|||||||III US005514680A United States Patent (19) 11 Patent Number: 5,514,680 Weber Et Al

|||||||III US005514680A United States Patent (19) 11 Patent Number: 5,514,680 Weber et al. (45) Date of Patent: May 7, 1996

54 GLYCINE RECEPTOR ANTAGONISTS AND 0377112 7/1990 European Pat. Off.. THE USE THEREOF 0511152 10/1992 European Pat. Off.. 572852 12/1993 European Pat. Off.. 2451049 4/1976 Germany. (75) Inventors: Eckard Weber, Laguna Beach, Calif.; 2446543 4/1976 Germany. John F. W. Keana, Eugene, Oreg. 2847285 5/1980 Germany. 72674 11/1974 Poland. 73) Assignees: State of Oregon, acting by and WO91/13878 9/1991 WIPO. through The Oregon State Board of WO92/07847 11/1991 WIPO. Higher Education, acting for and on WO92/O2487 2/1992 WIPO. behalf of The Oregon Health Sciences WO92/11012 7/1992 WIPO. University and The University of WO92/11245 7/1992 WIPO. Oregon, Eugene, Oreg.; The Regents WO92/14740 9/1992 WIPO : of the University of California, WO92/15565 9/1992 WIPO. Oakland, Calif. 94.07500 4/1994 WIPO. OTHER PUBLICATIONS (21) Appl. No.: 148,259. Allison et al., Polyfluoroheterocyclic Compounds, Part XX. 22 Filed: Nov. 5, 1993 Preparation and Nucleophilic Substitution of Hexafluoro quinoxaline, J. Fluorine Chem. 1:59-667 (1971). Related U.S. Application Data Burton et al., Halogeno-o-phenylenediamines and Derived Heterocycles. Part I. Reductive Fission of Benzotriazoles to 63 Continuation-in-part of PCT/US93/05859, Jun. 17, 1993, o-Phenylenediamines, J. Chem. Soc. (C) 10:1268-1273 which is a continuation-in-part of Ser. No. 69,274, May 28, (1968). 1993, abandoned, which is a continuation-in-part of Ser. No. Lutfy et al., Blockade of Morphine Tolerance by 995,167, Dec. 22, 1992, abandoned, which is a continuation ACEA-1328, a novel NMDA receptor/glycine site antago in-part of Ser. No. 903,080, Jun. 22, 1992, abandoned. nist, Eur: J. Pharmacol. 273:187-189 (Jan. 1995). (51) Int. Cl...... A61K 3/495 Warner et al., In Vivo Models of Cerebral Ischemia: Effects 52 U.S. C...... 514/249 of Parenterally Administered NMDA Receptor Glycine Site (58) Field of Search ...... 514/249 Antagonists, J. Cereb. Blood Flow Metab. 15:188-196 (Mar. 1995). (56) References Cited Copy of the Supplementary Partial European Search Report, mailed by the European Patent Office on Aug. 10, 1995. U.S. PATENT DOCUMENTS Derwent Abstract 93-387595 (Abstract of EP572 852). 3,962,440 6/1976 St. Clair et al...... 424/250 Adreasen et al., “Effects of Non-N-Methyl-D-Aspartate 3,992,378 11/1976 St. Clair et al. . 260/250 Q Antagonists on Synaptic Transmission in the In Vitro Rat 4,312,807 1/1982 Fuchs ...... 260/154 Hippocampus', J. Physiol. 414:317-336 (1989). 4,659,713 4/1987 Hass ..... 514/249 Bigge, C., "Structural Requirements for the Development of 4,803,270 2/1989 Takemoto...... 544/105 Potent N-Methyl-D-Aspartic Acid (NMDA) Receptor 4,812,458 3/1989 Honoré et al...... 514/249 Antagonists', Biochem. Pharm. 45:1547-1561 (1993). 4,889,855 12/1989 Jacobsen et al...... 54/250 4,912,108 3/1990 Jacobsen et al...... 514/250 (List continued on next page.) 4,948,794 8/1990 Honore et al...... 514/249 4,975,430 12/1990 Jahr et al...... 54/255 Primary Examiner-Glennon H. Hollrah 4,977,155 12/1990 Jacobsen et al...... 514/250 Assistant Examiner-Mary C. Cebulak 4,994,460 2/1991 Dextraze et al...... , 514/252 Attorney, Agent, or Firm-Sterne, Kessler, Goldstein & Fox 5,026,704 6/1991 Honore et al...... 514/250 5,055,465 10/1991 Davey ...... 54/228.2 57 ABSTRACT 5,055,470 10/1991 Boissard et al. . ... 514/252 Methods of treating or preventing neuronal loss associated 5,057,516 10/1991 Jacobsen et al...... 514/250 5,061,706 10/1991 Honoreé et al...... 514/249 with stroke, ischemia, CNS trauma, hypoglycemia and sur 5,075,306 12/1991 Jacobsen et al...... 514/250 gery, as well as treating neurodegenerative diseases includ 5,079,250 1/1992 Jacobsen et al...... 514/250 ing Alzheimer's disease, amyotrophic lateral sclerosis, Hun 5,081,123 1/1992 Honoré et al...... 514/250 tington's disease and Down's syndrome, treating or 5,109,001 4/1992 Jacobsen et al...... 514/250 preventing the adverse consequences of the hyperactivity of 5,166,155 11/1992 Jorgensen et al...... 514/249 the excitatory amino acids, as well as treating anxiety, 5,187,171 2/1993 Cordi ...... 514/359 chronic pain, convulsions, inducing anesthesia and treating 5,190,941 3/1993 Nozulak et al. ... 514/229.8 psychosis are disclosed by administering to an animal in 5,196.421 3/1993 McQuaid et al. ... 514/250 need of such treatment a compound having high affinity for 5,198,461 3/1993 Watjen et al...... 514/411 the glycine binding site, lacking PCP side effects and which 5,268,378 12/1993 Baker et al...... 514/312 crosses the blood brain barrier of the animal.

5,283,244 2/1994 Sakamoto et al... 514/249 5,308,845 5/1994 Honoré et al...... 514/250 5,352,683 10/1994 Mayer et al...... 514/289 Also disclosed are novel 1,4-dihydroquinoxaline-2,3-di ones, and pharmaceutical compositions thereof. Also dis FOREIGN PATENT DOCUMENTS closed are highly soluble ammonium salts of 1,4-dihydro 01.0722 5/1980 European Pat. Off.. quinoxaline-2,3-diones. 348872 1/1990 European Pat. Off.. 0374534 6/1990 European Pat. Off.. 26 Claims, 45 Drawing Sheets 5,514,680 Page 2

OTHER PUBLICATIONS Leeson et al., "Kynurenic Acid Derivatives. Structure-Ac tivity Relationships for Excitatory Amino Acid Antagonism Birch et al., “FG-9065 and FG-9041 antagonise responses and Identification of Potent and Selective Antagonists at the to NMDA via an action at the strychnine-insensitive glycine Glycine Site on the N-Methyl-D-aspartate Receptor”, J. receptor', Meeting of the British Pharmacological Society, Med. Chem, 34:1243-1252 (1991). Nottingham, England, Sep. 7-9, 1988, Br. J. Pharmacol. Leeson et al., “Amino Acid Bioisosteres: Design of 2-Qui 95:758P (1988). nolone Derivatives as Glycine-Site N-Methyl-D-aspartate Carling et al., "Anticonvulsant Activity of Glycine-site Receptor Antagonists', Bioorg. & Med. Chem. Lett. NMDA Antagonists", Bioorganic & Medicinal Chem. Lett. 3:299-304 (1993). 3:65-70 (1993). Leeson, P., "Glycine-Site N-Methyl-D-Aspartate Receptor Carling et al., "3-Nitro-3,4-dihydro-2(1H)-quinolones. Antagonists", pp. 339-381, in: Drug Design for Neurosci, Excitatory Amino Acid Antagonists Acting at Glycine-Site Kozikowski (ed.), Raven Press Ltd., New York (1993). NMDA and Lester et al., "Interaction of (RS)-O-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic 6-Cyano-7-nitroquinoxaline-2,3-dione with the N-Meth Acid Receptors', J. Med. Chem. 36:3397-3408 (1993). yl-D-aspartate Receptor-Associated Glycine Binding Site', Mol. Pharmacol. 35:565-570 (1989). Cheeseman, G. W. H., "Quinoxalines and Related Com Littman et al., "The quinoxalinediones DNQX, CNQX and pounds, Part VI. Substitution of 2,3-Dihydroxyquinoxaline two related congeners suppress hair cell-to-auditory nerve and its 1,4-Dimethyl Derivative', J. Chem. Soc. transmission', Hearing Res. 40:45-53 (1989). 223:1170-1176 (1962). Lodge & Jones, "Evidence for Glutamate Receptor Subtypes Davies & Collingridge, "Quinoxalinediones as Excitatory From In Vivo Electrophysiology: Studies with HA-966, Amino Acid Antigonists in the Vertebrate Central Nervous Quinoxalinediones and Philanthotoxin'", pp. 101-108 in: System”, Int. Rev. Neurobiol. 32:281-303 (1990). Excitatory Amino Acids and Neuronal Plasticity, Ben-Ari, Drejer & Honoré, "New quinoxalinediones show potent Y. (ed.), Plenum Press, New York (1990). antagonism of quisqualate responses in cultured mouse Lodge et al., "Excitatory amino acids:new tools for old cortical neurons', Neur: Lett. 87:104-108 (1988). stories or Pharmacological subtypes of glutamate recep Fletcher et al., "Quinoxalinediones selectively block tors:electrophysiological studies', Can. J. Physiol. Pharma quisqualate and kainate receptors and synaptic events in rat col. 69:1123-1128 (1991). Louvet et al., "Novel benzimidazoles as ligands for the neocortex and hippocampus and frog spinal cord in vitro', strychnine-insensitive N-methyl-D-aspartate-linked gly Br. J. Pharmacol. 95:585-597 (1988). cine receptor', Eur: J. Med. Chem, 28:71-75 (1993). Frenket al., “Absence of Side-Effects in the Anticonvulsant McFarlane & Smith, "A New Route to N-Hydroxyquinoxa Action of Cortically Applied Antagonists of N-Methyl line-2,3-diones and some AZA-Analogues', Tetrahedron -D-Aspartate', Brain Res. 373:222-226 (1986). Lett. 28:6363-6366 (1987). Hays et al., "N-Sulfonyl Derivatives of 6,7-Dichloro McQuaid et al., “Synthesis and Excitatory Amino Acid 3,4-Dihydro-3-Oxo-quinoxalinecarboxylate as Glycine-s- Pharmacology of a Series of Heterocyclic-Fused Quinox ite NMDA and AMPA Antagonists”, Bioorg. & Med. Chem. alinones and Quinazolinones', J. Med. Chem. Lett. 3:77-80 (1993). 35:3319-3324 (1992). Honoré et al., "Quinoxalinediones: Potent Competitive Non Moore et al., “Anticonvulsant Activity of Glycine-Site -NMDA Glutamate Receptor Antagonists', Science NMDA Antagonists', Bioorg. & Med. Chem. Lett. 3:61-64 241:701-703 (Aug. 5, 1988). (1993). Honoré et al., "Quinoxalinediones Non-N-Methyl-D-as Näsström et al., "Antinociceptive actions of different classes partate Receptor Antagonists as Potential Drug Candidates', of excitatory amino acid receptor antagonists in mice', Eur: J. Pharmacol. 212:21-29 (1992). pp. 451-460, in: Excitatory Amino Acids, Meldrum et al. Ogita & Yoneda, “6,7-Dichloroquinoxaline-2,3-Dione is a (eds.), Raven Press Ltd., New York (1991). Competitive Antagonist Specific to Strychnine-Insensitive Horner et al., “Derivatives of quinoxaline as isosteres of the HGlycine Binding Sites on the N-Methyl-D-Aspartate pteridines', Chem. Abst. 48:2692 (1953). Receptor Complex”, J. Neurochem. 54:699-702 (1990). Kay & Ikeda, "The quinoxalinediones antagonise the visual Patel et al., “6,7-Dinitroquinoxaline-2,3-Dione Blocks the firing of sustained retinal ganglion cells', Eur: J. Pharmacol. Cytotoxicity of N-Methyl-D-Aspartate and Kainate, but 164:381-384 (1989). Not Quisqualate, in Cortical Cultures', J. Neurochem. Kemp & Leeson, "The glycine site of the NMDA receptor 55:114-121 (1990). -five years on', TiPS 14:20-25 (Jan. 1993). Pellegrini-Giampietro et al., “Quinoxalines interact with the glycine recognition site of NMDA receptors: studies in Kessler et al., "Quinoxaline derivatives are high-affinity guinea-pig myenteric plexus and in rat cortical mem antagonists of the NMDA receptor-associated glycine branes', Br. J. Pharmacol. 98:1281–1286 (1989). sites', Brain Res. 489:377-382 (1989). Randle et al., "Quinoxaline Derivatives: Structure-Activity Kessler et al., "A Glycine Site Associated with N-Methyl Relationships and Physiological Implications of Inhibition -D-Aspartic Acid Receptors: Characterization and Identifi of N-Methyl-D-aspartate and Non-N-methyl-D-aspartate cation of a New Class of Antagonists', J. Neurochem. Receptor-Mediated Currents and Synaptic Potentials”, Mol. 52:1319-1328 (1989). Pharmacol. 41:337-345 (1991). Kleckner & Dingledine, "Selectivity of Quinoxalines and Randle et al., "Competitive inhibition of NBQX of kainate/ Kynurenines as Antagonists of the Glycine Site on N-Meth AMPA receptor currents and excitatory synaptic potential yl-D-aspartate Receptors', Mol. Pharmacol. 36:430-436 simportance of 6-nitro substitution', Eur: J. Pharmacol. (1989). 215:237-244 (1992). 5,514,680 Page 3

Rao et al., "6,7-Dinitroquinoxaline-2,3-Dione and 6-Nitro, Leeson and Iversen, "The Glycine Site on the NMDA 7-Cyanoquinoxaline-2,3-Dione Antagonize responses Receptor: Structure-Activity Relationships and Therapeutic mediated by N-methyl-D-aspartate and NMDA-associated Potential,' J. Med. Chem. 37:4053-4067 (1994). Glycine recognition sites in vivo: measurements of cerebel Salituro et al., "3-(2-Carboxyindol-3-yl)propionic Acid lar cyclic-GMP', Neuropharmacol. 29:1031-1035 (1990). -Based Antagonists of the N-Methyl-D-aspartic Acid Rowley et al., "3-Acyl-4-hydoxyquinolin-2(1H)-ones. Receptor Associated Glycine Binding Site", J. Med. Chem. Systemically Active Anticonvulsants Acting by Antagonism 35:1791-1799 (May, 1992). at the Glycine Site of the N-Methyl-D-Aspartate Receptor Turski et al., "Relief of Experimental Spasticity and Anxi Complex”, J. Med. Chem. 36:3386-3396 (1993). olytic/Anticonvulsant Actions of the Sheardown et al., "A potent antagonist of the strychnine Alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionate insensitive glycine receptor has anticonvulsant properties', Antagonist Eur: J. Pharmacol. 174: 197-204 (1989). 2,3-Dihydroxy-6-Nitro-7-Sulfamoyl-Benzo(F) Quinoxa Smith et al., "Structure-Activity Relationships of a Series of line,” J. Pharm. Exp. Ther. 260:742-747 (1992). Glycine antagonists related to 5,7-Dichlorokynurenic Acid White et al., "Anticonvulsant Profile and Therapeutic Poten and 3-(2-carboxy-6-chloroindol-3-yl)Acetic Acid', tial of N-Methyl-D-Aspartic Acid and Strychnine-Insensi Bioorg. & Med. Chem. Lett. 3:81–84 (1993). tive Glycine Antagonists,” Epilepsia 32, Suppl. 3:12 (1991). Yamada et al., "Quantitative Physiological Characterization Vaccarino et al., “NMDA Receptor Antagonists, MK-801 of a Quinoxalinedione non-NMDA Receptor Antagonist", J. and ACEA-1011, Prevent the Development of Tonic Pain Neurosci. 9:3230-3236 (Sep. 1989). Following Subcutaneous Formalin,' Brain Research Yoneda & Ogita, "Abolition of the NMDA-Mediated 615:331-334 (Jul. 2, 1993). Responses by a Specific Glycine Antagonist, 6,7-Dichloro Danysz, W. and Wroblewski, J.T., "Amnesic Properties of quinoxaline-2,3-Dione (DCQX)", Biochem. & Biophys. Glutamate Receptor Antagonists,' Neurosci. Res. Commun. Res. Comm. 164:841-849 (Oct. 31, 1989). 5(1):9-18 (1989). Honore et al., "Preparation and testing of 2,3(1H4H)-qui Jurson, P. A. and Freed, W. J., "A Slight Anticonvulsant noxalinediones as neuroleptics,” Chem. Abst. 111:232859a Effect of CNQX and DNQX as measured by Homocysteine (1989). and Quisqualate-Induced Seizures.' Pharmacol. Biochem. Honore et al., "Preparation and use of neuroleptic quinoxa Behav. 36:177-181 (1990). line compounds,” Chem. Abst. 114:81885q (1990). McQuaid, L. A. et al., Joergensen et al., "Preparation of 1-carboxyalkyl-2,3-di "3-Phenyl-4-hydroxyquinolin-2(1H)-ones: Potent and oxoquinoxalines as glycine antagonists,' Chem. Abst. Selective Antagonists at the Strychnine-Insensitive Glycine 115:280059u (1991). Site on the N-Methyl-D-aspartate Receptor Complex,” J. Kehne et al., "NMDA Receptor Complex Antagonists Have Med. Chem. 35:3423-3425 (1992). Potential Anxiolytic Effects as Measured With Separation Schoepp et al., "Excitatory Amino Acid-Induced Convul -Induced Ultrasonic Vocalizations,' Eur: J. Pharmacol. sions in Neonatal Rats Mediated by Distinct Receptor Sub 193:283-292 (1991). types,” Eur: J. Pharmacol. 182:421-427 (1990). Kulagowski et al., "3'-(Arylmethyl)- and Warner, D. S. et al., "Glycine NMDA Receptor Antagonists '-(Aryloxy)-3-phenyl-4-hydroxyquinolin-2(1H)-ones: Reduce Focal But Not Global Ischemic Brain Damage in the Orally Active Antagonists of the Glycine Site on the NMDA Rat,' Soc. Neurosci. Abs. 19:1646, Abstract No. 674.3 Receptor.” J. Med. Chem. 37:1402-1405 (1994). (1993). U.S. Patent May 7, 1996 Sheet 1 of 45 5,514,680

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5,514,680 1. 2 GLYCINE RECEPTOR ANTAGONSTS AND 85:1307-1311 (1988); MacDonald, J. F., et al., Neuro THE USE THEREOF physiol. 58:251-266 (1987)). In order for PCP to gain access to the PCP receptor, the channel must first be opened by This invention was made with government support under glutamate and glycine. In the absence of glummate and grant number NDA DAO 6726 awarded by the National glycine, PCP cannot bind to the PCP receptor although some Institutes of Health. The government has certain rights in the studies have suggested that a small amount of PCP binding invention. can occur even in the absence of glummate and glycine (Sircar and Zukin, Brain Res. 556:280-284 (1991)). Once CROSS REFERENCE TO RELATED PCP binds to the PCP receptor, it blocks ion flux through the APPLICATIONS 10 open channel. Therefore, PCP is an open channel blocker and a non-competitive glummate antagonist at the NMDA This is a continuation-in-part of International Application receptor/channel complex. PCT/US93/05859, Jun. 17, 1993, which is a continuation One of the most potent and selective drugs that bind to the in-part of U.S. application Ser. No. 08/069,274 filed May 28, PCP receptor is the anticonvulsant drug MK801. This drug 1993 now abandoned, which is a continuation-in-part of 15 has a K of approximately 3 nM at the PCP receptor (Wong, U.S. application Ser. No. 07/995,167, filed Dec. 22, 1992, E. H. F., et al., Proc. Natl. Acad. Sci. USA 83:7104-7108 now abandoned, which is a continuation-in-part of U.S. (1986)). application Ser. No. 07/903,080, filed Jun. 22, 1992, now Both PCP and MK801 as well as other PCP receptor abandoned, the contents of each of which are fully incor ligands (e.g. dextromethorphan, ketamine and N,N'-disub porated by reference herein. 20 stituted guanidines) have neuroprotective efficacy both in 1. Field of the Invention vitro and in vivo (Gill, R., et al., J. Neurosci. 7:3343-3349 The present invention is in the field of medicinal chem (1987); Keana, J. F. W., et al., Proc. Natl. Acad. Sci. USA istry. The present invention relates to compounds having 86:5631-5635 (1989); Steinberg, G. K., et al., Neuroscience high affinity for the glycine binding site, lacking PCP side Lett. 89:193-197 (1988); Church, J., et al., In: Sigma and effects and which cross the blood brain barrier at high levels. 25 Phencyclidine-Like Compounds as Molecular Probes in In particular, the present invention relates to novel 14 Biology, Domino and Kamenka, eds., Ann Arbor: NPP -dihydroquinoxaline-2,3-diones and their use to treat or Books, pp. 747-756 (1988)). The well-characterized neuro prevent neuronal degeneration associated with ischemia, protective efficacy of these drugs is largely due to their pathophysiologic conditions associated with neuronal capacity to block excessive Ca' influx into neurons through degeneration, convulsions, anxiety chronic pain and to 30 NMDA receptor channels which become over activated by induce anesthesia. The invention also relates to certain excessive glutamate release in conditions of brain ischemia highly soluble ammonium salts of 1,4-dihydroquinoxaline (e.g. in stroke, cardiac arrest ischemia etc.) (Collins, R. C., 2,3-diones. Metabol. Br. Dis. 1:231-240 (1986); Collins, R. C., et al., 2. Background of the Invention Annals Int. Med. 110:992-1000 (1989)). Glutamate is thought to be the major excitatory neu 35 However, the therapeutic potential of these PCP receptor rotransmitter in the brain. There are three major subtypes of drugs as ischemia rescue agents in stroke has been severely glutamate receptors in the CNS. These are commonly hampered by the fact that these drugs have strong PCP-like referred to as kainate, AMPA and N-methyl-D-aspartate behavioral side effects (psychotomimetic behavioral effects) (NMDA) receptors (Watkins and Olverman, Trends in Neu which appear to be due to the interaction of these drugs with rosci. 7:265-272 (1987)). NMDA receptors are found in the 40 the PCP receptor (Tricklebank, M. D., et al., Eur: J. Phar membranes of virtually every neuron in the brain. NMDA macol. 167:127-135 (1989); Koek, W., et at., J. Pharmacol. receptors are ligand-gated cation channels that allow Na', Exp. Ther. 245:969 (1989); Willets and Balster, Neurophar K" and Ca' to permeate when they are activated by macology 27:1249 (1988)). These PCP-like behavioral side glutamate or aspartate (non-selective, endogenous agonists) effects appear to have caused the withdrawal of MK801 or by NMDA (a selective, synthetic agonist) (Wong and 45 from clinical development as an ischemia rescue agent. Kemp, Ann. Rev. Pharmacol. Toxicol. 31:401-425 (1991)). Furthermore, these PCP receptor ligands appear to have Glutamate alone cannot activate the NMDA receptor. In considerable abuse potential as demonstrated by the abuse order to become activated by glutamate, the NMDA receptor liability of PCP itself. channel must first bind glycine at a specific, high affinity 50 The PCP-like behavioral effects of the PCP receptor glycine binding site which is separate from the glutamate/ ligands can be demonstrated in animal models: PCP and NMDA binding site on the receptor protein (Johnson and related PCP receptor ligands cause a behavioral excitation Ascher, Nature 325:329-331 (1987)). Glycine is therefore (hyperlocomotion) in rodents (Tricklebank, M. D., et al., an obligatory coagonist at the NMDA receptor/channel Eur: J. Pharmacol. 167:127-135 (1989)) and a characteristic complex (Kemp, J. A., et al., Proc. Natl. Acad. Sci. USA 55 katalepsy in pigeons (Koek, W., et al., J. Pharmacol. Exp. 85:6547-6550 (1988)). Ther. 245:969 (1989); Willets and Balster, Neuropharma Besides the binding sites for glutamate/NMDA and gly cology 27:1249 (1988); in drug discrimination paradigms, cine, the NMDA receptor carries a number of other func there is a strong correlation between the PCP receptor tionally important binding sites. These include binding sites affinity of these drugs and their potency to induce a PCP for Mg., Zn, polyamines, arachidonic acid and phencyc 60 appropriate response behavior (Zukin, S. R., et al., Brain lidine (PCP) (Reynolds and Miller, Adv. in Pharmacol. Res. 294:174 (1984); Brady, K. T., et al., Science 215:178 21:101-126 (1990); Miller, B., et al., Nature 355:722-725 (1982); Tricklebank, M. D., et al., Eur: J. Pharmacol. (1992)). The PCP binding site- now commonly referred to 141:497 (1987)). as the PCP receptor-is located inside the pore of the Drugs acting as competitive antagonists at the glummate ionophore of the NMDA receptor?channel complex (Wong, 65 binding site of the NMDA receptor such as CGS 19755 and E. H. F., et al., Proc. Natl. Acad. Sci., USA 83:7104-7108 LY274614 also have neuroprotective efficacy because these (1986); Huettner and Bean, Proc. Natl. Acad. Sci. USA drugs-like the PCP receptor ligands- can prevent exces 5,514,680 3 4 sive Ca' flux through NMDA receptor/channels in available glycine antagonists because they lack bio ischemia (Boast, C. A., et al., Brain Res. 442:345-348 availability in vivo. (1988); Schoepp, D. D., et al., J. Neural. Trans. 85:131-143 There have been a number of reports in the literature of (1991)). However, competitive NMDA receptor antagonists substituted 1,4-dihydroquinoxaline-2,3-diones which are also have PCP-like behavioral side-effects in animal models useful for treating pathophysiologic conditions mediated by (behavioral excitation, activity in PCP drug discrimination the, non-NMDA, NMDA and glycine receptors. For tests) although not as potently as MK801 and PCP (Trick example, U.S. Pat. No. 4,975,430 (1990), discloses 1,4 lebank, M. D., et al., Eur: J. Pharmacol. 167:127-135 -dihydroquinoxaline-2,3-dione compounds of the formula: (1989)). Y An alternate way of inhibiting NMDA receptor channel 10 activation is by using antagonists at the glycine binding site X N 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. X N O A., et al., Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)), 15 Y a glycine antagonist can completely prevention flux through the NMDA receptor channel-even in the presence of a wherein each X is independently nitro or cyano and wherein large amount of glummate. each Y is independently H, lower alkyl, lower alkoxy, or Recent in vivo microdialysis studies have demonstrated CF. These compounds are reportedly useful for the treat that in the rat focal ischemia model, there is a large increase 20 ment of neuronal conditions associated with stimulation of in glummate release in the ischemic brain region with no the NMDA receptor. significant increase in glycine release (Globus, M. Y.T., et U.S. Pat. No. 3,962,440 (1976), discloses 1,4-dihydroqui al., J. Neurochem. 57:470-478 (1991)). Thus, theoretically, noxaline-2,3-dione compounds having the formula: glycine antagonists should be very powerful neuroprotective Rl agents, because they can prevent the opening of NMDA 25 channels by glutamate non-competitively and therefore N O unlike competitive NMDA antagonists-do not have to overcome the large concentrations of endogenous glutamate that are released in the ischemic brain region. N O Furthermore, because glycine antagonists act at neither 30 H the glutamate/NMDA nor the PCP binding sites to prevent NMDA channel opening, these drugs might not cause the wherein, R' may be hydrogen or methyl, R may be low PCP-like behavioral side effect seen with both PCP receptor eralkyl, loweralkoxy, loweralkylthio, cyclopropyl, nitro, ligands and competitive NMDA receptor antagonists (Trick cyano, halogen, fluoroalkyl of C-C (trifluoromethyl) lebank, M. D., et al., Eur: J. Pharmacol. 167:127-135 35 amino or substituted amino, and n may be 0, 1 or 2. These (1989); Koek, W., et al., J. Pharmacol. Exp. Ther. 245:969 compounds are reportedly useful as hypnotic agents. (1989); Willets and Balster, Neuropharmacology 27:1249 U.S. Pat. No. 4,812,458 (1989), discloses 1,4-dihydroqui (1988); Tricklebank, M. D., et al., Eur: J. Pharmacol. noxaline-2,3-dione compounds having the formula: 167:127-135 (1989); Zukin, S.R., et al., Brain Res. 294:174 H (1984); Brady, K.T., et al., Science 215:178 (1982); Trick R1 N O R1 N OH lebank, M. D., et al., Eur. J. Pharmacol. 141:497 (1987)). W G YS 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 R2 N.H Yo R2 N oH into the brains of rodents without resulting in PCP-like 45 behaviors (Tricklebank, M. D., et al., Eur: J. Pharmacol. wherein R is halogen, cyano, trifluoromethyl, ethynyl or N. 167:127-135 (1989)). and R’ is SOC-alkyl, trifluoromethyl, nitro, ethynyl or However, there have been two major problems which cyano. These compounds are reportedly useful for treatment have prevented the development of glycine antagonists as of indications caused by hyperactivity of the excitatory clinically useful neuroprotective agents: 50 neurotransmitters, particularly the quisqualate receptors, and A. Most available glycine antagonists with relatively high as neuroleptics. receptor binding affinity in vitro such as 7-Cl-kynurenic U.S. Pat. No. 4,659,713 (1987), discloses 1,4-dihydroqui acid (Kemp, J. A., et al., Proc. Natl. Acad. Sci. USA noxaline-2,3-dione compounds having the formula: 85:6547-6550 (1988)), 5,7-dichlorokynurenic acid 55 H (McNamara, D., et al., Neuroscience Lett. 120:17-20 (1990)) and indole-2-carboxylic acid (Gray, N. M., et N O al., J. Med Chem. 34:1283-1292 (1991)) cannot pen 1 2 etrate the blood/brain barrier and therefore have no X. O 3 5 O utility as therapeutic agents; 60 B. The only available glycine antagonist that sufficiently NO penetrates the blood/brain barrier-the drug HA-966 (Fletcher and Lodge, Eur. J. Pharmacol. 151:161-162 wherein X represents hydrogen, chloro, bromo, fluoro, iodo, (1988)) is a partial agonist with only micromolar trichloromethyl, dichlorofluoromethyl, difluoromethyl or affinity for the glycine binding site. A neuroprotective 65 trifluoromethyl, and n represents 1 or 2. These compounds efficacy for HA-966 in vivo has therefore not been are reportedly useful for the control of coccidiosis in ani demonstrated nor has it been demonstrated for the other mals. 5,514,680 5 6 U.S. Pat. No. 4,948,794 (1990), discloses 1,4-dihydroqui dione antagonize responses mediated by NMDA-associated noxaline-2,3-dione compounds having the formula: glycine recognition sites in vivo. Pellegrini-Giampietro, D. E. et al., Br. J. Pharmacol. R8 R1 98:1281-1286 (1989), disclose that 6-cyano-7-nitro-1,4-di R N O hydroquinoxaline-2,3-dione and 6,7-dinitro-1,4-dihydro quinoxaline-2,3-dione may antagonize the responses to L-glutamate by interacting with the glycine recognition sites R6 N O of the NMDA receptor ion channel complex. Ogita and Yoneda, J. Neurochem. 54:699-702 (1990), R5 H disclose that 6,7-dichloro-1,4-dihydroquinoxaline-2,3-di 10 one is a competitive antagonist specific to the strychnine wherein insensitiveH)glycine binding sites on the NMDA receptor R" is C-2-alkyl, which may optionally be substituted by complex. According to the authors, the two chloride radicals hydroxy, formyl, carboxy, carboxylic esters, amides or at the 6- and 7-positions in the benzene ring of the quinoxa amines, Cas-cycloalkyl, aryl, aralkyl; and wherein R line are crucial for the antagonistic potency against the is, hydrogen, halogen, CN, CF, NO, or OR, wherein 15 glycine binding sites. R is C-alkyl and R, R and R is hydrogen, pro Kessler, M. et al., Brain Res. 489:377-382 (1989), dis vided R is not CF, OCH, NO, C or Br when R' is close that 6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione and CH; or 6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione inhibit R and Rindependently are NO, halogen, CN, CF, or 20 H. glycine binding to the strychnine-insensitive glycine OR', wherein R is C-alkyl, and R and Rare each binding sites associated with NMDA receptors. hydrogen; or European Patent Application Publication No. 0377 112, R and R together form a further fused aromatic ring, published Jul. 11, 1990, discloses 1,4-dihydroquinoxaline which may be substituted with halogen, NO, CN, CF 2,3-dione compounds having the formula: or OR, wherein R is C-alkyl; or 25 R and R together form a further fused aromatic ring, R8 R1 which may be substituted with halogen, NO, CN, CF R7 N O or OR, wherein R is C-alkyl, and R and R' independently are hydrogen, halogen, CN, CF, NO, or OR, wherein R is C-alkyl. These compounds are 30 R6 N O reportedly useful for the treatment of indications R5 H caused by hyperactivity of the excitatory neurotrans mitters, particularly the quisqualate receptors, and as wherein, inter alia, R' may be hydroxy, alkoxy, aryloxy, neuroleptics. aralkyloxy, cycloalkylalkoxy, cycloalkoxy or acyloxy; and oneda and Ogita, Biochem. Biophys. Res. Commun. 35 R, R, R and R may be independently hydrogen, nitro, 164:841-849 (1989), disclose that the following 1,4-dihyd halogen, cyano, trifluoromethyl, SONR'R', SOR or OR, roquinoxaline-1,2-diones competitively displaced the wherein R' is hydrogen or C alkyl. These compounds are strychnine-insensitive binding of Higlycine, without reportedly useful for the treatment of indications caused by affecting the other binding sites on the NMDA receptor hyperactivity of the excitatory neurotransmitters, particu complex: 40 larly the quisqualate receptors, and as neuroleptics. Lester, R. A. et al., Mol. Pharm. 35:565-570 (1989), H R. R. disclose that 6 -cyano-7-nitro-1,4-dihydroquinoxaline-2,3- R2 N O H H QX dione antagonizes NMDA receptor-mediated responses, by a e C. H. CQX competitive interaction of the glycine binding site. CI C, DCOX 45 Patel, J. et al., J. Neurochem, 55:114-121 (1990), disclose NO, CN CNQX that the neuroprotective activity of 6,7-dinitro-1,4-dihydro R1 N O NO NO DNQX quinoxaline-2,3-dione is due to antagonism of the coagonist H activity of glycine at the NMDA receptor-channel complex. Horner, L. et al., Chen. Abstracts 48:2692 (1953) disclose According to the authors, the structure-activity relation 50 6,8-dinitro-1,4-dihydroquinoxaline-2,3-dione. ships among quinoxalines clearly indicates that both chlo Cheeseman, G. W. H., J. Chem. Soc.:1170-1176 (1962), ride groups of the positions 6 and 7 in the benzene ring are discloses 6,7 -dibromo-2,3-dihydroxyquinoxaline (also crucial for the antagonist potency against the Gly sites. known as 6,7-dibromo-1,4-dihydroquinoxaline-2,3-dione). Removal of one chloride from the molecule results in a Honore, T. et al., Science 241:701-703 (1988), disclose 10-fold reduction in the affinity for Gly sites. 55 that 6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione and 7-cy Kleckner and Dingledine, Mol. Pharm. 36:430-436 ano-6-nitro-14 -dihydroquinoxaline-2,3-dione are potent (1989), disclose that 6,7-dinitro-1,4-dihydroquinoxaline-2, non-NMDA glutamate receptor antagonists. 3-dione and 6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3- Sheardown, M.J. et al., Eur: J. Pharmacol. 174: 197-204 dione are more potent antagonists of kainate than glycine, (1989), disclose that 5,7-dinitro-1,4-dihydroquinoxaline-2, but substitution of Cl at the 6-position and especially at the 60 3-dione is a potent antagonist of the strychnine insensitive 6- and 7-positions increases potency at the glycine site. In glycine receptor and has anticonvulsant properties. How addition, the authors suggest that antagonists of the glycine ever, Sheardown et al. also disclose that 5,7-dinitro-14 site might be effective against NMDA receptor-mediated -dihydroquinoxaline-2,3-dione as well as DNQX and neuropathologies. CNQX have poor access to the central nervous system. Rao, T. S. et al., Neuropharmacology 29:1031-1035 65 International Application Publication No. WO91/13878 (1990), disclose that 6,7-dinitro-1,4-dihydroquinoxaline-2, discloses the following N-substituted 1,4-dihydroquinoxa 3-dione and 7-cyano-6-nitro-1,4-dihydroquinoxaline-2,3- line-2,3-diones which bind to the glycine receptor: 5,514,680 7 8 cross the blood-brain barrier at levels sufficient for effi R NO cacy, have utility as novel anticonvulsants with less side-effects O1 (CH), than the PCP-like NMDA channel blockers or the competitive NMDA antagonists; R. Rg R10 help in defining the functional significance of the glycine R N binding site of the NMDA receptor in vivo.

R6 N O 10 SUMMARY OF THE INVENTION Rs R The invention relates to the discovery of a class of compounds which exhibit high affinity for the strychnine wherein R represents hydrogen, C alkyl or aralkyl and n insensitive glycine binding site, which do not exhibit PCP is an itegft from 0 to 5; R'represents hydrogen or hydroxy; side effects and which cross the blood brain barrier at high R, R, R and Rindependently represent hydrogen, nitro, 15 levels. This is in contrast to reports in the literature that other halogen, alkoxy, aryloxy, aralkoxy, Co-alkyl or aryl; R' compounds, e.g. particular 1,4-dihydroquinoxaline-2,3-di represents hydrogen, lower alkyl, or aryl; R' represents ones and HA-966, either do not cross the blood/brain barrier hydrogen, or alkyl, and pharmaceutically acceptable salts or do so at low levels. In addition, many of these compounds thereof. exhibit low binding affinity to other receptor sites. Thus, the Leeson et al., J. Med. Chen. 34:1243-1252 (1991), 20 present invention relates to compounds having high affinity disclose a number of derivatives of the nonselective exci for the glycine binding site, lack PCP side effects and which tatory amino acid antagonist kynurenic acid. Also disclosed cross the blood brain barrier at high levels; with the proviso are a number of structurally related quinoxaline-2,3-diones that the compound is not a substituted or unsubstituted which are also glycine/NMDA antagonists but which are not 2,5-dihydro-2,5-dioxo-3-hydroxy-1H-benzazepine. Thus, selective and are far less potent than the kynurenic acid 25 the compounds of the present invention are extremely useful derivatives. The quinoxaline-2,3-diones have the structure: for treating pathophysiologic conditions, without significant side effects or toxicity. H The invention also relates to a method of treating or 5 N O 30 preventing neuronal loss associated with stroke, ischemia, 6 CNS trauma, hypoglycemia and surgery, as well as treating R 7 neurodegenerative diseases including Alzheimer's disease, N O amyotrophic lateral sclerosis, Huntington's disease and 8 Down's syndrome, treating or preventing the adverse con H sequences of the overstimulation of the excitatory amino 35 acids, as well as treating anxiety, convulsions, chronic pain, wherein R is H, 5-Cl, 7-Cl, 5,7-Cl, 6,7-Cl, 6,7-(CH), psychosis and inducing anesthesia, comprising administer 6-NO or 6,7-(NO) . Also disclosed are a number of ing to an animal in need of such treatment a compound N-methyl derivatives. having high affinity for the glycine binding site, lacking PCP Swartz et al., Mol. Pharmacol. 41:1130-1141 (1992), 40 side effects and which crosses the blood brain barrier at high disclose that certain substituted and unsubstituted benza levels. zepines are competitive antagonists of glutamate receptor channels in cultured cortical neurons. See also, U.S. Pat. No. Preferably, such compounds have the Formula H: 5,254,683, which discloses that compounds of the formula Rl R I 45 R2 N O

