WO2013034755A1 - Triazolopyrazine derivatives and their use for treating neurological and psychiatric disorders - Google Patents

Abstract

The present invention is directed to triazolopyrazine compounds of Formula (I). Separate aspects of the invention are directed to pharmaceutical compositions comprising said compounds and uses of the compounds as therapeutic agents treating neurological and psychiatric disorders.

Description

TRIAZOLOPYRAZINE DERIVATIVES AND THEIR USE FOR TREATING

NEUROLOGICAL AND PSYCHIATRIC DISORDERS

FIELD OF THE INVENTION

The present invention is directed to compounds which are useful as therapeutic agents treating neurological and psychiatric disorders. Separate aspects of the invention are directed to pharmaceutical compositions comprising said compounds and uses thereof.

BACKGROUND ART

Cyclic-adenosine monophosphate (cAMP) and cyclic-guanosine monophosphate (cGMP) function as intracellular second messengers regulating an array of processes in neurons. Intracellular cAMP and cGMP are generated by adenyl and guanyl cyclases, and are degraded by cyclic nucleotide phosphodiesterases (PDEs). Intracellular levels of cAMP and cGMP are controlled by intracellular signaling, and stimulation/repression of adenyl and guanyl cyclases in response to GPCR activation is a well characterized way of controlling cyclic nucleotide concentrations (Antoni, Front. Neuroendocrinal. 2000, 21, 103-132).

Phosphodiesterase 2A (PDE2A) is a dual substrate enzyme with higher affinity for cGMP although it may metabolize either cAMP or cGMP depending on the tissue. cAMP is derived from adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway. Although expressed in the periphery, the highest expression levels of PDE2A are in the brain. A recent immunohistochemical study demonstrated a consistent pattern of PDE2A expression in the brain across mammalian species including humans (Stephenson, et al. J. Histochem. Cytochem. 2009, 57, 933). The enzyme expression was shown to be prominent in regions associated with cognitive function and mood control, including the cortex, striatum, hippocampus, amygdala and the habenula.

The selective PDE2A inhibitor, Bay 60-7550, preferentially increases cGMP in primary neuronal cultures and hippocampal slices. Bay 60-7550 also increases long term potentiation (LTP) induction in rat hippocampal slices. Consistent with its biochemical and electrophysiological effects, Bay 60- 7550 was found to be active in novel object and social recognition tasks (Boess, et al. Neuropharmacology 2004, 47, 1081). More recently, Bay 60-7550 was reported to reverse the deficit in object recognition produced by tryptophan depletion (van Donkelaar, et al. Eur. J. Pharmacol. 2008, 600, 98). These results are interesting in light of the PDE2 positive cells identified in the dorsal raphe, a region known to contain the cell bodies of the serotonergic neurons projecting to the forebrain (Stephenson, et al. J. Histochem. Cytochem. 2009, 57, 933). A similar study in aged rats demonstrated that the beneficial effect of Bay 60-7550 on object recognition could be reversed by a neuronal nitric oxide synthase (nNOS) inhibitor, suggesting that the effects of PDE2A inhibition in the central nervous system (CNS) are due to alterations in the levels of cGMP (Domek- Lopacinska and Strosznajder Brain Res. 2008, 1216, 68).

Recent studies indicate that PDE2A inhibition may also efficacy in the treatment of anxiety states (Masood, et al. J. Pharmacol. Exp. Ther. 2008, 326, 369; and Masood, et al. J. Pharmacol. Exp. Ther. 2009, 331, 699). Induction of oxidative stress in mice by depletion of central glutathione levels with buthionine sulfoximine (BSO) results in an increase in a number of anxiety-like behaviours assessed by open field time and the elevated plus maze assays. These effects were reversed by treatment with Bay 60-7550. Increased cGMP signaling, either by administration of the PDE2 inhibitors Bay 60-7550 or ND7001, or the NO donor detanonoate, antagonized the anxiogenic effects of restraint stress on behaviour in the elevated plus-maze, hole-board, and open-field tests, well established procedures for the evaluation of potential anxiolytics. These drugs also produced anxiolytic effects on behavior in non-stressed mice in the elevated plus-maze and hole-board tests. By contrast, administration of an NOS inhibitor, which reduces cGMP signaling, produced anxiogenic effects similar to restraint stress.

Phosphodiesterase 10A (PDEIOA) is another dual-specificity enzyme that can convert both cAMP to AMP and cGMP to GMP (Soderling, et al. Proc. Natl. Acad. Sci. 1999, 96, 7071). PDEIOA hydrolyses both cAMP and cGMP having a higher affinity for cAMP. PDEIOA is expressed in the neurons in the striatum, n. accumbens and in the olfactory tubercle (Seeger, et al. Brain Research, 2003, 985, 113-126) and the thalamus, hippocampus, frontal cortex and olfactory tubercle (Menniti et al., William Harvey Research Conference, Porto, December, 2001). All these brain areas are described to participate in the pathomechanism of schizophrenia (Lapiz, et al. Neurosci Behav Physiol 2003, 33, 13) so that the location of the enzyme indicates a predominate role in the pathomechanism of psychosis. In the striatum, PDEIOA is predominately found in the medium spiny neurons and they are primarily associated to the postsynaptic membranes of these neurons (Xie et al., Neuroscience 2006, 139, 597). In this location PDEIOA may have an important influence on the signal cascade induced by dopaminergic and glutamatergic input on the medium spiny neurons two neurotransmitter systems playing a predominate role in the pathomechanism of psychosis. Psychotic patients have been shown to have a dysfunction of cGMP and cAMP levels and their downstream substrates (Muly, Psychopharmacol Bull 2002, 36, 92). Additionally, haloperidol treatment has been associated with increased cAMP and cGMP levels in rats and patients, respectively (Leveque et al., J. Neurosci. 2000, 20,: 4011). As PDEIOA hydrolyses both cAMP and cGMP, an inhibition of PDEIOA would also induce an increase of cAMP and cGMP and thereby have a similar effect on cyclic nucleotide levels as haloperidol. The antipsychotic potential of PDE 10A inhibitors is further supported by studies of Kostowski et al. {Pharmacol Biochem Behav 1976, 5, 15) who showed that papaverine, a moderately selective PDEIOA inhibitor, reduces apomorphine- induced stereotypies in rats, an animal model of psychosis, and increases haloperidol-induced catalepsy in rats while concurrently reducing dopamine concentration in rat brain, activities that are also seen with classical antipsychotics. In addition to classical antipsychotics which mainly ameliorate the positive symptoms of psychosis, PDE10A also bears the potential to improve the negative and cognitive symptoms of psychosis.

Focusing on the dopaminergic input on the medium spiny neurons, PDE10A inhibitors by up- regulating cAMP and cGMP levels act as Dl agonists and D2 antagonists because the activation of Gs-protein coupled dopamine Dl receptor increases intracellular cAMP, whereas the activation of the Gi-protein coupled dopamine D2 receptor decreases intracellular cAMP levels through inhibition of adenylyl cyclase activity. Elevated intracellular cAMP levels mediated by Dl receptor signalling seems to modulate a series of neuronal processes responsible for working memory in the prefrontal cortex (Sawaguchi, Parkinsonism Relat. Disord. 2000, 7, 9), and it is reported that Dl receptor activation may improve working memory deficits in schizophrenic patients (Castner, et al., Science 2000, 287, 2020). Further indication of an effect of PDE10A inhibition on negative symptoms of psychosis was given by Rodefer et al. (Eur. J Neurosci 2005, 21, 1070) who could show that papaverine reverses attentional set-shifting deficits induced by subchronic administration of phencyclidine, an NMDA antagonist, in rats. Attentional deficits including an impairment of shifting attention to novel stimuli belongs to the negative symptoms of schizophrenia. In the study the attentional deficits were induced by administering phencyclidine for 7 days followed by a washout period. The PDE10A inhibitor papaverine was able to reverse the enduring deficits induced by the subchronic treatment.

These convergent findings indicate that the inhibition of PDE2A and/or PDE10A may be therapeutic targets for the treatment of certain neurological and psychiatric disorders. Accordingly, the present invention relates to triazolopyrazines, to their preparation, to their medical use and to medicaments comprising them.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide compounds that inhibit PDE2A and/or PDEIOA. Accordingly, the present .

wherein R¹ is Ci-Ce alkyl, C3-C₆cycloalkyl, tetrahydropyranyl, benzyl, phenyl and pyridyl, in which the benzyl, phenyl and pyridyl is optionally substituted with one or more halogen, CN, C -C4 alkyl/fluoroalkyl or C -C4 alkoxy/fluoroalkoxy; wherein R² is C -C4 alkyl or C3-C₆cycloalkyl; wherein R³ is halogen, CN, -C0₂H, -CON(H or C_rC₄ alkyl)₂ CHO, C_rC₄ alkyl/fluoroalkyl, C₂-C₄ alkenyl, C₂-C₄ alkenyl or C -C₄ alkoxy/fluoroalkoxy; and wherein n is 0-3; or a pharmaceutically acceptable salt thereof. In separate aspects of the invention, the compound is selected from one of the exemplified compounds of formula I.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I and a pharmaceutically acceptable carrier.

Methods of treating a subject suffering from anxiety, a cognitive disorder or schizophrenia comprising administering a therapeutically effective amount of a compound of formula I are provided. The present invention further provides uses of a compound of formula I in the manufacture of a medicament for treating anxiety, a cognitive disorder or schizophrenia.

Another aspect of the present invention provides a compound for use in treating anxiety, a cognitive disorder or schizophrenia. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of the compounds of Formula I which inhibit PDE2A and/or PDEIOA, and as such, are useful for the treatment of certain neurological and psychiatric disorders. Particular aspects of the invention are explained in greater detail below but this description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. Hence, the following specification is intended to illustrate some embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

It is understood by those practicing the art that compounds can exist in tautomeric forms. When any reference in this application to one of the specified tautomers is given, it is understood to encompass its tautomeric forms and mixtures thereof.

The subject invention is directed to compounds of formula I as defined in the summary of the invention, pharmaceutical compo

Formula I

In one embodiment, R is C₁-C₄ alkyl. In another embodiment, R is methyl or ethyl.

In a separate embodiment, R² is C₃-C₆cycloalkyl.

In yet another embodiment, R¹ is C₁-C₄ alkyl.

In yet another embodiment, R¹ is C₃-C₆cycloalkyl.

In one embodiment, R¹ is tetrahydropyranyl.

In one embodiment, R¹ is benzyl optionally substituted with one or two F, CI, C₁-C₃ alkyl or C₁-C₃ alkoxy/fluoroalkoxy. In a separate embodiment, the substitution occurs at the para position of the benzyl group.

In one embodiment, R¹ is phenyl optionally substituted with one or two F, CI, C₁-C₃ alkyl or C₁-C₃ alkoxy/fluoroalkoxy. In a separate embodiment, the substitution occurs at the ortho position of the phenyl group. In one embodiment, R¹ is pyridyl optionally substituted with one or two F, CI, C1-C3 alkyl or C1-C3 alkoxy. In a separate embodiment, the substitution occurs at the carbon atom adjacent to the triazole ring. In a separate embodiment, R¹ is selected from the group consisting of benzyl optionally substituted at the para position of the benzyl group; phenyl optionally substituted at the ortho position of the phenyl group; and pyridyl optionally substituted at the carbon atom adjacent to the triazole ring.

In one embodiment, R³ is halogen or CHO.

In one embodiment, R³ is C1-C4 alkyl or C -C4 alkoxy

In one embodiment, R³ is C2-C4 alkenyl and C2-C4 alkenyl. In a separate embodiment, R³ is C2-C4 alkenyl. In a separate embodiment, R³ is C2-C4 alkenyl.

In one embodiment, n is 0. In a separate embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3.

Racemic forms may be resolved into the optical antipodes by known methods, for example, by separation of diastereomeric salts thereof with an optically active acid, and liberating the optically active amine compound by treatment with a base. Separation of such diastereomeric salts can be achieved, e.g. by fractional crystallization. The optically active acids suitable for this purpose may include, but are not limited to d- or 1- tartaric, mandelic or camphorsulfonic acids. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optically active matrix. The compounds of the present invention may also be resolved by the formation and chromatographic separation of diastereomeric derivatives from chiral derivatizing reagents, such as, chiral alkylating or acylating reagents, followed by cleavage of the chiral auxiliary. Any of the above methods may be applied either to resolve the optical antipodes of the compounds of the invention per se or to resolve the optical antipodes of synthetic intermediates, which can then be converted by methods described herein into the optically resolved final products which are the compounds of the invention.

