WO2013034758A1 - Substituted triazolopyrazines and uses thereof - Google Patents

Abstract

The present invention is directed to substituted 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

SUBSTITUTED TRIAZOLOPYRAZINES AND USES THEREOF

FIELD OF THE INVENTION

The present invention is directed to substituted triazolopyrazine 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 PDE10A 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. JNeurosci 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 substituted 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 PDE10A. Accordingly, the present invention relates to compounds of formula I.

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, Q- C₄ alkyl/fluoroalkyl or C -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; wherein R⁴ is halogen, C₁-C4 alkyl/fluoroalkyl or C₁-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 compositions and uses thereof.

Formula I

In one embodiment, R² is C₁-C₄ alkyl.

In an embodiment, R² is methyl or ethyl.

In another embodiment, R² is C₃-C₆cycloalkyl. In one embodiment, R⁴ is halogen, C₁-C₂ alkyl or C₁-C₂ alkoxy. In one embodiment, R¹ is C₁-C₄ alkyl.

In one embodiment, R¹ is C3-C₆cycloalkyl or tetrahydropyranyl.

In yet another embodiment, R¹ is benzyl optionally substituted with one or two F, CI, C₁-C₃ alkyl or C₁-C₃ alkoxy/fiuoroalkoxy.

In one embodiment, R¹ is phenyl optionally substituted with one or two F, CI, C₁-C₃ alkyl or C₁-C₃ alkoxy/ fiuoroalkoxy. In one embodiment, R¹ is pyridyl optionally substituted with one or two F, CI, C1-C3 alkyl or Q- C3 alkoxy/fluoroalkoxy.

In another 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 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 C1-C4 alkoxy

In one embodiment, R³ is C2-C4 alkenyl or C2-C4 alkenyl.

In one embodiment, n is 0. In a separate embodiment, n isl . In another embodiment, n is 2.

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 fluoroalkyl" 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 C -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; post-traumatic 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.