R3 N O R4 H 50 or a tautomer thereof, wherein R is hydrogen, hydroxy, amino, -CHCONHAr, -NH CONHAr, -NHCOCHAr, -COCHAr, wherein Ar is an aryl group, or a radical having the formula: wherein R'-R may be hydrogen, C. perfluoroalkyl, halo, 55 R8 III nitro or cyano and R may be hydrogen or a C- alkyl YO group, are antagonists of the glycine binding site on the NMDA receptor complex. als (CH2) A need continues to exist for potent and selective glycine/ NMDA antagonists which: Re--R lack the PCP-like behavioral side effects common to the PCP-like NMDA channel blockers such as MK801 or wherein to the competitive NMDA receptor antagonists such as R is hydrogen, lower C, alkyl or aryl; R is hydrogen CGS19755; or lower C, alkyl; n is an integer from 0 to 5; and R' show potent anti-ischemic efficacy because of the non 65 is hydrogen, C alkyl or aralkyl; competitive nature of their glutamate antagonism at the R’ is hydrogen, amino, acylamino, halo, haloalkyl or NMDA receptor; nitro, 5,514,680 9 10 R’ is hydrogen, amino, acylamino, halo or haloalkyl; R is acylamino, hydrogen, halo, haloalkyl or nitro; R is hydrogen, amino, acylamino, halo or haloalkyl; and with hydroxylamine-O-sulfonic acid under basic conditions R is hydrogen, amino, acylamino, halo, haloalkyl or to give the corresponding N-amino-1,4-dihydroquinoxaline nitro. 2,3-dione. The present invention also relates to novel 1,4-dihydro The invention also relates to the use of the N-amino-1,4- quinoxaline2,3-diones, and pharmaceutical compositions quinoxaline-2,3-diones obtained according to the above thereof. method, either as a mixture of isomers or pure, in a method The present invention also relates to certain highly soluble of treating or preventing neuronal loss associated with ammonium salts of 1,4-dihydroquinoxaline-2,3-diones, in stroke, ischemia, CNS trauma, hypoglycemia and surgery, as particular, choline, Tris(2-amino-2-hydroxymethyl-1,3-pro 10 well as treating neurodegenerative diseases including Alzhe panediol, also known as Tromethamine), bis-TRIS propane, imer's disease, amyotrophic lateral sclerosis, Huntington's arginine and N-methyl-glucamine salts. disease and Down's syndrome, treating or preventing the The present invention also relates to a method for the adverse consequences of the overstimulation of the excita preparation of a 1,4-dihydroquinoxaline-2,3-dione, compris tory amino acids, as well as treating anxiety, convulsions, ing condensation of a compound having the formula: 15 chronic pain, psychosis and inducing anesthesia. R1 DESCRIPTION OF THE FIGURES FIG. 1 depicts graphs showing the relative potencies of 20 5-nitro-6,7 -dichloro-1,4-dihydroquinoxaline-2,3-dione (5-NO-6,7-Cl-QX) and 5,7-diClKA. Records from a single oocyte expressing rat whole brain poly(A) "RNA. In this and all following figures the holding potential was -70 with oxalic acid in an aqueous acid solution for a time and mV. Solution changes and duration of drug applications are temperature sufficient to effect a condensation reaction to 25 indicated by bars; dead time of the perfusion system 5-10 give a 1,4-dihydroquinoxaline-2,3-dione having the for sec. Inward current is denoted by downward deflection and mula individual responses were separated by approximately 10 min intervals of wash. For all pharmacological assays the Ri H initial spike of Cl current (due to underlying entry of Ca”) R2 N O 30 was ignored, and response amplitudes were measured under steady-state conditions at the peak of the slow phase (arrow). FIG. 2A (upper panel) depicts concentration-inhibition curves comparing potencies of 5-NO-6,7-Cl-QX, 5,7- DCIR4 H diClKA, DNQX and CNQX as inhibitors at NMDA recep 35 tors expressed by whole rat brain poly(A)RNA. Agonist wherein concentrations were NMDA 100 uM, glycine 10 uM. In R is hydrogen, acylamino, halo, amino, haloalkyl or FIGS. 2-11B, data are plotted as the mean+S.D. expressed nitro; either as a fraction of control responses (concentration R’ is hydrogen, acylamino, nitro, amino, haloalkyl or inhibition curves), or maximum responses (concentration halo; 40 response curves); n, indicates the number of experiments (each using separate oocytes). Data for each drug were R is hydrogen, acylamino, halo, amino or haloalkyl; and independently fitted to logistic equations (smooth curves) R is hydrogen, acylamino, halo, amino, haloalkyl or and ICso values obtained from these fits were used in nitro. estimation of Ks. In the case of CNQX dam was insufficient Where any one of R'-R' is amino, one starts with a 45 triaminobenzene where at least two of the amino groups are to plot a fitted curve. ortho with respect to each other. The product will be the FIG. 2B (lower panel) depicts the effects of 5-NO corresponding amino-substituted 1,4-dihydroquinoxaline ClQX on the concentration-response curve for glycine at 2,3-dione, where the two ortho-amino groups have become NMDA receptors expressed by rat whole brain poly(A) the 1,4-nitrogens of the quinoxalinedione. 50 RNA. NMDA concentration was fixed at 100 uM. Control The present invention also relates to an N-amino-1,4- curve gives an indication of the variance in 14 oocytes taken dihydroquinoxaline-2,3-dione obtained by reaction of a from four different frogs. 1,4-dihydroquinoxaline-2,3-dione of the formula FIG. 3A (upper panel) depicts a concentration-response curve for NMDA at NMDA receptors expressed by rat Rl R 55 whole brain poly(A) RNA. Glycine concentration was R2 N O fixed at 10 mM (n=3). The ECs from these curves was used to gauge the absolute minimum K values for 5-NO-6,7- Cl-QX at glummate binding sites on NMDA receptors. R3 N O FIG. 3B (lower panel) depicts a sample record from an R4 H 60 experiment designed to test whether 5-NO-6,7-Cl-QX had any detectable inhibitory effects at glummate binding or a tautomer thereof; wherein sites on NMDA receptors. Glycine was applied at high concentration (1 mM) to try and minimize inhibition at the R is hydrogen; glycine-site. Under these conditions glycine itself elicits R" is hydrogen, acylamino, halo, haloalkyl or nitro; 65 membrane current responses (Cl currents), not due to R’ is acylamino, hydrogen, nitro, haloalkyl or halo; effects at NMDA receptors, but due to activation of strych R is hydrogen, acylamino, halo or haloalkyl; and nine-sensitive glycine receptors co-expressed in the oocyte 5,514,680 11 12 membrane by the rat brain mRNA. In this particular experi combinations to 5-NO-6,7-Cl-QX (glycine=10 M, ment, the glycine response was not blocked by co-applica glummate=100 uM). tion of strychnine, but instead was allowed to desensitize, FIG. 10 depicts the effects of 100 nM 5-NO,-6,7-Cl and the plateau phase used as a baseline for the following QX on glycine concentration-response curves for three rat experiments. NMDA was applied at 1 mM, sufficient to elicit NMDA receptor subunit combinations (glutamate=100 uM). a threshold current, and 5-NO-6,7-Cl-QX was assayed FIG. 11A depicts the effects of 5-NO-6,7-Cl-QX on for inhibition of this response. At concentrations up to 1 glycine concentration-response curves for the NR1AJNR2C mM, 5-NO-6,7-Cl-QX showed any clear inhibition. At subunit combination. At concentrations ranging between concentrations>1 M, 5-NO-6,7-Cl-QXinhibition could 10-300 nM, 5-NO-6,7-Cl-QX inhibition is associated be predicted at the glycine site, even when using 1 mM 10 with progressive rightward shifts in concentration-response glycine, and this was indeed detected. FIG. 3A also shows application of a related compound, 5-nitro-6,7-dibromo-1, curve, without appreciable changes in slope, or maximum 4-dihydroquinoxaline-2,3-dione (5-NO-6,7-Br-QX), response. that was also largely inactive at glutamate binding sites. FIG. 11B depicts a graph showing the Schild regression of FIG. 4A depicts sample records illustrating inhibitory data depicted in FIG. 11A. effects of 5-NO-6,7-Cl-QX at non-NMDA receptors 15 FIG. 12 depicts a bar graph demonstrating normal, control expressed by rat whole brain poly(A)" where the currents are locomotor activity in gerbils pretreated with saline. Normal activated by kainic acid (control response is <10% of control gerbils were given intraperitoneal injections imme maximum response). diately prior to the six hour locomotor activity assessment period. Groups of 6 gerbils each were utilized and dam are FIG. 4B depicts inhibition of currents activated by AMPA 20 presented as the meanstandard error. (control response is approximately 50% of maximum FIG. 13 depicts a bar graph demonstrating the effects of response). Following application of high concentrations of 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di 5-NO-6,7-Cl-QX, oocytes needed to be washed for 2-3 one on locomotor activity, normal control gerbils (open bars) rain before responses fully returned to control levels (break and animals given 1.0 mg/kg of the compound (solid bars). in records). Records were taken from the same oocyte. 25 Gerbils were given intraperitoneal injections of the com FIGS. 5A and 5B depict concentration-inhibition curves pound immediately prior to the six hour locomotor activity comparing potencies of 5-NO-6,7-Cl-QX, 5,7-diClKA, assessment period. Control animals were given injections of DNQX and CNQX at non-NMDA receptors expressed by rat saline. Groups of 6 gerbils each were utilized and data are whole brain poly(A)" RNA. For FIG. 5A, currents were presented as the meantstandard error. activated by 10 m AMPA. For FIG. 5B, currents were 30 FIG. 14 depicts a bar graph demonstrating the effects of activated by 20 m kainic acid. 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di FIG. 6 depicts a graph showing the effects of 5-NO-6, one on locomotor activity, normal control gerbils (open bars) 7-Cl-QXon concentration-response curves for AMPA and and animals given 3.2 mg/kg of the compound (solid bars). kainic acid in oocytes expressing whole rat brain poly(A)" Gerbils were given intraperitoneal injections of the com RNA. For both agonists, 5-NO-6,7-Cl-QX (3 mM) 35 pound immediately prior to the six hour locomotor activity induces roughly parallel rightward shifts in concentration assessment period. Control animals were given injections of response curves. saline. Groups of 6 gerbils each were utilized and data are FIG. 7A depicts sample records illustrating membrane presented as the meant standard error. current responses elicited by 1S,3R-ACPD, a selective ago FIG. 15 depicts a bar graph demonstrating the effects of nist at metabotropic glutamate receptors, in an oocyte 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di expressing whole rat brain poly(A) RNA. Responses are one on locomotor activity in normal control gerbils (open the characteristic fluctuating (oscillatory) Cli currents, elic bars) and animals given 10 mg/kg of the compound. Gerbils ited through activation of the phosphoinositide/Ca" path were given intraperitoneal injections of the compound way. immediately prior to the six hour locomotor activity assess FIG. 7B depicts a simplified scheme of the intracellular 45 ment period. Control animals were given injections of messenger system underlying these responses. G-glum saline. Groups of 6 gerbils each were utilized and data are mate; MGR-metabotropic glutamate receptor; PLC presented as the meantstandard error. phospholipase C; IP, inositol 1,4,5-triphosphate; DAG, diacylglycerol; PKC-protein kinase C. FIG. 16 depicts a bar graph demonstrating the effects of 50 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di FIG. 8 depicts a concentration-response curve for 1S,3R one on locomotor activity in normal control gerbils (open ACPD at metabotropic glummate receptors expressed by rat bars) and animals given 32 mg/kg of the compound (solid whole brain poly(A) RNA. The EC from these curves was bars). Gerbils were given intraperitoneal injections of the used to gauge the absolute minimum K values for 5-NO compound immediately prior to the six hour locomotor 6,7-Cl-QX and 5-NO-6,7-Br-QX at glummate binding 55 activity assessment period. Control animals were given sites on the metabotropic receptors. injections of saline. Groups of 6 gerbils each were utilized FIG. 8B depicts a sample record from an experiment and data are presented as the mean-standard error. designed to test whether 5-NO-6,7-Cl-QX or 5-NO-6, FIG. 17 depicts a graph demonstrating the effects of 7-Br-QX showed any detectable inhibitory effects at pretreatment with saline immediately prior to the onset of a glummate binding sites on metabotropic receptors. The 5 minute period of bilateral carotid occlusion. Animals were oocyte was repeatedly exposed to 10 M 1S,3R-ACPD tested for changes in locomotor activity for 4 successive eliciting a reproducible threshold response. At 10 M, hours following ischemia reperfusion injury compared to neither 5-NO-6,7-Cl-QX nor the related compound control animals receiving no carotid artery occlusion. Data 5-NO-6,7-Br-QX, caused any appreciable reduction in are expressed as the mean value for each group of 6 gerbils this response. 65 given 5 minutes of bilateral carotid occlusion with saline FIG. 9 depicts concentration-inhibition curves showing pretreatment (closed symbol) or pretreated with saline but the relative sensitivities of three rat NMDA receptor subunit without bilateral carotid occlusion (open symbol). Animals 5,514,680 13 14 were placed in the locomotor activity chambers for the 4 of testing following 5 min. bilateral carotid occlusion. All successive hours as indicated. doses of the compound significantly protected against the FIG. 18 depicts a graph demonstrating the effects of locomotor activity increases routinely produced by bilateral pretreatment with 5-chloro-7-trifluoromethyl-1,4-dihydro carotid occlusions of 5 minutes or longer. Control animals quinoxaline-2,3-dione (0.32 mg/kg) immediately prior to the not exposed to ischemia demonstrated a low exploratory activity that is normal for healthy animals placed in the onset of a 5 minute period of bilateral carotid occlusion. locomotor activity chamber for 4 hours. Saline pre-treatment Animals were tested for changes in locomotor activity for 4 failed to interfere with the post-ischemic enhancement of successive hours following ischemia reperfusion injury. locomotor activity that is routinely observed. 0.32-32 mg/kg Data are expressed as the mean value for each group of 6 of the compound significantly reduced the ischemia elicited gerbils given 5 minutes of bilateral carotid occlusion with O increase in exploratory activity within the circular arena of drug pretreatment (closed symbol) or pretreated with saline the locomotor activity chamber. Data are expressed as the but without bilateral carotid artery occlusion (open symbol). mean of 6 subjects standard error. Animals were placed in the locomotor activity chambers for FIG.24 depicts a bar graph showing changes in locomotor the 4 successive hours as indicated. activity as a result of treatment with 5-chloro-7-trifluorom FIG. 19 depicts a graph demonstrating the effects of 15 ethyl-1,4-dihydroquinoxaline-2,3-dione (AG1) 24 hours pretreatment with 5-chloro-7-trifluoromethyl-1,4-dihydro after ischemia during a 1 hour testing session. Animals were quinoxaline-2,3-dione (3.2 mg/kg) immediately prior to the placed in the locomotor activity chambers 24 hours after the onset of a 5 minute period of bilateral carotid occlusion. ischemia reperfusion injury and evaluated for changes in Animals were tested for changes in locomotor activity for 4 locomotor activity. Control animals were saline control successive hours following ischemia reperfusion injury. 20 non-ischemic or drug control non-ischemic animals placed Data are expressed as the mean value for each group of 6 in the chamber 24hours after saline/drug injection. Ischemic gerbils given 5 minutes of bilateral carotid occlusion with (ISC) animals represent animals given saline pre-treatments drug pretreatment (closed symbol) or pretreated with saline and placed in the locomotor activity chamber 24 hours after but without bilateral carotid artery occlusion (open symbol). the onset of ischemia reperfusion. These animals were also 25 ones that were tested for the post ischemic hourly changes Animals were placed in the locomotor activity chambers for in locomotor activity represented in previous figures. Ani the 4 successive hours as indicated. mals given pre-treatment with the compound were animals FIG. 20 depicts a graph showing the effects of pretreat represented in previous figures for the immediate post ment with 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxa reperfusion changes in behavior. All animals were tested at line-2,3-dione (10 mg/kg) immediately prior to the onset of 30 24 hours for a 1 hour session. a 5 minute period of bilateral carotid occlusion. Animals FIG. 25 depicts a bar graph showing the effects of pre- and were tested for changes in locomotor activity for 4 succes post-treatment with 5-chloro-7-trifluoromethyl-1,4-dihydro sive hours following ischemia reperfusion injury. Data are quinoxaline-2,3-dione at 32 mg/kg. Control (CON) animals expressed as the mean value for each group of 6 gerbils received no ischemia, saline control animals (SAL) received given 5 minutes of bilateral carotid occlusion with drug ischemia but with saline pretreatment instead of drug. Pre pretreatment (closed symbol) or pretreated with saline but 35 treatment animals received multiple injections of 32 mg/kg without bilateral carotid artery occlusion (open symbol). at 6, 4, and 2 hours and 30 minutes prior to the onset of 5 Animals were placed in the locomotor activity chambers for minutes of bilateral carotid occlusion. Post-treatment ani the 4 successive hours as indicated. mals received doses at 30 minutes post reperfusion, 2 hours, FIG. 21 depicts a graph showing the effects of pretreat 4 hours and 6 hours. Testing was conducted at 24 hours ment with 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxa 40 post-reperfusion. Each group represents the mean-standard line-2,3-dione (32 mg/kg) immediately prior to the onset of error for 6 animals per treatment group. As can be seen, both a 5 minute period of bilateral carotid occlusion. Animals pre- and post-treatment produced a significant behavioral were tested for changes in locomotor activity for 4 succes protective effect. A clearindication of protection at 32 mg/kg sive hours following ischemia reperfusion injury. Data are 45 occurred. expressed as the mean value for each group of 6 gerbils FIG. 26 depicts a bar graph showing the post-ischemic given 5 minutes of bilateral carotid occlusion with no changes in radial arm maze performance of gerbils exposed pretreatment (closed symbol) or pretreated with saline but to 5 minutes of bilateral carotid occlusion 24 hours prior and without bilateral carotid artery occlusion (open symbol). pre-treated with the indicated doses of 5-chloro-7-trifluo Animals were placed in the locomotor activity chambers for 50 romethyl-1,4-dihydroquinoxaline-2,3-dione. Animals were the 4 successive hours as indicated. tested immediately after locomotor activity testing for FIG. 22 depicts a bar graph showing the effects of patrolling behavior in an 8 arm maze. This 8 arm maze is 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di located in a room in which no other research was conducted. one on first hour locomotor activity changes compared to All 8 arms were identical in dimensions and in figuration. saline and the non-ischemic control values. These data are 55 No arms were baited with food. Animals were tested until summarized from the preceding figures and indicate that they had entered and explored each of the 8 arms. Entry of there is a significant improvement in the spontaneous loco a previously entered arm defined an error. All animals motor activity of animals given 5-chloro-7-trifluoromethyl completed the entire exploratory task prior to being 1,4-dihydroquinoxaline-2,3-dione at doses above 0.32 removed. Each column represent the meantstandard error mg/kg in the first hour following ischemia. In contrast, 60 for 6 animals per treatment condition. Control animals control animals who were not exposed to ischemia reperfu received no ischemia, saline (SAL) animals received sion injury demonstrated a significantly high level of loco ischemia but no drug. Animals receiving ischemia plus motor activity in the first hour of exploration compared to treatment with 0.32-32 mg/kg drug showed a significant ischemic gerbils. behavioral protection from ischemia induced changes in FIG. 23 depicts a bar graph showing the effects of 65 patrolling behavior. pretreatment with 5-chloro-7-trifluoromethyl-1,4-dihydro FIG. 27 depicts a bar graph showing the effects of quinoxaline-2,3-dione on locomotor activity in the 4th hour 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di 5,514,680 15 16 one pretreatment doses on neuronal cell density in the dorsal were then observed and the time spent by the mice licking hippocampus (CA-1) of the gerbil. Gerbils were pretreated the injected hindpaw in five minute intervals during the time with 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxaline-2, periods indicated was recorded. The time spent licking the 3-dione doses as described in the previous figures and injected hindpaw is an indicator of the pain experienced by allowed to recover for the ischemia reperfusion injury for 7 the animal. 6,7-Dibromo-5-nitro-1,4-dihydroquinoxaline-2, days prior to anesthetization and fixation of the tissue. Tissue 3-dione inhibited the formalin-induced licking in a dose was frozen, sectioned and stained, neuronal nuclei were dependent manner both in the early phase (0-5 minutes, counted in discreet areas of the CA-1 region of the hippoc ampus. Dam are presented as the meantstandard error for 6 FIG.31A) of the pain (licking) response and in the late phase subjects (each subject was evaluated for a minimum of 3 (15-50 minutes, FIG. 31B) of the pain licking response successive sections of the dorsal hippocampus for neuronal 10 indicating potent antinociceptive efficacy in this animal loss). Pretreatment with 0.32-32 mg/kg of the drug provided model of chronic pain. a significant protection fromischemia-induced neuronal cell FIG. 32 depicts a diagram showing the solubilities of loss. 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di FIG. 28 depicts a bar graph showing the effects of one, the choline salt, the mono-potassium salt and the 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxaline-2,3-di 15 di-potassium salt thereof. one on neuronal density in the dorsal hippocampus (CA-1) FIG.33 depicts a graph showing the sedative activity (loss of the gerbil. Gerbils were pretreated and post treated with of righting reflex) of mice over time after i.p. injection of 3.2 mg/kg 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxa 5,7-dichloro-1,4-dihydroquinoxaline-2,3-dione (compound line-2,3-dione and allowed to recover for the ischemia no. 1), compared to 6,7-dichloro- 1,4-dihydroquinoxaline reperfusion injury for 7 days prior to anesthetization and 20 2,3-dione (compound no. 2; inactive) and ketamine (com fixation of the tissue. Tissue was frozen, sectioned and pound no. 3). stained, neuronal nuclei were counted in discreet areas of the FIGS. 34A and 34B depict graphs showing the effects of CA-1. Dam are presented as the meanstandard error for 6 various doses of PCP, 5-chloro-7-trifluoromethyl-1,4-dihy subjects (each subject was evaluated for a minimum of 3 droquinoxaline-2,3-dione and 5-nitro-6,7-dichloro-1,4-di successive sections of the dorsal hippocampus for neuronal 25 hydroquinoxaline-2,3-dione in rats trained to discriminate 2 loss). Both pre- and post-treatment with 3.2 mg/kg drug mg/kg PCP from saline. FIG. 34A shows degree of PCP-like provided a significant protection from ischemia-induced effects as shown in the mean percentage of PCP-lever neuronal cell loss. selection. FIG. 34B shows effects on overall rates of FIG. 29 depicts a bar graph showing the inhibition of responding. Values above SAL, PCP and DMSO show the formalin-induced pain in mice by 1, 5, 10, 20, 30 and 40 30 results of control tests with saline, 2 mg/kg PCP and 0.5 mg/kg of 5-chloro-7 -trifluoromethyl-1,4-dihydroquinoxa ml/kg DMSO conducted prior to the testing of PCP, line-2,3-dione. Mice were injected intraperitoneally with 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxaline-2,3-di either DMSO (vehicle control) or with the drug in DMSO 30 one and 5-nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3- minutes prior to subcutaneous injection of 20 ul of 5% 35 dione. Values represent the mean of six rats. formalin into the plantar surface of the hindpaw. The mice FIG. 35 depicts a bar graph showing the locomotor were then observed and the time spent by the mice licking activity of Swiss/Webster mice after intraperitoneal injection the injected hindpaw in five minute intervals during the time of 5-nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione periods indicated was recorded. The time spent licking the in DMSO or MK801 in saline. Groups of at least six mice injected hindpaw is an indicator of the pain experienced by each were injected with vehicle or with increasing doses of the animal. As shown in FIG. 29, 5-chloro-7-trifluorom 5 -nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione in ethyl-1,4-dihydroquinoxaline-2,3-dione inhibited the forma DMSO or MK801 in saline. Locomotor activity was then lin induced licking in a dose dependent manner indicating recorded for four successive 15 minute periods. The loco potent antinociceptive efficacy in this animal model of motor activity in the second fifteen minute interval is shown. chronic pain. 45 FIG. 30 depicts a bar graph showing the inhibition of formalin-induced pain in mice by 5, 10, 20 and 40 mg/kg of DESCRIPTION OF THE PREFERRED 6,7-dichloro-5-nitro-14 -dihydroquinoxaline-2,3-dione. EMBODIMENTS Mice were injected intraperitoneally with either DMSO The present invention relates to compounds having high (vehicle control) or with the drug in DMSO 30 minutes prior 50 affinity for the glycine binding site, lacking PCP side effects to subcutaneous injection of 20 ml of 5% formalin into the and which cross the blood brain barrier at high levels. planar surface of the hindpaw. The mice were then observed Examples of such compounds include 1,4-dihydroquinoxa and the time spent by the mice licking the injected hindpaw line-2,3-diones which are highly selective, competitive in five minute intervals during the time periods indicated antagonists of the glycine binding site of the NMDA recep was recorded. The time spent licking the injected hindpaw is 55 an indicator of the pain experienced by the animal. As shown tor. The 1,4-dihydroquinoxaline-2,3-diones of the invention in FIG. 30, 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2, have the following Formula (I): 3-dione inhibited the formalin induced licking in a dose 1 dependent manner indicating potent antinociceptive efficacy R H in this animal model of chronic pain. R2 N O FIGS. 31A and 31B depict line graphs showing the inhibition of formalin-induced pain in mice by 1, 5, 10, 20 R3 N O and 40 mg/kg 6,7-dibromo-5-nitro-1,4-dihydroquinoxaline R4 H 2,3-dione. Mice were injected intraperitoneally with either DMSO (vehicle control) or with the drug in DMSO 30 65 or a tautomer thereof, wherein minutes prior to subcutaneous injection of 20 ml of 5% R’ is hydrogen amino, hydroxylamino, acylamino, halo, formalin into the planar surface of the hindpaw. The mice haloalkyl or nitro; 5,514,680 17 18 R’ is amino, hydroxylamino, acylamino, hydrogen, nitro, carboxybutyl, 1-aryl-2-carboxypentyl, 1-aryl-2-carboxy haloalkyl or halo; hexyl, 1-aryl-3-carboxybutyl, 1-aryl-3-carboxypentyl, R is amino, hydroxylamino, acylamino, halo or 1-aryl-3-carboxyhexyl, 1-aryl-5-carboxypentyl, 1-aryl-5- haloalkyl; and carboxyhexyl, 2-aryl-2-carboxyethyl, 2-aryl-3-carboxypro R" is hydrogen amino, hydroxylamino, acylamino, hydro pyl, 2-aryl-4-carboxybutyl, 2-aryl-5-carboxypentyl, 2-aryl gen, halo, haloalkyl or nitro. 6-carboxyhexyl, 2-aryl-1-carboxyethyl, 2-aryl-1- Preferred compounds within the scope of Formula I are carboxypropyl, 2-aryl-1-carboxybutyl, 2-aryl-1 wherein R is halo or nitro, R is nitro or halo, R is halo or -carboxypentyl, 2-aryl-1-carboxyhexyl, 2-aryl-2-carbox haloalkyl and R' is hydrogen. Other preferred compounds ypropyl, 2-aryl-2-carboxybutyl, 2-aryl-2-carboxypentyl, are wherein one of R'-R' is amino. Other preferred com 10 2-aryl-2-carboxyhexyl, 2-aryl-3-carboxybutyl, 2-aryl-3-car pounds are when R is halo, nitro, haloalkyl or amino; R is boxypentyl, 2-aryl-3-carboxyhexyl, 2-aryl-5-carboxypentyl, hydrogen, halo, nitro, haloalkyl, or amino; R is halo, nitro, 2-aryl-5-carboxyhexyl, and the like. haloalkyl or amino; and R' is hydrogen or halo. Especially Typical C alkyl groups include methyl, ethyl, propyl, preferred compounds are where R' is amino or nitro, R is isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, halo, R is halo or haloalkyl and R' is hydrogen. Other 15 2-pentyl, 3-pentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, especially preferred compounds are where R' is halo, nitro, 2-methyl-1-pentyl, 3-methyl-l-pentyl, 4-methyl-1-pentyl or haloalkyl, R' is hydrogen, halo, nitro or haloalkyl, R is and the like. halo, nitro or haloalkyl and R is hydrogen or fluoro. Typical Caryl groups include phenyl, naphthyl, fluo The present invention also relates to certain N-substituted renyl, phenanthryl and anthracyl groups. 1,4-dihydroquinoxaline-2,3-diones which have the follow 20 Typical halo groups include fluorine, chlorine, bromine and iodine. ing Formula (II): Typical haloalkyl groups include C alkyl groups sub R 1 R stituted by one or more fluorine, chlorine, bromine or iodine R2 N O atoms, e.g. fluoromethyl, difluoromethyl, trifluoromethyl, 25 pentafluoroethyl, 1,1-difluoroethyl and trichloromethyl groups. R3 N O Typical amino groups include -NH2, NHR and R4 H -NR'R'', wherein R and R' are one of the C alkyl groups mentioned above. or a tautomer thereof, wherein 30 Typical acylamino groups include an amino group sub R is hydrogen, hydroxy, amino, -CHCONHAr, -NH stituted by a C- acyl group, e.g. acetyl, propionyl, CONHAr, -NHCOCHAr, -COCHAr, wherein Ar butanoyl, pentanoyl and hexanoyl groups. is an aryl group, or a radical having the Formula (III): Particularly preferred quinoxaline-2,3-diones of the present invention include, but are not limited to 6,7- R8 I 35 dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione, 6,7-dif No luoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione, 6-chloro als (CH2) 5-nitro- 7-bromo-1,4-dihydroquinoxaline-2,3-dione, 6,7- dichloro-5-bromo-14 -dihydroquinoxaline-2,3-dione, R--R 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxaline-2,3-di 40 one, 5-chloro-6-nitro-7-trifluoromethyl-1,4 -dihydroqui wherein R is hydrogen, lower C. alkyl or aryl; R is noxaline-2,3-dione, 6-chloro-5-nitro-7-trifluoromethyl-1,4 hydrogen or lower C. alkyl; n is an integer from 0 to -dihydroquinoxaline-2,3-dione, 5-chloro-8-nitro-7-trifluo 5; and Ris hydrogen, C. alkyl or aralkyl; romethyl-1,4-dihydroquinoxaline-2,3-dione, 5,7-dichloro R’ is hydrogen, amino, hydroxylamino, acylamino, halo, 1,4-dihydroquinoxaline-2,3-dione, 5-chloro-6,7-difluoro-1, 45 4-dihydroquinoxaline-2,3-dione, 5-bromo-6,7-difluoro-1,4 haloalkyl or nitro; -dihydroquinoxaline-2,3-dione, 5,6,7,8-tetrafluoro-1,4-di R’ is amino, hydroxylamino, acylamino, hydrogen, nitro, hydroquinoxaline-2,3-dione, 5-chloro-7-fluoro-1,4-dihyd haloalkyl or halo; roquinoxaline-2,3-dione, 5,7-dibromo- 1,4-dihydroquinoxa R is hydrogen, amino, hydroxylamino, acylamino, halo line-2,3-dione, 5-bromo-7-trifluoromethyl- 1,4- or haloalkyl; and 50 dihydroquinoxaline-2,3-dione, 5-bromo-7-fluoro-1,4- R" is amino, hydroxylamino, acylamino, hydrogen, halo, dihydroquinoxaline- 2,3-dione, 6,7-dibromo-5-nitro-1,4- haloalkyl or nitro. dihydroquinoxaline-2,3-dione, 4-carboxymethyl-1,4- Where the 1,4-dihydroquinoxaline-2,3-dione is substi dihydroquinoxaline-2,3-dione, 4-carboxymethyl-6,7 tuted by a radical having Formula III, the radical may be a -dibromo-1,4-dihydroquinoxaline-2,3-dione, 4-carboxym C-carboxyalkyl group including carboxymethyl, 2-car 55 ethyl-6,7-dichloro-1,4 -dihydroquinoxaline-2,3-dione, boxyethyl, 3-carboxypropyl, 4-carboxybutyl, 5-carboxypen 4-amino-6,7-dibromo-1,4-dihydroquinoxaline- 2,3-dione, tyl, 6-carboxyhexyl, 1-carboxyethyl, 1-carboxypropyl, 4-amino-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione, 4 1-carboxybutyl, 1-carboxypentyl, 1-carboxyhexyl, 2-car -carboxymethyl-6,7-dichloro-5-nitro-1,4-dihydroquinoxa boxypropyl, 2-carboxybutyl, 2-carboxypentyl, 2-carboxy line-2,3-dione, 4 -carboxymethyl-6,7-dichloro-5-bromo-1, hexyl, 3-carboxybutyl, 3-carboxypentyl, 3-carboxyhexyl, 60 4-dihydroquinoxaline-2,3-dione, 4 -carboxymethyl-5- 5-carboxypentyl, 5-carboxyhexyl, and the like. chloro-7-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione, Typical Cs carboxyaralkyl groups which are included 4-carboxymethyl-5-chloro-6-nitro-7-trifluoromethyl-1,4-di in Formula III include 1-aryl-2-carboxyethyl, 1-aryl-3-car hydroquinoxaline-2,3-dione, 4-carboxymethyl-5-chloro-8- boxypropyl, 1-aryl-4-carboxybutyl, 1-aryl-5-carboxypentyl, nitro-7-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione, 1-aryl-6-carboxyhexyl, 1-aryl-l-carboxyethyl, 1-aryl-1-car 65 4-carboxymethyl-5,7-dichloro-1,4-dihydroquinoxaline-2,3- boxypropyl, 1-aryl-1-carboxybutyl, 1-aryl-1-carboxypentyl, dione, 4-carboxymethyl-5-chloro-6,7-difluoro-1,4-dihydro 1-aryl-1-carboxyhexyl, 1-aryl-2-carboxypropyl, 1-aryl-2- quinoxaline-2,3-dione, 4-carboxymethyl-5-bromo-6,7-dif

5,514,680 21 22 droquinoxaline-2,3-dione, 7-chloro-5,6-dinitro-1,4-dihyd 7-nitro-1,4-dihydroquinoxaline-2,3-dione and 6,7-dinitro-1, roquinoxaline-2,3-dione, 6-chloro-5,7-dinitro-14 4-dihydroquinoxaline-2,3-dione and other 6,7-disubstituted -dihydroquinoxaline-2,3-dione, 7-trifluoromethyl-5,6-dini 1,4-dihydroquinoxaline-2,3-diones which are incapable of tro-1,4-dihydroquinoxaline-2,3-dione, and 6-trifluorom crossing the blood/brain barrier after i.p. administration (see ethyl-5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione. Turski, L. et al., J. Pharm. Exp. Ther. 260:742-747 (1992)). 6,7-Dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione, See also, Sheardown et al., Eur: J. Pharmacol. 174:197-204 6,7-dibromo-5-nitro-1,4-dihydroquinoxaline-2,3-dione and (1989), who disclose that 5,7-dinitro-1,4-dihydroquinoxa 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxaline-2,3-di line-2,3-dione, 6,7-dinitro-1,4-dihydroquinoxaline-2,3-di one prevent ischemia-induced nerve cell death in the gerbil one and 6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione ischemia model after i.p. administration. 6,7-Dichloro-5- nitro-1,4-dihydroquinoxaline-2,3-dione, 6,7-dibromo-5-ni 10 have poor access to the central nervous system. tro-1,4-dihydroquinoxaline-2,3-dione, 5-chloro-7-trifluo For a compound to begin to show in vivo efficacy and, romethyl-1,4-dihydroquinoxaline-2,3-dione, 5,6,7 thus, the ability to cross the blood-brain barrier, the com -trifluoro-1,4-dihydroquinoxaline-2,3-dione, 5,6,7,8-tet pound should exhibit an EDs of less than about 100 mg/kg rafluoro-14 -dihydroquinoxaline-2,3-dione, 7-chloro-6,8- body weight of the animal. Preferably, the compounds of the difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione, 6,7-dif 15 present invention exhibit an EDs of less than about 20 luoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione, mg/kg and, more preferably, less than about 10 mg/kg and 7-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-di most preferably, less than 1 mg/kg. one, 7-chloro-5-trifluoromethyl-1,4-dihydroquinoxaline-2, The compounds of the invention find particular utility in 3-dione, 6-chloro-7-bromo-5-nitro-1,4-dihydroquinoxaline treating or preventing the adverse neurological conse 2,3-dione, 6-chloro-7-fluoro-5(3)-nitro-14 20 quences of surgery. For example, coronary bypass surgery -dihydroquinoxaline-2,3-dione, 5,6,7-trichloro-1,4-dihydro requires the use of heart-lung machines which tend to quinoxaline-2,3-dione, and 5,7-dichloro-1,4-dihydroqui introduce air bubbles into the circulatory system which may noxaline-2,3-dione are potent anticonvulsants in the animal lodge in the brain. The presence of such air bubbles robs models after i.p. or i.v. administration. neuronal tissue of oxygen, resulting in anoxia and ischemia. 5-Chloro-7-trifluoromethyl-1,4-dihydro-1,4-dihydroqui 25 Pre- or post-surgical administration of the 1,4-dihydroqui noxaline-2,3-dione, 6,7-dichloro-5-nitro-1,4-dihydro-1,4- noxalines of the present invention will treat or prevent the dihydroquinoxaline-2,3-dione and 6,7-dibromo-5-nitro-1,4- resulting ischemia. In a preferred embodiment, the com dihydro-1,4-dihydroquinoxaline-2,3-dione were also found pounds of the invention are administered to patients under to exhibit analgesic efficacy after i.p. administration in an going cardiopulmonary bypass surgery or carotid endarter animal of chronic (formalin-induced) pain. 30 ectomy surgery. The compounds of the present invention show low cross The compounds of the present invention also find utility reactivity with kainate, AMPA, and quisqualate receptors in treating or preventing chronic pain. Such chronic pain and the glummate and PCP binding sites of the NMDA may be the result of surgery, trauma, headache, arthritis, or receptors and are, therefore, distinct from any previously other degenerative disease. The compounds of the present described 1,4-dihydroquinoxaline-2,3-diones, in particular, 35 invention find particular utility in the treatment of phantom 6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione and 6,7- pain that results from amputation of an extremity. In addition dinitro-1,4-dihydroquinoxaline-2,3-dione as disclosed in to treatment of pain, the compounds of the invention are also U.S. Pat. No. 4,975,430. useful in inducing anesthesia, either general or local anes The compounds of the present invention are active in thesia, for example, during Surgery. treating or preventing neuronal loss, neurodegenerative dis 40 A total of more than 100 1,4-dihydroquinoxaline-2,3- eases, chronic pain, are active as anticonvulsants and induc dione derivatives have been synthesized and tested for ing anesthesia without untoward side effects caused by glycine antagonist activity in vitro. From among these novel non-selective binding with other receptors, particularly, drugs, several compounds with a particularly high affinity kainate, AMPA, and quisqualate receptors and the PCP and for glycine/NMDA receptors have been identified. From an glutamate receptors associated with the NMDA receptor. In 45 analysis of the structure-activity relationship of the most addition, the compounds of the present invention are effec potent analogs, it appears that the combination of a NO in tive in treating or preventing the adverse consequences of the 5-position group with two halogen atoms, e.g., chloro, the hyperactivity of the excitatory amino acids, e.g. those bromo or fluoro; or with a halogen such as Cl and a CF which are involved in the NMDA receptor system, by group in the 1,4-dihydroquinoxaline-2,3-dione ring system blocking the glycine receptors and preventing the ligand 50 gives particularly potent glycine antagonists. It is notewor gated cation channels from opening and allowing excessive thy that the 5-NO, group in 5-nitro-6,7-disubstituted 1,4- influx of Ca' into neurons, as occurs during ischemia. dihydroquinoxaline-2,3-dione has increased generally, from Neurodegenerative diseases which may be treated with ten to hundredfold, the glycine receptor affinity of the parent the compounds of the present invention include those 6,7-disubstituted 1,4-dihydroquinoxaline-2,3-dione (Table selected from the group consisting of Alzheimer's disease, 55 II). This discovery is but one aspect of this invention. For amyotrophic lateral sclerosis, Huntington's disease and example, 6,7-dibromo-5-nitro-1,4-dihydroquinoxaline-2, Down's syndrome. 3dione and 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2, The compounds of the present invention find particular 3-dione, with affinities of 4.1 nM and 3.3 nM, respectively, utility in the treatment or prevention of neuronal loss asso for the glycine binding site, are among the most potent ciated with multiple strokes which give rise to dementia. 60 glycine/NMDA antagonists discovered to date. It is note After a patient has been diagnosed as suffering from a worthy, that the NO, group in 6,7-dichloro-5-nitro-1,4- stroke, the compounds of the present invention may be dihydroquinoxaline-2,3-dione compound has increased, by administered to ameliorate the immediate ischemia and several hundred fold, the glycine receptor affinity of its prevent further neuronal damage that may occur from recur parent compound, 6,7-dichloro-1,4-dihydroquinoxaline rent strokes. 65 2,3-dione (DCQX), which has been described by others as a Moreover, the compounds of the present invention are potent and selective glycine antagonist (Yoneda and Ogita, able to cross the blood/brain barrier, in contrast to 6-cyano Biochem. Biophy. Res. Commun, 164:841-849 (1989). Sur 5,514,680 23 24 prisingly, it is possible to reduce the nitro group of 6,7- 10 mg/kg). However, these two compounds exhibited dif dibromo-5-nitro-1,4-dihydroquinoxaline-2,3-dione and 6,7 fering propensities to cause ataxia side-effects as determined -dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione tO by the rotorod ataxia test in the mouse. In particular, obtain amino-substituted 1,4-dihydroquinoxaline-2,3-di 6,7-dibromo-5-nitro-1,4-dihydroquinoxaline-2,3-dione ones which also have high binding affinity to the glycine exhibited a TD=200 mg/kg in this test. Thus, this com binding site. pound is effective in preventing seizures at doses which are As discussed above, structure activity relationship of much lower than those which cause ataxia side-effects. This compares to a TD-27 mg/kg in the rotorod ataxia test for 1,4-dihydroquinoxaline-2,3-diones reveals that the combi 6,7-Dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione. nation of halogen atoms, CF group, or amino group in the Thus, the invention relates as well to compounds which 5, 6, and 7 positions in the benzene ring portion of the O exhibit ataxia side effects in the rotorod ataxia test at dosage 1,4-dihydroquinoxaline-2,3-diones gives particularly potent levels of greater than about 100 mg/kg, more preferably, glycine antagonists. In contrast, 5, 6 and 8 trisubstituted 1,4 greater than about 200 mg/kg. -dihydroquinoxaline-2,3-diones and 5,6,7,8-tetrasubstituted Two compounds (#1 (Table XVI) and 5-chloro-7-trifluo 1,4-dihydroquinoxaline-2,3-diones have a low affinity as romethyl-1,4-dihydroquinoxaline-2,3-dione (compound #2 glycine antagonists. The striking examples are 6,7,8- 15 (Table XVI)) were also tested for neuroprotective efficacy trichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione and after intraperitoneal injection in the gerbil global ischemia 8-bromo-6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3- model and were found to have potent neuroprotective effi dione, both are hundreds to thousands fold less active than cacy in this paradigm. The same compounds were also tested 6,7-dichloro-5-nitro-14 -dihydroquinoxaline-2,3-dione. in drug discrimination tests in rats trained to discriminate Therefore, substitution in both the 5 and 8 positions of 20 PCP from saline. Neither of the two compounds generalized 1,4-dihydroquinoxaline 2,3-dione generally resulted in com to PCP at any dose in these drug discrimination studies. In pounds with low activity as glycine antagonists. It is pro addition, none of the compounds produced a behavioral posed that at least one of the amide NH is pivotal for excitation in locomotor activity tests in the mouse. The interaction with the receptor and substituents in both 5 and results from these studies suggest that the novel glycine 8 positions of 1,4-dihydroquinoxaline-2,3-dione interfere or 25 antagonists of the present invention do not show the PCP block this crucial interaction and therefore resulted in mol like behavioral side effects that are common to NMDA ecules with low affinity in the glycine site of the receptor. channel blockers such as MK801 and PCP or to competitive Fluorine might be an exception due to its small size (Table NMDA antagonists such as CGS19755. III). The present invention is also directed to this discovery. It is important that the novel glycine antagonists showed Structure activity relationship of 1,4-dihydroquinoxaline 30 potent activity in vivo after intraperitoneal or intravenous 2,3-diones also reveals that 5,7-disubstituted 1,4-dihydro injection suggesting that these compounds can penetrate the quinoxaline-2,3-diones and 6,7-disubstituted 1,4-dihydro blood/brain barrier. Other investigators have reported that quinoxaline-2,3-diones generally have similar activity as the 1,4-dihydroquinoxaline-2,3-dione analogs CNQX and glycine antagonists (Table IV). DNQX cannot penetrate the blood/brain barrier (Turski, L., Because 1,4-dihydroquinoxaline-2,3-diones, especially 35 et al., J. Pharm. Exp. Ther. 260:742-747 (1992)), a finding CNQX and DNQX, are known to be potent kainate and that has been confirmed by the inventors. Apparently, alter AMPA antagonists, the new compounds of the present ations in the benzene ring substituents of 1,4-dihydroqui invention were tested in kainate and AMPA binding assays noxaline-2,3-diones not only result in a dramatic increase in to determine cross-reactivity at these non-NMDA receptors. glycine receptor affinity and loss in AMPA/kainate receptor The potent glycine antagonists were found to have no affinity but also can produce compounds with a quite satis significant cross-reactivity at the kainate and AMPA sites or factory ability to penetrate the blood/brain barrier (see only minor cross-reactivity. Thus, the invention also relates Turski, L. et al., J. Pharm. Exp. Ther. 260:742-747 (1992)). to compounds which are potent glycine antagonists but In contrast, 5-nitro-6,7-disubstituted 1,4-dihydroquinoxa which exhibit little or no cross-reactivity at the kainate and line-2,3-diones are generally able to cross the blood brain AMPA sites. 45 barrier at high levels as shown by their high in vivo activity Preferably, the compounds of the invention exhibit a (Table V). binding affinity to the glycine binding site of K=about 10 From analysis of structure activity relationship of 1,4 M or less, more preferably, 1 M or less, and more -dihydroquinoxaline-2,3-diones, it was found that although preferably, 500 nM or less and more preferably, 100 nM or 5,7-disubstituted 1,4-dihydroquinoxaline-2,3-diones and less and most preferably, about 10 nM or less. Also prefer 50 6,7-disubstituted 1,4-dihydroquinoxaline-2,3-diones have ably are compounds which exhibit binding at the kainate and similar activity as glycine antagonists in vitro (Table IV), AMPA sites of not less than K=1 M and, more preferably, 5,7-disubstituted 1,4-dihydroquinoxaline-2,3-diones are not less than 10 M. generally active in vivo (Table VI) while 6,7-disubstituted The novel glycine antagonists were then tested for in vivo 1,4-dihydroquinoxaline-2,3-diones are generally not active activity after intraperitoneal (i.p.) injection or intravenous 55 in vivo (Table XVII). Therefore, 5,7-disubstituted 1,4- (i.v.) injection using a number of anticonvulsant tests in mice dihydroquinoxaline-2,3-diones are generally able to cross (audiogenic seizure model in DBA-2 mice, pentylenetetra the blood brain barrier at high levels while 6,7-disubstituted Zol-induced seizures in mice, NMDA-induced death in mice, 1,4-dihydroquinoxaline-2,3-diones are generally notable to and maximum electroshock seizures (MES) in mice). All cross the blood brain barrier. The present invention is also compounds tested showed activity in one or more of the four directed to this discovery. models. 6,7-Dichloro-5-nitro-1,4-dihydroquinoxaline-2,3- The present invention also relates to the discovery that dione (compound #1, Table XVI) was the most potent one of certain fluorosubstituted 1,4-dihydroquinoxaline-2,3-diones the five, particularly in the audiogenic seizure model have high affinity for the glycine/NMDA receptor and have (ED=5 mg/kg) and the NMDA-induced death model unexpectedly high in vivo activity as anticonvulsant in the (ED=20 mg/kg). 6,7-Dibromo-5 -nitro-1,4-dihydroqui 65 MES experiment in mice (Table VII). Therefore, these noxaline-2,3-dione (compound #13, Table IV) was also very compounds are able to cross the blood brain barrier at high potent, particularly in the audiogenic seizure model (EDso levels. 5,514,680 25 26 6,7-Difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione dione (Ki-320 nM) and 7-chloro-5-trifluoromethyl- 1,4- was found to have high affinity for the glycine/NMDA dihydroquinoxaline-2,3-dione (Ki=395 nM). receptor with a Ki of 87 nM, which is more than 20 times The placement and type of electron withdrawing group on less active compared to one of the most active compounds, the ring seems to be important, as substitutions on the 5- and 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione (Ki 7-positions of the 1,4-dihydro-2,3-quinoxalinedione ring 3.3 nM). 6,7-Difluoro-5-nitro-1,4-dihydroquinoxaline-2,3 seem to enhance potency of the glycine binding site. Sub dione, however, was found to have surprisingly high in vivo stitution at the 6-position by ionizable groups such as activity. It has a EDs of 0.7- 0.8 mg/kg as an anticonvulsant sulfonates, primary sulfonamides and carboxylic acids in the MES experiment in mice. In comparison, 6,7- destroys the binding ability of the compounds to the glycine dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione has an 10 receptor. Neither alkylation of the sulfonamide nor methy EDs of 4-5 mg/kg as an anticonvulsant in the MES lation of the acid leads to increased activity. Methylation of experiment in mice. This means that 6,7-difluoro-5-nitro-1, the amide nitrogen in the quinoxaline ring also destroys the 4-dihydroquinoxaline-2,3-dione might be about 100 times binding ability of the compound, as evidenced by the lack of better in crossing the blood brain barrier than 6,7-dichloro-5 activity of the 6,7-dinitro-N-methyl-1,4-dihydro-2,3-qui -nitro-1,4-dihydroquinoxaline-2,3-dione. 5 noxalinedione. 7-Chloro-6,8-difluoro-5-nitro-1,4-dihydroquinoxaline-2, Another important aspect of the present invention relates 3-dione was found to have high affinity for the glycine/ to the discovery that amino-substituted 1,4-dihydroquinoxa NMDA receptor with Ki of 170 nM, which is about 50 times line-2,3-diones also have high binding affinity to the glycine less active compared to 6,7-dichloro-5-nitro-1,4-dihydro binding site. This was unexpected in view of the fact that quinoxaline-2,3-dione. 7-Chloro-6,8-difluoro-5-nitro-1,4 20 amino groups are electron donating and the expectation that -dihydroquinoxaline-2,3-dione also was found to have sur electron withdrawing groups are important for high binding prisingly high in vivo activity. It has a EDso of 2-3 mg/kg affinity. as an anticonvulsant in the MES experiment in mice. In Table VIII summarizes results of eight 1,4-dihydroqui comparison, 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline noxaline-2,3-diones tested i.v. as anticonvulsants in MES 2,3-dione has an EDso of 4-5 mg/kg as an anticonvulsant in experimentin mice. Most of the compounds tested have very the MES experiment in mice. This means that 7-chloro-6, fast peak of action, especially the fluoro substituted com 8-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione might pounds. The protecting effect of fluoro substituted com be about 100 times better in crossing the blood brain barrier pound against MES decreased very quickly. After 60 min., than 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-di no more protection was observed for 6,7-difluoro-5-nitro one. It is noted that certain CF substituted quinoxaline-2, 30 1,4-dihydroquinoxaline-2,3-dione and 5,6,7-trifluoro-1,4- 3-diones also have high in vivo activity (Table VII). dihydroquinoxaline-2,3-dione. Chloro substituted com Prior to the present invention, one of the primary phar pounds also have a fast peak of action but the protecting macophores for glycine antagonism was thought to be the effect lasts much longer than fluoro substituted compounds. amide group in the 1,4-dihydro-2,3-quinoxalinedione with Trifluoromethyl substituted compounds have relatively slow enhancement of potency occurring with the substitution of 35 peak of action and the protecting effect lasts longer than electron withdrawing substituents on the aromatic ring, thus fluoro substituted compounds. This different pattern of lowering the pKa of the amide hydrogen (Gray et al., J. Med. action of 1,4-dihydroquinoxaline-2,3-diones could be used Chem. 34:1283 (1991); Leeson, P. D., et al., J. Med. Chem. to apply individual compounds for different types of thera 34:1243 (1991)). While the studies described in this inven peutic treatment. The present invention is also directed to tion have generally borne out this proposal, it has now been this discovery. discovered that the relative positions of substituents on the The compounds synthesized for testing as potential gly ring as well as the identity of the electron withdrawing cine antagonists which are not substituted on the amide (1 or groups are also important. The present invention is also 4) position are summarized below (Table I). Most were directed to this discovery. available by simple condensation of diethyl oxalate with the It has further been discovered that one of the two amide 45 corresponding diaminobenzene according to Cheeseman, G. hydrogens may be replaced by amine, hydroxy, carboxy W. H., J. Chem. Soc. 1171 (1962). The 1,4-dihydro-2,3- alkyl, and carboxyaralkyl groups and that the resulting quinoxalinediones may be easily prepared in high yield by compounds retain high binding to the glycine receptor. The heating oxalic acid and the corresponding o-diamine to present invention is also related to this discovery. about 100 to 140° C. for 1 to 10 hr. in the presence of a The most potent novel antagonist that is a part of this 50 strong mineral acid such as HCl, HSO, HPO, and the invention is 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2, like. See, Mager and Berends, Rec. Trav. Chim. 77:842 3-dione (Ki=3.3 nM). The second most potent antagonist is (1958). In a preferred embodiment, oxalic acid and the 6,7-dibromo-5-nitro-1,4-dihydroquinoxaline-2,3-dione (Ki= o-diamine are heated to 125 C. in 2N HCl for 2.5 hours to 4.1 nM). Other potent antagonists include a mixture of give the corresponding 1,4-dihydro-2,3-quinoxalinedione in nitrated 5 -chloro-7-trifluoromethyl-1,4-dihydroquinoxa 55 high yield. line-2,3-diones. Other potent antagonists include 6-bromo The starting o-diaminobenzenes are either available 7-chloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione (Ki=5.3 directly from the manufacturer or were easily accessible via nM) and 7-bromo-6-chloro-5-nitro-1,4-dihydroquinoxaline the reduction of the corresponding o-nitroaniline (Scheme I, 2,3-dione (Ki-6.0 nM) and all the 5-nitro-6,7-disubstituted eq. 1), according to Bellamy, F. D. and Ou, K., Tetr. Lett. 1,4-dihydroquinoxaline- 2,3-diones prepared (Table II). 60 25:839 (1984). Nitrations were performed by treatment of Other 5,6,7-trisubstituted 1,4-dihydroquinoxaline-2,3-di the 1,4-dihydroquinoxaline-2,3-dione with KNO in conc. ones synthesized also are potent antagonists (Table III) such HSO (Scheme I, eq. 2). The 5-halo-6,7-fluoro-1,4-dihy as 5,6,7-trichloro-1,4-dihydroquinoxaline-2,3-dione (Ki=58 dro-2,3-quinoxalinediones were prepared by treatment of nM) and 5-bromo-6,7-dichloro-1,4-dihydroquinoxaline-2,3- the 4,5-difluoro-2-nitroaniline with N-bromosuccinimide or dione (Ki=53 nM). 5,7-Diisubstituted 1,4-dihydroquinoxa 65 N-chlorosuccinimide (Mitchell, R. H., et al., J. Org. Chem. line-2,3-diones are also potent antagonists (Table IV) such 44:4733 (1979)), followed by reduction and condensation as 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxaline-2,3- with diethyl oxalate. 5,514,680 27 28 Sulfonates and derivatives were prepared by treatment of -continued the parent 1,4-dihydro-2,3-quinoxalinedione with chlorosul Scheme I fonic acid and subsequent treatment with the desired amine H H to form the sulfonamide (Scheme I, eq. 3). See Mitchell et N O N O al., J. Org. Chem, 44:4733 (1979). The 5-bromo-7-fluoro / RNH W 1,4-dihydroquinoxaline-2,3-dione was prepared by treat - Ge. ment of the 4-fluoro-2-nitroaniline with N-bromosuccinim \ ide followed by reduction and condensation with diethyl CO2S N O R2NSO2 N O H H oxalate. 10 Scheme I R4 (1) to a Rs NH 15 H SnCl2, EtOAc O EtOH, 70° C. G N / R2 NO R1 R4 20 HOSO N O H R3 NH (EtOCO) A G For those o-nitroanilines that were not commercially R NH available, they were prepared from the corresponding 25 anilines in three steps. First, the amino groups of the anilines R were protected by reaction with acetic anhydride or trifluo R H roacetic anhydride to give the amides, which were then nitrated in HSO with KNO or HNO. Finally the amides were hydrolyzed to give the o-nitroanilines (Scheme II, eq R N r 30 4 and 5). R2 N O Schee II R H Cl (4) H (2) 35 CF N O FC NH KNO H2SO4 G Cl N O Cl HNOJH2SO4 Cl H FC NHCOCH H Cl NO CF N O HC G major 45 FC NHCOCH ON N O C NO C H 3y FC NH 50 -- F (5) (CFCO)2O ON H Ge. CF N O C1 NH minor 55 F

