixture remained cold during the addition (final volume 700 mL). 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 NaHCO3 (200 mL) and brine (2 x 100 mL). The organic portion was dried over anhydrous Na2SO4 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). The solid was crystallized by dissolving in boiling acetone (— 100 mL), decolorizing with activated charcoal (500 mg), adding hexanes to a total volume of 300 mL and concentrating to 100 mL to yield colorless prisms of unknown identity (1.9 g); mp 173-175°C (acetone/hexanes); Η NMR (CDC13) δ 3.06 (s, 4 H), 7.16 (d, 7=8.4 Hz, 1 H), 7.65 (dd 7=8.1, 2.1 Hz, 1 H), 8.29 (d, 7=2.1 Hz, 1 H); IR (KBr) 1700 s (C=O); MS (El) m/z 380 (M+, 35, [379Br], 382 (M+, 90, [279Br, 81Br], 384 (M+, 90, [79Br, 281BR], 386 (M+, 30, [381Br].
The solvent was removed from the hexanes portion in vacuo to yield a dark oil (29 g). This was subjected to vacuum distillation (Kugelrohr, 0.025 Torr). An initial fraction distilled at an oven temperature of 80-85°C, which proved to consist mostly of unreacted tetralone and small amounts of volatile products (7.7 g) as determined by TLC analysis (10% EtOAc, 90% hexanes).
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. Cooling in an ice bath promoted solidification. The colorless solid was collected, washed with cold petroleum ether (3 x 2 mL) and dried in vacuo to yield 5-bromo-l -tetralone (1.31 g, 4%); mp 47-48 °C (petroleum ether), lit. (Adamcyzk, M. et al., J. Org. Chem. 49:4627-4637 (1984)) 49- 50°C (hexanes); Η NMR (CDC13) δ 2.16 (p, 7=6.3 Hz, 2H), 2.65 (t, 7=6.3 Hz, 2 H), 3.02 (t, 7=6.3 Hz, 2H), 7.19 (t, 7=7.8 Hz, 1H), 7.73 (d, 7=8.1 Hz, 1H), 8.02 (d, 7=7.5 Hz). The slower eluting solid was crystallized from hexanes (25 mL) to yield 7-bromo-l -tetralone as colorless needles (1.38 g, 4%); mp 74-76°C,
(hexanes), lit. (Fieser, et al., A.M. J. Amer. Chem. Soc. 60: 170-176 (1938))
76-77°C, (ether/petroleum ether); Η NMR (CDC13) δ 2.13 (p, 7=6.3 Hz, 2 H), 2.65 (t, 7=6.3 Hz, 2 H), 2.91 (t, 7=6.0 Hz, 2 H), 7.14 (d, 7=8.1 Hz,
1 H), 7.56 (dd, 7=8.1, 2.1 Hz, 1 H), 8.14 (d, 7=2.1 Hz).
Example 12 Preparation of 3-hydroxy-7-bromonaptho-l,4-quinone.
To a 0.3 M solution of t-BuOK in t-BuOH (50 mL), that had been stirred under O2 for 15 min at 30-35°C, there was added a solution of 7-bromo-l -tetralone (1.20 g, 5.33 mmol) in t-BuOH (10 mL). The reaction mixture immediately turned red. The warmed reaction mixture was allowed to vigorously stir under O2 for 1.5 h at which point TLC analysis (10% MeOH, 90% CHC13) showed total consumption of the starting material. A red suspension was present. The reaction was added to 10% HCl (150 mL) to yield a yellow suspension. The suspension was extracted with CHC13 (2 x 100 mL). The organic portion was extracted with saturated NaHCO3 (1 x 150 mL,
2 x 50 mL). The organic portion was filtered to collect a portion of the sodium salt that failed to dissolve in the bicarbonate solution. The combined carbonate and solid portion was acidified to pH 2 with 6 N HCl. The resulting yellow suspension was extracted with CHC13 (3 x 100 mL). The extract was washed with H2O, filtered through a cotton plug and the solvent removed in vacuo to yield an orange solid (0.8 g). Crystallization from 95 % EtOH (dissolved in 50 mL, concentrated to 25 mL) yielded orange needles (465 mg, 34%); mp 212-213°C, (EtOH), lit (Buckle, D.R., et al., J. Med. Chem. 20: 1059-1064 (1977)) 216°C, (EtOH); Η NMR (CDC13) δ 6.83 (s,
1 H), 7.25 (s, 1 H), 7.92 (dd, 7 = 8.1, 1.8 Hz, 1 H), 7.99 (d, 7 = 8.1, 1.8 Hz, 1 H), 8.24 (d, 7 = 1.8 Hz, 1 H). Example 13 Preparation of 3-methoxy-7-bromonaptho-l,4-quinone.
To a suspension of 3-hydroxy-7-bromonaptho-l,4-quinone (500 mg, 1.97 mmol) in ether (35 mL) there was added etherial diazomethane (20 mL, — 0.3 M). The reaction was allowed to stir for 3 h at rt, while gas evolution was noted, to yield a yellow suspension. TLC analysis (10% MeOH, 90%
CHC13) indicated total consumption of the starting material. The ether was removed in vacuo and the residue crystallized from 95 % EtOH (dissolved in 100 mL, concentrated to 65 mL) to yield yellow needles (458 mg, 87%); mp 213-214°C, (EtOH); Η NMR (CDC13) δ 3.91 (s, 3 H), 6.19 (s, 1 H), 7.87 (dd, 1 H, 7 = 8.1, 1.8 Hz, 1 H), 7.95 (d, 7 = 8.1 Hz, 1 H), 8.25 (d, 7 =
1.5 Hz, 1 H).
Example 14 Preparation of 2, 5-dihydro-2, 5-dioxo-3-methoxy-7-bromo-lH- benzazepine.
To stirred, ice bath cold, coned H2SO4 (2.9 mL), there was added 3-methoxy-7-bromonaptho-l,4-quinone (440 mg, 1.65 mmol) in portions. A deep red solution resulted. To this cold solution, sodium azide (214 mg, 3.30 mmol, Aldrich) was added in portions. The reaction was allowed to slowly attain rt with stirring. Gas evolution was noted. After 48 h, a sample taken from TLC analysis (10% MeOH, 90% CHC13) showed only partial consumption of the quinone. An additional portion of azide was added and the reaction was allowed to stir an additional 24 h at rt. The analysis now showed total consumption of the quinone with a major product and a minor product being present (Rfs 0.9, 0.7 and 0.5, respectively). The reaction was added to crushed ice (75 mL) to yield a green suspension. The solid was collected, resuspended in water (75 mL) and neutralized with solid NaHCO3. The solid was collected by filtration and the filtrate extracted with 30% MeOH, 70%
CHC13 (5 x 75 mL). The extract was filtered through a cotton plug and the solvent removed in vacuo to yield a green solid. The two solid portions were combined and dried in vacuo to yield — 0.2 g of material. Crystallization from 95% EtOH (dissolved in 500 mL, concentrated to 100 mL) with charcoal decolorization (100 mg) yielded a shiny light green solid (25 mg, 5 %); mp decomposes without melting > 300°C; Η NMR (DMSO--76) δ 3.79 (s, 3 H), 6.35 (s, 1 H), 7.41 (dd, 1 H, 7 = 8.4, 1.2 Hz), 7.62 (d, 7 = 0.9 Hz), 7.83
(d, 7 = 8.7 Hz), 11.37 (s, 1 H).
Solvent removal from the mother liquor yielded an additional 89 mg of an unknown material (green solid), that was more soluble in EtOH and DMSO than the desired product; Η NMR (DMSO-< 6) δ 3.78 (s, 3 H), 6.12 (s, 1 H), 7.31 (d, 1 H, 7 = 8.7 Hz), 7.79 (dd, 7 = 8.7, 2.4 Hz), 7.93 (d, 7
= 2.4 Hz), 11.10 (s, 1 H).
Example 15 Preparation of2,5-dihydro-2,5-dioxo-3-hydroxy-8-bromo-lH- benzazepine.
