WO2005041957A1

WO2005041957A1 - Oxindole derivatives and their use as phosphodiesterase type 2 inhibitors

Info

Publication number: WO2005041957A1
Application number: PCT/IB2004/003404
Authority: WO
Inventors: Robert James Chambers; Kelvin T. Lam
Original assignee: Pfizer Products Inc.
Priority date: 2003-10-29
Filing date: 2004-10-18
Publication date: 2005-05-12

Abstract

The present invention provides compounds, methods and kits for treatment of disease states or disorders mediated by PDE2. Oxindole derivatives are provided which have therapeutic use and as PDE2 inhibitors.

Description

OXINDOLE DERIVATIVES AND THEIR USE AS PHOSPHODIESTERASΞ TYPE 2 INHIBITORS

FIELD OF THE INVENTION

The present invention relates to oxindole compounds which are Phosphodiesterase Type 2 (PDE 2) inhibitors. The PDE 2 inhibitors may be used as agents in the treatment of dementia, cancer and hematologic disorders among others mediated by PDE2 activity. The invention also relates to pharmaceutical compositions comprising oxindole compounds. BACKGROUND

The phosphodiesterase enzyme family controls intracellular levels of secondary messenger cAMP or cG P through regulation of their hydrolysis. PDE2 possesses a low affinity catalytic domain and an allosteric domain specific for cGMP. The low affinity catalytic site can hydrolyze both cAMP and cGMP with a lower apparent K_M for cGMP over cAMP. However, when cGMP binds to the allosteric site, the catalytic site undergoes a conformational change showing high affinity for cAMP. PDE2 exists as a homodimer that binds two molecules of cGMP per homodimer at the allosteric site. PDE2 shows the highest expression in the brain but is also found in many other tissues as well and therefore inhibitors to PDE2 have a broad array of function and potential therapeutic utility to these sites (Beavo, J. A. et al., Rev. Physio. Biochem. Pharm., 1999, 135, 67 and Soderling and Beavo, Curr. Opin. Cell Biol., 2000, 12, 174). Examples of PDE2 inhibitors include therapeutic potential in neuronal development, learning and memory (van Staveren, W. C. G. et al., Brain Res., 2001 , 888, 275 and O'Donnell, J. et al., J. Pharm. Exp. Ther., 2002, 302, 249); prolactin and aldosterone secretion (Velardez, M. O. et al., Eur. J. Endo., 2000, 143, 279 and Gallo-Payet, N. et al., Endo., 1999, 140, 3594); immunological response (Houslay, M. D. et al., Cell. Signal., 1996, 8, 97); vascular angiogenesis (Keravis, T. et al., J. Vase. Res., 2000, 37, 235); inflammatory cell transit (Wolda, S. L. et al., J. Histochem. Cytochem., 1999, 47, 895); cardiac contraction (Fischmeister, R. et al., J. Clin. Invest, 1997, 99, 2710; Donzeau-Gouge, P. et al., J. Physiol., 2001 , 533, 329 and Paterson, D. J. et al., Card. Res., 2001 , 52, 446); platelet aggregation (Haslam, R. J. et al., Biochem. J., 1997, 323, 371); hypoxic pulmonary vasoconstriction (Haynes, J. et al., J. Pharm. Exp. Ther., 1996, 276, 752) and olfactory signal transduction (Ma et al., J. Neurosci. 2003, 23, 317). It has been shown that EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), a potent adenosine deaminase inhibitor, selectively inhibits PDE2. However, the use of EHNA as a PDE2 based therapeutic agent may be limited due to low potency in inhibiting PDE2 and high potency in inhibiting adenosine deaminase (U.S. Patent No. 6,066,649, Fischmeister, R. et al., Mol. Pharm., 1995, 48, 121 and Michie A.M. et al., Cell Signal ,1996, 8, 97). U.S. Patent. No. 4,695,571 , , discloses tricyclic oxindole carboxamide derivatives useful as anti-inflammatory agents. The design, synthesis and screening of various PDE2 inhibitors have been reported in the periodical and patent literature. U.S. Patent Nos. 5,861 ,396, 5,965,619, 6,066,649, 6,465,494, 6,479,493 B1 , and published U.S. Patent applications US2002/0132754, US2003/0190686, and published PCT Applications WO 02/09713, and WO 03/001968 provide disclosure of compounds for use as phosphodiasterase inhibitors, including PDE2 inhibitors. Amino acid and nucleotide sequences for various phosphodiesterases including PDE2 have been provided by Charbonneau et al., Proc. Natl. Acad. Sci., 1986, 83, 9308; Trong et al., Biochemistry, 1990, 29, 10280; Sonnenberg, et al., J. Biol. Chem., 1991 , 266,17655 and in Rosman et al., Gene, 1997, 191 , 89. An assay to determine the catalytic activity of PDE2 is disclosed by Beavo, J.A., J. Biol. Chem., 1990, 245, 5649. Appropriate assays to screen compounds for therapeutic use as inhibitors of PDE2 may be implemented with the use of full length or regions, i.e. catalytic region, of synthetic, purified or isolated recombinant polypeptides of PDE2 (Beavo and Reifsnyder, TIPS, 1990, 11 , 150). A cellular based assay method for identifying inhibitors was reported in U.S. Patent No. 5,800,987. SUMMARY OF THE INVENTION

The present invention is directed to PDE2 inhibitors useful in treatment of diseases or disorders where inhibition of PDE2 activity is considered beneficial. In particular, the present invention is directed to the unexpected finding that oxindole compounds are PDE2 inhibitors. The invention further relates to the use of PDE2 inhibitors as pharmaceuticals. In a further aspect of the invention kits are provided for use in the treatment of disease states or disorders mediated by PDE2. This invention is also directed to pharmaceutical compositions which comprise a therapeutically effective amount of a compound of Formula I, as noted below, a tautomeric isomer, a ester or carbonate prodrug thereof, or a pharmaceutically acceptable salt of said compound, and a pharmaceutically acceptable carrier. The compounds of the invention are inhibitors of PDE2, and are therefore useful to treat a mammal, preferably human, to address those processes which involve the production and/or action of PDE2. The invention provides for the administration of an inhibitor of PDE2 to a mammal in need of such treatment or prevention of dementia. Dementia is characterized by the loss, usually progressive, of cognitive functions without impairment of perception of consciousness. Some effects of dementia include disorientation, impaired memory, judgment and intellect. Selective PDE2 inhibitors are particularly suitable for improving perception, concentration, learning or memory after cognitive disturbances as occur in particular in situations/diseases/syndromes such as mild cognitive impairment, age- associated learning and memory disturbances, age-associated memory losses, vascular dementia, craniocerebral trauma, stroke, dementia occurring after strokes (post stroke dementia), post-traumatic craniocerebral trauma, general disturbances of concentration, disturbances of concentration in children with learning and memory problems, Alzheimer's disease, dementia with Lewy bodies, dementia with degeneration of the frontal lobes including Pick's syndrome, Parkinson' s disease, progressive nuclear palsy, dementia with corticobasal degeneration, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob dementia, HIV dementia, schizophrenia with dementia or Korsakoff psychosis. The invention provides for the administration of an inhibitor of PDE2 to a mammal in need of such treatment for cancer. Further, the invention provides for the administration of an inhibitor of PDE2 to a mammal in need of such treatment for a hematologic disorder including the treatment and control of thromboses. Arterial thrombosis mediates tissue infarction in coronary artery disease, cerebrovascular disease, and peripheral vascular disease, and, thus is a common cause of morbidity and mortality. Platelets are key mediators of arterial thrombosis. Thus, the identification of compounds that inhibit platelet function, and thus thrombosis formation, is of great importance. In addition to coronary artery disease/myocardial infarction, cerebrovascular disease and peripheral vascular disease, diseases and disorders associated with inappropriate platelet activity and arterial thrombosis also include, for example, stable and unstable angina, transient ischemic attacks, placental insufficiency, unwanted thromboses subsequent to surgical procedures, e.g., aortocoronary bypass surgery, angioplasty, stent placement, and heart valve replacement, or thrombosis subsequent to atrial fibrillation. PDE2 inhibitors can provide therapeutic and preventive benefits for each of these diseases or disorders. Inappropriate platelet activation may also play a role in venous thrombosis, such that platelet inhibitors can be useful for the treatment or prevention of disorders associated with such thromboses. DETAILED DESCRIPTION OF THE INVENTION

