WO2012135084A1 - METABOLITES OF 2-(FURAN-2-YL)-7-(2-(4-(4-(2-METHOXYETHOXY)PHENYL)PIPERAZIN-1-YL)ETHYL)-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) or a pharmaceutically acceptable salt thereof in isolated and purified form. The compound is an 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 2-rFURAN-2-YL)-7-f2-(4-(4-(2- METHOXYETHOXY)PHENYL)PIPERAZIN-l-YL)ETHYLV7H- PYRAZOLOi4.3-elil.2.41TRIAZOLO[1.5-c1PYRIMIDIN-5-AMINE AND THEIR

UTILITY AS ADENOSINE A¾, RECEPTOR ANTAGONISTS

FIELD OF THE INVENTION:

The present invention relates to metabolites of 2-(furan-2-yl)-7-(2-(4-(4-(2- methoxyethoxy)phenyl)piperazin-l-yl)ethyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5- c]pyrimidin-5-amine referred to herein below as the compound of formula II

Formula II

(also known as Preladenant, a Merck compound, now in Phase III clinical studies for the treatment of Parkinson's disease), and the use of said compounds as adenosine A_2a receptor antagonists. These antagonists are useful 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: A_1? 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_{l s} A_2a, A¾ 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 Ai 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. Ai 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 restless 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,

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

The compound 2-(furan-2-yl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- l-yl)ethyl)-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 01/92264 and WO 2005/044245. 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 2007/038284, 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

Formula I

or a pharmaceutically acceptable salt thereof in isolated and purified form.

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- 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-methyitransferase (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 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 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 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 method of determining if a subject has been administered the compound of 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 selected from the group of compounds of Formula I, III, IV, V, and VI or a pharmaceutically acceptable salt or solvate thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the biotransformation of Preladenant in human, dog and rat following a single oral dose of ^I4C-Preladenant.

FIG. 2 shows a comparison of representative radiochromatographic profiles of pooled plasma extract following a single oral administration of ¹⁴C-Preladenant to healthy male volunteers, dogs, and rats.

FIG. 3 shows a comparison of representative radiochromatographic profiles of pooled urine following a single oral administration of ¹⁴C-Preladenant to healthy male volunteers, dogs, and rats. FIG. 4 shows a comparison of representative radiochromatographic profiles of pooled fecal extract following a single oral administration of ¹⁴C-Preladenant to healthy male volunteers, dogs, and rats.

DETAILED DESCRIPTION OF THE INVENTION

The compounds disclosed herein are metabolites of the compound of Formula II as set forth above.

In one embodiment, the metabolite is a compound having the structural formula

I

Formula I (Metabolite M9)

or a pharmaceutically acceptable salt thereof in isolated and purified form.

In another embodiment, the metabolite is a compound having the formula III:

Formula III (Metabolite M12)

or a pharmaceutically acceptable salt thereof in isolated and purified form. The structure of the compound of formula III has previous been disclosed (see compound lib in Table 1, page 1378 of B.R. Neustadt et al., Biorog. Med. Chem. Lett. 17 (2007) 1376-1380).

In another embodiment, the metabolite is a compound having the formula IV:

Formula IV (Metabolite M13) or a pharmaceutically acceptable salt thereof in isolated and purified form. The structure of the compound of formula IV has previous been disclosed (see compound 12c in Table 1, page 1379 of B.R. Neustadt et al., Biorog. Med. Chem. Lett. 17 (2007) 1376-1380).

In another embodiment, the metabolite is a compound having the formula V:

Formula V (Metabolite M7)

or a pharmaceutically acceptable salt thereof in isolated and purified form. The Li salts of the two enantiomeric forms of this compound are also disclosed:

Lithium (S)-4-(5-amino-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 -yl)ethyl)-7H- pyrazolo[4,3 -15S)

A-15S

and Lithium (R)-4-(5-amino-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 - yl)emyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-2-yl)-4-hydroxybutanoate

(A-15R)

A-15R

In another embodiment, the metabolite is a compound of formula VI:

Formula VI (A-9; Metabolite M2)

or a pharmaceutically acceptable salt thereof in isolated and purified form. The enantiomers of this compound are also disclosed herein:

R-4-(5-amino-7-(2-(4-(4-(carboxymethoxy)phenyl)piperazin-l-yl)ethyl)-7H- pyrazolo[4,3-e][l ,2,4]triazolo[l,5-c]pyrimidin-2-yl)-4-hydroxybutanoic acid (A-9R):

and S-4-(5-amino-7-(2-(4-(4-(carboxymethoxy)phenyl)piperazin- 1 -yl)ethyl)-7H- pyrazolo[4,3- -c]pyrimidin-2-yl)-4-hydroxybutanoic acid (A-9S):

In another embodiment, the metabolite is a compound of Formula VI:

Formula VII (Metabolite M5a)

or a pharmaceutically acceptable salt thereof in isolated and purified form.

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 the other metabolites (Compounds of Formula III, IV, V, VI and VII) 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.

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., 93(3), 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, MPS PharmSciTech., 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.

"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)", 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 J. 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.

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 dicyclohexylammes, 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 C_1-4alkoxy 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_6-24)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.

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 trifluoroacetic 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-l,13,3-tetramethyl uranium hexafluorophosphate, NaH is 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 RLS and/or PLMD/PLMS include phenytoin, carbamazepine and gabapentin.

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

Opioids useful in treating RLS and/or PLMD/PLMS include codeine, hydrocodone, oxycodone, propoxyphene and tramadol.

Benzodiazepines useful in treating RLS and/or PLMD/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).

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

Intermediate A-2

4-chloro- 1 H-pyrazo -d]pyrimidin-6-amine (A-2)

Reference : Hevitica Chimica Acta 69 1602-1613 (1986)

A mixture of 2-amino-4,6-dichloropyrimidine-5-carbaldehyde (A-1) (9.6 g, 50 mmol), THF (150 mL), and water (50 mL) at 50 °C was treated with a solution of hydrazine hydrate (5.1 mL, 100 mmol) in water (50 mL) at room temperature while stirring under nitrogen. Ten mins after addition, the reaction mixture was poured into ice-cold water (250 mL). The THF was evaporated and the remaining aqueous suspension was filtered to give 4-chloro-lH-pyrazolo[3,4-d]pyrimidin-6-amine (A-2) (7.86 g, 93% yield). A small portion was recrystallized from DMF/ water. 1H NMR (500 MHz, DMSO) δ ppm 7.15 (s, 2H), 7.95 (s, 1H), 13.25 (s, 1H). MS (M+l): 170, 172 (1 CI).

Intermediate A-3

4-chloro- 1 -(2-chloroeth l)- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-amine (A-3)

A-2 A-3

Sodium hydride (60% dispersion in mineral oil) (354 mg, 8.85 mmol) was added portionwise to a yellow suspension of 4-chloro- 1 H-pyrazolo [3 ,4-d]pyrimidin-6- amine (A-2) (1.25 g, 7.34 mmol) in dry DMF (5 mL). l-Bromo-2-chloroethane (3.68 mL, 44.2 mmol) was added dropwise. The resulting mixture was stirred for 2 h at room temperature. The DMF was evaporated. The residue was purified by chromatography on silica gel (eluant: 0-10% methanol/ CH₂C1₂ gradient). The product was washed with hexanes and filtered to give 4-chloro-l-(2-chloroethyl)-lH-pyrazolo[3,4-d]pyrimidin-6- amine (A-3) (409 mg, 24% yield). Ή NMR (500 MHz, CDC1₃) δ ppm 3.96 (t, 2H, J=6.5 Hz), 4.59 (t, 2H, J=6.5 Hz), 5.34 (s, 2H), 7.93 (s, 1H). MS (M+l): 232, 233, 234 (2 CI).

