WO2006008644A1

WO2006008644A1 - Antidiabetic compounds

Info

Publication number: WO2006008644A1
Application number: PCT/IB2005/002159
Authority: WO
Inventors: Shawn Cabral; Bernard Hulin; David Walter Piotrowski
Original assignee: Pfizer Products Inc
Current assignee: Pfizer Products Inc
Priority date: 2004-07-13
Filing date: 2005-07-01
Publication date: 2006-01-26
Anticipated expiration: 2007-01-13

Abstract

The invention provides compounds of formula (I), prodrugs and stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds, prodrugs, and stereoisomers, wherein R1 is hydrogen, -(C1-C6)alkyl, -(C1-C6)alkoxy, -(C1-C6)arylalkyl, -NRaRb, hydroxy, cyano, arly or heteroaryl, wherein said -(C1-C6)alkyl, said aryl, or said heteroaryl is optionally substituted independently with one to three -COOH, -C(O)(C1-C6)alkyl, -C(O)NRaRb cyano, halogen, nitro, trifluoromethyl, -(C1-C6)alkyl, --(C1-C6)alkoxy, -(C3-C6)cycloalkyl or phenyl, wherein: Ra and Rb are, independently, hydrogen, -(C1-C6)alkyl, aryl or heteroaryl or Ra and Rb, taken together with the nitrogen atom to which they are attached, form a four- to six- membered heterocyclic ring, wherein said ring optionally incorporates an additional one or two nitrogen, oxygen or sulfur ring heteroatoms; R2 and R3 are, independently, hydrogen, halogen, -(C1-C6)alkyl, or -(C3-C8)cycloalkyl; R4 is (i) hydrogen, (ii) - COOH, (iii) -C(O)(C1-C6)alkoxy, (iv) -C(O)(C1-C6)alkyl, (v) -C(O)NRaRb, (vi) cyano or (vii) -(C1-C6)alkyl, optionally substituted with one to six halogen atoms, -(C1-C6)alkoxy, cyano, hydroxy, or -NRaRb, (viii) -(C3-C6)cycloalkyl, (ix) -(C1-C6)arylalkyl, (x) aryl, or (xi) heteroaryl; Q is a covalent bond, -C(O)-, or -SO2-; HET is heterocycloalkyl ring, moiety, optionally substituted independently with: (A) one to four -(C1-C6)alkyl optionally substituted with one to six halogen atoms, -(C1-C6)alkoxy, cyano, halogen, hydroxy, or -NRaRb, or (B) -(C1-C6)aralkyl, optionally substituted with on to six halogen atoms, -(C1-C6)alkoxy, cyano, halogen, hydroxy, or -NRaRb; n is zero or one; X is hydrogen or cyano; and Y is -CH2-, -S-, -S(O)-, or -SO2; provided that R2, R3 and R4 are not all hydrogen; compositions thereof; and uses thereof in treating diabetic complications including diabetic neuropathy diabetic nephropathy, diabetic microangiopathy, and the like.

Description

ANTIDIABETIC COMPOUNDS

FIELD OF THE INVENTION

The invention relates to selective inhibitors of the enzyme dipeptidyl peptidase-IV (DPP-IV), pharmaceutical compositions thereof, and uses thereof for treating diseases associated with proteins that are subject to processing by DPP-IV, such as Type 2 diabetes, metabolic syndrome (Syndrome X or insulin resistance syndrome), hyperglycemia, impaired glucose tolerance, glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, Type 1 diabetes, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, acute coronary syndrome, infertility due to polycystic ovary syndrome, short bowel syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, eating disorders, chronic pain, alcohol addiction, diseases associated with intestinal motility, ulcers, irritable bowel syndrome, inflammatory bowel syndrome, and preventing disease progression in Type 2 diabetes.

BACKGROUND OF THE INVENTION

DPP-IV (EC 3.4.14.5) is a serine protease that preferentially hydrolyzes an N-terminal dipeptide from proteins having proline or alanine in the 2-position. The physiological roles of DPP-IV have not been fully elucidated, but it is believed to be involved in diabetes, glucose tolerance, obesity, appetite regulation, lipidemia, osteoporosis, neuropeptide metabolism and T-cell activation, among others.

DPP-IV has been implicated in the control of glucose homeostasis because its substrates include the incretin peptides glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). Cleavage of the N-terminal amino acids from these peptides renders them functionally inactive. GLP-1 has been shown to be an effective anti-diabetic therapy in Type 2 diabetic patients and to reduce the meal-related insulin requirement in Type 1 diabetic patients. GLP-1 and/or GIP are believed to regulate satiety, lipidemia and osteogenesis. Exogenous GLP-1 has been proposed as a treatment for patients suffering from acute coronary syndrome, angina, and ischemic heart disease.