The present invention includes the administration of an inhibitor of PDE2 in a therapeutically effective dose to a mammal in need of such treatment. Suitable inhibitors of PDE2 include the compounds of Formula I: Formula I

where R¹ is C C₆ alkyl, preferably a methyl or ethyl group; R² is a hydrogen atom or C C₆ alkyl, preferably a methyl group; R³ is a heterocyde selected from a group consisting of thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isooxazolyl, oxadiazolyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl, optionally substituted with hydrogen atom, C1-C3 alkyl or haloalkyl group; X is a sulfur or oxygen atom and Y is a carbon or nitrogen atom, or a tautomeric isomer or a pharmaceutically acceptable salt; ester; or carbonate prodrug thereof. Preferably R³ is selected from the group consisting of 2-thiadiazolyl, 5-methyl- 2-thiadiazolyl, 5-ethyl-2-thiadiazolyl, 5-trifluromethyl-2-thiadiazolyl, 2-thiazolyl, 5- methyl-2-thiazolyl, 3-methyl-5-isoxazolyl, 2-pyrazine, 5-pyrimidine, and 3-pyridine. Most preferable in the present invention are compounds comprising 5-Methyl- 6-oxo-6,7-dihydro-5H-1-oxa-5-aza-indacene-7-carboxylic acid [1 ,3,4]thiadiazol-2- ylamide, 5-Methyl-6-oxo-6,7-dihydro-5H-1 -oxa-5-aza-indacene-7-carboxylic acid [1 ,3,4]thiadiazol-2-ylamide, 5-Methyl-6-oxo-6,7-dihydro-5H-1 -oxa-5-aza- indacene-7-carboxylic acid(5-Methyl-[1 ,3,4]thiadiazol-2-yl)-amide; or a tautomeric isomer; a pharmaceutically acceptable salt; ester; or carbonate prodrug thereof. In a preferred embodiment the compounds of Formula I are useful as PDE2 inhibitors and particularly those that have a PDE2 inhibitor IC50 value of less than ' about 5.0 μM, preferably less than about 1.0 μM, and more preferably about 0.2 μM. As used herein, the following terms and abbreviations have the defined meanings unless otherwise stated. The abbreviation PDE2 means Phosphodiesterase Type 2. The abbreviation TEA means triethylamine. The abbreviation DMAP means dimethylaminopyridine. The abbreviation TFA means trifluoroacetic acid. The term "C C₆ alkyl" as used herein denotes any straight or branched alkyl chain containing 1 to 6 carbon atoms and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl or the like. The term "heterocyde" means an optionally substituted ring structure containing one or more heteroatoms (non-carbon ring atoms) selected from O, N, and S. The term "IC₅₀" is defined as the concentration of a compound that results in 50% enzyme inhibition, in a single dose response experiment. The IC₅₀ value as related to the present invention is a measure of the potency of a compound to inhibit PDE2. The terms "inhibiting" or "inhibits" as related to the present invention refer to blocking the enzymatic activity of PDE2 to a sufficient degree to reduce a clinical symptom in the patient or to prevent the recurrence of a clinical symptom in a patient; and a PDE2 mediated disease state or disorder. A PDE2 inhibitor useful in the present invention is a compound that inhibits PDE2 and preferably has an IC₅₀ against human recombinant or purified PDE2 of less than about 5.0 μM. Preferably, the IC₅₀ value of the PDE2 inhibitor is less than about 1.0 μM, more preferably about 0.2 μM or less. Preferred PDE2 inhibitors of the present invention do not significantly inhibit, i.e. are not specific to, other PDE enzymes. The term "hematologic disorder" includes a disease or condition such as anemia including beta-thalassemia, hemorrhage, thrombosis, embolism, lymphadenopathy, splenomegaly, phagocytic disorders, hematopoietic disorders, hemoglobin disorders including sickle cell anemia, bone marrow disorders, leukemia including chronic myelogenous leukemia and other myeloproliferative disorders, lymphoma including non-Hodgkin's lymphoma, Hodgkin's disease, complications related to blood transfusion, complications related to bone marrow transplantation, and clotting disorders including von Willebrand's disease and hemophilia; a karyotypic disorder associated with sex chromosome imbalance including Klinefelter syndrome and Turner syndrome. The term "thrombosis" (plural thromboses) refers to the formation or development of a blood clot whether the result of a disease or disorder or as may be associated with or caused by surgical procedure or otherwise by severe injury. The term "mammal" as used herein includes males and females, and encompasses humans, domestic animals (e.g., cats, dogs), livestock (e.g. cattle, horses, swine), and wildlife (e.g., primates, large cats), all of which may be referred to as a patient(s). The expression "pharmaceutically acceptable salts" includes both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable cationic salts. The expression "pharmaceutically acceptable cationic salts" is intended to define but is not limited to such salts as the alkali metal salts, (e.g. sodium and potassium), alkaline earth metal salts (e.g., calcium and magnesium), aluminum salts, ammonium salts, and salts with organic amines such as benzathine (N,N'- dibenzylethylenediamine), choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine), benethamine (N-benzylphenethylamine), diethylamine, piperazine, tromethamine (2-amino-2-hydroxymethyl-1 ,3-propanediol) and procaine. The expression "pharmaceutically acceptable acid addition salts" is intended to define but is not limited to such salts as the hydrochloride, hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogenphosphate, acetate, succinate, citrate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts. A "therapeutically effective amount" refers to that amount of the compound that results in achieving the desired effect. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the tested population) and the ED₅₀ (the dose therapeutically effective in 50% of the tested population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indices are preferred. The data obtained from such procedures can be used in formulating a dosage range for use in patients. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. The general range of effective administration rates of a compound of the present invention is from about 0.001 mg/day to about 200 mg/day. A preferred rate range is from about 0.010 mg/day to about 100 mg/day. Of course, it is often practical to administer the daily dose of compound in portions, at various hours of the day. However, in any given case, the amount of compound administered will depend on such factors as the potency of the PDE2 inhibitor, the solubility of the compound, the formulation used, the route of administration and the wellness of the patient. The terms "treat", "treatment", and "treating" include preventative (e.g., prophylactic) and palliative treatment or the act of providing preventative or palliative treatment. The term "tautomerization" refers to the phenomenon wherein a proton of one atom of a molecule shifts to another atom of the same molecule wherein two or more structurally distinct compounds are in equilibrium with each other. The term "tautomer" refers to the compounds produced by the proton shift. The present invention contemplates that particular compounds may exist as tautomers and isomes and mixtures thereof and all are contemplated to be within the scope of the present invention. One of ordinary skill in the art will recognize that certain compounds of this invention will contain one or more atoms which may be in a tautomeric, or geometric configuration, giving rise to tautomers and configurational isomers. Hydrates and solvates of the compounds of this invention are also included. The subject invention also includes isotopically-labeled compounds, which are structurally identical to those disclosed above, 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, phosphorous, sulfur, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸0, ⁷0, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶CI, respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug 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., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out known or referenced procedures and by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. Nonisotopically-labeled compounds of Formula I are also subject of the present invention. The term "label" or "labeled" is intended to encompass direct labeling by coupling a detectable substance as well as indirect labeling with another reagent that is itself directly labeled. Examples of detectable substances include radioactive isotopes, fluorescent, luminescent and others known by those of skill in the art. Those of ordinary skill in the art will recognize that physiologically active compounds which have accessible hydroxy groups can be administered in the form of pharmaceutically acceptable esters or carbonates. The compounds of this invention can be effectively administered as an ester or carbonate, formed on the hydroxy groups, just as one skilled in pharmaceutical chemistry would expect. It is possible, as has long been known in pharmaceutical chemistry, to adjust the rate or duration of action of the compound by appropriate choices of ester or carbonate groups. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995). Pharmaceutical compositions for use within the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art. Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances such as starch of many different kinds, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are substances such as starch, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidine and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders. A lubricant may be necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. Tablet disintegrators are substances that facilitate the disintegration of a tablet to release a compound when the tablet becomes wet. They include starches, clays, celluloses, algins and gums, more particularly, corn and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation- exchange resins, alginic acid, guar gum, citrus pulp and carboxymethylcellulose, for example, may be used as well as sodium lauryl sulfate. Tablets are often coated with sugar as a flavorant and sealant, or with film- forming protecting agents to modify the dissolution properties of the tablet. The compounds may also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well- established in the art. When it is desired to administer a compound as a suppository, the typical bases may be used. Cocoa butter is a traditional suppository base, which may be modified by addition of waxes to raise its melting point slightly. Water-miscible suppository bases comprising, particularly, polyethylene glycols of various molecular weights are in wide use. The effect of the compounds may be delayed or prolonged by proper formulation. For example, a slowly soluble pellet of the compound may be prepared and incorporated in a tablet or capsule. The technique may be improved by making pellets of several different dissolution rates and filling capsules with a mixture of the pellets. Tablets or capsules may be coated with a film which resists dissolution for a predictable period of time. Topical formulations may be designed to yield delayed and/or prolonged percutaneous absorption of a compound. Even the parenteral preparations may be made long-acting, by dissolving or suspending the compound in oily or emulsified vehicles which allow it to disperse only slowly in the serum. The term "prodrug" means a compound that is transformed in vivo to yield a compound of the present invention. The transformation may occur by various mechanisms, such as through hydrolysis in blood. A discussion of prodrugs is provided by T. Higuchi and W. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. For example, if a compound of the present invention contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group. Similarly, if a compound of the present invention comprises an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group. If a compound of the present invention comprises an amine functional group, a prodrug can be formed by the replacement of a hydrogen atom in the amine group. Advantageously, the present invention also provides kits for use by a consumer to treat a disease state or disorder mediated by PDE2. The kits comprise at least one pharmaceutical composition comprising a oxindole derivate having PDE2 inhibiting activity and a pharmaceutically acceptable carrier, vehicle or diluent; and instructions describing a method of using the pharmaceutical compositions. Preferably the kits would include a compound of Formula I defined above. Those skilled in the art will appreciate that the PDE2 inhibitors of the present invention may be concurrently administered with other drugs in the treatments of the disease state or disorder for which the patient is being treated. A "kit" as used in the instant application includes a container for containing the pharmaceutical compositions and may also include divided containers such as a divided bottle or a divided foil packet. The container can be in any conventional shape or form as known in the art which is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a re- sealable bag (for example, to hold a "refill" of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle or blister pack, which is in turn contained within a box. A general method for preparing oxindole compounds of the present invention is described, for example, in U.S. Patent No. 4,695,571. One of skill in the art will understand that other methods may be utilized to prepare the compounds of the present invention. The method for preparing compounds of Formula II is presented by the following scheme: Synthetic Scheme