Acetic acid (e.g. Sigma-Aldrich 242853). Acetonitrile (e.g. Aldrich 271004). Activated charcoal (e.g. Sigma-Aldrich 161551). 2-Amino-3-nitrophenol (e.g. Aldrich 297003). APPI = atmospheric pressure photo ionization. Aq = aqueous. 37% aq HC1 (e.g. Sigma-Aldrich 320331). B0C₂C di- tert-butyldicarbonate (e.g. Fluka 34660). Brine = saturated aq solution of sodium chloride (e.g. Aldrich S7653). Bromine (e.g. Sigma-Aldrich 277576). l-Bromo-3-fluoro-2-nitro-benzene (e.g. Matrix 054378 or Apollo PC9918). Butyryl chloride (e.g. Aldrich 236349). 2-Chlorobenzoyl chloride (e.g. Aldrich 103918). 3-Chloro-isonicotinic acid (e.g. Aldrich 633410). 2-Chloro-6- methyl-benzoic acid (e.g. Fluorochem 018392 or ABCR AB134559). Cyclopentanecarboxylic acid (e.g. Aldrich CI 12003). DCM = methylene chloride / dichloromethane (e.g. Aldrich 270997). 2-(Difluoromethoxy)benzaldehyde (e.g. Aldrich 470155). l,3-Difluoro-2-nitrobenzene (e.g. Aldrich 382957). 2,6-Dimethylbenzaldehyde (e.g. Aldrich 515159). 1,4-Dioxane (e.g. Sigma- Aldrich 296309). DIPEA = di-z o-propyl ethyl amine (e.g. Aldrich 387649). DMAP = 4- dimethylaminopyridine (e.g. Fluka 29224). DMF = dimethyl formamide (e.g. Sigma-Aldrich 227056). DMSO = dimethyl sulfoxide (e.g. Sigma D4540). ELS = evaporative light scattering. Ethanol (e.g. Sigma-Aldrich 459844). EtOAc = ethyl acetate (e.g. Fluka 34972). Ethyl pyruvate (e.g. Fluka 15960). 3-Fluoro-2-nitroanisole (e.g. ABCR AB230935 or Apollo PC3402). h = hour(s). Heptanes (e.g. Sigma-Aldrich 730491). HMBC = Heteronuclear Multiple Bond Correlation NMR experiment. HPLC = high performance liquid chromatography. HSQC = Heteronuclear Single Quantum Coherence NMR experiment. Hydrazine hydrate (e.g. Sigma- Aldrich 225819). Iron powder (e.g. Fluka 44900). K₂C0₃ (e.g. Sigma-Aldrich 209619). LC = liquid chromatography. LC/MS = liquid chromatography / mass spectrometry. 2M = 2 molar solution (similarly 8M = 8 molar solution etc). MgS0₄ (e.g. Sigma-Aldrich 246972). Methanol (e.g. Sigma-Aldrich 34860). 2-Methyl-benzaldehyde (e.g Aldrich 117552). 3-Methyl-benzene- 1,2-diamine (e.g. Aldrich 272361). Methyl iodide (e.g. Sigma-Aldrich 67692). 3-Methyl- isonicotinic acid (e.g. Matrix 020640 or Fluorochem 040096). min = minutes. MW = microwave. MW conditions = reactions performed in sealed tubes using a Biotage Initiator instrument or a CEM Explorer-48 instrument. NaOAc = sodium acetate (e.g. Sigma-Aldrich S2889). NaOH (e.g. Sigma-Aldrich S5881). NaHC0₃ (e.g. Sigma-Aldrich S6014). Na₂S0₄ (e.g. Sigma-Aldrich 238597). NBS = N-bromo-succinimide (e.g. Aldrich B81255). NMR = nuclear magnetic resonance spectroscopy. 2-Oxo-propanoic acid methyl ester (e.g. Aldrich 371173). 10% Palladium on charcoal (e.g. Aldrich 75990). PDA = photo diode array. PhI(OAc)₂ = iodobenzene diacetate (e.g. Fluka 31490). 5%> Platinum on charcoal (e.g. Aldrich 80982). POCl₃ = phosphoryl chloride (e.g. Aldrich 262099). Racemic alanine (e.g. Sigma A7502). RT = retention time. Sat = saturated. 96% Sulphuric acid (e.g. Sigma- Aldrich 320501). T = time. TBAI = tetra-n- butylammonium iodide (e.g. Aldrich 68694). Tetrahydro-pyran-4-carbaldehyde (e.g. Apollo OR9347 or BBB-SCI 3B3-084066). TFA = trifluoroacetic acid (e.g. Aldrich T6508). THF = tetrahydrofuran (e.g. Sigma- Aldrich 401757). TLC = thin layer chromatography. THP = tetrahydropyranyl. 1,3-Xylene (e.g. Sigma- Aldrich 95672). 2-(Trifluoromethoxy)benzaldehyde (e.g. Aldrich 529168). 2-Trifluoromethyl-benzaldehyde (e.g. Aldrich 250694).

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 nM) 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 mL/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.

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 RiCOCl in a suitable solvent such as acetonitrile at elevated temperature as described for example Ial . The acid chloride can be prepared in situ from the corresponding acid by the addition of phosphoryl chloride (POCI₃) as described for example Ial .

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) as described for example Ia2. 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-iso-propyl ethyl amine (DIPEA) as described for Urn.

Method 6 is the treatment of lactams IV 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.

