WO2012135083A1 - METABOLITES OF 7-(2-(4-(6-FLUORO-3-METHYLBENZO[d]ISOXAZOL-5-YL)PIPERAZIN-1-YL)ETHYL)-2-(PROP-1-YNYL)-7H-PYRAZOLO[4,3-e][1,2,4]TRIAZOLO[1,5-c]PYRIMIDIN-5-AMINE AND THEIR UTILITY AS ADENOSINE A2a RECEPTOR ANTAGONISTS - Google Patents

Abstract

The present invention provides a compound of the Formula I: Formula I or a pharmaceutically acceptable salt thereof in isolated and purified form wherein R, R1 and R2 are as described herein. The compounds are adenosine A2a receptor antagonists useful in treatment of central nervous system disorders, such as Parkinsons disease, Extra-Pyramidal Syndrome (EPS) caused by treatment with an antipsychotic agent, restless legs syndrome, Huntington s disease, attention disorders, depression, stroke and psychoses.

Description

METABOLITES OF 7-(2-(4-i6-FLUORO-3-METHYLBENZOrd1ISOXAZOL-5- YL⁾PIPERAZIN-l-YL⁾ETHYL)-2-(PROP-l-YNYL)-7H-PYRAZOLOi4,3- eiri.2,41TRIAZOLOri>5-c1PYRIMIDIN-5-AMINE AND THEIR UTILITY AS

ADENOSINE A2a RECEPTOR ANTAGONISTS

FIELD OF THE INVENTION:

The present invention relates to metabolites of 7-(2-(4-(6-fluoro-3- methylbenzo[d]isoxazol-5-yl)piperazin-l-yl)ethyl)-2-(prop-l-ynyl)-7H-pyrazolo[4,3- e][l,2,4]triazolo[l,5-c]pyrimidin-5-amine, having the structure of Formula II shown hereinbelow,

Formula II

the use of said compounds in the treatment of central nervous system disorders, including movement disorders, e.g., Parkinson's disease, Extra-Pyramidal Syndrome (EPS) caused by treatment with an antipsychotic agent, restless legs syndrome, essential tremor and Huntington's Disease; attention disorders, e.g., attention deficit hyperactivity disorder, cognitive impairment and negative symptoms of schizophrenia, and in the treatment of other central nervous system diseases such as depression, stroke and psychoses. The invention also relates to pharmaceutical compositions comprising said compounds.

BACKGROUND OF THE INVENTION:

Adenosine is known to be an endogenous modulator of a number of

physiological functions. At the cardiovascular system level, adenosine is a strong vasodilator and a cardiac depressor. On the central nervous system, adenosine induces sedative, anxiolytic and antiepileptic effects. On the respiratory system, adenosine induces bronchoconstriction. At the kidney level, it exerts a biphasic action, inducing vasoconstriction at low concentrations and vasodilation at high doses. Adenosine acts as a lipolysis inhibitor on fat cells and as an antiaggregant on platelets. Adenosine action is mediated by the interaction with different membrane specific receptors which belong to the family of receptors coupled with G proteins. Biochemical and pharmacological studies, together with advances in molecular biology, have allowed the identification of at least four subtypes of adenosine receptors: Aj, A_2a, A¾ and A₃. A] and A₃ are high-affinity, inhibiting the activity of the enzyme adenylate cyclase, and A_2a and A¾ are low-affinity, stimulating the activity of the same enzyme. Analogs of adenosine able to interact as antagonists with the A_l5 A_2a, A_2b and A₃ receptors have also been identified.

Selective antagonists for the A_2a receptor are of pharmacological interest because of their reduced level of side affects. In the central nervous system, A_2a antagonists can have antidepressant properties and stimulate cognitive functions.

Moreover, data has shown that A_2a receptors are present in high density in the basal ganglia, known to be important in the control of movement. Hence, A_2a antagonists can improve motor impairment due to neurodegenerative diseases such as Parkinson's disease, senile dementia as in Alzheimer's disease, and psychoses of organic origin. Some xanthine-related compounds have been found to be At receptor selective antagonists, and xanthine and non-xanthine compounds have been found to have high A_2a affinity with varying degrees of A_2a vs. Aj selectivity.

EPS is a collective term for a series of adverse neurological reactions associated with the use of antipsychotic drugs. There are six different categories of EPS-related neurological syndromes of which four, dystonia, akathisia, pseudoparkinsonism (parkinsonian syndrome), and tardive dyskinesia, are particularly prevalent in patients taking antipsychotic medication. Dystonia is a painful spasm of the muscle groups of, in particular, the neck, jaw, back, pharynx, and larynx. It is most common in young males being treated with antipsychotic drugs, but can also be associated with the use of cocaine, tricyclic antidepressants, lithium and anticonvulsants such as phenytoin and carbamazepine. Pseudoparkinsonism manifests itself as akinesia (rigidity, stiffness and slow voluntary motion, stooped, shuffling walk) and tremor and these symptoms develop within weeks or months after initiation of therapy. Akathisia manifests itself as strong, subjective inner feelings of distress or discomfort characterized by motor restlessness. Often mistaken for agitation or anxiety, this common syndrome is frequently under-diagnosed and is the least responsive to treatment. Tardive dyskinesia is a late-appearing syndrome associated with chronic use of neuroleptic drugs. It occurs more frequently in older patients and is characterized by stereotypical, repetitive, involuntary, quick choreiform movements of the face, eyelids, mouth, tongue, extremities and trunk.

EPS is more prevalent with the use of typical antipsychotic agents but has also been reported with the use of atypical agents. Typical antipsychotics include loxapine, haloperidol, chlorpromazine, prochlorperazine and thiothixene. Atypical antipsychotics include clozapine, olanzapine, loxapine, quetiapine, ziprasidone, risperidone and aripiprazole.

Akathisia is also a characteristic of resltless leg syndrome (RLS) and periodic leg (or limb) movement disorder (PLMD) or syndrome (PLMS). RLS is a common disorder that causes patients to have an irresistible and unpleasant desire to move their legs; it usually manifests during periods of inactivity and/or at night, and can disturb sleep. Patients who do not have the typical RLS symptoms, but who do exhibit periodic leg movements that adversely impact sleep, are diagnosed with PLMS.

Treatments for RLS and PLMS have included levodopa/carbidopa,

levodopa/benserazide, dopamine agonists such as pramipexole and ropinerole, benzodiazepines, opioids, anticonvulsants and iron (ferrous sulfate). RLS and PLMS have been extensively described in the literature, for example by Saletu et al,

Neuropsvchobioloev. 41, 4 (2000), p. 190-199.