A solution of 2,5-dihydro-2,5-dioxo-3-methoxy-8-bromo-lH- benzazepine (22 mg, 78 mmol) in 250 mL of boiling 95 % EtOΗ was prepared by boiling the suspended material for 30 min. The hot solution was stirred without further heating for two min and a solution of 10% aqueous Η2SO4 (25 mL) was added. The reaction was allowed to stir for 5 min at rt. An additional portion of the acid solution (225 mL) was then added and the reaction was allowed to stir in an ice bath. After 12 min of total reaction time, the mixture was added to crushed ice (200 mL). The EtOH was removed in vacuo at 35 to 40° C. The precipitate was collected and washed with water to neutrality (6 x 2 ml). The damp filter cake was crystallized from 95 % EtOH (dissolved in 30 mL, concentrated to 10 mL) to yield a light beige solid (12 mg, 57%); mp decomposes without melting >300°C,
Η NMR (DMSO-< 6) δ 6.40 (s, 1 H), 7.43 (dd, 7 = 8.4, 1.5 Hz, 1 H), 7.68
(d, 7 = 1.5 Hz, 1 H), 7.94 (d, 7 = 8.4 Hz, 1 H), 10.81 (bs, 1 H), 11.62 (s,
1 H); HRMS Cacld. for C10H6BrNO3:266.9532; Found: 266.9531. Example 16 Preparation of 4-azidonaptho-l,2-quinone.
Warning: This compound decomposes violently at its melting point and reacts violently when in contact with rt, coned acid. Caution is advised! To a vigorously stirred, aqueous solution of naptho-l,2-quinone-4- sulfonic acid, sodium salt (5.00 g, 19.2 mmol in 270 mL H2O, Eastman, used as received), there were added an aqueous solution of sodium azide (1.37 g, 21.1 mmol in 30 mL H2O, Aldrich) followed by CHC13 (200 mL). The reaction was allowed to stir at rt for 15 min so that the aqueous and organic portions were thoroughly mixed. The layers were separated and the aqueous portion extracted with CHC13 (100 mL). The combined organic portion was washed with half saturated brine (3 x 300 mL), filtered through a cotton plug, and the solvent removed in vacuo to yield an orange solid (1.7 g, 44%); mp 129-130°C dec; Η NMR (CDC13) δ 6.36 (1 Λ), 7.62 (t, 7 = 7.2Hz, 1 H), 7.70 (t, 7 = 7.5 Hz, 1 H), 7.78 (d, 7 = 7.5 Hz, 1 H), 8.16 (d, 7 = 7.2 Hz, 1 H); IR (KBr) 2138 s (N3); TLC Rf 0.6 (10% MeOH, 90% CHC13).
Example 17 Preparation of 2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine.
To stirred, ice bath cold, coned H2SO4, there was added 4-azidonaptho-l,2-quinone (1.7 g, 1.5 mmol) in small portions to yield a dark orange solution. The ice bath was removed and the reaction was allowed to slowly warm with stirring. The reaction began to vigorously froth as it approached rt and was controlled by re-cooling with an ice bath. After the initial reaction subsided, the reaction was allowed to stir at rt for a total time of 1.5 h. The dark brown/green reaction mixture was carefully added to ice water (250 mL) to yield a brown precipitate. The solid was collected and washed to neutrality with water (6 x 10 ml). Crystallization of the damp filter cake from 95% EtOH yielded yellow needles (1.3 g, 86%); mp 259-260°C dec, lit. (Moore, H.W.; Shelden, H.R; Weyler Jr., W. Tetrahedron Letts. 76: 1243-1246 (1969)) 260°C; Η NMR (DMSO--i6) δ 6.21 (d, 7 = 1.8, 1 H), 7.21 (t, 7 = 7.5 Hz, 1 H), 7.40 (d, 7 = 8.4 Hz, 1 H), 7.65 (t, 7 = 7.5 Hz), 8.07 (d, 7 = 8.1 Hz, 1 H), 10.55 (s, 1 H), 11.01 (s, 1 H); HRMS Cacld. for C10H7NO3: 189.0426; Found: 189.0413.
Example 18 Reaction of 2,5-dihydro-2,5-dioxo-3-methoxy-7-nitro-lH- benzazepine (9) with Diazomethane.
To a stirred solution of 9 in 1: 1 dichloromethane/methanol (5 mg in 4 mL) there was added a solution of diazomethane in ether (approximately 0.2 mL, approximately 0.3 M). The yellow color discharged immediately. TLC analysis (10% methanol, 90% chloroform) showed partial conversion of
9 to a spot that corresponded with authentic 8 (Rfs 0.02 and 0.5 respectively). Additional diazomethane solution was added until the characteristic yellow color persisted (approximately 0.5 mL). Analysis now showed no 9 present, the presence of 8 and the presence of a spectrum of side products. Additional diazomethane resulted in the total consumption of 8. Similar treatment of authentic 8 yielded the same spectrum of products.
Example 19 Direct Bromination and Chlorination of 2,5-dihydro- 2, 5-dioxo-3-methoxy-lH-benzazepine (5).
Reaction of 5 with NBS and NCS. A solution of 5 in DMF (10 mg/1 mL) was treated with approximately a molar eq of NBS or NCS at room temperature. The reactions were allowed to stir overnight. The DMF was removed from an aliquot in vacuo, the residue washed with water and dried in vacuo. The samples were analyzed by TLC (ethyl acetate) and Η NMR (DMSO--76) analyses. Reaction of 5 with bromine and silver sulfate. Compound 5 was prepared in situ as described above from 7 (1.00 g, 5.31 mmol, mp 177- 178°C) in sulfuric acid (6.6 mL) with sodium azide (380 mg, 5.84 mmol, Aldrich). To the reaction mixture, bromine (420 mg, 5.31 mmol) was added. The reaction was allowed to stir for 24 hours at room temperature. TLC analysis (10% methanol, 90% chloroform) of a hydrolyzed portion showed no reaction. Silver sulfate (828 mg, 2.66 mmol, Aldrich, 99%) was added and the reaction was allowed to stir an additional 24 hours at room temperature. TLC and lU NMR (DMSO-d6) analyses showed no discernible reaction.
Example 20 Competitive Antagonism of Binding by the Substituted 1H- benzazepine-2, 5-diones.
Compounds. L-Glutamate, glycine, kainate, NMDA, and D-serine were obtained from Sigma, whereas (- )-quisqualate was purchased from
Research Biochemicals Inc. The parent 2,4-dihydro-2,5-dioxy-3-hydroxy-lH- benzazepine (DDΗB), 8-Me-DDΗB, and 7-Me-DDHB were synthesized from the appropriately substituted 2-methoxy-l ,4-naphthoquinones, according to the method of Birchall and Rees (Birchall, G.R. and Rees, A.H., Can. J. Chem. 52:610-615 (1974)). The 3-acetyl ester and the 3-methyl ether of DDHB were synthesized from DDHB, as previously described (Birchall, G.R. and Rees, A.H., Can. J. Chem. 52:610-615 (1974)). 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 μM, 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.
52:610-615 (1974)) to undergo an intramolecular rearrangement to kynurenic acid, in the presence of aqueous base. The concentration of kynurenic acid in all of the solutions used for electrophysiological experiments was measured as previously described (Swartz et al , Anal. Biochem. 185:363-376 (1990)) and was found to be < 0.1 % , on a molar basis.
Cell culture and electrophysiology. Neurons from the visual cortex of PO-postnatal day 5 Long Evans rat pups were dissociated with papain (Worthington Biochemical Corp.), as previously described (Huettner, J.E. and
Baughman, R.W., 7. Neurosci. 6:3044-3060 (1986)). Cells were plated onto glial monolayers or directly onto glass coverslips coated with Cell-Tak (BioPolymers Inc.).