PREPARATION OF INTERMEDIATES

INTERMEDIATE 3-Hydrazinyl-5-methoxy-2-methylquinoxaline (Ila). To a solution of 3- fluoro-2-nitroanisole (5 g) in DMSO (60 mL) were added racemic alanine (5.8 g,) and K₂C0₃ (4.0 g). The reaction mixture was heated at 90 °C for 3h and then at 110 °C for 20h. The reaction mixture was then cooled to ambient temperature. The crude mixture was poured onto 100 mL ice and the acidified with 37% aq HC1. EtOAc was added, and the organic layer was washed 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). A stream of argon was bubbled through the mixture for 5 min after which 10% palladium on charcoal (600 mg) was added and mixture was treated with hydrogen gas (3 bar) for 23h using a Parr shaker. The catalyst was filtered off, and the filtrate was concentrated in vacuo. The residue was partitioned between DCM and a 1 : 1 mixture of sat. aq K2CO3 and water. The aq layer was extracted with DCM. The combined organic layers were washed with brine, dried over MgS0₄, filtered, and concentrated in vacuo to afford 8-methoxy-3-methyl-3,4-dihydro-lH-quinoxalin-2-one (4.9 g) as a black oil. This material was dissolved in 1,3-xylene (200 mL). 10% palladium on charcoal (600 mg) was added and the mixture was refluxed for 12h. The reaction was allowed to cool to ambient temperature and THF (200 mL) was added. The mixture was filtered through a plug of celite and the filtrate was concentrated in vacuo to afford 8-methoxy-3-methyl-lH-quinoxalin-2-one (3.91 g). This material was added to ice-cold phosphoryl chloride (60 mL). The reaction was then heated at 100 °C for 140 min. After cooling to ambient temperature the volatiles were removed in vacuo. The residue was diluted with DCM (100 mL) and poured carefully onto ice (150 mL). The pH was adjusted to neutral using K₂CO₃. The aq layer was extracted with DCM. The combined organic layers were washed with brine, dried over MgS0₄, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford 3-chloro- 5-methoxy-2-methyl-quinoxaline (2.8 g). This material was added to a mixture of ethanol (90 mL) and hydrazine hydrate (3.3 mL) and refluxed for 25h before it was cooled to ambient temperature. The volatiles were removed in vacuo. The resulting solid was washed with water and heptanes and dried to afford Ila (2.7 g) sufficiently pure for next step.

INTERMEDIATE (3,8-Dimethyl-quinoxalin-2-yl)-hydrazine (lib). 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 (l.lg 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 lib and its regioisomer (3,5-dimethyl-quinoxalin-2-yl)-hydrazine (0.6g in total). This mixture was used in the next step.

INTERMEDIATE (8-fluoro-3-methyl-quinoxalin-2-yl)-hydrazine (lie). 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 g). 2 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). 1.2g of this mixture was refluxed in phosphoryl chloride (20 rriL) for 2h. The volatiles were removed in vacuo. The residue was basified with aq NaHCC>3 and extracted with DCM. The organic layer was dried over Na₂S0₄, 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 rriL) at 110 °C for 2h. The precipitated solid was filtered off to afford an approximate 1:1 mixture of lie and its regioisomer (5-fiuoro-3-methyl- quinoxalin-2-yl)-hydrazine (0.4 g in total). This mixture was applied in the next step.

INTERMEDIATE (8-Bromo-3-methyl-quinoxalin-2-yl)-hydrazine (lid). A mixture of l-bromo-3- fluoro-2-nitro-benzene (99 g), racemic alanine (120 g) and CS₂CO₃ (440 g) in ethanol (1.2 L) and water (400 niL) was refluxed for 5h. After cooling to ambient temperature, the mixture was diluted with water (600 mL) and acidified to pH 3. The precipitate solid was collected and dried to afford racemic 2-(3-bromo-2-nitro-phenylamino)-propionic acid (110 g) as a yellow solid. lOg of this material in acetic acid (35 mL) and treated with iron powder (5.8 g) at 90 °C for 2h, cooled and filtered then most of the acetic acid was removed in vacuo. The remaining slurry was extracted with DCM, dried over Na₂S0₄, filtered, and concentrated in vacuo to afford 8-bromo-3- methyl-3,4-dihydro-lH-quinoxalin-2-one (6.4 g) as a yellow solid. A larger portion of this material prepared in a similar manner (29.2 g) was dissolved in 5% aq NaOH and treated with 30% aq hydrogen peroxide (140 mL) and water (180 mL). The mixture was stirred at 60 °C for 6h before it was cooled. The precipitate solid was filtered off, washed with water, and dried to afford 8-bromo-3 -methyl- lH-quinoxalin-2-one (28.0 g). 21 g of this material was stirred in PI1POCI₂ (80 mL) at 150 °C for 4h. After cooling, water was added to quench excess PI1POCI₂ and pH was subsequently adjusted to 7 with aq ammonia. The precipitated solid was filtered off, washed with water, and dried to afford 5-bromo-3-chloro-2-methyl-quinoxaline (17.7 g) as a yellow solid. This material was dissolved in ethanol (250 mL) was treated with hydrazine hydrate (160 mL) at reflux for 3h. Most of the volatiles were removed in vacuo. The precipitated solid was filtered off, washed with water, and dried to afford lid (14.8 g) as a yellow solid sufficiently pure for the next step.