The compound of Formula II, viz., 7-(2-(4-(6-fluoro-3- methy lbenzo [d] isoxazol-5 -yl)piperazin- 1 -yl)ethyl)-2-(prop- 1 -ynyl)-7H-pyrazolo [4,3 - e][l,2,4]triazolo[l,5-c]pyrimidin-5-amine and its utility to treat various central nervous system disorders has been disclosed in WO 2007/038284. Some xanthine-related compounds have been found to be A_\ receptor selective antagonists, and xanthine and non-xanthine compounds have been found to have high A_2a affinity with varying degrees of A_2a vs. A_\ selectivity. Triazolo-pyrimidine adenosine A_2a receptor antagonists have been disclosed, for example in WO 2005/044245, WO 01/92264, WO 95/01356; US 5,565,460; WO 97/05138; WO 98/52568, US 6,630,475, US 6,653,315, and US 6,897,217. SUMMARY OF THE INVENTION:

The present invention relates to the compound having the structural formula I

Formula I

or a pharmaceutically acceptable salt thereof in isolated and purified form, wherein:

R is selected from the group consisting of H, and OH;

R¹ is OH or

R² is -C(=0)CH₃; or

R¹ and R² taken together with the carbon atoms to which they are shown attached form

Another aspect of the invention is the compound of formula I or a

pharmaceutically acceptable salt thereof in isolated and purified form, wherein in formula I, R¹ is OH, R² is -C(=0)CH₃, or R¹ and R² taken together with the carbon atoms to which they are shown attached form 23034

Another aspect of the invention is the compound of formula I or a

pharmaceutically acceptable salt thereof in isolated and purified form, wherein in formula I, R is H, R¹ is OH, and R² is -C(=0)CH₃, i.e., the compound of formula I has the structure:

Another aspect of the invention is the compound of formula I or a

pharmaceutically acceptable salt thereof in isolated and purified form, wherein in f ² is -€(=0)CH₃, and R¹ is

₉ i.e., the compound of Formula I has the structure 23034

Another aspect of the invention is the compound of formula I or a

pharmaceutically acceptable salt thereof in isolated and purified form, wherein in formula I, R is OH and R¹ and R² taken together with the carbon atoms to which they are shown attached form

i.e., the compound of Formula I has the structure

Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of the compound of formula I in a pharmaceutically acceptable carrier.

Yet another aspect of the invention is a method of treating central nervous system disorders including movement disorders, e.g., Parkinson's Disease, Extra- 23034

7

Pyramidal Syndrome, restless legs syndrome, essential tremor, Huntington's Disease, dystonia, periodic limb movement in sleep; attention disorders, e.g., attention deficit hyperactivity disorder, cognitive impairment and negative symptoms of schizophrenia; and to other central nervous system diseases such as depression, stroke and psychoses, comprising administering the compound of formula I to a mammal in need of such treatment.

In particular, the invention is drawn to the method of treating movement disorders such as Parkinson's disease, essential tremor or Huntington's Disease comprising administering the compound of formula I to a mammal in need of such treatment.

Still another aspect of the invention is a method of treating Parkinson's disease with a combination of the compound of formula I and one or more agents useful in the treatment of Parkinson's disease, for example dopamine; L-DOPA; a dopaminergic agonist; an inhibitor of monoamine oxidase, type B (MAO-B); a DOPA decarboxylase inhibitor (DCI); or a catechol-O-methyltransferase (COMT) inhibitor.

Another aspect of the invention is a pharmaceutical composition comprising the compound of formula I and one or more agents known to be useful in the treatment of Parkinson's in a pharmaceutically acceptable carrier.

The invention also relates to the treatment or prevention of EPS (e.g., dystonia, akathisia, pseudoparkinsonism and tardive dyskinesia) comprising administering the compound of formula I to a mammal in need of such treatment. In particular, this method is for the treatment or prevention of EPS in patients treated with an

antipsychotic agent that has the side effect of inducing EPS. The compound of formula I can be administered after the symptoms of EPS have manifested, or the compound of formula I can be administered at the onset of administering an antipsychotic agent in order to prevent EPS from occurring. Thus, the invention also includes a method of treating or preventing EPS induced by an antipsychotic agent comprising administering a combination of an antipsychotic agent and the compound of formula I to a patient in need thereof.

The invention also relates to the treatment of primary (idiopathic) dystonia, and to the treatment or prevention of dystonia in patients who exhibit dystonia as a result of 23034

treatment with a tricyclic antidepressant, lithium or an anticonvulsant, or who have used cocaine, comprising administering a therapeutically effective amount of the compound of formula I to a patient in need thereof. When dystonia is caused by treatment with a tricyclic antidepressant, lithium or an anticonvulsant, the compound of formula I can be administered after the symptoms of dystonia have manifested, or the compound of formula I can be administered at the onset of administering a tricyclic antidepressant, lithium or an anticonvulsant in order to prevent dystonia from occurring. The invention, therefore, also includes a method of treating or preventing dystonia induced by a tricyclic antidepressant, lithium or an anticonvulsant comprising administering a combination of the compound of formula I and a tricyclic

antidepressant, lithium or an anticonvulsant to a patient in need thereof.

The invention further relates to treatment of abnormal movement disorders such as RLS or PLMS, comprising administering to a patient in need thereof a

therapeutically effective amount of the compound of formula I. The invention also comprises a method of treating RLS or PLMS comprising administering a combination of the compound of formula I with another agent useful in treating RLS or PLMS, such as levodopa/carbidopa, levodopa benserazide, a dopamine agonist, a benzodiazepine, an opioid, an anticonvulsant or iron, to a patient in need thereof.

The invention also relates to the treatment of attention related disorders such as attention deficit disorder (ADD) and ADHD, as well as cognitive impairment and negative symptoms of schizophrenia, comprising administering a therapeutically effective amount of the compound of formula I.

In another aspect, this invention relates to a kit comprising, in separate containers in a single package, pharmaceutical compositions for use in combination to treat Parkinson's Disease, wherein one container comprises a pharmaceutical composition comprising an effective amount of the compound of formula I in a pharmaceutically acceptable carrier, and wherein a separate container comprises a pharmaceutical composition comprising an effective amount of an agent useful in the treatment of Parkinson's disease.

In another aspect, this invention relates to a kit comprising, in separate containers in a single package, pharmaceutical compositions for use in combination to 23034

9 treat or prevent EPS caused by treatment with antipsychotic agent, wherein one container comprises a pharmaceutical composition comprising an effective amount of the compound of formula I in a pharmaceutically acceptable carrier, and wherein a separate container comprises a pharmaceutical composition comprising an effective amount of an antipsychotic agent.

In another aspect, this invention relates to a kit comprising, in separate containers in a single package, pharmaceutical compositions for use in combination to treat or prevent dystonia caused by treatment with a tricyclic antidepressant, lithium or an anticonvulsant, wherein one container comprises a pharmaceutical composition comprising an effective amount of the compound of formula I in a pharmaceutically acceptable carrier, and wherein a separate container comprises a pharmaceutical composition comprising an effective amount of a tricyclic antidepressant, lithium or an anticonvulsant.

In another aspect, this invention relates to a kit comprising, in separate containers in a single package, pharmaceutical compositions for use in combination to treat RLS or PLMS, wherein one container comprises a pharmaceutical composition comprising an effective amount of the compound of formula I in a pharmaceutically acceptable carrier, and wherein a separate container comprises a pharmaceutical composition comprising an effective amount of levodopa/carbidopa,

levodopa/benserazide, a dopamine agonist, a benzodiazepine, an opioid, an

anticonvulsant or iron.

In another aspect, this invention also relates to the use of the compound of formula I for the preparation of a medicament for treating or preventing Parkinson's Disease, EPS, idiopathic dystonia, dystonia associated with the use of cocaine, tricyclic antidepressants, lithium or anticonvulsants, restless leg syndrome (RLS), periodic limb movement disorder/syndrome (PLMD/PLMS), essential tremor, Huntington's Disease, cognitive impairment or negative symptoms of schizophrenia, alone or in combination with the other agents discussed above.

In another aspect, this invention relates to a method of determining if a subject has been administered the compound of formula II 23034

10

Formula II

or a pharmaceutically acceptable salt or solvate thereof, comprising the step of determining if a plasma, urine, bile or fecal sample obtained from the subject shows the presence of at least one compound of Formula I

Formula I

or a pharmaceutically acceptable salt or solvate thereof, wherein:

R is selected from the group consisting of H, and OH;

R¹ is OH or

R is -C(=0)CH₃; or

R¹ and R² taken together with the carbon atoms to which they are shown attached form

Another aspect of the invention relates to the above method of determining if a subject has been administered the compound of formula II or a pharmaceutically 23034

11 acceptable salt thereof in isolated and purified form, wherein in formula I, R is H, R¹ is OH, and R² is -C(=0)CH₃.