Tight-seal whole-cell recordings were obtained from cells that had been in culture for 6-14 days. Pipettes pulled from 100-μl Boralex micropipettes
(Rochester Scientific Co., Inc.) were coated with Sylgard (Dow Corning Corp.) and fire polished. Pipette resistance ranged from 2 to 6 MΩ with 140 mM CsCH3SO3, 5 mM CsCl, 10 mM EGTA, 10 mM HEPES, pH 7.4 (adjusted with CsOH), as the internal solution. The external solution for drug applications contained 160 mM NaCl, 2 mM CaCl2, 1 μM tetrodotoxin
(Sigma), and 10 mM HEPES, pH 7.4. Dizocilpine (MK-801; donated by Merck, Sharp & Dohme) was added to the external solution at 1 μM 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-μl 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 (Vyklicky et al. , 7. Physiol. (Lond.) 428:313-331 (1990)). The bath was perfused at 1-5 ml/min with Tyrode's solution (150 mM NaCl, 4 mM KC1, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4). Membrane potentials have been corrected for a junction potential of -10 mV between the internal solution and the Tyrode's solution in which seals were formed. Whole-cell currents recorded with a Dagan 3900 amplifier were filtered at 1 kHz (-3 dB, eight-pole Bessel) and digitized at 5 kHz. For storage and analysis, the data were compressed by averaging 3 msec of current at 0.1-0.5 sec intervals.
Experimental design and data analysis. Concentration-response curves were generated by applying a set of five to seven different agonist concentrations. In most experiments, each concentration was applied for
10-15 sec. Steady state currents were measured as the average current during the final third of each application. In most cells, the full set of applications was repeated several times. Because the absolute current levels varied from cell to cell, the values were normalized to the maximal current (7max) produced by a saturating concentration of agonist. This control dose of agonist was included in every set of applications. Currents evoked by a combination of agonist plus antagonist were normalized to the control (saturating) dose of agonist alone.
Nonlinear regression (Sigmaplot 4.1, Marquardt-Levenberg algorithm; Jandel Scientific) was used to fit the concentration-response data with the logistic equation (eq. 1):
max
EC 5, 0 (1)
1 +
[agonist]
where ECJ0 is the agonist concentration producing half-maximal activation and n is the slope factor. In several cases, the control dose-response relation, for agonist alone, together with one to three agonist dose-response curves obtained in the presence of an antagonist were fit simultaneously, with the model for simple competitive antagonism (Clark, A.J., J. Physiol. (Lond.) 67:547-556 (1926); Gaddam, J.H., J. Physiol. (Lond.) 67: 141-150 (1926); Arunlakshana, O., and Schild, H.O., Br. J. Pharmacol. 14:48-58 (1959)) embodied in eq. 2 (Waud, D.R., Methods Pharmacol. 5:471-506 (1975).
Figure imgf000107_0001
where the parameters EC50 (the half-maximal dose of agonist alone), KB (the antagonist dissociation constant), and n (the slope factor) were adjusted to provide an optimal fit of all of the curves at once. Eq. 2 constrains all of the concentration-response curves to be parallel and shifted from the control curve by the factor (1 + [antagonist ]/KB). These constraints correspond to assuming a Schild slope of exactly -1 (Arunlakshana and Schild, Br. J. Pharmacol. 74:48-58 (1959)). The principal advantage of simultaneous fitting with eq. 2, in comparison with standard Schild analysis, is that the control concentration- response relation (agonist alone) is given equal weight, relative to the relations obtained in the presence of antagonist (Waud, D.R., Methods Pharmacol.
3:471-506 (1975); Stone, M. and Angus, J.A., 7. Pharmacol. Exp. Therp. 207:705-718 (1978)). Furthermore, eq. 2 provides greater certainty in the value of KB, because it is obtained from a direct fit of the experimental data, rather than from regression of calculated dose ratios. Plots of concentration- response relations shown in the figures display I/Im∞ as the mean ± standard error for each agonist concentration. To ensure proper weighting, however, eqs. 1 and 2 were fit to all of the individual data points.
To test for statistically significant departure from the simple competitive model, the ratio of residual variance was calculated according to eq. 3 (Waud, D.R., Methods Pharmacol. 3:471-506 (1975); Stone, M. and
Angus, J.A., 7. Pharmacol. Exp. Therp. 207:705-718 (1978); De Lean et al., Mol. Pharmacol. 21:5-16 (1982)).
where SS2 is the sum of squared deviations for the simultaneous fit with eq. 2, SS! is the total sum of squared deviations obtained when eq. 1 was fit to each df -dfvdfχ (3)
Figure imgf000108_0001
concentration-response curve individually, and df and df2 are the degrees of freedom (number of data points - number of parameters) for individual fits with eq. 1 and the simultaneous fit with eq. 2, respectively. F values are given in the text in the form F^ -___■ ), dS ). Because EC50 an KB are expected to exhibit log-normal distributions (De Lean et al. , Mol. Pharmacol. 21:5-16
(1982)), the confidence intervals were obtained using the following substitutions in eqs. 1 and 2: ECJ0 = 10"pE, where pE = -log EC50, and KB = 10"pKB, where pKB = -log KB. The 95 % confidence intervals for pE and pKB were calculated as the product of the standard deviation for each parameter (given by the fitting program) times the appropriate value from the t distribution. Confidence limits, as given in the text, have been transformed to EC50 and ^B.
Results
In preliminary experiments, DDHB was found to produce voltage- independent blockade of currents activated by half-maximal concentrations of
L-glutamate, (-f)-quisqualate, kainate, and NMDA. Fig. 1 shows the inhibition of kainate- and NMDA-gated currents by DDHB at holding potentials of +50 mV and -80 mV. At 100 μM, DDHB blocked 40-45 % of the current evoked by 100μM kainate and produced complete block of current activated by 20 μM NMDA plus 300 nM glycine. Both the onset of and recovery from block were complete within seconds. An initial screen of available derivatives of DDHB revealed that 8-Me-DDHB was the most potent antagonist, and 4-Br-DDHB and 7-Me-DDHB were also active, whereas the 3-acetyl ester and the 3-methyl ether of DDHB produced little or no inhibition of currents gated by any of the agonists. 8-Me-DDHB was selected for detailed analysis.
Antagonism at non-NMDA receptors by 8-Me-DDHB. 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)). In order to evaluate quantitatively the affinity of 8-Me-DDHB for non-NMDA receptors, we determined the concentration-response relation for kainate and L-glutamate in the presence of 0, 8, 20, and 50 μM antagonist.
As shown in Figs. 2 and 3, 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. Following the method of 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
(1926); Arunlakshana, O., et al., Br. J. Pharmacol. 74:48-58 (1959)), because the addition of antagonist results in a parallel displacement of the control curve, obtained with agonist alone, by the factor (1 + [antagonist]/^), where KB is the antagonist dissociation constant. As shown in Fig. 3, the smooth curves defined by eq. 2 provide a good fit to the experimental data (see below; Fig. 8). The ratio of residual variance indicates that departure from the simple competitive model is not statistically significant at the 5 % level (F5 197 = 0.71). In agreement with previous work (Huettner, J., Neuron 5:255-366 (1990); Verdoorn, T.A., et al, Mol. Pharmacol. 34:298-307 (1988); Patneau, D.K., et al, J. Neurosci. 10:2385-
2399 (1990)), kainate alone produced half-maximal activation at a concentration of 120 μM (111-131 μM, 95 % confidence interval for EC50 from the fit of eq. 2). 8-Me-DDHB antagonized the current gated by kainate with a KB of 6.4 μM (5.5-7.5 μM).
Activation of non-NMDA receptors by L-glutamate was also inhibited by 8-Me-DDHB. 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 μM L-glutamate. (For all of the experiments with glutamate, 1 μM MK-801 was added to the external solutions, to suppress completely current through NMDA receptor channels.) The potency of 8-Me-DDHB as a non-NMDA receptor antagonist was evaluated quantitatively only for blockade of the sustained current. Antagonist affinity was not determined for the transient component of current because the null method used in eq. 2 requires that agonist and antagonist binding be at equilibrium (Colquhoun, D., Handb. Exp. Pharmacol. 59:59-113 (1986)). This is not likely to be the case at the peak of the transient current evoked by L-glutamate. Fig. 7 illustrates pooled steady state concentration-response data for the application of L-glutamate alone and in the presence of 8, 20, and 50 μM 8-Me-DDHB. In contrast to experiments with kainate, glycine and
NMDA, we observed considerable cell to cell variation in the EC50 values obtained for application of L-glutamate, both in the absence and in the presence of 8-Me-DDHB. For this reason, a larger number of cells were tested with L-glutamate than was necessary with the other agonists. Although the source of this variability is not clear, it could potentially be due to differences in the extent of desensitization from one cell to another. In order to compensate for the fact that a different number of applications were performed on each cell, we first calculated the average normalized currents evoked by each concentration of agonist, on a cell by cell basis. Then, the mean and standard deviation of these individual cellular averages were computed for each agonist concentration over all cells tested. Using this app roach, Fig. 7 shows that the control EC50 for L-glutamate alone was 17 μM (15-19 μM, 95 % confidence interval) and the ^B for 8-Me-DDHB, determined from the best fit of eq. 2, was 9.6 μM (7.8-11.8 μM). This control EC50 for activation of steady state current by L-glutamate is consistent with that previously reported by others (Verdoorn, T.A., et al, Mol Pharmacol.