R₃NH2 4A Sieves, PhH reflux

Formula II where R³ substituents in Formula II are as defined previously in Formula I provided herein. Examples of such substituents are noted in Table I along with their respective PDE2 activity levels. The preparation of precursors to compounds of Formula II are given in Preparations1-7, below. This invention includes both pharmaceutical and veterinary compositions consisting of compounds and a method of treating patients with dementia, cancer and/or hematologic disorders, such as thrombosis disorders or diseases, among others as noted herein, and administering a therapeutically effective amount or dose of those compounds to a mammal or patient in need of such treatment, Such compounds need to be effective as inhibitors of PDE2. Oxindole derivatives are known to have anti-inflammatory activity through the inhibition of 5-lipoxygenase (5-LO) and clyclooxygenase-1 (COX-1) enzymes. The compound exemplified below in Example 2 was assayed against these enzymes and no significant inhibition was found (<40%@10μM). These assays were performed by a commercial laboratory, Cerep, Celle L'Evescault, France. The optimal utility as PDE2 inhibitors is shown where these compounds have an IC₅₀ value for PDE2 of no more than 5 μM. The PDE2 activity for examples of Formula I are provided in Table I. Representative compounds of this invention may be prepared as provided below in Examples 1-17, and as shown in Table I or alternatively using methods known by those of skill in the art. PRECURSOR PREPARATION Suitable methods of preparing precursors to compounds of Formula II are prepared as follows:

Preparation 1 2-Chloro-N-(4-hydroxy-phenyl)-N-methyl-acetamide.