N O F Cl H HNOJHSO4. - H (3) Cl NHCOCF N lo F CISO F NO 60° C., 2h G K2CO3 N O 65 Cl NHCOCF H F 5,514,680 29 30 -continued (1993); Tian, W. et al., J. Chem, Soc. Perkin Trans. 1:258 Scheme II (1993)). Cyclization of the diaminobenzene gave the 1,4- dihydroquinoxaline-2,3-dione with the nitro group in pre F NO determined position. Bromination of the 1,4-dihydroqui 5 noxaline- 2,3-dione then produced 7-bromo-6-chloro-5- nitro-1,4-dihydroquinoxaline-2,3-dione. C1 NH F Scheme IV 6,7-Diisubstituted 1,4-dihydroquinoxaline-2,3-diones 10 were treated in concentrated HSO or trifluoroacetic acid Ge. with HNO or KNO to give 5 -nitro-6,7-disubstituted C1 NO2 1,4-dihydroquinoxaline-2,3-diones (Scheme III). For those compounds where the two substituents are different, the reaction could give a single product (eq6) but sometimes it 15 gave a mixture of two products (eq. 7). It was found that this type of mixtures were usually very difficult to separate. For Cl some compounds it is also difficult to determine the position of the nitro group. 20 Scheme III H (6) Cl N O 2 KNO/HSO4. Ge. 25 FC N O

H NO2 H 30 Cl N

FC N O 35 H NBSIDMF H (7) F N Neo NO2 KNO3 40 FCCO2H G NO H Cl N O Cl N O

H 45 Br C N O NO2 H H F N O -- The second procedure was based on the discovery that nitration of 3,4-dihydroquinoxaline-2(1H)-one resulted Cl N O 50 exclusively in the product with the nitro group in the H 5-position. For example, treatment of 1-bromo-2,4-difluoro 5-nitrobenzene with sodium glycinate gave the substituted H aniline. The nitro group was reduced by SnCl2 and the F N product spontaneously cyclized to give the 3,4-dihydroqui 55 noxaline-2(1H)-one. It was nitrated by HNO in trifluoro acetic acid which resulted in both nitration and oxidation of the compound to give 7-bromo-6-fluoro-5-nitro-1,4-dihyd Cl N O roquinoxaline-2,3-dione in a single step (Scheme V). The NO2 h F-H coupling constants measured from the "H NMR spec 60 trum confirmed that the fluoro was ortho to the nitro group. Two new procedures was developed for the preparation of these compounds. One involves the synthesis of substituted 3-nitro-1,2-diaminobenzenes using a selenium heterocycle F F approach (Scheme IV). Nitration of the selenium hetero KNO3, cycle is known to give exclusively the product with the nitro 65 H2SO4 G group ortho to the nitrogen (Bird, C. Wet al., J. Chem. Soc. Br 4767 (1963); Tian, W. et al., J. Heterocycl. Chem. 29:1305 5,514,680 31 32 -continued Because nitro substituted 1,4-dihydroquinoxaline-2,3-di Scheme W ones are easily accessible and the nitro group could be

F F reduced to an amino group, diazotization of amino substi H2NCH2CONa 5 tuted 1,4-dihydroquinoxaline-2,3-diones followed by treat DMF/H2O Ge ment with reagents such as sodium iodide and copper(I) Br NO2 chloride provided a new avenue to a series of 1,4-dihydro F NHCHCO2H quinoxaline-2,3-diones (Scheme VII, eq 10). SnCl2 O of S Schene VII B NO, H Cl H (10) F N HN N O 1. NaNOf fuming HNO3 15 H2SO4. CFCO2H Ge 2 Se Br N O FC N O HC H H NO H H F N o 202 Cl Cl N O

Br N O H FC N O 25 1,2-Diamino-3-nitrobenzenes also were prepared by H mono-reduction of one of the nitro group in 2,6-dinitroa nilines (Scheme VI, eq8). This method is useful especially for the preparation of 5-nitro-7-substituted quinoxaline-2,3- 30 diones, such as 5-nitro-7-fluoroquinoxaline-2,3-dione. Scheme VI NO NO (8) 35 NH NH (NH4)2S Ge. X NO X NH 40 X = F, Cl X = F, Cl

TABLE I Structures of 1,4-dihydroquinoxaline-2,3-dione

Rs H N

R N O Rs H compd R. R R Rs mp (°C) for Inula 1 NO Br Br H 355-357 CHBrNO 2 NO, Cl C H 354 CHCl2N3O4. 3 NO, C1 Br H s350 CHBrCNO, 4 NO, Br Cl H 338-343 CHBrCNO 5 NO, Br CF, H 333-335 CHBrFNO 6 NOOH) Br NO, H(NO) 249-251 CHBrNO 7 NO,(H) Cl F H(NO) ND CHCIFNO 8 NO, C CF, H 342-345(d) CHCIFNO, 9 NO NO, Cl H 298-300 CHCNO 10 NO,(H) Br F H(NO) 250-252 CHBrFNO 11 NH Br Br H 325(d) CHBr,N.O. 12 NH Cl C H 350 CHCl2NO

5,514,680 35 36

TABLE I-continued Structures of 1,4-dihydroquinoxaline-2,3-dione

Rs H N O

R7 N O Rs H compd Rs R6 R Rs mp (°C) formula 77 H F F H 360 CHFNO2 78 Br H Br H 356-358 CHBr,NO, 79 NO, CF, NO C 322-323 CHCIFNO 80 H. H H H >300 CHNO, 81 NO, H C H 315-317 CHCINO 82 Br H F H ND CHBrFNO 83 F OCH, F H 310-314(d) CHFNO 84 H NH NH H ND CHNO2 85. F NH, F H >360 CHFNO, 86 F F F H 360 CHFNO2 8 H NH H H 300 CHNO 88 C. Cl Cl C 326-328 CHCNO, 89 I NO, C1 H 288-290 CHCINO 90 I H F H 310-312 CHFINO 9. I Cl Cl I 353-354(d) CH.ClNO2 92 NO, Cl NO I 240-242 CH.ClNO 93 Br Br I >350 CHBr2N2O2 94 NO, H H H 333-335 CHNO 95 NO, CF, H Cl 305 CHCIFNO 96 NH CF H Cl 360 CH.ClFNO ND, not determined, d, decomposed.

TABLE II 35 TABLE II Structure Activity Relationship of 5-Nitro-6,7-Disubstituted 1,4- Structure Activity Relationship of Dihydroquinoxaline-2,3-diones and 6,7-Diisubstituted 5,6,7-Trisubstituted 5,7,8-Trisubstituted and 1,4-Dihydroquinoxaline-2,3-diones 5,6,7,8-Tetrasubstituted 1,4-Dihydroquinoxaline-2,3-diones

H 40 H Rs R5 N

R N O 45 R N O Rg H R8 R R R Rs K (nM) R R R Rs K (nM) NO C Cl H 3.3 50 NH C Cl H 35 NO Br Br H 4.1 Br C Cl H 53 NO, Br Cl H 5.3 Cl Cl C1 H 58 NO, C Br H 6.0 C NO CF H 95 NO, Br CF, H 14 C C CF H 70 NO,(H) Br NO H(NO) 16 C F Cl H 135 NO,(H) C F H(NO) 18 55 NO, CF, H Cl inactive NO C CF, H 8 NH CF, H Cl inactive NO NO Cl H 20 NO C Cl Cl 180 NO(H) Br F H(NO) 32 NO, C Cl Br 6700 NO F F H 87 C. C C Cl inactive H C C H 320 C C I inactive H Br Br H 360 NO, F Cl F 170 H Br C1 H 30b 60 F F F F 2540 H Br NO H 200b H C CF H 420 From electrophysiology using Xenopus oocytes unless otherwise noted. From binding assays. From electrophysiology using Xenopus oocytes unless otherwise noted. From binding assays. 5,514,680 37 38

TABLE IV TABLE VI Structure Activity Relationship of 5,7-Diisubstituted and 6,7- In Vivo Activity of 5,7-Disubstituted Disubstituted 1,4-Dihydroquinoxaline-2,3-diones 1,4-Dihydroquinoxaline-2,3-diones

Rs R5 H N O

10 R R N O Rs R8 H R R Ra EDs (DBA- EDs (MES) 15 Rs R. R. Rs K (nM) 2)" mg/kg mg/kg NO H CF, 95 CF, H C 395 NO H. CF, H 95 NDb 17 Cl H Cl 386 CF, H Cl H 395 ND 10 Br H CF, 366 Cl H C H 386 9 14 C H CF, 320 Cl H CF, H 320 17 O H Br Br 360b 20 H Cl Cl 320 i.p. injection. H NO, CF, 246 ND, not determined. H Br NO, 200b i.v. injection unless otherwise noted. H Cl CF 420 "From electrophysiology using Xenopus oocytes unless otherwise noted. 25 TABLE VI From binding assays. In Vivo Activity of Fluoro Substituted 1,4-Dihydroquinoxaline-2,3-diones TABLE V In vivo Activity of 5-Nitro-6,7-disubstituted 1,4-Dihydroquinoxaline-2,3-diones 30 R5 H N O R5 H N O R N O C 35 R. R N O Ki EDs (DBA- EDs (MES) R R. R. Rs (nM) 2)" mg/kg mg/kg R8 H F F F F F 9 20a K, ED (DBA- EDso (MES) 40 NO F C F 170 NDb 2-3 R R. R. Rs (nM) 2)" mg/kg mg/kg F F F H. 2966 ND 4-5 NO(H) C F HONO) 18 ND 0.5-0.7 NO, C. C. H. 3.3 5 4-5 Br F F H 2300 30 ND NO Br Br H 4.1 0 40 NO F F H 87 ND 0.7-08 NO, C Br H 6.0 Nob 40 NO2(H) Cl F H(NO) 18 NED 0.5-0.7 45 i.p. injection. NO F F H 87 ND 0.7-0.8 ND, not determined. i.v. injection unless otherwise noted. "i.p. injection. ND, not determined. i.v. injection unless otherwise noted. 5,514,680 39 TABLE VIII 1,4-Dihydroquinoxaline-2,3-diones Tested i.v. for Protection Against MES

R5 H N O

R N C O R. h. IV peak of EDs (MES) ED (MES) 96 protection R R. R. Rs K (nM) action (min) mg/kg Ing/kg after 60 min. NO C1 C1 H 3.3 5 4-5 7-10 55 (10 mg/kg) NO F Cl F 170 2-5 2-3 5 50 (5 mg/kg) F F F H 2966 2 4-5 15-20 0 (10 mg/kg) NOOH) C1 F HONO) 18 5 0.5-0.7 4. 14 (10 mg/kg) CF, H. C. H 395 30 10 30 87 (30 mg/kg) C1 H CF, H 320 5 10 20–25 62 (20 mg/kg) NO, H CF, H 95 30 17 40 62 (40 mg/kg) NO, F F H 87 0.7-0.8 2.5 0 (2.5 mg/kg)

The compounds having Formula VI (R-CHCONHAr) 25 Alternatively, the compounds having Formula VI may be prepared by reaction of the corresponding substi (R=CHCONHAr) may be prepared by condensation of the tuted o-phenylenediamine with aqueous sodium chloroac o-phenylenediamine with glyoxalic acid in ethanol to give etate solution followed by acidification to give the corre the corresponding quinoxaline-3(2H)-3-one. See, Barton, D. sponding N-carboxymethylquinoxalin-3(1H)-one. E.; Lamb, A. J.; Lane, D. L. J.; Newbold, G. T.; Percival, D., Oxidation of this product with alkaline KMnO, gives the 30 J. Chem. Soc. (C), 1268 (1968). This product may be N-alkylated with a sodium alkoxide and a reactive O-halo N-carboxymethyl-1,4-dihydroquinoxaline-2,3-dione. This ester to give the N-carboxylmethylquinoxaline-3(2H)-one compound may be converted to the aryl amide by conden ethyl ester. Finally, oxidation with hydrogen peroxide gives sation with an arylamine in the presence of dicyclohexyl the N-carboxymethyl-1,4-dihydroquinoxaline-2,3-dione. carbodiimide in DMF (See, Scheme IX.) 35 (See, Scheme X.) Scheme X Scheme X R1 R1 O O R2 NH R2 NH, 1. CICHCONat H-C-COH G 2. Hit G EtOH R3 NH R3 NH R4 R4 Rl Chicoi 45 R H R2 N R2 N O e l 1. KMnO, OH Ge. NaOR G X-CHCOR1 R3 N O R3 N CO2 50 R4 R4 Ri (H-coh R1 (HCOR R2 N O R2 N O a Ar-NH2, DCC 55 DMF G H2O2 R3 N O R3 N r > R4 R4 Rl (HCONHA 60 R (H-coh R2 N O R2 N O

R3 N I. R3 N O 65 R4 5,514,680 41 42 Alternatively, compounds having Formula VI -continued (R=CHCONHAr) may be prepared by N-alkylation of the Scheme XI corresponding anion with a reactive halide (see Scheme XI). NO2 H For example, deprotonation of 6,7-dichloro-5-nitro-1,4-di- Cl hydroquinoxaline-2,3-dione (III) with a base such as lithium 5 O diisopropylamide will give the corresponding anion (IV). Alkylation with an o-haloester such as methyl bromoacetate Cl N O followed by ester hydrolysis will give the corresponding acid (V). Condensation of the acid with an arylamine in the VI CH2CONHAr presence of a dehydrating agent such as DCC gives the anilide (VI). Where R=-NHCONHAr (VII), the compound may be Scheme XI prepared by reaction of the aminate anion IV with chloram H 15 ine or mesitylenesulfonyloxyamine (Tamura, Y. et al., Syn NO2 thesis 1, 1977) to give the N-amino 1,4-dihydroquinoxaline Cl N O 2.1. BrCH2CO2MeBase 23 -rinnadione intermediatei VIII (Scheme XII). Alternatively, the cc s nitrogen may be amidated by reaction of the 1,4-quinoxa Cl N O line-2,3-dione with hydroxylamine-O-sulfonic acid in aque III h ous sodium hydroxide according to Shin, S.C. and Lee, Y. H Y., J. Korean Chem. Soc. 27:382–384 (1983) to give the N NO2 and/or N-amino-1,4-quinoxaline-2,3-diones. Cl C N C O 25 Cl N O V CHCO2H 30 1. H2N-Air 2. DCC

No. No. Cl N Neo Cl N Neo 2 1. Cl-NH O Cl N O CH3 Cl N O IV H VIII NH2 2. CH3 -S-O-NH, NCO O PhCh-COCl CH3 O 3. a) HONO b) H NO H NO2 H N C N O Cl C O Cl N O Cl N O VII NHCONHPh IX NHCOCHPh

65 The present invention also relates to the N-amino-14 -dihydroquinoxaline-2,3-diones obtained by reaction of a 1,4-dihydroquinoxaline-2,3-dione of the formula 5,514,680 44 The anxiolytic activity of any particular compound of the present invention may be determined by use of any of the s recognized animal models for anxiety. A preferred model is described by Jones, B. J. et al., Br. J. Pharmacol. 93:985-993 (1988). This model involves administering the N compound in question to mice which have a high basal level H 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 or a tautomer thereof, wherein white and brightly lit. The test box has two compartments, R is hydrogen; 10 one white and brightly illuminated and one black and R" is hydrogen, acylamino, haloalkyl, halo or nitro; non-illuminated. The mouse has access to both compart ments via an opening at floor level in the divider between the R’ is hydrogen, nitro, haloalkyl or halo; two compartments. The mice are placed in the center of the R is hydrogen, acylamino, halo or haloalkyl; and brightly illuminated area. After locating the opening to the R is hydrogen, acylamino, halo, haloalkyl or nitro, 15 dark area, the mice are free to pass back and forth between with hydroxylamine-O-sulfonic acid under basic conditions the two compartments. Control mice tend to spend a larger to give the corresponding N-amino-1,4-dihydroquinoxaline proportion of time in the dark compartment. When given an 2,3-dione. Such basic conditions may include aqueous anxiolytic agent, the mice spend more time exploring the KOH, NaOH, LiOH, and the like. more novel brightly lit compartment and exhibit a delayed Alternatively, compounds having Formula VI may be 20 latency to move to the dark compartment. Moreover, the prepared from the N-alkylated phenylenediamine X by mice treated with the anxiolytic agent exhibit more behavior condensation with diethyl oxalate to give the intermediate in the white compartment, as measured by exploratory XI. Nitration of XI with nitric acid/sulfuric acid gives the rearings and line crossings. Since the mice can habituate to isomeric nitro-1,4-dihydroquinoxaline-2,3-diones IV and the test situation, naive mice should always be used in the XII which may then be separated, for example, by column 25 test. Five parameters may be measured: the latency to entry chromatography (see Scheme XIII). See also, International into the dark compartment, the time spent in each area, the Application Publication No. WO91/13878, the contents of number of transitions between compartments, the number of which arefully incorporated by reference hereinformethods lines crossed in each compartment, and the number of rears of preparing such N-substituted carboxyalkyl and car in each compartment. The administration of the compounds boxyaralkyl 1,4-dihydroquinoxaline-2,3-diones as well as 30 is expected to result in the mice spending more time in the the N-hydroxy-1,4-dihydroquinoxaline-2,3-diones. larger, brightly lit area of the test chamber.