Additional methods for the resolution of optical isomers, known to those skilled in the art, may be used. Such methods include those discussed by J. Jaques, A. Collet and S. Wilen in Enantiomers, Racemates, and Resolutions, John Wiley and Sons, New York, 1981. Optically active compounds can also be prepared from optically active starting materials. Definitions

As used herein, the term "Ci-Ce alkyl" refers to a straight chained or branched saturated hydrocarbon having from one to six carbon atoms inclusive. Examples include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-butyl, 2-methyl-2-propyl, 2-methyl-l -propyl, n-pentyl and n- hexyl. Similarly, the term "straight chained or branched C₁-C4 alkyl" refers to a saturated hydrocarbon having from one to four carbon atoms. Examples include methyl, ethyl and n-propyl.

Likewise, the term "C -C4 alkoxy" refers to a straight chained or branched saturated oxygen containing hydrocarbon group having from one to four carbon atoms with the open valency on the oxygen. Examples include, but are not limited to, methoxy, ethoxy, n-butoxy, and t-butoxy.

The term "Ci-Ce fiuoroalkyl" refers to a straight chained or branched saturated hydrocarbon having from one to six carbon atoms inclusive substituted with one or more fluorine atoms. Examples include trifluoromethyl, pentafluoroethyl, 1-fluoroethyl, monofluoromethyl, difluoromethyl, 1,2- difluoroethyl and 3,4 difluorohexyl. Similarly, the term "straight chained or branched C₁-C4 fluoroalkoxy" refers to a saturated hydrocarbon having from one to four carbon atoms substituted with one or more fluorine atoms with the open valency on the oxygen.

The term "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "C₂-C4-alkenyl" refers to a branched or unbranched alkenyl group having from two to four carbon atoms and one double bond, which includes ethenyl, propenyl, and butenyl. The term "C₂-C4-alkynyl" shall mean a branched or unbranched alkynyl group having from two to four carbon atoms and one triple bond, which includes ethynyl, propynyl and butynyl.

The term "CON(H or C₁-C4 alkyl)₂ refers to an amido group in which the substituents off the amido moiety are each independently selected from the group consisting of H or C₁-C4 alkyl. Examples include -CONH₂, -CONHCH3, -CON(CH₃)₂ and -CON(CH₃)CH₂CH₃. The term "treatment" or "treating" as used herein means ameliorating or reversing the progress or severity of a disease or disorder, or ameliorating or reversing one or more symptoms or side effects of such disease or disorder. "Treatment" or "treating", as used herein, also means to inhibit or block, as in retard, arrest, restrain, impede or obstruct, the progress of a system, condition or state of a disease or disorder. For purposes of this invention, "treatment" or "treating" further means an approach for obtaining beneficial or desired clinical results, where "beneficial or desired clinical results" include, without limitation, alleviation of a symptom, diminishment of the extent of a disorder or disease, stabilized (i.e., not worsening) disease or disorder state, delay or slowing of a disease or disorder state, amelioration or palliation of a disease or disorder state, and remission of a disease or disorder, whether partial or total, detectable or undetectable. As used herein, the phrase "effective amount" when applied to a compound of the invention, is intended to denote an amount sufficient to cause an intended biological effect. The phrase "therapeutically effective amount" when applied to a compound of the invention is intended to denote an amount of the compound that is sufficient to ameliorate, palliate, stabilize, reverse, slow or delay the progression of a disorder or disease state, or of a symptom of the disorder or disease. In an embodiment, the method of the present invention provides for administration of combinations of compounds. In such instances, the "effective amount" is the amount of the combination sufficient to cause the intended biological effect.

Pharmaceutically Acceptable Salts

The present invention also comprises salts of the present compounds, typically, pharmaceutically acceptable salts. Such salts include pharmaceutically acceptable acid addition salts. Acid addition salts include salts of inorganic acids as well as organic acids.

Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, sulfamic, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, itaconic, lactic, methanesulfonic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methane sulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, theophylline acetic acids, as well as the 8-halotheophyllines (for example, 8-bromotheophylline and the like). Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in S. M. Berge, et al., J. Pharm. Sci., 1977, 66, 2.

Furthermore, the compounds of this invention may exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. Pharmaceutical compositions

The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I and a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one of the specific compounds disclosed in the Experimental Section and a pharmaceutically acceptable carrier.

The compounds of the invention may be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19^th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, the compositions may be prepared with coatings such as enteric coatings or they may be formulated so as to provide controlled release of the active ingredient such as sustained or prolonged release according to methods well known in the art. Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs.

Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use.

Oral dosages are usually administered in one or more dosages, typically, one to three dosages per day. The exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art. The formulations may also be presented in a unit dosage form by methods known to those skilled in the art. For illustrative purposes, a unit dosage form for oral administration may contain from about 0.01 to about 1000 mg, from about 0.05 to about 500 mg, or from about 0.5 to about 200 mg. The compounds of this invention are generally utilized as the free substance or as a pharmaceutically acceptable salt thereof. One example is an acid addition salt of a compound having the utility of a free base. When a compound of formula I contains a free base such salts are prepared in a conventional manner by treating a solution or suspension of a free base of formula I with a molar equivalent of a pharmaceutically acceptable acid. Representative examples of suitable organic and inorganic acids are described above.

For parenteral administration, solutions of the compounds of formula I in sterile aqueous solution, aqueous propylene glycol, aqueous vitamin E or sesame or peanut oil may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The compounds of formula I may be readily incorporated into known sterile aqueous media using standard techniques known to those skilled in the art. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers include lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The pharmaceutical compositions formed by combining the compounds of formula I and a pharmaceutically acceptable carrier are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

If a solid carrier is used for oral administration, the preparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it may be in the form of a troche or lozenge. The amount of solid carrier will vary widely but will range from about 25 mg to about 1 g per dosage unit. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

Therapeutic Uses

Methods of treating a subject suffering from anxiety, a cognitive disorder or schizophrenia comprising administering a therapeutically effective amount of a compound of formula I are provided in this invention.

The present invention further provides uses of a compound of formula I in the manufacture of a medicament for treating an anxiety disorder, a cognitive disorder or schizophrenia. Another aspect of the present invention provides a compound for use in treating an anxiety disorder, a cognitive disorder or schizophrenia. The present invention provides a method of treating anxiety, a cognitive disorder or schizophrenia comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

The present invention provides a method of treating an anxiety disorder is selected from anxiety; panic disorder; agoraphobia; a specific phobia; social phobia; obsessive-compulsive disorder; posttraumatic stress disorder; acute stress disorder; and generalized anxiety disorder. The present invention further provides a method of treating a subject suffering from a cognition disorder comprising administering to the subject a therapeutically effective amount of a compound of formula I. Examples of cognition disorders that can be treated according to the present invention include, but are not limited to, Alzheimer's disease, multi-infarct dementia, alcoholic dementia or other drug-related dementia, dementia associated with intracranial tumors or cerebral trauma, dementia associated with Huntington's disease or Parkinson's disease, or AIDS-related dementia; and age-related cognitive decline. This invention also provides a method of treating a movement disorder comprising administering to the subject a therapeutically effective amount of a compound of formula I. Examples of movement disorders that can be treated according to the present invention include, but are not limited to, Huntington's disease and dyskinesia associated with dopamine agonist therapy. This invention further provides a method of treating a movement disorder selected from Parkinson's disease and restless leg syndrome, which comprises administering to the subject a therapeutically effective amount of a compound of formula I.

The present invention provides a method of treating schizophrenia, for example of the paranoid, disorganized, catatonic, undifferentiated, or residual type; schizophreniform disorder; schizoaffective disorder, for example of the delusional type or the depressive type; delusional disorder; substance- induced psychotic disorder, for example psychosis induced by alcohol, amphetamine, cannabis, cocaine, hallucinogens, inhalants, opioids, or phencyclidine; personality disorder of the paranoid type; and personality disorder of the schizoid type; and wherein the drug addiction is an alcohol, amphetamine, cocaine, or opiate addiction.

EXPERIMENTAL SECTION

The compounds of formula I can be prepared by the methods outlined in the following methods and in the examples. In the methods below, it is possible to make use of variants or modifications, which are themselves known to chemists skilled in the art or could be apparent to the person of ordinary skill in this art. Furthermore, other methods for preparing compounds of the invention will be readily apparent to the person skilled in the art in light of the following reaction schemes and examples. For example, the methods describe the use of selective protecting groups during the synthesis of the compounds of the invention. One skilled in the art would be able to select the appropriate protecting group for a particular reaction. Methods for protection and deprotection of such groups are well known in the art, and may be found in Watts and Green, et al., Protective Groups in Organic Synthesis, 2006, 4^th Edition, Wiley Ineterscience, New York.

Abbreviations & chemicals used.

AcOH = acetic acid (e.g. Sigma-Aldrich 320099). Acetonitrile (e.g. Aldrich 271004). Activated charcoal (e.g. Sigma-Aldrich 161551). APPI = atmospheric pressure photo ionization. Aq = aqueous. 37% aq HCl (e.g. Sigma-Aldrich 320331). 1,2-benzenediamine (e.g. Aldrich P23938). Benzoyl chloride (e.g. Sigma-Aldrich 259950). Brine = saturated aq solution of sodium chloride (e.g. Aldrich S7653). 4-Bromo-l-fluoro-2-nitro-benzene (e.g. Aldrich 680931). 4-Bromo-2-fluoro-l-nitro-benzene (e.g. Aldrich 680931). 3-Bromo-2-nitro-phenylamine (e.g. BBB-SCI 3B-19030 or CombiBlocks AN- 1330 or Apollo OR2214). Butyryl chloride (e.g. Aldrich 236349). Chloramine-T = N-Chloro-4- toluenesulfonamide sodium salt (e.g. Sigma-Aldrich 402869 for the trihydrate). (4-Chloro-benzene- 1,2-diamine (e.g. Aldrich 108871). 2-Chlorobenzhydrazide (e.g. Aldrich 259993). 2-Chlorobenzoyl chloride (e.g. Aldrich 103918). 2-Chloro-3-ethyl-quinoaxline (e.g. Apollo OR25841 or Maybridge 13- 31). 3-Chloro-isonicotinic acid (e.g. Aldrich 633410). 2-Chloro-6-methyl-benzoyl chloride (e.g. Fluorochem 38160 or Betapharm 15-47106). 2-Chloro-3-methoxyquinoxaline (e.g. BBB-SCI 3B- 16097/3B3-015501 or Anichem T14691). 4-Chloro-3-nitroanisole (e.g. Aldrich 116289). 4-Chloro-3- nitrotoluene (e.g. Aldrich 213055). (4-Chloro-phenyl)-acetyl chloride (e.g. Aldrich 638951). Chloroform (e.g. Sigma-Aldrich 650498). Copper(I) bromide-dimethyl sulfide complex (e.g. Aldrich 230502). Cuprous monochloride (e.g. Sigma-Aldrich 224332). DCM = methylene chloride / dicholormethane (e.g. Aldrich 270997). 2,3-Dicholorobenzoyl chloride (e.g. ABCR L09506 or BBB- SCI 3B4-0231). 2,6-Dichlorobenzoyl chloride (e.g. Fluka 35423). Diethyl amine (e.g. Sigma-Aldrich 471216). Diethyl ether (e.g. Sigma-Aldrich 346136). l,3-Difluoro-2-nitrobenzene (e.g. Aldrich 382957). 1,2-Dimethylbenzene (e.g. Sigma-Aldrich 95662). DIPEA = di-z^' o-propyl ethyl amine (e.g. Aldrich 387649). DMSO = dimethyl sulfoxide (e.g. Sigma D4540). 1,4-Dioxane (e.g. Sigma-Aldrich 296309). ELS = evaporative light scattering. Ethanol (e.g. Sigma-Aldrich 459844). Ethyl pyruvate (e.g. Fluka 15960). EtOAc = ethyl acetate (e.g. Fluka 34972). 3-Fluoro-4-nitroanisole (e.g. BBB-SCI 3B3- 013992 or ABCR AB229166). 4-Fluoro-benzene-l,2-diamine (e.g. Aldrich 653586). h = hour(s). Heptanes (e.g. Sigma-Aldrich 730491). HPLC = high performance liquid chromatography. Hydrazine hydrate (e.g. Sigma-Aldrich 225819). 30% aq hydrogen peroxide (e.g. Sigma-Aldrich H3410). Iron powder (e.g. Fluka 44900). Isobutyryl chloride (e.g. Aldrich 139122). K₂C0₃ (e.g. Sigma-Aldrich 209619). KI (e.g. Sigma-Aldrich 60400). LC = liquid chromatography. LC MS = liquid chromatography / mass spectrometry. 2M = 2 molar solution (similarly 8M = 8 molar solution etc). Methanol (e.g. Sigma-Aldrich 34860). 3 -Methyl-benzene- 1,2-diamine (e.g. Aldrich 272361). 4- methyl-benzene-l,2-diamine (e.g. Aldrich 339938). 2-Methyl-nicotinic acid (e.g. Aldrich 325228). 3- Methyl-isonicotinic acid (e.g. Matrix 020640 or Fluorochem 040096). 2-Methoxy-6-nitro-phenylamine (e.g. BBB-SCI 3B3-069963 or Synchem inc. SC-24920). 4-Methoxybenzhydrazide (e.g. Aldrich 558346). 4-Methoxy-benzoic acid chloride (e.g. Aldrich A88476). MgS0₄ (e.g. Sigma-Aldrich 246972). min = minutes. Mn0₂ (e.g. Aldrich 377201). MTBE = methyl tert-butyl ether (e.g. Sigma- Aldrich 306975). MW = microwave. MW conditions = reactions performed in sealed tubes using a Biotage Initiator instrument or a CEM Explorer-48 instrument. Na₂C0₃ (e.g. Sigma-Aldrich S7795). NaHC0₃ (e.g. Sigma-Aldrich S6014). NaOH (e.g. Sigma-Aldrich S5881). NaN0₂ (e.g. Sigma-Aldrich 237213). Na₂S0₄ (e.g. Sigma-Aldrich 238597). N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (e.g. Sigma-Aldrich E6383). NH₄CI (e.g. Aldrich 254134). 2-Oxo-propanoic acid methyl ester (e.g. Aldrich 371173). 5% Palladium on charcoal (e.g. Aldrich 75991). 10% Palladium on charcoal (e.g. Aldrich 75990). PDA = photo diode array. Pd(Ph₃P)₄ (e.g. Fluka 87645). Pentane (e.g. Sigma-Aldrich 236705). PhI(OAc)₂ = iodobenzene diacetate (e.g. Fluka 31490). PhPOCl₂ = phenylphosphonic dichloride (e.g. Aldrich 389560). POCI₃ = phosphoryl chloride (e.g. Aldrich 262099). 2-Propanol (e.g. Sigma-Aldrich 34959). PyBroP = benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (e.g. Fluka 12809). Pyridine (e.g. Sigma P3776). Racemic alanine (e.g. Sigma A7502). RT = retention time. Sat = saturated. SFC = supercritical fluid chromatography. 96%> Sulphuric acid (e.g. Sigma-Aldrich 320501). T = time. Tetrahydro-pyran-4- carbonyl chloride (e.g. BBB-SCI 3B3-077297 or Maybridge CC29902CB). TFA = trifluoroacetic acid (e.g. Sigma-Aldrich 302031). THF = tetrahydrofuran (e.g. Sigma-Aldrich 401757). THP = tetrahydropyranyl. (Trimethylsilyl)acetylene (e.g. Aldrich 218170).