INTERMEDIATE (7-Bromo-8-methoxy-3-methyl-quinoxalin-2-yl)-hydrazine (He). 3-Fluoro-2- nitroanisole (5 g) was dissolved in dimethyl sulfoxide (60 mL). Racemic alanine (5.8 g) and K₂CO₃ (4.0 g) were added, and the mixture was heated at 90 °C for two days. The mixture was cooled to ambient temperature, and poured into 200 mL ice and then acidified with 36% aq HC1. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo to afford 2-(3-methoxy-2-nitro-phenylamino)- propionic acid (3 g) sufficiently pure for the next step. A larger portion of this material (6.18 g) prepared in a similar manner was mixed with NBS (4.6 g) and refluxed in acetonitrile (100 mL) for 2h. The crude mixture was partitioned between 7.5% aq sodium thiosulfate solution and EtOAc. The organic layer was washed with brine, dried over MgSO i, filtered, and concentrated in vacuo. The residue was dissolved refluxing AcOH (200 mL) and treated with iron (5.75 g; added portion- wise). After 0.5h the mixture was cooled to ambient temperature, diluted with water and extracted with EtOAc. The organic layer was washed sequentially with dilute aq K2CO3, 2M aq NaOH and brine before it was concentrated in vacuo. The residue was dissolved in refluxing methanol and treated with activated charcoal and filtered. The filtrate was concentrated in vacuo to afford 7-bromo-8-methoxy-3-methyl-3,4-dihydro-lH-quinoxalin-2-one (6.97 g) sufficiently pure for the next step. 2.8 g of this material was dissolved in xylenes (100 mL) and refluxed in the presence of 10%> palladium on charcoal (0.55 g) overnight. The crude mixture was cooled to ambient temperature, diluted with methanol and then filtered through a plough of celite. The filtrate was concentrated in vacuo to afford 7-bromo-8-methoxy-3-methyl-lH-quinoxalin-2-one (2.2 g) sufficiently pure for the next step. 2.11 g of this material was dissolved in ice-cold phosphoryl chloride (20 mL). The mixture was heated 100 °C for lh before the volatiles were removed in vacuo. The residue was partitioned between DCM and ice/water, and the mixture was neutralized with K2CO3. The aq layer was extracted with DCM, and 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 gel (eluent: heptanes→ EtOAc) to afford 6-bromo-3-chloro- 5-methoxy-2-methyl-quinoxaline (965 mg) sufficiently pure for the next step. 960 mg of this material was dissolved in ethanol (20 mL) and refluxed with hydrazine hydrate (0.62 mL) overnight. The mixture was cooled to ambient temperature. The solid was filtered off and washed with water and heptanes to afford He (396 mg) sufficiently pure for the next step.

INTERMEDIATE 6-Bromo-8-methoxy-3-methyl-quinoxalin-2-yl)-hydrazine (Ilf).

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 B0C2O (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 (tert- 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 MgS0₄, 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 ^N-'H HSQC and comparing with the shift of the nitrogen having long range correlation to an aromatic proton in 2D ^N-'H HMBC. 6-bromo- 8-methoxy-3-methyl-lH-quinoxalin-2-one (1.7 g) was dissolved in phosphoryl chloride (23 mL) was refluxed for 2h. The volatiles were removed in vacuo. The residue was partitioned between DCM and a little ice. The biphasic mixture was basified with 2M aq Na₂C03 and was filtered. The organic part of the filtrate was dried over MgSO i, filtered, and concentrated in vacuo to afford 7- bromo-3-chloro-5-methoxy-2-methyl-quinoxaline (1.1 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 residue was suspended in water (50 mL). The solid was filtered off, washed with heptanes and dried to afford Ilf (1.6 g) sufficiently pure for the next step.