Another aspect of the invention relates to the above method of determining if a subject has been administered the compound of formula II or a pharmaceutically acceptable salt thereof in isolated and purified form, wherein in formula I, R is H, R² is -C(=0)CH₃, and R¹ is

Another aspect of the invention relates to the above method of determining if a subject has been administered the compound of formula II or a pharmaceutically acceptable salt thereof in isolated and purified form, wherein in formula I, R is OH and R¹ and R² taken together with the carbon atoms to which they are shown attached form

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 refers to Human Plasma Metabolite Profiles on Day 1 and Steady State (Day 10) following Multiple Oral Administration of 50 mg (Cohort 3) and 100 mg (Cohort 4) of the compound of Formula II.

Figure 2 refers to Human Urinary Metabolite Profiles on Day 1 and Steady State (Day 10) following Multiple Oral Administration of 50 mg (Cohort 3) and 100 mg (Cohort 4) of the compound of Formula II.

Figure 3 refers to Rat Plasma Metabolite Profiles from Pooled (0-24 hr) Male and Female Rat Plasma Following a Once Daily Oral Administration of 30 mg (Group 23034

12

2), 100 mg (Group 3) and 1000 mg (Group 4) of the compound of Formula II for 14 Days.

Figure 4 refers to Metabolite Profiles from Pooled (0-24 hr) Female Rabbit Plasma on Gestation Day 19 Following a Once Daily Oral Administration of 30 mg/kg (Group 2), 100 mg/kg (Group 3) and 300 mg/kg (Group 4) of the compound of Formula II.

Figure 5 refers to the Chemical Structures of the compound of Formula II and Its Major Circulating Metabolites in Humans, Rats and Female Rabbits.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The term "purified", "in purified form" or "in isolated and purified form" for a compound refers to the physical state of said compound after being isolated from a synthetic process (e.g. from a reaction mixture), or natural source or combination thereof. Thus, the term "purified", "in purified form" or "in isolated and purified form" for a compound refers to the physical state of said compound after being obtained from a purification process or processes described herein or well known to the skilled artisan (e.g., chromatography, recrystallization and the like), in sufficient purity to be characterizable by standard analytical techniques described herein or well known to the skilled artisan. In particular, the term "purified", "in purified form" or "in isolated and purified form" for a compound that is a referred to herein as a metabolite means that the compound/metabolite is free from the presence of other metabolites disclosed herein. Thus, for example, "the compound of Formula I in isolated and purified form" would refer to a physical state of the compound of Formula I wherein other metabolites are not present.

It should also be noted that any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples and Tables herein is assumed to have the sufficient number of hydrogen atom(s) to satisfy the valences. 23034

13

One or more compounds of the invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms. "Solvate" means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate wherein the solvent molecule is H₂0.

One or more compounds of the invention may optionally be converted to a solvate. Preparation of solvates is generally known. Thus, for example, M. Caira et al, J. Pharmaceutical Sci., 930}, 601-611 (2004) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar

preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al, AAPS PharmSciTec , 5(1}, article 12 (2004); and A. L. Bingham et al, Chem. Commun., 603-604 (2001). A typical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example I. R. spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

"Effective amount" or "therapeutically effective amount" is meant to describe an amount of compound or a composition of the present invention effective in inhibiting the above-noted diseases and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect.

"At least one", as used in reference to the number of compounds of this invention means for example 1-6, generally 1-4, more generally 1, 2 or 3, and usually one or two, and more usually one. 23034

14

"At least one", as used in reference to the number of "other" agents useful for treating a particular disease mentioned herein, means for example 1-6, generally 1-4, and more generally 1, 2 or 3, and usually one or two, or one.

The compounds of Formula I can form salts which are also within the scope of this invention. Reference to a compound of Formula I is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)^M, as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. In addition, when a compound of Formula I contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein.

Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Salts of the compounds of Formula I may be formed, for example, by reacting a compound of Formula I, with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates,

camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates,) and the like. Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley- VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International! of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website). These disclosures are incorporated herein by reference thereto. 23034

15

Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quartemized with agents such as lower alkyl halides (e.g. methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g. decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.

All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the invention and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.

Pharmaceutically acceptable esters of the present compounds include the following groups: (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n- propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, C_1-4alkyl, or Ci^alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example,

methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a C_1-20 alcohol or reactive derivative thereof, or by a 2,3-di (C _-24)acyl glycerol.

Compounds of Formula I, and salts, solvates, esters and prodrugs thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.

If the compounds of Formula I may exist in different tautomeric forms, all such forms are embraced within the scope of the invention. Also, for example, all keto-enol and imine-enamine forms of the compounds are included in the invention. 23034

16

The present invention also embraces isotopically-labelled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸0, ¹⁷0, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶C1, respectively.

Certain isotopically-labelled compounds of Formula I (e.g., those labeled with ³H and ¹⁴C) are useful in compound and/or substrate tissue distribution assays.

Tritiated (i.e., ³H) and carbon- 14 (i.e., ¹⁴C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labeled compounds of Formula I can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples hereinbelow, by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent.

Polymorphic forms of the compounds of Formula I, and of the salts, solvates, esters and prodrugs of the compounds of Formula I, are intended to be included in the present invention.

The following abbreviations are used below and have the following meanings:

Boc is tert-butoxycarbonyl, dba is dibenzylideneacetone, DMF is N,N - dimethylformamide, DMSO is dimethylsulfoxide, EtOAc is ethyl acetate, LCMS is liquid chromatography mass spectrometry, MeOH is methanol, NMR is nuclear magnetic resonance, PBS is phosphate buffered saline, SPA is scintillation proximity assay, Tf is triflate, TFA is trifiuoroacetic acid and Xantphos is 9,9-Dimethyl-4,5- bis(diphenylphosphino)xanthene. Me₄Si is tetramethyl silane, DIEA is diisopropyl ethylamine,SGC is silicagel column, TMSCHN₂ is trimethylsilyl diazomethane, BBr₃ is tribromoborane,m-CPBA is m-chloro perbenzoic acid, CDI is carbodiimidazole,HATU is 2-(lH-azabenzotriazol-l-yl- 1,13, 3 -tetramethyl uranium hexafluorophosphate, NaH is 23034

17 sodium hydride, Si0₂ is silica, CBZ is benzyloxy carbonyl, Tos is p-toluene sulfonyl, and CH₃CN is acetonitrile.

"Patient" includes both human and animals.

"Mammal" means humans and other mammalian animals.

The other agents known to be useful in the treatment of Parkinson's disease that can be administered in combination with the compounds of formula I include:

L-DOPA; dopaminergic agonists such as quinpirole, ropinirole, pramipexole, pergolide and bromocriptine; MAO-B inhibitors such as deprenyl and selegiline; DOPA decarboxylase inhibitors such as carbidopa and benserazide; and COMT inhibitors such as tolcapone and entacapone.

Antipsychotic agents causing the EPS treated by adenosine A_2a receptor antagonists and for use in combination with adenosine A_2a receptor antagonists include typical and atypical antipsychotic agents. Typical antipsychotics include loxapine, haloperidol, chlorpromazine, prochlorperazine and thiothixene. Atypical antipsychotics include clozapine, olanzapine, loxapine, quetiapine, ziprasidone, risperidone and aripiprazole.

Tricyclic antidepressants causing dystonia treated by adenosine A_2a receptor antagonists include perphenazine, amitriptyline, desipramine, doxepin, trimipramine and protriptyline. Anticonvulsants which may cause dystonia, but which also may be useful in treating ERLS or PLMS include phenytoin, carbamazepine and gabapentin.