Analytical LC-MS data were obtained using one of the following methods:

LC/MS Method 131 : LC/MS were run on a Sciex API150EX equipped with APPI-source operating in positive ion mode. The HPLC consisted of Shimadzu LClO-ADvp LC pumps, SPD-M20A PDA detector (operating at 254 tiM) and SCL-IOA system controller. Autosampler was Gilson 215, Column oven was a Jones Chromatography 7990R and ELS detector was a Sedere Sedex 85. LC- conditions: The column was a Waters Symmetry C-18, 4.6 x 30 mm, 3.5 microm operating at 60 °C with 3.0 rnL/min of a binary gradient consisting of water + 0.05 % TFA (A) and methanol + 0.05 % TFA. Gradient: 0.01 min: 17% B; 0.27 min 28% B; 0.53 min 39% B; 0.80 min 50% B; 1.07 min 59% B; 1.34 min 68% B; 1.60 min 78% B; 1.87 min 86% B; 2.14 min 93% B; 2.38 min 100% B; 2.40 min 17% B; 2.80 min 7% B; Total run time: 2.8 min.

LC/MS Method 132: same hardware as LC/MS method 131. LC-conditions: The column was a Waters Symmetry C-18, 4.6 x 30 mm, 3.5 microm operating at 60 °C with 2.5 mL/min of a binary gradient consisting of water + 0.05 % TFA (A) and methanol + 0.05 % TFA. Gradient: 0.01 min 5% B; 2.38 min 100% B; 2.40 min 5% B; 2.80 min 5% B. Total run time: 2.8 min.

LC/MS Method 350: LC/MS were run on a Sciex API300 equipped with APPI source operating in positive ion mode. The UPLC consisted of Waters Aquity including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM) and ELS detector. LC-conditions: The column was a Waters Aquity UPLC BEH C-18, 2.1 x 50 mm, 1.7 microm operating at 60 °C with 1.2 mL/min of a binary gradient consisting of water + 0.05 % TFA (A) and 95 % acetonitrile containing 5 % water + 0.03 % TFA. Gradient: 0.00 min 10.0% B; 1.00 min 100.0% B; 1.01 min 10.0% B; 1.15 min 10.0% B. Total run time 1.15 min.

Preparative LC/MS was performed using either of the following methods. Prep LC-MS method 1: The prep station was based on a Sciex API150EX equipped with APPI source operating in positive ion mode. The LC system consisted of Gilson 333 and 334 pumps, Gilson UV/VIS 155 detector Gilson GX 281 autosampler/fraction collector, and water bath for column and solvent heating. MS was controlled by Analyst (Sciex) and LC by Trilution (Gilson).

LC-conditions (polar methods, generic description). The column was a Waters Atlantis Prep dC18, 5 microm, 10 x 100 mm (19 x 50 for large scale) operating at 40 °C with 15-40mL/min of a binary gradient consisting of water + 0.05 % TFA (A) and methanol + 0.05 % TFA (B). Gradient: O.OOmin 5 or 10% B; 1.20 min 5 or 10% B; 4.00 min 100% B; 4.70 min 100% B; 4.75 min 5 or 10% B; 5.20 min 5 or 10%> B. Total runtime 8.0 min. Injection volume 0-200 microL. Injection into mobile phase B - time from injection to application on column/mixture with mobile phase A: 1.7 min (prior to T=0 minute in gradient program).

LC-conditions (normal methods, generic description). The column was a Waters Symmetry Prep C18, 5 microm, 10 x 100 mm (19 x 50/100 for large scale) operating at 40 °C with 15-40 mL/min of a binary gradient consisting of water + 0.05 % TFA (A) and methanol + 0.05 % TFA (B). Gradient: 0.00 min 10%B, 1.20 min 10%B, 4.00 min 100% B, 4.70 min 100% B, 4.75 min 10%B, 5.20 min 10%) B. Total runtime 8.0 min. Injection volume 0-200 microL. Injection into mobile phase B - time from injection to application on column/mixture with mobile phase A: 0.7 minute (prior to T=0 minute in gradient program).

Prep LC-MS method 2: The prep station consisted of the following Waters components: SQ 3100 with APPI source working in positive ion mode, Binary Gradient Module 2545, HPLC Pumps 515 (x2), Sample Manager 2767, Photodiode Array Detector 2998, and System Fluidics Organizer SFO.

LC-conditions

The column was a Waters Symmetry Prep C18, 10 microm, 30 x 100 mm operating at 40 °C with 90 mL/min of a binary gradient consisting of water + 0.1 %> TFA (A) and methanol + 0.1 %> TFA (B). Gradient: 0.00 min 20% B; 1.20 min 20%B, 5.00 min 100% B, 5.40 min 100%B, 5.50 min 20% B. Total runtime 6.5 min. Injection volume 0-400 microL. Injection into mobile phase B - time from injection to application on column/mixture with mobile phase A: 1.2 minute (from T=0).

GENERAL METHODS

In brief, the compounds of the invention I can be prepared from quinoxalin-2-yl-hydrazines II under the conditions described as method 1 or method 2, respectively. Hydrazines II can be prepared from III under the conditions described as method 3. In some cases, it is also possible to convert III directly to I using method 4. Precursors III can be obtained from lactams IV using either method 5 or method 6.

Method 1 consists of the treatment of hydrazines II with the appropriate acid chloride R COCI in a suitable solvent such as acetonitrile at elevated temperature as described for example Ial. Sometimes it can be an advantage to add POCI₃ or PI1POCI2. The acid chloride can be prepared in situ from the corresponding acid by the addition of POCI₃ as described for example Id8.

Method 2 is an alternative to method 1 in which the hydrazine II is condensed with the appropriate aldehyde R CHO in a suitable solvent like methylene chloride (DCM) to form the corresponding hydrazones. Subsequent addition of a suitable oxidant affords the compounds of the invention for example under the conditions reported by Sadana et al (A.K. Sadana, Y. Mirza, K.R. Aneja, O. Prakash European Journal of Medicinal Chemistry 2003, 38, 533) in which the oxidant is iodobenzene diacetate (PhI(OAc)2). Alternatively it is possible to use the procedure reported by Mogilaiah et al (K. Mogilaiah, T. Kumara Swamy, K. Shiva Kumar J Heterocyclic Chem. 2009, 46, 124) in which the intermediate hydrazones are cyclized oxidatively by treatment with chloramine-T.

Method 3 involves the displacement by hydrazine of X in compounds III wherein X is either a chlorine atom or another leaving group such as the phosphonium species drawn in the reaction scheme. The reaction typically occurs with hydrazine hydrate in a suitable solvent such as ethanol at elevated temperature as described for the synthesis of lid.

Method 4 is the direct conversion of compounds III to the compounds of the invention I by reaction with the appropriate acid hydrazide R₁CONHNH₂ in a suitable solvent such as acetonitrile at elevated temperatures as described for example Ia4. Method 5 is the conversion of lactams IV to compounds III wherein X is a chlorine atom by heating the substrate in excess phosphoryl chloride or PhPOCl₂ as described for lid; sometimes it can be an advantage to add a suitable base such as triethyl amine or di-z o-propyl ethyl amine (DIPEA) as described for Ilm.

Method 6 is the treatment of lactams TV with benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBroP) or a similar peptide coupling agent in the presence of a suitable base such as DIPEA to provide compounds III wherein X is the phosphonium species drawn in the reaction scheme. This method is known for other lactams in the literature (T.D. Ashton, P.J. Scammells Australian Journal of Chemistry 2008, 61, 49).

One skilled in the art would recognize the value and subsequent functionalization of a halogen, cyano or like group in connection with the further derivatization of a compound of the invention. For example, the skilled artisan would recognize the value of the halogen (bromo) moiety on the compound of Example Iol as opening the avenue to the synthesis further variants such as the alkynyl compound of Examp

Example Iol Example Io2

PREPARATION OF INTERMEDIATES

INTERMEDIATE: (3-Methyl-quinoxalin-2-yl)-hydrazine (Ha). 2-Oxo-propanoic acid methyl ester (9.0 mL) was added to a solution of 1,2-benzenediamine (10.8 g) in methanol (80 mL). The resulting suspension was refluxed for 10 min before it was cooled to ambient temperature. The precipitated solid was filtered off and dried to afford 3-methyl-lH-quinoxalin-2-one (15.3 g) sufficiently pure for the next step. 1.60 g of this material was dissolved in phosphoryl chloride (10 mL) and heated under MW conditions at 130 °C for 0.5h. The volatiles were removed in vacuo, and the residue was treated with ice/water to quench excess phosphoryl chloride. Diethyl ether and brine were added and the organic layer was dried over Na₂SO i, filtered, and concentrated in vacuo to afford 2-chloro-3- methyl-quinoxaline (1.7 g) sufficiently pure for the next step. This material was dissolved in ethanol (150 mL) and hydrazine hydrate (2.43 mL) was added. The mixture was refluxed for 1.5h. The volatiles were removed in vacuo, and the residual solid was washed with water, filtered off and dried Ila (1.2 g) sufficiently pure for the next step.