Intermediate A-4

tert-butyl 2-(6-arnino- 1 -(2-chloroethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-4- yl)hydrazinecarbox late (A-4)

A-3 A-4

4-Chloro-l-(2-chloroethyl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine (A-3) (360 mg, 1.55 mmol), and tert-butyl carbazate (205 mg, 1.55 mmol) in DMF (4 mL) was heated at 80 °C for 24 h then allowed to stand at room temperature for 24 h. The DMF was evaporated, and the residue (862 mg) was purified by chromatography on silica gel (eluant: 0-10% methanol/ CH₂C1₂) to give tert-butyl 2-(6-amino-l -(2-chloroethyl)- 1H- pyrazolo[3,4-d]pyrimidin-4-yl)hydrazinecarboxylate (A-4) (185 mg, 37% yield). 1H NMR (500 MHz, CDC1₃) δ ppm 3.33 (broad s, 2H), 3.89 - 3.96 (m, 2H), 4.55 (t, 2H, J=6.5 Hz), 4.78 (broad s, 1H), 4.95 (broad s, 1H), 7.81 and 7.84 (2s, 1H). MS (M+l): 328, 330 (1 CI).

Intermediate B-3

Ethyl 2-(4-(piperazin-l-yl)phenoxy)acetate (B-3)

Step 1 : tert-butyl 4-(4-(2-ethoxy-2-oxoethoxy)phenyl)piperazine-l-carboxylate (B-2) tert-Butyl 4-(4-hydroxyphenyl)piperazine-l-carboxylate (B-1) (11.13 g, 0.040 mol), ethyl bromoacetate (20 g, 0.120 mol) and cesium carbonate (13 g, 0.040 mol) were stirred together in DMF (160 mL) under nitrogen. The reaction mixture was heated at 80 °C for 20 h then cooled to RT and poured into water (1 L). The aqueous mixture was extracted twice with ethyl acetate. The combined organic extract was washed once with IN NaOH and twice with brine, dried (Na₂S0₄), filtered, and concentrated. Trituration in 1:1 ether:hexanes provided 8.55 g of product. The mother liquor was concentrated to a residue which was purified by chromatography on silica gel (eluant: 30% EtOAc- hexanes) to give 3.59 g of product. The two batches were combined to give tert-butyl 4- (4-(2-ethoxy-2-oxoethoxy)phenyl)piperazine-l-carboxylate (B-2) (12.14 g, 83% yield). 1H NMR (500 MHz, CDC1₃) δ ppm 1.32 (t, 3H, J=7.0 Hz), 1.51 (s, 9H), 3.04 (broad t, 4H, J= 5.0 Hz), 3.59 (broad t, 4H, J= 5.0 Hz), 4.29 (q, 2H, J=7.0 Hz), 4.59 (s, 2H), 6.88 (d, 2H, J=9.0 Hz), 6.91 (d, 2H, J=9.0 Hz). MS (M+l): 365.

Step 2: ethyl 2-(4-(piperazin-l-yl)phenoxy)acetate (B-3)

To compound B-2 (926 mg, 2.54 mmol) dissolved in CH₂CI₂ (15 mL) was added 4N HC1 in dioxane (50 mL). The resulting reaction mixture was stirred at RT for 4 h then concentrated to give the crude product as the dihydrochloride salt (853 mg, 100% yield) white solid. MS (M+l): 265.

Intermediate A-5

7¾rt-butyl 2-(6-amino- 1 -(2-(4-(4-(2-ethoxy-2-oxoethoxy)phenyl)piperazin- 1 - yl)ethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-4-yl)hydrazinecarboxylate (A-5)

tert-Butyl 2-(6-amino- 1 -(2-chloroethyl)- 1 H-pyrazolo[3 ,4-d]pyrimidin-4- yl)hydrazinecarboxylate (A-4) (800 mg, 2.44 mmol), ethyl 2-(4-(piperazin-l- yl)phenoxy)acetate dihydrochloride (B-3) (2.46 g, 7.31 mmol), anhydrous potassium iodide (813 mg, 4.87 mmol) and diisopropylethylai ine (2.9 mL, 17.55 mmol) were stirred together in DMF (15 mL) under nitrogen in a sealed tube. The reaction mixture was heated at 100 °C for 24 h then stirred at RT for 4 days. The reaction mixture was poured into half saturated brine (200 mL). The aqueous mixture was extracted twice with ethyl acetate. The combined organic extract was washed twice with brine, dried (Na₂S0₄), filtered, and concentrated. The crude product was purified by

chromatography silica gel (eluant: 2 - 3.5% gradient of 7N ammonia/methanol in dichloromethane) to give the product (A-5) (500 mg, 37% yield). 1H NMR (500 MHz, CDC1₃) δ ppm 1.32 (t, 3H, J=7.0 Hz), 1.49 (s, 9H), 2.72 (broad t, 4H, J= 4.8 Hz), 2.92 (t, 2H, J= 7.0 Hz), 3.10 (broad t, 4H, J= 4.8 Hz), 4.29 (q, 2H, J=7.0 Hz), 4.40 (t, 2H, J=7.0 Hz), 4.58 (s, 2H), 5.16 (broad s, 2H), 6.83 - 6.90 (m, 4H), 7.29 (s, 1H), 7.82 (s, 1H). MS (M+l): 556.

Intermediate A-6

Ethyl 2-(4-(4-(2-(6-amino-4-hydrazinyl- 1 H-pyrazolo[3 ,4-d]pyrimidin- 1 - yl)ethyl)piperazin- 1 -yl)phenoxy)acetate (A-6)

To compound A-5 (100 mg, 0.18 mmol) dissolved in CH₂CI₂ (1 mL) was added ethanol (1 mL) and 4N HC1 in dioxane (1 mL). The resulting reaction mixture was stirred at RT for 3 h then concentrated to give the crude product as the trihydrochloride salt (102 mg, 100% yield). ). 1H NMR (500 MHz, DMSO) δ ppm 1.21 (t, 3H, J=7.0 Hz), 3.00-3.40 (broad s, 4H),3.41-3.54 (m, 2H),

3.63 (broad t, 2H, J= 6.5 Hz), 3.66- 3.75 (m, 2H), 4.16 (q, 2H, J=7.0 Hz), 4.64 (broad t, 2H, J=6.5 Hz), 4.70 (s, 2H), 6.86 (d, 2H, J- 9.0 Hz), 6.96 (d, 2H, J= 9.0 Hz), 8.30 (s, 2H), 11.91 (s, 2H), 11.40 (s, 2H). MS (M+l): 456.

Intermediate A-7

Ethyl 2-(4-(4-(2-(6-ammo-4-(2-(5-oxotetxahydrofuran-2-carbonyl)hydrazinyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 -yl)ethyl)piperazin- 1 -yl)phenoxy)acetate (A-7)

Step 1 : 5-Oxo-2-tetrahydrofuran carboxylic acid (1.77 g, 13.6 mmol) was dissolved in CH₂C1₂ (8 mL). DMF (30 uL) was added, and the solution cooled in an ice bath.

Oxalyl chloride (2.97 mL, 34 mmol) was added dropwise over 20 min. The ice bath was then removed, and the reaction mixture was stirred for 2 h. The solvent was removed, and the residue crude 5-oxo-2-tetrahydrofuran carbonyl chloride was used in step 2. Step 2: A 0.5 mL solution of CH₂C1₂ containing 5-oxo-2-tetrahydrofuran carbonyl chloride (32.8 mg, 0.22 mmol) from step 1 was added dropwise to a mixture of A-6 trihydrochloride (102 mg, 0.18 mmol), and diisopropylethylamine (119 uL, 0.72 mmol) in CH₂C1₂ (1.5 mL) cooled in an ice bath. The bath was removed, and the mixture was stirred at room temperature for 40 min. The solvent was evaporated, and the residue partitioned between ethyl acetate and half saturated brine. The organic extract was dried (Na₂S0₄), filtered, and concentrated to give 53 mg solid. The solid was dissolved in DMF (2 mL) and purified on a C₁₈ reverse phase column (eluant: acetonitrile / water gradient) to give the product (A-7) as a solid (27 mg, 27% yield). 1H NMR (500 MHz, CDC1₃) δ ppm 1.30 (t, 3H, J=7.3 Hz), 2.30-3.20 (m, 15H), 4.26 (q, 2H, J=7.2 Hz), 4.33 (broad s, 2H), 4.56 (s, 2H), 5.05 (broad s, 1H), 5.68 (broad s, 2H), 6.82 (broad s, 4H), 7.70 (broad s, 1H). MS (M+l): 568.