Dopamine agonists useful in treating RLS and PLMS include pergolide, pramipexole, ropinerole, fenoldopam and cabergoline.

Opioids useful in treating PRLS and PLMS include codeine, hydrocodone, oxycodone, propoxyphene and tramadol.

Benzodiazepines useful in treating PRLS and PLMS include clonazepam, triazolam and temazepam.

The antipsychotics, tricyclic antidepressants, anticonvulsants, dopamine agonists, opioids and benzodiazepines are commercially available and are described in the literature, e.g., in The Physicians' Desk Reference (Montvale: Medical Economics Co., Inc., 2001). 23034

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It is contemplated that the compound of formula I could be administered in combination with one or more other agents (e.g., antipsychotics, tricyclic

antidepressants, anticonvulsants, dopamine agonists, opioids or benzodiazepines), although administration of the compound of formula I in combination with one other agent is preferred for each of the indications. While administration of separate dosage forms of the compound of formula I and the other agent(s) are preferred, it is also contemplated that the other agent(s) could be combined in a single dosage form with the compound of formula I for the treatment or prevention of Parkinson's disease, EPS, dystonia, RLS or PLMS. It is also contemplated that the compound of formula I could be administered in combination with another adenosine A_2a antagonist.

Compounds of formula I can be prepared by known methods from starting materials either known in the art or prepared by methods known in the art, or by the methods described hereinbelow.

General Methods

The general methods described in this paragraph were used unless stated otherwise in the experimental procedures below. All solvents and reagents were used as received. Proton NMR spectra were obtained using a Varian XL-400 (400 MHz) or a Bruker Avance (500 MHz) instruments. 1H chemical shifts are reported in parts per million (ppm), measured relative to residual solvent peaks as an internal standard set to δ 7.26 ppm for chloroform-d, 3.34 for methanol-c¾ and 2.50 ppm for DMSO-£¾. LCMS analysis was performed using a PE SCIEX API-150EX single quadrupole mass spectrometer equipped with a Phenomenex Gemini Ci₈ column (5.0 μπι, 50 x 4.6 mm); mobile phase A: 0.05 % trifluoroacetic acid in water, B: 0.05% trifluoroacetic acid in CH₃CN; gradient: 90 % A and 10 % B to 5 % A and 95 % B in 5 minutes. Flash column chromatography was performed using Teledyne Isco RediSep Normal Phase Columns and C₁₈ Reverse Phase Columns. Alternatively, HPLC separation was performed on a Gilson instrument (Gilson 322 pump, 156 UV-vis detector 215 liquid handler and 845Z injector) using a Phenomenex Gemini C₁₈ column (5.0 μπι, 100 x 21.1 mm or 150 x 21.2 mm); mobile phase A: 0.05 % trifluoroacetic (or formic acid) in water, B: 0.05% trifluoroacetic (or formic acid) in CH₃CN. Microwave-mediated 23034

19 reactions were performed using a Biotage Initiator Synthesis System and using the standard 2 mL, 5 mL or 20 mL vials and lids. Preparative and analytical TLC were performed using Analtech Silica gel GF plates.

23034

20

Scheme 1 3-(5-amino-7-(2-(4-(6-fluoro-3-methylbenzo[d]isoxazol-5-yl)piperazin-l- yl)ethyl)-7H-pyrazolo[4,3-e] [1 ,2,4]triazolo[l ,5-c]pyrimidin-2-yl)prop-2-yn- 1 -ol (9)

Step 1 : 4-chloro-lH-pyrazolo[3,4-d]pyrimidin-6-amine (2)

To a solution of 2-amino-4,6-dichloropyrimidine-5-carbaldehyde (25.0 g, 130 mmol) in DMF (100 mL) was added N, N-diisopropylethylamine (28.4 mL, 163 mmol). Then hydrazine hydrate (6.32 mL, 130 mmol) was added slowly. The reaction mixture was stirred at room temperature for 24 h and concentrated under vacuum. Water (100 mL) was added, the aqueous solution was filtered, and the filtrate was washed with water. The solid was dried under vacuum to give 4-chloro-lH- pyrazolo[3,4-d]pyrimidin-6-amine (2) as a yellow solid (18.9 g, 85% yield). Ή NMR (500 MHz, OMSO-de) δ ppm 7.10 (br s), 7.95 (s, 1 H); MS (M+l): 170. Step 2: 4-chloro- 1 -(2-chloroethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-amine (3)

To 4-chloro- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-amine (2) (15.0 g, 88 mmol) dissolved in DMF (150 mL) was added 60% NaH in mineral oil (4.25 g, 106 mmol) slowly. l-Bromo-2-chloroethane (22.1 mL, 265 mmol) was added slowly. The resulting reaction mixture was stirred at RT for 2 h then concentrated. The residue was purified by silica gel chromatography (eluant: 3 : 1 CH₂C1₂ : methanol) to obtain the product 4- chloro-1 -(2-chloroethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-amine (3) as a white solid (10.9 g; 53% yield). Ή NMR (500 MHz, CDC1₃) δ ppm 3.74 (t, J=6.0 Hz, 2 H), 4.18 (br s), 4.36 (t, J=6.0 Hz, 2 H), 7.71 (s, 1 H); MS (M+l): 232.

Step 3 : tert-butyl 2-(6-amino- 1 -(2-chloroethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-4- yl)hydrazinecarboxylate (4)

To 4-chloro-l-(2-chloroethyl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine (3) (12.2 g, 52.5 mmol) dissolved in DMF (70 mL) was added t-butyl carbazate (8.3 g, 63 mmol). The resulting reaction mixture was heated at 80 °C for 24 h then cooled to RT and concentrated. The residue was purified by chromatography on silica gel (eluant: 3 : 1 CH₂C1₂ : methanol) to give the product tert-butyl 2-(6-amino- 1 -(2-chloroethyl)- 1 H- pyrazolo[3,4-d]pyrimidin-4-yl)hydrazinecarboxylate (4) as a white solid (13.8 g, 80% yield). 1H NMR (500 MHz, CDC1₃) δ ppm 1.44 (s, 9H), 3.86 (t, J=6.0 Hz, 2 H), 4.48 (t, J=6.0 Hz, 2 H), 7.80 (s, 1 H); MS (M+l): 328.

Step 4: 1 -(2-cliloroethyl)-4-hydrazinyl-lH-pyrazolo[3,4-d]pyrimidin-6-arnine (5)

To tert-butyl 2-(6-amino- 1 -(2-chloroethyl)- 1 H-pyrazolo[3 ,4-d]pyrimidin-4- yl)hydrazinecarboxylate (4) (5.0 g, 15.0 mmol) suspended in 1 : 1 MeOH: CH₂C1₂ (100 mL) was added 4M HC1 in dioxane (50 mL). The resulting reaction mixture was stirred at RT for 16 h. Cone, ammonium was added until the pH was raised to 11. The resulting mixture was concentrated under vacuum, water was added, and the solid was filtered. The filtrate was washed with water and then dried to give the product l-(2- chloroethyl)-4-hydrazinyl-l H-pyrazolo [3 ,4-d]pyrimidin-6-amine (5) as a brown solid (3.2 g, 95% yield). 1H NMR (500 MHz, DMSO- ) δ ppm 3.91 (t, J=6.0 Hz, 2 H), 4.31 (t, J=6.0 Hz, 2 H), 7.81 (s, 1 H); MS (M+l): 228. Step_5_ N'-(6-amino- 1 -(2-chloroethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-4-yl)-4-(tert- butyldimethylsilyloxy)but-2-ynehydrazide (6)

To 1 -(2-chloroethyl)-4-hydrazinyl-l H-pyrazolo [3 ,4-d]pyrimidin-6-amine (5) (2.0 g, 8.8 mmol) dissolved in DMF (10 mL) and cooled to 0 °C was added freshly prepared 4-(tert-butyldimethylsilyloxy)but-2-ynoic (isopropyl carbonic) anhydride (2.7 g, 9.0 mmol) in DMF (5 mL) drop wise. The resulting reaction mixture was stirred at 0 °C to RT for 2 h then concentrated to dryness under vacuum. Water was added, and the solid was removed by filtration. The filtrate was dried under vacuum to give the product N'-(6-amino- 1 -(2-chloroethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-4-yl)-4-(tert- butyldimethylsilyloxy)but-2-ynehydrazide (6) as a yellow solid (3.2 g, 85% yield). This solid was used directly in the next step without purification; MS (M+l): 424.