INTERMEDIATE: (5-Fluoro-3-methyl-quinoxalin-2-yl)-hydrazine (lib). l,3-Difluoro-2- nitrobenzene (5 g) was dissolved in THF (180 mL) and treated with hydrazine hydrate (1.57 g) at ambient temperature overnight. The volatiles were removed in vacuo to afford (3-fluoro-2-nitro- phenyl)-hydrazine (3.0 g). 2.0 g of this material was dissolved in methanol (15 mL). 10% Palladium on charcoal (0.3 g) was added and the mixture was treated with hydrogen (1 bar) for 12h at ambient temperature. The catalyst was filtered off, and the filtrate was concentrated in vacuo to afford 3- fluoro-benzene-l,2-diamine (1.4 g). This material was dissolved in methanol, ethyl pyruvate (1.2 g) was added and the mixture was stirred at ambient temperature for 2h. The precipitated solid was filtered off to afford an approximate 1 :1 mixture of 5-fluoro-3 -methyl- lH-quinoxalin-2-one and 8- fluoro-3-methyl-lH-quinoxalin-2-one (1.6 g in total). 1.2 g of this mixture was refluxed in phosphoryl chloride (20 mL) for 2h. The volatiles were removed in vacuo. The residue was basified with aq NaHCOs and extracted with DCM. The organic layer was dried over Na₂SO i, filtered, and concentrated in vacuo to afford an approximate 1 :1 mixture of 2-chloro-5-fluoro-3-methyl- quinoxaline and 3-chloro-5-fluoro-2-methyl-quinoxaline (1.1 g in total). This mixture was reacted with hydrazine hydrate (10 mL) at 110 °C for 2h. The precipitated solid was filtered off to afford an approximate 1 :1 mixture of lib and its regioisomer (8-fluoro-3-methyl-quinoxalin-2-yl)-hydrazine (0.4 g in total). This mixture was applied in the next step.

INTERMEDIATE: (5-Methoxy-3-methyl-quinoxalin-2-yl)-hydrazine (lie). 2-Methoxy-6-nitro- phenylamine (25.0 g) was dissolved in 37% aq HC1, and the mixture ws cooled on an ice/water bath. A solution of NaN0₂ (11.8 g) in water (3 mL) was added, and the resulting mixture was stirred at 0 °C for 15 min. The reaction mixture was added to a solution of cuprous monochloride (14.7 g) in 37% aq HC1 (10 mL) under stirring at 45-50 °C. The resulting mixture was stirred at 50 °C for 15 min, cooled at 5 °C for 15 min. The solid was filtered off and dried to give 2-chloro-l-methoxy-3- nitrobenzene (16.5 g). A mixture of 16 g of this material, racemic alanine (17 g), and K₂CO₃ (12 g) was heated in DMSO (180 mL) at 100 °C for 24h. The volatiles were removed using a freeze dryer. The residue was acidified with 2M aq HC1 (50 mL) and extracted into EtOAc. The organic extract was extracted with 2M aq Na₂C0₃ and water. The combined aq extracts were acidified with 2M aq HC1 and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo to afford 2-(2-methoxy-6-nitro-phenylamino)-propionic acid. This material was dissolved in ethanol (600 mL) and 96%> sulphuric acid (6 mL) was added. The mixture was heated at 80 °C overnight. The volatiles were removed in vacuo and the residue was dissolved in EtOAc and washed with 2M aq Na₂C0₃. The organic layer was dried over MgSO i, filtered, and concentrated in vacuo to afford 2-(2-methoxy-6-nitro-phenylamino)-propionic acid ethyl ester (8.0 g). This material was dissolved in ethanol (300 mL) and 5% palladium on carbon was added. The mixture was treated with 3 bars of hydrogen pressure on a Parr shaker for 3h. The catalyst was filtered off, and the filtrate was concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford 5-methoxy-3-methyl-3,4-dihydro- lH-quinoxalin-2-one (3.0 g). This material was dissolved in ethanol (300 mL) and treated with 30% aq hydrogen peroxide at 80 °C overnight. Most of the volatiles were removed in vacuo. The residue was suspended in ethanol (10 mL) and cooled on an ice/water bath before the solid was filtered off, washed with ice-cold ethanol, and dried to afford 5-methoxy-3-methyl-lH-quinoxalin-2-one (2.2 g). This material was dissolved in phosphoryl chloride (24 mL) and heated at 130 °C for 2h. The volatiles were removed in vacuo. The residue was partitioned between chloroform and ice + 2M aq NaOH. The organic layer was dried over MgSO i, filtered, and concentrated in vacuo to afford 2- chloro-5-methoxy-3-methyl-quinoxaline (2.5 g). This material was dissolved in ethanol (22 mL), hydrazine hydrate (2.9 mL) was added, and the mixture was refluxed for 2h. The volatiles were removed in vacuo, and water was added. The solid was filtered off, washed with water and heptanes and dried to afford lie (1.70 g) sufficiently pure for the next step.

INTERMEDIATE: (6-Fluoro-3-methyl-quinoxalin-2-yl)-hydrazine (lid) and (7-fluoro-3-methyl- quinoxalin-2-yl)-hydrazine (He). Ethyl pyruvate (8.69 mL) was added to a solution of 4-fluoro- benzene-l,2-diamine (9.91 g) in methanol (150 mL). The resulting suspension was refluxed for 10 min and cooled to ambient temperature. The solid was filtered off, washed with diethyl ether, and dried to afford an approximate 3:2 or 2:3 mixture of 6-fluoro-3 -methyl- lH-quinoxalin-2-one and 7- fluoro-3-methyl-lH-quinoxalin-2-one (7.3 g in total). 356 mg of this mixture was dissolved in acetonitrile (4 mL) and phosphoryl chloride (373 microL) was added. The resulting mixture was heated at 125 °C for 0.5h under MW conditions. The volatiles were removed in vacuo. The residue was partitioned between DCM and 5% aq NaHC0₃. The organic layer was dried over Na₂S0₄, filtered through a short silica plug, and concentrated in vacuo to afford an approximate 4:3 or 3:4 mixture of 2-chloro-6-fluoro-3-methyl-quinoxaline and 3-chloro-6-fluoro-2-methyl-quinoxaline (366 mg in total). 710 mg of this mixture prepared in a similar manner was dissolved in ethanol (10 mL) and hydrazine hydrate (1.05 mL) was added. The mixture was heated at 130 °C for 0.5h under MW conditions. The volatiles were removed in vacuo. The residue was triturated with 2% aq ammonia. The solid was filtered off and dried to afford an approximate 4:5 or 5:4 mixture of lid and He (0.66 for the next step.

INTERMEDIATE: (6-Chloro-3-methyl-quinoxalin-2-yl)-hydrazine (Ilf) and (7-Chloro-3-methyl- quinoxalin-2-yl)-hydrazine (Ilg). Ethyl pyruvate (12.2 mL) was added to a solution of 4-chloro- benzene-l,2-diamine (14.2 g) in methanol (150 mL). The resulting suspension was refluxed for 10 min. After cooling to ambient temperature the solid was filtered, washed with ether and dried. This material was an approximate 1 :1 mixture of 6-chloro-3-methyl-lH-quinoxalin-2-one and 7-chloro-3- methyl-lH-quinoxalin-2-one (13 g in total). 1.17 g of this mixture was dissolved acetonitrile (4 mL) and heated with phosphoryl chloride (1.12 mL) under MW conditions at 125 °C for 0.5h. The volatiles were removed in vacuo. The residue was partitioned between DCM and 5% aq NaHC0₃. The organic layer was dried over Na₂S0₄, filtered through a short silica plug, and concentrated in vacuo to afford an approximate 1 :1 mixture of the two regioisomers 2,6-dichloro-3-methyl- quinoxaline and 3,6-dichloro-2-methyl-quinoxaline (1 g in total). A larger portion (5.0 g) prepared in a similar manner was dissolved in ethanol (100 mL) and refluxed with hydrazine hydrate (6.85 mL) for 3h. The volatiles were removed in vacuo. The residue was triturated with 2% aq ammonia (25 mL). The solid was filtered off and dried to afford an approximate 1 :1 mixture of Ilf and Ilg (3.90 g ently pure for the next step.

INTERMEDIATE: (3,6-Dimethyl-quinoxalin-2-yl)-hydrazine (Ilh). 2-Oxo-propanoic acid methyl ester (10.45 g) was added to solution of 4-methyl-benzene-l,2-diamine (10 g) in methanol (200 mL). The mixture was stirred at ambient temperature for 4h. The precipitated solid was filtered off to afford an approximate 1:2 or 2:1 mixture of 3,6-dimethyl-lH-quinoxalin-2-one and 3,7-dimethyl- lH-quinoxalin-2-one (9.5 g in total). This mixture was treated with PI1POCI₂ (21.3 g) at 150 °C for 4h. The crude mixture was cooled to ambient temperature and treated with water (100 mL) before the mixture was basified with ammonia. The precipitated solid was filtered off and purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford an approximate 1 :2 or 2:1 mixture of 2-chloro-3,6-dimethyl-quinoxaline and 3-chloro-2,6-dimethyl-quinoxaline (5 g in total) as a yellow solid. This material was dissolved in ethanol (50 mL) and refluxed overnight with hydrazine hydrate (30 mL). The volatiles were removed in vacuo. The residue was purified by chromatography on silica (eluent: pentane/EtOAc 20:1→ 5:1) to afford an approximate 2:1 or 1 :2 mixture of Ilh and its regioisomer (3,7-dimethyl-quinoxalin-2-yl)-hydrazine (4.8 g in total). This material was used in the next step.

INTERMEDIATE: 6-Methoxy-3-methyl-lH-quinoxalin-2-one (IVi). 3-Fluoro-4-nitroanisole (2.15 g) was dissolved in DMSO (30 mL). Racemic alanine (2.5 g) and K2CO3 (1.7 g) were added and the mixture was heated at 50 °C for 1.5h. More racemic alanine (2.2 g) was added and the mixture was allowed to stir at 50 °C overnight before it was heated at 75 °C for 3h. The crude mixture was partitioned between 2M aq HCl and EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was refluxed lh in a mixture of methanol (50 mL) and sulfuric acid (2 mL). The volatiles were removed in vacuo. The residue was partitioned between sat. aq Na₂C0₃ and EtOAc. The organic layer was with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was dissolved in a mixture of THF (20 mL), ethanol (100 mL) and 2M aq sulfuric acid (7 mL). The mixture was de-gassed with argon. 10% Palladium on charcoal (300 mg) was added and the mixture was treated with hydrogen gas (3 bar) using a Parr shaker for 5h. The crude mixture was filtered through a pad of celite and concentrated in vacuo. The residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was dissolved in a mixture of ethanol (100 mL) and 30% aq hydrogen peroxide and heated at 50 °C overnight. The volatiles were removed in vacuo. The residue was refluxed in a mixture of ethanol (80 mL) and 30% aq hydrogen peroxide for 5h. The volatiles were removed in vacuo. The residue was re-precipitated from ethanol to afford fficiently pure for the next step.

INTERMEDIATE: (3,7-Dimethyl-quinoxalin-2-yl)-hydrazine (Ilj). 4-Chloro-3-nitrotoluene (19 mL) was dissolved in DMSO (300 mL). Racemic alanine (45 g) and K₂C0₃ (20 g) were added, and the mixture was stirred overnight at 150 °C. The crude mixture was poured into water (300 mL) and acidified with 37% aq HC1. The product was extracted into EtOAc. The organic layer was washed with brine and concentrated in vacuo. The residue was refluxed for 0.5h in a mixture of methanol (300 mL) and 96% sulfuric acid (20 mL). The volatiles were removed in vacuo. The residue was partitioned between water and EtOAc after basification with NaOH. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was dissolved in a mixture of ethanol (500 mL), THF (100 mL) and 2M aq sulfuric acid (35 mL). The mixture was degassed with Ar. 10% Palladium on charcoal (1 g) was added, and the mixture was treated with hydrogen gas (3 bar) using a Parr shaker for 3 h. More 10% palladium on charcoal (1 g) was added and the mixture was treated with hydrogen gas (3 bar) using a Parr shaker overnight. The catalyst was filtered off, and the filtrate was concentrated in vacuo. The residue was partitioned between water and EtOAc. The aq layer was basified with 28% aq NaOH and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford 3,7-dimethyl-3,4-dihydro-lH-quinoxalin-2-one (6.12 g) as a pale brown solid that was used directly in the next step. A suspension of this material (5.98 g) and 10% palladium on charcoal (0.60 g) in 1,2-dimethylbenzene (200 mL) refluxed overnight. The crude mixture was cooled to 50 °C and diluted with THF (200 mL) before it was filtered through a plug of celite. The filtrate was concentrated in vacuo to yield 3,7-dimethylquinoxalin-2(lH)-one (5.9 g) as a brown solid that was used in the next step. A portion of this material (1.08 g) was added to an ice-cold mixture of phosphoryl chloride (20 mL) and DIPEA (2.16 mL). The mixture was heated at 100 °C for 2h. The volatiles were removed in vacuo. The residue was diluted with DCM (50 mL) and poured carefully onto ice (200 mL) to quench excess phosphoryl chloride. The aq layer was extracted with DCM. The combined organic layers were washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford 3-chloro-2,6-dimethyl-quinoxaline (879 mg) sufficiently pure for the next step. A larger portion of this material (3.31 g) prepared in a similar manner was dissolved in ethanol (100 mL). Hydrazine hydrate (3.2 mL) was added, and the mixture was refluxed for 8h before the heat was turned off and the mixture was allowed to stand at ambient temperature overnight. Next morning, hydrazine hydrate (1 mL) added and the mixture was refluxed for 8h. The mixture was allowed to cool to ambient temperature. The volatiles were removed in vacuo. The residual solid was washed with water and heptanes before it was dried to afford Ilj (2.57 g) as a slightly red solid sufficiently pure for the next step.