Intermediate A-8

Ethyl 2-(4-(4-(2-(5-amino-2-(5-oxotetrahydrofuran-2-yl)-7H-pyrazolo[4,3- -c]pyrimidin-7-yl)ethyl)piperazin- 1 -yl)phenoxy)acetate (A-8)

Compound A-7 (22 mg, 0.039 mmol) was suspended in N,0-bis(trimethylsilyl) acetamide (1 mL) under a nitrogen atmosphere in a sealed tube. The reaction mixture was heated at 100 °C for 10.5 h then stirred at RT for 12 h. The resulting reaction mixture was cooled in an ice bath and quenched with excess methanol. The methanol was evaporated, and the residue partitioned between CH₂C1₂ and half saturated brine. The aqueous solution was extracted three more times with CH₂C1₂. The combined organic extract was dried (Na₂S0₄), filtered, and concentrated. The solid was dissolved in DMF (1 mL) and purified on a C₁₈ reverse phase column (eluant: acetonitrile / water gradient) to give the product (A-8) as a solid (6.5 mg, 30% yield). 1H NMR (500 MHz, CDC1₃) δ ppm 1.31 (t, 3H, J-7.3 Hz), 2.64-2.73 (m, 3H), 2.75 (broad t, 4H, J= 4.5 Hz), 2.84-2.94 (m, lH), 3.00 (t, 2H, J=7.0 Hz), 3.11 (broad t, 4H, J= 4.5 Hz), 4.28 (q, 2H, J=7.0 Hz), 4.55 (t, 2H, J=7.0 Hz), 4.58 (s, 2H), 5.70- 5.77 (m, 1H), 6.26 (broad s, 2H), 6.84- 6.94 (m, 4H), 8.17 (s, 1H). MS (M+l): 550.

Compound A-9

4-(5-amino-7-(2-(4-(4-(carboxymethoxy)phenyl)piperazin-l-yl)ethyl)-7H- pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-2-yl)-4-hydroxybutanoic acid (A-9)

Compound A-8 (31.4 mg, 0.057 mmol) was stirred with lithium hydroxide monohydrate (5.7 mg, 0.136 mmol), THF (500 uL), methanol (250 uL), and water (250 uL) and heated at 40 °C for 1 h. The solvent was evaporated to give A-9 (31.6 mg, 100% yield) as the dilithium salt. 1H NMR (500 MHz, CD₃OD) δ ppm 2.22-2.35 (m, 2H), 2.38 (t, 2H, J= 7.0 Hz), 2.75 (broad t, 4H, J= 4.5 Hz), 2.96 (t, 2H, J=6.8 Hz), 3.03 (broad t, 4H, J= 4.5 Hz), 3.36 (s, 1H), 4.32 (s, 2H), 4.54 (t, 2H, J=6.8 Hz), 4.97 (t, 1H, J=6.5 Hz), 6.86 (d, 2H, J= 9.0 Hz), 6.91 (d, 2H, J= 9.0 Hz), 8.10 (s, 1H). MS (M+l): 540.

Intermediate A-7R

R-ethyl 2-(4-(4-(2-(6-amino-4-(2-(5-oxotetrahydrofuran-2- carbonyl)hydrazinyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 -yl)ethyl)piperazin- 1 - yl)phenoxy)acetate (A-7R)

Step 1: Compound A-6 trihydrochloride (100 mg, 0.177 mmol), and sodium carbonate (56.3 mg, 0.531 mmol) were stirred and sonicated together in ethanol (3 mL) then filtered. The solid was suspended a second time in ethanol (2 mL) then filtered. The solid was suspended a third time in methanol (2 mL) then filtered. The combined three filtrates were evaporated to give compound A-6 as the free base (78 mg, 0.171 mmol, 97% yield).

Step 2: Compound A-6 free base from step 1 (302 mg, 0.664 mmol ), R(-)-5-oxo-2- tetrahydrofuran carboxylic acid (94 mg, 0.712 mmol), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (137 mg, 0.712 mmol),

1-hydroxybenzotriazole (95 mg, 0.712 mmol), and 4-methylmorpholine (78 uL, 0.712 mmol) were stirred together in DMF (7.5 mL) under nitrogen for 17 h. The solvent was evaporated, and the residue was dissolved into ethyl acetate and quickly washed with brine. The organic extract was dried (Na₂S0₄), filtered, and concentrated. The solid was dissolved in DMF (6 mL) and purified on a Cis reverse phase column (eluant:

acetonitrile / water gradient) to give the product (A-7R) as a solid (51 mg, 14% yield). MS (M+l): 568.

Intermediate A-7S

S-ethyl 2-(4-(4-(2-(6-amino-4-(2-(5-oxotetrahydrofuran-2- carbonyl)hydrazinyl)- 1 H-pyrazolo[3 ,4-d]pyrimidin- 1 -yl)ethyl)piperazin- 1 - yl)phenoxy)acetate (A-7S)

Compound A-7S was synthesized by coupling S(+)-5-oxo-2-tetrahydrofuran carboxylic acid (91.6 mg, 0.704 mmol) with compound A-6 free base (321 mg, 0.704 mmol), in the same manner as above (see Intermediate A-7R, step 2 above) to give the product A-7S (64 mg, 16% yield). MS (M+l): 568.

Intermediate A-8R

R-ethyl 2-(4-(4-(2-(5-amino-2-(5-oxotetrahydrofuran-2-yl)-7H-pyrazolo[4,3- e] [1 ,2,4]triazolo[ 1 ,5-c]pyrimidin-7-yl)ethyl)piperazin- 1 -yl)phenoxy)acetate (A-8R)

Compound A-7R (51 mg, 0.089 mmol) was suspended inN, O- bis(trimethylsilyl) acetamide (2.2 mL) under a nitrogen atmosphere in a sealed tube. The reaction mixture was heated at 110 °C for 20 h. The Ν,Ο- bis(trimethylsilyl)acetamide was evaporated under high vacuum at 80 °C, and the residue was partitioned between CH₂C1₂ and half saturated brine. The aqueous solution was extracted four more times with CH2CI2 and once with ethyl acetate. The CH2C1₂ and ethyl acetate extracts were dried separately (Na₂S0 ), filtered, and concentrated together. The solid was dissolved in DMF (2 mL) and purified on a C₁₈ reverse phase column (eluant: acetonitrile / water gradient) to give the product (A-8R) as a solid (12.5 mg, 26% yield). MS (M+l): 550.

Intermediate A-8S

S-ethyl 2-(4-(4-(2-(5-amino-2-(5-oxotetrahyckofuran-2-yl)-7H-pyrazolo[4,3- e] [1 ,2,4]triazolo[ 1 ,5-c]pyrimidin-7-yl)ethyl)piperazin- 1 -yl)phenoxy)acetate (A-8S)

Compound A-7S (53 mg, 0.093 mmol) was suspended in Ν,Ο- bis(trimethylsilyl) acetamide (2.7 mL) under a nitrogen atmosphere in a sealed tube. The reaction mixture was heated at 110 °C for 20.5 h. The Ν,Ο- bis(trimethylsilyl)acetamide was evaporated under high vacuum at 75 °C, and the residue was partitioned between CH₂C1₂ and half saturated brine. The aqueous solution was extracted four more times with CH₂C1₂ and once with ethyl acetate. The CH₂C1₂ and ethyl acetate extracts were dried separately (Na₂S0₄), filtered, and concentrated together. The solid was dissolved in DMF (2 mL) and purified on a C₁₈ reverse phase column (eluant: acetonitrile / water gradient) to give the product (A-8S) as a solid (13 mg, 25% yield). MS (M+l): 550.