Step 6: 2-(3 -(tert-butyldimethylsilyloxy)prop- 1 -ynyl)-7-(2-chloroethyl)-7H- pyrazolo[4,3-e] [1 ,2,4]triazolo[l ,5-c]pyrimidin-5-amine (7)

The solution of N'-(6-amino- 1 -(2-chloroethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-4- yl)-4-(tert-butyldimethylsilyloxy)but-2-ynehydrazide (6) (2.0 g, 4.7 mmol) in N, O- bis(trimethylsilyl)acetamide(10 mL) was heated at 120 °C for 2 h. The reaction was concentrated to dryness under vacuum. Methanol was added, and the resulting mixture was stirred at room temperature for 1 h. The residue was purified by chromatography on silica gel (eluant: 5 : 1 CH₂C1₂ : methanol) to give the product 2-(3-(tert- butyldimethylsilyloxy)prop- 1 -ynyl)-7-(2-chloroethyl)-7H-pyrazolo [4,3- e] [1 ,2,4]triazolo[l ,5-c]pyrimidin-5-amine (7) as a brown solid (1.3 g, 70% yield). H NMR (500 MHz, DMSO-i ) 5 ppm 0.15 (s, 6 H), 1.01 (s, 9 H), 3.90 (t, J=6.0 Hz, 2 H), 4.35 (t, J=6.0 Hz, 2 H), 4.50 (s, 2 H), 8.0 (s, 1 H); MS (M+l): 406.

StepJZ: 2-(3-(tert-butyldimethylsilyloxy)prop-l-ynyl)-7-(2-(4-(6-fluoro-3- methylbenzo [d]isoxazol-5 -yl)piperazin- 1 -yl)ethyl)-7H-pyrazolo [4,3 - e] [ 1 ,2,4]triazolo[l ,5-c]pyrimidin-5-amine (8)

To a solution of 2-(3-(tert-butyldimethylsilyloxy)prop-l-ynyl)-7-(2- chloroethyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-5-amine (7) (0.34 g, 0.84 mmol) and 6-fluoro-3-methyl-5-(piperazin-l-yl)benzo[d]isoxazole (0.20 g, 0.84 23 mmol) in DMF (10 mL) was added DIPEA (0.15 mL, 0.84 mmol) and potassium iodide (0.14 g, 0.84 mmol). The resulting mixture was heated in the microwave at 130 °C for 4 h. The reaction was concentrated to dryness under vacuum. The residue was purified by chromatography on silica gel (eluant: 10 : 1 CH₂C1₂ : methanol) to give the product 2-(3 -(tert-butyldimethylsilyloxy)prop- 1 -ynyl)-7-(2-(4-(6-fluoro-3 - methylbenzo [d] isoxazol-5 -yl)piperazin- 1 -yl)ethyl)-7H-pyrazolo [4,3 - e][l,2,4]triazolo[l,5-c]pyrimidin-5-amine (8) as a brown solid (0.36 g, 72% yield). MS (M+l): 605.

Step 8: 3-(5-amino-7-(2-(4-(6-fluoro-3-methylbenzo[d]isoxazol-5-yl)piperazin- 1 - yl)emyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-2-yl)prop-2-yn-l-ol (9)

To 2-(3-(tert-butyldimethylsilyloxy)prop- 1 -ynyl)-7-(2-(4-(6-fluoro-3- methylbenzo [d] isoxazol-5 -yl)piperazin- 1 -yl)ethyl)-7H-pyrazolo [4,3 - e][l,2,4]triazolo[l,5-c]pyrimidin-5-amine (8) (0.5 g, 0.8 mmol) dissolved in THF (10 mL) was added 1.0 M tetrabutylammonium fluoride in THF (0.83 mL, 0.83 mmol). The resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum, and DMF was added. The solution was purified by chromatography on Gilson (eluant: water : acetonitrile) to give the product 3-(5-amino-7-(2-(4-(6-fiuoro-3-methylbenzo[d]isoxazol-5-yl)piperazin-l-yl)ethyl)-7H- pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-2 -yl)prop-2-yn-l-ol (9) as a white solid (0.24 g, 60% yield). 1H NMR (500 MHz, DMSO-rf₆) δ ppm 2.50 (s, 3 H), 2.66 (br s, 4 H), 2.86 (t, J=6.5 Hz, 2 H), 2.94 (br s, 4 H), 4.38 (d, J= 6.0 H z, 2 H), 4.43 (t, J=7.0 Hz, 2 H), 5.58 (t, J=6.5 Hz, 1 H), 7.40 (d, J=12.0 Hz, 1 H), 7.65 (d, J=12.0 Hz, 1 H), 8.16 (s, 3 H); MS (M+l): 491. 23034

24

Scheme 2

Step 1 : l-(5-(4-(2-(5-amino-2-(prop-l-ynyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5- c]pyrimidin-7-yl)ethyl)piperazin-l-yl)-4-fluoro-2-hydroxyplienyl)ethanone (11)

A microwave reaction vial was charged with 7-(2-(4-(6-fluoro-3- methylbenzo [d]isoxazol-5-yl)piperazin- 1 -yl)ethyl)-2-(prop- 1 -ynyl)-7H-pyrazolo [4,3 - e][l,2,4]triazolo[l,5-c]pyrimidin-5-amine (2) (0.78 g, 1.6 mmol), Mo(CO)₆ (0.48 g, 1.8 mmol), water (0.06 mL) and DMF (12 mL). The vial was capped, and air was removed by vacuum and back-filled with nitrogen (3 times). The reaction mixture was heated by a microwave reactor to 90 °C for 30 mins. Water (1 mL) was added and mixture was heated again to 100 °C for 10 mins. The mixture was poured into water, and stirred at room temperature for 2 h. The precipitate was collected by filtration, which was then partially dissolved in THF and mixed with silica gel. After concentrating to dryness, the silica gel was loaded onto the top of a flash chromatography column, then eluted with 0-5% MeOH/dichloromethane solvent gradient to give the product l-(5-(4-(2-(5- arnino-2-( rop-l-ynyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-7- yl)ethyl)piperazin- 1 -yl)-4-fluoro-2-hydroxyphenyi)ethanone (11)

as a pale solid (0.51 g, 65% yield). H-NMR (DMSO-6d) ) δ ppm 2.14 (s, 3H), 2.60 (s, 3H), 2.61(m, br, 4H), 2.83 (t, 2H), 2.89 (m, 4H), 4.40 (t, 2H), 6.80 (d, 1H), 7.38 (d, 1H), 8.11 (s, br, 2H), 8.12 (s, 1H), 11.95 (s, 1H); MS (M+l): 478.