To a stirred solution of p-methylaminophenol sulfate (90.0 g, 0.26 mol), triethylamine (144 ml, 1.0 mol.) and 4-dimethylaminopyridine (2.9 g, 0.03 mol.) in dimethylforamide (200 ml) at 0°C was added a solution of chloroacetyl chloride (41 ml, 0.52 mol.) in dimethylforamide (40 ml) over 45 minutes. The reaction mixture was allowed to slowly warm to room temperature over 2 hours then poured into water (1400 ml) and extracted with ethyl acetate (2 X 1400 ml). The organic extracts were combined and washed successively with 1 N hydrochloric acid (1 X 200 ml), water (2 X 200 ml) and brine (2 X 200 ml) then dried (sodium sulfate) and concentrated in vacuo to give 71.4 g 2-chloro-N-(4-hydroxy-phenyl)-N-methyl-acetamide as a tan solid (69%). ¹H-NMR (CDCI3): 7.1 (d, 2H), 6.9 (d, 2H), 3.9 (s, 2H), 3.3 (s, 3H). MS (m/z, %): 200/202 (M⁺+1 , 100)

Preparation 2 2-Hvdroxy-1 -methyl-1 ,3-dihvdro-indol-2-one

A mixture of 2-chloro-N-(4-hydroxy-phenyl)-N-methyl-acetamide (25.0 g, 0.13 mol.) and aluminum chloride (13.4 g, 0.50 mol.) was thoroughly mixed then heated at 170°C for 4 hours. The reaction mixture was cooled and quenched with 400 g crushed ice. The aqueous mixture was extracted with ethyl acetate (2 X 800 ml) and the organic extracts were combined, washed with brine (2 X 500 ml) then dried (sodium sulfate) and concentrated in vacuo to give 17.0 g 2-hydroxy-1-methyl-1 ,3- dihydro-indol-2-one as a brown solid (83%). ¹H-NMR (d₆-DMSO): 6.7 (m, 2 H), 6.6 (m, 2H), 3.5 (m, 2H), 3.1 (s, 3H). MS (m/z, %): 164 (M⁺+1 , 100)

Preparation 3 Chloro acetic acid 1-methyl-2-oxo-2,3-dihvdro-1H-indol-5-yl ester

To a solution of 2-hydroxy-1-methyl-1 ,3-dihydro-indol-2-one (17.0 g, 0.10 mol.), and pyridine (17 ml, 0.21 mol.) in methylene chloride (150 ml) and tetrahydrofuran (50ml) at 0°C was added drop wise a solution of chloroacetyl chloride (17 ml, 0.21 mol.) in methylene chloride (50 ml). After 30 minutes at 0°C, the reaction mixture was stirred at room temperature for 90 minutes, and then quenched with water (200 ml). The mixture was diluted with ethyl acetate (1000 ml) and the organic extract was washed with 1N hydrochloric acid (1 X 100 ml), 1N sodium hydroxide (1 X 100 ml), brine (1 X

100 ml) then dried (sodium sulfate) then concentrated to give 26 g crude product.

Chromatography on Silica Gel eluting with ethyl acetate/hexanes (1:1) afforded 15.4 g chloro acetic acid 1-methyl-2-oxo-2,3-dihydro-1H-indol-5-yl ester as a tan solid

(62%). ¹H-NMR (CDCI₃): 7.1 (m, 2H), 6.8 (M, 1 H), 4.3 (s, 2H), 3.6 (s, 2H), 3.2 (s, 3H). MS (m/z, %): 240/242 (M⁺+1 , 100).

Preparation 4 5-Methyl-5,7-dihvdro-1-oxa-5-aza-s-indacene-3,6-dione

A mixture of chloroacetic acid 1-methyl-2-oxo-2,3-dihydro-1H-indol-5-yl ester (26.0 g, 0.11 mol.) and aluminum chloride (58.0 g, 0.43 mol.) was thoroughly mixed and heated at 170°C for 30 minutes. The reaction flask was cooled in an ice bath and the reaction mixture quenched by the careful addition of water (300 ml). To the aqueous mixture was added a solution of ethyl acetate/tetrahydrofuran (1 :1 , 100 ml) and the resulting solid was filtered and dried in vacuo to give 16.5 g 5-methyl-5,7-dihydro-1- oxa-5-aza-s-indacene-3,6-dione (76%) as a brown solid. ¹H-NMR (CDCI₃): 7.1 (s, 1 H), 7.0 (S, 1 H), 4.7 (s, 2H), 3.6 (s, 2H), 3.2 (s, 3H). MS (m/z, %): 204 (M⁺+1 , 100).

Preparation 5 3-Hvdroxy-5-methyl-2,3,5,7-tetrahvdro-1-oxa-5-aza-s-indacen-6-one

To a stirred solution of 5-methyl-5, 7-dihydro-1-oxa-5-aza-s-indacene-3,6-dione (5.3 g, 0.03 mol.) in methanol (50 ml) and tetrahydrofuran (50 ml) at 0°C was added sodium borohydride (1.0 g, 0.03 mol.). The mixture was allowed to slowly come to room temperature while stirring overnight and after 22 hours the reaction mixture was quenched with brine (15 ml). The reaction mixture was concentrated in vacuo then diluted with brine (150 ml) and methylene chloride (150 ml). A solid formed and was filtered and dried to give 3.6 g brown solid. The layers of the filtrate were separated and the aqueous layer was extracted with methylene chloride (3 X 50 ml). The organic extracts were combined and dried (sodium sulfate) and concentrated to give 2.0 g orange solid. The solids were combined to afford 5.6 g 3-hydroxy-5-methyl- 2,3,5,7-tetrahydro-1-oxa-5-aza-s-indacen-6-one (100%). ¹H-NMR (d₆-DMSO): 7.0 (s, 1H), 6.8 (s, 1H), 5.3 (t, 1H), 4.5 (dd, 1H), 4.2 (dd, 1H), 3.5 (s, 2H), 3.1 (s, 3H). MS (m/z, %): 206 (M⁺+1 , 100).

Preparation 6 5-Methyl-5,7-dihvdro-1-oxa-5-aza-s-indacene-6-one

To a stirred suspension of 3-hydroxy-5-methyl-2,3,5,7-tetrahydro-1-oxa-5-aza-s- indacen-6-one (14.3 g, 0.07 mol.) in acetonitrile (250 ml) at room temperature was added portion wise a solution of trifluoroacetic acid (0.3 ml, 0.004 mol.) in acetonitrile (10 ml) over 90 minutes. The reaction mixture was stirred at room temperature for 18 hours then heated at 40°C for 4 hours. To this was added a solution of trifluoroacetic acid (0.3 ml, 0.004 mole) in acetonitrile (10 ml) drop wise and then the mixture was stirred at 40°C for 60 hours. The mixture was concentrated in vacuo to give 13.5 g brown solid. Chromatography on Silica Gel eluting with ethyl acetate/hexanes yielded 9.2 g 5-methyl-5,7-dihydro-1-oxa-5-aza-s-indacene-6-one as a tan solid (71%). ¹H- NMR (CDCI₃): 7.6 (s, 1H), 7.4 (s, 1H), 7.0 (s, 1H), 6.8 (s, 1H), 3.6 (s, 2H), 3.3 (s, 3H). MS (m/z, %): 188 (M⁺+1 , 100).