Compound A-9R

R-4-(5-amino-7-(2-(4-(4-(carboxymethoxy)phenyl)piperazin-l-yl)ethyl)-7H- pyrazolo[4,3-e][l ,2,4]triazolo[l ,5-c]pyrimidin-2-yl)-4-hydroxybutanoic acid (A-9R)

Compound A-8R (12.5 mg, 0.023 mmol) was stirred with lithium hydroxide monohydrate (2.1 mg, 0.050 mmol), THF (200 uL), methanol (100 uL), and water (100 uL) and heated at 40 °C for 1 h. The solvent was evaporated to give impure A-9R (15.2 mg) as the dilithium salt. This material was taken into hot 2-propanol and a little methanol, and filtered hot. The volume was reduced by evaporation, and the solution was cooled in an ice bath. The precipitate was removed by filtration, and the mother liquor was concentrated to give compound A-9R as the dilithium salt (5.4 mg, 43% yield) Ή NMR (500 MHz, CD₃OD) δ ppm 2.24-2.36 (m, 2H), 2.39 (t, 2H, J= 7.3 Hz), 2.76 (broad t, 4H, J- 4.5 Hz), 2.97 (t, 2H, J=6.8 Hz), 3.05 (broad t, 4H, J= 4.5 Hz), 3.37 (s, 1H), 4.33 (s, 2H), 4.55 (t, 2H, J=6.8 Hz), 4.98 (t, 1H, J=6.5 Hz), 6.87 (d, 2H, J= 9.0 Hz), 6.92 (d, 2H, J= 9.0 Hz), 8.12 (s, 1H). MS (M+l): 540.

Compound A-9S

S-4-(5-amino-7-(2-(4-(4-(carboxymethoxy)phenyl)piperazin-l-yl)ethyl)-7H-

Compound A-8S (13 mg, 0.024 mmol) was stirred with lithium hydroxide monohydrate (2.2 mg, 0.052 mmol), THF (200 uL), methanol (100 uL), and water (100 uL) and heated at 40 °C for 1 h. The solvent was evaporated to give impure A-9S (13.3 mg) as the dilithium salt. This material was taken into hot 2-propanol and a little methanol, and filtered hot. The volume was reduced by evaporation, and the solution was cooled in an ice bath. The precipitate was removed by filtration, and the mother liquor was concentrated to give compound A-9S as the dilithium salt (5.4 mg, 41.4% yield) Ή NMR (500 MHz, CD₃OD) δ ppm 2.23-2.35 (m, 2H), 2.39 (t, 2H, J= 7.5 Hz), 2.76 (broad t, 4H, J= 4.5 Hz), 2.97 (t, 2H, J=6.8 Hz), 3.05 (broad t, 4H, J= 4.50 Hz), 3.37 (s, IH), 4.33 (s, 2H), 4.55 (t, 2H, J=6.8 Hz), 4.98 (t, IH, J=7.0 Hz), 6.87 (d, 2H, J= 9.0 Hz), 6.92 (d, 2H, J= 9.0 Hz), 8.12 (s, IH). MS (M+l): 540

Compound A-15S

Lithium (S)-4-(5-amino-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 - -7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-2-yl)-4- (A-15)

Step 1 : tert-butyl 2-(6-amino- 1 -(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 - yl)ethyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-4-yl)hydrazinecarboxylate (A-11)

To a solution of A-4 in DMF (14 mL) was added l-(4-(2- methoxyethoxy)phenyl)-piperazine (2.83 g, 12 mmol), KI (1.01 g, 6.1 mmol), and iPr₂NEt (1.12 mL, 6.45 mmol). The reaction mixture was heated to 100°C for 20 h then cooled to room temperature. To the reaction mixture was added a saturated NaCl solution and then extracted with EtOAc for four times. The combined organic extracts were washed with brine, dried (Na₂S0₄), filtered, and concentrated. Purification by silica gel column chromatography on an ISCO system (eluant: CH₂Cl₂:MeOH gradient) gave compound A-11 as a white solid (0.75 g, 48% yield). 1H NMR (500 MHz, CDC1₃) δ ppm 1.51 (s, 9H), 2.73 (m, 4H), 2.91 (m, 2H), 3.10 (m, 4H), 3.46 (s, 3H), 3.74 (m, 2H), 4.08 (m, 2H), 4.40 (m, 2H), 5.48 (s, 2H), 6.86 (m, 4H), 7.80 (s, 2H). MS (M+l): 528.

Step 2: 4-hydrazinyl-l -(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin-l -yl)ethyl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine (A-12)

To a solution of compound A-11 (0.51 g, 0.97 mmol) dissolved in 1 :1 MeOH: CH₂C1₂ (10 mL) was added 4N HC1 in dioxane (4.5 mL) at room temperature. After stirring for 5.5 h, the reaction mixture was concentrated, IN NaOH was added, and extracted with CH₂C1₂ (6 times). The combined organic extracts were washed with brine, dried (Na₂S0₄), filtered, and concentrated to give compound A-12 as a pale dark solid (0.26 g, 63% yield). MS (M+l): 428

Step 3: (S)-N'-(6-amino- 1 -(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 -yl)ethyl)- 1 H- pyrazolo[3,4-d]pyrimidin-4-yl)-5-oxotetrahydrofuran-2-carbohydrazide (A-13S)

To a solution of A-12 (0.10 g, 0.234 mmol) in DMF (2 mL) was added (S)-5- oxotetrahydrofuran-2-carboxylic acid (0.047 g, 0.351 mmol), HOBT (0.047 g, 0.351 mmol), EDCI (0.068 g, 0.351 mmol), and NMM (0.030 mL, 0.70 mmol). The reaction mixture was stirred at room temperature for 14 h. Saturated NaCl solution (40 mL) was added, and the aqueous solution was extracted with EtOAc six times. The combined organic extracts were washed with brine, dried (Na₂S0₄), filtered, and concentrated. Purification by a reverse phase HPLC Gilson system (eluant:

CH₃CN:H₂0 gradient) gave compound A-13S as a white solid (0.05 g, 40% yield). 1H NMR (500 MHz, DMSO-d₆) δ ppm 2.57 (m, 2H), 2.73 (m, 2H), 2.94 (m, 4H), 3.28 (s, 3H), 3.61 (m, 2H), 3.99 (m, 2H), 4.23 (m, 2H), 5.04 (s, 1H), 6.31 (s, 2H), 6.89-6.79 (m, 4H). MS (M+l): 540.

Step 4: (S)-5-(5-arnino-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 -yl)ethyl)-7H- pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidm-2-yl)dihydroto (A-14S) To a flame-dried 25 mL flask equipped with a reflux condenser under nitrogen was added compound A-13S (0.120 g, 0.222 mmol) and (Z)-trimethylsilyl N- trimethylsilylacetimidate (5.5 mL). The reaction mixture was heated to 110°C via oil bath for 20 h then cooled to room temperature. The reaction mixture was quenched with MeOH (25 mL) then concentrated. Saturated NaCl (50 mL) was added, and the aqueous solution was extracted with EtOAc. The combined organic extracts were washed with brine, dried ( a₂S0₄), filtered and concentrated to a pale white solid which was dissolved in DMF (6 mL) and purified on a reverse phase HPLC Gilson system (eluant: CH₃CN / H₂0 gradient) to give compound A-14S as an off white solid (0.050 g, 43% yield). 1H NMR (500 MHz, DMSO-de) δ ppm 2.62 (m, 4H), 2.82 (m, 1H), 2.94 (m, 4H), 3.28 (s, 3H), 3.61 (m, 2H), 3.99 (m, 2H), 4.41 (m, 2H), 6.77 (m, 1H), 6.84-6.79 (m, 4H), 8.11 (br, 2H), 8.16 (s, 1H). MS (M+l): 522.