34:298-307 (1988); Patneau, D.K., et al, J. Neurosci 70:2385-2399 (1990); O'Dell, T.J., et al, J. Neurophysiol. 67: 162-172 (1989)).
The two plots shown in Fig. 8 disclose the extent to which the concentration-response relations for kainate and L-glutamate diverge from the equation for competitive antagonism. The points show the control agonist
ECJO along with the agonist concentration required to produce half-maximal activation (ECJO0 in the presence of each antagonist concentration, obtained from the individual fits of the logistic equation (eq. 1). These values are plotted against the sum (K + [antagonist]), using the K given by the simultaneous fit of eq. 2. The straight lines in Fig. 8 represent the competitive relationship defined by EC50' = (β>C50IK^(KB + [antagonist]). For both kainate and L-glutamate, the points are well described by the theory. This form of display is similar to the Clark plot developed by Stone and Angus (Stone, M., et al. J. Pharmacol. Exp. Ther. 207:705-718 (1978); see also ref. Stone, M., J. Pharm. Pharmacol. 32:81-86 (1980)). In summary, these results are consistent with the action of 8-Me-DDHB as a simple competitive antagonist at the agonist recognition site on non-NMDA receptors, holding true when the channel is activated by either kainate (KB = 6.4 μM) or L-glutamate (KB = 9.6 μM). Antagonism at the glycine allosteric site by 8-Me-DDHB. In preliminary experiments, benzazepines were found to inhibit currents elicited by submaximal concentrations of NMDA and glycine. Further work revealed that antagonism occurred both at the glycine allosteric site and at the transmitter binding site recognized by NMDA (see below). In order to study antagonism of the glycine site selectively, concentration-response relations for glycine were determined using a saturating level of NMDA (1 mM; see Figs. 13 and 14). A few of the cells in the cultures expressed strychnine-sensitive glycine receptors that activated a chloride-selective current; however, cells that displayed this current with high concentrations of glycine were excluded from the analysis. As shown in Fig. 9, 8-Me-DDHB at 2, 10 and 50 μM displaced the glycine dose-response relation toward progressively higher concentrations.
The magnitudes of the shifts indicate a KB of 470 nM (410-540 nM) for 8-Me- DDHB at the glycine site. The action of the drug is fully consistent with a mechanism of simple competitive antagonism, as shown by the parallel displacement of the glycine dose-response relations in Fig. 10 (FJ 217 = 2.03; not significant at 5 %) and by the plot in Fig. 11.
In the absence of antagonist, glycine potentiated the response to NMDA, with an EC50 of 770 nM (690-850 nM). This value is somewhat higher than previously reported EC50 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);
Henderson, G., et al, J. Physiol. (Lond.) 430: 189-212) (1990); Kleckner, N.W., et al, Mol. Pharmacol. 36:430-436 (1989); Vyklicky, L., Jr., et al, J. Physiol. (Lond.) 428:313-331 (1990); McBain, C.J., et al, Mol. Pharmacol 36:556-565 (1989)). Mayer and colleagues (Vyklicky, L., Jr., et al, J. Physiol. (Lond.) 428:313-331 (1990); Benveniste, M., et al, J.
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 EC50 for potentiation of steady state current by glycine was sensitive to the concentration of NMDA. Glycine potentiated the current evoked by 25 μM NMDA with an EC50 of 310 nM, compared with an ECJ0 of nearly 800 nM when 1 mM NMDA was used. These results, which were obtained from sister cultures after 7 days in vitro, are in fairly close agreement with the -I l l-
model of Mayer and colleagues. The dotted lines shown in Fig. 12 represent the concentration-response relations for glycine with 5 μM, 25 μM, and 1 mM NMDA predicted by scheme 2 of Benveniste et al. (Benveniste, M., et al, J. Physiol. (Lond.) 428:333-357 (1990)). In the presence of 1 mM NMDA, the glycine EC50 of 853 nM predicted by their model falls just outside the 95 % confidence interval of our experimental EC50 (690-850 nM). Consistent with previous reports (Huettner, J.E., Science (Washington, D. C.) 243: 1611-1613 (1989); Kleckner, N.W., et al, Mol. Pharmacol. 36:430-436 (1989); Vyklicky, L., Jr., et al, J. Physiol. (Lond.) 428:313-331 (1990); McBain, C.J., et al, Mol. Pharmacol. 36:556-565 (1989); Benveniste, M., et al, J.
Physiol. (Lond.) 428:333-357 (1990)), we obtained a significantly higher affinity for glycine potentiation of current gated by 25 μM NMDA. However, our experimental EC50 for glycine of 308 nM (279-339 nM) was closer to the EC50 predicted by the model of Benveniste et al. (Benveniste, M., et al, J. Physiol. (Lond.) 428:333-357 (1990)) for 5 μM NMDA (294 nM) than to the value of 586 nM expected for 25 μM NMDA. The deviation of our results from their model may be due to the fact that our external solution contained 2 mM calcium, compared with 0.2 mM in the experiments modeled by Benveniste et al. (Benveniste, M., et al, J. Physiol. (Lond.) 428:333-357 (1990)). Calcium is known to influence the rate and extent of NMDA receptor desensitization (Mayer, M.L., et al, J. Physiol. (Lond.) 367:65-90 (1985); Vyklicky, L., Jr., et al, J. Physiol. (Lond.) 428:313-331 (1990)). There might also be differences as a result of age (Embryonic day 16-18 versus Postnatal day 0-5), species (mouse versus rat), or cell type (hippocampus versus cortex) . It is interesting that the microscopic reversibility of the model of Benveniste et al. (Benveniste, M., et al, J. Physiol. (Lond.) 428:333-357 (1990)) requires that binding of glycine cause a reduction in the affinity for NMDA; however, such a reduction has not been observed, to our knowledge (Kleckner, N.W., et al, Science (Washington, D.C.) 241 :835-837 (1988); Huettner, J.E., Science (Washington, D.C.) 243: 1611-1613 (1989)). Antagonism at the NMDA recognition site by 8-Me-DDHB. In addition to its action at the glycine allosteric site, 8-Me-DDHB also inhibits activation of the receptor by NMDA. As shown in Fig., 14, antagonist potency at the NMDA recognition site is approximately 60-fold lower than at the glycine allosteric site. Inhibition produced by 50 μM 8-Me-DDHB was completely overcome by increasing the concentration of NMDA. On the assumption that the interaction is competitive, the shift in the EC50 for NMDA from 13 to 28 μM after the addition of 50 μM 8-Me-DDHB indicates a KB of 27 μM (23-32 μM). The control EC50 for NMDA (13 μM) is consistent with previous findings (Huettner, J.E. , Science (Washington, D. C.) 243: 1611-1613
(1989); Verdoorn, T.A., et al, Mol. Pharmacol 35:360-368 (1989); Verdoorn, T.A., et al., Mol. Pharmacol. 34:298-307 (1988); Patneau, D.K., et al., J. Neurosci 70:2385-2399 (1990)). D-Serine (1 mM) (Kleckner, N.W. , et al, Science (Washington, D.C.) 247:835-837 (1988); Snell, L.D., et al, Eur. J. Pharmacol. 756: 105-110 (1988)) was used in place of glycine for the experiments shown in Fig. 13 and 14, to avoid activation of chloride channels by strychnine-sensitive glycine receptors. Preliminary experiments with 1 mM glycine revealed a similar shift in the ECJ0 for NMDA in cells that lacked the strychnine-sensitive glycine receptor. The fact that the inhibition produced by 50 μM 8-Me-DDHB was completely overcome by high concentrations of
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. DDHB, 4-Br-DDHB, and 7- Me-DDHB were each tested, at a concentration of 100 μM, 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 μM (22-32 μM), compared with 63 μM (53-74 μM) for 4-Br-DDHB and 65 μM (53-80 μM) for DDHB. All three compounds were less potent than 8-Me-DDHB, which showed a K of 6.4 μM against kainate (Figs. 3 and 8). The four smooth curves fit to the data in Fig. 15 were constrained to be parallel, in accordance with the model for simple competitive antagonism. This constraint did not significantly reduce the goodness of fit, as determined by the ratio of residual variance (F3 185 = 0.63).