Isolation and Characterization of the Metabolites

Chemicals: 7-[2-[4-(6-fluoro-3-methyl-l,2-benzisoxazol-5-yl)-l-piperazinyl]ethyl]-2- (1 -propynyl)-7H-pyrazolo[4,3-e] [1 ,2,4]triazolo[l ,5-c]pyrimidin-5-amine was synthesized at Merck Research Laboratories. All other compounds/reference standards 23034

25 were obtained from Chemical Research at Merck Research Laboratories. HPLC grade methanol and acetonitrile were from Burdick and Jackson (Muskegon, MI). Water was purified using Millipore Milli-Q water purification system (Bedford, MA).

Test Species:

M = Male;

F = Female;

n = number of animals/subjects

Sample Collection: Blood at selected time points and urine (human only) over selected time intervals were collected from healthy human volunteers, rats and rabbits.

Sample Processing for Profiling and Characterization of Metabolites: 23034

26

Sample Pooling:

For each species, plasma samples were pooled across subjects/animals using a scheme to generate one sample for Day 1 and/or at Steady State that has concentrations of parent and its metabolites proportional to their pharmacokinetic area under the curve (AUC).

Sample Processing:

Mobile Phase and HPLC Conditions:

The mobile phase consisted of 10 mM ammonium acetate adjusted to pH 7.0 with ammonium hydroxide (A) and methanol (B) and was maintained at a constant flow rate (1 mL/min). HPLC column [Luna Phenyl-hexyl, 250x4.6 mm, 5-μιη particle size (Phenomenex)] temperature was maintained at 40°C. Satisfactory separation of the compound of Formula II and its metabolites in rat matrices was achieved using programmed linear changes in mobile phase composition as shown in the following table:

Time (min) % A % B

0 95 5

3 95 5

5 85 15

30 60 40

40 25 75

48 15 85

48.1 5 95

50 5 95

50.1 95 5

60 95 5 23034

27

For all LC-MS experiments, the column effluent was split to divert -17% into mass spectrometer and the balance into waste.

LC-MS System:

The mass spectrometer was nominally operated under the conditions listed below:

Total ion chromatograms from study samples were examined and compared with those from the control samples using background subtraction. After background subtraction, each peak was examined for possible molecular ion related to drug and/or its putative metabolites. Based on the elution order, metabolite peak labels were assigned as M0 to Ml 8 where M0 is the first and Ml 8 is the last to elute from the column (see Figures 1- 5). When available, synthetic reference standards were used to confirm the structural assignments.

Results

As shown in Figure 1, after repeated administration of the compound of Formula II, the compound of Formula II, M5 (an N-dealkylated metabolite, M-239), Ml 5 (a hydroxylated metabolite, M+16), and Ml 8 (oxazol ring opening, M+3) were the major circulating drug-derived material at steady state. In contrast to plasma profiles, metabolites dominated the urinary profile (Figure 2) and the compound of Formula II was only a minor component excreted in urine. The proposed biotransformation pathways for the major urinary metabolites included cleavage (Ml, M5 and M8) and opening of the oxazol ring and glucuronization (M7). Ml corresponded to the other half of M5 that resulted from N-dealkylation.

Representative rat plasma metabolite profiles [expressed as extracted ion

chromatograms (XICs)] at steady state for both sexes and all dosing levels are shown in Figure 3. There was no significant sex-related difference in the plasma metabolite profiles. The plasma metabolite profiles were also qualitatively similar for all dosing groups. After adjusting the intrinsic MS response factors of the compound of Formula II relative to M5, M15 and M18; Compound of Formula II, M18 and M15 were the major circulating drug-derived material in plasma at steady state for both sexes and all dosing levels following 14-day oral administration of the compound of Formula II in rats.

Representative rabbit plasma metabolite profiles [expressed as extracted ion chromatograms (XICs)] at steady state for all dosing levels are shown in Figure 4. The plasma metabolite profiles were also qualitatively similar for all dosing groups. After adjusting the intrinsic MS response factors of the compound of Formula II relative to M5, M15 and M18, Compound of Formula II, M5, M7, M15 and M18 were the major circulating drug-derived material in plasma on Gestation Day 19 (steady state) for all dosing levels following once daily oral administration of the compound of Formula II in female rabbits.

Metabolites detected in humans and animals are summarized in Table 1. All metabolites detected in humans were also detected in rats or rabbits. The structures of major circulating metabolites in humans, rats and rabbits are shown in Figure 5.

Table 1. Metabolites detected in plasma and urine (human only) in human, rat and rabbit species.

Compound of Formula II and Metabolites (Plasma)

Species

Major Minor Trace

Human Compound of Formula M7 (m/z 654), Ml l Ml (m/z 272), M4

II, M5 (m/z 236), Ml 5 (m/z 612), M13 (m/z (m/z 670), M6 (m/z (m/z491),M18 (m/z 467), M16 (m/z 491), 670), M8 (m/z 326), 478) M17(m/z494) M9(m/z669),M10

(mz 667), Ml 2 (m/z 507),

Rat Compound of Formula M5 (m/z 236), Ml 6 M0 (m/z 288), Ml

II,M15(m/z491), (m/z 491), (m/z 272), M2 (m/z

M18(m/z478) 342), M3 (m/z 252),

M4 (m/z 670), M7 (654), M8 (m/z 326), M9 (mz 669), M9A (mz 798), Ml 0 (m/z 667), Mil (m/z 612), M12 (m/z 507), M14 (m/z 491), Ml 7 (m/z 494)

Rabbit Compound of Formula MO (m/z 288), Ml (m/z M0A (m/z 428), MOB

II, M5 (m/z 236), M7 272), M4 (m/z 670), (m/z 282), M0C (m/z (m/z 654), Ml 5 (m/z M5A (mz 656), M8 428), MIA (m/z 268), 491), M18 (m/z 478) (m/z 326), M9A (m/z M2 (m/z 342), M3

798), Mil (m/z 612), (m/z 252), M3A (m/z M17(m/z494) 428), M3B (m/z 268),

M6 (m/z 670), M6A (m/z 642), M6B (m/z 656), M9 (m/z 669), M10 (m/z 667), Ml OA (m/z 782), Ml 0B (m/z 640), Ml 3 (m/z 467), M14(m/z491),M16 (m/z 491)

Compound of Formula II and Metabo ites (Urine)

Species

Major Minor Trace 30

compound of Formula II in urine.

Minor: Components between 1 and 10% of the compound of Formula II in plasma or between 1000 and 100% of the compound of Formula II in urine.

Trace: Components <1% of the compound of Formula II in plasma or <100% of the compound of Formula II in urine.

Note: It is arbitrarily set that any urinary metabolite between 1- 10X the abundance of the compound of Formula II was minor, >10X of the compound of Formula II was major, <1X of the compound of Formula II was trace. Since no radiolabel study was done with the compound of Formula II, the numbers in the table are semi-quantitative.

Because of their adenosine A_2a receptor antagonist activity, the compounds of the present invention is useful in the treatment of central nervous system diseases such as Parkinson's Disease, Extra-Pyramidal Syndrome, restless legs syndrome, essential tremor, Huntington's Disease, attention deficit hyperactivity disorder, cognitive impairment, negative symptoms of schizophrenia, depression, stroke or psychoses. In particular, the compound of the present invention can improve motor-impairment due to neurodegenerative diseases such as Parkinson's disease.

The pharmacological activity of the compounds of the invention can be determined by the following in vitro and in vivo assays to measure A_2a receptor activity.

Human Adenosine A? and Αχ Receptor Competition Binding Assay Protocol

Membrane sources:

A_2a: Human A_2a Adenosine Receptor membranes, Catalog #RBHA2AM, Perkin Elmer Life Sciences, Shelton CT. Dilute to 17 μg/100 μΐ in membrane dilution buffer (see below).