Step 5: Lithium (S)-4-(5-amino-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 - yl)ethyl)-7H-pyrazolo [4,3 -e] [ 1 ,2,4]triazolo [1,5 -c]pyrimidin-2-yl)-4-hydroxybutanoate (A-15S)

To a stirred solution of compound A-14S (48 mg, 0.092 mmol) in MeOH (2 mL), THF (2 mL), and water (2 mL) was added lithium hydroxide (4 mg, 0.092 mmol). The reaction mixture was heated at 50 °C for 105 min then cooled to room temperature. The solution was concentrated to yield compound A-15S as an off white solid (0.049 g, 98% yield). Ή NMR (500 MHz, CD₃OD) δ ppm 2.38-2.29 (m, 4H), 2.74(m, 4H), 2.97 (m, 2H), 3.03 (m, 4H), 3.42 (s, 3H), 3.71 (m, 2H), 4.06 (m, 2H), 4.55 (m, 2H), 4.98 (m, 2H), 6.94-6.85 (m, 4H), 8.11 (s, 1H). MS (M+l): 540. S/R ratio (60:40) was determined by HPLC analysis. HPLC condition: normal phase Chiral AD column (EtOH:hexane with 0.1% DEA, 30 min).

Compound A-15R

The synthesis of final product A-15R was carried out according the same procedures used for the synthesis of A-15S.

Step 3: (R)-N'-(6-amino- 1 -(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 -yl)ethyl)- lH-pyrazolo[3,4-d]pyrimidin-4-yl)-5-oxotetrahydrofuran-2-carbohydrazide (A-13R) To a solution of A-12 (0.29 g, 0.54 mmol) in DMF (4 mL) was added (R)-5- oxotetrahydrofuran-2-carboxylic acid (0.105 g, 0.81 mmol), HOBT (0.10 g, 0.81 mmol), EDCI (0.156 g, 0.81 mmol), and iPr₂NEt (0.422 mL, 2.43 mmol). The reaction mixture was stirred at room temperature for 14 h. Saturated NaCl solution (40 mL) was added, and the aqueous solution was extracted with EtOAc six times (125 mL total). The combined organic extracts were washed with brine, dried (Na₂S0₄), filtered, and concentrated. Purification by reverse phase HPLC Gilson system (eluant: CH₃CN:H₂0 gradient) gave compound A-13R as a white solid (0.110 g, 38% yield). MS (M+l): 540.

Stej (R)-5-(5-amino-7-(2-(4-(4-(2-methoxyemoxy)phenyl)piperazm-l-yl)ethyl)-7H- pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidm^ (A-14R) To a flame-dried 25 mL flask equipped with a reflux condenser under nitrogen was added compound A-13R (0.104 g, 0.193 mmol) and (Z)-trimethylsilyl N-trimethylsilyl acetimidate (4.5 mL). The reaction mixture was heated to 110°C via oil bath for 20 h. After cooling to room temperature, MeOH (25 mL) was added and then concentrated. Aqueous saturated NaCl (50 mL) was added, and the aqueous solution was extracted with EtOAc (40 mL, 3x30 mL). The combined organic extracts were washed with brine, dried (Na₂S0₄), filtered and concentrated to give a pale white solid which was dissolved in DMF (6 mL) and purified by reverse phase HPLC Gilson system (eluant: CH₃CN/H₂0 gradient) to provide compound A-14R as an off white solid (0.028 g, 28% yield). 1H NMR (500 MHz, CDC13) δ 2.68 (m, 4H), 2.78 (m, 1H), 3.10 (m, 4H), 3.43 (s, 3H), 4.06 (m, 2H), 4.57 (m, 2H), 5.74 (m, 1H), 6.07 (broad s, 2H), 6.85 (m, 3H), 8.11 (broad s, 2H), 8.15 (s, 1H). MS (M+l): 522.

Step 11 : Lithium (R)-4-(5-amino-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin- 1 - yl)ethyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidin-2-yl)-4-hydroxybutanoate

(A-15R)

To a stirred solution of compound A-14R (24 mg, 0.046 mmol) in MeOH (1 mL), THF (1 mL) and water (1 mL) was added lithium hydroxide (2 mg, 0.051 mmol). The reaction mixture was heated at 50 °C for 2 h then cooled to room temperature. The solution was concentrated to yield compound A-15R as an off white solid (0.028 g, 98% yield). 1H NMR (500 MHz, CD₃OD) δ 2.36-2.27 (m, 4H), 2.74 (m, 4H), 2.97 (m, 2H), 3.03 (m, 3H), 3.40 (s, 3H), 3.69 (m, 2H), 4.02 (m, 2H), 4.52 (m, 2H), 4.97 (m, 1H), 6.89-6.85 (m, 4H), 8.09 (s, 1H). MS (M+l): 540. R/S ratio (60:40) was determined by HPLC analysis. HPLC condition: normal phase Chiral AD column (EtOH:hexane with 0.1% DEA, 30 min).

Compound A-18

Step 1 : ethyl 2-(5-amino-2-(furan-2-yl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5- c]pyrimidin-7-yl)acetate (A-17)

Compound A-16 (0.800 g, 3.32 mmol), prepared according to US

2005/0239795, was combined with ethyl bromoacetate (0.52 mL, 4.6 mmol) and K₂C0₃ (0.69 g, 5.0 mmol) in DMF (8 mL). The mixture was stirred 24 h, concentrated, and purified by silica column chromatography followed by preparative layer chromatography to give intermediate A-17 as a yellow film (0.11 g, 10% yield). 1H NMR (CDC1₃) δ ppm 1.23 (t, 3H, J=7 Hz), 4.21 (q, 2H, J=7 Hz), 5.07 (s, 2H), 6.12 (broad s, 2H), 6.55 (d, IH, J=4 Hz), 7.21 (d, IH, J=4 Hz), 7.58 (d, IH, J=2 Hz), 8.10 (s, IH). MS (M+l): 328.

Step 2: 2-(5-amino-2-(furan-2-yl)-7H-pyrazolo[4,3-e] [1 ,2,4]triazolo[l ,5-c]pyrimidin- 7-yl)acetic acid (A-18)

Compound A-17 (0.099 g, 0.30 mmol) was partially dissolved in ethanol (10 mL) and 1.0N NaOH (0.60 mL, 0.60 mmol) added. The mixture was heated at 60 °C for 2 h, cooled to room temperature, and acidified with 1.0N HC1 (1.2 mL, 1.2 mmol). The mixture was concentrated and treated with water (6 mL). Filtration provided compound A-18 as a yellow solid (0.070 g, 78% yield). 1H NMR (DMSO) δ ppm 4.98 (s, 2H), 6.68 (d, IH), 7.18 (d, IH), 7.89 (d, IH), 8.10 (broad s, IH), 8.13 (s, IH). MS (M+l): 300. ompound A-25

Step 1: ethyl 2-(4-(4-acetylpiperazin-l-yl)phenoxy)acetate (A-20)

To anhydrous DMF (50 mL) was added NaH (60% in oil, 1.44 g, 36 mmol), followed by compound A-19 (6.60 g, 30.0 mmol) portionwise over 5 min. The suspension was stirred under nitrogen for 5 min, and ethyl bromoacetate (6.02 g, 36 mmol) was added dropwise over 5 min. After stirring for 16 h at room temperatuare, the solution was concentrated and partitioned between 200 mL each of EtOAc and water. The EtOAc solution was washed with 2x200 mL water, then brine, dried (MgS0₄), and

concentrated to a solid. The solid was warmed in hexane (40 mL), allowed to cool, filtered, and washed with hexane. Drying gave compound A-20 as an off-white solid (7.2 g, 78% yield). 1H NMR δ ppm 1.23 (t, 3H, J=7 Hz), 2.13 (s, 3H), 3.05 (m, 4H), 3.61 (m, 2H), 3.76 (m, 2H), 4.26 (q, 2H, J=7 Hz), 4.57 (s, 2H), 6.87 (m, 4H).