3-Chloro-6-methoxy-2-methyl-quinoxaline (Illk). 4-Chloro-3-nitroanisole (5 g) was dissolved in DMSO (60 mL). Racemic alanine (4.7 g) and K₂C0₃ (3.7 g) were added. The mixture was stirred at 80 °C for 0.5h, then at 130 °C for lh, then at 150 °C for 3h. Then the mixture was cooled to 100 °C overnight. Next day it was heated at 150 °C for 5h before it was cooled to ambient temperature and poured into water (400 mL). The pH adjusted to 2 with 2M aq HC1. The desired intermediate was extracted into EtOAc, and the organic layer was washed with brine, dried over MgS0₄, filtered, and concentrated in vacuo. The residue was dissolved in a mixture of methanol (100 mL) and 96% sulfuric acid (5 mL). The mixture was refluxed for lh. The volatiles were removed in vacuo. The residue was suspended in water and basified with K₂CO₃. The desired intermediate was extracted into EtOAc and the organic layer was washed with brine, dried over MgS0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography to afford methyl-2-[(4-methoxy- 2-nitrophenyl)amino]propanoate (870 mg) as a red solid. This material was dissolved in a mixture of ethanol (60 mL), THF (15 mL) and 2M aq sulfuric acid (5 mL). 10% Palladium on charcoal (300 mg) was added and the mixture was treated with hydrogen gas (3 bar) for 3h using a Parr shaker. The catalyst was filtered off, and the filtrate was concentrated in vacuo. The residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried over MgS0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes → EtOAc) to afford 7-methoxy-3-methyl-3,4-dihydro-lH-quinoxalin-2-one that was carried on directly in the synthesis. This material was dissolved in ethanol (100 mL) and 30% aq hydrogen peroxide was added and the mixture was heated at 60 °C for 3h. The volatiles were removed in vacuo. The residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried over MgS0₄, filtered, and concentrated in vacuo. The residue was dissolved in ethanol (100 mL) and treated with 30% aq hydrogen peroxide overnight at 50 °C. The volatiles were removed in vacuo. The residue was partitioned between sat. aq K₂CO₃ and EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo to afford 7-methoxy- 3-methyl-lH-quinoxalin-2-one (445 mg). A portion of this material (326 mg) was suspended in acetonitrile (15 mL). Phosphoryl chloride (0.32 mL) was added and the mixture was heated at 110 °C for 15 min under MW conditions and subsequently at 120 °C for 10 min under MW conditions and finally at 130 °C for 5 min under MW conditions. The crude mixture was poured into water (20 mL) and extracted with EtOAc. The organic layer was washed with brine, dried over MgS0₄, concentrated in vacuo to afford Illk (370 mg) sufficiently pure for the next step.

INTERMEDIATE: (3,5-Dimethyl-quinoxalin-2-yl)-hydrazine (Ilm). To a solution of 3-methyl- benzene-l,2-diamine (1.5 g) in methanol (15 mL) was added ethyl pyruvate (1.4 g), and the mixture was stirred at ambient temperature for 2h. The precipitated solid was filtered off to afford an approximate 1 :6 or 6:1 mixture of 3,5-dimethyl-lH-quinoxalin-2-one and 3,8-dimethyl-lH- quinoxalin-2-one (1.9 g in total). 1.0 g of this mixture was refluxed in phosphoryl chloride (30 mL) for 2h. The volatiles were removed in vacuo. The residue was basified with aq NaHC0₃ and extracted with DCM. The organic layer was dried over Na₂S0₄, filtered, and concentrated in vacuo to afford an approximate 1 :6 or 6:1 mixture of 2-chloro-3,5-dimethyl-quinoxaline and 3-chloro-2,5- dimethyl-quinoxaline (1.1 g in total). This material was heated in hydrazine hydrate (20 mL) at 110 °C for 2h. The precipitated solid was filtered off and dried to afford an approximate 1 :6 or 6:1 mixture of Ilm and its regioisomer (3,8-dimethyl-quinoxalin-2-yl)-hydrazine (0.6 g in total). This mixture was used in the next step.

INTERMEDIATE: 4-Chloro-l -(2-chloro-phenyl)-[l ,2,4]triazolo[4,3-a]quinoxaline (IIx).

A mixture of 2-chloro-3-methoxyquinoxaline (455 mg) and 2-chlorobenzhydrazide (439 mg) in acetonitrile (10 mL) was heated at 150 °C for 15 min under MW conditions. The reaction mixture was cooled in an ice/water bath and the precipitated solid was collected by filtration. This material was suspended in acetonitrile (10 mL) and phosphoryl chloride (1.09 mL) was added. The mixture was heated at 150 °C for lh under MW conditions, before another 0.5 mL of phosphoryl chloride was added and the mixture was heated for an additional lh at 150 °C under MW conditions. The reaction mixture was poured onto ice and diluted with ice/water to a final volume of 50 mL. It was neutralized with solid NaHC0₃ and the resulting yellow precipitate was collected by filtration, water and dried to afford IIx (409 mg).

INTERMEDIATE: (7-Bromo-3-methyl-quinoxalin-2-yl)-hydrazine (Iln). A mixture of 4-bromo-l- fluoro-2-nitn benzene (99 g), racemic alanine (120 g), and CS2CO3 (440 g) were refluxed for 5h in a mixture of in ethanol (1.2 L) and water (400 mL). After cooling to ambient temperature the mixture was diluted with water (600 mL) and acidified to pH 3. The solid was filtered off and dried to afford 2-(4-bromo-2-nitro-phenylamino)-propionic acid (110 g) as yellow solid. A portion of this material (10 g) was dissolved in AcOH (35 mL), and iron powder (5.8 g) was added. The mixture was stirred at 90 °C for 2h, cooled to ambient temperature, and filtered. Most of the volatiles in the filtrate were removed in vacuo. The remaining slurry was diluted in DCM. The organic layer was dried over Na₂S0₄, filtered, and concentrated in vacuo to afford 7-bromo-3-methyl-3,4-dihydro H-qumoxalin- 2-one (6.4 g) as a yellow solid. A larger portion of this material prepared in a similar manner (45 g) was mixed with water (180 mL) and 30% aq hydrogen peroxide (140 mL). The mixture was stirred at 60 °C for 6h, cooled and the solid was filtered off, washed with water, and dried to afford 7- bromo-3-methyl-lH-quinoxalin-2-one (36 g) as a yellow solid. A portion of this material (12 g) was stirred in PI1POCI₂ (80 mL) at 150 °C for 4h. After cooling to ambient temperature, water was added and pH was adjusted to 7 with aqueous ammonia. The precipitated solid was filtered off, washed with water, and dried to afford 6-bromo-3-chloro-2-methyl-quinoxaline (8.0 g) as a yellow solid. A larger portion of this material prepared in a similar manner (16 g) was dissolved in ethanol (250 mL). Hydrazine hydrate (160 mL) was added, and the mixture was refluxed for 3h, cooled to ambient temperature, and most of the volatiles were removed in vacuo. The residue was suspended in water, the solid was filtered off, washed with water, and dried to afford Iln (13 g) as a yellow solid pure for the next step.

INTERMEDIATE: (6-Bromo-3-methyl-quinoxalin-2-yl)-hydrazine (IIo). 4-Bromo-2-fluoro-l- nitro-benzene (40 g), racemic alanine (16.2 g), and K₂CO₃ (30 g) were refluxed overnight in a mixture of in ethanol (200 mL) and water (200 mL). After cooling to ambient temperature, the mixture was diluted with water, and acidified with 1M aq HCl. The precipitated solid was collected and dried to afford 2-(5-bromo-2-nitro-phenylamino)-propionic acid (41 g). A larger portion of this material prepared in a similar manner (60 g) was dissolved in methanol (250 mL) and treated with SOCI₂ (30 mL, added drop-wise). The reaction mixture was stirred at ambient temperature overnight. The volatiles were removed in vacuo. The residue was partitioned between EtOAc and aq NaHC0₃. The organic layer was dried over MgSO i, filtered, and concentrated in vacuo to afford (5- bromo-2-nitro-phenylamino)-propionic acid methyl ester (60 g). This material was dissolved in AcOH (400 mL), iron powder (55 g) was added, and the mixture was refluxed for 4h. After cooling to ambient temperature, the solid was filtered off and the filtrate was concentrated in vacuo. The residue was partitioned between EtOAc and sat. aq NaHC0₃. The organic layer was dried over Na₂S0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford 6-bromo-3-methyl-3,4-dihydro-lH-quinoxalin-2-one (47.7 g) as a pale yellow solid. A portion of this material (10 g) was dissolved in THF (150 mL), and the solution was cooled on an ice/water bath. Mn0₂ (19.3 g) was added. The resulting mixture was stirred at ambient temperature overnight. EtOAc (100 mL) was added to the mixture. The solid was filtered off. The filtrate was concentrated in vacuo to afford 6-bromo-3-methyl-lH-quinoxalin-2-one (8.8 g). A larger portion of this material prepared in a similar manner (10 g) was stirred in PI1POCI₂ (80 mL) at 150°C for 3h. After cooling to ambient temperature, water was added and pH was adjusted to 7 with aqueous ammonia. The precipitated solid was filtered off, washed with water and dried to afford 6-bromo-2-chloro-3-methyl-quinoxaline (6.67 g). A larger portion of this material prepared in a similar manner (14 g) was dissolved in ethanol (250 mL) and hydrazine hydrate (180 mL) was added. The mixture was refluxed for 3h, cooled to ambient temperature, and most of the volatiles were removed in vacuo. The residue was diluted with water, and solid was filtered off, washed with water, and dried to afford IIo (11.2 g) as a yellow solid sufficiently pure for the next step.

INTERMEDIATE: (5-Bromo-3-methyl-quinoxalin-2-yl)-hydrazine (Hp). 3-Bromo-2-nitro- phenylamine (1.2 g) and K₂CO₃ (0.8 g) in DCM (100 mL) were treated with 2-chloroacetyl chloride (0.8 g) for 0.5h at ambient temperature. The mixture was refluxed for 4h and then stirred for additional 0.5h at ambient temperature. The reaction mixture was poured into water. The aq layer was extracted with DCM. The combined organic layers were washed with water and dried over MgS0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes/EtOAc 1 :1) to afford N-(3-bromo-2-nitro-phenyl)-2-chloro-propionamide (1.6 g) as a white solid. Iron powder (7.5 g) and NH₄C1 (0.4 g) were suspended in a mixture of AcOH (1.9 mL) and water (105 mL). The mixture was at 50 °C and stirred vigorously for approximately 15 min. N-(3-Bromo-2-nitro-phenyl)-2-chloro-propionamide (4.52 g prepared in a similar manner to the one described above) was dissolved in DMF (38 mL) and added to the aforementioned vigorously stirred mixture. The mixture was stirred at 50 °C. The resulting slurry was basified with 10% aq Na₂C0₃ solution to pH 8-9. The crude product was re-precipitated from EtOAc/pentane (4:1) to afford N-(2- amino-3-bromo-phenyl)-2-chloro-propionamide (1.8 g). A mixture of this material, KI (0.54 g), and NaHC0₃ (0.9 g) in acetonitrile (100 mL) was refluxed for 15h. The volatiles were removed in vacuo. The residue was stirred in water (200 mL) for 10 min. The resulting suspension was acidified with 2M aq HC1 to pH = 5-6. The solid was re-precipitated from EtOAc to afford 5-bromo-3-methyl-3,4- dihydro-lH-quinoxalin-2-one (0.6 g). A larger portion of this material (6.6 g) prepared in a similar manner was dissolved in 5% aq NaOH (250 mL). 30% Aq hydrogen peroxide (24 mL) and water (6 mL) were added, and the resulting mixture was stirred at 60 °C for 6h. The mixture was cooled to ambient temperature, and the solid was collected by filtration, washed with water, and dried to afford a crude material that was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford almost pure 5-bromo-3-methyl-lH-quinoxalin-2-one as white a solid that was re-precipitated from acetone/pentane (4:1) to afford 5-bromo-3-methyl-lH-quinoxalin-2-one (3.6 g). 0.5 g of this material was stirred in PhPOC12 (1.1 mL) at 150 °C for 3h. After cooling to ambient temperature, the mixture was poured into water, and pH was adjusted to 8 with aqueous ammonia. The product was extracted into DCM and purified by preparative TLC (eluent: pentane/EtOAc 2: 1) to afford 5-bromo-2-chloro- 3-methyl-quinoxaline (272 mg) as a yellow solid. This material was dissolved in ethanol (4 mL) and refluxed with hydrazine hydrate (2.3 mL) for lh. Most of the volatiles were removed in vacuo. The residue was suspended in water, and the solid was filtered off, washed with water, and dried to afford

) as a red solid sufficiently pure for the next step.