COMPOUNDS OF THE INVENTION

Example Ial l-(2-Chlorophenyl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. 2- Chlorobenzoyl chloride (0.74 mL) and Ila (1 g) were mixed in acetonitrile (18 mL). The reaction was heated at 150 °C for 0.5h under MW. The crude mixture was poured into 2M aq NaOH 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). The resulting solid was dissolved in methanol (200 mL) and refluxed with activated charcoal for 5 min. The charcoal was filtered off, and the filtrate was concentrated in vacuo. The residue was precipitated from methanol to afford example Ial (389 mg). LC/MS (method 131): RT(PDA)=1.45 min; PDA / ELS-purities 97.4% / 100%; mass observed 325.2.

Example Ia2 9-Methoxy-4-methyl-l-(o-tolyl)-[l,2,4]triazolo[4,3-a]quinoxaline. 2- Methylbenzaldehyde (0.117 mL) and Ila (206 mg) were mixed in DCM (10 mL). The suspension was briefly heated at reflux and then stirred at ambient temperature overnight before PhI(OAc)2 (325 mg) was added. The mixture was stirred at ambient temperature for 3h. The crude reaction mixture was purified by chromatography on silica (eluent: heptanes→ EtOAc) to afford example Ia2 (211 mg). LC/MS (method 131): RT(PDA)=1.45 min; PDA / ELS-purities 99.2% / 100%; mass observed 305.1.

Example Ia3 l-(2-Chloro-6-methylphenyl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline. To a solution of Ila (0.3 g) and 2-chloro-6-methyl-benzoic acid (377 mg) in 1,4- dioxane (5 mL) was added phosphoryl chloride (0.3 mL). The mixture was refluxed for 1.5h. The crude mixture was partitioned between water and DCM. The organic layer was concentrated in vacuo. The residue was purified by preparative TLC (eluent: pentane/EtOAc 1 :1) to give example Ia3 (130 mg) as a white solid. LC/MS (method 132): RT(PDA)=1.89 min; PDA / ELS-purities 98.0% / 100%; mass observed 339.2.

Example Ia4 1 -(2,6-Dimethylphenyl)-9-methoxy-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline. Prepared as described for example Ia2 using 2,6-dimethylbenzaldehyde (136 mg) and Ila (206 mg) in DCM (5 mL) to afford example Ia4 (236 mg). LC/MS (method 131): RT(PDA)=1.52 min; PDA / ELS-purities 95.3% / 100%; mass observed 319.0.

Example Ia5 l-(3-Chloropyridin-4-yl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. Prepared as described for example Ia3 using Ila (0.3 g) and 3-chloro-isonicotinic acid (347 mg) to afford example Ia5 (108 mg) as a yellow solid. LC/MS (method 132): RT(PDA)=1.63 min; PDA / ELS-purities 80.9% / 100%; mass observed 326.2.

Example Ia6 9-Methoxy-4-methyl-l-(3-methylpyridin-4-yl)-[l,2,4]triazolo[4,3-a]quinoxaline. A mixture of Ila (200 mg), 3-methyl-isonicotinic acid (134 mg), PyBroP (476 mg), and DIPEA (253 mg) was stirred in DMF (5 mL) at ambient temperature overnight. The crude mixture was partitioned between water and EtOAc. The organic layer was concentrated in vacuo to afford isonicotinic acid N'-(8-methoxy-3-methyl-quinoxalin-2-yl)-hydrazide (200 mg) as a white solid. A larger portion of this material prepared in a similar manner (300 mg) was dissolved in 1,4-dioxane (5 mL) and refluxed with phosphoryl chloride (0.3 mL) for 1.5h. The crude mixture was partitioned between water and EtOAc. The organic layer was concentrated in vacuo. The residue was purified by preparative TLC (eluent: pentane/EtOAc 1 :1) to afford example Ia6 (90 mg) as a white solid. LC/MS (method 132): RT(PDA)=1.19 min; PDA / ELS-purities 85.1% / 100%; mass observed 306.1.