Fig. 16 shows the antagonism produced by 100 μM DDHB, 4-Br- DDHB, and 7-Me-DDHB at the glycine allosteric site on the NMDA receptor.
All three compounds reduced the apparent affinity for glycine, and in each case the inhibition was overcome by adding a sufficiently high concentration of glycine (400 μM). To be fully consistent with the simple competitive mechanism of inhibition, the four concentration-response relations in Fig. 16 should be parallel. However, slight differences in the slopes of the four curves produced a significant departure from parallelism. The ratio of residual variance for comparing the fit obtained with parallel curves and individual fits of each dose-response relation with the logical equation (eq. 1) was F3 271 = 4.64 (significant at the 1 % level). The four smooth curves shown in Fig. 16 represent individual fits of eq. 1 to the control data and to the data points for each of the three antagonists. Although the curves are not parallel, KB 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 EC50 for glycine, the KB for DDHB is estimated at 3.0 μM, compared with 9.5 μM for 7-Me-DDHB and 25 μM for 4-Br-DDHB. These values are all significantly higher than the KB of 470 nM obtained for 8-Me-DDHB (Fig.
11).
At the NMDA recognition site, antagonism by DDHB was found to be slightly more potent than that produced by 8-Me-DDHB. Fig. 17 shows the shifts produced by 100 μM 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 smooth curves shown in Fig. 17 are individual fits of eq. 1. When the four curves were constrained to have the same slope, the ratio of residual variance was significant at the 1 % level (F3 229 = 6.19), which indicates that the data are not well described by four parallel curves. Nevertheless, K values were calculated for DDHB, 4-Br-DDHB, and 7-Me- DDHB using the assumption of competitive inhibition, in order to make a comparison with 8-Me-DDHB. The KB calculated for DDHB from the data in Fig. 17 was 16 μM, which is lower than the KB value of 27 μM obtained for 8-Me-DDHB (Fig. 14). 7-Me-DDHB displayed a K of 108 μM, whereas that for 4-Br-DDHB was 81 μM.
Figure imgf000117_0001
Table 3 presents some additional binding data for some 7- and 8-substituted benzazepines.
cr- 1
Figure imgf000118_0001
Discussion
This study has shown that substituted benzazepines constitute a novel class of broad spectrum excitatory amino acid antagonists. Table 2 summarizes the structure-activity relationships for the four compounds DDHB, 7-Me-DDHB, 8-Me-DDHB, and 4-Br-DDHB. All four compounds showed high potency as antagonists of the glycine allosteric site on the NMDA receptor, with 8-Me-DDHB being the most potent derivative of the four ( ^B = 470 nM versus glycine). 8-Me-DDHB was approximately 14-fold less potent as an antagonist of non-NMDA receptors (K = 6.4 μM versus kainate) and nearly 60-fold less effective at blocking the NMDA recognition site (K
= 27 μM versus NMDA). The parent compound, DDHB, demonstrated slightly higher affinity for the NMDA recognition site (KB = 16 μM) than did the 8-methyl derivative, but DDHB was less potent than 8-Me-DDHB against glycine and kainate (DDHB KB = 3 and 65 μM, respectively). 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. Such a relief from blockade with saturating doses of agonist was observed for steady state activation of non-NMDA receptors by kainate or glutamate and for activation of NMDA receptors by both NMDA and glycine. Second, in the case of 8-Me-DDHB, which was tested at three different concentrations, the shifts produced in the concentration-response relations for kainate, glutamate, and glycine were well described by the simple competitive model of inhibition (Clark, A.J., 7. Physiol. (Lond.) 67:547-556
(1926); Gaddum, J.H., J. Physiol. (Lond.) 67: 141-150 (1926); Arunlakshana, O., et al, Br. J. Pharmacol. 74:48-58 (1959)). For all three agonists, the dose-response relations in the presence of 8-Me-DDHB were fit well by parallel curves shifted, relative to the control dose-response relation, by the factor (1 + [δ-Me-DDHBjVT . Separate fits of the logistic equation to each dose-response relation individually were not significantly better, at the 5 % confidence level, than the simultaneous fit with parallel curves that conformed to the simple competitive relationship.
The logistic equation has been used widely to provide an empirical description of concentration-response relations (Verdoorn, T.A., et al, Mol. Pharmacol. 34:298-307 (1988); Patneau, D.K., et al, J. Neurosci 10:2385-
2399 (1990); Werman, R., Comp. Biochem. Physiol. 30:997-1017 (1969)). Although this equation does not correspond to any specific reaction scheme for channel activation (except for the special case of integral slope factor) (Werman, R., Comp. Biochem. Physiol. 30:997-1017 (1969)), it has the advantages that the half-maximal point of the relationship is one of the fitted parameters and that the two parameters, EC50 and n, are not interdependent. When the logistic equation is used, slope factor values greater than 1 indicate that the receptor must bind more than one agonist molecule before the channel will open efficiently. In our experiments, the slope factors for the various agonists ranged from approximately 1.3 to 1.7. Similar results have been obtained in physiological experiments from several other laboratories (Verdoorn, T.A., et al, Mol. Pharmacol. 34:298-307 (1988); Patneau, D.K., et al, J. Neurosci 70:2385-2399 (1990); O'Dell, T.J., et al, J. Neurophysiol. 67:162-172 (1989)). Although the simple competitive model of antagonism was developed with the assumption of a single agonist binding site, Colquhoun
(Colquhoun, D., "The Relation Between Classical and Cooperative Models For Drug Action," in Drug Receptors (H.P. Rang, ed.), Macmillan, London (1973) 149-182) and Thron (Thron, CD., Mol. Pharmacol. 9:1-9 (1973)) have shown that it also holds for many receptor mechanisms that involve binding of more than one agonist molecule. Colquhoun (Colquhoun, D.,
Handb. Exp. Pharmacol. 59:59-113 (1986)) has further elaborated the inhibition expected for a receptor with two nonequivalent sites, which may deviate from the simple competitive relationship in some cases.