INTERMEDIATE (7-Bromo-5 -methoxy-3 -methyl-quinoxalin-2-yl)-hydrazine (Ilq).

2-Amino-3-nitrophenol (25 g) and NaOH (25.0 g) were dissolved in a mixture of THF (500 mL) and water (200 mL). TBAI (2.5 g) and methyl iodide (21.2 mL) were added. The mixture was stirred overnight at ambient temperature. Most of the THF was removed in vacuo. The residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo to afford 2-methoxy-6-nitro-phenyl amine (29.4 g). 22 g of this material and NaOAc (17.8 g) were mixed in acetic acid (300 mL) at ambient temperature. Bromine (6.9 mL) in acetic acid (5 mL) was added drop-wise over 15 min. The precipitated solid was filtered off, washed with water and heptanes, and dried to afford 4-bromo-2-methoxy-6-nitro-phenylamine (25.5 g). 2.47 g of this material dissolved in DCM (100 mL). DMAP (1.22 g) and Boc₂0 (2.62 g) were added, and the mixture was overnight at ambient temperature. The volatiles were removed in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→ EtOAc). Fractions containing the desired material were pooled and most of the EtOAc was removed in vacuo. The residual solution was diluted with heptanes, and the resulting mixture was allowed to stand at ambient temperature overnight. The precipitated solid was filtered off and dried to afford (4-bromo- 2-methoxy-6-nitro-phenyl)-iminocarbonicacid-bis-(tert-butyl ester) (3.94 g). A larger portion of this material (25.6 g) prepared in a similar manner was dissolved in ethanol (700 mL). 5% Platinum on charcoal (4.0 g) was added, and the mixture was treated with hydrogen gas (1 bar) for 45 min using a Parr shaker instrument. The catalyst was filtered off. The filtrate was concentrated in vacuo. The residual solid was suspended in heptanes, filtered, and dried to afford 6-amino-4-bromo-2-methoxy- phenyl)-iminocabonicacid-bis (fert-butyl ester) (22.0 g). This material was dissolved in DCM (250 mL) and treated with TFA (5 mL) overnight at ambient temperature. The volatiles were removed in vacuo. The residue was partitioned between EtOAc and 2M aq NaOH (until pH was 9). The organic layer was dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→ EtOAc) to afford the mono-deprotected material (ca 11 g). This material was dissolved in a mixture of DCM (100 mL) and TFA (50 mL) and stirred overnight at ambient temperature. The volatiles were removed in vacuo. The residue was partitioned between EtOAc and 2M NaOH (until pH 9). The organic layer was dried over MgSO i, filtered, and concentrated in vacuo to afford 5-bromo-3-methoxy-benzene-l,2-diamine (3.63 g). This material was dissolved in methanol (200 mL). 2-Oxo-propanoic acid methyl ester (2.0 g) was added, and the mixture was stirred overnight at ambient temperature. The precipitated 7-bromo-5-methoxy-3- methyl-lH-quinoxalin-2-one was filtered off. The filtrate was concentrated in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→ EtOAc) to afford more 7-bromo- 5-methoxy-3-methyl-lH-quinoxalin-2-one as the first eluting isomer followed by 6-bromo-8- methoxy-3-methyl-lH-quinoxalin-2-one as the second eluting isomer. The two crops of 7-bromo-5- methoxy-3-methyl-lH-quinoxalin-2-one were mixed and washed with a little acetone to afford 7- bromo-5-methoxy-3 -methyl- lH-quinoxalin-2-one (1.7 g) sufficiently pure for the next step. The fractions containing the second eluting isomer were pooled and concentrated in vacuo; the residual solid was washed with a little acetone to afford 6-bromo-8-methoxy-3-methyl-lH-quinoxalin-2-one (0.61 g) sufficiently pure for the next step. The structure of the isomers were elucidated by identifying the nitrogen carrying a proton by 2D HSQC and comparing with the shift of the nitrogen having long range correlation to an aromatic proton in 2D HMBC. 7-Bromo-5- methoxy-3-methyl-lH-quinoxalin-2-one (1.7 g) was refluxed in phosphoryl chloride (23 mL) for 2h. The volatiles were removed in vacuo. The residue was partitioned between DCM and ice/water. The mixture was basified with 2M aq Na₂C03. The organic layer was dried over MgSO i, filtered, and concentrated in vacuo to afford 7-bromo-2-chloro-5-methoxy-3-methyl-quinoxaline (0.61 g). This material was dissolved in ethanol (38 mL). Hydrazine hydrate (3 mL) was added and the mixture was refluxed for 2h. The volatiles were removed in vacuo. The residual solid was suspended in water, filtered off, washed with heptanes, and dried to afford Ilq (0.58 g) sufficiently pure for the next step.

COMPOUNDS OF THE INVENTION

Example Ial l-(2-Chloro-phenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ila (250 mg) and 2-chlorobenzoyl chloride (220 microL) were mixed in acetonitrile (3 mL) and heated at 150 °C for 0.5h. The crude mixture was concentrated in vacuo. The residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried over Na₂S0₄, filtered, and concentrated in vacuo. 200 mg of the residue was purified by preparative LC/MS to afford example Ial (90 mg). LC/MS (method 350): RT(PDA)=0.60 min; PDA / ELS-purities 99.1% / 100%; mass observed

295.3.

Example Ia2 4-Methyl-l-phenyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ila (60 mg) and benzoyl chloride (50 microL) were treated under the conditions described for example Ial using 140 °C instead of 150 °C to afford example Ia2 (2.7 mg) after preparative LC/MS purification of a 10 mg portion of the crude product. LC/MS (method 350): RT(PDA)=0.56 min; PDA / ELS-purities 100% / 100%; mass observed 261.2.

Example Ia3 l-(4-Methoxy-phenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ila (175 mg) and 4-methoxybenzoic acid chloride (162 microL) were treated under the conditions described for example Ia2. The crude product was purified by chromatography on silica (eluent: DCM→ 20% EtOAc in DCM) to afford example Ia3 (130 mg) as a beige solid. LC/MS (method 350): RT(PDA)=0.58 min; PDA / ELS-purities 92.4% / 100%; mass observed 290.9.

Example Ia4 l-(3-Chloro-pyridin-4-yl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. A mixture of 3-chloroisonicotinic acid (49 mg), Ila (49 mg), and N-(3-dimethylaminopropyl)-iV- ethylcarbodiimide hydrochloride (59 mg) in acetonitrile (2 mL) was stirred at ambient temperature overnight. The mixture was then heated at 200 °C for 0.5h under MW conditions. Water (15 mL) was added and the mixture was partially evaporated to about half the original volume. A red precipitate formed, and this was collected by filtration, washed with water and dried to yield example Ia4 (45.2 mg) as a brick-red solid. LC/MS (method 350): RT(PDA)=0.49 min; PDA / ELS-purities 94.4% / 100%; mass observed 296.3.

Example Ia5 4-Methyl-l-propyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ila (1.0 g) was dissolved in acetonitrile (40 mL) and butyryl chloride (0.73 g) was added. The mixture was refluxed for 16h. The volatiles were removed in vacuo. The residue was partitioned between EtOAc and water. The organic layer was washed with brine, dried over Na₂S0₄, filtered, and concentrated in vacuo. The residue was washed with MTBE to afford example Ia5 (880 mg) as a yellow solid. LC/MS (method 132): RT(PDA)=1.70 min; PDA purity 99.0%; mass observed 226.9.

Example Ia6 l-Isopropyl-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. Prepared as described for example Ia5 using Ila (1.0 g) and isobutyryl chloride (0.74 g) to afford example Ia6 (970 mg). LC/MS (method 132): RT(PDA)=1.66 min; PDA purity 100%; mass observed 226.8.

Example Ia7 4-Methyl-l-(tetrahydro-pyran-4-yl)-[l,2,4]triazolo[4,3-a]quinoxaline. Prepared as described for example Ia5 using Ila (300 mg) and tetrahydro-pyran-4-carbonyl chloride (307 mg) to afford the crude product which was purified by chromatography on silica (eluent: pentane→ pentane/EtOAc 1 :1)= to afford example Ia7 (89 mg). LC/MS (method 132): RT(PDA)=1.46 min; PDA purity 93%; mass observed 269.2.

Example Ia8 4-Methyl-l-(2-methyl-pyridin-3-yl)-[l,2,4]triazolo[4,3-a]quinoxaline. 2-Methyl- nicotinic acid (103 mg), Ila (125 mg), acetonitrile (3 mL), and phosphoryl chloride (67 microL) were added to a MW vial. The mixture was heated at 140 °C for 0.5h under MW conditions. The precipitated solid was filtered off and dissolved in hot methanol, and the black oily gum was decanted off. The solution was treated with activated charcoal, filtered, and concentrated in vacuo to afford example Ia8 (87 mg) as a beige semisolid. LC/MS (method 131): RT(PDA)=0.83 min; PDA / ELS-purities 91.1% / 100%; mass observe

Example Ia9 4-Methyl-l-(3-methyl-pyridin-4-yl)-[l,2,4]triazolo[4,3-a]quinoxaline. 3-Methyl- isonicotinic acid (103 mg), Ila (125 mg), and phosphoryl chloride (67 mg) were reacted as described for example Ia8. The crude mixture was filtered to afford example Ia9 (27 mg) as a solid. LC/MS (method 131): RT(PDA)=0.89 min; PDA / ELS-purities 84.1% / 81.7%; mass observed 276.1.

Example IalO l-(4-Chloro-benzyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. (4-Chloro- phenyl)-acetyl chloride (122 microL) and Ila (174 mg) were suspended in acetonitrile (3.0 mL) and the mixture was heated under MW conditions at 150 °C for 0.5h. The black solid was filtered off from the crude mixture, dissolved in ethanol, and treated with activated charcoal. The mixture was filtered, and the filtrate was concentrated in vacuo to afford example IalO (120 mg) as a pale purple solid. LC/MS (method 131): RT(PDA)=1.58 min; PDA / ELS-purities 73% / 100%; mass observed 309.3.

Example Ibl l-(2-Chloro-phenyl)-6-fluoro-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. An approximate 1 :1 mixture of lib and its regioisomers (8-fluoro-3-methyl-quinoxalin-2-yl)-hydrazine (0.4 g in total) was dissolved in 1,4-dioxane (20 mL) and refluxed with 2-chloro-benzoyl chloride (365 mg) and phosphoryl chloride (0.3 mL) for 2h. The volatiles were removed in vacuo. The residue was partitioned between sat as NaHC0₃ and DCM. The organic layer was concentrated in vacuo. The residue was purified by preparative TLC (eluent: pentane/EtOAc 1 :1) to afford example Ibl (40 mg). LC/MS (method 131): RT(PDA) 1.44 min; PDA/ELS purities 62.0% / 100.0%; mass observed 313.0.