Example Ia7 9-Methoxy-4-methyl-l-propyl-[l,2,4]triazolo[4,3-a]quinoxaline. To a solution of Ila (300 mg) and butyryl chloride (235 mg) in 1,4-dioxane (5 mL) was added phosphoryl chloride (0.3 mL) and the mixture was refluxed for 1.5h. The crude mixture was partitioned between water and DCM. The organic layer was concentrated in vacuo. The residue was purified by preparative TLC (eluent: pentane/EtOAc 1 :1) to give example Ia7 (158 mg) as a white solid. LC/MS (method 131): RT(PDA)=1.33 min; PDA / ELS-p / 100%; mass observed 257.3.

Example Ia8 9-Methoxy-4-methyl-l-(2-trifluoromethyl-phenyl)-[l,2,4]triazolo[4,3- ajquinoxaline. Prepared as described for example Ia9 using 2-trifluoromethyl-benzaldehyde (59 mg) instead of 2-(difluoromethoxy)benzaldehyde to afford example Ia8 (70 mg). LC/MS (method 131): RT(PDA)=1.50 min; PDA / ELS-purities 93.9% / 100%; mass observed 359.0.

Example Ia9 1 -(2-Difluoromethoxy-phenyl)-9-methoxy-4-methyl-[l ,2,4]triazolo[4,3- a]quinoxaline. To a suspension of Ila (68 mg) and 2-(difluoromethoxy)benzaldehyde (57 mg) in DCM (1.65 mL) was heated at 75 °C under MW conditions for 15 min. PhI(OAc)₂ (107 mg) was added, and the mixture was heated at 75 °C under MW conditions for 20 min. The volatiles were removed in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→EtOAc) to afford example Ia9 82 mg. LC/MS (method 131): RT(PDA)=1.38 min; PDA / ELS-purities 92.4% / 100%; mass observed 357.2.

Example IalO l-(2-Trifluoromethoxy-phenyl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline. Prepared as described for example Ia9 using 2-(trifluoromethoxy)benzaldehyde (63 mg) instead of 2-(difluoromethoxy)-benzaldehyde. Yield of example IalO (83 mg). LC/MS (method 131): RT(PDA)=1.55 min; PDA / ELS-purities 91.8% / 100%; mass observed 375.0.

Example Iall 9-Methoxy-4-methyl-l-(tetrahydro-pyran-4-yl)-[l,2,4]triazolo[4,3-a]quinoxaline. Prepared as described for example Ia9 using tetrahydro-pyran-4-carbaldehyde (38 mg) instead of 2-(difluoromethoxy)-benzaldehyde. Yield of example Iall (77 mg). LC/MS (method 131): RT(PDA)=1.15 min; PDA / ELS-purities / 100%; mass observed 299.3.

Example Ial2 l-Cyclopentyl-9-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. To a suspension of Ila (68 mg) in acetonitrile (1.65 mL) was added cyclopentanecarboxylic acid (30 mg) and phosphoryl chloride (51 mg). The mixture was heated at 140 °C for 25 min under MW conditions. The supernatant of the resulting suspension was decanted off and concentrated in vacuo. The residue was purified by chromatography on silica gel (eluent: EtOAc/heptanes 1 :9→3:7) to afford example Ial2 (13 mg). LC/MS (method 131): RT(PDA)=1.54 min; PDA / ELS-purities 94.4% / 100%; mass observe

Example Ibl l-(2-Chlorophenyl)-4,9-dimethyl-[l,2,4]triazolo[4,3-a]quinoxaline. To a suspension of an approximate 1 :6 or 6:1 mixture of lib and its regioisomer (3,5-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), and the mixture was refluxed overnight. The volatiles were removed in vacuo. The residue was partitioned between sat. aq NaHCC>3 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 give example Ibl (140 mg). LC/MS (method 131): RT(PDA)=1.54 min; PDA / ELS-purities 98.5% / 100%; mass observed 309.3.