Preparation 7 5-Methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid ethyl ester

To ethanol (75 ml) with stirring was added sodium (3.6 g, 0.16 mol.) portion wise at 0°C. To this was added diethyl carbonate (19 ml, 0.16 mol.) followed by a solution of

5-methyl-5,7-dihydro-1-oxa-5-aza-s-indacene-6-one (9.2 g, 0.05 mol.) in ethanol (40 ml) at 0°C. The mixture was warmed to room temperature then heated at 80°C for 2 hours during which a thick solid mass formed, then allowed to stand at room temperature for 18 hours. The reaction mixture was diluted with methylene chloride (250 ml), 2N hydrochloric acid (500 ml) and brine (300 ml) and the undissolved solids were filtered. The filtrate layers were separated and the filtrant was resuspended in the aqueous extract and methylene chloride (300 ml) was added and stirred until the filtrant was dissolved. The organic extracts were combined and washed with brine (1

X 400 ml), dried (sodium sulfate) and concentrated in vacuo to give 12.3 g 5-methyl- 6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid ethyl ester (97%) as an amber solid. ¹H-NMR (CDCI₃): 7.6 (s, 1H), 7.5 (s, 1H), 7.0 (s, 1 H), 6.7 (s, 1H), 4.5 (s, 1 H), 4.3 (q, 2H), 3.3 (s, 3H). MS (m/z, %): 260 (M⁺+1 , 100).

Preparation 8 5-Ethyl-6-oxo-6 -dihvdro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid ethyl ester

A suitable preparation of this compound has been previously documented in U.S. Patent No. 4,695,571 , Preparation C.

Preparation 9

5-Ethyl-2-methyl-6-oxo-6.7-dihydro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid ethyl ester

A suitable preparation of this compound has been previously documented in U.S. Patent No. 4,695,571 , Preparation P.

Preparation 10 5-Ethyl-6-oxo-6,7-dihvdro-5H-1-thia-5-aza-s-indacene-7-carboxylic acid ethyl ester

A suitable preparation of this compound has been previously documented in U.S. Patent No. 4,695,571 , Preparation M.

Preparation 11

5-Ethyl-2-methyl-6-oxo-6,7-dihvdro-5H-1-oxa-3,5-diaza-s-indacene-7-carboxylic acid ethyl ester

A suitable preparation of this compound has been previously documented in U.S. Patent No. 4,695,571, Preparation Z. PREPARATION OF COMPOUNDS OF FORMULA I

The methods of preparing compounds of Formula I are provided as follows:

Example 1

5-Methyl-6-oxo-6,7-dihvdro-5H-1-oxa-5-aza-indacene-7-carboxylic acid (5-methyl-H ,3,41thiadiazol-2-yl)-amide (Compound 1)

A mixture of 5-methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid ethyl ester (150 mg, 0.6 mmol.) and 2-amino-5-methyl-1 ,3,4-thiadiazole (67 mg, 0.6 mmol.) in benzene (10 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 10 hours. The resulting precipitate was filtered and washed with additional benzene and dried to give 97 mg tan solid. Trituration with hot acetone afforded 47 mg 5-methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-indacene-7- carboxylic acid(5-methyl-[1 ,3,4]thiadiazol-2-yl)-amide (25%). ¹H-NMR (CDCI₃): 8.0 (s, 1H), 7.7 (s, 1H), 7.1 (s, 1H), 6.8 (s, 1H), 4.7 (s, br, 1H), 3.4 (s, 3H) 2.8 (s, 3H) MS (m/z, %): 329 (M⁺+1 , 100).

Example 2

5-Methyl-6-oxo-6,7-dihvdro-5H-1-oxa-5-aza-indacene-7-carboxylic acid f1 ,3,41thiadiazol-2-ylamide (Compound 2)

A mixture of 5-methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid ethyl ester (300 mg, 1.2 mmol.) and 2-amino-1 ,3,4-thiadiazole (59 mg, 0.6 mmol.) in benzene (30 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 20 hours. The resulting precipitate was filtered and washed with additional benzene (30 ml) and dried to give 193 mg 5-methyl-6-oxo-6,7-dihydro- 5H-1-oxa-5-aza-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2-ylamide (53%), mp 223-5°C. ¹H-NMR (d₆-acetone): 9.1 (s, 1 H), 7.9 (s, 1H), 7.8 (s, 1 H), 7.3 (s, 1 H), 6.9 (s, 1H), 5.0 (s, 1 H), 3.3 (s, 3H) MS (m/z, %): 315 (M⁺+1 , 30), 214 (50), 188 (100).

5-Methyl-6-oxo-6,7-dihydro-5H-1 -oxa-5-aza-s-indacene-7-carboxylic acid thiazol-2- ylamide (Compound 3)

A mixture of 5-methyl-6-oxo-6,7-dihydro-5H-1-oxa-s-indacene-7-carboxylic acid ethyl ester (100 mg, 0.4 mmol.) and 2-aminothiazole (39 mg, 0.4 mmol.) in benzene (10 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 18 hours. The reaction mixture was filtered and the filtrant triturated with hexanes/diethyl ether to give 15 mg 5-methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s- indacene-7-carboxylic acid thiazol-2-ylamide. (12%), mp 175-8°C. ¹H-NMR (CD₂CI₂): δ 8.0 (s, 1 H), 7.7 (s, 1H), 7.5 (s, 1 H), 7.1 (s, 1 H), 7.0 (s, 1H), 6.8 (s, 1H), 4.6 (s, 1 H), 3.4 (s, 3H). MS (m/z, %): 314 (M⁺+1 , 100).

Example 4

5-Ethyl-6-oxo-6,7-dihvdro-5H-1-oxa-5-aza-indacene-7-carboxylic acidH ,3,41thiadiazol-2-ylamide (Compound 4)

A suitable preparation of this compound has been previously documented in U.S. Patent No. 4,695,571 , Example 4.

Example 5 5-Ethyl-2-methyl-6-oxo-6,7-dihvdro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid f 1 ,3,41thiadiazol-2-ylamide (Compound 5)

A mixture of 5-ethyl-2-methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s-indacene-7- carboxylic acid ethyl ester (200 mg, 0.5 mmol.) and 2-amino-1 ,3,4-thiadiazole (57 mg, 0.6 mmol.) in benzene (20 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 4 hours. The resulting precipitate was filtered and washed with additional benzene (5 ml) and dried to give 107 mg 5-ethyl-2-methyl-6- oxo-6,7-dihydro-5H-1 -oxa-5-aza-s-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2- ylamide (60%) as a yellow orange solid, mp 208-10°C. ¹H-NMR (CDCI₃): 8.8 (s, 1 H), 7.8 (s, 1H), 7.0 (s, 1H), 6.4 (s, 1 H), 4.6 (s, 1H), 3.9 (q, 2H) 2.5 (s, 3H), 1.3 (t, 3H). MS (m/z, %): 329 (M⁺+1 , 100).