Example Icl l-(2,6-Dichloro-phenyl)-6-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. lie (0.300 g), acetonitrile (10 mL), and 2,6-dichlorobenzoyl chloride (252 microL) were added to a MW vial. A Stir bar was added, the vial was capped, and the mixture was heated at 150 °C for 0.5h under MW conditions. Phosphoryl chloride (274 microL) was added and the resulting mixture was heated at 150 °C for 0.5h. The volatiles were removed in vacuo, and the residue was partitioned between DCM and 2M aq NaOH. The organic layer was dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford purified example Icl. This material was stirred in heptanes to afford example Icl (140 mg) as a solid. LC/MS (method 131): RT(PDA)=1.35 min; PDA / ELS-purities 97.9% / 100%; mass observed 358.8.

Example Ic2 1 -(2-Chloro-6-methyl-phenyl)-6-methoxy-4-methyl-[l ,2,4]triazolo[4,3- a]quinoxaline. lie (306 mg) and 2-chloro-6-methyl-benzoyl chloride (233 microL) were treated as described for example 111 to afford purified example Ic2 after chromatographic purification. The obtained solid was stirred in a 1 : 1 mixture of EtOAc and heptanes to afford example Ic2 (44 mg) as a solid. LC/MS (method 131): RT(PDA)=1.41 min; PDA / ELS-purities 96.6% / 100%; mass observed 339.2.

Example Ic3 l-(2,3-Dichloro-phenyl)-6-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. lie (306 mg) and 2,3-dicholorobenzoyl chloride (377 mg) were treated as described for example Ic2 to afford example Ic3 (0.3 g) as a solid. LC/MS (method 131): RT(PDA)=1.53 min; PDA / ELS- purities 99.3% / 100%; mass observed 358.8.

Example Ic4 l-(2-Chloro-phenyl)-6-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. lie (300 mg) and 2-chlorobenzoyl chloride (223 microL) were treated as described for example Ic2 with the exception that the fractions containing the title compound after chromatography were pooled and treated with activated charcoal, filtered, and concentrated in vacuo. The resulting solid was suspended in a 1 :1 mixture of EtOAc and heptanes to afford example Ic4 (0.25 g) as a solid. LC/MS (method 131): RT(PDA)=1.31 / 100%; mass observed 325.2.

Example Idl l-(2-Chloro-phenyl)-7-fluoro-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline and example Iel l-(2-chloro-phenyl)-8-fluoro-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. 2- Chlorobenzoyl chloride (1.34 mL) was added to an approximate 4:5 or 5:4 mixture of lid and He (1.0 g in total) dissolved in a mixture of pyridine (0.85 mL) and acetonitrile (10 mL). The mixture was stirred at ambient temperature for lh. Phosphoryl chloride (0.98 mL) was added, and the mixture was heated at 135 °C for 0.5h under MW conditions. The volatiles were removed in vacuo. The residue was triturated with 5% aq ammonia. The precipitated solid was filtered off and dried to afford an approximate 1 :1 mixture of l-(2-chloro-phenyl)-7-fluoro-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline and l-(2-chloro-phenyl)-8-fluoro-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline (2.40 gram in total). 75 mg of this mixture was purified by preparative SFC to afford example Iel (25 mg) as the second eluting isomer and example Idl (50 mg) as the first eluting isomer using a Berger Multigram II system fitted with a Princeton SFC CN 6A 5u (250 x 21.2 mm) column operating at 50 mL/min at 35 °C and 100 bar back-pressure using stacked injections. The eluent was C0₂ (90 %>) and 2-propanol + 0.1 % diethylamine (30%). LC/MS (method 131) for example Idl: RT(PDA)=1.54 min; PDA / ELS-purities 95.6% / 96.7%; mass observed 312.9. LC/MS (method 131) for example Iel: RT(PDA)=1.52 min; PDA / ELS-purities 99.1% / 99.7%; mass observed 313.0.

Example Ifl 7-Chloro-l-(2-chloro-phenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline and example Igl 8-chloro-l-(2-chloro-phenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. 2- Chlorobenzoyl chloride (1.34 mL) was added to an approximate 1 :1 mixture of Ilf and Ilg (2.20 g) dissolved in a mixture of pyridine (0.85 mL) and acetonitrile (100 mL). The resulting mixture was stirred at ambient temperature for 2h. The volatiles were removed in vacuo. The residue was triturated with water and the solid (3.6 g after drying) was filtered and dried. This material was suspended in acetonitrile (12 mL) and treated with phosphoryl chloride (0.98 mL) at 135 °C for 0.5h under MW conditions. The volatiles were removed in vacuo. The residue was dissolved in EtOAc (100 mL) and treated with activated charcoal. The mixture was filtered and the filtrate was concentrated in vacuo to afford an approximate 1 :1 mixture of 7-chloro-l-(2-chloro-phenyl)-4- methyl-[l,2,4]triazolo[4,3-a]quinoxaline and 8-chloro-l-(2-chloro-phenyl)-4-methyl-

[l,2,4]triazolo[4,3-a]quinoxaline (2.2 g in total). 100 mg of this material was subjected to preparative LC/MS purification. The second eluting isomer was example Ifl (9 mg) and the first eluting isomer was example Igl (11 mg). LC/MS (method 131) for example Ifl: RT(PDA)=1.74 min; PDA / ELS- purities 99.4% / 100%; mass observed 329.2. LC/MS (method 131) for example Igl: RT(PDA)=1.66 min; PDA / ELS-purities 100% / 100%; mass observed 329.1.

Example Ihl 1 -(2-Chloro-phenyl)-4,7-dimethyl-[l ,2,4]triazolo[4,3-a]quinoxaline.

An approximate 2:1 or 1 :2 mixture of Ilh and its regioisomer (3,7-dimethyl-quinoxalin-2-yl)- hydrazine (500 mg, 2.66 mmol) was dissolved in 1,4-dioxane (15 mL) and 2-chloro-benzoyl chloride (0.5 mL) was added. The mixture was refluxed overnight. The crude mixture was cooled, basified with aq NaHCC and extracted with DCM. The organic layer was concentrated in vacuo. The residue was purified by chromatography on silica (eluent: pentane/EtOAc 2:1→ 1.5:2) to a mixture of example Ihl and example Ijl. This mixture was separated by preparative SFC on a Thar SFC 80 instrument fitted with a OJ (250mm*30mm, 20 microm) column using ethanol containing 0.05% diethylamine as eluent. Example Ihl (90 mog) as the second peak. LC/MS (method 131): RT(PDA)=1.81 min; PDA / ELS-purities 100% / 100%; mass observed 309.3.

Example Iil l-(2-Chloro-phenyl)-7-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. IVi (966 mg) was suspended in acetonitrile (44 mL). Phosphoryl chloride (0.95 mL) was added. The mixture was divided into three MW vials and each of them was heated at 130 °C for 15 min under MW conditions. The crude mixtures were pooled and poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO filtered, and concentrated in vacuo. The residual material was dissolved in ethanol (20 mL), and hydrazine hydrate (0.94 mL) was added before the mixture was refluxed for 5h. The crude mixture was cooled to ambient temperature and concentrated in vacuo. The residue was dissolved in acetonitrile (15 mL), 2-chlorobenzoyl chloride (0.77 mL) was added. The mixture was heated at 150 °C for 0.5h under MW conditions. The crude mixture was partitioned between EtOAc and water. The aq layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford example Iil (55 mg) as a yellow solid. LC/MS (method 131): RT(PDA)=1.53 min; PDA / ELS- purities 89.3% / 100%; mass observed 325.3.

Example Ijl l-(2-Chloro-phenyl)-4,8-dimethyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ilj (1.00 g) was suspended in acetonitrile (18 mL), 2-chlorobenzoyl chloride (0.74 mL) was added, and the mixture was heated at 150 °C for 0.5h under MW conditions.

The crude mixture was allowed to stand at ambient temperature overnight. The precipitated solid was filtered off and washed with 1M aq NaOH. Then the filter was transferred to another suction flask, and solid was washed with acetonitrile and heptanes. The filtrate was concentrated and the precipitated solid was washed with heptanes. The combined solids were washed with heptanes and dried to afford example Ijl (1.195 g). LC/MS (method 131): RT(PDA)=1.56 min; PDA / ELS- purities 98.1% / 100%; mass observed 309.0.

Example Ij2 l-(2,6-Dichloro-phenyl)-4,8-dimethyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ilj (42 mg) and 2,6-dichlorobenzoyl chloride (36 microL) were mixed in acetonitrile (2 mL). The mixture was heated at 150 °C for 0.5h under MW conditions. Phosphoryl chloride (21 microL) was added and the mixture heated at 150 °C for 15 min under MW conditions. The crude mixture was partitioned between sat. aq K₂CO₃ and EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes → EtOAc) to afford example Ij2 (21 mg) as yellow solid. LC/MS (method 131): RT(PDA)=1.60 min; PDA / ELS-purities mass observed 342.7.

Example Ij3 l-(2,3-Dichloro-phenyl)-4,8-dimethyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ilj (50 mg) and 2,3-dichlorobenzoyl chloride (57 mg) were mixed in acetonitrile (2 mL) and heated at 150 °C for 0.5h under MW conditions. The crude mixture was poured into sat. aq K2CO3 (20 mL) and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford example Ij3 (30 mg) as a yellow solid. LC/MS (method 131): RT(PDA)=1.74 min; PDA / ELS-purities 88% / 100%; mass observed 344.9.

Example Ikl l-(2-Chloro-phenyl)-8-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. Illk (370 mg), ethanol (10 mL), and hydrazine hydrate (0.33 mL) were mixed and refluxed overnight. After cooling to ambient temperature the volatiles were removed in vacuo. The residue was dissolved in acetonitrile (10 mL) and 2-chlorobenzoyl chloride (0.27 mL) was added and the mixture heated at 150 °C for 2x0.5h under MW conditions. The crude mixture was partitioned between 2M aq NaOH and DCM. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography (eluent: heptanes→ EtOAc) to afford example Ikl (81 mg. LC/MS (method 131): RT(PDA)=1.50 min; PDA / ELS-purities 83% / 99.2%; mass observed 325.4.

Example 111 l-(2-Chloro-phenyl)-4-ethyl-[l,2,4]triazolo[4,3-a]quinoxaline.

A mixture of 2-chloro-3-ethylquinoxaline (24 mg) and 2-chlorobenzhydrazide (24 mg) in acetonitrile (0.6 mL) was heated at 150 °C for 15 min under MW conditions. The volatiles were removed in vacuo evaporated and the residue was dissolved in DMSO (0.50 mL). The solution was filtered and purified by preparative LC/MS to afford example 111 (3.5 mg). LC/MS (method 131): RT(PDA) 1.65 min; PDA ELS purities 99.3% / 100.0%; mass observed 309.3.

Example 112 4-Ethyl-l-(4-methoxy-phenyl)-[l,2,4]triazolo[4,3-a]quinoxaline. Prepared as described for example 111 using 4-methoxybenzhydrazide (24 mg) instead of 2-chlorobenzhydrazide to afford example 112 (2.2 mg). LC/MS (method 131): RT(PDA) 1.65 min; PDA ELS purities 99.2% / 100.0%; mass observed 305.1.

Example Iml l-(2-Chloro-phenyl)-4,6-dimethyl-[l,2,4]triazolo[4,3-a]quinoxaline. To a suspension of an approximate 1 :6 or 6:1 mixture of Urn and its regioisomer (3,8-dimethyl- quinoxalin-2-yl)-hydrazine (0.3 g in total) in 1,4-dioxane (15 mL) was added 2-chloro-benzoyl chloride (280 mg) and phosphoryl chloride (0.25 mL). The mixture was refluxed overnight. The volatiles were removed in vacuo. The residue was partitioned between sat aq NaHC0₃ and DCM. The aq layer was extracted with DCM. The combined organic layers were concentrated in vacuo. The residue was purified by preparative TLC (eluent: pentane/EtOAc 1 :1) to afford example Iml (20 mg). LC/MS (method 131): RT(PDA) 1.65 min; PDA ELS purities 95.9% / 100.0%; mass observed 309.2.

Example Inl 8-Bromo-l-(2-chloro-phenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. A solution of Iln (18 g) in 1,4-dioxane (370 mL) was mixed with 2-chloro benzoyl chloride (12.4 g) and phosphoryl chloride (10 mL). The mixture was stirred at 80 °C for 2h, cooled to ambient temperature. The mixture was poured into ice-water and extracted with DCM. The organic layer was dried over Na₂S0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: pentane/EtOAc 10:1→ 1 :1) to give example Inl (8.7 g) as a white solid. LC/MS (method 131): RT(PDA) 1.70 min; PDA/ 96.3% / 100.0%; mass observed 374.7.