Assay Buffers: 23034

31

Membrane dilution buffer: Dulbecco's Phosphate Buffered Saline (Gibco/BRL) + 10 mM MgCl₂.

Compound Dilution Buffer: Dulbecco's Phosphate Buffered Saline (Gibco/BRL) + 10 mM MgCl₂ supplemented with 1.6 mg/ml methyl cellulose and 16% DMSO.

Prepared fresh daily.

Ligands:

A_2a: [3H]-SCH 58261, custom synthesis, AmershamPharmacia Biotech, Piscataway, NJ. Stock is prepared at 1 nM in membrane dilution buffer. Final assay concentration is 0.5 nM.

A_\: [3H]- DPCPX, AmershamPharmacia Biotech, Piscataway, NJ. Stock is prepared at 2 nM in membrane dilution buffer. Final assay concentration is 1 nM. Non-specific Binding:

A_2a: To determine non-specific binding, add 100 nM CGS 15923 (RBI, Natick, MA). Working stock is prepared at 400 nM in compound dilution buffer.

A_\: To determine non-specific binding, add 100 μΜ NECA (RBI, Natick, MA). Working stock is prepared at 400 μΜ in compound dilution buffer.

Compound Dilution:

Prepare 1 mM stock solutions of compounds in 100% DMSO. Dilute in compound dilution buffer. Test at 10 concentrations ranging from 3 μΜ to 30 pM. Prepare working solutions at 4X final concentration in compound dilution buffer. Assay procedure:

Perform assays in deep well 96 well plates. Total assay volume is 200 μΐ. Add 50 μΐ compound dilution buffer (total ligand binding) or 50 μΐ CGS 15923 working solution (A_2a non-specific binding) or 50 μΐ NECA working solution (A! non-specific binding) or 50 μΐ of drug working solution. Add 50 μΐ ligand stock ([3HJ-SCH 58261 for A_2a, [3H]- DPCPX for A^. Add 100 μΐ of diluted membranes containing the appropriate receptor. Mix. Incubate at room temperature for 90 minutes. Harvest using a Brandel cell harvester onto Packard GF/B filter plates. Add 45 μΐ Microscint 20 (Packard), and count using the Packard TopCount Microscintillation Counter.

Determine IC₅o values by fitting the displacement curves using an iterative curve fitting program (Excel). Determine IQ values using the Cheng-Prusoff equation. Haloperidol-induced catalepsy in the rat

Male Sprague-Dawley rats (Charles River, Calco, Italy) weighing 175-200 g are used. The cataleptic state is induced by the subcutaneous administration of the dopamine receptor antagonist haloperidol (1 mg/kg, sc), 90 min before testing the animals on the vertical grid test. For this test, the rats are placed on the wire mesh cover of a 25x43 plexiglass cage placed at an angle of about 70 degrees with the bench table. The rat is placed on the grid with all four legs abducted and extended ("frog posture"). The use of such an unnatural posture is essential for the specificity of this test for catalepsy. The time span from placement of the paws until the first complete removal of one paw {descent latency) is measured maximally for 120 sec.

The selective A₂A adenosine antagonists under evaluation are administered orally at doses ranging between 0.03 and 3 mg/kg, 1 and 4 h before scoring the animals.

In separate experiments, the anticataleptic effects of the reference compound, L- DOPA (25, 50 and 100 mg/kg, ip), were determined.

6-OHDA Lesion of the Middle Forebrain Bundle in Rats

Adult male Sprague-Dowley rats (Charles River, Calco, Como, Italy), weighing 275-300 g, are used in all experiments. The rats are housed in groups of 4 per cage, with free access to food and water, under controlled temperature and 12 hour light/ dark cycle. The day before the surgery the rats are fasted over night with water ad libitum.

Unilateral 6-hydroxy dopamine (6-OHDA) lesion of the middle forebrain bundle is performed according to the method described by Ungerstedt et al. (Brain Research, 1971, 6-OHDA and Cathecolamine Neurons, North Holland, Amsterdam, 101-127), with minor changes. Briefly, the animals are anaesthetized with chloral hydrate (400 mg/kg, ip) and treated with desipramine (10 mpk, ip) 30 min prior to 6-OHDA injection in order to block the uptake of the toxin by the noradrenergic terminals. Then, the animals are placed in a stereotaxic frame. The skin over the skull is reflected and the stereotaxic coordinates (-2.2 posterior from bregma (AP), +1.5 lateral from bregma (ML), 7.8 ventral from dura (DV) are taken, according to the atlas of Pellegrino et al (Pellegrino L.J., Pellegrino A.S. and Cushman A.J.. A Stereotaxic Atlas of the Rat Braia 1979, New York: Plenum Press). A burr hole is then placed in the skull over the lesion site and a needle, attached to a Hamilton syringe, is lowered into the left MFB. Then 8 μg 6-OHDA-HCI is dissolved in 4 μΐ of saline with 0.05% ascorbic acid as antioxidant, and infused at the constant flow rate of 1 μΐ /l min using an infusion pump. The needle is withdrawn after additional 5 min and the surgical wound is closed and the animals left to recover for 2 weeks.

Two weeks after the lesion the rats are administered with L-DOPA (50 mg/kg, ip) plus Benserazide (25 mg kg, ip) and selected on the basis of the number of full contralateral turns quantified in the 2 h testing period by automated rotameters (priming test). Any rat not showing at least 200 complete turns /2h is not included in the study.

Selected rats receive the test drug 3 days after the priming test (maximal dopamine receptor supersensitivity). The A₂A receptor antagonists of the invention are administered orally at dose levels ranging between 0.1 and 3 mg/kg at different time points (i.e., 1, 6, 12 h) before the injection of a subthreshold dose of L-DOPA (4 mpk, ip) plus benserazide (4 mpk, ip) and the evaluation of turning behavior.

EPS Assay

The following procedure describes the use of an adenosine A_2a antagonist to attenuate the Extra-Pyramidal Syndrome (EPS) displayed in cebus apella monkeys sensitized to the dopamine D₂ receptor antagonist, haloperidol.

A colony of Cebus apella monkeys previously sensitized to the chronic effects of haloperidol exhibits EPS when administered haloperidol acutely (0.3 mg/kg, p.o.). A test compound is administered orally (p.o.) at a dose ranging from 0.3-30 mg/kg, in conjunction with haloperidol. The studies are conducted using a within-subjects design such that each monkey receives all treatments (vehicle and doses of test compound) in a crossover, balanced design. The reduction in the maximum EPS score, as well as the dose-dependent delay in the onset of EPS are determined.

Clinical guidelines for the treatment of RLS and PLMS have been established: see A. L. Chesson et al, Sleep, 22, 7 (1999), p. 961-8. Efficacy of adenosine A_2a antagonists in treating RLS and PLMS can be determined by a method analogous to the clinical method described in the literature for pramipexole and ropinerole by

Weimerskirch et al, Annals of Pharmacotherapy, 35, 5 (2001), p. 627-30.

Using the above test procedures, the following results were obtained for the compound of the invention. 34

Results of the binding assay on the compounds of the invention showed an A_2a K, value as follows:

23034

35

Selectivity is determined by dividing ¾ for Ai receptor by ¾ for A_2a receptor.

In the 6-OHDA lesion test, test animals are administered a combination of a compound of formula I and a sub-threshold amount of L-DOPA to demonstrate if there is a significantly higher contralateral turning.

In the haloperidol-induced catalepsy assay in rats, at a particular given time (e.g., @ 4 h) the % inhibition of catelepsy at certain concentrations (e.g., 0.3 mpk, 1 mpk, 3 mpk) of the compounds of the invention can be observed to determine their efficacy in this assay.