Example 6

5-Ethyl-6-oxo-6,7-dihvdro-5H-1 -oxa-5-aza-s-indacene-7-carboxylic acid pyrazine-2- ylamide (Compound 6)

A mixture of 5-ethyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid ethyl ester (200 mg, 0.7 mmol.) and aminopyrazine (70 mg, 0.7 mmol.) in benzene (40 ml) was refluxed over a Dean-Stark trap fro 1 hour. The mixture was cooled to room temperature and diluted with hexanes (35 ml). The resulting precipitate was filtered, washed with hexanes and dried to give 166 mg 5-ethyl-6-oxo-6,7-dihydro-5H- 1-oxa-5-aza-s-indacene-7-carboxylic acid pyrazine-2-ylamide, mp 185-7°C. ¹H-NMR (CDCI₃): 9.5 (s, 1 H), 8.3 (d, 2H), 8.0 (s, 1 H), 7.7 (s, 1 H), 7.1 (s, 1H), 6.8 (s, 1 H), 4.5 (s, 1 H), 3.9 (q, 2H) 1.4 (t, 3H). MS (m/z, %): 323 (M⁺+1 , 100).

Example 7

5-Methyl-6-oxo-6.7-dihvdro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid (5-ethyl-ri ,3,41thiadiazol-2-yl)-amide (Compound 7) A mixture of 5-methyl-6-oxo-6,7-dihydro-5H-1-oxa-s-indacene-7-carboxylic acid ethyl ester (100 mg, 0.4 mmol.) and 2-amino-5-ethyl-1 ,3,4-thiadiazole (50 mg, 0.4 mmol.) in benzene (10 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 18 hours. The reaction mixture was filtered and the filtrant triturated with 2-propanol to give 92 mg 5-methyl-6-oxo-6,7-dihydro-5H-1-oxa-5- aza-s-indacene-7-carboxylic acid (5-ethyl-[1 ,3,4]thiadiazol-2-yl)-amide (69%), mp 226-8°C. ¹H-NMR (CD₂CI₂): δ 8.0 (s, 1 H), 7.8 (s, 1H), 7.2 (s, 1 H), 6.9 (s, 1H), 4.6 (s, 1H), 3.4 (s, 3H), 3.1 (q, 2H), 2.3 (s, 3H), 1.4 (t, 3H). MS (m/z, %): 343 (M⁺+1 , 100).

Example 8 5-Ethyl-6-oxo-6,7-dihvdro-5H-1 -oxa-5-aza-s-indacene-7-carboxylic acid pyrimidin-5- ylamide (Compound 8)

A mixture of 5-ethyl-6-oxo-6,7-dihydro-5H-1-oxa-s-indacene-7-carboxylic acid ethyl ester (150 mg. 0.6 mmol) and 5-aminopyrimidine (52 mg, 0.6 mmol.) in benzene (40 ml) was refluxed over a Dean-Stark trap for 1 hour. The mixture was cooled to room temperature, diluted with hexanes and the resulting precipitate filtered. The filtrant was dissolved in ethyl acetate (75 ml) and methanol (3 ml) and washed with 1N hydrochloric acid (1 X 5 ml), brine (1 X 5 ml), dried (sodium sulfate) then concentrated in vacuo. The residue was dissolved in methylene chloride (2 ml), diluted with diethyl ether (5 ml) and triturated with hexanes to give 54 mg 5-ethyl-6- oxo-6, 7-dihydro-5H-1 -oxa-5-aza-s-indacene-7-carboxylic acid pyrimidin-5-ylamide (31%) as an orange solid, mp 100-3°C. ¹H-NMR (CDCI₃): δ 8.9 (s, 1 H), 8.6 (d, 1 H), 8.1 (d, 1H), 7.9 (s, 1H), 7.7 (s, 1H), 7.1 (s, 1H), 6.8 (s, 1H), 4.5 (s, 1H), 3.9 (q, 2H), 1.3 (t, 3H). MS (m/z, %): 321 (M*-1 , 100).

Example 9 5-Ethyl-6-oxo-6.7-dihvdro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid (3-methyl-isoxazol-5-yl)amide (Compound 9)

A mixture of 5-ethyl-6-oxo-6,7-dihydro-5H-1-oxa-s-indacene-7-carboxylic acid ethyl ester (200 mg, 0.7 mmol.) and 5-amino-3-methylisoxazole (72 mg, 0.7 mmol.) in benzene (20 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 18 hours. The suspension was poured into 1 N hydrochloric acid (100 ml) and extracted with chloroform (2 X 100 ml). The organic extracts were combined, washed with 1N hydrochloric acid (1 X 20 ml), water (1 X 20 ml), dried (magnesium sulfate) and concentrated to give an oil. Trituration with cold ethyl acetate gave 33 mg 5-ethyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s-indacene-7- carboxylic acid (3-methyl-isoxazol-5-yl)amide (14%) as a light tan solid, mp 186-8°C. ¹H-NMR (CDCI₃): δ 7.9 (s, 1H), 7.7 (s, 1 H), 7.1 (s, 1H), 6.8 (s, 1 H), 6.2 (s, 1 H), 4.5 (s, 1H), 3.9 (q, 2H), 2.3 (s, 3H), 1.3 (t, 3H). MS (m/z, %): 326 (M⁺+1 , 50), 243 (100).

Example 10

5-Ethyl-6-oxo-6.7-dihvdro-5H-1 -oxa-5-aza-s-indacene-7-carboxylic acid pyridin-3- ylamide (Compound 10)

A mixture of 5-ethyl-6-oxo-6,7-dihydro-5H-1-oxa-s-indacene-7-carboxylic acid ethyl ester (250 mg. 0.9 mmol) and 3-aminopyridine (86 mg, 0.9 mmol.) in benzene (40 ml) was refluxed over a Dean-Stark trap for 1 hour. The mixture was cooled to room temperature, diluted with hexanes and the resulting precipitate filtered. The filtrant was dissolved in methanol: chloroform (1:9), hexanes added and the resulting precipitate filtered and dried to give 219 mg 5-ethyl-6-oxo-6,7-dihydro-5H-1-oxa-5- aza-s-indacene-7-carboxylic acid pyridin-3-ylamide (75%) as a red solid, mp 175-8°C. ¹H-NMR (CDCI₃ + 5% CD₃OD): δ 8.7 (s, 1H), 8.3 (d, 1 H), 8.2 (d, 1 H), 7.9 (s, 1 H), 7.7 (s, 1H), 7.3 (m, 1H), 7.1 (s, 1H), 6.8 (s, 1H), 3.9 (q, 2H), 1.3 (t, 3H). MS (m/z, %): 326 (M⁺+1 , 50), 243 (100).

Example 11

5-Methyl-6-oxo-6.7-dihydro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid (5-methyl- thiazol-2-yl)amide (Compound 11 )

A suitable preparation of this compound has been previously documented in patent U.S. Patent No. 4,695,571 , Example 4.