Scheme XIII

CHCO2H O O Cl NH Cl Et-O-C-C-OEt G

C1 NH Cl X

NO2 CH-coh Cl N O C1 a --

Cl N O C N

H IV

The compounds were tested for potential glycine antago In the light/dark exploration model, the anxiolytic activity nist activity by observing the inhibition of binding of 1 M of a putative agent can be identified by the increase of the glycine-stimulated (H)-MK-801 in rat or guinea pig brain 60 numbers of line crossings and rears in the light compartment membrane homogenates. The more potent the glycine at the expense of the numbers of line crossings and rears in antagonist, the less H-MK-801 can bind since the H the dark compartment, in comparison with control mice. MK801 binding side (PCP receptor) is accessible only upon A second preferred animal model is the rat social inter opening of the ion channel by glutamate and glycine action test described by Jones, B.J. et al., supra, wherein the (Fletcher, E. L., et al., in Glycine Neurotransmission, Otter 65 time that two mice spend in social interaction is quantified. son, P., et al. (eds.), John Wiley and Sons (1990); Johnson, The anxiolytic activity of a putative agent can be identified J. W., et al., Nature 325:529 (1987)). by the increase in the time that pairs of male rats spend in 5,514,680 45 46 active social interaction (90% of the behaviors are investi from about 0.01 to about 50 mg/kg of body weight, or an gatory in nature). Both the familiarity and the light level of equivalent amount of the pharmaceutically acceptable salt the test arena may be manipulated. Undrugged rats show the thereof, on a regimen of 1-4 times per day. Of course, it is highest level of social interaction when the test arena is understood that the exact treatment level will depend upon familiar and is lit by low light. Social interaction declines if the case history of the animal, e.g., human being, that is the arena is unfamiliar to the rats or is lit by bright light. treated. The precise treatment level can be determined by Anxiolytic agents prevent this decline. The overall level of one of ordinary skill in the art without undue experimenta motor activity may also be measured to allow detection of tion. drug effects specific to social behaviors. The unit oral dose may comprise from about 0.01 to about The present invention also relates to the use of the 10 50 mg, preferably about 0.1 to about 10 mg of the com compounds disclosed herein as sedative-hypnotics. It was pound. The unit dose may be administered one or more times found that the glycine/NMDA antagonist 5,7-dichloro-1,4- daily as one or more tablets each containing from about 0.1 dihydroquinoxaline-2,3-dione has potent sedative/hypnotic to about 10, conveniently about 0.25 to 50 mg of the activity after i.v. injection in mice. In contrast, 6,7-dichloro compound or its solvates. 1,4-dihydroquinoxaline-2,3-dione is completely devoid of 15 In addition to administering the compound as a raw sedative-hypnotic activity (see FIG. 22). 5,7-Dichloro-1,4- chemical, the compounds of the invention may be admin dihydroquinoxaline-2,3-dione is considerably more potent istered as part of a pharmaceutical preparation containing and long lasting as a sedative-hypnotic than ketamine, an suitable pharmaceutically acceptable carriers comprising NMDA channel blocker used as an anesthetic in humans. excipients and auxiliaries which facilitate processing of the The binding affinity of 5,7-dichloro-1,4-dihydroquinoxa 20 compounds into preparations which can be used pharma line-2,3-dione (Ki-0.9 M) at the glycine receptor is not ceutically. Preferably, the preparations, particularly those substantially different from that of 6,7-dichloro-1,4-dihyd preparations which can be administered orally and which roquinoxaline-2,3-dione (Ki-0.33 uM). Also, the kainate can be used for the preferred type of administration, such as and AMPA binding affinity of 5,7-dichloro-1,4-dihydroqui tablets, dragees, and capsules, and also preparations which noxaline-2,3-dione and 6,7-dichloro-1,4-dihydroquinoxa 25 can be administered rectally, such as suppositories, as well line-2,3-dione are not substantially different. Thus, it is as suitable solutions for administration by injection or orally, likely that the difference in sedative/hypnotic activity contain from about 0.01 to 99 percent, preferably from about between the two compounds is due to the fact that 5.7 0.25 to 75 percent of active compound(s), together with the -dichloro-1,4-dihydroquinoxaline-2,3-dione readilypen excipient. etrates the blood/barrier, while the 6,7-dichloro-1,4-dihyd 30 Also included within the scope of the present invention roquinoxaline-2,3-dione does not. The same difference in in are the non-toxic pharmaceutically acceptable salts of the vivo efficacy between these two compounds has also been compounds of the invention. Acid addition salts are formed observed in the anti-convulsant efficacy tests. by mixing a solution of the compound with a solution of a Compositions within the scope of this invention include pharmaceutically acceptable non-toxic acid such as hydro all compositions wherein the compounds of the present 35 chloric acid, fumaric acid, maleic acid, succinic acid, acetic invention are contained in an amount which is effective to acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, achieve its intended purpose. While individual needs vary, oxalic acid, and the like. For example, basic salts are formed determination of optimal ranges of effective amounts of each by mixing a solution of the 1,4-dihydroquinoxaline-2,3- component is with the skill of the art. Typically, the com dione with a solution of a pharmaceutically acceptable pounds may be administered to mammals, e.g. humans, 40 non-toxic base such as sodium hydroxide, sodium carbonate orally at a dose of 0.0025 to 50 mg/kg, or an equivalent and the like. amount of the pharmaceutically acceptable salt thereof, per Preferred salts of the 1,4-dihydroquinoxaline-2,3-diones day of the body weight of the mammal being treated for of the invention are the highly soluble salts comprising a anxiety disorders, e.g., generalized anxiety disorder, phobic C2 ammonium counter ion, wherein one of the alkyl disorders, obsessional compulsive disorder, panic disorder, 45 groups may be substituted with a hydroxy group. Most and post traumatic stress disorders. Preferably, about 0.01 to 1,4-dihydroquinoxaline-2,3-diones are highly insoluble in about 10 mg/kg is orally administered to treat or prevent aqueous solution. Thus, the i.v. administration of the 14 such disorders. For intramuscular injection, the dose is dihydroquinoxaline-2,3-diones is limited by the relative generally about one-half of the oral dose. For example, for insolubility of the active agent. The highly soluble ammo treatment or prevention of anxiety, a suitable intramuscular 50 nium salts may be prepared, for example, by dissolution of dose would be about 0.0025 to about 15 mg/kg, and most the 1,4-dihydroquinoxaline-2,3-dione in a solution of one preferably, from about 0.01 to about 10 mg/kg. molar equivalent of the corresponding ammonium hydrox In the method of treatment or prevention of neuronal loss ide or amino compound. inischemia, brain and spinal cord trauma, hypoxia, hypogly The ammonium counter ions may be quaternary ammo cemia, and surgery, as well as for the treatment of Alzhe 55 nium cations or mono-, di- or tri-substituted amines that imer's disease, amyotrophic lateral sclerosis, Huntington's form protonated ammonium salts when admixed with a disease and Down's Syndrome, or in a method of treating a 1,4-dihydroquinoxaline-2,3-dione in solution. disease in which the pathophysiology of the disorder The mono-choline salt of the 1,4-dihydroquinoxaline-2, involves hyperactivity of the excitatory amino acids or 3-diones has a pH of about 7.8-9.8, depending on the NMDA receptor-ion channel related neurotoxicity, the phar 60 1,4-dihydroquinoxaline-2,3-dione used. Alternatively, the maceutical compositions of the invention may comprise the 1,4-dihydroquinoxaline-2,3-dione may first be dissolved in a compounds of the present invention at a unit dose level of solution containing 2 molar equivalents of the ammonium about 0.01 to about 50 mg/kg of body weight, or an hydroxide. The di-choline salt may be isolated or the pH of equivalent amount of the pharmaceutically acceptable salt the solution may be adjusted to about 7.8-9.8 with 1 thereof, on a regimen of 1-4 times per day. When used to 65 equivalent of acetic acid. treat chronic pain or to induce anesthesia, the compounds of The ammonium salt of the 1,4-dihydroquinoxaline-2,3- the invention may be administered at a unit dosage level of diones can readily be isolated in pure form by lyophilizing 5,514,680 47 48 the solution to give a dry powder that is highly soluble in second equivalent of acetic acid causes a precipitate to form. water. Up to 90 mg/ml or more of the mono-choline salt of By the time the pH reaches 11, the precipitation is complete. a 1,4-dihydroquinoxaline-2,3-dione will dissolve in water to It has been unexpectedly discovered that 5-chloro-7-trifluo give a clear solution. The salt can also be dissolved in an romethyl-1,4-dihydroquinoxaline-2,3-dione readily dis isotonic glucose solution suitable for i.v. injection. W solves in only 1 equivalent of choline hydroxide. When Examples of 1,4-dihydroquinoxaline-2,3-diones which acetic acid is added, a precipitate does not start to form until can be solubilized according to the present invention include the pH reaches about 9.2. Similarly, 6,7-dichloro-5-nitro-1, those disclosed herein as well as those disclosed in U.S. Pat. 4-dihydroquinoxaline-2,3-dione can be dissolved in 1 Nos. 5,109,001, 5,081,123, 5,079,250, 5,075,306, 5,057, equivalent of choline hydroxide in water. The pH can be 516, 5,026,704, 5,061,706, 4,977,155, 4,975,430, 4,889,855, 10 adjusted to about 8 without appearance of a precipitate. The 4,812,458, 3,992,378, 3,962,440, 4,812,458, 4,659,713,4, dry mono-choline salt may be isolated in pure form, for 948,794, International Application Publication No. WO91/ example, by lyophilization of an aqueous solution, and is 13878, Yoneda and Ogita, Biochem. Biophys. Res. Commun. soluble at very high concentrations (at least 90 mg/ml). 164:841-849 (1989), Kleckner and Dingledine, Mol. Thus, this aspect of the invention is a great advance in the Pharm. 36:430-436 (1989), Rao, T. S. et al., Neurophar 15 art as it allows the preparation of concentrated aqueous macology 29:1031-1035 (1990), Pellegrini-Giampietro, D. solutions of 1,4-dihydroquinoxaline-2,3-diones for intrave E. et al., Br. J. Pharmacol. 98:1281-1286 (1989), Ogita and nous administration. Yoneda, J. Neurochem. 54:699-702 (1990), Kessler, M. et Some of the ammonium counter ions useful for solubi al., Brain Res. 489:377-382 (1989), European Patent Appli lizing 1,4-dihydroquinoxaline-2,3-diones may show toxic cation Publication Nos. 0.377 112,0315959, and 260,467, 20 ity after i.v. administration, for example, tetramethylammo Lester, R. A. et al., Mol. Pharm. 35:565-570 (1989), Patel, nium hydroxide and tetraethylammonium hydroxide. It has J. et al., J. Neurochem, 55:114-121 (1990), Horner, L. et al., been discovered that many 1,4-dihydroquinoxaline-2,3-di Chem. Abstracts 48:2692 (1953), Cheeseman, G. W. H., J. ones can be readily solubilized in 0.05M-0.5M solutions of Chem. Soc.:1170-1176 (1962), Honore, T. et al., Science trishydroxymethylamino-methane (TRIS, Tromethamine 241:701-703 (1988), and Sheardown, M. J. et al., Eur: J. 25 USP). Tromethamine is virtually non-toxic when adminis Pharmacol. 174:197-204 (1989), the disclosures of which tered intravenously in humans. Thus, a Tromethamine solu are fully incorporated by reference herein. tion of 1,4-dihydroquinoxaline-2,3-diones is highly useful Examples of ammonium hydroxides which can be used to for i.v. administration to humans and overcomes the major prepare the ammonium salts of the present invention include obstacle in achieving a solution of 1,4-dihydroquinoxaline any tetra-C-alkyl ammonium hydroxide, e.g. tetramethy 30 2,3-diones for human i.v. use. The present invention is lammonium hydroxide, tetraethylammonium hydroxide, tet directed in part towards this discovery. As an alternative to rabutylammonium hydroxide, tetrapentylammonium Tromethamine, one may use bis-tris-propane (1,3-bis(tris hydroxide, tetrahexylammonium hydroxide, as well as any (hydroxymethyl)methylaminopropane), an analog of tri-C-alkyl-C alkanol ammonium hydroxide, e.g. cho Tromethamine which also shows low toxicity in mammals. line hydroxide, (3-hydroxypropyl)trimethylammonium 35 Bis-tris-propane has a higher pK than Tromethamine and is hydroxide, (4-hydroxybutyl)trimethylammonium hydrox therefore useful for dissolving some 1,4-dihydroquinoxa ide, (2-hydroxyethyl)triethylammonium hydroxide, (2-hy line-2,3-diones which are not as readily soluble in droxyethyl)tripropylammonium hydroxide, and (2-hydroxy Tronethamine. ethyl)tributylammonium hydroxide. Preferably, the The pharmaceutical compositions of the invention may be ammonium hydroxide is choline hydroxide. In addition, any 40 administered to any animal which may experience the C-2 aralkyl C-trialkylammonium hydroxide may be beneficial effects of the compounds of the invention. Fore employed, e.g. benzyltrimethyl ammonium hydroxide. most among such animals are humans, although the inven Examples of amino compounds which may be used to tion is not intended to be so limited. prepare salts of the 14-quinoxaline-2,3-diones include, but The pharmaceutical compositions of the present invention are not limited to ethylenediamine, diethylenetriamine, 45 may be administered by any means that achieve their N-methylethanolamine, di-(2-ethanol)amine, tri-(2-ethano intended purpose. For example, administration may be by lamine, spermidine, spermine, and aminocarbohydrates such parenteral, subcutaneous, intravenous, intramuscular, intra as glucosamine, N-methyl-glucamine, galactosamine, man peritoneal, transdermal, buccal or ocular routes. Alterna nosamine, xylosamine, cellobiosamine, and maltosamine. tively, or concurrently, administration may be by the oral Other amino compounds which can be used to prepare 50 route. The dosage administered will be dependent upon the ammonium salts of the 1,4-dihydroquionoxaline-2,3-diones age, health, and weight of the recipient, kind of concurrent of the present invention include mono-N-, di-(N,N and treatment, if any, frequency of treatment, and the nature of N,N), tri-(N,N,N') and tetra-(N,N,N',N') Calky the effect desired. lguanidines as well as biguanidine, poly Calkyl-substi When the compositions of the invention are administered tuted biguanidines, amidine, arginine, N-C-alkyl amidines, 55 ocularly, one may achieve either local or systemic admin 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), tris(hydroxym istration. For example, the compositions of the present ethyl)aminomethane (TRIS, Tromethamine) and bis-tris invention may be administered in the form of eye drops propane. which are substantially isotonic with tear fluid to achieve As shown in FIG. 21, when the mono-potassium salt of systemic administration. Preferably, such compositions will 5-chloro-7-trifluoromethyl-1,4-dihydroquinoxaline-2,3-di also comprise a permeation-enhancing agent which aids the one is prepared, it is insoluble in water. However, the systemic absorption of the compounds of the present inven di-potassium salt of 5-chloro-7-trifluoromethyl-1,4-dihyd tion. See, U.S. Pat. No. 5,182,258. Alternatively, the com roquinoxaline-2,3-dione is soluble in water, but requires the positions of the invention may be administered ocularly to addition of 4 equivalents of KOH to give a solution having treat or prevent optic nerve degeneration. In this embodi a pH of 12.7. The 1,4-dihydroquinoxaline-2,3-dione will 65 ment, the compounds of the present invention are adminis stay soluble when the pH is lowered to 11.9 by the addition tered in the form of eye drops, as disclosed above, or may of 1 equivalent of acetic acid. However, the addition of a be injected into the vicinity of the optic nerve. In the 5,514,680 49 50 alternative, thin ocular implants may be employed which example, natural or synthetic triglycerides, or paraffin slowly release the compounds of the present invention. hydrocarbons. In addition, it is also possible to use gelatin In addition to the pharmacologically active compounds, rectal capsules which consist of a combination of the active the new pharmaceutical preparations may contain suitable compounds with a base. Possible base materials include, for pharmaceutically acceptable carriers comprising excipients example, liquid triglycerides, polyethylene glycols, or par and auxiliaries which facilitate processing of the active affin hydrocarbons. compounds into preparations which can be used pharma Suitable formulations for parenteral administration ceutically. Preferably, the preparations, particularly those include aqueous solutions of the active compounds in water preparations which can be administered orally and which soluble form, for example, water-soluble ammonium (espe can be used for the preferred type of administration, such as 10 cially tris, bis-tris-propane and choline) salts and alkaline tablets, dragees, and capsules, and also preparations which solutions. In addition, suspensions of the active compounds can be administered rectally, such as suppositories, as well as appropriate oily injection suspensions may be adminis as suitable solutions for administration by injection or orally, tered. Suitable lipophilic solvents or vehicles include fatty are present at a concentration of from about 0.01 to 99 oils, for example, sesame oil, or synthetic fatty acid esters, percent, together with the excipient. 15 for example, ethyl oleate or triglycerides or polyethylene The pharmaceutical preparations of the present invention glycol-400 (the compounds are soluble in PEG-400). Aque are manufactured in a manner which is itself known, for ous injection suspensions may contain substances which example, by means of conventional mixing, granulating, increase the viscosity of the suspension include, for dragee-making, dissolving, or lyophilizing processes. Thus, example, sodium carboxymethyl cellulose, sorbitol, and/or pharmaceutical preparations for oral use can be obtained by 20 dextran. Optionally, the suspension may also contain stabi combining the active compounds with solid excipients, lizers. optionally grinding the resulting mixture and processing the The characterization of glycine binding sites in vitro has mixture of granules, after adding suitable auxiliaries, if been difficult because of the lack of selective drug ligands. desired or necessary, to obtain tablets or dragee cores. Thus, the glycine ligands of the present invention may be Suitable excipients are, in particular, fillers such as sac 25 used to characterize the glycine binding site. Particularly charides, for example lactose or sucrose, mannitol or sorbi preferred substituted 1,4-dihydroquinoxaline-2,3-diones tol, cellulose preparations and/or calcium phosphates, for which may be used for this purpose are isotopically radio example tricalcium phosphate or calcium hydrogen phos labelled derivatives, e.g. where one or more of the atoms are phate, as well as binders such as starch paste, using, for replaced with H, C, C, N, or 'F. In addition, positron example, maize starch, wheat starch, rice starch, potato 30 emitters such as 'C and F may be incorporated into the starch, gelatin, tragacanth, methyl cellulose, hydroxypropy 1,4-dihydroquinoxaline-2,3-dione for use in positron emis lmethylcellulose, sodium carboxymethylcellulose, and/or sion tomography (PET) for the localization of glycine bind polyvinyl pyrrolidone. If desired, disintegrating agents may ing sites. Moreover, 'I-substituted 1,4-dihydroquinoxa be added such as the above-mentioned starches and also line-2,3-diones may be used for single photon emission carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, 35 computed tomography (SPECT) imaging of the glycine agar, or alginic acid or a salt thereof, such as sodium binding site. In addition, one may prepare isotopically alginate. Auxiliaries are, above all, flow-regulating agents labelled compounds which are not radioactive for use in and lubricants, for example, silica, talc, stearic acid or salts metabolic studies, e.g. wherein one or more of the hydrogens thereof, such as magnesium stearate or calcium stearate, and/or carbons are enriched in H or 'C. and/or polyethylene glycol. Dragee cores are provided with 40 The following examples are illustrative, but not limiting, suitable coatings which, if desired, are resistant to gastric of the method and compositions of the present invention. juices. For this purpose, concentrated saccharide solutions Other suitable modifications and adaptations of the variety may be used, which may optionally contain gum arabic, talc, of conditions and parameters normally encountered in clini polyvinyl pyrrollidone, polyethylene glycol and/or titanium cal therapy and which are obvious to those skilled in the art dioxide, lacquer solutions and suitable organic solvents or 45 are within the spirit and scope of the invention. solvent mixtures. In order to produce coatings resistant to EXAMPLES gastric juices, solutions of suitable cellulose preparations such as acetyl-cellulose phthalate or hydroxypropylmethyl Methods and Materials cellulose phthalate, are used. Dye stuffs or pigments may be In the following Examples 1-27, all 'H NMR were run at added to the tablets or dragee coatings, for example, for 50 300 MHz and CNMR at 75 MHz on a QE-300 instrument identification or in order to characterize combinations of and are referenced to the residual protio solvent. 'F NMR active compound doses. were run at 339 MHz on an NT 360 instrument and are Other pharmaceutical preparations which can be used referenced to external CF. Melting points were taken on a orally include push-fit capsules made of gelatin, as well as Mel-Temp melting point apparatus and are uncorrected. soft, sealed capsules made of gelatin and a plasticizer such 55 Samples were placed in the block when the temperature was as glycerol or sorbitol. The push-fit capsules can contain the >250° C. in order to minimize decomposition prior to active compounds in the form of granules which may be melting. DMF was distilled before use. All other reagents mixed with fillers such as lactose, binders such as starches, were used as received from the manufacturer. Compounds and/or lubricants such as talc or magnesium stearate and, were purchased from Aldrich Chemical Co. unless otherwise optionally, stabilizers. In soft capsules, the active com 60 indicated below. 12 -Dinitro-3,4,5,6-tetrachlorobenzene, pounds are preferably dissolved or suspended in suitable 5,6-diamino-1,3-dimethyluracil hydrate and 1,2-diamino-3- liquids, such as fatty oils, or liquid paraffin. In addition, chloro-5-trifluoromethylbenzene were purchased from May stabilizers may be added. bridge Chemical. 3-Fluoro-6-nitroaniline was obtained from Possible pharmaceutical preparations which can be used Dr. Michael Scherz. 1,2-Diamino-4-chloro-5-fluorobenzene, rectally include, for example, suppositories, which consist of 65 1,2-diamino-3-chloro-5-trifluoromethylbenzene and 1,2-di a combination of one or more of the active compounds with amino-3-bromo-5-trifluoromethylbenzene, were purchased a suppository base. Suitable suppository bases are, for from PCR Chemical. 5,514,680 51 S2 Example 1 basified to pH 13 with 4N aq. NaOH (125 mL). The Preparation of 6,7-Dichloro-5-nitro-1,4-dihydro-2,3 -qui precipitate was collected on a sintered funnel by vacuum noxalinedione filtration, washed with HO (5x10 mL), dried at 40° C. Method A: under 0.1 mmHg for 16 h giving 3.72 g (70%) of the crude 6,7-Dichloro-1,4-dihydro-2,3-quinoxalinedione. (1) A 5 product as a coffee colored powder. mixture of 422 mg (2.5 mmol) of 4,5-dichloro-o-phenylene The crude product (3.70 g) obtained above was purified diamine (Pfaltz & Bauer, Inc., used as received) and 1.10 g by crystallization from benzene (60 mL) affording 3.17 g (7.5 mmol) of diethyl oxalate (Sigma, used as received) was stirred under Ar at 160° C. for 2 h, then at 180 C. for 7 h. (85% recovery) as slightly purple scales, which was pure by The reaction mixture was allowed to cool to rit. (22 C.), 10 TLC (CHCI: EtOH-9:1). Mp 159°-60° C. (Aldrich: diluted with hexanes (10 mL), the precipitate was collected 159°-62° C). by centrifuging and washed with hexanes (2x10 mL). The 6,7-Dichloro-1,4-dihydroquinoxaline-2,3-dione: A sus grey solid was stirred with 40 mL of aq. NaOH (about 1N) pension of 2.655 g (15.0 mmol) of 4,5-dichlorophenylene and activated charcoal (0.4 g) at r,t. for 30 min, the charcoal 1,2-diamine and 1.986 g (15.75 nmol) oxalic acid dihydrate was removed by vacuum filtration and washed with distilled 15 (Fisher Scientific Co., used as received) in 22.5 mL of 2Naq. HO (6x10 mL), which were combined with the original HCl was refluxed with stirring at 125°C. (bath temperature) filtrate, acidified without about 4N aq. HCl (about 20 mL). The white precipitate was collected by vacuum filtration, for 2.5 h, (during the first 5 min heating, the suspension washed with distilled HO (5x10 mL), EtOH (3x5 mL), then almost turned into a solution, then began to form a precipi dried at 60° C. under 0.1 mmHg for 8 h affording 426 mg 20 tate). The reaction mixture was allowed to cool to 22°C., (73.8%) of 1 as a cream solid, mp.>400° C. H NMR and HO (50 mL) was added. The precipitate was collected (DMSO-d6) 12.010 (s, 2H) 7.226 (s, 2H) ppm. on a Hirsh funnel by vacuum filtration, washed with HO 5-Nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione. (6x25 mL) and dried at 60° C. under 0.1 mmHg for 12 h (2) Compound 1 (416 mg, 1.80 mmol) was dissolved in 5.5 affording 3.39 g (98%) of 6,7-dichloroquinoxaline-2,3- mL of conc. HSO at 0°C. with stirring. To this resulting 25 dione as a deep pink powder. Mp. >400° C. "H NMR deep grey solution was added 202 mg (2.22 mmol) of finely (DMSO-d): 812.016 (s, 2H), 7.234 (s, 2H). This product ground KNO (Baker, used as received) at 0° C. with was used in the next reaction without further purification. stirring. The mixture was stirred at 0°C. for 3 h, then at r,t. 5-Nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione: (22 C.) for 30 h. The reaction mixture was added to ice-HO (60 g), the precipitate was collected by vacuum 30 3.335 g (14.5 mmol) of 6,7-dichloro-1,4-dihydroquinoxa filtration, washed with distilled HO (5x10 mL), EtOH (3x5 line-2,3-dione was dissolved in 65 mL of conc. HSO with mL), then dried at under 0.1 mm Hg at 80° C. for 2 h stirring and cooling in an ice-HO bath, then 2.20 g (21.76 affording 443.5 mg (89.6%) of crude 2 as a cream amor mmol) of KNO (Baker, used as received) was added in phous solid (The "H NMR indicated that it was 95-98% portions over 10 min, with stirring. The resulting mixture pure). 35 was stirred at 22° C. under N. for 20 h. then was slowly The further purification was as following: poured into ice-HO (400 mL) with stirring. The precipitate 443 mg of the crude 2 (obtained above) was dissolved in was collected on a sintered funnel by vacuum filtration, about 1N aq. NaOH (120 Ml) at rit., activated charcoal (1 g) was added, then stirred at r,t, for 15 min. The charcoal was washed with HO (5x10 mL), and dried at 60° C. under 0.1 removed by vacuum filtration, washed with distilled HO mmHg for 12 h affording 3.39 g (85%) of the crude (2x10 mL). The combined filtrate was acidified to pH 2 with 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione as a about 4N aq. HCl (about 50 mL). The precipitate was grey-yellow powder. Purity: >98.5% based on HPLC analy collected by vacuum filtration, washed with distilled HO S.S. (5x10 mL), EtOH (2x5 mL), dried under 0.1 mmHg at 80° The compound was purified as follows. 3.365g of 6,7- C. for 2 h affording 327 mg (74% recovery) of essentially 45 dichloro-5 -nitro-1,4-dihydroquinoxaline-2,3-dione pure 2, mp. 350°–4° C. (dec.). (The "H NMR indicated that obtained above was added to 550 mL of 1N aq. NaOH and there was almost no impurities present). stirred vigorously for 20 min. The resulting mixture was Recrystallization: 315 mg of the purer 2 (obtained above) filtered by vacuum filtration through a sintered funnel to was dissolved in 45 ml of DMSO, to this solution was added remove a small amount of insoluble material. To the filtrate HO (about 1 mL) was added dropwise until a precipitate 50 was slowly added conc. HCl (about 43 mL) (dropwise) with was produced. The mixture was heated (in a 100-105 C. vigorous stirring to adjust the pH from 13 to 11 (using pH oil bath) with stirring (using a small magnetic bar), HO meter to monitor). (A blank experiment showed that the 6,7 (about 1 mL) was added dropwise until a cloudy mixture -dichloroquinoxaline-2,3-dione, the starting material for the was formed, and DMSO was added dropwise to produce a nitration reaction, could be precipitated from its 1N aq. clear solution. After standing at r,t. for 8 h, the yellow 55 microcrystals were collected by vacuum filtration, washed NaOH solution only in such condition that the pH was with HO (5x10 mL), EtOH (2x3 mL), dried under 0.1 mm within 9.5-8.) The precipitate was collected on a sintered Hg at 80° C. for 3 h affording 286 mg (90.8% recovery) of funnel by vacuum filtration and washed with HO (5x50 pure 2, mp. 354-7 C. mL). The moist product was added to 200 mL of HO, and Method B: to this resulting suspension was slowly added conc. HCl 4,5-Dichloro-1,2-phenylenediamine (1) To a suspension (dropwise) to adjust the pH to 5. The precipitate was of 6.21 g (30.0 mmol) of 4,5-dichloro-2-nitroaniline (Ald collected on a Hirsh funnel by vacuum filtration, washed rich, used as received) in EtOH (100 mL) was added 310 mg with HO (8x50 mL), and dried at 60° C. under 0.1 mmHg of 5% Pd/C, the mixture was hydrogenated at 30-20 parr of for 16 h affording 3.12 g (92.9% recovery) of the purer H for 4 h, then filtered. The filtrate was roto-evaporated to 65 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione. dryness. The black solid residue was stirred with 250 mL of Mp.347–8° C. "HNMR(DMSO-d): 12.265 (bs, 2H), 7.379 2N aq. HCl for 20 min., then filtered. The filtrate was (s, 1H). Purity: >99.2% based on HPLC analysis. 5,514,680 53 54 Example 2 (s, 1H, ArH), 7.53 (s, 1H, ArH), 11.9 (brs, 2H, NH). 'F Preparation of 5-Chloro-7-trifluoromethyl-1,4-dihydroqui NMR (CF external standard, 8-1629) 8-58.43 (s). noxaline-2,3-dione EIHRMS calc. for CHCIFNO, 263.9912, found 439.0 mg (3.0 mmol) of diethyl oxalate (from Sigma, used 263.9891. as received) was added to 210.6 mg (1.0 mmol) of 1,2- diamino-3-chloro-5-trifluoromethylbenzene (from PCR Example 3 Inc., used as received), and the resulting yellow solution was heated at 180° C. (bath temperature) under Ar with stirring Preparation of 5-Chloro-7-fluoro-1,4-dihydroquinoxaline for 3.5h, the solution became thick with formation of cream 2,3-dione precipitate and hard to stir. The reaction mixture was 10 2-Chloro-4-fluoro-6-nitroaniline allowed to cool to rit., and triturated with hexane (10 mL). 2-Chloro-4-fluoro-6-nitroaniline was prepared using an The precipitate was collected by vacuum filtration, washed adaptation of the method of Mitchell, et al. (Mitchell, R. H. with hexane (2x10 mL) (the combined hexane filtrate was et al., J. Org. Chem. 44:4733 (1979)). To a solution of saved), dried under 0.1 mmHg for 4 h affording 134.6 mg 4-fluoro-2-nitroaniline (500 mg, 3.2 mmol) in dry DMF (10 (58%) of the crude desired product as a yellow powder 15 mL) under N, was added dropwise a solution of N-chloro (95-98% pure by NMR), mp.330-2° C. (dec.) (preheated succinimide (426 mg, 3.2 mmol) in dry DMF (16 mL). The block). reaction was allowed to stir overnight. The solution was then A portion (125.5 mg) of the crude product obtained above poured into 100 mL HO and the resulting cloudy suspen was stirred with 1N aq. NaOH (10 mL) at rit., the resulting sion extracted with 4x25 mL ethyl acetate. The combined clear yellow basic solution was acidified to pH 2 by the 20 organic phases were washed with 4x25 mL HO and 25 mL addition of 5N aq. HCl (about 1.2 mL) dropwise with saturated NaCl solution and dried (MgSO). The MgSO shaking at rit. The white precipitate was collected by cen was vacuum filtered and the solvent rotary evaporated to trifuging, washed with HO (5x10 mL), then dried by yield a brown crystalline solid which was purified further by co-evaporation with EtOH (2x 10 mL) at 40° C. giving flash chromatography (2:1 hexanes:ethyl acetate) to yield 118.8 mg (94.6% recovery) of pure (by NMR) compound as 25 424 mg of an orange crystalline solid (69.5%) H NMR an off-white powder, mp. 334°14 6° C. (dec.) (preheated (CDCl) 86.46 (br s, 2H, NH2), 7.42 (dd, Jrs-3 Hz, block). Attempts to recrystallize from EtOH, EtOH-HO and J-F-7.2 Hz, H-4), 7.85 (dd, Jrs-3 3 Hz, Jrs--8.7 Hz, DMSO-HO failed (those solutions only gave either a pre H-5) cipitate or not). HNMR(DMSO-d) 8:7.349(s, 1H), 1,2-Diamino-3-chloro-5-fluorobenzene 7.604(s, 1H), 11.724(s, 1H), 12.222(s, 1H) ppm. 30 1,2-Diamino-3-chloro-5-fluorobenzene was prepared The combined hexane filtrate obtained above was rotary using an adaptation of the method of Bellamy, et al., evaporated at 40° C. to dryness, the residue (viscous orange (Bellamy, F. D. et al., Tetrahedron Lett. 25:839 (1984)). A oil) was heated at 180° C. for 5h. The resulting brown bulk mixture of 2-chloro-4-fluoro-6-nitroaniline (424 mg, 2.22 solid was triturated with hexane (5 mL), the precipitate was mmol) and SnCl2.H2O (2.50 g, 11.1 mmol) dissolved in 7 collected by centrifuging, washed with hexane (4x5 mL), 35 mL ethyl acetate and 3 mL absolute ethanol under Na was the stirred with 1N aq. NaOH (4.5 mL), centrifuged to heated at 70° C. for 2.5 h. All the starting material had remove a small amount of black solid, the supernatant was reacted as evidenced by TLC (silica gel, 2:1 hexanes:ethyl acidified to pH2 by the addition of 5Naq. HCl (about 1 mL) acetate). The reaction was allowed to cool to room tempera dropwise with shaking at r,t. The yellowish precipitate was ture and poured into 20 mL crushed ice. Sufficient solid collected by centrifuging, washed with H2O (5X5 mL), then 40 NaHCO was added (foaming!) to bring the pH to 6. The dried by co-evaporation with EtOH (3x5 mL) at 40° C. thick yellow white emulsion was then extracted with 3X25 affording 51.8 mg (22.3%, based on the starting diamine in mL ethyl acetate and the combined organic extracts washed the first reaction) of the title compound as a yellowish with sat'd NaCl solution. The organic phase was dried powder (essentially pure by NMR), mp. 334°-6° C. (pre (MgSO), vacuum filtered and the solvent rotary evaporated heated block). 45 to yield a dark brown oil which crystallized on standing to After the recovery mixed material was reused, the total yield 290 mg (82%). "H NMR (CDCl) 83.59 (brs, 4H, yield was about 77%. 20NH)); 6.37 (dd, 1H, H5, J-2.7, J =9.3); 6.55 (dd, The result reported hereby was from one of three experi 1H, H4, J-5-2.7, Jar-8.4). ments and was reproducible. The reaction with 1:2 ratio of 5-Chloro-7-fluoro-1,4-dihydro-2,3-quinoxalinedione the diamine to the oxalate using same procedure as that for 50 5-Chloro-7-fluoro-1,4-dihydro-2,3-quinoxalinedione was 1:3 ratio was also tried, but the product was complicated. prepared using an adaptation of the method of Cheeseman. Alternatively, the title compound was prepared using an (Cheeseman, G. W. H. J. Chem. Soc. 1171 (1962)). A adaptation of the method of Cheeseman, G. W. H., J. Chem. mixture of diethyl oxalate (2.64 g, 18.1 mmol) and 1,2 Soc. 1171 (1962). A mixture of diethyl oxalate (1.34g, 9.60 -diamino-3-chloro-5-fluorobenzene (290 mg, 1.81 mmol) mmol) and 1,2-diamino-3-chloro-5-trifluoromethylbenzene 55 was heated to reflux under N2 for 10 h. The reaction was (200 mg, 0.95 mmol) was heated to reflux under N for 2 h. allowed to cool to room temperature and the shiny yellow The reaction was allowed to cool to room temperature and brown crystals collected by vacuum filtration and rinsed the solid collected by vacuum filtration and rinsed with ice with EtOH (10 mL) and air dried to give 164.1 mg (42%). cold EtOH (10 mL). This yellow white solid was dissolved A portion of this solid was taken and dissolved in 10 mL 1N in 1N NaOH (15 mL) and a few insoluble particles were 60 NaOH. The solution was treated with activated charcoal and removed by gravity filtration. The solution was treated with filtered through a pad of Celite. The resulting solution was decolorizing carbon and the mixture was filtered through a carefully acidified with 1NHCl (pH=6). Pale yellow needles pad of celite and the resulting solution acidified with 4N slowly formed in the solution and were collected by vacuum HCl. A precipitate formed that was isolated by vacuum filtration, rinsed with 20 mL of HO and further dried under filtration and washed with water (20 mL). This white solid 65 vacuum (0.1 torr, 78° C) to yield 67.8 of powdery pale was dried in drying pistol (0.05 torr, 78° C) to yield 91.9 mg yellow crystals. mp 306°-308° C. (dec), H NMR (d- (36.0%), mp3469-348° C. (dec). "H NMR (d-dimso) 87.30 DMSO) 67.28 (s, 1H, ArH), 7.56 (s, 1H, ArH), 11.7 (brs, 5,514,680 55 56 2H, NH), EIMS m/z 216 (M+2,34, 214 (M+, bp), 188 (21), difluoro-1,4-dihydro-2,3-quinoxalinedione present by 186 (68), 123 (61), 131 (62). EIHRMS calc. for NMR. CHCIFNO, 213.9945, found 213.9961. Example 4 Example 5 Preparation of 5-Chloro-6,7-difluoro-1,4-dihydroquinoxa Preparation of 5-Bromo-6,7-difluoro-1,4-dihydro-2,3 -qui line-2,3-dione noxalinedione 2-Chloro-3,4-difluoro-6-nitroaniline 2-Bromo-3,4-difluoro-6-nitroaniline 2-Chloro-3,4-difluoro-6-nitroaniline was prepared using 10 2-Bromo-3,4-difluoro-6-nitroaniline was prepared using an adaptation of the method of Mitchell et al. (Mitchell, R. an adaptation of the method of Mitchell et al. (Mitchell, R. H. et al., J. Org. Chem. 44:4733 (1979)). To a solution of H. et al., J. Org. Chem. 44:4733 (1979)). To a solution of 4,5-difluoro-2-nitroaniline (500 mg, 2.87 mmol) in DMF (16 4,5-difluoro-2-nitroaniline (500mg, 2.87 mmol) in DMF (25 mL) under N, was added N-chlorosuccinimide (401 mg, mL) under N: was added all at once N-bromosuccinimide 3.00 mmol) in DMF. The reaction was allowed to stir 48 h. 15 (511 mg, 2.87 mmol) in dry DMF (16 mL). The reaction was The solution was then poured into 75 mL H.O. the cloudy allowed to stir overnight. TLC (1:1 hexanes:ethyl acetate) orange suspension which formed was then extracted with showed still some starting material present. Additional 4x25 mL of methylene chloride. The combined organic N-bromosuccinimide (100 mg) was added and the reaction extracts were washed with 5x20 mL of HO and 25 mL sat'd stirred another 12 h. The solution was then poured into 100 NaCl solution. The organic phase was dried (MgSO) and 20 ml HO and the resulting cloudy suspension extracted with the drying agent removed by vacuum filtration. The solvent 3x20 mL methylene chloride. The combined organic phases was rotary evaporated to yield a yellow orange oil which were washed with 4x25 mL HO and 25 mL saturated NaCl crystallized on standing. "H NMR showed this solid to be solution and dried (MgSO). The MgSO was vacuum mixture of chlorinated product and starting material. The filtered and the solvent rotary evaporated to yield a yellow mixture was separated by flash chromatography (silica gel, 25 brown oil which slowly crystallized to yield 700 mg (96%). 