Step 2: 2-(4-(piperazin-l-yl)phenoxy)acetic acid (A-21)

Compound A-20 (7.2 g, 24 mmol) was dissolved in 6.0N HCl (50 mL) and heated at 80 °C for 3 h under nitrogen. The cooled solution was concentrated to give a solid. The solid was suspended in EtOH (75 mL) and concentrated to give compound A-21 as the dihydrochloride salt (7.7 g, >100% yield).

Step 3: ethyl 2-(4-(piperazin-l-yl)phenoxy)acetate (A-22)

Compound A-21 dihydrochloride salt (7.7 g, 24 mmol) was suspended in EtOH (225 mL) and cooled in an ice bath. SOCl₂ (25 mL, 340 mmol) was added dropwise over 10 min. The mixture was heated in a 90 °C oil bath for 16 h, then allowed to cool and filtered. The solid was washed with EtOH and dried to give compound A-22 as the dihydrochloride salt as an off-white powder (7.59 g, 94% yield). 1H NMR (DMSO) δ ppm 4.68 (s, 2H). A 4.50 g portion was dissolved in water (40 mL) and basified to pH=8 with satd. NaHC0₃. The mixture was saturated with NaCl and extracted with CH₂C1₂ (4x40 mL). The organic extracts were dried (MgS0 ) and concentration to give compound A-22 as the free base as a brown oil (2.81 g, 80% yield).

Step 4: ethyl 2-(4-(4-(2-(5-amino-2-(furan-2-yl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5- c]pyrimidin-7-yl)ethyl)piperazin- 1 -yl)phenoxy)acetate (A-24)

Compound A-22 free base (2.81 g, 10.6 mmol) was combined with compound A-23 (prepared according to US 2005/0239795, 2.74 g, 6.24 mmol) and KI (0.518 g, 3.12 mmol) in anhydrous DMF (40 mL). The mixture was heated at 100 °C for 22 h under nitrogen. The cooled mixture was concentrated and adsorbed onto silica gel. Column chromatography on silica gel (eluant: MeOH/ CH₂CI₂ gradient) gave compound A-24 as an off-white solid (1.62 g, 49% yield). Ή NMR (CDC1₃) δ ppm 1.28 (t, 3H, J=7 Hz), 2.74 (m, 4H), 2.97 (t, 2H, J=6 Hz), 3.09 (m, 4H), 4.23 (q, 2H, J=7 Hz), 4.52 (t, 2H, J=6 Hz), 4.56 (s, 2H), 6.21 (broad s, 2H), 6.58 (t, 1H), 6.83 (m, 4H), 7.22 (d, 1H, J=4 Hz), 7.58 (d, 1H, J=2 Hz), 8.20 (s, 1H).

Step 5: 2-(4-(4-(2-(5-amino-2-(furan-2-yl)-7H-pyrazolo[4,3-e][ 1 ,2,4]triazolo[l ,5- c]pyrimidin-7-yl)ethyl)piperazin-l-yl)phenoxy)acetic acid (A-25)

Compound A-24 (1.62 g, 3.05 mmol) was combined with EtOH (90 mL) and 1.0N NaOH (6.1 mL, 6.1 mmol). The yellow suspension was heated at 60 °C for 20 h under nitrogen. The cooled mixture was treated with 1.0N HC1 (6.1 mL) and concentrated. The resulting white solid was treated with water (50 mL) and filtered. The solid was boiled in EtOH (500 mL) to give a white suspension. On cooling, the solid was collected and treated with hot EtOH as above four times. The combined filtrates were concentrated to 100 mL volume. After cooling, the solid was filtered and washed with EtOH to give compound A-25 as an off-white solid (0.91 g, 59% yield). 1H NMR (DMSO): δ ppm 2.48 (m, 4H), 2.80 (t, 2H, J=6 Hz), 2.95 (m, 4H), 4.39 (t, 2H, J=6 Hz), 4.52, (s, 2H), 6.71 (d, 1H, 3=4 Hz), 6.75 (d, 2H, J=8 Hz), 6.81 (d, 2H, J=8 Hz), 7.21 (d, 1H, J=4 Hz), 7.92 (d, 1H, J=2 Hz), 8.07 (broad s, 1H), 8.18 (s, 1H). MS (M+l): 504. Calc. for C₂₄H₂₅N₉0₄: C 57.25, H 5.00, N 25.04. Found: C 57.03, H 4.94, N 24.64. Isolation of Metabolites

Chemicals: ¹⁴C-Preladenant [7-[2-[4-[4-(2-methoxyethoxy)phenyl]-l- piperazinyl]ethyl]-2-(2-ruranyl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimi amine, i.e., ¹⁴C-Preladenant shown below]

C-Preladenant (* designates position of 14C radiolabel) shown above had >97% radiochemical purity. HPLC grade acetonitrile and methanol were from Burdick and Jackson (Muskegon, MI). Water was purified using the Milli-Q water purification system (Bedford, MA).

Test Species:

Species Age Weight or Body Oral Dose

Mass Index (BMI)

Human (M) 18-50 yr BMI = 19-29 kg/m 50 mg dose (n = 8) containing approximately 55

μϋϊ of total radioactivity

Dog (M & F) At least 5 7-13 kg (male) 10 mg/kg (25 (n = 4) months 7-12 (female) μα/kg)

(Strain: beagle)

Rat (M&F) 7-10 wk 175-250 g 10 mg/kg (25 (n=3) μα/kg)

(Strain: Sprague Dawley)

M = Male; F = Female; n = number of animals/subjects per gender.

Sample Collection: Urine and feces over selected intervals and blood at selected time points were collected from healthy male volunteers, dogs, and rats through 336-hr, 168- hr, and 168-hr post dose, respectively.

Radioactivity: Total radioactivity was measured using liquid scintillation spectrometer (LSS).

Sample Pooling:

For each species, plasma samples were pooled across subjects/animals by time point. All the other matrices were first pooled for a desired collection interval within each subject/animal and then across subjects/animals to obtain a composite sample containing >90% of the radioactivity excreted in each respective matrix.

Sample Processing

Matrix Methods

Plasma Solvent extraction with protein precipitation (SEPP) using

methano acetonitrile (v:v, 50:50) containing 0.1% TFA

Urine Direct injection after centrifugation

Feces Solvent extraction using methanol :acetonitrile (v:v, 50:50) containing 0.1% TFA

Mobile Phase and HPLC Conditions:

The HPLC column and guard column temperature was maintained at 40°C for all LC-MS and LC-MS/MS experiments. The mobile phase consisted of (A) 99% 20 mM ammonium acetate and 1% acetonitnle (pH adjusted to 5.0 with acetic acid) and (B) 99% acetonitnle and 1% acetic acid. The combined flow was maintained at 1.0 mL/min. The column effluent was split to divert 15-25% into mass spectrometer and the balance into a radiometric detector.

Mobile Phase Gradient:

Separation of metabolites was achieved using programmed linear changes in mobile phase composition as summarized in the following table:

HPLC-MS/FSA Systems:

Two HPLC-MS/FSA systems were used in the studies. The components of system 1 are summarized in the table below.