Example 12

5-Ethyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-indacene-7-carboxylic acid (5-methyl-π .3.41thiadiazol-2-yl)-amide (Compound 12)

Example 13

5-Ethyl-6-oxo-6,7-dihvdro-5H-1-oxa-5-aza-indacene-7-carboxylic acid (5-trifluoromethyl-H ,3.41thiadiazol-2-yl)-amide (Compound 13)

Example 14

5-Ethyl-6-oxo-6,7-dihydro-5H-1-thia-5-aza-indacene-7-carboxylic acidH ,3.41thiadiazol-2-ylamide (Compound 14)

A mixture 5-ethyl-6-oxo-6,7-dihydro-5H-1-thia-5-aza-s-indacene-7-carboxylic acid ethyl ester (300 mg, 1.0 mmol.) and 2-amino-1 ,3,4-thiadiazole (52 mg, 0.5 mmol.) in benzene (30 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 24 hours. The reaction mixture was filtered and the filtrate poured into 1 N hydrochloric acid (100 ml) and extracted with methylene chloride (2 X

100 ml). The organic extracts were combined, washed with 1N hydrochloric acid (1 X 40 ml), water (1 X 40 ml), dried (magnesium sulfate) and concentrated to give an oil.

Crystallization from 2-propyl ether/methylene chloride gave 27 mg 5-ethyl-6-oxo-6,7- dihydro-5H-1-thia-5-aza-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2-ylamide (15%) as a dark tan solid, mp 206-8°C. An additional 25 mg 5-ethyl-6-oxo-6,7-dihydro-5H-1- thia-5-aza-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2-ylamide (14%) was obtained as a second crop, mp 204-6°C. ¹H-NMR (CDCI₃): δ 8.9 (s, 1 H), 8.3 (s, 1H), 7.6 (d,

1H), 7.4 (d, 1H), 7.3 (s, 1H), 4.5 (s, 1H), 3.9 (q, 2H), 1.3 (t, 3H). MS (m/z, %): 345

(M⁺+1 , 50), 218 (100).

Example 15

5-Ethyl-2-methyl-6-oxo-6,7-dihydro-5H-1-oxa-3,5-diaza-s-indacene-7-carboxylic acidH .3,41thiadiazol-2-ylamide (Compound 15)

A mixture 5-ethyl-2-methyl-6-oxo-6,7-dihydro-5H-1 -oxa-3,5-diaza-s-indacene-7- carboxylic acid ethyl ester (150 mg, 0.5 mmol.) and 2-amino-1 ,3,4-thiadiazole (63 mg, 0.6 mmol.) in benzene (15 ml) was refluxed over a soxhlet extractor filled with activated 4A molecular sieves for 2 hours. The reaction mixture was filtered and the filtrate concentrated to give a solid. Trituration with hot acetone gave 15 mg 5-ethyl-2- methyl-6-oxo-6,7-dihydro-5H-1-oxa-3,5-diaza-s-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2-ylamide (8%) as a solid, mp 218-220°C. ¹H-NMR (CDCI₃): δ 8.9 (s, 1 H), 7.9 (s, 1 H), 7.2 (s, 1 H), 4.6 (s, 1H), 3.9 (q, 2H), 2.7 (s, 3H), 1.3 (t, 3H). MS (m/z, %): 344 (M⁺, 100).

Example 16

5-Ethyl-6-oxo-6,7-dihvdro-5H-1-oxa-5-aza-s-indacene-7-carboxylic acid (5-ethyl-π ,3,41thiadiazol-2-yl)-amide (Compound 16)

The preparation of this compound has been previously documented in patent U.S. Patent No. 4,695,571 , Example 4.

Example 17

5-Ethyl-6-oxo-6,7-dihvdro-5H-1 -oxa-5-aza-s-indacene-7-carboxylic acid thiazol-2- ylamide (Compound 17)

A suitable preparation of this compound has been previously documented in patent U.S. Patent No. 4,695,571 , Example 4. The preparation of Compounds 18-24 could be prepared by those of skill in the art based on the above teaching and/or by reference to the literature. Biological Examples PDE II Inhibition Activity Compounds 1-24 of Formula I as described in Table I when assayed exhibited PDE2 inhibition activity. Table I. PDE2 Inhibition Activity