3:1 hexanes:ethyl acetate) to yield 162 mg of a yellow H NMR (CDC1,) 86.70 (brs, 2H, NH,), 7.99 (m, 1H, H-5). crystalline solid (27%). H NMR (CDCl) 66.60 (brs, 2H, 3-Bromo-4,5-difluoro-1,2-diaminobenzene NH), 8.00 (m, 1H, H-5). There was 17% starting material 3-Bromo-4,5-difluoro-1,2-diaminobenzene was prepared contamination by NMR. using an adaptation of the method of Bellamy et al. (Bel 3-Chloro-4,5-difluoro-1,2-diaminobenzene 30 lamy, F. D. et al., Tetrahedron Lett. 25:839 (1984)). A 3-Chloro-4,5-difluoro-1,2-diaminobenzene was prepared mixture of 2-bromo-3,4-difluoro-6-nitroaniline (700 mg, using an adaptation of the method of Bellamy et al. (Bel 2.78 mmol) and SnCl2.H2O (3.14g, 13.9 mmol) dissolved lamy, F. D. et al., Tetrahedron Lett. 25:839 (1984)) Amixture in 7 mL ethyl acetate and 3 mL absolute ethanol under N of 2-chloro-3,4-difluoro-6-nitroaniline (160 mg, 0.767 was heated at 75 C. for 2 h. All the starting material had mmol) and SnCl2.H2O (0.863 g, 3.84 mmol) was dissolved 35 reacted as evidenced by TLC (silica gel, 2:1 hexanes:ethyl in 5 mL ethyl acetate and 2 mL absolute ethanol under N acetate). The reaction was allowed to cool to room tempera and heated at 75 C. for 5 h. The reaction was allowed to ture and poured into 20 mL crushed ice. Sufficient sat'd cool to room temperature and poured into 50 mL. H.O. NaHCO solution was added (foaming!) to bring the pH to Sufficient sat'd NaHCO, solution was added (foaming) to 5. The thick yellow white emulsion was then extracted with bring the pH to 7. The resulting mixture was extracted with 3x25 mL ethyl acetate and the combined organic extracts 3x20 mL ethyl acetate and the combined organic extracts washed with sat'd NaCl solution. The organic phase was washed with 20 mL sat'd NaCl solution. The organic phase dried (MgSO), vacuum filtered and the solvent rotary was dried (MgSO), vacuum filtered and the solvent rotary evaporated to yield a dark brown oil which crystallized on evaporated to yield a brown solid, 124 mg (91%). H NMR standing to yield 410 mg (66%). H NMR (CDC) 83.59 (CDCl) 83.52 (brs, 4H, 20NH)); 6.49 (dd, 1H, J-7.5, 45 (brs, 4H, 20NH)); 6.52 (m, 1H, H-6). 10.8 Hz, H-6). There was 13% 4,5-difluoro-1,2-diaminoben 5-Bromo-6,7-difluoro-1,4-dihydro-2,3-quinoxalinedione zene present by NMR. The title compound was prepared using an adaptation of 5-Chloro-6,7-difluoro-1,4-dihydro-2,3-quinoxalinedione the method of Cheeseman. (Cheeseman, G. W. H. J. Chem. The title compound was prepared using an adaptation of Soc. 1171 (1962)). A mixture of diethyl oxalate (2.70 g, 18.5 the method of Cheeseman. (Cheeseman, G. W. H. J. Chem. 50 mmol) and 3-bromo-4,5-difluoro-1,2-diaminobenzene (410 Soc. 1171 (1962)). A mixture of diethyl oxalate (981 mg, mg, 1.85 mmol) was heated to reflux under N, for 15h. The 6.72 mmol) and 3-chloro-4,5-difluoro-1,2-diaminobenzene reaction was allowed to cool to room temperature and the (120 mg, 0.670 mmol) was heated to reflux under N. for 15 dark-brown solid collected by vacuum filtration and rinsed h. The reaction was allowed to cool to room temperature and with EtOH (20 mL) and air dried to give 215 mg (42%). A the gray solid collected by vacuum filtration and rinsed with 55 portion of this solid (150mg) was taken and dissolved in 20 ice-cold EtOH (10 mL) and air dried. The solid was taken mL 1N NaOH with heating. The solution was treated with and dissolved in 5 mL 1N NaOH with heating. The solution activated charcoal and filtered through a pad of Celite. The was treated with activated charcoal and filtered through a resulting solution was carefully acidified with 1N HCl pad of Celite. The resulting solution was carefully acidified (pH=1). Pale yellow needles slowly formed in the solution with 1N HCl (pH=1). A white powderformed in the solution and were collected by vacuum filtration, rinsed with 20 mL at pH-6, but a few drops 1N NaOH cleared the solution and of HO and dried under vacuum (0.1 torr, 78 C.) to yield upon addition of a few drops 1N HCl white needles slowly 67.8 mg of powdery pale yellow crystals, mp306–310 C. formed in the solution. These were collected by vacuum (dec). H NMR (d-DMSO) 8 7.09 (dd, 1H, J=7.5, H-8), filtration, rinsed with 20 mL of HO and dried under vacuum 11.3 (brs, 1H, NH), 12.1 (brs, 1H, NH). EMS m/z 278 (0.1 torr, 78° C) to yield 24.9 (16%) of pale yellow needles. 65 (M+2, 75), 276 (M+, 77), 250 (56), 248 (57), 141 (bp). H NMR (d-DMSO) 87.05 (dd, 1H, J=10.5, H-8), 11.6 (br EIHRMS calc. for CHBrF.N.O., 275.9346, found s, 1H, NH), 12.0 (bris, 1H, NH). There was 13%. 6,7- 275.9331. 5,514,680 57 S8 Example 6 Example 7 Preparation of 6-Bromo-7-fluoro-1,4-dihydroquinoxaline Preparation of 5,7-Dichloro-1,4-dihydroquinoxaline-2,3- 2,3-dione dione 4-Bromo-3-fluoro-6-nitroaniline 3,5-Dichloro-1,2-diaminobenzene 4-Bromo-3-fluoro-6-nitroaniline was prepared using an 3,5-Dichloro-1,2-diaminobenzene was prepared using an adaptation of the method of Mitchell et al. (Mitchell, R. H. adaptation of the method of Bellamy, et al. (Bellamy, F. D. et al., J. Org. Chem. 44:4733 (1979)). To a solution of et al., Tetrahedron Lett. 25:839 (1984)). A mixture of 2,4- 3-fluoro-6-nitroaniline (500 mg, 3.2 mmol) in dry DMF (15 dichloro-6-nitroaniline (1.00 g, 4.8 mmol) and SnCl2.H2O mL) under N, was added dropwise a solution of N-bromo 10 (5.41 g, 24.1 mmol) dissolved in 10 mL ethyl acetate and 5 succinimide (626 mg, 3.2 mmol) in dry DMF. The reaction mL absolute ethanol under N was heated at 70° C. for 1 h. was allowed to stir 48 h. The solution was then poured into All the starting material had reacted as evidenced by TLC 100 mL HO. The cloudy yellow suspension which formed (silica gel, 3:1 hexanes:ethyl acetate). The reaction was was then extracted with 4x25 mL of methylene chloride. The allowed to cool to room temperature and poured into 40 mL combined organic extracts were washed with 4X 25 mL of 15 crushed ice. Sufficient sat'd NaHCO solution was added HO and 25 mL sat'd NaCl solution. The organic phase was (foaming) to bring the pH to 5. The orange oil/thick white dried (MgSO) and the drying agent removed by vacuum emulsion was extracted with 3x25 mL ethyl acetate and the filtration. The solvent was rotary evaporated to yield a combined organic extracts washed with sat'd NaCl solution. yellow orange oil which crystallized on standing. "H NMR The organic phase was dried (MgSO), vacuum filtered and showed this solid to be a mixture of mono- and dibrominated 20 the solvent rotary evaporated to yield a pale orange oil which products in a 3.8:1 ratio. The mixture was separated by flash crystallized on standing to yield 789 mg (93%). H NMR chromatography (2:1 hexanes:ethyl acetate) to yield 324 mg (CDCl) 83.69 (brs, 4H, 20H)); 6.61 (s, 1H, H-6); 6.82 (s, 1FH, H-4). of a yellow solid (43%). "H NMR (CDCl) 86.19(brs, 2H, 5,7-Dichloro-1,4-dihydro-2,3-quinoxalinedione NH); 6.58(d, 1H, J–9.6, ArH); 8.39(d, 1H, J =6.9, ArH). 25 The title compound (Leeson, P. D. et al., J. Med. Chem 4-Bromo-5-fluoro-1,2-diaminobenzene 34:1243 (1991) was prepared using an adaptation of the 4-Bromo-5-fluoro-1,2-diaminobenzene was prepared method of Cheeseman. (Cheeseman, G. W. H. J. Chem. Soc. using an adaptation of the method of Bellamy et al. (Bel 1171 (1962)). A mixture of diethyl oxalate (4.12 g, 28.2 lamy, F. D. et al., Tetrahedron Lett. 25:839 (1984)). A mmol) and 3,5-dichloro-1,2-diaminobenzene (500 mg, 2.82 mixture of 4-bromo-3-fluoro-6-nitroaniline (320 mg, 1.36 30 mmol) was heated to reflux under N for 6 h. The reaction mmol) and SnCl2H2O (1.53g, 6.81 mmol) dissolved in 7 was allowed to cool to room temperature and the pale yellow mL ethyl acetate and 3 mL absolute ethanol under N, was shiny solid collected by vacuum filtration and rinsed with heated at 75°C. for 8h. Some starting material had remained EtOH (20 mL) and air dried to give 286 mg (44%). mp (by TLC) after only 1 h heating. All the starting material had 326°-328°C. (dec) Lit3379–340°C). HNMR (d-DMSO) reacted after 8 h as evidenced by TLC (silica gel, 3:1 35 87.05 (d, 1H, J=1.8, H-8), 7.32 (d, 1H, J=1.8, H-6), 11.5 (br hexanes:ethyl acetate). The reaction was allowed to cool to s, 1H, NH), 12.1 (brs, 1H, NH). EIMS mile 234 (M-4, 12), room temperature and poured into 50 mL H.O. Sufficient 232 (M+2, 67), 230 (M+, bp), 204 (52), 202 (77), 141 (19), sat'd NaHCO solution was added (foaming) to bring the 142 (59) EIHRMS calc. for CHCNO, 229.9650, found pH to 5. The resulting mixture was extracted with 3X.25 mL. 229.9646. ethyl acetate and the combined organic extracts washed with Example 8 20 mL sat'd NaCl solution. The organic phase was dried (MgSO), vacuum filtered and the solvent rotary evaporated Preparation of 5,7-Dibromo-1,4-dihydroquinoxaline-2,3- to yield a white powder 277 mg (99%). H NMR (CDC1) dione 83.22 (brs, 2H, NH); 3.60(brs, 2H, NH); 6.50 (d. 1H, J, 3,5-Dibromo-1,2-diaminobenzene =9.6 Hz, H-6); 6.83 (d, 1H, J-6.6 Hz, H-3). 45 3,5-Dibromo-1,2-diaminobenzene was prepared using an 6-Bromo-7-fluoro-1,4-dihydro-2,3-quinoxalinedione adaptation of the method of Bellamy, et al. (Bellamy, F. D. The title compound was prepared using an adaptation of et al., Tetrahedron Lett. 25:839 (1984)). A mixture of 2,4- the method of Cheeseman. (Cheeseman, G. W. H. J. Chem. dibromo-6-nitroaniline (500 mg, 1.69 mmol) and Soc. 1171 (1962)). A mixture of diethyl oxalate (1.97 mL, SnCl2HO (1.90 g, 8.45 mmol) dissolved in 5 mL ethyl 13.5 mmol) and 4-bromo-5-fluoro-1,2-diaminobenzene (277 50 acetate and 2 mL absolute ethanol under Na was heated at mg, 1.35 mmol) was heated to reflux under N for 12 h. The 70° C. for 1 h. All the starting material had reacted as reaction was allowed to cool to room temperature and the evidenced by TLC (silica gel, 3:1 hexanes:ethyl acetate). dark-brown solid collected by vacuum filtration and rinsed The reaction was allowed to cool to room temperature and with ethanol (20 mL) and air dried to give 233 mg (67%) of poured into 20 mL crushed ice. Sufficient sat'd NaHCO a powdery brown solid. A portion of this solid (100mg) was 55 solution was added (foaming!) to bring the pH to 5. The taken and dissolved in 5 mL 1N NaOH. The solution was thick yellow white emulsion was vacuum filtered and the treated with activated charcoal and filtered through a pad of filtrate extracted with 3X25 mL ethyl acetate and the com Celite. The Celite was rinsed with 10 mL additional NaOH bined organic extracts washed with sat'd NaCl solution. The solution. The resulting solution was carefully acidified with organic phase was dried (MgSO), vacuum filtered and the 1N HCl (pH=5). Bright yellow needles slowly formed in the 60 solvent rotary evaporated to yield a pale yellow oil which solution and were collected by vacuum filtration, rinsed with crystallized on standing to yield 400 mg (89%). "H NMR 15 mL of HO and dried under vacuum (0.1 torr, 78° C) to (CDCl3) 83.62 (brs, 4H, 20NH)); 6.78 (d, J=1.8, 1H, H-6); yield 40.0 mg of yellow crystals. H NMR (d-DMSO) 7.01 (d, J=1.8, 1H, H-4). 86.99 (d.1H, J=9.3, H-8), 7.29 (d, 1H, J=6.3, H-6), 11.95 (br 5,7-Dibromo-1,4-dihydro-2,3-quinoxalinedione s, 2H, 20NH)). EIMS m/z 260 (M--2,96), 258 (M--(bp)), 232 65 The title compound was prepared using an adaptation of (51), 230 (52), 123 (83). EIHRMS calc: for CHBrFNO, the method of Cheeseman. (Cheeseman, G. W. H. J. Chem. 256.9941, found 257.9441. Soc. 1171 (1962)). A mixture of diethyl oxalate (2.19 g, 15.0 5,514,680 59 60 mmol) and 3,5-dibromo-1,2-diaminobenzene (400 mg, 1.50 Soc. 1171 (1962)). 6-Chloro- 7-trifluoromethyl-1,4-dihydro mmol) was heated to reflux under N for 6 h. The reaction 2,3-quinoxalinedione (500 mg, 1.89 mmol) was dissolved in was allowed to cool to room temperature and the pale brown 15 mL of concentrated HSO, and the clear solution was shiny solid collected by vacuum filtration and rinsed with cooled to 0° C. with stirring. To this was added in small EtOH (20 mL) and air dried to give 264 mg (55%). A portion portions KNO (191 mg, 1.89 mmol). The reaction was of this solid (150mg) was taken and dissolved in 20 mL 1N allowed to stir 1 h at 0° C. and then was allowed to come to NaOH with heating. The solution was treated with activated room temperature and stir overnight. The pale yellow reac charcoal and filtered through a pad of Celite. The resulting tion mixture was then poured into 50 mL ice-HO. The solution was carefully acidified with 2N HCl (pH=1). Pale product was isolated by vacuum filtration as a white solid yellow needles slowly formed in the solution and were 10 which was rinsed with a small amount of cold HO and air collected by vacuum filtration, rinsed with 20 mL of HO) dried. The white solid was dissolved in a minimum amount and dried under vacuum (0.1 torr, 78° C) to yield 50.0 mg of hot DMSO. Boiling HO was added dropwise, with of powdery white solid, mp 356-358 C. (dec). H NMR heating after each addition, until the precipitate could not be (d-DMSO) 87.21 (d, 1H, J= 2.1, H-8), 7.53 (d. 1H, J=2.1, dissolved. A few drops of DMSO were added until the H-6), 11.1 (brs, 1H, NH), 12.1 (brs, 1H, NH). EIMS m/z 15 solution was clear, and the solution was allowed to cool 322 (M-4, 51.3),320 (M+2, bp),318 (M+, 53.9), 294 (32.2), slowly to room temperature. The white solid was isolated by 292 (62.6), 290 (28.7), 185 (24.3), 183 (25.2). EIHRMS vacuum filtration and allowed to air dry. The solid was calc. for CHBrNO 317.8641, found 317.8642. further dried under vacuum (0.1 torr, 25° C) to yield 241.6 mg of a mixture of the 6-nitro and 8-nitro products in a 3.3:1 Example 9 20 ratio by H NMR. The precipitate which formed in the Preparation of 5,6,7,8-Tetrachloro-1,4-dihydro-2,3 -qui filtrate above was isolated by vacuum filtration, rinsed with noxalinedione 50 mLHO and dried as above to yield a whim powder (80.7 1,2-Diamino-3,4,5,6-tetrachlorobenzene was prepared mg) which was a mixture of the 6-nitro and the 8-nitro using an adaptation of the method of Bellamy et al. (Bel products in a 12.6:1 ratio. Combination of the above samples lamy, F. D. et al., Tetrahedron Lett. 25:839 (1984)). A 25 resulted in a 55% yield, correcting for unreacted starting mixture of 1,2-dinitro-3,4,5,6-tetrachlorobenzene (1.00 g, material. A portion of the 3.3:1 mixture was taken and 3.27 mmol) and SnCl2.H2O (3.69 g, 16.4 mmol) dissolved recrystallized from DMSO:HO as described above, to give in 10 mL ethyl acetate and 5 mL absolute ethanol under N small, needle-like crystals which were isolated by vacuum was heated at 80° C. for 1 h. The reaction was allowed to filtration to yield 21.1 mg of a mixture of the 6-nitro and cool to room temperature and poured into 20 mL crushed 30 8-nitro products in a 1:1.76 ratio. Ratios of product mixtures ice. Sufficient sat'd NaHCO solution was adding (foam were determined by H NMR, observing the integration of ing) to bring the pH to 6. The thick white emulsion was the aromatic hydrogens at 67.45 and 7.84 Substitution extracted with 3x25 mL ethyl acetate and the combined positions are tentative and are based on relative chemical organic extracts washed with sat'd NaCl solution. The shifts of the aromatic hydrogens. H NMR (d-DMSO) organic phase was dried (MgSO), vacuum filtered and the 35 87.45 (s, 8-H, 6-nitro product), (s, 6-H, 8-nitro product), solvent rotary evaporated to yield a brown solid 569 mg 12.18 (s, N-H), 12.41 (s, N-H). EIMS m/z. 311 (M--2,35), (71%). "H NMR (CDC) 83.96 (brs, 201H)). CNMR 309 (M+, bp), 251 (60), 235 (95). EIHRMS calc. for (CDC1,) 5118.2, 127.0, 132.0. CHCIFNO:308.9733, found 308.9768. 5,6,7,8-Tetrachloro-1,4-dihydro-2,3-quinoxalinedione (Bar The major isomer, 5-chloro-6-nitro-7-trifluoromethyl-1, ton, D. E.; Lamb, A. J.; Lane, D. L. J.; Newbold, G. T.; 4-dihydro-2,3-quinoxalinedione, was isolated in pure form Percival, D., J. Chem. Soc. (C), 1268 (1968). in 64% yield by crystallization from DMSO-water. Mp 5,6,7,8-Tetrachloro-1,4-dihydro-2,3-quinoxalinedione 343-347°C. The structure was confirmed by X-ray analy was prepared using an adaptation of the method of Cheese SS man. (Cheeseman, G. W. H. J. Chem. Soc. 1171 (1962)). A Method B mixture of diethyl oxalate (2.97 g, 20.0 mmol) and 1,2- 45 To a solution of 661 mg (2.5 mmol) of 5-chloro-7- diamino-3,4,5,6-tetrachlorobenzene (500 mg, 2.03 mmol) trifluoro-1,4-dihydroquinoxaline-2,3-dione in conc. HSO was heated to reflux under N. for 6 h. The reaction was (15 mL) was added portionwise KNO (380 mg, 3.75 mmol) allowed to cool to room temperature and the orange solid at 0°C. The resulting solution was stirred at room tempera collected by vacuum filtration and rinsed with cold EtOH ture for 22 h, then poured into ice-water (150 mL), and the (10 mL) and air dried. This solid was recrystallized from 50 precipitate was collected by vacuum filtration and washed abs. ethanol to give 97.0 mg (16%) of an orange solid mp with water (6x15 mL). The wet solid was dissolved in 0.5N 326-328° C. (dec). (Lit., >360°). FT-IR: 3198, 3135 aq NaOH (100 mL), then to the clear yellow basic solution cm (N-H), 1750, 1623 cm (C=O). H NMR 8 (d. was added 2N aq. HCl slowly to take the pH to 8.0 (pH DMSO) 811.7 (brs, 2H, N-H) EIMS m/z 306 (M+8, 1.7), meter). The precipitate was collected by vacuum filtration 55 and the filtrate was saved. The wet solid was suspended in 304 (M+6, 11.5), 302 (M-4, 474), 300 (M+2,939), 288 water (about 50 mL), then the pH was taken to 4 by the (M+, 76.7), 274 (50.0), 272 (bp), 270 (79.0), 209 (61.0), 207 addition of 2Naq HCl. The solid was collected, washed with (64.0). EIHRMS calc. for CHCINO, 297.8870, found water and dried to give 50 mg (14%) of 5-chloro-6 -nitro 297.8864. 1,4-dihydro-2,3-quinoxalinedione as a yellow powder, Mp 305° C. (dec.). IR (KBr) 3492, 1720, 1556, 1337, 1144. Example 10 cm'. 'HNMR (DMSO-d) 12.431 (bs, 1H), 12.028 (s, 1H), Preparation of 5-Chloro-6-nitro-7-trifluoromethyl-1,4-dihy 7.895 (s, 1H). The filtrate (pH=8) obtained above was droquinoxaline-2,3-dione and 5-Chloro-8-nitro-7 -trifluo acidified with 2N aq. HCl to pH 4.5. The precipitate was romethyl-1,4-dihydroquinoxaline-2,3-dione collected by filtration, washed with water and dried to give Method A 65 535 mg (70%) of 5-chloro- 8-nitro-1,4-dihydro-2,3-qui The title compounds were prepared using an adaptation of noxalinedione as a yellowish powder. Mp 345 C. (dec). IR the method of Cheeseman. (Cheeseman, G. W. H. J. Chem. (KBr) 3442,3243, 1732, 1716, 1563, 5,514,680 61 62 1390, 1357, 1178, 1158 cm. H NMR (DMSO-d) 12.429 with cold HO and dried in a vacuum desiccator (0.5 torr, (s, 1H), 12.202 (s, 1H), 7.479 (s, 1H). 25° C) over CaSO, to yield 275 mg (67%) of tan crystals. "H NMR (CDC) 83.42 (brs, 2H, NH,), 3.86 (brs, 2H, Example 11 NH), 6.70 (d, J=8, 1H, ArH), 6.96 (d, J=1, 1H, ArH), 7.06 (dd, J-8, J=1, 1H, ArH). Preparation of 6-Fluoro-1,4-dihydro-2,3-quinoxalinedione 6-Cyano-1,4-dihydro-2,3-quinoxalinedione 4-Fluoro-1,2-diaminobenzene The title compound was prepared using an adaptation of 4-Fluoro-1,2-diaminobenzene was prepared using an the method of Cheeseman. (Cheeseman, G. W. H. J. Chem. adaptation of the method of Tsuji et al. (Tsuji, Y. et al., J. Soc. 1171 (1962)). A mixture of diethyl oxalate (3.90g, 27.6 Org. Chem. 55:580 (1990)). A suspension of Zn powder 10 mmol) and 3,4-diaminobenzonitrile (275 mg, 2.06 mmol) (10.5g, 0.160 mol), CaCl (1.05 g) and HO in 40 ml EtOH was heated to reflux under N for 2 h. The reaction was was heated to reflux with stirring under N. To this was allowed to cool to room temperature and the solid collected added slowly dropwise a solution of 4-fluoro-2-nitroaniline by vacuum filtration and rinsed with EtOH. The yellow (2.00 g, 12.8 mmol) in 10 mL EtOH. The reaction mixture brown solid was air dried to yield 156.6 mg (40.8%) which was refluxed 8 h. TLC analysis (silica gel, 2:1 benzene:E- 15 was >98% pure by "H NMR. An analytically pure sample tOAc) indicated complete disappearance of the starting was prepared by recrystallization of 100 mg in 10 mL. glacial material. The Zn was removed by vacuum filtration, and the acetic acid to give 21.2 mg of fine white crystals. "H NMR solvent rotary evaporated. The residue was dissolved in 50 (d-DMSO) 87.20 (d, J=8.1, 1H, ArH), 7.39 (d, J=1.2, 1H, mL EtO and the solution extracted with 3x25 mL 1N HCl. ArH), 7.50 (dd, J=1.2, J-8.4, 1H, ArH), 12.09 (s, 1H, NH), The aqueous layers were combined and basified with 50% 20 12.22 (s, 1H, NH). EIMS m/z 187(87, M+), 159 (100, bp), aq. NaOH (6 g) and the resulting solution extracted with 131(83), 104 (77), 77 (65), 53 (43), 28 (30). EIHRMS calc. 3x25 mL EtO. The EtO layers were combined and dried (MgSO). The solvent was rotary evaporated to yield 1.36 g for CHN.O. 187.0381, found 187.0377. (84%) of a brown solid. mp 9092 C. "H NMR (CDC1) Example 13 83.18 (brs, 2H, NH); 3.58 (brs, 2H, NH); 6.44 (m, 2H, 25 Preparation of 6-Trifluoromethyl-1,4-dihydroquinoxaline-2, ArH), 6.61 (m, 1H, ArH). 3-dione 6-Fluoro-1,4-dihydro-2,3-quinoxalinedione 1,2-Diamino-4-benzotrifluoride The title compound (Sarges, R. et al., J. Med Chem. 1,2-Diamino-4-benzotrifluoride was prepared using an 33:2240 (1990)) was prepared using an adaptation of the adaptation of the method of Tsujietal. (Tsuji, Y. etal., J. Org. method of Cheeseman. (Cheeseman, G. W. H. J. Chem. Soc. 30 Chem. 55:580 (1990)). Zn powder (3.93 g, 60.1 mmol), 1171 (1962)). A mixture of diethyl oxalate (17.4 g, 0.100 CaCl2 (393 mg), H2O (4.65 mL) and 14.1 mL EtOH were mol) and 4-fluoro-1,2-diaminobenzene (1.00 g, 7.93 mmol) combined and brought to reflux as described for 4-fluoro was heated to reflux with stirring under N for 2 h. The 1,2-diaminobenzene (see Example 10) and to this mixture reaction was cooled to room temperature and the solid was was added slowly dropwise a solution of 4-amino-3-ni collected by vacuum filtration and rinsed with EtOH (50 35 trobenzotrifluoride in 5 mL EtOH. Analysis and workup mL) to give a gray-brown solid which was further dried were described for 4-fluoro-1,2-diaminobenzene (see under vacuum (0.1 torr, 25° C) to yield 1.06 g (74.4%) Example 10) except that the reaction residue was dissolved which was >98% pure by NMR. An analytically pure sample in 30 mL 1NHCl. This solution was then washed with 3x35 was prepared by dissolution of 60 mg of the solid in 1.0N mL EtO. The aqueous layer was then basified with 50% NaOH and treatment of this solution with activated charcoal. NaOH and the resulting solution extracted with 3x25 mL Filtration of this mixture through a pad of Celite and EtO. The organic layers were combined and dried (MgSO) acidification with 1N HCl gave fine white needles which and the solvent evaporated at reduced pressure to yield 641 were collected by vacuum filtration, rinsed with HO and mg (75.9%) of a dark brown solid. "H NMR (CDC) 83.54 further dried under vacuum (0.1 torr, 25° C) to give 25.5 mg (brs, 4H, NH), 6.73 (m, 1H, ArH), 6.93 (m, 2H, ArH). of white needles, mp. 375°-380° C. (dec) (lit. >300° C. 45 6-Trifluoromethyl-1,4-dihydro-2,3-quinoxalinedione (Sarges, R., et al., J. Med. Chem. 33:2240 (1990)). H NMR The title compound was prepared using an adaptation of (d-DMSO) 86.90 (m, 2H, ArH), 7.09 (dd, J=9, J=5.4), 11.9 the method of Cheeseman. (Cheeseman, G. W. H. J. Chem. (s, 1H, NH), 11.96 (s, 1H, NH). EIMS m/z180(100M+), Soc. 1171 (1962)). A mixture of diethyl oxalate (3.72g, 25.5 152(44), 124(63), 97(43), 28(53). EIHRMS calc. for mmol) and 1,2-diamino-4-benzotrifluoride (300 mg, 1.70 CHFNN.O., 180,0334, found 180,0337. 50 mmol) was heated to reflux under N for 2 h. The reaction was cooled to room temperature and the solid collected by Example 12 vacuum filtration. This yellow brown solid was rinsed with Preparation of 6-Cyano-1,4-dihydroquinoxaline-2,3-dione hexanes and air dried. Further drying under vacuum (0.1 3,4-Diaminobenzonitrile torr, 25° C) yielded 240.1 mg (61.4%) that was >95% pure 3,4-Diaminobenzonitrile was prepared using an adapta 55 by "H NMR. An analytical sample was obtained by recrys tion of the method of Tsuji et al. (Tsuji, Y. et al., J. Org. tallization from acetone-ether to give a yellow-white solid. Chem, 55:580 (1990)). Zn powder (2.51 g, 38.3 mmol), H NMR (d-DMSO) 8744 (t, 2H, ArH), 7.56 (s, 1H, ArH), CaCl (251 mg), HO (3.0 mL) and 9.0 mL EtOH were 10.98 (brs, 1H NH), 11.08 (brs, 1H, NH). EIMS m/z 230 combined and brought to reflux as described for 4-fluoro-1,2 (100, bp, M-), 202(46). -diaminobenzene (Example 10) and to this mixture was 60 Example 14 added slowly dropwise a solution of 4-amino-3-nitroben zonitrile (500 mg, 3.06 mmol) in 20 mLEtOH. Analysis and Preparation of 6,7-Difluoro-1,4-dihydro-2,3-quinoxalinedi workup were described for 3-fluoro-1,2-diaminobenzene OC except that the reaction residue was dissolved in 20 mL 1N Method A HCl. This solution was then made basic by the addition of 65 4,5-Difluoro-1,2-diaminobenzene 20 mL 1.5M NaOH. A precipitate separated as fine tan 4,5-Difluoro-1,2-diaminobenzene was prepared using an needles which were collected by vacuum filtration, rinsed adaptation of the method of Tsuji et al., (Tsuji, Y. et al., J. 5,514,680 63 64 Org. Chem. 55:580 (1990)). Zn powder (942 mg, 14.4 matography (basic alumina, benzene). Purification of this mmol), CaCl2 (94.4 mg), HO (1.0 mL) and 4.0 mL EtOH sample was achieved by column chromatography (basic were combined and brought to reflux as described for alumina, activity II) on a 1.5"X20" column. The first band 4-fluoro-1,2-diaminobenzene (see Example 11) and to this was collected and concentrated to yield a yellow solid 1.88 mixture was added slowly dropwise a solution of 4,5- g(63.0%), mp 44.5-45 (lit. 42.5°-43.5° C). 'FNMR (CF difluoro-2-nitroaniline (200 mg, 1.15 mmol) in 2 mL EtOH. external standard, 6-162.9) 8-149.9(m), -1.56.6(m), Analysis and workup were as described for 4-fluoro-1,2 -162.0 (m), -178.3(m). -diaminobenzene (Example 11) except that the reaction was 3,4,5,6-Tetrafluoro-1,2-diaminobenzene dissolved in 5 mLHO and the solution extracted with 3x10 3,4,5,6-Tetrafluoro-1,2-diaminobenzene was prepared mL EtO. The organic layers were combined and treated O using an adaptation of the method of Tsuji et al., J. Org. with activated charcoal, dried (MgSO) and filtered through Chem. 55:580 (1990). A suspension of Zn powder (1.95 g, a pad of Celite. The solvent was evaporated at reduced 29.8 mmol), CaCl (195 mg) and HO (2.3 mL) in 7 mL pressure to yield 111.5 mg (67.3%) of a brown crystalline EtOH was heated to reflux with stirring under N. To this solid. "H NMR (CDCl) 83.34 (brs, 4H, NH,), 6.53 (t, 2H, was added slowly dropwise a solution of 2-nitro-3,4,5,6- ArH). 15 tetrafluoroaniline (2 g, 12.8 mmol) in 5 mL EtOH. The 6,7-Difluoro-1,4-dihydro-2,3-quinoxalinedione reaction mixture was refluxed 5 h. TLC analysis (silica gel, The title compound (Sarges, R. et al., J. Med. Chem. 2:1 benzene:EtOAc) indicated complete disappearance of 33:2240 (1990)) was prepared using an adaptation of the the starting material. The Zn was removed by vacuum method of Cheeseman. (Cheeseman, G. W. H. J. Chem. Soc. filtration, and the Zn rinsed with EtOH (30 mL). The solvent 1171 (1962)). A mixture of diethyl oxalate (1.11 g, 7.63 20 was rotary evaporated and the residue was dissolved in 20 mmol and 4,5-difluoro-1,2-diaminobenzene (110 mg, 0.763 mL EtO and the solution washed with 2x15 mL HO and mmol) was heated to reflux under N, for 2 h. The reaction 15 mL sat'd NaCl. The organic layer was dried (MgSO) and was allowed to cool to room temperature and the solid vacuum filtered. The solvent was rotary evaporated to yield collected by vacuum filtration and rinsed with hexanes and 396.6 mg (92.5%) of a purple brown solid, mp 120-125 air dried. This gray brown solid was recrystallized from 20 25 C., that was used without further purification. ml of EtOH and the brown-white crystals collected by 5,6,7,8-Tetrafluoro-1,4-dihydro-2,3-quinoxalinedione vacuum filtration and the crystals further dried under The title compound (Allison, et al., J. Fluorine Chem. vacuum (0.5 torr, 25°C) to yield 45.3 mg (30.0%) mp>360° 1:59 (1971)) was prepared using an adaptation of the method C. (lit.>310° C). H NMR (d-acetone) 87.19 (t, 2H, ArH, of Cheeseman (Cheeseman, G. W. H., J. Chem. Soc. 1171 J=9.3), 10.9 (brs, 2H, NH). 30 (1962)). A mixture of diethyl oxalate (2.86 mL, 4.1 mmol) Method B and 3,4,5,6-tetrafluoro-1,2-diaminobenzene (380 mg, 2.11 To a solution of 2.0 g (11.5 mmol) 4,5-difluoro-2-nitroa mmol) was heated to reflux with stirring under N for 8 h. niline (Aldrich, used as received) in EtOH (20 mL) was The reaction was cooled to room temperature and a small added 100 mg of 10% Pd/C. The suspension was shaken amount of purple precipitate was observed. The excess under H. (40-20 psi) for 3 h. The catalyst was removed by 35 diethyl oxalate was evaporated and the resulting solid was filtration and washed with EtOH (2x15 mL). The EtOH suspended in 20 ml hexanes, vacuum filtered and the solid solution was rota-evaporated to dryness. To the residual rinsed with hexanes (20 mL) and ethyl acetate (10 mL). The black solid was added oxalic acid dihydrate (1.74 g, 13.8 solid was airdried to yield 244.8 mg (49.9%). mp330-331 mmol) and 2N aq. HCl (18 mL). The mixture was heated at C. (lit. ca. 300° C. dec.). H NMR (d-DMSO)812.33 (brs, 125°C. with stirring for 3 h, then cooled to 25°C. The black NH). 'F NMR (CF external standard, 8-1629), precipitate was collected by vacuum filtration, and washed 8-157.7(m), -167.9(m). with water (5x5mL). The wet product was dissolved in 0.2N aq NaOH (100 mL) with stirring, then filtered. The clear Example 16 slightly orange-yellow filtrate was acidified by the addition of 2Naq HCl with stirring to pH4. The off-white precipitate 45 Preparation of 5-Bromo-7-fluoro-1,4-dihydroquinoxaline-2, was collected by vacuum filtration, washed with water and 3-dione dried under 1 mmHg at 40° C. to give 1.83 g (89%) of the 6-Bromo-4-fluoro-2-nitroaniline title compound as a cream colored powder. Mp2360° C. IR 6-Bromo-4-fluoro-2-nitroaniline was prepared using an (KBr) 3454, 3120, 1708, 1530, 1400, 1298 cm. H NMR adaptation of the method of Mitchell et al., J. Org. Chem. (DMSO-d) 11.939 (s, 2H), 7.054 (m, 2H). 50 44:4733 (1979). To a solution of 4-fluoro-2-nitroaniline (500 mg, 3.2 mmol) in dry DMF (16 mL) under N, was added Example 15 dropwise a solution of N-bromosuccinimide (570 mg, 3.2 mmol) in dry DMF (16 mL). The reaction was allowed to stir Preparation of 5,6,7,8-Tetrafluoro-1,4-dihydroquinoxaline 24 h. The solution was then poured into 100 mL HO and 2,3-dione 55 this aqueous phase extracted with 4x25 mL CHCl2. The 2-Nitro-3,4,5,6-tetrafluoroaniline combined organic phases were washed with 3x4 mL HO 2-Nitro-3,4,5,6-tetrafluoroaniline was prepared using an and dried (MgSO). The MgSO was vacuum filtered and adaptation of the method of Brooke et al., J. Chem. Soc. 802 the solvent rotary evaporated to yield a brown oil which (1961). Ammonia gas was bubbled through a solution of crystallized on standing. 642 mg (85.4%). "HNMR (CDCl) pentafluoronitrobenzene (3.00 g, 114.1 mmol) in 200 mL 86.51 (brs, 2H NH), 7.58 (dd, J =3 Hz, J-6.5 Hz, anhydrous diethyl ether for 4 h. During this time the color H-4), 7.92 (dd, J-3 Hz, J-8.7 Hz, H-3). changed from clear white to a deep yellow and a white 1,2-Diamino-3-bromo-5-fluorobenzene precipitate formed. The precipitate (ammonium fluoride) 1,2-Diamino-3-bromo-5-fluorobenzene was prepared was separated by vacuum filtration and washed with ether using an adaptation of the method of Bellamy et al., Tetra (30 mL). The filtrate was rotary evaporated and the resulting 65 hedron Lett. 25:839 (1984). A mixture of 6-bromo-4-fluoro orange crystals gave 3 spots on TLC (alumina, benzene). 2-nitroaniline (673 mg, 2.87 mmol) and SnCl2.H2O (3.23 Purification of this sample was achieved by column chro g, 14.3 mmol) dissolved in 6 mL ethyl acetate and 3 mL 5,514,680 65 66 absolute ethanol was heated at 70° C. for 30 min. All the 1H), 11.933 (s, 1H), 7.103 (d, 1H, J=6.6 Hz), 7.082 (d, 1H, starting material had reacted as evidenced by TLC (silica J=9.6 Hz). gel, 2:1 hexanes:ethyl acetate). The reaction was allowed to cool to room temperature and poured into 20 mL crushed Example 18 ice. Sufficient solid NaHCO was added to bring the pH to 7.5. The reaction mixture was then extracted with 3X20 mL Preparation of 5-Bromo-7-trifluoromethyl-1,4-dihydro-2,3- ethyl acetate and the combined organic phases washed with quinoxalinedione sat'd NaCl solution. The organic phase was dried (MgSO), The title compound was prepared using an adaptation of vacuum filtered and the solvent rotary evaporated to yield a the method of Cheeseman, G. W. H., J. Chem. Soc. 1171 dark brown liquid 510 mg (86.7%). "HNMR (CDC) 83.64 10 (1962). A mixture of diethyl oxalate (1.15g, 7.91 mmol) and (brs, 4H, 2ONH)), 6.44 (dd, 1H, H5, J-2.7, J =9.8), 1,2-diamino-3-bromo-5-trifluoromethylbenzene (200 mg, 6.72 (did, 1H, H4, J = 2.7, J-8.4). 0.95 mmol) was heated to reflux under N for 2 h. The 5-Bromo-7-fluoro-1,4-dihydro-2,3-quinoxalinedione reaction was allowed to cool to room temperature and the The title compound was prepared using an adaptation of solid collected by vacuum filtration and rinsed with EtOH the method of Cheeseman (Cheeseman, G. W. H., J. Chem. 15 (15 mL). This white solid was dried in a drying pistol (0.05 Soc. 1171 (1962)). A mixture of diethyl oxalate (3.31 mL, torr, 78° C) to yield 148.3 mg (60.7%). mp301-304 (dec). 24.4 mmol) and 1,2-diamino-3-bromo-5 -fluorobenzene H NMR (d-DMSO) 87.28 (s, 1H, ArH), 7.56 (s, 1H, ArH), (500 mg, 2.44 mmol) was heated to reflux under N for 5 h. 11.7 (bris, 2H, NH). 'F NMR (CF external standard, The reaction mixture was dark brown. The reaction was 8-1629) 8-57.97 (s). EIHRMS calc. for CHBrFNO, allowed to cool to room temperature and the solid collected 20 307.9408, found 307.9411. by vacuum filtration and rinsed with EtOH (30 mL). The brown solid was air dried for 1 hr to give 250 mg (39.6% Example 19 crude yield). A portion of this solid was removed and Preparation of 6-Fluoro-7-nitro-1,4-dihydro-2,3-quinoxa dissolved in 1N NaOH (10 mL) and a few insoluble particles linedione were removed by gravity filtration. The solution was treated 25 The title compound was prepared using an adaptation of with decolorizing carbon and the mixture was filtered the method of Cheeseman (Cheeseman, G. W. H., J. Chem. through a pad of celite and the resulting solution acidified Soc. 1171 (1962)). 6-Fluoro-1,4-dihydroquinoxaline-2,3- with 1N HCl. White crystals formed that were isolated by dione (200 mg, 1.10 mmol) was dissolved in 3 mL of vacuum filtration and washed with water (20 mL). The concentrated H2SO and the bluegreen solution was cooled crystals were dried in a drying pistol (0.05"torr, 78° C) to 30 to 0°C. with stirring. To this was added in small portions yield 54.1 mg, mp 308-310° C. (dec). "H NMR (d KNO (110 mg, 1.10 mmol). The reaction was allowed to DMSO) 86.2 (dd, 1H, J-2.7, J =9.3, ArH), 7.35 (dd, stir 1 h at 0° C. and then was allowed to come to room 1H, J-24, J-8.4 Hz), 11.1 (brs, 1H, NH), 12.1 (brs, temperature and stir overnight. The brown-orange reaction 1H, NH). EIHRMS calc. for CHBrFNO: 257.9440, mixture was then poured into 10 mL ice/HO. The product found 257.9455. 35 was isolated by vacuum filtration as brown crystals which were rinsed with a small amount of cold H2O and air dried. Example 17 The crystals were further dried under vacuum (0.1 torr, 25 C.) to yield 173.4 mg (70.0% yield). Preparation of 6-chloro-7-fluoro-1,4-dihydroquinoxaline-2, An analytical sample was prepared by dissolution of 75 3-dione 40 mg of the crude product in 5 mL of 1NNaOH and treatment Method A of this solution with activated charcoal. This suspension was The title compound was prepared using an adaptation of vacuum filtered through a pad of Celite and the solution the method of Cheeseman, G. W. H., J. Chem. Soc. 1171 carefully acidified with concentrated HCl to give yellow (1962). A mixture of diethyl oxalate (1.83 g, 12.5 mmol) and crystals as the precipitate. The crystals were isolated by 1,2-diamino-4-chloro-5-fluorobenzene (200 mg, 1.25 mmol) 45 vacuum filtration and dried as described above to give 38.9 was heated to reflux under N for 2 h. The reaction was of bright yellow crystals. mp 348-350 (dec.) "H NMR allowed to cool to room temperature and the solid collected (d-DMSO) 812.4 (s, 1H, NH), 12.1 (s, 1H, NH), 7.8 (d, Jr. by vacuum filtration and rinsed with EtOH. This solid was meta=7.2, 1H, ArH), 7.0 (d, Jel2, 1H, ArH). EMS dissolved in 1NNaOH (20 mL) and the solution treated with m/z 225 (100, M, 167 (10), 45 (41), 28 (96). EIHRMS calc. decolorizing carbon. This mixture was filtered through a pad 50 of celite and the resulting pale orange solution acidified with for CHFNO, 225.0185, found 225.0196. 4N HCl. A precipitate formed that was isolated by vacuum Example 20 filtration and washed with water. This tan solid was dried in a drying pistol (0.05 torr, 78° C) to yield 121.7 mg (45.5%). Preparation of 6-Trifluoromethyl-7-nitro-1,4-dihydro-2,3- mp3449-348° C. (dec) H NMR (d-DMSO) 66.93 (d, 1H, 55 quinoxalinedione J–10.2 Hz, H-8), 7.08 (d, 1H, J-7.2 Hz, H-4). 'F The title compound was prepared using an adaptation of NMR (CF external standard, 8-162.9) 8-124.7 (s). the method of Cheeseman (Cheeseman, G. W. H., J. Chem. EIHRMS calc. for CHCIFNO 213.9945, found Soc. 1171 (1962)). 6-Trifluoromethyl-1,4-dihydro-2,3-qui 213.9961. noxalinedione (200 mg, 0.869 mmol) was dissolved in 8 mL Method B 60 of concentrated HSO and the yellow-green solution was A mixture of 1,2-diamino-4-chloro-5-fluorobenzene (3.78 cooled to 0°C. with stirring. To this was added in small g, 23.54 mmol), oxalic acid dihydrate (3.65g, 28.24 mmol) portions KNO (87.8 mg, 1.10 mmol). The reaction was and 2Naq HCl (38 mL) was heated at 125° C. with stirring allowed to stir 1 h at 0° C. and then was allowed to come to for 1 h. The precipitate was collected by vacuum filtration, room temperature and stir overnight. The brown-orange washed with water and dried at 40 C. under 1 mmHg for 12 65 reaction mixture was then poured into 10 mL ice H.O.The h, affording 4.68 g (93%) of the title compound as a cream product was isolated by vacuum filtration as pale yellow powder. Mp2360° C. (dec). H NMR (DMSO-d) 12.011 (s, crystals which were rinsed with a small amount of cold H2O 5,514,680 67 68 and air dried. The crystals were further dried under vacuum mmol) in 0.5 mL pyridine at 0° C. was added in small (0.1 torr, 25° C) to yield 121.1 mg (50.6% yield). H NMR portions 6-chlorosulfonyl-1,4-dihydro-2,3-quinoxalinedi (d-DMSO) 87.53 (s, 1H, ArH), 7.80 (s, 1H, ArH), 12.4 (s, one (100 mg, 0.385 mmol). The solution was allowed to 2H, NH). EIMS m/z 275 (81, M+), 217 (36), 201 (100, bp), warm to room temperature and stir 8 h under N. The EIHRMS calc. for CHN.O.F, 275.0153, found 275,0170. reaction mixture was poured into a mixture of 5 mL 1:1 HO:conc. HCl and 3 g ice. Crystals began to form after 1 Example 21 h and the solution was allowed to stand overnight. The crystals were isolated by vacuum filtration as pale yellow Preparation of 6-Sulfonyl-1,4-dihydro-2,3-quinoxalinedi needles, rinsed with cool H2O and further dried under One 10 vacuum (0.1 torr, 25°C.) to yield 47.5 mg (44.1%). HNMR 6-Chlorosulfonyl-1,4-dihydro-2,3-quinoxalinedione was (d-DMSO)80.74 (t,3H, CH,), 1.32 (m, 2H, CH), 2.62 (m, prepared using an adaptation of the method of Keana et al. 2H, CH), 7.20 (d, J-84, 1H, H.), 742 (d, J-74, 1H, H.), (J. Org. Chem. 48:3654 (1983)). To 1,4-dihydro-2,3-qui 7.50 (m,2H, NH, H), 12.07 (s, 1H, NH), 12.14 (s, 1H, NH). noxalinedione (1.0 g, 6.2 mmol) was added all at once 3.0 mL of chlorosulfonic acid. The mixture was stirred under N 15 Example 24 at 60° C. for 2 h, allowed to come to room temperature and the solution added slowly dropwise to 25 mL of crushed ice. Preparation of 6-(N,N-Dimethylsulfonyl)-1,4-dihydro-2,3- The resulting solid was vacuum filtered and rinsed with quinoxalinedione ice/HO. The white solid was further dried at 0.5 torr (25 The title compound was prepared using an adaptation of C.) over CaSO to yield 1.1 g (68%). H NMR (d-DMSO) 20 the method of Adams et al. (J. Am. Chen. Soc. 73:1147 57.07 (d, J =8.1, 1H, Hs), 7.29 (dd, J =8.1, J-1.0, 1H, (1951)). To a mixture of dimethylamine (40% solution, 52.2 H), 7.40 (d, J =1.0, 1H, H). EMS m/z 262 (M+2, 15), mg, 131 L, 1.16 mmol) and 0.5 mL pyridine at 0°C. was 260 (M+, 40), 225 (35), 105 (43), 36 (100, bp). EIHRMS added in small portions 6-chlorosulfonyl-1,4-dihydro-2,3- calc. for CHCINOS 259.9687, found 259.9645. quinoxalinedione (100 mg, 0.385 mmol). The solution was 6-Sulfonyl-1,4-dihydro-2,3-quinoxalinedione allowed to warm to room temperature and stir 8 hunder N. 6-Sulfonyl-1,4-dihydro-2,3-quinoxalinedione was pre 25 The reaction mixture was poured into a mixture of 5 mL 1:1 pared using an adaptation of the method of Keana et al. (J. HO:conc HCl and 3 g ice. Crystals began to form after 1 h Org. Chem. 48:3654 (1983)). A suspension of 6-chlorosul and the solution was allowed to stand overnight. The crystals fonyl-1,4-dihydro-2,3-quinoxaline (142.7 mg, 0.546 mmol) were isolated by vacuum filtration as white needles, rinsed in 5.0 mLHO was stirred at 50° C. for 8 h. The solvent was 30 with cool HO and further dried under vacuum (0.1 torr, 25 removed at reduced pressure and the resulting solid further C) to yield 44.2 mg (42.6%). HNMR (d-DMSO)82.57 (s, dried under vacuum (0.5 torr, 50° C.) to give a powdery pale 6H, CH), 7.28 (d, J =8.4, 1H, Hs), 7.44 (dd, J-8.7, J-1.8, orange solid 121.6 mg (91.8%). "H NMR (DO) 7.2 (brm, 2HHH), 12.03 (s, 1H, NH), 12.22 (s, 1H, NH). EIMS m/z. 2H, ArH), 7.5 (brm, 1H, ArH). EMS m/z 242 (M+, 5), 162 269 (M+, 83), 225 (26), 161 (bp, 100), 106 (94). (37), 106 (100), 80 (89). 