A key feature of the simple competitive model is that a competitive inhibitor will exhibit the same KB 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
(1990)), which suggests that these two agonists activate the same population of receptors. 8-Me-DDHB inhibited kainate current with a K of 6.4 μM (5.5- 7.5 μM, 95% confidence interval) and blocked steady state responses to glutamate with a KB of 9.6 μM (7.8-11.8 μM), 95 % confidence interval). Although the difference between these values reached statistical significance
(Student's t test), the two ^B values are nearly the same. The K against kainate is considered to be the more reliable indicator of antagonist affinity for non-NMDA receptors, because of the greater variability that was observed in the concentration-response relations for glutamate. The reason for this variability is not clear. It could be due to the strong desensitization produced by glutamate or might possibly arise from heterogeneity in the expression of non-NMDA receptor subunits (Boulter, J., et al, Science (Washington, D.C.) 249:1033-1037 (1990); Keinanen, K., et al, Science (Washington, D.C.) 249:556-560 (1990)). Structure-activity relations. The promise that glutamate receptor antagonists show as neuroprotective agents (Meldrum, B., et al, Trends Pharmacol. Sci. 77:379-387 (1990)) has spurred an increasing effort to develop additional antagonists and to understand the structural determinants of antagonist affinity. With this aim, a large number of compounds have recently been synthesized and tested for antagonist activity at the binding sites for glycine, NMDA, and kainate or AMPA [see Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252 (1991); Gray, N., et al., 7. Med. Chem. 34: 1283-1292 (1991); Harrison, B.L., et al., J. Med. Chem. 33:3130-3132 (1990); Salituro, F.G., et al., J. Med. Chem. 33:2944-2946 (1990)). 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 μM, respectively (Table 1). For kynurenic acid (Birch, P.J., et al, Eur. J. Pharmacol. 754:85-87 (1988); Kemp, J.A., et al, Proc. Natl. Acad. Sci. USA
85:6547-6550 (1988); Kessler, M., et al, J. Neurochem. 52: 1319-1328 (1989); Leeson, P.D., et al, J. Med. Chem. 34:1243-1252 (1991)), inhibition constants of 15-41 μM have been measured against glycine, 154-325 μM against NMDA, and 82-132 μM against kainate, quisqualate, or AMPA. Unsubstituted quinoxaline-2,3-dione displays the following potency (Kessler,
M., et al, J. Neurochem. 52: 1319-1328 (1989); Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252 (1991)); 26-39 μM versus glycine, 52 μM versus NMDA, and 120 μM versus kainate, quisqualate, or AMPA. Indole-2- carboxylic acid binds to the glycine site with a KB of approximately 25 μM (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). Taken together, these values indicate that the antagonist potency of DDHB is equal to or greater than kynurenic acid, quinoxaline-2,3-dione and indole-2-carboxylic acid. Our results with substituted derivatives of DDHB fit quite well with observations made by Leeson et al. (Leeson, P.D., et al, J. Med. Chem. 34:1243-1252 (1991)) on the potency of kynurenic acid derivatives. They found that methylation at the 7-position of kynurenic acid (which corresponds to the 8-position of DDHB) significantly improved the affinity for the glycine modulatory site. In contrast, addition of a 6-methyl group to kynurenic acid
(corresponding to 7-Me-DDHB) reduced the antagonist potency against glycine but increased the affinity for non-NMDA receptors (Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252 (1991)). Table 1 shows that 8-Me-DDHB was roughly 6-fold more potent against glycine than was DDHB, whereas 7-Me- DDHB was less potent than the parent compound at both the glycine and
NMDA recognition sites, but roughly twice as potent as DDHB against currents activated by kainate. Halogen substitution of the benzene ring significantly enhances the affinity of kynurenic acid (Kemp, J.A., et al, Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988); Baron, B.M., et al, Mol. Pharmacol. 38:554-561 (1990); Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252 (1991); Kleckner, N.W., et al, Mol. Pharmacol. 36:430-436 (1989)), indole-2-carboxylic acid
(Huettner, J.E., Science (Washington, D.C.) 243:1611-1613 (1989); Salituro, F.G., et al, J. Med. Chem. 33:2944-2946 (1990)), and quinoxaline-2,3-dione (Kleckner, N.W., et al, Mol. Pharmacol. 36:430-436 (1989)), both for the glycine modulatory site and for non-NMDA receptors. 4-Br-DDHB, which is substituted on the heterocyclic ring, was unchanged, relative to DDHB, in its potency against kainate, but was somewhat reduced in potency against both glycine and NMDA. 8-Br-DDHB was found to be the most potent glycine antagonist (Kj = 60 nM).
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,
B.M., et al, Mol. Pharmacol. 38:554-561 (1990); Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252 (1991)) and 4,6-dichloroindole-2-carboxylic acid (Salituro, F.G., et al, J. Med. Chem. 33:2944-2946 (1990)). Leeson et al. (Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252 (1991)) have emphasized the importance of hydrophobic interactions in the enhancement of potency by substituents of the benzene ring, but electronegativity of the substituent groups may also play a role in determining antagonist affinity. Both energy calculations (Harrison, B.L., et al, J. Med. Chem. 33:3130-3132 (1990)) and spectroscopic data (Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252 (1991); El-Ezaby, M.S., et al, Indian J. Chem. 77:1142-1145 (1973); Pileni, M.P., et al, Photochem. Photobiol. 30:251-256 (1979)) support the proposal (Huettner, I.E., Biochem. Pharmacol. 47:9-16 (1991)) that the 4-keto tautomer of kynurenic acid predominates in aqueous solutions and may be the most likely form to interact with the receptor sites. Donation of a hydrogen bond by the 1-NH group (Leeson, P.D., et al, J. Med. Chem. 34: 1243-1252
(1991)) appears to be essential for receptor binding by all of these antagonists. The lack of antagonism by the 3-acetyl ester and the 3-methyl ether of DDB (Table 1) suggests that the 3-hydroxyl group of DDHB is required for receptor binding. It seems likely that in DDHB, as well as the quinoxaline- 2,3-diones, the oxygens at positions 2 and 3 exhibit partial anionic character (Leeson, P.D., et al., 7. Med. Chem. 34: 1243-1252 (1991); Huettner, J.E.,
Biochem. Pharmacol. 41:9-16 (1991)) and can, therefore, substitute for the carboxylate group of kynurenic acid, indole-2-carboxylic acid, and the various amino acid agonists. Finally, the oxygen at position 5 of DDHB may accept a hydrogen bond when binding to the glycine modulation site, as has been proposed for the 4-keto group of kynurenic acid (Leeson, P.D., et al, J. Med.
Chem. 34: 1243-1252 (1991)), for various C-3 derivatives of indole-2- carboxylic acid (Gray, N., et al, J. Med. Chem. 34: 1283-1292 (1991)), and for several small agonist compounds (McBain, C.J., et al, Mol. Pharmacol. 36:556-565 (1989)). Bioavailability. Excessive activation of glutamate receptors causes damage to neurons and eventually leads to cell death (Choi, D.W., Neuron 7:623-634 (1988)). These neurotoxic actions of L-glutamate, and possibly other endogenous excitatory amino acids, have been implicated in a number of pathological conditions, including ischemia, epilepsy, and Huntington's disease (Choi, D.W., Neuron 7:623-634 (1988); Meldrum, B., et al, Trends
Pharmacol. Sci. 77:379-387 (1990)). Work on animal model systems suggests that glutamate receptor antagonists can protect neurons from the harmful effects of hyperstimulation; such neuroprotection has been observed with selective antagonists of both NMDA and non-NMDA receptors (Meldrum, B., et al, Trends Pharmacol. Sci. 77:379-387 (1990); Sheardown, M.J., et al,
Science (Washington, D.C.) 247:571-574 (1990)). In order to be therapeutically useful, however, antagonists must gain access to the CNS, usually by entry from the periphery through the blood-brain barrier. Most competitive antagonists possess ionized groups at physiological pH and, therefore, penetrate the blood-brain barrier very poorly. In contrast, DDHB and its derivatives are uncharged and highly lipophilic at neutral pH, which suggests that they may enter the brain much more readily than many antagonists. In the case of NMDA receptors, the necessity for passage into the CNS has focused attention on hydrophobic noncompetitive antagonists, such as phencyclidine and dizocilpine, which act by blocking the ion channel that is gated by NMDA. Although these compounds are neuroprotective, they also exhibit adverse side effects (Willetts, J., et al, Trends Pharmacol. Sci.
77:423-428 (1990)), including reinforcement of self-administration and possible direct toxic actions (Olney, J.W., et al, Science (Washington, D.C.) 244: 1360-1362 (1989)). Recent studies (reviewed by Willets et al , Trends Pharmacol. Sci. 77:423-428 (1990)) suggest that competitive NMDA antagonists exhibit a different behavioral profile with fewer of the undesirable psychotomimetic effects that are characteristic of phencyclidine and related compounds.
As mentioned above, recent work (Meldrum, B., et al, Trends Pharmacol. Sci. 77:379-387 (1990); Sheardown, M.J., et al, Science (Washington, D. C.) 247:571-574 (1990)) indicates that both NMDA and non-
NMDA receptor antagonists may contribute separately to neuroprotection. Of the four unsubstituted parent compounds kynurenic acid, indole-2-carboxylic acid, quinoxaline-2,3-dione and 2,5-dihydro-2,5-dioxo-3-hydroxy-7H- benzazepine (DDΗB), DDΗB has the highest apparent affinity at the glycine allosteric site, at the NMDA recognition site, and at non-NMDA receptors.