> PDE2 Assays a. Isolation of PDE2 Enzyme PDE2 enzyme was isolated from human platelets with approximately 1.4L of blood from multiple donors used to make the platelet pellets. Platelets were resuspended in approximately 75ml of Lysis Buffer [20mM Tris pH7.2, 5mM MgCI₂, 250mM Sucrose, 1 mM DTT, 1 μ!/2ml Sigma Protease inhibitor #8340] and lysed by sonication at 4°C, with 3 rounds of 1 minute bursts then spun at 4°C overnight at 100,000x g. Cleared lysates are loaded to AKTA explorer FPLC (AP Biotech) in a series of three chromatographic separations, with an average load volume of 25ml. A 5ml HiTrap Q anion exchange column (AP Biotech) is used. A buffer [20mM Tris pH7.2, 5mM MgCI₂, 1 μl/2ml Sigma Protease inhibitor #8340] is mixed with a gradient of B Buffer [20mM Tris pH7.2, 500mM NaCI, 5mM MgCI₂, 1μl/2ml Sigma Protease inhibitor #8340] over 20 column volumes from an initial B Buffer concentration of 0% to a final concentration of 100%. cGMP-hydrolyzing peaks are noted with average resolution at low salt- 125mM NaCI (PDE5) and high salt- 325mM NaCI (PDE2). Two major cGMP activity fractions (PDE5 and PDE2) are isolated and pooled separately. The PDE2 pooled fraction total is approximately 40ml and dispensed in cryovials of 200ul/vial and placed in -80°C storage. Preferably, recombinant production of PDE2 enzyme may be produced as previously published for use in binding assays noted below. b. PDE2 Binding Assay The activity of the test substances on human recombinant produced or isolated PDE2, and other PDE's is determined using the [³H]cAMP scintillation proximity assay (SPA) kits from Amersham International (Little Chalfont, England). The SPA assays were performed using 96 well plates. The PDE SPA yttrium silicate beads (Amersham Biosciences®) bind preferentially to the linear nucleotide, GMP, compared to the cyclic nucleotide, cGMP. ³H-cGMP is added to the reaction and when the product, ³H-GMP, is in close proximity to the beads, the scintillant within the bead is excited, which is detected using a Packard scintillation counter. The enzyme concentration used is in the linear range and the K_m of the enzyme was determined (15 μ). The final substrate concentration is <1/3 of K_m (1 μM) so that IC₅₀ values would approximate the Kj values. The assay was validated using literature compounds as controls before testing the representative compounds of the present invention. In an assay system for inhibition of 5-lipoxygenase (5-LO) (Coffey, M. et al., J. Biol. Chem. 1992, 267, 550) and cyclooxygenase (COX-1) (Mitchell, J. A. et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 11693), 5-methyl-6-oxo-6,7-dihydro-5H-1- oxa-5-aza-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2-ylamide (Example 2) showed no significant activity (<40% inhibition @ 10 μM). Thus, 5-methyl-6-oxo-6,7- dihydro-5H-1 -oxa-5-aza-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2-ylamide (Example 2) selectivity inhibits cGMP stimulated Phosphodiestease Type II (PDE2) enzyme over that of 5-lipoxygenase (5-LO) and cyclooxygenase (COX-1 ) and therefore, is expected to lack anti-inflammatory activity through the later pathway. Phosphodiesterase catalytic activity measurements obtained in the presence of the test compound and those obtained in the absence of the test compound are compared and the IC₅₀ value is determined. The IC₅o value represents the concentration of the test compound tested which leads to a 50% inhibition of the ^• cAMP hydrolysis, as compared with the cAMP hydrolysis obtained in control experiments performed in the absence of said test compound. The IC₅₀ values may be calculated using a sigmoidal model with Hill slope as known by those skilled in the art. c. Assay for PDE2 Activity and Determination of IC₅₀ Values Assays to determine the specific activity of PDE2s are known in the art, and may be used to identify the PDE2 inhibitors useful in the therapeutic methods or administration. The PDE activity was measured with an off line liquid scintillation spectrometry using HPLC. The reaction mixture consisted of (final concentrations): 1 μM c[8^"3H]AMP or 1 μM cβ^KIGMP (9.25 GBq/mmol) (approximately 35,000 cpm), 1 μM cGMP (if existing), 5 mM MgCI₂, 5 x 10^"7M to 10^"3M of inhibitor (if usable) and 20 mM tris-HCI, pH 7.5, in a total volume of 200 μl. The enzyme activity was measured at 25° C. with 10 minute incubations. The reaction was started with the addition of 50 μl of enzyme solution and terminated through injection of 20 μl of 60% perchloric acid. Aliquots of 20 μl of acidic supernatant were siphoned off and injected into an automated HPLC system (column LiChrospher RP-18e) (250x4 mm; 5 μm packing). The radioactive substrates and the products of the PDE reaction were determined quantitatively using a RACK-BETA 1219 liquid scintillation counter (LKB Wallac, Freiburg, FRG). The IC₅₀ values were determined with 1μM cAMP or cGMP using the peak fractions. The data were fitted with four parameters with the aid of the sigmoidal logistic function. Using the previously described PDE2 enzyme preferably recombinantly produced or isolated from human platelets and the method for assaying test compounds for inhibition of the enzyme, for the assay of 5-Methyl-6-oxo-6,7-dihydro- 5H-1-oxa-5-aza-indacene-7-carboxylic acid[1 ,3,4]thiadiazol-2-ylamide (Example 2, Compound 2) resulted in an IC₅₀=0.1 μM and for EHNA (Erythro-9-(2-hydroxy-3- nonyl)adenine, resulted in an IC₅₀=1.7 μM. In further comparison, IC₅₀ values of Compound 2 against purified PDE3 subtypes and PDE4 subtypes were all greater than 16.0 μM (data not shown). PDE2 Expression in Normal and Diseased Human Tissues Expression profiling of PDE2 was performed against a panel of RNAs from normal and diseased human tissues. The RNA panel is composed of samples from three donors per tissue and each RT-PCR reaction is performed with cDNA represents 40 ng of total RNA. A standard curve was run using varying quantities of PDE2 plasmid to quantify the amount of cDNA copies per sample. The average expression levels per tissue were calculated and binned into high (> 10⁴ copies), medium (10³ to 10⁴ copies), and low (>10³ copies) expression categories. The data for this experiment is provided in Table II below and would support alternate sites of action for PDE2 inhibitors. Table II. PDE2 Human Tissue Expression

All periodical literature references and patents and patent applications noted in this disclosure are incorporated herein by reference in their entireties.

Claims

What is claimed is:

1. A method for inhibiting PDE2 in mammals, the method comprising administering to said mammal a therapeutically effective amount of a compound of Formula I:

Formula I

where R¹ is C C₆ alkyl;

R² is hydrogen or C C₆ alkyl; R³ is a heterocyde selected from a group consisting of thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isooxazolyl, oxadiazolyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl, optionally substituted with hydrogen atom, C1-C3 alkyl or haloalkyl group.

X is sulfur or oxygen; and

Y is carbon or nitrogen; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

2. The method of claim 1 wherein R³ includes one of 2-thiadiazolyl; 5-methyl-2- thiadiazolyl; 5-ethyl-2-thiadiazolyl; 5-trifluromethyl-2-thiadiazolyl; 2-thiazolyl; 5-methyl- 2-thiazolyl; 3-methyl-5-isoxazolyl; 2-pyrazine; 5-pyrimidine; or 3-pyridine; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

3. The method of claim 1 wherein R¹ is methyl or ethyl; R₂ is hydrogen or methyl; R³ is a heterocyde selected from a group consisting of 2-pyrazine; 5-pyrimidine; and 3- pyridine; X is sulfur or oxygen; and Y is carbon or nitrogen; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

4. The method of claim 1 wherein R¹ is methyl, R² is hydrogen and R³ is 5-methyl-2- thiadiazolyl; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

5. The method of claim 1 wherein R¹ is methyl, R²is hydrogen and R³ is 2- thiadiazolyl; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

6. The method of claim 1 wherein R¹ is methyl, R² is hydrogen and R³ is 2-thiadiazolyl; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

7. The method of claim 1 wherein R¹ is ethyl, R² is hydrogen and R³ is 2-thiadiazolyl; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

8. The method of claim 1 wherein R¹ is ethyl, R² is methyl, and R³ is 2-thiadiazoyl; or a tautomeric isomer thereof; or a pharmaceutically acceptable salt; ester; carbonate or prodrug thereof.

9. The method of any one of claims 1 to 8 for the treatment of disease states or disorders mediated by PDE2.

10. A compound comprising 5-Methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza- indacene-7-carboxylic acid [1 ,3,4]thiadiazol-2-ylamide or a tautomeric isomer thereof; or a pharmaceutically acceptable salt, ester, carbonate or prodrug thereof.

11. A compound comprising 5-Methyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza- indacene-7-carboxylic acid(5-methyl-[1 ,3,4]thiadiazol-2-yl)-amide or a tautomeric isomer thereof; or a pharmaceutically acceptable salt, ester, carbonate or prodrug thereof.

12. A compound comprising 5-Ethyl-6-oxo-6,7-dihydro-5H-1-oxa-5-aza-s- indacene-7-carboxylic acid pyrazine-2-ylamide or a tautomeric isomer thereof; or a pharmaceutically acceptable salt, ester, carbonate or prodrug thereof.

13. A kit comprising: a) a pharmaceutical composition comprising an oxindole derivative having PDE2 inhibiting activity; and b) instructions for use of said composition.

14. A kit of claim 27 wherein said composition is an oxindole derivative of Formula I or a tautomeric isomer, pharmaceutically acceptable salt, ester, carbonate or prodrug thereof.