Equipment Model and Vendor HPLC System

System Controller Model SCL-IOA VP (Shimadzu Corporation,

Kyoto, Japan)

Liquid Model LC-10AD VP (Shimadzu Corporation)

Chromatograph

Degasser Model DGU-14A (Shimadzu Corporation)

UV-Vis Detector Model SPD-10AV VP (Shimadzu Corporation)

Autoinjector Model SIL-10AD VP (Shimadzu Corporation)

Column Oven Model CTO-IOA VP (Shimadzu Corporation)

Radioactivity Detector Model D525 (Packard Instrument Co., Meriden,

CT)

Radioactivity Detector 250 μΐ, (Packard Instrument Co.)

Cell

Column Symmetry C8, 4.6 x 250 mm, 5-μιη particle size

(Waters Corporation, Milford, MA)

Guard Column Symmetry C8, 3.9 x 20 mm, 5-μπι particle size

(Waters Corporation)

LC-MS and LC-MS/MS experiments were performed using a PE Sciex QSTAR/Pulsar (Quadrupole Time-of-Flight) mass spectrometer (PE Sciex, Toronto, Ontario, Canada), equipped with a Turbo lonSpray® source. Typical operational conditions are provided as follows. Parameter Setting

Ionization Source Turbo IonSpray

Ionization Mode Positive

IonSpray Voltage 5 kV

TurboProbe Temperature 350°C

Curtain Gas 25^a

Ion Source Gas 1 (Nebulizer 55^a

Gas)

Ion Source Gas 2 (Heater Gas) 85^a

Collision Gas (MS/MS) 5^a

Collision Energy 35 eV

a: All gas flow settings represent arbitrary units.

The components of system 2 are summarized in the following table:

Component Model and Vendor

HPLC System HPLC: Acella including Pumps, and Autosampler

(Thermo Fisher Scientific, San Jose, CA)

Mass Spectrometer LTQ Orbitrap Discovery (Thermo Fisher Scientific, San

Jose, CA)

MSC 1450 MicroBeta TriLux Liquid Scintillation and

Luminescence Counter (PerkinElmer Life and Analytical

Sciences, Boston, MA)

Fraction Collector FC204 (Gilson, Middleton, WI)

MSC 96-well plates Scintiplate® (PerkinElmer Life and Analytical Sciences)

FSA β-Ram Model 4, with a 250 detector cell (IN/US

Systems, Tampa, FL)

Column Symmetry C8, 4.6 x 250 mm, 5-μπι particle size (Waters

Corp, Milford, MA.) Guard Column Symmetry C8, 3.9 x 20mm, 5-μπι particle size (Waters

Corp)

The LTQ Orbitrap Discovery mass spectrometer was equipped with an electrospray ionization source and nominally operated under the conditions listed following table:

Radiochromatograms from study samples were examined to locate radioactive peaks corresponding to metabolites. Each radiolabeled peak examined for possible molecular ions related to the drug and/or its putative metabolites. Based on the elution order, metabolite peak labels assigned as Ml to Ml 7, where Ml is the first eluting compound and Ml 7 is the last to elute from the column (see FIGS. 1-4 below). When available, synthetic standards were used to confirm the structural assignment.

As shown in Table 1, following 50 mg single oral administration of ¹⁴C- Preladenant to healthy male volunteers, on average, 7.81% and 82.8% of the dose was excreted in urine and feces, respectively. In preclinical species, 1.66% and 74.5% of the dose was excreted in urine and feces of male dogs and 2.44% and 64.9% of the dose was excreted in urine and feces of female dogs, respectively, while 5.47% and 86.4% of the dose was excreted in urine and feces of male rats and 2.41% and 89.3% of the dose was excreted in urine and feces of female rats, respectively. As shown in FIG. 1 , Preladenant was extensively metabolized in human, dog, and rat after a single 50 mg, 10 mg/kg, and 10 mg/kg oral administration of C-Preladenant, respectively. The metabolite profiles from human, dog, and rat are shown in FIGS 2-4.

Table 1- Excretion of radioactivity as % of dose in human (50 mg), dog (10 mg/kg), and rat (10 mg/kg) following a single oral administration of ¹⁴C-Preladenant.

The following table describes the distribution of compound 29A (Preladenant) and its metabolites in plasma in human, male dog, and male rat species.

Preladenant and Metabolites (Plasma)

Species Major Minor Trace

Human (2-hr) Preladenant (m z M9 (m/z 504), Ml 3 M2 (m/z 540), M4

504) (m/z 490) (m/z 237), M5a

(m/z 300), M6 (m/z 354), M7 (m/z 540), M8 (m/z 526), M12 (m/z 446), M14 (m/z 478)

Dog (0-24 hr) Preladenant (m/z M2 (m z 540), M3 Mia (m/z 223), M4

504), Ml 3 (m/z (m/z 526), M3a (m z 237), M5c 490) (m/z 512), M5 (m/z (m/z 496), M5d

524), M5a (m/z (m/z 622), M6 (m/z 300), M7 (m/z 354), M7aa (m/z 540), M9 (m/z 698), M7a (m/z 504), M10 (m/z 666), M8 (m/z

538), 526), Ml 2 (m/z

446), M14 (m/z 478), Ml 7 ((m/z 500)

Rat (0-24 hr) Preladenant (m/z M5a (m/z 300), M9 Mia (m/z 223), M4

504) (m/z 504), Ml 3 (m/z 237), M6 (m/z

(m/z 490), Ml 7 354), M7 (m/z (m/z 500) 540), M8 (m/z

526), Ml 0 (m/z 538), M14 (m/z 478)

Major: components with >20% of the total chromatographic radioactivity (TCR).

Minor: components between 3 and 20% of the TCR.

Trace: components <3% of the TCR and/or only detected with a mass spectrometer.

Based on the above, the following observations can be made:

The major circulating drug derived components in human, dog, and rat were Preladenant, M9 and Ml 3.

There was no human specific circulating metabolite.

The following table describes the distribution of the compound of Formula II (Preladenant) and its metabolites in urine of human, male dog, and male rat species.

Preladenant and Metabolites (Urine)

Species Major Minor Trace

Human M9 (m/z 504) M2 (m/z 540), M5a M3 (m/z 526), M4

(m/z 300) (m/z 237), M6 (m/z

354), M7 (m/z 540), M8 (m/z 526), M10 (m/z 538), Mi l (m/z 524), Ml 3 (m/z 490), M14

(m/z 478)

Dog Ml (m/z 237), Mia

(m/z 223), M2 (m/z

540), M3 (m/z

5267), M3a (m/z

512), M4 (m/z 237),

M5 (m/z 524), M5a

(m/z 300), M5c

(m/z 496), M5d

(m/z 622), M6 (m/z

354), M7 (m/z 540),

M7aa (m/z 698),

M7a (m/z 666), M8

(m/z 526), M9 (m/z

504), Ml 0 (m/z

538), Mi l (m/z

524), M13 (m/z

490), Ml 5 (m/z

520), Preladenant

(m/z 504)

Rat M5a (m/z 300) M9 (m/z 490) Ml (m/z 237), Mia

(m/z 223), M2 (m/z 540), M4 (m/z 237), M5 (m/z 524), M6 (m/z 354), M7 (m/z 540), M7a (m/z 666), M8 (m/z 526), M10 (m/z 538), Mi l (m/z 524), M13 (m/z 490), M14 (m/z 478),

M15 (m/z 520), Preladenant (m/z 504), M17 (m/z 500)

Major: components with >3% of the administrated dose.

Minor: components between 0.5 and 3% of the administrated dose.

Trace: components <0.5% of the administrated dose and/or only detected with a mass spectrometer.

Based on the above, the following observations can be made:

M9 was the most prominent urinary metabolite in human urine, representing 3.53% of the dose. M5a was the major urinary metabolite in male rat urine, representing 3.42% of the dose. However, no metabolite in dog urine accounted for >0.5% of the dose.

The following table describes the distribution of the compound of Formula II (Preladenant) and its metabolites in feces of human, male dog, and male rat species.