In the EPS assay, four haloperidol-sensitized monkeys are co-administered a compound of formula I (30 mg/kg) and haloperidol (0.3 mg/kg) in a banana. A scoring system to rate the severity of each symptom is employed over a certain period of observation (e.g., 6 hour observation period). The compounds of formula I are evaluated for determining when they completely block haloperidol-induced EPS in the subjects during the observation period or when they delay the onset and reduce the severity of EPS compared to that observed in monkeys dosed with haloperidol alone. 23034

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Ex vivo binding study to show duration of receptor occupancy:

Rats are dosed with 1 mg/kg of a compound of formula I for 4, 8, 12, and 16 hours prior to sacrifice and removal of brains. The A_2a receptor-rich striatal nucleus is dissected and homogenized in buffer solution. Striatal homogenate is incubated with the A_2a antagonist radioligand ³H-SCH 58261 (see WO 96/38728) prior to separation of bound and free radioactivity by filtration. Bound radioligand on filters is dried, soaked with scintillation fluid, and counted. Homogenates from striata of vehicle-treated rats treated with the same experimental conditions define the quantity of bound radioligand in the absence of test compound. The time it takes for the receptors to be occupied by the test compound is determined by the decrease in ³H-SCH 58261 binding. The time period required to demonstrate exhibition of sustained displacement of radioligand (corresponding to a certain % displacement of radiolabel) is noted.

For preparing pharmaceutical compositions from the compounds of this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 70 percent active ingredient. Suitable solid carriers are known in the art, e.g. magnesium carbonate, magnesium stearate, talc, sugar, lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.

Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection.

Liquid form preparations may also include solutions for intranasal

administration.

Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas. 23034

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Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral

administration. Such liquid forms include solutions, suspensions and emulsions.

The compound of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

Preferably the compounds are administered orally.

Preferably, the pharmaceutical preparation is in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.

The quantity of an active compound of formula I in a unit dose of preparation may be varied or adjusted from about 0.1 mg to 1000 mg, more preferably from about 1 mg to 300 mg, according to the particular application.

The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

The amount and frequency of administration of the compound of the invention and the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated. A typical

recommended dosage regimen for a compound of formula I is oral administration of from 10 mg to 2000 mg/day preferably 10 to 1000 mg/day, in two to four divided doses to provide relief from central nervous system diseases such as Parkinson's disease or the other disease or conditions listed above.

The doses and dosage regimen of the other agents used in combination with the compound of formula I, i.e., the Parkinson's Disease agents, the antipsychotics, 23034

38 tricyclcic antidepressants, anticonvulsants, dopamine agonists, benzodiazepines, opioids, lithium or iron, will be determined by the attending clinician in view of the approved doses and dosage regimen in the package insert, taking into consideration the age, sex and condition of the patient and the severity of the disease. When

administered in combination, the compound of formula I and the other agent can be administered simultaneously or sequentially. This is particularly useful when the components of the combination are preferably given on different dosing schedules, e.g., one component is administered daily and the other every six hours, or when the preferred pharmaceutical compositions are different, e.g. one is preferably a tablet and one is a capsule. It is therefore advantageous to provide the compound of formula I and the other agent in a kit comprising, in separate containers in a single package, pharmaceutical compositions for use in combination to treat or prevent Parkinson's disease, EPS, dystonia, RLS or PLMS, wherein one container comprises a

pharmaceutical composition comprising an effective amount of a compound of formula I in a pharmaceutically acceptable carrier, and wherein a separate container comprises a pharmaceutical composition comprising an effective amount of another agent appropriate to treat the indicated condition.

Those skilled in the art will recognize that a dosage form for one of the components of the combination can be modified to contain both the compound of formula I and another agent, e.g., the compound of formula I and an antipsychotic or the compound of formula I and a dopamine agonist.

While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives,

modifications and variations are intended to fall within the spirit and scope of the present invention.

Claims

23034

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WHAT IS CLAIMED IS:

1. A compound of Fo:

Formula I

or a pharmaceutically acceptable salt or solvate thereof in isolated and purified form, wherein:

R is selected from the group consisting of H, and OH;

R¹ is OH or

R² is -C(=0)CH₃; or

R and R taken together with the carbon atoms to which they are shown attached form

The compound of claim 1, wherein R is OH, R is -C(=0)CH₃, or taken together with the carbon atoms to which they are shown attached form

3. The compound of claim 1, wherein R is H, R 1 is OH, and R 2 i ·s 23034

40

-C(=0)C¾.

4. The compound of claim 1 , wherein R is H, R² is

-C(=0)CH₃, and R¹ is

5. The compound of claim 1 , wherein R is OH and R¹ and R² taken together with the carbon atoms to which they are shown attached form

6. A pharmaceutical composition comprising at least one compound of claim 1 or a pharmaceutically acceptable salt thereof and at least one carrier.

7. The pharmaceutical composition of claim 6, further comprising 1 to 3 other agents useful in treating Parkinson's disease in a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the other agents are selected from the group consisting of L-DOPA, dopaminergic agonists, MAO-B inhibitors, DOPA decarboxylase inhibitors and COMT inhibitors.

9. A method of treating central nervous system diseases or stroke, comprising administering an effective amount of a compound of claim 1 to a patient in need of such treatment.

10. The method of claim 9 for treating depression, a cognitive disease or a neurodegenerative disease.

11. The method of claim 10 for treating for treating Parkinson's disease, senile dementia or psychoses of organic origin.

12. A method of treating Extra-Pyramidal Syndrome (EPS) caused by treatment with an antipsychotic agent, comprising administering to a patient in need thereof, a therapeutically effect amount of at least one compound of claim 1. 23034

41

13. The method of claim 12, wherein the antipsychotic agent is a typical antipsychotic agent or an atypical antipsychotic agent.

14. The method of claim 13, wherein the typical antipsychotic agent is selected from the group consisting of loxapine, haloperidol, chlorpromazine, prochlorperazine and thiothixene, and the atypical antipsychotic agent is selected from the group consisting of clozapine, olanzapine, loxapine, quetiapine, ziprasidone and risperidone

15. The method of claim 12, further comprising adrninistering an antipsychotic agent.

16. The method of claim 14, wherein the antipsychotic agent is a typical antipsychotic agent selected from the group consisting of loxapine, haloperidol, chlorpromazine, prochlorperazine and thiothixene, or an atypical antipsychotic agent selected from the group consisting of clozapine, olanzapine, loxapine, quetiapine, ziprasidone and risperidone.

17. A method of treating a disease selected from the group consisting of idiopathic dystonia, dystonia associated with the use of cocaine, tricyclic antidepressants, lithium or anticonvulsants, restless leg syndrome (RLS), and periodic limb movement disorder/syndrome (PLMD/PLMS) comprising administering a therapeutically effect amount of the compound of claim 1.

18. A method of determining if a subject has been administered the compound of formula II

Formula II

or a pharmaceutically acceptable salt or solvate thereof, comprising the step of determining if a plasma, urine, bile or fecal sample obtained from the subject shows the presence of at least one compound of Formula I 23034

42

Formula I

or a pharmaceutically acceptable salt or solvate thereof, wherein:

R is selected from the group consisting of H, and OH;

R¹ is OH or

R is -C(=0)CH₃; or

R¹ and R² taken together with the carbon atoms to which they are shown attached form

19. The method of claim 16, wherein R is H, R¹ is OH, and R² is

-C(=0)CH₃.

20. The method of claim 16, wherein R is OH and R and R taken together with the carbon atoms to which they are shown attached form