35 Example 25 Example 22 Preparation of N-methyl-6,7-dinitro-1,4-dihydro-2,3-qui The Preparation of 6-Sulfonamide-1,4-dihydro-2,3-qui noxalinedione noxalinedione N-Methyl-1,2-diaminobenzene To 6-chlorosulfonyl-1,4-dihydro-2,3-quinoxalinedione N-Methyl-1,2-diaminobenzene was prepared using an (200 mg, 0.770 mmol) was added all at once 2 mL conc. adaptation of the method of Tsuji et al. (J. Org. Chem. NHOH. The mixture was gently warmed on a steam bath 55:580 (1990)). Zn powder (8.07 g., 0.123 mol), CaCl (807 with occasional swirling. As the solution heated, a white mg), HO (9.9 mL) and 30 mL EtOH were combined and precipitate formed. The solution was heated for 20 min and brought to reflux as described for 4-fluoro-1,2-diaminoben cooled to room temperature and the mixture acidified with 45 Zene (see Example 10), and to this mixture was added slowly 1N HCl. The solid was collected by vacuum filtration and dropwise a solution of N-methyl-2-nitroaniline (1.50 g, 9.86 then rinsed with cold HO. The solid was further dried under mmol) in 15 mL EtOH. Analysis and workup were as vacuum (0.5 torr, 25° C) to yield 98.2 mg (53.0%) that was described for 4-fluoro-1,2-diaminobenzene except that the >95% pure by H NMR. An analytical sample was prepared reaction residue was dissolved in 50 ml EtO. This EtO by dissolution of 45 mg of the solid in 1 mL 1N NaOH 50 solution was then extracted with 3x25 mL 1N HCl and the followed by acidification of the solution with 1N HCl. The aqueous layers combined and basified with 5 g of 50% sulfonamide precipitated as pale yellow needles which were NaOH solution. This solution was then extracted with 3x25 isolated by vacuum filtration, rinsed with cool H2O and mL EtO. These EtO layers were then combined, dried dried under vacuum (0.5 torr, 25° C) to yield 28.9 mg. "H (MgSO) and the solvent evaporated at reduced pressure to NMR (d-DMSO) 7.20 (d, J =8.4, 1H, Hs), 7.34 (s, 2H, 55 yield 911.1 mg (76.1%) of a dark brown oil. "H NMR NH), 7.50 (dd, J =1.8, J–8.4, 1H, H), 7.56 (d, J-7-1.8, (CDC1,) 82.87 (s, 3H, CH,), 3.31 (brs, 3H, NH), 6.67 (m, 3H, ArH), 6.86 (m, 1H, ArH). 1H, H), 12.14 (s, 1H, NH), 12.11 (s, 1H, NH). EIMS m/z N-Methyl-1,4-dihydro-2,3-quinoxalinedione 241 (M+, 83), 133 (63), 105 (bp, 100), 64 (75), 28 (80). N-Methyl-1,4-dihydro-2,3-quinoxalinedione was pre EIHRMS calc. for CHNOS 241.0156, found 241.0139. pared using an adaptation of the method of Cheeseman (J. Chem. Soc. 1171 (1962)). A mixture of diethyl oxalate (3.23 Example 23 g, 22.1 mmol) and N-methyl-1,2-diaminobenzene (332 mg, Preparation of 6-(N-Propylsulfonyl-1,4-dihydro-2,3-qui 2.72 mmol) was heated to reflux under N for 2 h. The noxalinedione reaction was allowed to cool to room temperature and the The title compound was prepared using an adaptation of 65 solid collected by vacuum filtration and rinsed with EtOH. the method of Adams et al. (J. Am. Chem. Soc. 73:1147 The gray brown solid was further dried under vacuum (0.1 (1951)). To a mixture of n-propylamine (50 mg, 0.846 torr, 25°C.) to give 274.3 mg (57.2%). A portion of this solid 5,514,680 69 70 was further purified by recrystallization from 50 mL EtOH temperature was >250 C. in order to minimize decompo and treatment with activated charcoal to give fluffy pale sition prior to melting. Column chromatography was per yellow crystals which were collected by vacuum filtration formed in the flash mode on Davisil silica gel (200-425 and rinsed with cold EtOH to yield 104.9 mg. "H NMR mesh), unless otherwise indicated. Analytical thin layer (d-DMSO) 83.50 (s, 3H, CH), 7.16 (m,3H, ArH), 7.36 (m, chromatography was performed on aluminum-backed silica 1H, ArH), 12.01 (s, 1H, NH). EIMS 176 (M+,52), 148 (33), gel 60 Fis plates and visualization was effected with an 119 (100, bp). ultraviolet lamp. H NMR spectra were recorded on a 300 N-Methyl-6,7-dinitro-1,4-dihydro-2,3-quinoxalinedione MHz. General Electric QE-300; chemical shifts are reported The title compound was prepared using an adaptation of in delta units referenced to residual proton signals of the the method of Cheeseman (J. Chem. Soc. 1171 (1962)). 10 deuterated solvents (CHCl, 67.26; CHDOD, 53.30; N-Methyl-1,4-dihydro-2,3-quinoxalinedione (200 mg, 1.13 CDSOCDH, 82.49; CDCOCDH, 62.04. CNMR spec mmol) was dissolved in 3 mL of concentrated HSO and tra were run at 75 MHz. Infrared spectra were obtained on the blue green solution was cooled to 0°C. with stirring. To a Nicolet 5DXB FT-IR spectrometer. Absorptions recorded this was added in small portions KNO (228 mg, 2.26 in wavenumbers (cm) and the intensity of the absorptions mmol). The dark orange solution was allowed to stir 1 h at 15 are indicated by the letters s (strong), m (medium), and 0° C. and then was allowed to come to room temperature and w(weak). Mass spectra were recorded on a VGZAB-2-HF stir overnight. The brown-orange reaction mixture was then mass spectrometer with a VG-11-250 data system, in the poured into 10 mL ice H.O. The product was isolated by electronionization mode (70 eV) unless otherwise indicated. vacuum filtration as a yellow white solid which was rinsed Microanalyses were performed by Desert Analytics of Tus with a 10 mL of cold HO and air dried. The crystals were 20 con, Ariz. further dried under vacuum (0.1 torr, 25°C.) to yield 220 mg (72.0%). An analytical sample was prepared by recrystalli Example 28 zation of 120 mg of the crude sample from glacial acetic Preparation of 1-amino-1,4-dihydro-2,3-quinoxalinedione acid. The resulting crystals were collected by vacuum fil The procedure of Wallace, R. G., Org. Prep. Proc. Int. tration and rinsed with HO and dried under vacuum (0.1 25 14:269 (1982) was adapted. To a stirred suspension of torr, 60° C) to yield 34.2 mg yellow crystals. "H NMR 1,4-dihydro-2,3-quinoxalinedione (162 mg, 1.00 mMol, (d-DMSO) 83.52 (s, 3H, CH), 7.80 (s, 1H, ArH), 8.10 (s, Aldrich) in distilled water (4 ml) at 25°C. was added NaOH 1H, ArH), 12.6 (s, 1H, NH). (100 mg, 2.5 mMol). After 5 min, the resulting solution was Example 26 treated portionwise over 10 min. with hydroxylamino-o- 30 sulphonic acid (113 mg, 1.00 mMol, Aldrich). Reaction was Preparation of 2,3-(4N)-1,4-dihydro-2,3-quinoxalinedione carried out at room temperature. A white precipitate came The title compound was prepared using an adaptation of out after 1 h, continually stirred another 1 h, it was collected the method of Cheeseman (J. Chem. Soc. 1171 (1962)). A to get 70 mg (40%) of crude 1-amino-1,4-dihydroquinoxa mixture of diethyl oxalate (6.22 mL, 45.5 mmol) and 1,2- line-2,3-dione (free base Na', ratio of starting material to diaminopyridine (500 mg, 4.58 mmol) was heated to reflux 35 product=5:95 by NMR, DO). The filtrate was acidified to with stirring under N for 2 h. The reaction was cooled to pH=2 by 1N HCl (-1.5 mL), giving a white precipitate 50 room temperature and the solid was collected by vacuum mg, which was a 1:1 mixture of 1,4-dihydro-2,3-quinoxa filtration and rinsed with EtOH (20 mL) to give a yellow linedione and the hydrochloride salt of 1-amino-2,3-qui white solid which was further dried under vacuum (0.1 torr, noxalinedione (by NMR, DMSO). Total yield of 1-amino 25° C) to yield 693 mg (93.0%). "H NMR (d-DMSO) 40 1,4-dihydroquinoxaline-2,3-dione is 51%. 87.11 (dd, 1H, H-7), 7.43 (dd, 1H, J=8.1 Hz, 1.2 Hz, H-8), A70 mg sample of the crude 1-amino-2,3-quinoxalinedi 8.05 (dd, 1H, J=5.1 Hz, J-1.2 Hz, H-6). one was dissolved into distilled water (5 mL) and then acidified to pH-5 with AcOH and then the solution was Example 27 45 allowed to stand at 25°C. one day, giving white needles. The Preparation of 2,3,6,8-Tetraketo-5,6-dimethylpteridine crystals were collected by filtration and then washed with The title compound was prepared using an adaptation of distilled water (2x2 mL) followed by ethanol (2x1 mL) the method of Cheeseman (J. Chem. Soc. 1171 (1962)). A giving 61 mg (34.5%) of pure 1-amino-2,3-quinoxalinedi mixture of diethyl oxalate (1.75 mL, 12.8 mmol) and 5,6- one (free base H) as a white needles; mp:226-8 C. diamino-1,3-dimethyluracil hydrate (200 mg, 1.18 mmol) 50 (sublime); 260-2 C. (dec.) (lit., Shin, S. C. and Lee, Y.Y. Taehan Hwahakhoe Chi 27(5):382-4 (1983), 228° C. sub was heated to reflux with stirring under N for 8 h. The lime). IR (KBr, cm):3306, 1687, 1631, 1587. NMR (H, reaction was cooled to room temperature and the solid was DMSO-d): 85.880 (s.2H); 7.143 (m, 3H); 7.601 (d, 1H); collected by vacuum filtration and rinsed with EtOH (10 12.061 (s, 1H). HRMS, calcd. for CHNO (M+) mL) to give a yellow solid which was air dried to yield 76.0 m/z:177.0537; found: 177.0536. mg (66.6%) further dried under vacuum (0.1 torr, 25 C.) to 55 yield 1.06 g (74.4%) which was >98% pure by NMR. A Example 29 portion of this solid (100 mg) was dissolved in 1.0N NaOH (5 mL). Acidification of this solution with 6N HCl gave a Preparation of 1-amino-6,7-dichloro-1,4-dihydro-2,3-qui bright yellow solid which was collected by vacuum filtra noxalinedione tion, rinsed with HO (10 mL) and dried in a drying pistol 60 The procedure of Wallace, R. G., Org. Prep. Proc. Int. (0.05 torr, 78° C) to give 42.4 mg. "H NMR (d-DMSO) 14:269 (1982) was adapted. To a stirred suspension of 83.20 (S, 6H, CH,), 11.59 (brs, 2H, NH). mp370-372 6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione (189 mg, (dec.), EIMS m/z 224 (100, M-), 196 (80). 0.82 mMol) in distilled water (15 mL) at 60° C. was added In the following Examples 28-44, reagents were used as NaOH (335 mg, 8.37 mMol). After 30 min. the resulting received unless otherwise indicated. Melting points were 65 solution was treated portionwise over 10 min. with hydroxy taken on a Mel-Temp melting point apparatus and are lamino-o-sulphonic acid (111 mg, 0.98 mMol, Aldrich). uncorrected. Samples were placed in the block when the Reaction was carried out at 60° C. A white precipitate came 5,514,680 71 72 out after 10 min. The mixture was stirred at 25 C. for 8 h, 1-(o-tolylamino)carbonyl)aminol-1,4-dihydro-2,3-qui it was collected by filtration at 50° C., affording 180 mg noxalinedione as a white powder (60%). Mp:decomposed (90%) of crude 1-amino-6,7-dichloro-1,4-dihydro-2,3-qui from 265° C. IR (KBr, cm): 3375; 3237; 1718; 1693; noxalinedione, as a white amorphous solid (ratio of starting 1675. NMR (H, DMSO-d) 82.161 (s, 3H); 6.996-7.380 material to product=10:90 by H, NMR, DO). Yield is 81%. (m, 8H); 8.530 (s, 1H), 9.343 (s, 1H); 12.155(s, 1H). A 109 mg sample of the crude 1-amino-6,7-dichloro-1,4- dihydro-2,3-quinoxalinedione (free base Na+, 0.445 mMol) Example 32 was suspended into 1N NaOH (10 mL) at 50 C. for 30 min. and collected by filtration. The precipitate (87 mg) was Preparation of 6,7-dibromo-1,4-dihydro-2,3-quinoxalinedi dissolved into hot distilled water (80 mL) and the insoluble 10 One material was removed by filtration. The filtrate was then 6,7-Dibromo-1,4-dihydroquinoxaline-2,3-dione (Method acidified with AcOH to pH-5. The resulting suspension was A): The procedure of Jorgenson, A. K. et al., International heated at 60'-0 C. to effect a clear solution and then it was Patent Application Publication No. WO91/13878 and Chem. cooled it slowly to 25°C., giving white needles. The crystals Abstr. 115(25):280059u (1991), was adapted. To a stirred were collected by filtration and then washed with distilled 15 suspension of 1,4-dihydro-2,3-quinoxalinedione (1.62 g, water (2x2 mL) followed by ethanol (2x1 mL), dried by 10.00 mMol, Aldrich) and AgSO (3.43 g, 11.00 mMol) in rotavapor at 60° C. for 4 h, giving 73 mg (84%) of pure concentrated HSO (10 mL) at RT was added bromine 1-amino-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione (3.52g, -1.13 mL, 22.00 mMol) over 30 min. The mixture (free base H). The mp was then measured. The color of was then stirred at room temperature for 24 h. Tetrachlo 1-amino-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione 20 romethane (10 mL) was then added and the reaction mixture changed to yellow at 335 C.; the decomposition of was stirred at 50 C. for 2 h. The insoluble material was 1-amino-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione is removed by filtration, the filtrate was poured into ice water obvious at 340 C. and it melted to a black liquid at 343 (200 mL), and the separated yellow solid was collected by 5° C. IR (KBr, cm):3337; 3225; 3056; 1706; 1668; 1581. filtration and dried in air. The crude 6,7-dibromo-1,4-dihy NMR (H, DMSO-d): 85.791(s, 2H); 7.271 (s, 1H); 7.721 25 droquinoxaline-2,3-dione was dissolved into 1N NaOH (20 (s, 1H); 12.115 (s, 1H). HRMS: calcd for CHNOCl, ml) and water (20 mL), and the yellow precipitate was (M) m/z:244.9756; found:244.9767. removed by filtration. The filtrate was acidified with 4NHCl to pH=2 to give a white precipitate, which was washed with Example 30 distilled water (2x2 mL) and ethanol (2x1 mL), affording 30 1.082 of 6,7-dibromo-1,4-dihydroquinoxaline-2,3-dione as Preparation of 1-Acetamido-1,4-dihydro-2,3-quinoxalinedi a gray fine powder (95% purity by NMR). Second crop from O yellow precipitate with above procedure, to give 927 mg of A mixture of 1-amino-1,4-dihydro-2,3-quinoxalinedione 6,7-Dibromo-1,4-dihydroquinoxaline-2,3-dione. Total yield (53 mg, 0.30 mMol) and pyridine (1 mL, fresh distilled from is 65%. Crystallization from DMSO/HO furnished white KOH before use) and acetic anhydride (1 mL, Aldrich) was 35 microcrystals (recovered 85%). Mp: decomposed from 335 stirred under nitrogen at 60° C. for 4 hrs. The reaction C. IR (KBr, cm): 3200; 1718; 1693. NMR (H, DMSO mixture became a clear solution. All of solvent and reagent d): 87.336 (s, 2H); 11.962 (s.2H). HRMS:calcd for were then evaporated under reduced pressure and washed CHNOBr. (M-) m/z:317.8639, found:317.8619. with benzene:cyclohexane=1:1(2x2 mL); ether (2x2 mL), (SHARON) dried at 60° C. with rotavapor for 2h, affording 57 mg (86%) 6,7-Dibromo-1,4-dihydroquinoxaline-2,3-dione (Method of pure 1-acetamido-1,4-dihydro-2,3-quinoxalinedione as a B): The procedure of Mitchell, R. H. et al., J. Org. Chem. white powder; mp:211-213° C. IR (KBr, cm): 3430; 44(25):4733 (1979) was adapted. To a stirred suspension of 3127; 1741; 1717; 1668. NMR (H, DMSO-d) 8:2.326 (s, 1,4-dihydro-2,3-quinoxalinedione (3.24 g, 20.00 mMol, 3H); 7.121-7.287 (n, 4H); 12.345 (s, 1H). Mass:calcd for Aldrich) in dried DMF (100 mL) was added N-bromosuc CHNO (M) m/z:219.0641; found: 219.0651. 45 cinimide (14.24 g, 80.00 mMol, Aldrich) and the mixture Example 31 was stirred at 25°C. for 0.5 h to give a light yellow solution. The reaction was carried out continually at 25°C. for 24h Preparation of 1-(o-Tolylamino)carbonyl)amino)-1,4-di to give a white precipitate, which was collected by filtration hydro- 2,3-quinoxalinedione and then washed with distilled water (2x1 mL) followed by The procedure of Leeson, P. D. et at., J. Med. Chem 50 95% ethanol (2x2 mL) to afford 2.216 g of pure 6,7- 35:1954-68 (1992) was adapted. A suspension of 1-amino dibromo-1,4-dihydro-2,3-quinoxalinedione (by NMR) as a 1,4-dihydro-2,3-quinoxalinedione (37 mg, 0.21 mMol) in white powder. The filtrate was poured into 200 ml ice water pyridine (2.5 mL) was stirred at 70° C. until dissolving was and the precipitate was collected by filtration, then washed completed. To the solution was then added o-tolyl isocyan with distilled water (2x2 mL) followed by 95% ethanol (2x2 ate (27.8 mg, 0.21 mMol, Aldrich), which was stirred at 60 55 mL) to afford 3.627 g of 6,7-dibromo-1,4-dihydro-2,3-qui C. for 2 h, then overnight at room temperature. The solvent noxalinedione (with 1% impurity by NMR) which was was evaporated under reduced pressure, the residue was dissolved into 1N NaOH (50 mL) and then acidified to pH=2 washed by ether (2x2 mL) to give 59 mg of crude 1-(o- with 4N HCl to give a white cream precipitate. The precipi tolylamino)carbonyl)amino)-1,4-dihydro-2,3-quinoxa tate was collected by filtration and washed with distilled linedione (66% product; 30% tolyl-byproduct; 4%. 1-amino water (2x2 mL) followed by 95% ethanol (2x2 mL) and 1,4-dihydro-2,3-quinoxalinedione by H NMR). Separated dried in the air at 50° C. for 8 h, giving 3.517 g (total yield by chromatograph with silica gel (2 g) column, eluted with 89%) of pure 6,7-dibromo-1,4-dihydro-2,3-quinoxalinedi 100% benzene (20 mL) and benzene: acetone=1:1 (20 mL) one (by NMR) as a white powder. Recrystallization from and 100% acetone (20 mL) to remove most of impurity. The DMSO/HO) furnished white microcrystals. M.p. of 5,6- residue (43 mg of 1-(o-tolylamino)carbonyl)amino-1,4- 65 dibromo-1,4-dihydro-2,3-quinoxalinedione was measured: dihydro-2,3-quinoxalinedione) was washed with ethanol decomposed from 335° C. IR (KBr, cm): 3200, 1718, (2x2 mL) and ether (2x1 mL) to give 38 mg of pure 1693. NMR (H, DMSO-d): 87.336 (s.2H); 11.962 (s.2H). 5,514,680 73 74 HRMS: calcd. for CHBrNO, (M) m/z:317.8639 found: 4,5-Dibromo-1,2-phenylenediamine. To a solution of 4,5- 317.8619. dibromo-2-nitroaniline (3.35g, 11.32 mmol) in EtOAc (150 6,7-Dibromo-1,4-dihydro-2,3-quinoxalinedione (Method mL) was added stannous chloride dihydrate (12.8 g., 56.74 C): To a stirred suspension of 1,4-dihydro-2,3-quinoxa mmol), the mixture was stirred at 75° C. for 16 h, then linedione (550 mg, 3.39 mMol, Aldrich) in dry DMF (10 allowed to cool to room temperature and poured into ice mL) was dropwise added a solution of bromine (1.07 g., 6.77 water (about 150 mL). To the mixture was added 1N aq mMol, Aldrich) in dried DMF (0.5 mL) within 1 h and then NaOH with vigorous stirring to take the pH to 9 (pH paper). the reaction was stirred at 25 C. for 30 h. Then, tetrachlo The resulting thick yellow white emulsion was extracted romethane (10 mL) was added and the reaction mixture was with EtOAc (3x100 mL). The organic extracts were com stirred at 50 C. for 2 h. The reaction mixture was poured 10 bined, washed with brine (2x50 mL), dried (over NaSO) into ice water (50 mL) and the precipitate was collected by and evaporated to dryness. The residual solid was dried filtration and washed with distilled water (2x1 mL) followed under 1 mmHg at 40° C. for 14 h giving 2.92 g (97%) of by 95% ethanol (2x1 mL) to afford crude 6,7-dibromo-1,4- 4,5-dibromo-1,2-phenylenediamine as a yellowish powder. dihydro-2,3-quinoxalinedione (with 1% impurity by NMR) Mp 149°-50° C. IR (KBr) 3400, 1623, 1489, 1394, 1271, as a white powder. The crude product was dissolved into 1N 5 868 cm. HNMR (DMSO-d) 6.754 (s, 2H), 4.867 (s, 4H). NaOH (10 mL) and then acidified to pH=2 with 4N HCl to 6,7-Dibromo-1,4-dihydroquinoxaline-2,3-dione. A mix give a white cream precipitate. The precipitate was collected ture of 4,5-dibromo-1,2-phenylenediamine (1.862 g, 7.0 by filtration and washed with distilled water (2x1 mL) mmol) and oxalic acid dihydrate (1.06 g, 8.4 mmol) in 2N followed by 95% ethanol (2x1 mL) and dried in the air at 50 HCl (100 mL) was heated at 125° C. for 2 h then allowed to C. for 8 h, to give 770 mg (yield 72%) of pure 6,7-dibromo 20 cool to 25°C. The solid was collected by vacuum filtration, 1,4-dihydro-2,3-quinoxalinedione (by NMR) as a white washed with water and dried at 40°C. under 1 mmHg for 10 powder. Recrystallization from DMSO/HO) furnished h affording 2.053 g (92%) of the title compound as a light white microcrystals. M.p. of 6,7-dibromo-1,4-dihydro-2,3- brown powder. 'H NMR (DMSO-d) 11.995 (s, 2H), 7.361 quinoxalinedione was measured: decomposed from 335 C. (s, 2H). IR (KBr, cm): 3200, 1718, 1693. NMR (H, DMSO-d): 25 67.336 (s, 2H); 11.962 (s, 2H). HRMS: calcd for Example 33 CHBrNO, (M) m/z: 317.8639 found: 317.8619. Preparation of 1-Amino-6,7-dibromo-1,4-dihydro-2,3-qui Preparation of 6,7-Dibromo-1,4-dihydro-2,3-quinoxalinedi noxalinedione one (Method D) The procedure of Wallace, R. G., Org. Prep. Proc. Int. 3,4-Dibromo-(trifluoroacetamido)benzene. To 30 mL of 30 14:269 (1982) was adapted. To a stirred suspension of trifluoroacetic anhydride (Aldrich used as received) was 6,7-dibromo-1,4-dihydroquinoxaline- 2,3-dione (100 mg, added 10.0 g (39.8 mmol) of 3,4-dibromoaniline (Lancaster, 0.312 mMol) in distilled water (10 mL) at 60° C. was added used as received) in portions at 0° C. with stirring. The NaOH (400 mg, 10 mMol). After 30 min., the resulting resultant mixture was stirred at room temperature for 2.5 h, solution was treated dropwise over 10 min with hydroxy then poured into ice-HO (about 300 mL) with stirring. The 35 lamino-o-sulphonic acid (40 mg, 0.35 mMol, Aldrich) in solid was collected by vacuum filtration, washed with HO water (0.5 mL). Reaction was carried out at 60° C. A white (5x20 mL), and dried at 40° C. under 1 mmHg for 16 h to precipitate came out after 15 min. The mixture was stirred at give 13.0 g (94%) of 3,4-dibromo-(trifluoroacetamido)ben 60° C. for 1 h. The white precipitate was collected by Zene as an off-white powder, which was used for the next filtration, and washed with distilled water (2x2 mL) and reaction without further purification. "H NMR (DMSO-d): 40 ethanol (2x1 mL), affording 72 mg (68%) of 1-amino-6,7- 11.474 (s, 1H), 8.098 (s, 1H), 7.935 (d. 1H, J=8.7 Hz), 7.607 dibromo-1,4-dihydroquinoxaline-2,3-dione as a white pow (d, 1H, J=8.7 Hz). der after dying at 60° C. for 2 h. Acidification of the filtrate 3,4-Dibromo-2-nitro-(trifluoroacetamido)benzene. To a with 4N HCl gave 35 mg of a mixture consisting of 25% solution of 12.95 g (37.3 mmol) of 3,4-dibromo-(trifluoro 1-amino-6,7-dibromo-14 -dihydroquinoxaline-2,3-dione acetamido)benzene in 150 mL of conc. HSO cooled at 8 45 and 75%. 6,7-dibromo-1,4-dihydroquinoxaline-2,3-dione by C. was added 7.0 mL of 70% HNO (110 mmol) over 30 NMR. The 72 mg sample of crude 6,7-dibromo-1,4-dihy min. After the addition, the stirring was continued within droquinoxaline-2,3-dione was dissolved in distilled water 10-12°C. for 1 h and then poured into ice-water (600 mL). (15 mL) at 60° C., filtered to remove the insoluble material, The precipitate was collected by vacuum filtration, washed and the filtrate was acidified with AcOH to pH=5 giving a with HO (6x50 mL), dried at 40° C. under 1 mmHg for 12 50 white precipitate, which was collected by filtration and h, affording 13.90 g (95%) of the crude product. It was washed with distilled water (2x2 mL) followed by ethanol crystallized from hexanes (about 180 mL) to give 10.05 g (2x 1 mL). The solid was dried on a rotavapor at 60° C. for (69%) of 3,4-dibromo-2-nitro-(trifluoroacetamido)benzene. 2 h affording 43 mg of pure 1-amino-6,7-dibromo-1,4- mp. 107-9° C. IR (KBr) 3331, 3256, 1745, 1551, 1203, dihydroquinoxaline-2,3-dione as a white powder; 1141 cm. H NMR (DMSO-d) 11.834 (s, 1H), 8.448 (s, 55 mp335°-338°C. (dec.). IR (KBr, cm): 3337; 3212;3062; 1H), 8,082 (s, 1H). 4,5-Dibromo-2-nitroaniline. 5.88 g (15.0 mmol) of 3,4- 1706; 1668; 1575. NMR (H, DMSO-d): 85.784 (s, 2H); dibromo-2 -nitro-(trifluoroacetamido)benzene was dis 7.391 (s, 1H); 7.841 (s, 1H); 12.158 (s, 1H). HRMS:calcd solved in MeOH (60 mL), to this solution was added 2N for CHNOBr, (M+) m/z: 332.8746; found: 332.8741. NaOH (20 mL) and it was heated to boil, then allowed to 60 Example 34 cool to 25 C. The mixture was rota-evaporated to remove the MeOH. The yellow solid was collected by vacuum Preparation of 5-Nitro-6,7-dibromo-1,4-dihydro-2,3-qui filtration, washed with water (5x10 mL), dried under 1 noxalinedione mmHg at 40° C., affording 3.52 g (80%) of 4,5-dibromo The procedure of Cheeseman, G. W. H., J. Chem. Soc. 2-nitroaniline as a yellow powder. Mp 1989 C. IR (KBr) 65 1170 (1962) was adapted. To a stirred suspension of 6,7- 3488, 3359, 1612, 1551, 1489, 1251, 1128 cm. H NMR dibromo-1,4-dihydro-2,3-quinoxalinedione (576 mg, 1.8 (DMSO-d) 8.184 (s, 1H), 7.584 (bs, 2H), 7.444 (s, 1H). mMol) in concentrated HSO (6 mL) at 0°C. for 30 min. 5,514,680 75 76 was added KNO (220 mg, 2.18 mMol, Baker) in one distilled water (0.5 mL) with stirring, whereupon a yellow portion. The mixture was stirred at 0°C. for 3 hthen atroom precipitate came out after 5 mins. The mixture was stirred at temperature for one day. The color of mixture was changed 65° C. for 1 h and allowed to stand at room temperature from red to yellow brown. Then it was poured into ice (60 overnight, then the precipitate was collected by filtration at g) resulting in the separation of a bright yellow precipitate, 50° C., washed with distilled water (2 mL), then air dried at which was collected by filtration and washed with distilled 50° C. overnight, affording 80 mg (64%) of crude 1-amino water (2x2 mL) followed by ethanol (2x1 mL) to get 498 mg 5-nitro-6,7-dibromo-1,4-dihydro-2,3-quinoxalinedione (free of crude 5-nitro-6,7-dibromo- 1,4-dihydro-2,3-quinoxa base Na' by NMR, DO), as a yellow amorphous solid (80% linedione (76%, contains minor impurities by NMR). Crys desired 1 -amino-5-nitro-6,7-dibromo-1,4-dihydro-2,3-qui tallization from DMSO/HO gave pure 5-nitro-6,7-dibromo 10 noxalinedione with 20% starting material by NMR). (It is 1,4-dihydro- 2,3-quinoxalinedione as bright yellow not known if the reaction actually gave the 1-amino or microcrystals, mp. 352-354 C. (dec.). IR (KBr, cm): 4-amino isomer.) A 80 mg sample of crude 1(4)-amino-5- 3387; 3256; 1756; 1700; 1537. NMR (H, DMSO-d): nitro-6,7 -dibromo-1,4-dihydro-2,3-quinoxalinedione 57.475 (s, 1H); 12.217 (s, 1H); 12.265 (s, 1H). HRMS: calcd (0.211 mMol) was dissolved into distilled water (10 mL) at for CHNOBr. (M-) m/z: 362.8489; found: 362.8509. 5 50° C., then acidified with AcOH to pH=5. After the insoluble material was removed by filtration, it was heated Example 35 at 60-70° C. until a clear solution was obtained, then Preparation of 1-Amino-5-nitro-6,7-dichloro-1,4-dihydro-2, slowly cooled, and a yellow precipitate came out. The 3-quinoxalinedione precipitate was crystallized from hot EtOH, collected by The procedure of Shin, S. C. and Lee, Y. Y., Taehan 20 filtration, and washed with cold ethanol (2 mL), then dried Hwahakhoe Chi 27(5):382–4 (1983) was adapted. A red in the air at 60° C. for 4 h, affording 57 mg (45.6%) of pure solution of 6,7-dichloro-5-nitro-1,4-dihydro-2,3-quinoxa 1(4)-amino-5-nitro-6,7 -dibromo-1,4-dihydro-2,3-quinoxa linedione (100 mg, 0.36 mMol, Cheeseman, supra) and 3N linedione (free base H). NHOSOH (40 mg, 0.35 mMol) KOH (2 mL) in distilled water (5 mL) at 65° C. was was added to the mother liquid, and allowed to react at dropwise added to a colorless solution of NHOSOH (75 25 65–70° C. for 30 min to give a second crop of pure mg, 0.66 mMol, Aldrich) in distilled water (0.5 mL) with 1(4)-amino-5-nitro-6,7-dibromo-1,4-dihydro-2,3-quinoxa stirring. A yellow precipitate came out after 10 mins. The linedione (26 mg, 21%), using the same procedure as above. mixture was stirred at 65 C. for 1 h and allowed to stand at The total yield is 66%. Mp was measured: the color of room temperature overnight, then the precipitate was col 1(4)-amino-5 -nitro-6,7-dibromo-1,4-dihydro-2,3-quinoxa lected by filtration at 50° C. and was washed with distilled 30 linedione changed to dark yellow at 301 C.; the decompo water (2 ml), then dried at 50° C. overnight, affording 85 mg sition of 1(4)-amino-5-nitro-6,7-dibromo-1,4-dihydro- 2,3- (80%) of crude 1-amino-5-nitro-6,7-dichloro-1,4-dihydro-2, quinoxalinedione is obvious at 310 C. and it melted to a 3-quinoxalinedione, as a yellow amorphous solid (80% of black liquid at 320-1 C. IR (KBr, cm): 3414; 3211; desired 1-amino-5-nitro-6,7-dichloro-1,4-dihydro-2,3-qui 1745; 1728; 1682; 1631; 1546. NMR (H, DMSO-d): noxalinedione with 20% starting material by NMR). A 85 35 85.832 (s, 2H); 8.047 (s, 1H); 12.565 (s, 1H). HRMS: calcd mg sample of crude 1-amino-5-nitro-6,7-dichloro-1,4-di for CHNOBr. (M-) m/z. 377.8613; found: 377.8583. hydro-2,3-quinoxalinedione (free base Na', 0.293 mMol) Example 37 was dissolved into distilled water (10 mL) at 50 C., then was acidified with AcOH to pH=5. After removal of the Preparation of 6-Nitro-5,7-dichloro-1,4-dihydro-2,3-qui insoluble material by filtration, the mixture was heated at noxalinedione -70 C. until a clear solution was obtained, which was The method of Cheeseman, supra was adapted. 5,7- slowly cooled, whereupon a yellow precipitate came out. Dichloro-1,4-dihydroquinoxaline-2,3-dione (239 mg, 1.03 The precipitate was crystallized from hot EtOH and the mMol) was dissolved in concentrated HSO (3 mL) at 0°C. yellow microcrystals were collected by filtration, washed for 30 min, and KNO (125.3 mg, 1.24 mMol, Baker) was with cold ethanol (2 mL), and dried in the air at 60° C. for 45 added to this solution. The mixture was stirred at 0°C. for 4 h, affording 31 mg (29%) of pure 1-amino-5-nitro-6,7- 3h and then was stirred at room temperature for 30 h. It was dichloro-1,4-dihydro-2,3-quinoxalinedione. Mp was mea then poured into ice water (15 g). A precipitate came out and sured: the color of 1-amino-5-nitro-6,7-dichloro-1,4-dihy was collected by filtration, was dissolved in 1N KOH (10 dro-2,3-quinoxalinedione changed to dark yellow at 275 mL) and the red precipitate was removed by filtration. The C.; the decomposition of 1-amino-5-nitro-6,7-dichloro-1,4- 50 solution then was acidified to pH-2 with 4N HCl to give a dihydro-2,3-quinoxalinedione is obvious at 280 C. and it cream precipitate, which was collected by filtration, then melted to a black liquid at 290-1 C.IR (KBr, cm): 3442; was dried in the air at 50 C. for 4 h to afford the pure 3315; 3231; 1747; 1723; 1680; 1632; 1547. NMR (H, 6-nitro-5,7-dichloro-1,4-dihydro-2,3-quinoxalinedione (231 DMSO-d): 85.848 (s, 2H); 7,951 (s, 1H); 12.595 (s, 1H). mg, 80%), as yellow microcrystals. mp: 320-25° C. (dec. HRMS: calcd for CHNOCl (M-) m/z:289.9623; found 55 from 290° C.). IR (KBr, cm): 3467, 3140, 3055, 2946, 289.9616. 1717, 1693, 1541. NMR (H, DMSO-d): 87.221 (s, 1H), 11.932 (s, 1H), 12.312 (s, 1H). HRMS: calcd. for Example 36 CHNOCl (M-) m/z: 274.9499, found: 274.9509. Preparation of 1(or 4-)-Amino-5-nitro-6,7-dibromo-1,4-di hydro-2,3-quinoxalinedione Example 38 The procedure of Shin, S. C. and Lee. Y. Y., Taehan Preparation of 6-Nitro-5,7-dibromo-1,4-dihydro-2,3-qui Hwahakhoe Chi 27(5):382–4 (1983) was adapted. To a noxalinedione stirred red solution of 5-nitro-6,7-dibromo-1,4-dihydro-2, The method of Cheeseman, supra. was adapted. 5,7- 3-quinoxalinedione (120 mg, 0.33 mMol) and 3N KOH (2 65 Dibromo-1,4-dihydroquinoxaline-2,3-dione (74 mg, 0.23 mL) in distilled water (5 mL) at 65° C. was dropwise added mMol) was dissolved in concentrated HSO (1 mL) at 0°C. a colorless solution of NHOSOH (56 mg, 0.50 mMol) in for 30 mins., and then KNO (28 mg, 0.27 mMol, Baker) 5,514,680 77 78 was added to this solution. The mixture was stirred at 0°C. mg, 0.28 mMol, Baker) at 0°C. with stirring. The mixture for 3 hand then at room temperature for 30 h. It was poured was stirred at 0°C. for 2 h. then at room temperature for 1 into ice water (8 g) and the precipitate was collected by day. The color of mixture was changed to yellow brown. filtration, was dissolved into iN KOH (5 mL), and the red Then it was poured into ice water (10 g) to separate the light precipitate was removed by filtration. The solution then was yellow precipitate. The precipitate was collected by filtration acidified to pH=2 with 4N HCl to obtain a cream precipitate, and washed with distilled water (1 mL) followed by ethanol which was collected by filtration, then was dried in the air (2x1 mL) to give crude 5-bromo-6(8)-nitro-7-trifluorom at 50° C. for 4 h, affording the pure 6-nitro-5,7-dibromo-1, ethyl-1,4-dihydro-2,3-quinoxalinedione (78 mg, 92%) 4-dihydro-2,3-quinoxalinedione (71 mg, 84.5%), as a yel which contains some isomer by NMR. Crystallization from low powder; mp: 318-20°C. (dec.). IR (KBr, cm): 3468, 10 DMSO/HO gave pure product as a white microcrystal. mp: 3131,3062,2931, 1712, 1593, 1537. NMR (H, DMSO-d): 290°-2°C. IR (KBr, cm): 3435; 3143; 1713; 1613; 1555. 57.392 (s, 1H), 11.566 (s, H), 12.274 (s, 1H). HRMS: calcd. NMR ('H, DMSO-d): 8.7.519 (s, 1H) for 6-nitro; 7,960 (s, for C.H.N.OBr, m/z. 362.8489, found: 62.8478. 1H) for 8-nitro (6:8=70:30); 11.811 (s, 1H); 12.391 (s, 1H). HRMS: calcd for CHNOFBr (M+) m/z: 352.9258; Example 39 15 found: 352.92.70. Preparation of 5-Chloro-6-nitro-7-fluoro-1,4-dihydro-2,3- quinoxalinedione Example 42 The Method of Cheeseman, supra was adapted. Preparation of 1(4)-Amino-5,7-dibromo-1,4-dihydro-2,3- 5-Chloro-7-fluoro- 1,4-dihydro-2,3-quinoxalinedione (30 20 quinoxalinedione mg, 0.14 mMol) was dissolved in concentrated H2SO4 (0.5 The procedure of Shin, S. C. and Lee. Y. Y., Taehan ml) at 0° C. for 30 min, and KNO (17 mg, 0.17 mmole, Hwahakhoe Chi 27(5):382–4 (1983) was adapted. 5,7-Di Baker) was added in one portion to this solution. The bromo-1,4-dihydroquinoxaline-2,3-dione (46 mg, 0.144 mixture was stirred at 0°C. for 3 h, then at room temperature mmole) was dissolved into 3N KOH (2 mL) at 60° C. for 1 for 30h. It was poured into ice water (5g) and the precipitate 25 h, and NHOSOH (20 mg, 0.172 mmole, Aldrich) in was collected by filtration. The precipitate was dissolved in distilled water (0.5 mL) was dropwise added into above 1N NaOH (5 mL), then was acidified to pH-2 with 4N HCl solution with stirring at 60° C. Some precipitate came out to give a cream precipitate, which was collected by filtration, after 15 mins, then a second 20 mg NHOSOH portion was then was dried in the air at 50° C. for 4 h, affording the pure added. The mixture was stirred at room temperature for 1 h. (by NMR) title compound 5-chloro-6-nitro-7-fluoro-1,4- 30 The white precipitate was collected by filtration, washed dihydro-2,3-quinoxalinedione (31 mg, 85.4%), as a white with cold distilled water (0.5 mL), affording crude 1-amino amorphous solid. mp: decomposed from 280° C. IR (KBr, 5,7-dibromo-1,4-dihydro-2,3-quinoxalinedione (38 mg, cm): 3600, 3462,3131, 1712, 1612, 1550. NMR (H, 79%) after drying in the air at 60° C. for 2 h (contains the DMSO-d): 87.106 (d, J=102 Hz, 1H), 11.800 (s, 1H), isomeric 4 -amino-5,7-dibromo-2,3-quinoxalinedione, by 12.371 (s, 1H). HRMS: calcd. for CHNOCIF (M+) m/z: 35 NMR, but it is not known which is produced in a greater 258.9795, found: 258.9790. amount). A 38 mg sample of crude 1-amino-5,7-dibromo 1,4-dihydro-2,3-quinoxalinedione was dissolved into dis Example 40 tilled water (4 mL) at 60° C., the insoluble material was Preparation of 5-Bromo-6-nitro-7-fluoro-1,4-dihydro-2,3- removed by filtration, and the filtrate was acidified with quinoxalinedione 40 AcOH to pH-5 to give a white precipitate, which was The method of Cheeseman, supra. was adapted. 5-Bromo collected by filtration and washed with cold distilled water 7-fluoro-1,4-dihydroquinoxaline-2,3-dione (77 mg, 0.30 (2x1 mL). The precipitate was dried at 60° C. for 2 h mMol) was dissolved in concentrated HSO (1 mL) at 0°C. affording 1-amino-5,7-dibromo-14 -dihydro-2,3-quinoxa for 30 min, and KNO (35 mg, 0.346 mMol, Baker) was linedione (28 mg, 58.5%) as a white powder with some added to this solution. The mixture was stirred at 0° C. for 45 isomer. Mp: 273°-5° C. (dec. from 270° C). IR (KBr, 3 hand then at room temperature for 30 h. The mixture was cm): 3435; 3289; 3190; 1719, 1672; 1625; 1584. NMR poured into ice water (10 g) and the precipitate was collected (H, DMSO-d): 85.831 ((s, 2H); 7.672 (d, J=15 Hz, 1H); by filtration. The precipitate was dissolved in 1N NaOH (10 7.810 (d, J=15 Hz, 1H); 11.275 (s, 1H). HRMS: calcd for mL), then was acidified to pH-2 with 4NHCl to give cream CHNOBr, (M+) m/z: 332.8746; Found: 332.8744. precipitate, which was collected by filtration, and was dried 50 in the air at 50° C. for 4 h affording pure (by NMR) Example 43 5-bromo-6-nitro-7-fluoro- 1,4-dihydro-2,3-quinoxalinedi one (81 mg, 90%), as a white amorphous solid; mp: Preparation of 1(4)-Amino-5,7-dichloro-1,4-dihydro-2,3- 320°-25° C. (dec. from 290° C). IR (KBr, cm): 3416, quinoxalinedione 3071, 2952, 1721, 1609, 1546. NMR (H, DMSO-d): 55 The procedure of Wallace, R. G., Org. Prep. Proc. Int. 87.146 (d, J-10.2 Hz, 1H), 11.432 (s, 1H), 12.340 (s, 1H). 14:269 (1982) was adapted. 5,7-Dichloro-1,4-dihydroqui HRMS: calcd. for CHNOBrF (M+) m/z: 302,9290, noxaline-2,3-dione (52 mg, 0.225 mmole) was dissolved in found: 302,9290. 3N KOH (1 mL) at 60° C. for 0.5h, and NHOSOH (30 mg, 0.265 mmole, Aldrich) in distilled water (0.5 mL) was Example 41 60 dropwise added into above solution with stirring at 60° C. Some precipitate came out after 15 mins. The mixture was Preparation of 5-Bromo-6(8)-nitro-7-trifluoromethyl-1,4-di stirred at room temperature overnight. The white precipitate hydro-2,3-quinoxalinedione was collected by filtration, washed with cold distilled water The procedure of Cheeseman, supra, was adapted. (0.5 mL) and dried on rotavapor at 60° C. for 2 h affording 5-Bromo-7-trifluoro-1,4-dihydroquinoxaline-2,3-dione (75 65 crude 1-amino-5,7-dichloro-1,4-dihydro-2,3-quinoxalinedi mg, 0.24 mmole) was dissolved in concentrated H2SO4 (1 one (38 mg, 69%), which included a little of the isomer mL) at 0°C. with stirring, to which was added KNO (30 (4-amino-5,7-dichloro-2,3-quinoxalinedione) by H NMR 5,514,680 79 80 (it is not known which isomer is present in a greater gave pure 5,6-dichloro-2-mercaptobenzimidazole (545 mg, amount). 86%) as white long needles. Mp was measured: the color of A 38 mg sample of crude 1(4)-amino-5,7-dichloro-1,4- 5,6-dichloro-2-mercaptobenzimidazole changed to yellow at dihydro-2,3-quinoxalinedione was dissolved into distilled 303 C.; the decomposition of 5,6-dichloro-2-mercaptoben water (4 mL) at 60° C., the insoluble material was removed Zimidazole is obvious at 305 C. and it melted to a black by filtration, the filtrate was acidified with AcOH to pH=5 to give a white precipitate, which was collected by filtration liquid at 308°-10° C. IR (KBr, cm): 3447; 3107; 3043; and washed with cold distilled water (2x1 mL). The pre 1607; 1496; 1461. NMR (H, DMSO-d): 87.305 (s, 2H); cipitate was dried on rotavapor at 60° C. for 2 h, affording 12.781 (s, 2H). HRMS: calcd for CHCNS (M) m/z: 1(4)-amino-5,7-dichloro-1,4-dihydro-2,3-quinoxalinedione 10 217.9425, Found: 217.9482. (29 mg, 53.5%) as a white powder. mp: 294°-6° C. (with In the following Examples 46-56, melting points are dec.) IR (KBr, cm): 3450; 3325; 3200, 3075; 1693; 1625; determined in open capillary tubes on Thomas Hoover and 1593; 1500; 1368. NMR (N, DMSO-d): 85.838 (s, 2H); Mel-Temp apparatuses and are uncorrected. IR and HNMR 7.454 (d, J-2.1 Hz, 1H); 7.639 (d, J=2.1 Hz, 1H); 11.691 ((s, spectra of all compounds were consistent with the structure 1H). HRMS: calcd for CNCNO (M-) m/z: 244.9757; 15 assigned and matched with previously reported data wher found: 244.97.69. ever available. "H NMR spectra were recorded on a 300M Hz. General Electric QE-300; chemical shifts are reported in Example 44 delta units referenced to the residual proton signal of the Preparation of 1-Amino-5-bromo-7-fluoro-1,4-dihydro-2,3- deuterated solvent (CHSOCHD, d 2.49). Infrared spectra quinoxalinedione 20 were recorded on a Nicolet 5DXB FT-IR spectrometer. The procedure Shin, S. C. and Lee, Y.Y., Taehan Hwa Absorptions recorded in wavenumbers (cm). Mass spectra hakhoe Chi 27(5):382–4 (1983) was adapted. 5-Bromo-7- were recorded on a VGZAB-2-HF mass spectrometer with fluoro-1,4-dihydroquinoxaline- 2,3-dione (85 mg, 0.33 a VG-11-250 DATA system, in the electron ionization mode mmole) was dissolved in 3N KOH (1.5 mL) at 60° C. for 0.5 25 (70 eV) unless otherwise indicated. All solvents were h to give a clear brown solution, and NHOSOH (45 mg, reagent grade. Reagents were used as received unless oth 0.396 mmole, Aldrich) in distilled water (0.5 mL) was erwise indicated. dropwise added into above solution with stirring at 60° C. Some precipitate came out after 15 mins. The mixture was then stirred at room temperature overnight. The brown Example 46 precipitate was collected by filtration, washed with cold 30 distilled water (0.5 mL) and dried on rotavapor at 60° C. for Preparation of 1-Carboxymethyl-1,4-dihydro-2,3-quinoxa 2 h, affording crude 1-amino-5-bromo- 7-fluoro-1,4-dihy linedione dro-2,3-quinoxalinedione (59 mg, 65.5%, 5% isomer of 4 1-Carboxymethyl-1,2,3,4-tetrahydroquinoxaline-3-one. -amino-5-bromo-7-fluoro-2,3-quinoxalinedione by NMR). The procedure of Borthakur, N. et al., Ind. J. Chem, 20B:822 A 59 mg sample of crude 1-amino-5-bromo-7-fluoro-1, 35 (1981) was adapted. The stirred solution of chloroacetic acid 4-dihydro-2,3-quinoxalinedione was dissolved in distilled (19.000 g, 0.200 mol) in water (100 mL) was neutralized water (5 mL) at 60° C., the insoluble material was removed with sodium carbonate (10.600 g, 0.100 mol) and o-phe by filtration, and the filtrate was acidified with AcOH to nylenediamine (10.800 g, 0.100 mol, Aldrich) was added to pH=5 to give brown precipitate, which was collected by it. The clear solution was refluxed for 4 h, cooled and made filtration and washed with cold distilled water (2x1 mL). alkaline (pH-10) with 0.3M aqueous sodium carbonate The precipitate was dried on rotavapor at 60° C. for 2 h, affording pure 1-amino-5-bromo-7-fluoro-1,4-dihydro-2,3- solution (150 mL). A small amount of residual solid was quinoxalinedione (49 mg, 54.5%) as a brown powder, mp. removed by filtration. The clear filtrate was acidified (pH-2) 293°-5° C. (with dec.) IR (KBr, cm): 3443; 3318; 3206; with concentrated HCl. The grey colored solid precipitated. 