Example Icl l-(2-Chlorophenyl)-9-fluoro-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. To a suspension of an approximate 1 :1 mixture of lie and its regioisomer (5-fluoro-3-methyl- quinoxalin-2-yl)-hydrazine (0.4 g in total) in 1,4-dioxane (20 mL) was added 2-chloro-benzoyl chloride (365 mg) and phosphoryl chloride (0.3 mL), and the mixture was refluxed for 2h. The volatiles were removed in vacuo. The residue was partitioned between sat. aq NaHC0₃ and DCM. The aq layer was extracted by DCM. The combined organic layers were concentrated in vacuo. The residue was purified by preparative TLC (eluent: pentane/EtOAc 1 : 1) to give example Icl (39 mg). LC/MS (method 131): RT(PDA)=1.50 min; PDA / ELS-purities 99.1% / 100%; mass observed 313.0.

Example Idl 9-Bromo-l-(2-chlorophenyl)-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline. A solution of lid in anhydrous 1,4-dioxane (370 mL) was treated with 2-chlorobenzoyl chloride (10.2 g) and phosphoryl chloride (8.9 mL) at 80 °C for 2h. After cooling to ambient temperature the reaction mixture was poured into ice- water and stirred for 10 min 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 Idl (10.6 g) as white solid. LC/MS (method 131): RT(PDA) = 1.58; PDA / ELS purities 96.6% / 100%; mass observed 374.8.

Example Iel 8-Bromo-l-(2-chloro-phenyl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline. He (100 mg) and 2-chlorobenzaldehyde (40 microL) were mixed in DCM (2 mL) and stirred at ambient temperature for 3h. More DCM (2 mL) was added and the mixture was stirred for an additional 2.5h. Then PhI(OAc)2 (0.1 g) was added, and the resulting mixture was stirred overnight at ambient temperature. The volatiles were removed in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→ EtOAc) to afford example Iel (96 mg). LC/MS (method 131): RT(PDA)=1.69 min, PDA/ELS-purities 93.4% / 100%, mass observed = 403.2.

Example 111 7-Bromo-l-(2-chloro-phenyl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3- ajquinoxaline. Ilf (0.8 g) and 2-chlorobenzaldehyde (0.35 mL) were dissolved in DCM (10 mL), and the mixture was stirred at ambient temperature overnight. Next morning PhI(OAc)2 (1.00 g) was added, and the mixture was stirred for 3h at ambient temperature. The volatiles were removed in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→ EtOAc) to afford a solid that was washed with a little heptanes/EtOAc (9:1) to afford example 111 (0.58 g). LC/MS (method 131): RT(PDA)=1.78 min, PDA/ELS-purities 97.3% / 100%, mass observed = 405.4.

Example I£2 7-Bromo-9-methoxy-4-methyl-l-o-tolyl-[l,2,4]triazolo[4,3-a]quinoxaline. Ilf (0.8 g) and 2-methylbenzaldehyde (0.36 mL) were dissolved in DCM (10 mL), and the mixture was stirred overnight at ambient temperature. Next morning, PhI(OAc)2 (1.00 g) was added, and the reaction was stirred at ambient temperature for 3h. The volatiles were removed in vacuo. The residue was purified by chromatography on silica gel (eluent: heptanes→ EtOAc) to afford a solid material that was suspended in hot heptanes (100 mL); this mixture was allowed to cool to ambient temperature and then left to stand overnight. The solid was filtered off to afford example Η2 (0.61 g). LC MS (method 131): RT(PDA)=1.77 min, PDA/ELS-purities 96.4% / 100%, mass observed = 383.2.

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; lO 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).

Data obtained for the compounds of the invention are listed in the table below.