Although antagonists with dual action at NMDA (glycine) and non-NMDA receptors are likely to cause more depression of neuronal function, they may prove especially valuable in countering the heterogeneity of pathological mechanisms that occur during ischemia. These experiments indicate that DDΗB and derivatives thereof are neuroprotective in both in vitro and in vivo assays.
Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. All patents and publications cited herein are fully incorporated by reference herein in their entirety.

Claims

WHAT IS CLAIMED IS:
1. A method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia or surgery, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound of the Formula
Figure imgf000126_0001
or a tautomer thereof; wherein:
R. is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R, and R5 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, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
2. The method of claim 1 , wherein said compound is 2,5-dihydro- 2, 5-dioxo-3-hydroxy- IH-benzazepine.
3. The method of claim 1 , wherein said compound is 8-methyl-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
4. The method of claim 1 , wherein said compound is 8-bromo-2,5- dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
5. A method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia or surgery, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000127_0001
II
or a tautomer thereof; wherein:
R. is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
6. The method of claim 5, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4-hydroxy- lH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy- IH- benzazepine, 6-bromo-7,8-dichloro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6,7,8,9-tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- lH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,7-bromo-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, and 8-fluoro- 7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine.
7. A method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia or surgery, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000128_0001
III
or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R, and R5 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;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are C alkyl groups and Ar is an aryl group which may be substituted by a haio group.
8. A method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia or surgery, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000129_0001
IV
or a tautomer thereof; wherein:
Rj is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are C1-4 alkyl groups and Ar is an aryl group which may be substituted by a halo group.
9. A method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia or surgery, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000130_0001
or a tautomer thereof; wherein:
Rj is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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
Rg is hydrogen.
10. The method of claim 1, 5, 7, 8 or 9, wherein said neuronal loss occurs as a result of air bubbles that lodge in the brain during or immediately after surgery.
11. The method of claim 1, 5, 7, 8 or 9, wherein said neuronal loss occurs as a result of cardiopulmonary bypass surgery.
12. The method of claim 1, 5, 7, 8 or 9, wherein said neuronal loss occurs as a result of carotid endarterectomy surgery.
13. The method of claim 1, 5, 7, 8 or 9, wherein said neuronal loss occurs as a result of multiple strokes resulting in dementia.
14. A method of treating or preventing a neurodegenerative disease selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula
Figure imgf000131_0001
or a tautomer thereof; wherein:
Rj is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
R6 is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
15. The method of claim 14, wherein said compound is 2,5-dihydro- 2, 5-dioxo-3-hydroxy- IH-benzazepine.
16. The method of claim 14, wherein said compound is 8-methyl- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
17. The method of claim 14, wherein said compound is 8-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
18. A method of treating or preventing a neurodegenerative disease selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000132_0001
or a tautomer thereof; wherein:
R, is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, RΛ and R5 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, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
19. The method of claim 18, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-bromo-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo-
4-hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6,7,8,9-tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- lH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,7-bromo-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, and 8-fluoro- 7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine.
20. A method of treating or preventing a neurodegenerative disease selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000133_0001
III
or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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; Rβ is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are Cw alkyl groups and Ar is an aryl group which may be substituted by a halo group.
21. A method of treating or preventing a neurodegenerative disease selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000134_0001
IV
or a tautomer thereof; wherein:
R] is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R, and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are C1 alkyl groups and Ar is an aryl group which may be substituted by a halo group.
22. A method of treating or preventing a neurodegenerative disease selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000135_0001
or a tautomer thereof; wherein:
Ri is H2, H(OH), H (acyloxy), or oxo;
R2, R3, R4 and R5 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
Re is hydrogen.
23. A method of treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula
Figure imgf000135_0002
or a tautomer thereof; wherein
R, is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
24. The method of claim 23, wherein said compound is 2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine.
25. The method of claim 23, wherein said compound is 8-methyl- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
26. The method of claim 23, wherein said compound is 8-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
27. A method of treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000136_0001
or a tautomer thereof; wherein
R- is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, cyano, acylamino, azido, hydroxy, thiol, acyloxy, alkoxy, carboxy, carbonylamido or alkylthiol;
R6 is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
28. The method of claim 27, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-bromo-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7 , 8-difluoro-2 ,5-dihydro-2 ,5-dioxo- 4-hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6,7,8,9-tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,7-bromo-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, and 8-fluoro-
7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine.
29. A method of treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000138_0001
III
or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
30. A method of treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000139_0001
IV
or a tautomer thereof; wherein:
R, is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are C_ alkyl groups and Ar is an aryl group which may be substituted by a halo group.
31. A method of treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, comprising administering to an animal in need of such treatment an effective amount of a compound having the formula
Figure imgf000139_0002
V
or a tautomer thereof; wherein: R] is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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
Re is hydrogen.
32. A method of treating or preventing the adverse consequences of the hyperactivity of the NMDA receptor, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula
Figure imgf000140_0001
or a tautomer thereof; wherein
R. is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
33. The method of claim 32, wherein said compound is 2,5-dihydro- 2, 5-dioxo-3-hydroxy- IH-benzazepine.
34. The method of claim 32, wherein said compound is 8-methyl- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
35. The method of claim 32, wherein said compound is 8-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
36. A method of treating or preventing the adverse consequences of the hyperactivity of the NMDA receptor, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000141_0001
or a tautomer thereof; wherein
R_ is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CΗ2CONΗAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
37. The method of claim 36, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo- 4-hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6,7,8,9-tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- lH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine, 6, 8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,7-bromo-8- chloro-6-nitro-2 , 5 -dihydro-2 , 5 -dioxo-4-hydroxy- 1 H-benzazepi ne , 7-fl uoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, and 8-f uoro- 7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine.
38. A method of treating or preventing the adverse consequences of the hyperactivity of the NMDA receptor, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000142_0001
III or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
39. A method of treating or preventing the adverse consequences of the hyperactivity of the NMDA receptor, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000143_0001
IV
or a tautomer thereof; wherein:
Rj is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
40. A method of treating or preventing the adverse consequences of the hyperactivity of the NMDA receptor, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000144_0001
V
or a tautomer thereof; wherein:
Rj is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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
Re is hydrogen.
41. A method of treating or preventing chronic pain, comprising administering to an animal in need of such treatment a compound of the Formula
Figure imgf000144_0002
or a tautomer thereof; wherein
Rj is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
42. The method of claim 41, wherein said compound is2,5-dihydro- 2 ,5-dioxo-3-hydroxy- IH-benzazepine.
43. The method of claim 41, wherein said compound is 8-methyl-
2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
44. The method of claim 41, wherein said compound is 8-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
45. A method of treating or preventing chronic pain, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000145_0001
Rt is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
R6 is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and R7 is hydrogen, acyl or alkyl.
46. The method of claim 45, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-bromo-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo- 4-hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6,7,8,9-tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- lH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 7-bromo-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, and 8-fluoro- 7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine.
47. A method of treating or preventing chronic pain, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000147_0001
III
or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NΗCO-Ar, NΗCOCΗ2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are C^ alkyl groups and Ar is an aryl group which may be substituted by a halo group.
48. A method of treating or preventing chronic pain, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000148_0001
IV
or a tautomer thereof; wherein:
R, is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
49. A method of treating or preventing chronic pain, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000148_0002
or a tautomer thereof; wherein:
R, is H2, H(OH), H(acyloxy), or oxo; R2, R3, R4 and R5 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
Re is hydrogen.
50. The method of claim 41 , 45, 47, 48 or 49, wherein said chronic pain is the result of surgery on said animal.
51. A method of treating or preventing anxiety, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound of the Formula
Figure imgf000149_0001
or a tautomer thereof; wherein
R_ is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
52. The method of claim 51 , wherein said compound is 2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine.
53. The method of claim 51 , wherein said compound is 8-methyl- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
54. The method of claim 51, wherein said compound is 8-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
55. A method of treating or preventing anxiety, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000150_0001
or a tautomer thereof; wherein R: is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, azido, hydroxy, thiol, acyloxy, alkoxy, carboxy, carbonylamido or alkylthiol; Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CΗ2CONΗAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and R7 is hydrogen, acyl or alkyl.