3081; 1731, 1668; 1612; 1600; 1506; 1343. NMR (H, 45 It was filtered and dried under vacuum (water aspirator) to DMSO-d): 85.832 (s, 2H); 7.439-7.519 (m.2H); 11.207 (s, obtain 17.2 g (83%, pure by HNMR) of product as a light 1H). HRMS: calcd for CHBrFNO, (M+) m/z: 272.9548; grey powder, m.p. 227-230° C. (lit. m.p. 228-230° C., found: 272.9569. Cheeseman, supra). It was sufficiently pure for use in the next reaction. Example 45 50 1-Carboxymethyl-1,4-dihydroquinoxaline-2,3-dione. The procedure of Borthakur, N. et at., Ind. J. Chem. 20B:822 Preparation of 5,6-Dichloro-2-mercaptobenzimidazole (1981) was adapted. To a stirred solution of 1-carboxym The procedure of Van Allan, J. A. W. and Deacon, B. D., ethyl-1,2,3,4-tetrahydroquinoxaline-3-one (15.400 g, 0.075 Organic Synthesis. IV:569 was adapted. A mixture of 1,2- mol) and sodium hydroxide (5.20 g, 0.13 mol) in water (250 diamino-4,5-dichlorobenzene (510 mg, 2.88 mMol, Ald 55 rich), potassium hydroxide (190 mg, 3.40 mMol), carbon mL), was added gradually, a solution of KMnO, (20.800 g disulfide (260 mg, 3.40 mMol), 95% ethanol (3 mL) and 0.132 mol) in aq. NaOH (4% why, 120 mL) and the dark water (0.45 ml) was heated under reflux for 3 h. Activated purple colored solution was refluxed for 4 h, cooled and charcoal (120 mg) was then added cautiously, and after the filtered. The clear filtrate was acidified (pH-2) with con mixture has been heated at the refluxing temperature for 10 centrated HCl. Filtration under vacuum (water aspirator) min the activated charcoal was removed by filtration. The afforded 9,200 g (56%, pure by "H NMR) of 1-carboxym filtrate was heated to 60-70 C., warm water (3 mL) was ethyl-1,4-dihydro-2,3-quinoxalinedione as a cream colored added, and then acetic acid (0.25 mL) in water (0.5 mL) was powder, m.p.>300° C. (decomposes) (lit. m.p.>300 C., added with good stirring overnight. The mixture was placed Borthakur et al., supra). It was sufficiently pure for use in the in a refrigerator for 3 h to get two crystals (brown and 65 next reaction; H NMR: 84.84 (s, 2H), 7.13-7.27 (m, 4H), white). The brown crystals were removed by washing with 12.16 (s, 1H); IR (KBr, cm): 3431, 1743, 1687, 1481, chloroform (5 mL). Recrystallization from hot EtOH/H2O 1406, 1393, 1250. 5,514,680 81 82 Example 47 0.63M aq. NaOH solution (25 mL) and 30% HO, (0.700 mL) was added to it. The suspension was stirred at 70-80 Preparation of 1-Carboxymethyl-6,7-dibromo-1,4-dihydro C. for 5 h, during which time, it formed a dark red colored 2,3-quinoxalinedione solution. It was then cooled in an ice-bath and acidified The procedure of Jorgensen et al., supra. was adapted. 1 5 (pH-2) with concentrated HCl. The precipitated solid was -Carboxymethyl-1,4-dihydro-2,3-quinoxalinedione (1.500 filtered to yield 484 mg light purple colored powder. Crys g, 0.068 mol) and AgSO (2.232 g, 0.071 mol) were tallization from DMF-water furnished 457 mg (85%, pure suspended in concentrated HSO (7.5 mL). Bromine (0.75 by H NMR) acid 1 -carboxymethyl-6,7-dichloroquinoxa mL, 0.014 mol, Aldrich) was added to it at 28° C. and the line-2,3(1H4H)-dione as a grey colored powder; m.p. suspension was stirred at 28°C. for 24 h. Carbon tetrachlo 317-320° C. (lit. m.p.317-319 C., Jorgesen et al., supra); ride (7.5 mL) was then added to the suspension and heated H NMR: 84.85 (s, 2H), 7.31 (s, 1H), 7.67 (s, 1H), 12.28 (s, at 50° C. for 2 h. It was then poured into ice-water (75 g). The precipitated white solid was filtered and washed with 1H); IR (KBr, cm): 3428, 3129, 1694, 1489, 1396. water (10 mL) and dried under vacuum (water aspirator). It Example 49 was then treated with 4M aq. NaOH (60 mL). The residue 15 was filtered off and the clear filtrate was acidified (pH-3) Preparation of 6,7-Dichloro-5-nitroquinoxaline-2(1H)-one with concentrated HCl. The precipitated white solid was To a stirred suspension of 6,7-dichloroquinoxaline-2(1H)- filtered and dried to yield 1.81 g (70%, pure by "H NMR) one (100 mg, 0.465 mmol) in concentrated HSO (1.5 mL), acid 1 -carboxymethyl-6,7-dibromo-1,4-dihydro-2,3-qui water (1.0 mL) was added to form a solution. It was then noxalinedione as a white powder, m.p.>300° C. (decom 20 cooled to 5°-10° C. and KNO (50 mg, 0.46 mmol) was poses) (lit. m.p.>300° C., Jorgesen et al., supra); H NMR: added in one portion. The addition of KNO results in a dark 84.85 (s, 2H), 7.44 (s, 1H), 7.73 (s, 1H), 12.28 (s, 1H); IR green colored solution which was stirred at 5-10°C. for 3 (KBr, cm): 3437, 1687, 1481, 1406, 1393, 1250. h and then at 28° C. for 60 h. The yellow suspension thus obtained was poured into ice-water (15g) and the resulting Example 48 25 yellow solid was filmed and dried in the air overnight to obtain 78 mg crude product as a yellow powder. Crystalli Preparation of zation from DMF-water furnished 45 mg (37%, pure by 'H 1-Carboxymethyl-6,7-dichloro-1,4-dihydro-2,3- NMR) 6,7-dichloro-5-nitroquinoxaline-2(1H)-one as a yel quinoxalinedione low powder; m.p. 330°-332° C. (decomposes); H NMR: 6,7-Dichloroquinoxaline-2(1H)-one. The procedure of 30 87.59 (s, 1H), 8.28 (s, 1H); IR (KBr, cm): 1695, 1654, Kazimierczuk, Z. et al., Liebigs Ann. Chem 75 (1982) was 1555, 1367; HRMS calcd for CHCl2NO (M-) m/z. adapted. A solution of 4,5-dichloro-1,2-phenylenediamine 258.9551, found m/z 258.9550. (500 mg, 2.82 mmol, Pfaltz and Bauer), glyoxalic acid Example 50 monohydrate (389 mg, 4.23 mmol, Aldrich) in ethanol (8 35 mL) was refluxed for 12 h. Upon cooling to 28 C., a purple Preparation of 1-Carboxymethyl-6,7-dibromo-5-nitro-1,4- colored solid precipitated which was filtered under vacuum dihydro-2,3-quinoxalinedione (water aspirator), washed with cold ethanol (20 mL) and To a stirred solution of 1-carboxymethyl-6,7-dibromo-14 dried further under vacuum to obtain 575 mg (94%, pure by -dihydroquinoxaline-2,3-dione (100 mg, 0.264 mmol) in "H NMR) 6,7-dichloroquinoxaline-2(1H)-one as a light 40 concentrated HSO (1.5 mL) at 5°-10 C., KNO (28 mg, purple colored powder, m.p. 325–328° C. (lit. m.p.>300 0.28 mmol) was added in one portion. The resulting dark C.; Kazimierczuk, et al., supra). It was sufficiently pure for green solution was stirred at 5°-10°C. for 30 min and at 28 use in the next reaction. H NMR: 87.40 (s, 1H), 8.02 (s, C. overnight. The yellow colored suspension thus obtained 1H),8.18 (s, 1H), 12.52 (s, 1H). IR (KBr, cm): 1668, 1606, was poured into ice-water (15 g) and the resulting shining 1468, 1387. 45 yellow solid was filtered and dried under vacuum to obtain 6,7-Dichloro-1-ethoxycarbonylmethylquinoxaline 53 mg (47%, pure by H NMR) 1-carboxymethyl-6,7-di 201H)-one. The procedure of Jorgensen et al., supra. was bromo-5-nitro-1,4-dihydro-2,3-quinoxalinedione as a shin adapted. Under a nitrogen atmosphere, sodium (60 mg, 2.60 ing yellow powder; M. p. 260°-264° C. "H NMR: 84.91 (s, mmol) was dissolved in absolute ethanol (20 mL) and 1H), 7.98 (s, 1H). IR (KBr, cm): 1701, 1543, 1391, 1244. 6,7-dichloroquinoxaline-2(1H)-one (520 mg, 2.420 mmol) 50 HRMS calcd for CHBrNO (M+) m/z 420.8546, found was added to it. The dark purple colored solution was n/Z 420.8559. refluxed for 30 min., cooled to 28° C. and ethyl bromoac etate (485 mg, 2.900 mmol, Aldrich) was added to it and refluxed further for 2 h. During this time, a purple colored Example 51 solid separated which was filtered, washed with absolute 55 Preparation of 6,7-Dichloro-N-hydroxy-1,4-dihydro-2,3- ethanol (10 mL) and dried in the air overnight to afford 636 quinoxalinedione mg (91%, pure by H NMR) 6,7-dichloro-1-ethoxycarbo All reactions were run under a nitrogen atmosphere. nylmethylquinoxaline-2(1H)-one as a light purple colored Reagents were used as received unless otherwise indicated. powder, m.p. 207-210° C. (lit. m.p. not reported). It was Melting points were taken on a Mel-Temp melting point sufficiently pure for use in the next reaction. 'HNMR: 81.18 60 apparatus and are uncorrected. Samples were placed in the (t,3H, J=6.9Hz), 4.14 (q, 2H, J=6.9 Hz), 5.04 (s, 2H), 8.01 block when the temperature was >250° C. in order to (s, 1H), 8.13 (s, 1H), 8.33 (s, 1H). IR (KBr, cm): 1737, minimize decomposition prior to melting. Tetrahydrofuran 1662, 1400, 1231. (THF) was distilled from blue sodium benzophenone ketyl 1-Carboxymethyl-6,7-dichloro-1,4-dihydroquinoxaline solution. DMF was dried over molecular sieves. "H NMR 2,3-dione. The procedure of Jorgensen et al., supra. was 65 spectra were recorded on a 300 MHz. General Electric adapted. 6,7-Dichloro-1 -ethoxycarbonylmethylquinoxa QE-300; chemical shifts are reported in delta units refer line-2(1H)-one (528 mg, 1.840 mmol) was suspended in enced to residual proton signals of the deuterated solvents 5,514,680 83 84 (CDC1, 57.26; CHSOCHD, 82.49). Infrared spectra were (some of the product also ends up being extracted by hot obtained on a Nicolet 5DXB FT-IR spectrometer. ethanol which accounts for the low yield of the pure prod Ethyl-N-(4,5-dichloro-2-nitro-phenyl)oxamate. Ethyl-N- uct). M.p.>300° C. (decomposes). H NMR: 84.92 (s, 2H), (4,5-dichloro-2-nitro-phenyl)oxanate was prepared using 7.01-7.30 (m, 7H), 7.51 (d, 2H, J-6.9 Hz), 10.26 (s, 1H), an adaptation of the method of Loev, et al., J. Med. Chem. 12.13 (s, 1H). IR (KBr, cm): 3148, 1715, 1671, 1600, 28:363 (1985). To a stirred solution of 4,5-dichloro-2- 1557. HRMS: Calculated for C, H, N, O, 295.0951; nitroaniline (2.07 g., 0.01 mol) in dry THF (15 mL) and Observed, 296.1033. triethylamine (1.5 mL, 0.011 mol) at 0° C. was added dropwise ethyl oxalyl chloride (4.6 g., 0.015 mol). The resulting yellow suspension was allowed to warm to 25 C. 10 Example 53 in the bath and then stirred for 3 h. The resulting brown suspension was poured into 75 mL of ice water. A brown Preparation of precipitate formed. The mixture was vacuum filtered, and N-(N'-(p-nitrophenyl)carboxamidyl)methyl-1,4- the solid was air dried for 1 h to yield 3.27g of a dark brown dihydroquinoxaline-2,3-dione solid which was dissolved in ethanol (38 mL) with heating 15 to 70° C. Water (6 mL) was added until precipitation To a stirred solution of N-carboxymethyl-1,4-dihydroqui appeared. The mixture was heated again until the precipitate noxaline-2,3-dione (250mg, 1.140 mmol) and p-nitroaniline redissolved. The brown solution was allowed to cool slowly, (156 mg, 1.140 mmol) in DMF (3 mL) under N, at 0°C., giving yellow needle-like crystals. The mixture was vacuum DCC (233 mg, 1.140 mmol) was added in one portion. The filtered and the crystals were air dried for 2 h to yield 1.7322 20 solution was allowed to warm to 28° C. and stirred at that g (5.64 mmol) of ethyl-N-(4,5-dichloro-2-nitro-phenyl)ox temperature overnight. The precipitated solid was filtered amate as pale yellow needles (56.4%): mp 95-97° C. "H and the clear filtrate was poured into water (30 mL). The NMR (300 MHz, CDC1,) 811.897 (brs, 1H, NH), 9.067 (s, solid thus obtained was filtered and dried under vacuum 1H, H-3), 8,412 (s, 1H, H-6), 4.463 (q, 2H CH), 1.435 (t, (water aspirator) to obtain 100 mg crude product as a yellow 3H, CH,). 25 powder. The crude product was purified by Soxhlet extrac 6,7-Dichloro-N-hydroxy-1,4-dihydroquinoxaline-2,3-di tion in boiling ethanol (20 mL), keeping the oil bath tem one. This compound was prepared using an adaptation of the perature at 120° C., for 4 h. The insoluble material (in the method of Loev, et al., supra. A mixture of ethyl-N-(4,5- thimble) was dried under vacuum to obtain 23 mg pure ('H dichloro-2-nitro-phenyl)oxamate (0.307 g, 1 mmol) and NMR) N-(N'-(p-nitrophenyl)carboxamidyl)methyl-1,4-di 0.04 g of 5% Pd-C in 5 mL of DMF was hydrogenated at 45 30 hydroquinoxaline-2,3-dione as a light yellow powder (some psi for 1.5h. The reaction mixture was filtered and the liquid of the product also ends up being extracted by hot ethanol was added to water (18 mL). A white precipitation formed. which accounts for the low yield of the pure product). The mixture was vacuum filtered. The solid was rinsed with M.p.2300° C. (decomposes), "H NMR: 85.02 (s, 1H), water (5x2 mL), and air dried for 1 h to yield 215.14 mg 7.13-7.3 (m, 4H), 7.78 (d, 1H, J-84 Hz), 8.21 (d, 1H, J=8.4 (0.87 mmol)of 6,7-dichloro-N-hydroxy-1,4-dihydroqui 35 Hz), 10.92 (s, 1H), 12.16 (s, 1H). IR (KBr, cm): 3453, noxaline-2,3-dione as a pale yellow solid (87%). The pale 1701, 1684, 1625, 1572, 1509. HRMS: Calculated for C yellow solid (215 mg, 0.87 mmol) was dissolved in 4 mL of H NO. 340,0808; Observed, 340.0811. DMSO with heating. Water (0.8 mL) was added until precipitation appeared. The mixture was heated again to Example 54 redissolve the precipitate. The yellow solution was cooled down. Pale yellow crystals came out which were collected Preparation of N-(N'-(p-Aminophenyl)carboxamidyl)m- by vacuum filtration and rinsed with water (5x2 mL) and ethyl-1,4-dihydroquinoxaline-2,3-dione dried in vacuum to yield 173 mg (0.70 mmol) of 6,7- To a stirred solution of acid N-carboxymethyl-1,4-dihy dichloro-N-hydroxy-1,4-dihydroquinoxaline-2,3-dione as droquinoxaline-2,3-dione (200 mg, 0.910 mmol) and p-phe pale yellow crystals. Mp >300° C. (dec). H NMR (300 45 nylenediamine (98 mg, 0.91 mmol) in DMF (2 mL) under MHz, DMSO-d) 612.244 (brs, 1H, NH), 11.953 (brs, 1H, N2 at 0°C., DCC (190 mg, 0.910 mmol) was added in one N-OH), 7.562 (s, 1H, H-8), 7.318 (s, 1H, H-5). HRMS calcd. portion. The solution was allowed to warm to 28° C. and for CHCNO, 245.9599; found, 245.9600. stirred at that temperature overnight. The precipitated solid was filtered and the clear filtrate was poured into water (10 50 mL). The solid thus obtained was filtered and dried under Example 52 vacuum (water aspirator) to obtain 180 mg crude product as Preparation of N-(N-Phenylcarboxamidyl)methyl-1,4-dihy a brown powder. The product was purified by Soxhlet droquinoxaline-2,3-dione extraction in boiling ethanol (20 mL), keeping the oil bath To a stirred solution of 1-carboxymethyl-1,4-dihydroqui temperature at 120°C., for 4 h. The insoluble material (in the noxaline-2,3-dione (100 mg, 0.450 mmol) and aniline (62 55 thimble) was dried under vacuum to obtain 135 mg pure ('H mg, 0.66 mmol) in dry DMF (2 mL) under N at 28° C., NMR) N-(N'-(p-aminophenyl)carboxamidyl)methyl-1,4-di DCC (95 mg, 0.46 mmol, Aldrich) was added in one portion. hydroquinoxaline-2,3-dione as a white powder. M.p.>300 The solution was stirred for 4h at 28 C. The insoluble solid C. (decomposes). H NMR: 84.84 (s, 2H), 493 (s.2H), 6.5 was filtered and washed with DMF (1 mL). The clear filtrate (d, 2H, J=8.4Hz), 7.15-7.25 (m, 6H), 9.83 (s, 1H), 12.15 (s, was poured into water (30 mL). The solid thus obtained was 1H). IR (KBr, cm): 3462, 3143, 1793, 1693, 1593, 1443. filtered and dried under vacuum (water aspirator) to yield HRMS: Calculated for CHNO 310.1066; Observed, 133 mg crude product as a grey powder. It was purified by 10.1071. Soxhlet extraction in boiling ethanol (20 mL), keeping the oil bath temperature at 120° C., for 4 h. The insoluble Example 55 material (in the thimble) was dried under vacuum to obtain 65 55 mg of pure (H NMR) N-(N-phenylcarboxamidyl)m- Preparation of N-(N-Phenylcarboxamidyl)methyl-6,7-di ethyl- 1,4-dihydroquinoxaline-2,3-dione as a white solid bromo-1,4-dihydroquinoxaline-2,3-dione 5,514,680 85 86 To a stirred solution of N-carboxymethyl-6,7-dibromo-1, absorptions are indicated by the letters s (strong), m 4-dihydroquinoxaline-2,3-dione (100 mg, 0.250 mmol) and (medium), and w (weak). Mass spectra were recorded on a aniline (25 mg, 0.25 mmol) in dry DMF (1.5 mL) under N VGZAB-2-HF mass spectrometer with a VG-11-250 data at 28° C., DCC (55 mg, 0.25 mmol) was added in one system, in the electron ionization mode (70 eV) unless portion. The solution was stirred at 28°C. for 18 h. The otherwise indicated. Microanalyses were performed by insoluble white solid was filtered and the clear filtrate was Desert Analytics of Tuscon, Ariz. poured into water (6 mL). The precipitated solid was filtered and air dried to obtain 113 mg crude product as a grey Example 57 powder. The crude product was then purified by Soxhlet extraction in boiling ethanol (20 mL), keeping the oil bath 10 Preparation of 5-Amino-6,7-dibromo-1,4-dihydro-2,3-qui temperature at 120° C., for 5h. The residue (in the thimble) noxalinedione was dried in the oven (approx. 70-80 C.) overnight to To a stirred mixture of 5-nitro-6,7-dibromo-1,4-dihydro furnish 35 mg pure (HNMR) N-(N-phenylcarboxamidyl 2,3-quinoxalinedione (327 mg, 0.89 mMol) in ethanol (10 )methyl-6,7-dibromo-1,4-dihydroquinox-aline-2,3-dione as mL) was added SnCl2.H2O (1.0 g, 4.45 mMol) in one a lightgrey powder; m.p.>300° C. (decomposes). H NMR: 15 portion. The mixture was refluxed at 80° C. (oil bath 90° C.) with stirring for 4 h. The mixture was then cooled to room 84.97 (s, 1H), 699-7.11 (m, 1H), 7.23-7.37 (m, 2H), 7.44 temperature and the yellow precipitate was collected by (s, 1H), 7.52 (d, 2H, J-84 Hz), 7.73 (s, 1H), 10.25 (s, 1H), filtration, followed by washing with cold ethanol (2x1 mL), 12.25 (s, 1H). IR (KBr, cm): 3620, 3468, 1714, 1694, to get 227 mg (76%) of crude title product (contains minor 1595, 1542. HRMS:Calculated for CHBrNO, impurities by NMR). Crystallization from DMSO/HO gave 450.9168; Observed, 450.9177. 193 mg of pure title product as bright yellow needles; mp: 324°-6° C. (dec.), changed color from 270° C. IR (KBr, Example 56 cm): 3456; 3281; 1700; 1643. NMR (H, DMSO-d): Preparation of N-(N'-(m-nitrophenyl)carboxamidyl)methyl 85.844 (s, 2H); 6.732 (s, 1H); 11.257 (s, 1H); 11.810 (s, 1H). 6,7-dibromo-1,4-dihydroquinoxaline-2,3-dione 25 Purity:>96.96% by HPLC. HRMS: calcd for CHNOBr, To a stirred solution of N-carboxymethyl-6,7-dibromo (M+) m/z 332.8747; found: 332.8754. Potency relative to 1,4-dihydroquinoxaline-2,3-dione (100 mg, 0.260 mmol) DCK: 341%. and m-nitroaniline (40 mg, 0.29 mmol) in dry DMF (2 mL) under N at 28°C., DCC (60 mg, 0.29 mmol) was added in Example 58 one portion. The solution was stirred for 4 h at 28°C. The 30 Preparation of 5-Amino-6,7-dichloro-1,4-dihydro-2,3-qui insoluble solid was filtered and washed with DMF (1 mL). noxalinedione The clear filtrate was then poured into water (30 mL). The To a stirred mixture of 5-nitro-6,7-dichloro-1,4-dihydro precipitated solid was filtered and dried under vacuum 2,3-quinoxalinedione (110 mg, 0.40 mMol) in ethanol (6 (water aspirator) to obtain 65 mg crude product as a yellow mL) was added SnCl2.H2O (448 mg, 2.0 mMol) in one powder. The product was purified by Soxhlet extraction in 35 portion. The mixture was refluxed at 80° C. (oil bath 90° C) boiling ethanol (20 mL), keeping the oil bath temperature at with stirring for 1 h to form a clear solution and continually 120° C., for 4 h. The insoluble material (in the thimble) was refluxed for another 3 h. The mixture was then cooled to dried under vacuum to obtain 30 mg pure ("H NMR) room temperature and the yellow precipitate was collected N-(N'-(m-nitrophenyl)carboxamidyl)-methyl-6,7-dibromo by filtration and washed with cold ethanol (2x1 mL) to give 1,4-dihydroquinox-aline-2,3-dione as a white solid (some of 40 61 mg (62%) of crude title compound (contains minor the product also ends up being extracted by hot ethanol impurities by NMR). Crystallization from DMSO/HO gave which accounts for the low yield of pure product). 43 mg of pure title compound as bright yellow needles, Mp2300° C. (decomposes). H NMR: 84.98 (s, 1H), 7.45 mp350° C. (dec.), changed color from 320° C. IR (KBr, (s, 1H), 7.61 (d of d seen as at, J=8.1 Hz) 7.79 (s, 1H), 7.86 cm): 3468; 3389; 3057; 1695; 1636; 1596; 1397. NMR (d. 1H, J=9 Hz), 7.91 (d, 1H, J-84 Hz), 8.54 (s, 1H), 10.76 45 (H, DMSO-d): 85.935 (s, 2H); 6.595 (s, 1H); 11.317 (s, (s, 1H), 12.28 (s, 1H). IR (KBr, cm): 3444, 3289, 1707, 1H); 11.868 (s, 1H). Purity>98.95% by HPLC. HRMS: 1686, 1672, 1602. HRMS: Calculated for CHBFN.O.s, calcd for CHNOBr, (M+) m/z: 244,9757; found 495.9018; Observed, 495.9008. 244.9740. Potency relative to DCK: 323%. General In the following Examples 57-70, reagents were used as 50 Example 59 received unless otherwise indicated. Melting points were taken on a Mel-Temp melting point apparatus and are Preparation of 5-Acetamido-6,7-dichloro-1,4-dihydro-2,3- uncorrected. Samples were placed in the block when the quinoxalinedione temperature was >250° C. in order to minimize decompo To a stirred solution of 5-amino-6,7-dichloro-1,4-dihy sition prior to melting. Column chromatography was per 55 dro-2,3-quinoxalinedione (61.5 mg, 0.25 mMol) in dried formed in the flash mode on Davisil silica gel (200-425 DMF (7 mL) was added triethylamine (33 mg, 32 mMol, mesh), unless otherwise indicated. Analytical thin layer distilled before use) and acetyl chloride (20 mg, 0.255 chromatography was performed on aluminum-backed silica mMol, distilled before use). The mixture became a yellow gel 60 Fis plates and visualization was effected with an solution after 2 min. After 2 h at 25 C., a precipitate ultraviolet lamp. "H NMR spectra were recorded on a 300 60 appeared and stirring was continued overnight during which MHz. General Electric QE-300; chemical shifts are reported time more white precipitate came out. The precipitate was in delta units referenced to residual proton signals of the collected by filtration and washed with water (2x1 mL), deuterated solvents (CHCH, d 7.26; CHDOD, d 3.30; affording a white powder which was 34 mg of crude title CDSOCDH, d 2.49; CD.COCDH, d 2.04). 'C NMR compound (contains some impurity by NMR). The filtrate spectra were run at 75 MHz. Infrared spectra were obtained 65 was added to water (15 mL) to get a precipitate and this was on a Nicolet 5DXB FT-IR spectrometer. Absorptions collected by filtration and washed with water (2x1 mL), recorded in wavenumbers (cm) and the intensity of the affording 29 mg of pure product (by NMR). The total yield 5,514,680 87 88 was 88%. Crystallization from DMSO/HO gave 25 mg of (H, DMSO-d): d 5.257 (s, 2H); 6.558 (s, 1H); 7.087 (s, pure title compound as white microcrystals; mp: 320-2 C. 1H); 11.599 (s, 1H); 11.792 (s, 1H). HRMS: calcd for (dec. from 315° C). IR (KBr, cm): 3500, 3162, 3056, CHN.O.Br (M+) m/z: 2549642; found 2549630. 1706, 1606, 1531. NMR (H, DMSO-d): d 2,065 (s, 3H); Potency relative to DCK; 7.1%. 7.236 (s, 1H); 9.621 (s, 1H); 11.655 (s, 1H); 12.096 (s, 1H). HRMS: calcd for C.H.N.OCl (M+) m/z: 286,9863; Example 63 found: 286,9859. Potency relative to DCK: 44%. Preparation of 5-Iodo-7-chloro-1,4-dihydro-2,3-quinoxa linedione Example 60 A. Synthesis of 4-chloro-2-iodo-6-nitroaniline: The pro Preparation of 6-Amino-5,7-dichloro-1,4-dihydro-2,3-qui 10 cedure of Leeson, P. D. et al. (J. Med. Chem. 34:1243-1252 noxalinedione (1991)) was adapted. To a solution of 4-chloro-2-nitroaniline To a stirred mixture of 6-nitro-5,7-dichloro-1,4-dihydro (2.15g, 12.45 mMol, Aldrich, used as received) in glacial 2,3-quinoxalinedione (81 mg, 0.295 mMol) in ethanol (3 AcOH (16 mL) was added iodine monochloride (2.114 g, mL) was added SnCl2.H2O (331 mg, 1.47 mMol) in one 15 12.90 mMol, Aldrich). The mixture was heated at 120 C. portion. The mixture was refluxed at 80°C. (oil bath90 C.) for 5 h, then cooled and poured into ice water (30 g). The with stirring to 0.5h, to form a clear solution and continually precipitate was collected and washed with 10% sodium refluxed for another 0.5 h. It was then cooled to room sulfite solution (20 mL), then it was crystallized from MeOH temperature and the yellow precipitate was collected by to give 4-chloro-2-iodo-6-nitroaniline (1.05g, 28%), as long filtration, followed by washing with cold ethanol (2x1 mL) brown needles, m.p. 134°-5° C. NMR (H, CDCl): 86.660 to give 70 mg (97%) of crude title compound (contains 20 (s, 2H); 7.906 (d, 1H, J=2.1 Hz); 8.188 (d. 1H, J=1.8 Hz). minor impurities by NMR). Crystallization from DMSO/ B. Synthesis of 4-chloro-6-iodo-1,2-phenylenediamine: HO gave 32 mg of pure title compound as bright yellow To a stirred mixture of 4-chloro-2-iodo-6-nitroaniline (389 needles; mp: 342-5° C. (dec. from 335° C), changed color mg, 1.305 mMol) in ethanol (10 mL) was added from 325°C. IR (KBr, cm): 3468,3362,3193, 1693, 1631, SnCl2HO (1468 g, 6.526 mMol) in one portion. The 1493, 1375; NMR (H, DMSO-d): 85.418 (s, 2H); 6.999 (s, 25 mixture was refluxed at 80 C. (oil bath90 C.) with stirring 1H); 11.238 (s, 1H); 11.776 (s, 1H). HRMS: calcd for for 0.5 h to form a clear solution and then refluxing was CHN.OCl, (M+) m/z: 244.9757; found 244.9769. continued for another 0.5h. The solution was cooled to room Potency relative to DCK: 8.6%. temperature and ice water (20g) was added. The pH was adjusted to pH-7 and the mixture was extracted with ethyl Example 61 30 acetate. The extract was dried over MgSO, and evaporated to dryness to give 336 mg (96%) of 4-chloro-6-iodo-1,2- Preparation of 6-Amino-7-chloro-1,4-dihydro-2,3-quinoxa phenylenediamine, as a brown solid. NMR (H, CDCl3): linedione 83.536 (s, 2H); 3.763 (s, 2H); 7.165 (d, 1H, J=1.8 Hz); 6.671 To a stirred mixture of 6-nitro-7-chloro-1,4-dihydro-2,3- (d, 1H, J=1.8 Hz). quinoxalinedione (35 mg, 0.145 mMol) in ethanol (2 mL) 35 C. Synthesis of 5-Iodo-7-chloro-1,4-dihydro-2,3-qui was added SnCl2.H2O (163 mg, 0.724 mMol) in one noxalinedione: The procedure of Foged, C. and Journal, P. portion. The mixture was refluxed at 80°C. (oil bath90° C.) (J. of Lab. Compa, and Radiopharmac. XXXI (5):365-373 with stirring for 0.5 h to form a clear solution and continu (1992)) was adapted. To a stirred mixture of 4-chloro-6- ally refluxed for another 0.5 h. It was then cooled to room iodo-1,2-phenylenediamine (366 mg, 1.253 mMol) in 2N temperature and the yellow precipitate was collected by HCl (30 mL) was added oxalic acid (160 mg, 1.269 mMol, filtration, followed by washing with cold ethanol (1x1 mL) used as received) in one portion. The mixture was refluxed to give 25 mg (82%) of crude title compound (contains at 120°-5°C. for 3 h, then cooled to room temperature minor impurities by NMR). Crystallization from DMSO/ overnight. The mixture was centrifuged and the liquid layer HO gave 14 mg of pure title compound as bright yellow was removed. The red solid was washed twice with cold needles, mp.>350° C. (dec. from 335° C). IR (KBr, cm): 45 water (2x2 mL), collected by filtration, and dried at 60° C. 3406, 3356, 3212, 1668, 1637, 1518, 1437. NMR (H, with reduced pressure for 2 h, giving 300 mg of crude DMSO-d): d 5.306 (s, 2H); 6.551 (s, 1H); 6.940 (s, 1H); 5-iodo-7-chloro-1,4-dihydro-2,3-quinoxalinedione (74%), 11.606 (s, 1H); 11.788 (s, 1H). HRMS: calcd for as a red powder containing some impurities (by NMR). A CHN.O.C. (M+) m/z: 2110147; found 211.0159. Potency 300 mg sample of crude product was dissolved in 1N NaOH relative to DCK: 28.0%. 50 (10 mL). Some insoluble material was removed by filtration, and the filtrate was then acidified to pH-6, giving 260 mg of Example 62 purer product. Crystallization from DMSO/HO, gave 169 Preparation of 6-Amino-7-bromo-1,4-dihydro-2,3-quinoxa mg of pure product (42%), as red microcrystals, mp:>350 linedione 55 C. (dec. from 295° C). IR (KBr, cm) 3443, 3212, 1750, To a stirred mixture of 6-nitro-7-brono-1,4-dihydro-2,3- 1706, 1606, 1587, 1393. NMR (H, DMSO-d): 87.133 (d, quinoxalinedione (87 mg, 0.30 mMol) in ethanol (3 mL) and 1H, J=1.5Hz); 7.628 (d, 1H, J=1.5Hz); 10.386 (d, 1H, J=1.5 DMSO (0.5 mL) was added SnCl2HO (343 mg, 1.50 Hz); 12.015 (d, 1H, J=1.8 Hz). HRMS: calcd for mMol) in one portion. The mixture was refluxed at 80° C. CHNOCII (M+) m/z: 321.9004; found: 321.8995. (oil bath90° C) with stirring for 1 h to form a clear solution Potency relative to DCK: 28.4%. which was refluxed for another 1 h. It was then cooled to Example 64 room temperature and the yellow precipitate was collected by filtration, followed by washing with cold ethanol (2x1 Preparation of 5-Iodo-7-fluoro-1,4-dihydro-2,3-quinoxa mL) to give 50 mg (67%) of crude title compound (contains linedione minor impurities by NMR). Crystallization from DMSO/ 65 A. Synthesis of 4-Fluoro-6-iodo-2-nitroaniline: The pro HO gave 21 mg of pure as bright yellow needles, mp.2300 cedure of Sy, W. W. (Synthetic Communications C. (dec. from 315° C), changed color from 300° C. NMR 22(22):3215-19 (1992)) was adapted. To a solution of 5,514,680 89 90 4-fluoro-2-nitroaniline (312 mg, 2.0 mMol, Aldrich, used as 22(22):3215-19 (1992)) was adapted. To a solution of received) in EtOH (40 mL) was added iodine (0.508 g, 2.0 4,5-dichloro-2-nitroaniline (4.14 g, 2.0 mMol, used as mMol, used as received) and AgSO4 (622 mg, 2.0 mMol, received) in EtOH (40 mL) was added iodine (521 g, 2.05 used as received). The mixture was stirred at room tempera mMol, used as received) and AgSO (622 mg, 2.0 mMol, ture for one day. TLC (CHCl) of the mixture showed that used as received). The mixture was stirred at room tempera it consisted of 40% starting material and 60% product. ture for one day (monitored by TLC), then the yellow Additional iodine (127mg, 0.5 mMol) and AgSO (311 mg, precipitate that formed was removed by filtration. The 1 mMol) was added. The mixture was stirred at room filtrate was evaporated to dryness under reduced pressure temperature for another day, then the yellow precipitate that giving 600 mg of crude 4,5-dichloro-6-iodo-2-nitroaniline. formed was removed by filtration and the filtrate was O This was dissolved in dichloromethane and washed with 5% evaporated to dryness under reduced pressure, giving 774 sodium hydroxide solution (20 mL) followed by water. The mg of crude 4-fluoro-6-iodo-2-nitroaniline. This was dis organic layer was dried over MgSO and evaporated to solved in dichloromethane and washed with 5% sodium dryness. The residue was chromatographed over silica gel hydroxide solution (20 mL), followed by water. After sepa and eluted with chloroform, giving crude product. This was ration of the layers, the organic layer was dried over MgSO4. 15 purified by preparative TLC (elution with chloroform) to and evaporated to dryness. The residue was chromato give pure 4,5-dichloro-6-iodo-2-nitroaniline (356mg, 53%), graphed on silica gel and eluted with chloroform, giving as a yellow powder. NMR (H, CDCl); 86.940 (s, 2H); crude 4-fluoro-6-iodo-2-nitroaniline. The sample was puri 8.378 (s, 1H). fied by preparative TLC (eluted with chloroform) to give B. Synthesis of 1,2-Diamino-4,5-dichloro-6-iodoben pure 4-fluoro-6-iodo-2-nitroaniline (466 mg, 83%), as a 20 Zene: To a stirred mixture of 4,5-dichloro-6-iodo-2-nitroa yellow powder. NMR (H, CDC1): 86.538 (o, 2H); 7.768 niline (216 mg, 0.648 mMol) in ethanol (5 mL) was added (q, JE3 Hz, J-6.9 Hz, 1H); 7,939 (q, J=3 Hz, J-6.9 Hz, SnCl2.H2O (730 mg, 3.24 mMol) in one portion. The 1H). mixture was refluxed at 80° C. (oil bath90° C) with stirring B. Synthesis of 4-fluoro-6-iodo-1,2-phenylenediamine: for 0.5 h to form a clear solution and then reflux was To a stirred mixture of 4-fluoro-6-iodo-2-nitroaniline (359 25 continued for another 0.5h. The solution was cooled to room mg, 1.273 mMol) in ethanol (10 mL) was added temperature and ice water (10 g) was added. The pH was SnCl2.H2O (1432 g, 6.365 mMol) in one portion. The adjusted to pH-7 with aqueous 5% NaHCO3 solution and mixture was refluxed at 80° C. (oil bath 90° C.) with stirring the mixture was extracted with ethyl acetate. The extract was for 0.5 h to form a clear solution, and then reflux was dried over MgSO and concentrated to dryness to give 156 continued for another 0.5h. The solution was cooled to room 30 mg (80%) of 1,2-diamino-4,5-dichloro-6-iodobenzene as a temperature and ice water (20 g) was added. The pH was brown solid. NMR (H, DMSO-d): 84.954 (s, 2H); 5.162 adjusted to pH-7 and the mixture was extracted with ethyl (s, 2H); 6.722 (s, 1H). acetate. The extract was dried over MgSO and evaporated C. Synthesis of 5-iodo-6,7-dichloro-1,4-dihydro-2,3-qui to dryness to give 232 mg (73%) of 4-fluoro-6-iodo-1,2- noxalinedione: The procedure of Foged, C. and Journal, P. phenylenediamine as a brown solid. NMR (H, CDCl3): 35 (J. of Lab. Compa, and Radiopharmac. XXXI (5):365-373 84.366 (s, 2H); 5.149 (s, 2H); 6.368 (tetra, 1H, J-3 Hz, (1992)) was adapted. To a stirred mixture of 1,2-diamino J=6.9 Hz); 6.655 (tetra, J-3.0 Hz, J-6.9 Hz, 1H). 4,5-dichloro-6-iodobenzene (70 mg, 0.23 mMol) in 2N HCl C. Synthesis of 5-iodo-7-fluoro-1,4-dihydro-2,3-quinoxa (10 mL) was added oxalic acid (32 mg, 0.25 mMol, used as linedione: The procedure of Foged, C. and Journal, P. (J. of received) in one portion. The mixture was refluxed at Lab. Compa, and Radiopharmac. XXXI (5):365-373 (1992)) 40 120°-5° C. for 3 h, then cooled to room temperature was adapted. To a stirred mixture of 4-fluoro-6-iodo-1,2- overnight. The mixture was centrifuged and the red solid phenylenediamine (232 mg, 0.92 mMol) in 2N HCl (10 mL) was washed twice with cold water (2x1 mL), collected by was added oxalic acid (126 mg, 1.0 mMol, used as received) filtration, and dried at 60° C. with reduced pressure for 2 h, in one portion. The mixture was refluxed at 120°-5°C. for giving 60 mg of crude title compound (73%), as a red 3 h, then cooled to room temperature overnight. The mixture 45 powder. The sample was dissolved in 1N NaOH (8 mL) and was centrifuged and the liquid layer was removed. The red the insoluble material was removed by filtration. The filtrate precipitate was washed with cold water (2x2 mL), collected was acidified to pH-6, giving 46 mg of title compound. by filtration, and dried at 60° C. with reduced pressure for 2 Crystallization from DMSO/HO gave 19 mg of pure prod h, giving 160 mg of crude title compound (57%), as a red uct (23%) as red microcrystals, mp: 335-8°C. (dec. began powder containing some impurities (NMR). The sample of 50 at 330° C). IR (KBr, cm): 3437, 3325, 1750, 1712, 1475, crude product was dissolved in 1NNaOH (10 mL) and some 1393. NMR (H, DMSO-d): 87.273 (s, 1H); 10.282 (s, 1H); insoluble material was removed by filtration. The filtrate was 12.038 (s, 1H). HRMS, calcd for CHNOClI (M-) m/z: acidified to pH=6, giving 156 mg of purified product. 355.8614; found: 355.8603. Potency relative to DCK: Crystallization from DMSO/HO gave 149 mg of pure title 152%. compound (51%) as red microcrystals, mp. 310-2 C. 55 (changed color, from 242° C). IR (KBr, cm) 3431, 3350, 3062, 1743, 1718, 1606, 1518, 1400. NMR (H, DMSO-d): Example 66 86.947 (q, 1H, J-2.7 Hz, J-93 Hz); 6.963 (q, 1H, J-2.7 Preparation of 5-Iodo-6-nitro-7-chloro-1,4-dihydro-2,3-qui Hz, J-9.3 Hz); 10.313 (s, 1H); 12.054 (s, 1H). HRMS calcd noxalinedione for CHNOFI (M+) m/z: 305.9301; found: 3059288. 60 The method of Cheeseman, G. W. H. (J. Chem. Soc. 1170 Potency relative to DCK: partially active. (1962)) was adapted. 5-Iodo-7-chloro-1,4-dihydro-2,3-qui noxalinedione (96 mg, 0.33 mMol) was dissolved in con Example 65 centrated HSO (1.0 mL) at 0° C. for 30 min and then Preparation of 5-Iodo-6,7-dichloro-1,4-dihydro-2,3-qui KNO (36 mg, 0.36 mMol, Baker) was added into this noxalinedione 65 solution. The mixture was stirred at 0°C. for 0.5 h and then A. Synthesis of 4,5-dichloro-6-iodo-2-nitroaniline: The at room temperature for 30 h. It was poured into ice water procedure of Sy, W. W. (Synthetic Communications (5g). A precipitate came out and was collected by filtration 5,514,680 91 92 giving 101 mg of crude title compound. The sample was pressure giving 2.74 g of crude product. The sample was dissolved in 1N KOH (5 mL) and then red precipitate was dissolved in dichloromethane and washed with 5% sodium removed by filtration. The filtrate was acidified to pH=2 with hydroxide solution (40 mL), followed by water. The organic 4N HCl to give a brown precipitate which was collected by layer was dried over MgSO and evaporated to dryness. The filtration and then dried in air at 50 C. for 4 h. The title residue was chromatographed over silica gel and eluted with compound (75 mg, 67%) was obtained as a brown powder. chloroform. Preparative TLC (elution with chloroform) gave Crystallization from DMSO/HO afforded pure compound pure 4-iodo-2-nitroaniline (1.8 g, 68.0%) as a yellow pow (34 mg) as brown microcrystals, mp: 388°-90° C. IR (KBr, der. NMR (H, CDCl): 84.832 (s, 2H); 6.658 (d, J=8.7 Hz, cm): 3462, 3200, 3050, 1712, 1587, 1537. NMR (H, 1H); 7.595 (dd, J-1.5 Hz, J-8.7 Hz, 1H); 8.442 (d, J=1.5 DMSO-d): 87.289 (s, 1H), 10.846 (s, 1H), 12.225 (s, 1H). 10 Hz, 1H). HRMS: calcd for CHNOCII (M+) m/z: 366.8855, found B. Synthesis of 6-Iodo-1,4-dihydro-2,3-quinoxalinedi pending. Potency relative to DCK: partially active. one: The procedure of Foged, C. and Journal, P. (J. of Lab. Compa and Radiopharmac. XXXI (5):365-373 (1992)) was Example 67 adapted. To a stirred mixture of 2-nitro-4-iodoaniline (1.8 g., 15 6.9 mMol) in ethanol (40 mL) was added SnCl2.H2O (7.8 Preparation of 5-Iodo-7-chloro-6,8-dinitro-1,4-dihydro-2,3- g, 34.6 mMol) in one portion. The mixture was refluxed at quinoxalinedione 80° C. (oil bath 90° C) with stirring for 0.5 h to form a clear The method of Cheeseman, G. W. H. (J. Chem. Soc. 1170 solution and the reflux was continued for another 1.5h. The (1962) was adapted. 5-Iodo-7-chloro-1,4-dihydro-2,3-qui solution was cooled to room temperature and ice water (100 noxalinedione (74 mg, 0.23 mMol) was dissolved in con 20 g) was added. The pH was adjusted to pH-7 and the mixture centrated HSO, (1.0 mL) at 25° C. for 30 min and then was extracted with ethyl acetate. The extract was dried over KNO (116 mg, 1.17 mMol, Baker) was added into the MgSO and concentrated to dryness to give 1.235 g (78%) solution. The mixture was stirred at 25 C. for 12 h and at of title compound as a brown solid. To a stirred mixture of 100° C. for 4 h. After the mixture cooled to room tempera this product (450 mg, 1.92 mMol) in 4N HCl (20 mL) was ture, it was poured into ice water (5 g). A precipitate came 25 added oxalic acid (267 mg, 2.115 mMol, used as received) out and was collected by filtration. It was dissolved in 1N in one portion. The mixture was refluxed at 120°-5° C. for KOH (10 mL), filtered, and the filtrate was acidified to pH=5 3 h, then cooled to room temperature overnight. The mixture with 4N HCl to give a red precipitate. This was dried in air was centrifuged and the red precipitate was washed with at 50 C. for 4 h affording the title compound (27 mg, 28%) cold water (2x2 mL), collected by filtration and dried at 60 as a dark red powder, mp: 240-2 C. (with dec, change 30 C. at reduced pressure for 2 h, affording 140 mg (25%) of color from 180°-5° C). IR (KBr, cm): 3431, 3218, 3143, crude product as a white powder. The sample was dissolved 1712, 1550, 1400, 559. HRMS: calcd. for CHNOCII in 1N NaOH (10 mL), filtered, and the filtrate was acidified (M-) m/z.: 411.8705, found: 411.8713. to pH-6, affording 130 mg of product which was washed with EtOH (2 mL). Crystallization from DMSO/HO gave Example 68 35 19 mg of pure title compound as white microcrystals, mp. Preparation of 5,8-Diiodo-6,7-dichloro-1,4-dihydro-2,3-qui 355° 7° C. IR (KBr, cm) 3459, 3148, 1750, 1704, 1392. noxalinedione NMR (H, DMSO-d): 86.727 (d, J=8.1 Hz, 1H), 7.053 (dd, The method of Leeson, P. D. et al. (J. Med. Chem. J=1.8 Hz, J-8.4 Hz, 1H), 11.952 (s, 1H), 11.998 (s, 1H). 34:1243-1252 (1991)) was adopted. 6,7-Dichloro-1,4-dihy HRMS: calcd for CHNOI (M+) m/z 287.9393. Found: dro-2,3-quinoxalinedione (92 mg, 0.40 mMol) was dis 287.9390. Potency relative to DCK: 4.7%. solved in concentrated HSO (2.0 mL) at room temperature for 30 min and then ICl (383 mg, 2.16 mMol, Aldrich) was Example 70 added to this solution. The mixture was stirred at 115-20 Preparation of 6,7-Dibromo-5,8-diiodo-1,4-dihydro-2,3- C. for 14 h. It was cooled to room temperature and poured quinoxalinedione into ice water (10 g). A precipitate came out and was 45 The method of Leeson, P. D. et al. (J. Med Chem. collected by filtration. The precipitate was dissolved in 1N 34:1243–1252 (1991)) was adopted. 