Example Iol 7-Bromo-l-(2-chloro-phenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. A solution of IIo (7 g) in anhydrous 1,4-dioxane (300 mL) was mixed with 2-chloro benzoyl chloride (4.84 g) in the presence of phosphoryl chloride (35 mL). The mixture was stirred at 80 °C for 2h. After cooling to ambient temperature the reaction mixture was poured into ice-water and extracted with DCM. The organic layer was dried over Na₂S0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: pentane/EtOAc 10:1→ 1 :1) to afford example Iol (3.74 g). LC/MS (method 131): RT(PDA) 1.73 min; PDA/ELS purities 94% / 100%; mass observed 374.8.

Example Io2 1 -(2-Chloro-phenyl)-7-ethynyl-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline.

Example Iol (300 mg), copper(I) bromide-dimethyl sulfide complex (33 mg) and Pd(Ph₃P)₄ (93 mg) were mixed in triethylamine (10 mL). (Trimethylsilyl)acetylene (0.17 mL, 1.2 mmol) was added and the reaction was stirred overnight at 70 °C. The mixture was cooled to ambient temperature and partitioned between 2M aq NaOH and EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→ EtOAc) to afford l-(2-chloro-phenyl)-4-methyl-7-trimethylsilanylethynyl- [l,2,4]triazolo[4,3-a]quinoxaline (229 mg). This material was suspended in methanol (10 mL) and treated with K2CO3 (162 mg) for lh at ambient temperature. The volatiles were removed in vacuo. The residue was partitioned between EtOAc and water. The aq layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO i, filtered, and concentrated in vacuo to afford example Io2 (187 mg) as a yellow solid. LC/MS (method 131): RT(PDA) 1.57 min; PDA/ELS purities 92.5% / 100%; mass observed 318.9.

Example Ipl 6-Bromo-l-(2-chloro-phenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. To a solution of Hp (1.375 g) in 1,4-dioxane (34 mL) was added 2-chloro benzoyl chloride (0.95 g) and phosphoryl chloride (0.83 mL). The mixture was stirred at 80 °C for 2h, cooled to ambient temperature, and poured into ice- water. The product was extracted into DCM. The organic layer was dried over Na₂S0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: pentane/EtOAc 5:1→ 1 :1) to afford example Ipl as a pink solid. LC/MS (method 131): RT(PDA) 1.66 min; PDA/ELS purities 91.9% / 100%; mass observed 374.8.

Example Iql 8-Bromo-l-(2-chloro-phenyl)-6-methoxy-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline. 2-Chlorobenzaldehyde (0.21 mL) and Ilq (0.48 g) were suspended in DCM (8 mL). The suspension was briefly refluxed using a heatgun before it was allowed to stir overnight at ambient temperature. PhI(OAc)2 (0.60 g) was added, and the mixture was stirred for lh at ambient temperature. The volatiles were removed in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes/EtOAc 3:1→ EtOAc) to afford example Iql (126 mg). LC/MS (method 131): RT(PDA) 1.57 min; PDA/ELS purities 97.9% / 100%; mass observed 405.4.

PDE in-vitro assays

The inhibitory activities of the compound of the invention were determined in connection with the following methods: PDEIOA enzyme

Active PDEIOA enzyme is prepared in a number of ways for use in PDE assays (Loughney, K. et al. Gene 1999, 234, 109-117; Fujishige, K. et al. Eur J Biochem. 1999, 266, 1118-1127 and Soderling, S. et al. Proc. Natl. Acad. Sci. 1999, 96, 7071-7076). PDEIOA can be expressed as full-length proteins or as truncated proteins, as long as they express the catalytic domain. PDEIOA can be prepared in different cell types, for example insect cells or E. coli. An example of a method to obtain catalytically active PDEIOA is as follows: The catalytic domain of human PDEIOA (amino acids 440-779 from the sequence with accession number NP 006652) is amplified from total human brain total RNA by standard RT-PCR and is cloned into the BamHl and Xhol sites of the pET28a vector (Novagen). Expression in coli is performed according to standard protocols. Briefly, the expression plasmids are transformed into the BL21 (DE3) E. coli strain, and 50 mL cultures inoculated with the cells allowed to grow to an OD600 of 0.4-0.6 before protein expression is induced with 0.5mM IPTG. Following induction, the cells are incubated overnight at room temperature, after which the cells are collected by centrifugation. Cells expressing PDEIOA are resuspended in 12 mL (50 mM TRIS-HCl-pH8.0, 1 mM MgCl₂ and protease inhibitors). The cells are lysed by sonication, and after all cells are lysed, TritonXlOO is added according to Novagen protocols. PDEIOA is partially purified on Q sepharose and the most active fractions were pooled.

PDEIOA inhibition assay

A typical PDEIOA assay was performed as follows: the assay was performed in 60 μΕ samples containing a fixed amount of the PDE2A enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl₂; 0.02% Tween20), 10 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 20 μΕ (0.2 mg) yttrium silicate SPA beads (Amersham). The beads were allowed to settle for 1 h in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC₅o values were calculated using XlFit (model 205, IDBS). PDE2Aenzyme

Likewise, active human PDE2A enzyme (ATCC68585) is prepared in a number of ways for use in PDE assays and procedures are well known to those skilled in the art.

PDE2A inhibition assay

A typical PDE2A assay was performed as follows: the assay was performed in 60 μΕ samples containing a fixed amount of the PDE2A enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; lO mM MgCl₂; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 20 μΕ (0.2 mg) yttrium silicate SPA beads (Amersham). The beads were allowed to settle for 1 h in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC₅o values were calculated using XlFit (model 205, IDBS).

Each of the exemplified compounds of the invention were tested in the assays above and were found to possess IC₅o values of less than about 10 micromolar.

Data obtained for selected examples are listed in the table below.

Compound PDE2A IC₅₀ PDE2A IC₅₀

Example Ial 21 nM 506 nM

Example Ia9 396 nM 4823 nM

Example lb 1 27 nM 291 nM

Claims

A compound of Formula I:

wherein R¹ is Ci-Ce alkyl, C₃-C₆Cycloalkyl, tetrahydropyranyl, benzyl, phenyl and pyridyl, in which the benzyl, phenyl and pyridyl is optionally substituted with one or more halogen, CN, C₁-C4 alkyl/fluoroalkyl or C₁-C4 alkoxy/fluoroalkoxy; wherein R² is C₁-C4 alkyl or C₃-C₆ cycloalkyl; wherein R³ is halogen, CN, C0₂H, CON(H or C_rC₄ alkyl)₂,CHO, C_rC₄ alkyl/fluoroalkyl, C₂-C₄ alkenyl, C₂-C4 alkenyl or C₁-C4 alkoxy/fluoroalkoxy; and wherein n is 0-3; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein R is C₁-C₄ alkyl.

3. The compound of claim 2, wherein R is methyl or ethyl.

4. The compound of claim 1, wherein R is C3-C6 cycloalkyl.

5. The compound of anyone of claims 1-4, wherein R is C₁-C₄ alkyl.

6. The compound of anyone of claims 1-4, wherein R is C₃-C6 cycloalkyl.

7. The compound of anyone of claims 1-4, wherein R is tetrahydropyranyl.

8. The compound of anyone of claims 1-7, wherein R is benzyl optionally substituted with one or two F, CI, C₁-C₃ alkyl or C₁-C₃ alkoxy/fluoroalkoxy.

9. The compound of anyone of claims 1-7, wherein R is phenyl optionally substituted with one or two F, CI, C₁-C₃ alkyl or C₁-C₃ alkoxy/fluoroalkoxy.

10. The compound of anyone of claims 1-7, wherein R¹ is pyridyl optionally substituted with one or two F, CI, C₁-C₃ alkyl or C₁-C₃ alkoxy/fluoroalkoxy.

11. The compound of anyone of claims 8-10 wherein R¹ is selected from the group consisting of benzyl optionally substituted at the para position of the benzyl group; phenyl optionally substituted at the ortho position of the phenyl group; and pyridyl optionally substituted the carbon atom adjacent to the triazole ring.

12. The compound of anyone of one of claims 1-11, wherein R³ is halogen or CHO.

13. The compound of anyone of claims 1-11, wherein R³ is C -C4 alkyl or C -C4 alkoxy

14. The compound of anyone of claims 1-11, wherein R³ is C2-C4 alkenyl or C2-C4 alkenyl.

The compound of anyone of claims 1-14, wherein n is 0.

The compound of anyone of claims 1-14, wherein n is 1.

The compound of claim 1, wherein the compound is selected from the group consisting of 1 -(2-chloro-phenyl)-4-methyl- [ 1 ,2,4]triazolo [4,3 -a] quinoxaline; 4-methyl- 1 -phenyl-

[1 ,2,4]triazolo[4,3-a]quinoxaline; 1 -(4-methoxy-phenyl)-4-methyl-[l ,2,4]triazolo[4,3- ajquinoxaline; l-(3-chloro-pyridin-4-yl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline; 4- methyl-l-(2-methyl-pyridin-3-yl)-[l,2,4]triazolo[4,3-a]quinoxaline; 4-methyl- 1 -(3 -methyl- pyridin-4-yl)-[l,2,4]triazolo[4,3-a]quinoxaline; l-(4-chloro-benzyl)-4-methyl- [l,2,4]triazolo[4,3-a]quinoxaline; l-(2-chloro-phenyl)-6-fluoro-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline; 1 -(2,6-dichloro-phenyl)-6-methoxy-4-methyl-[l ,2,4]triazolo[4,3- ajquinoxaline; l-(2-chloro-6-methyl-phenyl)-6-methoxy-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline; 1 -(2,3-dichloro-phenyl)-6-methoxy-4-methyl-[l ,2,4]triazolo[4,3- ajquinoxaline; l-(2-chloro-phenyl)-6-methoxy-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline; l-(2-chloro-phenyl)-4,7-dimethyl-[l,2,4]triazolo[4,3-a]quinoxaline; l-(2- chloro-phenyl)-7-methoxy-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2-chloro- phenyl)-4,8-dimethyl-[l,2,4]triazolo[4,3-a]quinoxaline; l-(2,6-dichloro-phenyl)-4,8- dimethyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2,3-dichloro-phenyl)-4,8-dimethyl-

[1 ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2-chloro-phenyl)-8-methoxy-4-methyl- [1 ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2-chloro-phenyl)-4-ethyl-[l ,2,4]triazolo[4,3- ajquinoxaline; 4-ethyl-l-(4-methoxy-phenyl)-[l,2,4]triazolo[4,3-a]quinoxaline; l-(2- chloro-phenyl)-4,6-dimethyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 8-bromo-l -(2-chloro- phenyl)-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 7-bromo-l -(2 -chloro-phenyl)-4-methyl- [1 ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2-chloro-phenyl)-7-ethynyl-4-methyl-

[l,2,4]triazolo[4,3-a]quinoxaline; 6-bromo-l-(2-chloro-phenyl)-4-methyl- [1 ,2,4]triazolo[4,3-a]quinoxaline and 8-bromo- 1 -(2-chloro-phenyl)-6-methoxy-4-methyl- [l,2,4]triazolo[4,3-a]quinoxaline or a pharmaceutically acceptable salt thereof.

The compound of claim 1, wherein the compound is l-(2-chloro-phenyl)-7-fluoro-4-methyl- [1 ,2,4]triazolo[4,3-a]quinoxaline or 1 -(2-chloro-phenyl)-8-fluoro-4-methyl-

[ 1 ,2,4]triazolo [4,3 -a]quinoxaline.

The compound of claim 1, wherein the compound is7-chloro-l-(2-chloro-phenyl)-4-methyl- [l,2,4]triazolo[4,3-a]quinoxaline or 8-chloro-l-(2-chloro-phenyl)-4-methyl-

[ 1 ,2,4]triazolo [4,3 -a]quinoxaline.

The compound of claim 1, wherein the compound is 4-methyl-l-propyl-[l,2,4]triazolo[4,3- ajquinoxaline; 1 -isopropyl-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline; and

4-methyl-l-(tetrahydro-pyran-4-yl)-[l,2,4]triazolo[4,3-a]quinoxaline.

A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

A method of treating an anxiety disorder comprising administering a therapeutically effective amount of the compound of anyone of claim 1-20.

A method of treating a cognitive disorder comprising administering a therapeutically effective amount of the compound of anyone of claim 1-20.

A method of treating schizophrenia comprising administering a therapeutically effective amount of the compound of anyone of claim 1 -20.

A compound of anyone of claims 1-20, for use in therapy.