Preladenant and Metabolites (Feces)

Species Major Minor Trace

Human M2 (m/z 540), M7 M3 (m/z 526), M5a M6 (m/z 354), M8

(m/z 540), M9 (m/z (m/z 300), (m/z 526), Ml 0

504), Preladenant (m/z 538), Mi l

(m/z 504) (m/z 524), M12

(m/z 446), Ml 3 (m/z 490), M14 (m/z 478)

Dog Preladenant (m/z M2 (m/z 540), M3

504) (m/z 5267), M3a

(m/z 512), M5 (m/z 524), M5a (m/z 300), M5c (m/z

496), M6 (m/z

354), M7 (m/z

540), M8 (m/z

526), M9 (m/z

504), Ml 0 (m/z

538), Mi l (m/z

524), M12 (m/z

446), Ml 3 (m/z

490), Ml 4 (m/z

478), M14a (m/z

550), Ml 5 (m/z

520)

Rat M5a (m/z 300), M7 M2 (m/z 540), M3

(m/z 540), M9 (m/z (m/z 526), M5 (m/z

504), Ml 0 (m/z 524), M5aa (m/z

538), Ml 3 (m/z 550), M5b (m/z

490), Preladenant 604), M6 (m/z

(m/z 504) 354), M8 (m/z

526), Ml 0b (m/z

680), Mi l (m/z

524), M12 (m/z

446), M14 (m/z

478), M14a (m/z

550), Ml 7 (m/z

500)

Major: components with >5% of the administrated dose.

Minor: components between 1 and 5% of the administrated dose.

Trace: components <1% of the administrated dose and/or only detected with a mass spectrometer. Based on the above, the following observations can be made:

In human feces, Preladenant accounted for 14% of the dose. Major metabolites included M2, M7, and M9, representing 22%, 6% and 29% of the dose, respectively. In male dog feces, Preladenant was the only drug-derived material detected by radiometric detector with integrated radioactivity, and accounted for 71.0% of the dose. In rat feces, Preladenant was the most prominent drug-related compound and accounted for 26.3% of the dose. Other major fecal metabolites in male rats included M5a, M7, M9, M10, and M13.

Following a single oral administration of Preladenant, the radioactivity was predominantly eliminated in feces (>65%) while <10% of the dose was eliminated in urine.

In all species investigated, the primary biotransformation pathways of

Preladenant included O-demethylation and subsequent oxidation to a carboxylic acid, N-dealkylation, and alteration(s) of the furan ring.

No human specific metabolites were observed following a single oral administration of 50 mg Preladenant to healthy male volunteers.

Because of their adenosine A_2a receptor antagonist activity, the compounds of the present invention are 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 reduce 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 A¾ and A^ Receptor Competition Binding Assay Protocol

Materials

• Membranes from HEK293 cells expressing the human adenosine 2a receptor

(Perkin-Elmer RBHA2AM400UA)

• Membranes from CHO-K1 cells expressing the human adenosine 1 receptor

(Perkin-Elmer ES-010-M400UA) • [³H]SCH58261 for the A2a receptor (custom synthesis; Merck Research

Laboratories)

• [³H]-Cyclopentyl-l,3-dipropylxanthine (DPCPX) for the Al receptor (Perkin-Elmer NET 1026)

• Wheatgerm agglutinin-coated yttrium silicate SPA beads (GE Healthcare

RPNQ0023).

• Assay Buffer-Dulbecco's phosphate buffered saline without calcium, without

magnesium + 10 mM MgCl₂

• Adenosine deaminase from calf intestine (Roche # 10 102 105 001).

• Dimethylsulfoxide (DMSO)

• CGS15943 (A2a antagonist) Tocris #1699

• DPCPX (Al antagonist) Tocris #0439

Compound Dilution

• Dilute compounds to 1 mM in 100% DMSO

• Make 1 :10 serial dilutions in 100% DMSO.

• Dilute 1 :20 into assay buffer (i.e. 3 ul into 57 ul buffer). This is a 5X solution in 5% DMSO.

Radioisotopes

• For the A2a receptor: Dilute [³H]SCH58261 to 5 nM in assay buffer. The final concentration in the assay is 2 nM.

• For the Al receptor: Dilute [³H]DPCPX to 1.75 nM in assay buffer. The final concentration in the assay is 0.7 nM.

Membrane Preparation

• For the A2a receptor: Use 1 ug of membrane/well. Dilute membranes to 50 ug/ml in assay buffer.

• For the Al receptor: Use 5 ug of membrane/well. Dilute membranes to 250 ug/ml in assay buffer.

• For both receptors, add adenosine deaminase (ADA) to the diluted membranes to a final concentration of 20 ug/ml and incubate for 15 minutes at room temperature.

Membrane-Bead Mixture • For the A2a receptor, use 25 ug/well wheatgerm agglutinin-coated yttrium silicate SPA beads.

• For the Al receptor, use 100 ug/well wheatgerm agglutinin-coated yttrium silicate SPA beads.

• Mix ADA-treated membranes and SPA beads together for 30 min prior to assay.

For the A2a receptor, mix beads and membranes at a ratio of 25 ug/beads to 1 ug membranes. For the Al receptor, mix beads and membranes at a ratio of 20 ug beads to 1 ug membranes.

Assay Assembly

Add in order to a Perkin-Elmer white opaque Optiplate-384 (Catalog #6007299):

• For A2a: 10 ul of 5X compound, 20 ul of 5 nM [³H] SCH 58261 , 20 ul membrane- bead mixture. To determine total binding, substitute assay buffer + 1% DMSO for compound. To determine non-specific binding, substitute 2.5 uM CGS 15943 in place of compound. Incubate for one hour at room temperature with agitation.

• For Al : 10 ul of 5X compound, 20 ul of 1.75 nM [³H]DPCPX, 20 ul membrane- bead mixture. To determine total binding, substitute assay buffer + 1% DMSO for compound. To determine non-specific binding, substitute 2.5 uM DPCPX in place of compound. Incubate for one hour at room temperature with agitation.

At the end of the incubation allow the beads to settle for one hour then measure the fluorescence in a Perkin-Elmer TopCount scintillation counter. ICs₀ values are determined by fitting the displacement curves using an iterative curve fitting program (GraphPad Prism). K, values are calculated 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-hydroxydopamine (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 Brain. 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-HCl 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.

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

Compound Human A?„ Ki (nM)

M9 1.4

M13 0.6 M12 1.3

M5a >1000

M2 >1000

M7 >1000

Selectivity is determined by dividing ¾ for A_\ receptor by Kj 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.

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 H-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.

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, 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 PLMD/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

WHAT IS CLAIMED IS:

1. A compound of the formula:

in isolated and purified form or a pharmaceutically acceptable salt thereof.

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

3. The pharmaceutical composition of claim 2, further comprising at least one other agent useful in treating Parkinson's disease in a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein the other agent is selected from the group consisting of L-DOPA, dopaminergic agonists, MAO-B inhibitors, DOPA decarboxylase inhibitors and COMT inhibitors.

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

6. The method of claim 5 for treating depression, a cognitive disease or a neurodegenerative disease.

7. The method of claim 6 for treating for treating Parkinson's disease, senile dementia or psychoses of organic origin.

8. A method of treating Extra-Pyramidal Syndrome (EPS) caused by treatment with an antipsychotic agent, comprising administering a therapeutically effect amount of the compound of claim 1.

9. The method of claim 8, wherein the antipshychotic agent is a typical antipsychotic agent or an atypical antipsychotic agent.

10. The method of claim 9, 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

11. The method of claim 8, further comprising administering an antipsychotic agent.

12. The method of claim 11 , 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.

13. 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.

14. 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 selected from the group of compounds of Formula I, III, IV, V, VI and VII:

Formula I Formula III Formula IV

Formula V Formula VI Formula VII rmaceutically acceptable salt or solvate thereof.