56. The method of claim 55, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-bromo-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo- 4-hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6,7,8,9-tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- lH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,7-bromo-8- chloro-6-.nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, and 8-fluoro- 7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine.
57. A method of treating or preventing anxiety, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000151_0001
III
or a tautomer thereof; wherein: R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
58. A method of treating or preventing anxiety, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000152_0001
IV
or a tautomer thereof; wherein:
R, is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R, and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
59. A method of treating or preventing anxiety, comprising adminis¬ tering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000153_0001
or a tautomer thereof; wherein:
Ri is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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
Re is hydrogen.
60. A method of treating or preventing convulsions, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula
Figure imgf000153_0002
or a tautomer thereof; wherein
Ri is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
61. The method of claim 60, wherein said compound is 2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine.
62. The method of claim 60, wherein said compound is 8-methyl- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
63. The method of claim 60, wherein said compound is 8-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
64. A method of treating or preventing convulsions, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000154_0001
or a tautomer thereof; wherein Rj is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
65. The method of claim 64, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-bromo-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo- 4-hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6,7 , 8 ,9-tetrafluoro-2 ,5-dihydro-2 ,5-dioxo-4-hydroxy- IH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2 ,5-dihydro-2 ,5-dioxo-4-hydroxy- IH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,7-bromo-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, and 8-fluoro-
7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine.
66. A method of treating or preventing convulsions, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000156_0001
III
or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
67. A method of treating or preventing convulsions, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000156_0002
IV or a tautomer thereof; wherein:
R_ is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
68. A method of treating or preventing convulsions, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000157_0001
or a tautomer thereof; wherein:
Ri is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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
Re is hydrogen.
69. A method of inducing anesthesia, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula
Figure imgf000158_0001
or a tautomer thereof; wherein
R, is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
70. The method of claim 69, wherein said compound is 2,5-dihydro- 2,5-dioxo-3-hydroxy-lH-benzazepine.
71. The method of claim 69, wherein said compound is 8-methyl- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
72. The method of claim 69, wherein said compound is 8-bromo- 2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
73. A method of inducing anesthesia, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000159_0001
or a tautomer thereof; wherein
Ri is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, > and R5 are hydrogen, halo, haloalkyl, aryl, fused aryl, a heterocyclic group, a heteroaryl group, alkyl, nitro, amino, azido, cyano, acylamino, hydroxy, thiol, acyloxy, alkoxy, carboxy, carbonylamido or alkylthiol;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
74. The method of claim 73, wherein said compound is selected from the group consisting of 6-nitro-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-nitro-7,8-dibromo-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 6-chloro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-bromo-7,8-dichloro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6-chloro-7-nitro-8-trifluoromethyl-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-9-nitro-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo- 4-hydroxy- IH-benzazepine, 6-bromo-7,8-difluoro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 6,7,8, 9-tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy- lH-benzazepine, 6-chloro-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH- benzazepine,6,8-dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- dibromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8- difluoro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 7-chloro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine,7-bromo-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 8-fluoro-7- chloro-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 7-fluoro-8- bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, and 8-fluoro- 7-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine.
75. A method of inducing anesthesia, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000160_0001
III
or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R, and R5 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; Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
76. A method of inducing anesthesia, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000161_0001
IV
or a tautomer thereof; wherein:
R, is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
77. A method of inducing anesthesia, comprising administering to an animal in need of such treatment an effective amount of a compound of the Formula:
Figure imgf000162_0001
or a tautomer thereof; wherein:
Ri is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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
R6 is hydrogen.
78. The method of any one of claims 1, 5, 7, 8, 9, 14, 18, 20-23, 27, 29-32, 36, 38-41, 45, 47-49, 51, 55, 57-60, 64, 66-69, 73, and 75-77, wherein said compound is administered as part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
79. A compound having the Formula:
Figure imgf000162_0002
or a tautomer thereof; wherein
Ri is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R, and R5 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;
Re is hydrogen, aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl; with the proviso that R2 is halo or nitro, R3 is halo or nitro, and R4 is halo or haloalkyl.
80. A compound having the Formula:
Figure imgf000163_0001
or a tautomer thereof; wherein
Ri is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CH2CONHAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
81. 8-Methyl-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
82. 6,8-Dichloro-2,5-dihydro-2,5-dioxo-3-hydroxy- IH-benzazepine.
83. 8-Bromo-2,5-dihydro-2,5-dioxo-3-hydroxy-lH-benzazepine.
84. A compound of the Formula:
Figure imgf000164_0001
or a tautomer thereof; wherein
Ri is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido;
R2, R3, R4 and R5 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;
Re is aryl, a heterocyclic group, a heteroaryl group, alkyl, amino, -CΗ2CONΗAr, -NHCONHAr, -NHCOCH2Ar, -COCH2Ar, hydroxy, alkoxy, aryloxy, aralkyloxy, cycloalkylalkoxy or acyloxy, wherein Ar is an aryl group or a heteroaryl group; and
R7 is hydrogen, acyl or alkyl.
85. A compound selected from the group consisting of 6-nitro-7,8- dichloro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-nitro-7,8- dibromo-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-chloro-8- trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo- 7,8-dichloro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-chloro-7- nitro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6- chloro-9-nitro-8-trifluoromethyl-2,5-dihydro-2,5-dioxo-4-hydroxy- IH- benzazepine, 6,7,8-trichloro-2,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-chloro-7,8-difluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6- bromo-7 , 8-difluoro-2 ,5-dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6,7,8,9- tetrafluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-chloro-8- fluoro-2,5-dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6,8-dibromo-2,5- dihydro-2,5-dioxo-4-hydroxy-lH-benzazepine, 6-bromo-8-trifluoromethyl-2,5- dihydro-2,5-dioxo-4-hydroxy- IH-benzazepine, 6-bromo-8-fluoro-2,5-dihydro- 2,5-dioxo-4-hydroxy-lH-benzazepine, 7,8-dibromo-6-nitro-2,5-dihydro-2,5- dioxo-4-hydroxy- IH-benzazepine, 7,8-difluoro-6-nitro-2,5-dihydro-2,5-dioxo- 4-hydroxy- IH-benzazepine, 7-chloro-8-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine,7-bromo-8-chloro-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 7-fluoro-8-chloro-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, 8-fluoro-7-chloro-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy-lH-benzazepine, 7-fluoro.-8-bromo-6-nitro-2,5-dihydro-2,5-dioxo-4- hydroxy- IH-benzazepine, and 8-fluoro-7-bromo-6-nitro-2,5-dihydro-2,5-dioxo- 4-hydroxy- IH-benzazepine.
86. A compound having the formula
Figure imgf000165_0001
III
or a tautomer thereof; wherein:
R1 is hydrogen, halo, haloalkyl, alkyl, aryl, a heterocyclic group, a heteroaryl group, nitro, amino, hydroxy, alkoxy or azido; R2, R3, R4 and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
X is -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
87. A compound having the formula
Figure imgf000166_0001
IV
or a tautomer thereof; wherein:
R_ is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R, and R5 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;
Re is hydrogen;
R7 is hydrogen, acyl or alkyl; and
Y is alkyl, -NHCO-Ar, NHCOCH2-Ar, NHCONH-Ar, -NHCONH2, -NHCONHR8 or -NHCONR8R9, wherein R8 and R9 are CM alkyl groups and Ar is an aryl group which may be substituted by a halo group.
88. A compound of the formula
Figure imgf000167_0001
or a tautomer thereof; wherein:
R, is H2, H(OH), H(acyloxy), or oxo;
R2, R3, R4 and R5 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
Re is hydrogen; with the proviso that at least one of R1, R2, R3 and R4 is other than hydrogen.
89. A pharmaceutical composition, comprising the compound of any one of claims 79-88 and a pharmaceutically acceptable carrier.
PCT/US1993/009288 1992-09-30 1993-09-30 2,5-dihydro-2,5-dioxo-1h-azepines and 2,5-dihydro-2-oxo-1h-azepines and the use thereof as excitatory amino acid and glycine receptor antagonists WO1994007500A1 (en)

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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
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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
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