PDE2A IC₅₀ PDE10 IC₅₀

Example Ia1 6.3 nM 9.5 nM

Example Ia2 41 nM 36 nM

Example Ia3 5.7 nM 31 nM

Example Ia4 8.3 nM 31 nM

Example Ia5 25 nM 67 nM

Example Ia6 71 nM 99 nM

Example Ia7 660 nM 210 nM

Example Ia8 15 nM 29 nM

Example Ia9 41 nM 76 nM

Example Ia10 71 nM 260 nM

Example Ia11 1000 2200 nM

Example Ia12 540 190 nM

Example Ib1 29 nM 83 nM

Example Id 7.2 nM 143 nM

Example Id1 32 nM 140 nM

Example Ie1 54 nM 580 nM

Example If 1 47 nM 72 nM

Example If2 51 nM 43 nM

Claims

1. A compound of Formula I:

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, Q- 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; wherein R⁴ is halogen, C₁-C4 alkyl/fluoroalkyl or C₁-C4 alkoxy/fluoroalkoxy; and wherein n is 0- 3; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein R² is C₁-C4 alkyl.

3. The compound of claim 2, wherein R² is methyl or ethyl.

4. The compound of claim 1, wherein R² is C3-C6 cycloalkyl.

5. The compound of anyone of claims 1-4, wherein R⁴ is halogen, C₁-C₂ alkyl or C₁-C₂ alkoxy.

6. The compound of anyone of claims 1-5, wherein R¹ is C₁-C4 alkyl.

7. The compound of anyone of claims 1-5, wherein R¹ is C3-C6 cycloalkyl or

tetrahydropyranyl.

8. The compound of anyone of claims 1-7, wherein R¹ 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 R¹ 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 C₁-C₄ alkyl or C₁-C₄ alkoxy

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

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

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

17. The compound of claim 1, wherein the compound is selected from the group consisting of 1 -(2-chlorophenyl)-9-methoxy-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 9-methoxy-4- methyl-l-(o-tolyl)-[l,2,4]triazolo[4,3-a]quinoxaline; l-(2-chloro-6-methylphenyl)- 9-methoxy-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2,6-dimethylphenyl)-9- methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline; l-(3-chloropyridin-4-yl)-9- methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline; 9-methoxy-4-methyl-l-(3- methylpyridin-4-yl)-[l,2,4]triazolo[4,3-a]quinoxaline; 9-methoxy-4-methyl-l- propyl-[l,2,4]triazolo[4,3-a]quinoxaline; 9-methoxy-4-methyl-l-(2-trifluoromethyl- phenyl)-[l,2,4]triazolo[4,3-a]quinoxaline; l-(2-difluoromethoxy-phenyl)-9-methoxy-4- methyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2-trifluoromethoxy-phenyl)-9-methoxy-4- methyl-[l,2,4]triazolo[4,3-a]quinoxaline; 9-methoxy-4-methyl-l-(tetrahydro-pyran-4- yl)-[l ,2,4]triazolo[4,3-a]quinoxaline; 1 -cyclopentyl-9-methoxy-4-methyl- [1 ,2,4]triazolo[4,3-a]quinoxaline; 1 -(2-chlorophenyl)-4,9-dimethyl-[l ,2,4]triazolo[4,3- ajquinoxaline; l-(2-chlorophenyl)-9-fluoro-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline;

9-bromo-l -(2-chlorophenyl)-4-methyl-[l ,2,4]triazolo[4,3-a]quinoxaline; 8- bromo-l-(2-chloro-phenyl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline; 7- bromo-l-(2-chloro-phenyl)-9-methoxy-4-methyl-[l,2,4]triazolo[4,3-a]quinoxaline; and 7-bromo-9-methoxy-4-methyl-l-o-tolyl-[l,2,4]triazolo[4,3-a]quinoxaline; or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition comprising the compound of claim 1 or a

pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

19. A method of treating an anxiety disorder comprising administering a therapeutically effective amount of the compound of anyone of claim 1-17.

20. A method of treating a cognitive disorder comprising administering a therapeutically effective amount of the compound of anyone of claim 1-17.

21. A method of treating schizophrenia comprising administering a therapeutically effective amount of the compound of anyone of claim 1-17.

22. A compound of anyone of claims 1-17, for use in therapy.