6,7-Dibromo-1,4-dihy KOH (10 mL), filtered, and the filtrate was acidified to pH=5 dro-2,3-quinoxalinedione (105 mg, 0.33 mMol) was dis with 4N HCl to give ared precipitate. This was collected by solved in concentrated HSO (2.0 mL) at 0°C. for 30 min. filtration and dried in air at 50° C. for 4 h affording the title Then ICI (358 mg, 2.19 mMol, Baker) was added. The compound (147 mg, 75%) as a white powder. Crystallization 50 mixture was stirred at 120° C. for 14 h and then cooled to from DMSO/HO gave white microcrystals (96 mg, 50%), room temperature and poured into ice water (5 g). The mp: 353°-4° C. (dec. from 315° C). IR (KBr, cm): 3428, precipitate was collected by filtration and dissolved in 1N 3189, 3142, 1741, 1688, 1462, 1396,579. HRMS: calcd. for KOH (10 mL). A red precipitate was removed by filtration CHNOCl (M+) m/z: 481.7579, found 481.7575. and the filtrate was acidified to pH=2 with 4N HCl to give 55 a white precipitate. This was collected by filtration and dried Example 69 in air at 50° C. for 4h affording the title compound (240 mg, Preparation of 6-Iodo-1,4-dihydro-2,3-quinoxalinedione >100%) as a white powder. Crystallization from EtOH gave A. Synthesis of 4-Iodo-2-nitroaniline: The procedure of pure title compound (140 mg, 75%), mp.>350° C. IR (KBr, Sy, W. W. (Synthetic Communications 22(22):3215-19 60 cm): 3437,3287, 1718, 1593, 1550. NMR ('H, DMSO-d) (1992)) was adapted. To a solution of 2-nitroaniline (1.38g, 812.297 (s, 2H). 10.0 mMol, Aldrich, used as received) in EtOH (100 mL) Example 71 was added iodine (2.54 g, 10.0 mMol, used as received) and AgSO, (3.11 g, 10.0 mMol, used as received). The mixture Preparation of 6-Amino-5-chloro-7-trifluoromethyl-1,4-di was stirred at room temperature for 1 h (monitored by TLC). 65 hydroquinoxaline-2,3-dione The yellow precipitate thatformed was removed by filtration A mixture of 5-chloro-6-nitro-7-trifluoromethyl-1,4-dihy and the filtrate was evaporated to dryness under reduced droquinoxaline- 2,3-dione (230 mg, 0.743 mmol), 5,514,680 93 94 SnCl2HO (1.90 g, 7.43 mmol) and EtOH (300 mL) was mmol, used as received) in 2N HCl (4 mL) was refluxed at refluxed for 24 h, then rota-evaporated to dryness. The 120°-5° C. for 3 h, then cooled to room temperature. The residual solid was washed on a Bursch funnel with water mixture was centrifuged and the liquid layer was removed. (6x15 mL), and dried to give 153 mg (74%) of the title The yellow solid was washed by cold water (2x2 mL), compound as a yellow powder. Mp2360° C. (H NMR collected by filtration, and dried at 60° C. for 2 h, affording (DMSO-d) 11.777 (s, 1H), 11.360 (s, 1H), 7.140 (s, 1H), 136 mg of crude title compound (53%) as a yellow powder. 5.499 (s, 2H). The crude title compound was dissolved in 1N NaOH (6 mL) and filtered. The filtrate was acidified to pH=6, afford Example 72 ing 115 mg of title compound. Recrystallization from Preparation of 8-Amino-5-chloro-7-Trifluoromethyl-1,4-di 10 DMSO/HO gave 95 mg of pure title compound (37%) as hydroquinoxaline-2,3-dione yellow microcrystals. Mp: 302-4°C. (dec. from 295 C.). A mixture of 5-chloro-3-nitro-7-trifluoromethyl-1,4-dihy IR (KBr, cm) 3401, 3255, 1754, 1694, 1528. 'H NMR droquinoxaline-2,3-dione (25 mg, 0.08mmol), SnCl2.H2O (DMSO-d): 57.374 (d, 1H, J=1.8 Hz); 7.474 (d, 1H, J=1.8 (97 mg, 0.34 mmol) and EtOH (3 mL) was heated at 75° C. Hz); 11.347 (s, 1H); 12.215 (s, 1H). HRMS: calcd for with stirring for 12 h. Work-up as above gave 21 mg (93%) 15 CHCIFNO, (M) m/z: 263.9912; found: 263.9909. of the title compound as a yellow powder. MpD360° C. "H Example 74 NMR (DMSO-d) 11.441 (s, 1H), 11.324 (s, 1H), 7.241 (s, 1H), 5.916 (s, 2H). Preparation of 7-Chloro-6-nitro-5-trifluoromethyl-1,4-dihy dro-2,3-quinoxalinedione Example 73 20 To a solution of 7-chloro-5-trifluoromethyl-1,4-dihydro 2,3-quinoxalinedione (29 mg, 0.16 mmol) in concentrated Preparation of 7-Chloro-5-trifluoromethyl-1,4-dihydro-2,3- HSO, (0.5 mL) at 0°C. was added KNO (11.6 mg, 0.115 quinoxalinedione mmol). The mixture was stirred at 0°C. for 0.5 h, then at A. 2-Acetamido-5-chlorobenzotrifluoride room temperature for 12 h and it was poured into ice water A solution of 2-amino-5-chlorobenzotrifluoride (1.01 g, 25 (2 g). The precipitate was collected by filtration, affording 31 5.16 mmol, Aldrich) in acetic anhydride (5 mL) was stirred mg (91.4%) of crude title compound. It was dissolved in 1N at room temperature for 12 h to produce white needle KOH (1 mL) and filtered. The filtrate was acidified to pH=2 precipitate. It was filtered to give 1.123 g (91.7%) of with 4N HCl to give a yellow precipitate, which was 2-acetamido-5-chlorobenzotrifluoride. "H NMR (CDCl3): collected by filtration, and dried at 50° C. for 4 h, affording 82.214 (s, 3H); 7.359 (br, 1H); 7.512 (d, 1H, J=9 Hz), 7.581 30 the title compound (23 mg, 67%) as a yellow powder. (s, 1H); 7.163 (d. 1H, J=8.4 Hz). B. 2-Acetamido-5-chloro-3-nitrobenzotrifluoride Recrystallization from DMSO/HO gave pure title com To a solution of 2-acetamido-5-chlorobenzotrifluoride pound as yellow microcrystals. Mp: 278-80° C., IR (KBr, (890 mg, 3.74 mmol) in concentrated HSO (4 mL) at 0°C. cm): 3425, 3150, 3125, 1712, 1618, 1556; H NMR was added dropwise 70% HNO (0.5 mL, Baker). The 35 (DMSO-d): 87.531 (s, 1H), 11.803 (s, 1H), 12.459 (s, 1H). mixture was stirred at 0° C. for 0.5 h, then at room HRMS: calcd. for CHCIF.N.O. (M) m/z: 308.9763, temperature for 3 hand it was poured into ice water (15g). found: 308.9759. The precipitate was collected by filtration, affording 700 mg Example 75 of crude 2-acetamido-5-chloro-3 -nitrobenzotrifluoride. It was crystallized from EtOH/HO to give 559 mg of pure 40 Preparation of 7-Bromo-5-trifluoromethyl-1,4-dihydro-2,3- 2-acetamido-5-chloro-3-nitrobenzotrifluoride as yellow quinoxalinedione needles. Mp 190°-2°C., H NMR (CDC): 82.210 (s, 3H), A. 2-Acetamido-5-bromobenzotrifluoride 7.357 (s, 1H), 7.896 (d, 1H, J-2.1 Hz), 8.105 (d. 1H, J-2.1 A solution of 2-amino-5-bromobenzotrifluoride (1.623 g, Hz). 6.76 mmol, Aldrich) in acetic anhydride (10 mL) was stirred C. 2-Amino-5-chloro-3-nitrobenzotrifluoride 45 at room temperature for 12 h to produce a white needle A mixture of 2-acetamido-5-chloro-3-nitrobenzotrifluo precipitate. It was filtered to give 1.840 g (99.7%) of title ride (354 mg, 1.252 mmol) in concentrated HCl (3 mL) was compound. Mp: 140°-2° C., HNMR (CDC): 82.212 (s, refluxed overnight, and it was extracted by ethyl acetate 3H); 7.358(s, 1H); 7.659(d, 1H, J=8.7 Hz); 7.726(d, 1H, (2x3 mL). The extract was dried (MgSO) and evaporated J=1.8 Hz); 8.118 (d. 1H, J=8.4 Hz). to give 250 mg (83%) of 2-amino-5-chloro-3-nitrobenzot 50 B. 2-Acetamido-5-bromo-3-nitrobenzotrifluoride rifluoride. "H NMR (CDC): 86.663 (s, 2H), 7.717 (s, 1H), To 2-acetamido-5-bromobenzotrifluoride (665 mg, 2.436 8.360 (s, 1H). mmol) in concentrated HSO (4 mL) at 0°C. was added D. 2,3-Diamino-5-chlorobenzotrifluoride dropwise 70% HNO (0.4 mL, Baker). The mixture was To a stirred mixture of 2-amino-5-chloro-3-nitrobenzot stirred at 0°C. for 0.5 h, then at room temperature for 3 h rifluoride (250 mg, 1.043 mmol) in ethanol (4 mL) was 55 and it was poured into ice water (15g). The precipitate was added SnCl2.H2O (1.174g, 5.217 mmol) in one portion. collected by filtration, affording 700 mg (90%) of crude The mixture was refluxed at 80° C. (oil bath 90° C.) for 1 h. 2-acetamido-5-bromo-3-nitrobenzotrifluoride. It was crys The solution was cooled to room temperature and ice water tallized from EtOH/HO to offer 610 mg (78.8%) of pure (20g) was added. It was adjusted to pH=7 and extracted with compound as white needles. Mp: 193°-5° C., H NMR ethyl acetate. The extract was dried (MgSO) and evapo 60 (CDC): 82.250 (s, 3H), 7.346 (s, 1H), 8.039 (s, 1H); 8.247 rated to give 181 mg (83%) of 2,3-diamino-5-chlorobenzot (s, 1H). rifluoride as a brown solid. "H NMR (CDC): 83.515 (s, C. 2-Amino-5-bromo-3-nitrobenzotrifluoride 2H); 3.878 (s, 2H); 6.845 (s, 1H); 6.988 (s, 1H). A mixture of 2-acetamido-5-bromo-3-nitrobenzotrifluo E. 7-Chloro-5-trifluoromethyl-1,4-dihydro-2,3-quinoxa ride (400 mg, 1.258 mmol) in concentrated HCl (3.5 mL) linedione 65 was refluxed overnight, then it was extracted by ethyl acetate A mixture of 2,3-diamino-5-chlorobenzotrifluoride (180 (2x3 mL). The extract was dried over MgSO and evapo mg, 0.86 mmol) and oxalic acid dihydrate (108 mg, 0.86 rated to give 261 mg (75.6%) of 2-amino-5-bromo-3-ni 5,514,680 95 96 trobenzotrifluoride. "H NMR (CDCl): 86.663 (S, 2H); To 2-acetamido-5-fluorobenzotrifluoride (1.369 g, 6.19 7.825 (d, 1H, J=1.2 Hz); 8.487 (d, 1H, J=1.2 Hz). mmol) in concentrated H2SO (6 mL) at 0° C. was added D. 2,3-Diamino-5-bromobenzotrifluoride dropwise 70% HNO (0.8 mL, Baker). The mixture was To a stirred mixture of 2-amino-5-bromo-3-nitrobenzot stirred at 0°C. for 0.5 h, then at room temperature for 3 h rifluoride (261 mg, 0.949 mmol) in ethanol (4 mL) was and it was poured into ice water (25 g). The precipitate was added SnCl2.H2O (1.076 g, 4.745 mmol) in one portion. collected by filtration, affording 2.1 g of crude 2-acetamido The mixture was refluxed at 80° C. (oil bath 90° C.) for 1 h. 5-fluoro-3-nitrobenzotrifluoride. It was crystallized from The solution was cooled to room temperature and ice water EtOH/HO to afford 1.50 g of pure compound as yellow (20g) was added. It was adjusted to pH=7 with NaHCO and needles. extracted by ethyl acetate (2x4 mL). The extract was dried 10 C. 2-Amino-5-fluoro-3-nitrobenzotrifluoride over MgSO and evaporated to give 150 mg (64.6%) of A mixture of 2-acetamido-5-fluoro-3-nitrobenzotrifluo 2,3-diamino-5-bromobenzotrifluoride as a brown solid. H ride (1.50 g, 5.64 mmol) in concentrated HCl (10 mL) was NMR (CDC1): 83.503 (s, 2H); 3.898 (s, 2H); 6.987 (d, 1H, refluxed overnight and it was extracted by ethyl acetate J=1.8 Hz); 7.123 (d, 1H, J=1.8 Hz). (2x10 mL). The extract was dried over MgSO, and evapo E. 7-Bromo-5-trifluoromethyl-1,4-dihydro-2,3-quinoxa 15 rated to give 624 mg of 2-amino-5-fluoro-3-nitrobenzotrif. linedione luoride (50%). H NMR (CDCl): 86.547 (s, 2H), 7.570 (dd, A mixture of 2,3-diamino-5-bromobenzotrifluoride (150 1H, J-2.7 Hz, J-5.7 Hz), 8.114 (dd, 1 H, J-2.7 Hz, J-5.7 mg, 0.612 mmol) and oxalic acid dihydrate (81 mg, 0.643 Hz). mmol, used as received) in 2N HCl (4 mL) was refluxed at D. 2,3-Diamino-5-fluorobenzotrifluoride 120°-5 C. for 3 h, then cooled to room temperature. The 20 To a stirred mixture of 2-amino-5-fluoro-3-nitrobenzotri mixture was centrifuged and the liquid layer was removed. fluoride (554 mg, 2.47 mmol) in ethanol (10 mL) was added The yellow solid was washed twice by cold water (2x2 mL), SnCl2.H2O (2.78 g, 12.36 mmol) in one portion. The collected by filtration, and dried at 60° C. for 2 h, affording mixture was refluxed at 80° C. (oil bath 90° C) for 1 h, 150 mg of crude title compound (81.9%) as a red powder. cooled to room temperature and ice water (20g) was added. The crude compound was dissolved in 1NNaOH (6 mL) and 25 It was adjusted to pH=7 and extracted with ethyl acetate. The filtered. The filtrate was acidified to pH=6, affording 135 mg extract was dried over MgSO and evaporated to give 148 of title compound. Recrystallization from DMSO/HO gave mg (30%) of 2,3-diamino-5-fluorobenzotrifluoride as a 125 mg of pure title compound (68.3%) as red microcrystals. brown solid. 'H NMR (CDC1): 83.666 (s, 2H); 3.676 (s, Mp: 307-9 C. (dec. from 290° C). IR (KBr, cm) 3412, 2H); 6.610 (dd, 1H, J-2.4 Hz, J=9.0 Hz), 6.703 (dd, 1H, 3250, 1756, 1706, 1606; H NMR (DMSO-d): 87.514 (d, 30 J=24 Hz, J-9.0 Hz). 1H, J=1.5 Hz); 7.556 (d, 1H, J=1.5 Hz); 11.342 (s, 1H); E. 7-Fluoro-5-trifluoromethyl-1,4-dihydro-2,3-quinoxa 12.220 (s, 1H). HRMS: calcd for CHBrFNO, (M) m/z: linedione 307.9407; found: 307.9420, A mixture of 2,3-diamino-5-fluorobenzotrifluoride (148 mg, 0.76 mmol) and oxalic acid dihydrate (96 mg, 0.76 Example 76 35 mmol, used as received) in 4N HCl (2 mL) was refluxed at 120°-5°C. for 3 h, then cooled to room temperature. The Preparation of 7-Bromo-6-nitro-5-trifluoromethyl-1,4-dihy mixture was centrifuged and the liquid layer was removed. dro-2,3-quinoxalinedione The yellow solid was washed by cold water (2x2 mL), To a solution of 7-bromo-5-trifluoromethyl-1,4-dihydro collected by filtration, and dried at 60° C. for 2 h, affording 2,3-quinoxalinedione (31 mg, 0.10 mmol) in concentrated 120 mg of crude title compound (64%) as a yellow powder. HSO (0.5 mL) at 0°C. was added KNO (11 mg, 0.109 mmol, Baker). The mixture was stirred at 0° C. for 0.5 h, The crude product was dissolved in 1N NaOH (5 mL) and then at room temperature for 12 h and it was poured into ice filtered. The filtrate was acidified to pH-6, affording 110 mg water (2 g). The precipitate was collected by filtration, of pure title compound as a yellow powder, Mp: 300-2 C. affording 32 mg (91.4%) of crude title compound. It was IR (KBr, cm): 3408, 3122, 1754, 1721, 1621, 1492. H dissolved in lN KOH (1 mL) and filtered. The filtrate was 45 NMR (DMSO-d): 87.178 (dd, 1H, J-2.7 Hz, J=8.7 Hz), acidified to pH 2 with 4N HCl to give a brown precipitate, 7.359 (dd, 1H, J-2.7 Hz, J-8.7 Hz); 11.270 (s, 1H); 12.237 which was collected by filtration, and dried at 50° C. for 4 (s, 1H). HRMS: calcd for CHNO (M) m/z: 248.0208; h, affording pure title compound (28 mg, 79.3%) as a yellow found: 248.0220. Example 78 powder. Mp: 292-94 C., IR (KBr, cm): 3412, 3068, 50 3937, 1718, 1556, 1387. H NMR (DMSO-d): 87.644 (s, Preparation of 7-Fluoro-6-nitro-5-trifluoromethyl-1,4-dihy 1H), 11.721 (s, 1H), 12.423 (s, 1H). HRMS: calcd for dro-2,3-quinoxalinedione CHCIFNO (M) m/z: 352.9258, found: 352.92.59. To a solution of 7-fluoro-5-trifluoromethyl-1,4-dihydro Example 77 2,3-quinoxalinedione (60 mg, 0.24 mmol) in concentrated 55 HSO (1 mL) at 0°C. was added KNO (27 mg, 0.26 mmol, Preparation of 7-Fluoro-5-trifluoromethyl, 1,4-dihydro-2,3- Baker). The mixture was stirred at 90-100° C. for 14 h, then quinoxalinedione cooled to room temperature and poured into ice water (2 g). A. 2-Acetamido-5-fluorobenzotrifluoride The precipitate was collected by filtration, affording 64 mg A solution of 2-amino-5-fluorobenzotrifluoride (2.025 g, (90%) of crude title compound. It was dissolved in 1N KOH 11.31 mmol, Aldrich) in acetic anhydride (10 mL) was (2 mL) and filtered. The filtrate was acidified to pH=2 with stirred at room temperature for 12 h, and then most of the 4N HCl to give a yellow precipitate, which was collected by acetic anhydride was removed. The white needle crystals filtration, then was dried in the air at 50° C. for 4h, affording were collected by filtration, affording 2.445 g (98%) of pure title compound (54 mg, 76%) as a yellow powder. Mp: 2-acetamido-5-fluorobenzotrifluoride. "H NMR (CDC): 305-307° C.; IR (KBr, cm): 3418, 3124, 2971, 1717, 82.205 (s, 3H); 7.229-7.335 (m, 3H); 8.055 (dd, 1H, J,-4.8 65 1624, 1556; H NMR (DMSO-d): 87,422 (d. 1H, J=102 Hz, J=8.4 Hz). Hz), 11.765 (br, 1H), 12.532 (s, 1H). HRMS: calcd for B, 2-Acetamido-5-fluoro-3-nitrobenzotrifluoride C.H.F.N.O. (M) m/z: 293.0086, found: 293,0071. 5,514,680 97 98 Example 79 H2SO4 (0.3 mL) at 0°C. was added KNO (14 mg, 0.141 mmol, Baker). The mixture was stirred at 0°C. for 0.5 h and Preparation of 6-Chloro-7-trifluoromethyl-1,4-dihydro-2,3- then at room temperature for 20h and it was poured into ice quinoxalinedione water (2 g). The precipitate was collected by filtration, A. 3-Acetamido-6-chlorobenzotrifluoride giving 31 mg of crude title compound. The sample was A solution of 3-amino-6-chlorobenzotrifluoride (1.045 g, dissolved in 1N KOH (1 mL) and filtered. The filtrate was 5.34 mmol, Aldrich) in acetic anhydride (5 mL) was stirred acidified to pH=2 with 4N HCl to give a yellow precipitate at room temperature for 12 h to produce a white needle which was collected by filtration and then dried in air at 50 precipitate. It was filtered to give 1.250 g (98.5%) of C. for 4 h to give the title compound (29 mg, 80%) as a 3-acetamido-6-chlorobenzotrifluoride. "H NMR (CDC): 10 yellow powder. Crystallization from DMSO/HO gave pure 82.193 (s, 3H); 7.420 (d, 1H, J=8.7 Hz); 7.726 (m, 2H), compound (18 mg) as yellow microcrystals. mp: 342°-5°C., 7.804 (s, 1H). B. 3-Acetamido-6-chloro-4-nitrobenzotrifluoride IR (KBr, cm): 3394,3301,3248,3209, 1761, 1701, 1628, To 3-acetamido-6-chlorobenzotrifluoride (908 mg, 3.82 1542. "HNMR (DMSO-d): 87.618 (s, 1H), 12.351 (s, 1H), mmol) in concentrated HSO (4 mL) at 0°C. was added 12.552 (s, 1H). HRMS: calcd for CHCIF.N.O. (M) m/z: dropwise 70% HNO (0.5 mL, Baker). The mixture was 15 308.9763, found: 308.9759. stirred at 0°C. for 0.5h, then at room temperature 3 h, and Example 81 it was poured into ice water (15 g). The precipitate was collected by filtration, affording 990 mg (91%) of crude Preparation of 6-Bromo-7-trifluoromethyl-1,4-dihydro-2,3- 3-acetamido-6-chloro-4-nitrobenzofluoride. It was crystal quinoxalinedione lized. from EtOH/HO to give 770 mg of pure compound as 20 A. 6-Bromo-3-(ethyl oxalamido)benzotrifluoride yellow needles. HNMR (CDC): 82.326 (s, 3H), 8.345 (s, To a stirred solution of 3-amino-6-bromobenzotrifluoride 1H), 9.293 (s, 1H), 10.222 (s, 1H). (2.40 g, 10.00 mmol, Aldrich) in dried THF (10 mL) and C. 3-Amino-6-chloro-4-nitrobenzotrifluoride triethylamine (1 mL) in an ice bath was dropwise added A mixture of 3-acetamido-6-chloro-4-nitrobenzotrifluo ethyl oxalyl chloride (1.7 mL, 16 mmol, Aldrich). The ride (780 mg, 2.76 mmol) in concentrated HCl (3 mL) was 25 resulting yellow suspension was stirred at 25 C. for 3 hand refluxed overnight and it was extracted by ethyl acetate (2x3 it was poured into ice water (50 mL). The precipitate was mL). The extract was dried over MgSO and evaporated to collected by filtration to give 3.304 g (97%) of crude give 517 mg (78%) of 3-amino-6-chloro-4-nitrobenzofluo 6-bromo-3-(ethyl oxalamido)benzotrifluoride. Crystalliza ride. "H NMR (CDC1): 86.205 (s, 2H); 7.206 (s, 1H); 8.263 30 tion from ethanol/water gave 2.95 g (85%) of pure com (s, 1H). pound. H NMR (CDC1): 81.434 (t, 3H, J=6.9 Hz); 4.432 D. 3,4-Diamino-6-chlorobenzotrifluoride (q, 2H, J=6.9 Hz), 7.715 (d, 1H, J=8.7 Hz); 7.802 (d, 1H, To a stirred mixture of 3-amino-6-chloro-4-nitrobenzot J=8.7 Hz); 7.946 (d, 1H, J=1.8 Hz), 8.991 (s, 1H). rifluoride (300 mg, 1.25 mmol) in ethanol (5 mL) was added B. 6-Bromo-3-(ethyl oxalamido)-4-nitrobenzotrifluoride SnCl2H2O (1.40 g, 6.20 mmol) in one portion. The mix To 6-bromo-3-(ethyl oxalamido)benzotrifluoride (2.324 ture was refluxed at 80°C. (oil bath, 90° C.) for 1 h and the 35 g, 6.815 mmol) in concentrated HSO (4 mL) at 0°C. was solution was cooled to room temperature and ice water (20 added KNO (757 mg, 7.496 mmol, Baker). The mixture g) was added. It was adjusted to pH=7 and extracted with was stirred at 0°C. for 0.5 h, then at room temperature for ethyl acetate. The extract was dried over MgSO and 13 hand it was poured into ice water (15g). The precipitate evaporated to give 185 mg (71%) of 3,4-diamino-6-chlo was collected by filtration, affording 2.5 g (95%) of crude robenzotrifluoride as a brown solid. 'H NMR (CDC1): 40 6-bromo-3-(ethyl oxalamido)-4-nitrobenzotrifluoride. It was 83.404 (s, 2H); 3.717 (s, 2H); 6.754 (s, 1H); 6.974 (s, 1H). crystallized from EtOH/HO, affording 2.10 g (80%) of pure E. 6-Chloro-7-trifluoromethyl, 4-dihydro-2,3-quinoxa compound as yellow needles. "H NMR (CDC1): 81.460 (t, linedione 3H, J=7.2 Hz), 4.478 (q, 2H), 8.602 (s, 1H), 9.297 (s, 1H); A mixture of 3,4-diamino-6-chlorobenzotrifluoride (185 11.806 (s, 1H). mg, 0.88 mmol) and oxalic acid dihydrate (117 mg, 0.93 45 C. 6-Bromo-7-trifluoromethyl-1,4-dihydro-2,3-quinoxa mmol, used as received) in 2N HCl (4 mL) was refluxed at linedione 170°-5°C. for 3 h, then cooled to room temperature. The To a stirred mixture of 6-bromo-3-(ethyl oxalamido)-4- mixture was centrifuged and the liquid layer was removed. nitrobenzotrifluoride (102 mg, 0.26 mmol) in DMF (3 mL) The yellow solid was washed twice by cold water (2x2 mL), was added SnCl2H2O (300 mg, 1.33 mmol) in one portion. collected by filtration, and dried at 60° C. with reduced 50 The mixture was refluxed at 80° C. (oil bath 90° C) for 5.5 pressure for 2 h, affording 180 mg of crude title compound h. The solution was cooled to room temperature and ice (77.3%) as a light yellow powder. The crude product was water (5 g) was added. The precipitate was collected by dissolved in 1N NaOH (6 mL) and filtered. The filtrate was filtration and the solid was dissolved in 1N NaOH (5 mL). acidified to pH=6, affording 138 mg of title compound. It The solution was adjusted to pH-5 to give a precipitate was recrystallized from DMSO/HOtwice to give 102 mg of 55 which was filtered, washed by water (1 mL) and dried at 60 pure compound (43.9%) as yellow microcrystals. mp:>360° C. under reduced pressure for 2 h, affording 50 mg of pure C. (dec. from 295° C). IR (KBr, cm) 3425, 3200, 1731, title compound (62%) as a light yellow powder. Mp: >360° 1706, 1625; 1400. "H NMR (DMSO-d): 87.274 (s, 1H); C. (dec. from 295° C). IR (KBr, cm) 3420, 3071, 1700, 7.478 (s, 1H); 12.145 (s, 2H). HRMS: calcd for 1618; 1302. HNMR (DMSO-d): 87.543 (s, 1H); 7.735 (s, CHCIF.N.O. (M) m/z: 263.9912; found: 263.9919. 60 1H); 12.046 (s, 1H), 12.305 (s, 1H). Example 80 Example 82 Preparation of 6-Chloro-5-nitro-7-trifluoromethyl-1,4-dihy Preparation of 6-Bromo-5-nitro-7-trifluoromethyl-1,4-dihy dro-2,3-quinoxalinedione 65 dro-2,3-quinoxalinedione To a solution of 6-chloro-7-trifluoromethyl-1,4-dihydro To a solution of 6-bromo-7-trifluoromethyl-1,4-dihydro 2,3-quinoxalinedione (31 mg, 0.12 mmol) in concentrated 2,3-quinoxalinedione (40 mg, 0.13 mmol) in concentrated 5,514,680 99 100 HSO, (0.5 mL) at 25°C. was added KNO (16 mg, 0.15 The filtrate was acidified to pH=5, affording 138 mg of title mmol, Baker). The mixture was stirred at 25°C. for 30 hand compound as a yellow powder. Mp: 330-333°C. IR (KBr, poured into ice water (2 g). The yellow precipitate was cm) 3398, 3170,2959, 1701, 1641, 1515; 1398. H NMR collected by filtration and the solid was dissolved in 1N (DMSO-d): 87.079 (d. 1H, J=11.4 Hz); 7.376 (d, 1H, J=5.1 KOH (10 mL). It was filtered and the filtrate was acidified 5 Hz); 12.022 (s, 1H); 12.225 (s, 1H). HRMS: calcd for to pH=5 with 4N HCl (2.5 mL) to give a yellow precipitate. CHF.N.O. (M) m/z: 248,0208; found: 248,0220. The precipitate was collected and dried in the air at 50° C. for 4 h, affording the title compound (34 mg, 74%) as a Example 84 yellow powder. Recrystallization from DMSO/HO gave 18 mg (40%) of the title compound as yellow needles. Mp: 10 Preparation of 5,6-Dinitro-7-bromo-1,4-dihydro-2,3-qui 333°-5° C. (dec.); IR (KBr, cm): 3428, 3308, 3251,3207, noxalinedione and 5,7-dinitro-6-bromo-1,4-dihydro-2,3- 1761, 1698, 1537, 600. "H NMR (DMSO-d): 812.601 (br, quinoxalinedione 1H), 12.341 (s, 1H), 7.606 (s, 1H). To a solution of 6-bromo-7-nitro-1,4-dihydro-2,3-qui noxalinedione (73 mg, 0.255 mmol) in concentrated HSO 15 (0.5 mL) at 0°C. was added KNO (38 mg, 0.376 mmol, Example 83 Baker). The mixture was stirred at 100° C. for 48 h and then Preparation of 6-Fluoro-7-trifluoromethyl-1,4-dihydro-2,3- cooled to room temperature. It was poured into ice water (5 quinoxalinedione g) and the yellow precipitate was collected by filtration. It A. 3-Acetamido-6-fluorobenzotrifluoride was dissolved in 1N NaOH (5 mL) and filtered. The filtrate A solution of 3-amino-6-fluorobenzotrifluoride (2.0 g, 20 was acidified to pH=2 with 4N HCl to give a yellow 11.17 mmol, Aldrich) in acetic anhydride (10 mL) and Etn precipitate, which was collected by filtration, then was dried (1 mL) was stirred at room temperature for 24 h, and then at 50° C. for 4 h, affording crude title compound mixture (49 most of the acetic anhydride was removed. The white needle mg, 58%) as a yellow powder. It was dissolved in acetone (1 crystals were collected by filtration and washed with water mL) and centrifuged to remove the insoluble material, then (2x2 mL), affording 2.4 g (97%) of 3-acetamido-6-fluo 25 water (-1 mL) was added to the clear filtrate until the robenzotrifluoride. "H NMR (CDC1): 82.184 (S,3H); 7.136 solution became cloudy and then the mixture was allowed to (t, 1H, J=99Hz); 7.733 (m, 3H). stand at room temperature overnight. The yellow crystals B. 3-Acetamido-6-fluoro-4-nitrobenzotrifluoride were collected by filtration, affording an the 5,6-dinitro To 3-acetamido-6-fluorobenzotrifluoride (2.4 g, 10.85 compound (a) and its isomer (b) (21 mg, 25%, a:b=1:2 by mmol) in concentrated HSO, (10 mL) at 0°C. was added 30 NMR). Mp:296-8° C. (dec.); IR (KBr, cm): 3422,3244, dropwise 70% HNO (1.5 mL, Baker). The mixture was 1755, 1711, 1552. "H NMR (DMSO-d): 811.445 (br, 2H), stirred at 0°C. for 0.5 h, then at room temperature for 3 h 8.080 (s, 0.7 H) for (a), 7.869 (s, 0.3 H) for (b). and it was poured into ice water (20g). The precipitate was collected by filtration, affording 2.6 g (90%) of crude Example 85 3-acetamido-6-fluoro-4-nitrobenzotrifluoride. It was crystal 35 Preparation of 5,6-Dinitro-7-chloro-1,4-dihydro-2,3-qui lized from EtOH/HO to give 2.1 g of pure compound as noxalinedione yellow needles. HNMR (CDC): 82.318 (s, 3H), 8.062 (d, To a solution of 6-nitro-7-chloro-1,4-dihydro-2,3-qui 1H, J-9.6 Hz), 9.196 (s, 1H), 10.152 (s, 1H). noxalinedione (120 mg, 0.50 mmol) in concentrated HSO C. 3-Amino-6-fluoro-4-nitrobenzotrifluoride (1.0 mL) was added KNO (61 mg, 0.60 mmol, Baker). The A mixture of 3-acetamido-6-fluoro-4-nitrobenzotrifluo mixture was stirred at 4 C. for 2 days, then ice (2 g) was ride (593 mg, 2.23 mmol) in concentrated HCl (5 mL) and added. The precipitate was collected by filtration and the EtOH (5 mL) was refluxed overnight and it was extracted by solid was dissolved in 1N NaOH (5 mL) and filtered. The ethyl acetate (2x5 mL). The extract was dried over MgSO filtrate was acidified to pH-2 with 4N HCl (-1.2 mL) to give and evaporated to give 328 mg (66%) of 3-amino-6-fluoro a yellow precipitate, which was collected by filtration and 4-nitrobenzotrifluoride. "H NMR (CDC1,): 86.105 (s, 2H); 45 was washed with water (2x1 mL), then was dried at 50° C. 7.110 (d, 1H, J=6.0 Hz); 7,979 (d, 1H, J=10.2 Hz). for 4 h, affording crude title compound (101 mg, 70%) as a D. 3,4-Diamino-6-fluorobenzotrifluoride yellow powder. The crude compound (100 mg) was dis A mixture of 3-amino-6-fluoro-4-nitrobenzotrifluoride solved into acetone (2 mL) and the insoluble material was (328 mg, 1.45 mmol) and 10% Pd/C (50mg) in ethanol (15 removed by centrifugation. Water (1 mL) was added to the mL) was hydrogenated for 2 hat 25°C. under 25 psi H. The 50 clear solution of acetone and the resultant solution was catalyst was removed by filtration with celite and the solvent allowed to stand at 4°C. overnight to give purer compound. was removed by rota-evaporation to give 270 mg of 3,4- Recrystallization from acetone/water again gave pure title diamino-6-fluorobenzotrifluoride (95%) as a brown solid. compound (55 mg). Mp: 298-300° C. (dec.); ER (KBr, H NMR (CDC1): 83.432 (br,4H), 6.469 (d, 1H, J-84 Hz); cm): 3434, 3295, 3111, 2920, 1716, 1571. H NMR 6.860 (d, 1H, J=66 Hz). 55 (DMSO-d): 812.560 (s, 1H), 12.421 (s, 1H), 7.442 (s, 1H). E. 6-Fluoro-7-trifluoromethyl-1,4-dihydro-2,3-quinoxa HRMS; calcd for CHCINO (M) m/z: 285.9740, found: linedione 285.974. A mixture of 3,4-diamino-6-fluorobenzotrifluoride (270 mg, 1.38 mmol) and oxalic acid dihydrate (200 mg, 1.60 Example 86 mmol, used as received) in 4N HCl (5 mL) was refluxed at 120°-5° C. for 3 h, then cooled to room temperature. The Preparation of 5-Nitro-6-bromo-7-fluoro-1,4-dihydro-2,3- mixture was centrifuged and the liquid layer was removed. quinoxalinedione and 8-nitro-6-bromo-7-fluoro-1,4-dihy The yellow solid was washed twice with cold water (2x2 dro-2,3-quinoxalinedione mL), collected by filtration, and dried at 60° C. under To the stirred solution of 6-bromo-7-fluoro-1,4-dihydro reduced pressure for 2 h, affording 152 mg of crude title 65 2,3-quinoxalinedione (201 mg, 0.77 mmol) in concentrated compound (40%) as a light yellow powder. The crude HSO (2.0 mL) at 0°C. was added KNO (94 mg, 0.93 compound was dissolved in 1N NaOH (4 mL) and filtered. mmol, Baker). The mixture was stirred at 25°C. for 30 hand 5,514,680 101 102 it was poured into ice water (2 g). The precipitate was a white solid: 'HNMR (CDCl), 2.28 (s, 3H), 6.95 (m, 1H), collected by filtration, and the solid was dissolved in 1N 7.31 (m, 1H), 7.97 (m, 1H). NaOH (2 mL) and filtered. The filtrate was acidified to pH=2 3,4,5-Trifluoro-1,2-phenylenediamine with 4N HCl (-0.5 mL) to give a yellow precipitate, which To 2,3,4-trifluoroacetanilide (1.35 g, 7.09 mmol) is added was collected by filtration and was washed with water (2x1 5 concentrated sulfuric acid (8 mL). While the flaskis in an ice mL), then was dried at 50° C. for 4 h, affording crude title bath, KNO was slowly added, giving a tan mixture, which compound (120 mg, 51%) as a yellow powder. Crystalliza was stirred overnight. The reaction mixture, now dark red, tion from EtOH twice and recrystallization from acetone was then added into ice water (45 mL), instantly giving an twice gave a mixture of the 5-nitro (a) and 8-nitro (b) orange precipitate. The volatile compound, presumably 2,3, isomers (a:b=5:1, 20%). Mp: 250-251° C. (dec.); IR (KBr, O 4-trifluoro-6-nitroaniline, is dissolved in ethyl acetate (18 cm): 3512, 3434, 3272, 1687, 1631, 1532. NMR (H, mL) and ethyl alcohol (12 mL). To the orange solution is DMSO-d): 811.685 (br, 2H), 6.968 (d, J-93 Hz, 1H) for added stannous chloride dihydrate (7.6 g., 34 mmol). The (a); 7.166 (d, J=7.2 Hz, 1H) for (b). resulting mixture is stirred and brought to reflux under N for 4 h. The mixture is added into ice water (40 mL) and Example 87 15 basified with 2NNaOH (40 mL). It was extracted with ethyl acetate (3x20 mL) and the combined extracts were washed Preparation of 6-Chloro-7-fluoro-5-nitro-1,4-dihydroqui with water (20 mL) and brine (20 mL). This dark red noxaline-2,3-dione and 7-Chloro-6-fluoro-5-nitro-1,4-dihy solution is dried (MgSO) and evaporated to give 285 mg droquinoxaline-2,3-dione (25%) of the diamine as a dark red solid. "H NMR (CDCl), In a 50 mL flask equipped with a reflux condenser, a 20 3.22 (brs, 2H), 3.46 (brs, 2H), 6.32 (m, 1H). mixture of 6-chloro-7-fluoro-1,4-dihydroquinoxaline-2,3- 5,6,7-Trifluoro-1,4-dihydro-2,3-quinoxalinedione dione (644 mg, 2.0 mmol), FCCOOH (10 mL) and KNO To a brown solution of 3,4,5-trifluoro-1,2-phenylenedi (490 mg, 3.8 mmol) was heated at 65° C. with stirring for 14 amine (285 mg, 1.75 mmol) in aqueous 2N HCl (10 mL) is h. The resulting solution was allowed to cool to room added oxalic acid (221 mg, 1.75 mmol). The brown mixture temperature, and rota-evaporated to dryness. The residual 25 is brought to reflux and stirred under N overnight. The solid was washed on a Busch funnel with water (5x5 mL), mixture is filtered to yield 139 mg (37%) of crude title dried under 1 mmHg at 40°C. for 12 h giving 440 mg (85%) compound. An analytical sample is prepared by dissolving of yellow powder, which is a mixture of the 6-chloro-7- 42 mg of this brownpowder in 2.5 mL boiling ethanol. Upon fluoro-5-nitro (a) and 7-chloro-6-fluoro-5-nitro (b) isomers cooling, brown rod-like crystals were formed which are by the chemical shift of aromatic proton in the "H NMR 30 filtered and dried in vacuo to yield 15 mg (36%) of pure title (DMSO-d): for (a): 7.239 (d, J=9.6 Hz); for (b): 7.384 (d, compound: H NMR (DMSO-d), 6.91 (m, 1H), 12.02 (s, J=6.6 Hz); the ratio of aib is about 4:1 from the NMR 1H), 12.19 (s, 1H); analysis calculated for CHFNO: C, integration. 44.46; H, 1.40; N, 12.96. Found: C, 44.42; H, 1.16; N, 12.76. Example 88 35 Example 90 Preparation of 5-Nitro-6,7,8-trifluoro-1,4-dihydro-2,3-qui Preparation of 6,7-Difluoro-5-nitro-1,4-dihydro-2,3-qui noxalinedione noxalinedione To 5,6,7-trifluoro-1,4-dihydro-2,3-quinoxalinedione (93 To a suspension of 6,7-difluoro-1,4-dihydro-2,3-quinoxa mg, 0.43 mmol) is added concentrated sulfuric acid (0.5 linedione (837 mg, 4.23 mmol) intrifluoracetic acid (30 mL) 40 mL). While the flask is an ice bath, KNO is slowly added, was added KNO (512 mg, 5.07 mmol). The mixture was giving a brown mixture, which is stirred overnight. The stirred at 55° C. for 20 hat the end of this time, 256 mg (2.50 reaction mixture, now dark red, is then added into ice water mmol) of KNO was added and the reaction mixture was (5 mL), instantly giving an orange precipitate, which is stirred at 55° C. for 20h, then another 256 mg (2.50 mmol) collected by centrifugation. The powder was crystallized of KNO was added and the mixture was stirred at 55° C. for 45 20 h. The reaction mixture was then rota-evaporated to from EtOH (5 mL) and dried in vacuo to yield 24 mg (20%) dryness. To the residual solid was added ice-cold water of pale orange microcrystals. HNMR (DMSO-d), 11.9 (br (about 15 mL), the solid was collected by vacuum filtration, s, 1H), 12.6 (brs, 1H). washed with ice-cold water (5X5 mL), and dried at 40 C. Example 91 under 1 mm Hg for 14 h, giving 700 mg (68%) of the title 50 compound as a yellow powder. Mp 288°-90° C. (dec.). IR Preparation of 6-Chloro-5,7-difluoro-1,4-dihydroquinoxa (KBr) 3424, 3226, 1752, 1717, 1554, 1356, 1304 cm. H line-2,3-dione NMR (DMSO-d): 12.249 (s, 1H), 11.864 (bs, 1H), 7.330 3-Chloro-2,4-difluoro-(trifluoroacetamido)benzene (dd, 1H, J-10.5, 7.8 Hz). Analysis for CHFNO, calcd: To a solution of 10.5 g (64.3 mmol) of 3-chloro-2,4- C, 39.50, H, 1.24, N, 17.29; found: C, 39.42, H, 1.26, N, 55 difluoroaniline in 25 mL of dioxane kept in an ice-bath was 17.08. added dropwise 10 mL (14.8 g., 70.4 mmol) of trifluoroacetic anhydride. The solution was stirred at room temperature for Example 89 20 h. It was then added into 150 mL of ice-water and the mixture was stirred for 1 h. It was filtered and washed by Preparation of 5,6,7-Trifluoro-1,4-dihydro-2,3-quinoxa 60 water, and dried to leave almost colorless solid 16.1 g (96%), linedione mp 73°-74° C. "H NMR (CDCl), 7.068 (m, 1), 7.976 (mb, 2,3,4-Trifluoroacetanilide 1), 8.146 (m, 1). To a pink solution of 2,3,4-trifluoroaniline (1.04 g, 9.45 3-Chloro-2,4-difluoro-6-nitro-(trifluoroacetamido)ben mmol) in chloroform (12 mL) was added acetic anhydride ZCC (1.63 g, 16.0 mmol), giving a pale pink solution which was 65 To a solution of 15.1 g (58.1 mmol) of 3-chloro-2,4- stirred overnight under nitrogen. The chloroform was difluoro(trifluoroacetamido)benzene in 80 mL of HSO removed in vacuo to give 1.35 g (99%) of the acetanilide as kept in an ice-bath was added dropwise 10 mL of HNO. 5,514,680 103 104 Solid precipitate was observed during addition of HNO. C. and the precipitated solid was filtered and dried under The mixture was stirred in an ice-bath for 4 h. It was added vacuum to yield 146 mg (65%) of pure 5-chloro-3-nitro-1, into 600 mL of ice-water. The precipitate was filtered and 2-phenylenediamine as a shining red solid, "H NMR washed by water, and dried to leave almost colorless solid (DMSO-d): 5.031 (brs, 2H), 6.687 (brs, 2H), 6.912 (d, 1H, (16.8 g., 95%), mp 124°-125° C. "H NMR (CDC), 7.69 J=2.4 Hz), 7.413 (d, 1H, -2.1 Hz). (did, 1), 8.936 (mb, 1). 7-Chloro-5-nitro-1,4-dihydro-2,3-quinoxalinedione 3-Chloro-2,4-difluoro-6-nitroaniline A suspension of the diamine as synthesized above (0.100 A solution of 6.35 g (20.8 mmol) of 3-chloro-2,4-dif g, 0.553 mmol) and oxalic acid dihydrate (0.073 g, 0.58 luoro-6-nitro-(trifluoroacetamido)benzene in 60 mL of 7% mmol) in 2N HCl (1.5 mL) was refluxed for 2.5 h. The KCO, methanol/water (3:2) was stirred at 25° C. for 4 h. O suspension was cooled to room temperature and the solid The solution was evaporated to remove the methanol. Solid was filtered, washed with water (5 mL) and dried under was observed in the residue and it was filtered and washed vacuum to afford 0.100 g (78%) of pure title compound as by water, and dried to leave 301 mg of crystalline yellow yellow brown powder, mp: 315-317 C.; H NMR solid, mp 96°-97° C.H NMR (CDCl), 6.07 (mb, 2), 7.815 (DMSO-d): 7.397 (d. 1H, J=1.5 Hz), 7.896 (d. 1H, J=1.8 (dd, 1, J=1.92, 9.13). More solid was observed after the 15 Hz), 11.19 (s, 1H), 12,341 (s, 1H). mother aqueous solution was allowed to stand at room temperature overnight. It was filtered and washed by water, Example 94 and dried to leave yellow solid (1.01 g). More solid was Preparation of 7-Fluoro-5-nitro-1,4-dihydro-2,3-quinoxa crystallized from the mother solution. It was collected three linedione more times to leave 2.48g of a yellow solid. 'H NMR is 20 identical with above, total yield 3.80 g (87%). A, 4-Fluoro-2,6-dinitroaniline 4-Chloro-3,5-difluoro-1,2-phenylenediamine A solution of 4-fluoro-2,6-dinitro-(trifluoroacetamido )benzene (297 mg, 1.00 mmol) in 10% KCO (10 mL) was A solution of 348 mg (1.67 mmol) of 3-chloro-2,4- refluxed for 1 h, then cooled to room temperature to give difluoro-6-nitroaniline and 1.57 g (8.28 mmol) of SnCl2 in yellow crystals. It was filtered and washed with cool water 8 mL of ethanol was heated at 70° C. for 2 h. The solution 25 (2x1 mL), affording 105 mg of 13c (52%). "H NMR was evaporated to remove the ethanol. The residue was (DMSO-d): 68.254 (s, 2H), 8.460 (d, 2H, J=8.4). treated with 2N NaOH to pH-13. White precipitate was B. 1,2-Diamino-4-fluoro-6-nitrobenzene observed. The mixture was extracted by CHCl (3x10 mL). A solution of 4-fluoro-2,6-dinitroaniline (125 mg, 0.62 The extract was dried (MgSO) and evaporated to leave a mmole) in freshly prepared 6% (NH)S (5 mL) and EtOH red solid (285 mg,95%), mp 77-78° C. "HNMR (CDCl), 30 3.156 (b, 2), 3.704 (b, 2), 6.352 (did, 1, J-1.86, 9.95). (5 mL) was refluxed for 30 min, diluted with water (10 mL) 6-Chloro-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dione and kept at 4 C. for several hours. The precipitate was collected and washed with cold water (2x1 mL), affording A mixture of 230 mg (1.29 mmol) of 4-chloro-3,5- 53 mg of 1,2-diamino-4-fluoro-6-nitrobenzene (50%) as red difluoro-1,2-phenylenediamine and 125 mg (1.38 mmol) of crystals. H NMR (CDCl: 83.619 (s, 2H), 5.724 (s, 2H), oxalic acid in 4 mL of 2N HCl was refluxed for 3 h and 35 6.732 (dd, 1H, J-24 Hz, J-8.4 Hz), 7.403 (dd, 1H, J-24 cooled to room temperature. The mixture was filtered and Hz, J-8.4 Hz). washed by water, dried to leave a brown solid (245 mg, C. 7-Fluoro-5-nitro-1,4-dihydro-2,3-quinoxalinedione 82%), mp. 250° C. "H NMR (DMSO-d), 6.921 (d, 1, A mixture of 1,2-diamino-4-fluoro-6-nitrobenzene (80 J=9.61), 12.143 (s, 1), 12.168 (s, 1). MS, 232 (M, 100), 204 mg, 0.46 mmole) and oxalic acid dihydrate (70 mg, 0.56 (80), 176 (40), 149 (70), 171 (80). HRMS calcd for mmole, used as received) in 4N HCl (4 mL) was refluxed at CH.ClFNO, 231.9848, found 231.9851. 120°-5° C. for 3 h, then cooled to room temperature. The Example 92 mixture was centrifuged and the supernatant was removed. The yellow solid was washed by cold water (2x2 mL), Preparation of 7-Chloro-6,8-difluoro-5-nitro-1,4-dihydro collected by filtration, and dried in vacuo for 2 h, affording quinoxaline-2,3-dione 45 45 mg of crude 7-fluoro-5-nitro-1,4-dihydro-2,3-quinoxa To a solution of 120 mg (5.16 mmol) of 6-chloro-5,7- linedione (42%) as a yellow powder. The crude product was difluoro-1,4-dihydroquinoxaline-2,3-dione in 1 mL of taken up in 1N NaOH (1 mL) and filtered. The filtrate was HSO (97%) kept in ice bath was added portionwise 60 mg acidified to pH-3, affording 35 mg of 7-fluoro-5-nitro-1,4- (0.59 mmol) of KNO. The solution was stirred at room dihydro-2,3-quinoxalinedione (33%), mp: 333°-335° C. temperature for 14h and 60 mg of KNO was added and it 50 (dec.), IR (KBr, cm): 3427,3328,3104,3072, 1716, 1545. was stirred at room temperature for 24 h. It was diluted by HNMR (DMSO-d): 812418(s, 1H), 11.149 (s, 1H), 7.819 ice-water (4 mL) and the mixture was filtered and washed by (dd, J=24 Hz, J-9.0Hz, 1H), 7.297 (dd, J=2.4 Hz, J-9.0 water, and dried to leave a yellow solid (94 mg, 65%). The Hz, 1H); HRMS: calcd for CHFN.O. (M) m/z: 225.0185; solid was purified by NaOH/HCl precipitate to leave 63 mg found: 225.0188. of yellow solid, mp.2250° C. H NMR (DMSO-d), 12.11 55 (mb, 1), 12.503 (s, 1). Example 95 Preparation of 5,7-Bis(trifluoromethyl)-1,4-dihydro-2,3- Example 93 quinoxalinedione Preparation of 7-Chloro-5-nitro-1,4-dihydro-2,3-quinoxa A. 1-Acetamido-3,5-bis(trifluoromethyl)benzene linedione A solution of 3,5-bis(trifluoromethyl)aniline (1.28 g, 5 5-Chloro-3-nitro-1,2-phenylenediamine mmol, Aldrich) in acetic anhydride (5 mL) was stirred at A suspension of 4-chloro-2,6-dinitroaniline (0.261 g, 1.19 room temperature for 12 h, then all of the solvent was mmol, Aldrich, used as received) in 6.66% aq. (NH)2S (8.0 removed to give 1.479 g (98%) of 1-acetamido-3,5-bis(tri mL, prepared from 20% aq. solution available from Aldrich) 65 fluoromethyl)benzene as white needles. "H NMR (CDC1): and ethanol (8.0 mL) was refluxed for 45 min during which 82.229 (s, 3H); 7.589 (s, 1H); 8.048 (s, 2H). time it formed dark red solution. It was then cooled to 28 B. 1-Acetamido-2-nitro-3,5-bis(trifluoromethyl)benzene