Administration of DPP-IV inhibitors in vivo prevents N-terminal degradation of GLP-1 and GIP, resulting in higher circulating concentrations of these peptides, increased insulin secretion and improved glucose tolerance. On the basis of these observations, DPP-IV inhibitors are regarded as agents for the treatment of Type 2 diabetes, a disease in which glucose tolerance is impaired. In addition, treatment with DPP-IV inhibitors prevents degradation of Neuropeptide Y (NPY), a peptide associated with a variety of central nervous system disorders, and Peptide YY which has been linked to gastrointestinal conditions such as ulcers, irritable bowel disease, and inflammatory bowel disease. In spite of the early discovery of insulin and its subsequent widespread use in the treatment of diabetes, and the later discovery of and use of sulfonylureas (e.g. chlorpropamide, tolbutamide, acetohexamide, biguanides (e.g., phenformin), metformin, thiazolidinediones (e.g., rosiglitazone), and pioglitazone as oral hypoglycemic agents, the treatment of diabetes remains less than satisfactory. The use of insulin, necessary in Type 1 diabetic patients and about 10% of Type 2 diabetic patients in whom currently available oral hypoglycemic agents are ineffective, requires multiple daily doses, usually by self-injection. Determination of the appropriate dosage of insulin necessitates frequent estimations of the glucose concentration in urine or blood. The administration of an excess dose of insulin causes hypoglycemia, with consequences ranging from mild abnormalities in blood glucose to coma, or even death.

Treatment of Type 2 diabetes usually comprises a combination of diet, exercise, oral agents, and in more severe cases, insulin. However, the clinically available hypoglycemics can have side effects that limit their use. A continuing need for hypoglycemic agents, which may have fewer side effects or succeed where others fail, is clearly evident.

Poorly controlled hyperglycemia is a direct cause of the multiplicity of complications (cataracts, neuropathy, nephropathy, retinopathy, cardiomyopathy) that characterize advanced Type 2 diabetes. In addition, Type 2 diabetes is a comorbid disease that frequently confounds hyperlipidemia, atherosclerosis and hypertension, adding significantly to the overall morbidity and mortality attributable to those diseases.

Epidemiological evidence has firmly established hyperlipidemia as a primary risk factor for cardiovascular disease (CVD) due to atherosclerosis. Atherosclerosis is recognized to be a leading cause of death in the United States and Western Europe. CVD is especially prevalent among diabetic subjects, at least in part because of the existence of multiple independent risk factors such as glucose intolerance, left ventricular hypertrophy and hypertension in this population. Successful treatment of hyperlipidemia in the general population, and in diabetic subjects in particular, is therefore of exceptional medical importance. Hypertension (high blood pressure) is a condition that can occur in many patients in whom the causative agent or disorder is unknown. Such "essential" hypertension is often associated with disorders such as obesity, diabetes, and hypertriglyceridemia and it is known that hypertension is positively associated with heart failure, renal failure, and stroke. Hypertension can also contribute to the development of atherosclerosis and coronary disease. Hypertension, together with insulin resistance and hyperlipidemia, comprise the constellation of symptoms that characterize metabolic syndrome, also known as insulin resistance syndrome (IRS) and Syndrome X.

Obesity is a well-known and common risk factor for the development of atherosclerosis, hypertension, and diabetes. The incidence of obesity and its related sequelae is increasing worldwide.

Currently, few pharmacological agents are available that reduce adiposity effectively and acceptably. Osteoporosis is a progressive systemic disease characterized by low bone density and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporosis and the consequences of compromised bone strength are a significant cause of frailty, and of increased morbidity and mortality.

Heart disease is a major health problem throughout the world. Myocardial infarctions are a significant source of mortality among those individuals with heart disease. Acute coronary syndrome denotes patients who have or are at high risk of developing an acute myocardial infarction (Ml). Though there are therapies available for the treatment of diabetes, hyperglycemia, hyperlipidemia, hypertension, obesity, and osteoporosis there is a continuing need for alternative and improved therapies. Various indications for DPP-IV inhibitors are discussed in Augustyns, et al., Curr. Medicinal

Chem., 6, 311 (1999); Ohnuki, et al., Drugs of the Future, 1999, 24, 665-670 (1999); Villhauer, et al., Annual Reports in Medicinal Chemistry, 3JJ₁ 191-200 (2001); Drucker, Expert Opin. Invest. Drugs, V≥, 87- 100 (2003); and Weideman, et al., Curr. Opin. Invest. Drugs, 4, 412-420 (2003).

SUMMARY QF THE INVENTION The invention provides compounds of formula (I),

prodrugs and stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds, prodrugs, and stereoisomers, wherein R¹, R², R³, R⁴, HET, n, Q, X, and Y are as described hereinbelow; compositions thereof; and uses thereof.

DETAILED DESCRIPTION OF THE INVENTION The invention provides compounds of formula (I),

the prodrugs and stereoisomers thereof, and the pharmaceutically acceptable salts of said compounds, prodrugs, and stereoisomers, wherein:

R¹ is hydrogen, -(CrC₆)aIkyl, -(CrC₆)alkoxy, -(C_rC₆)arylalkyl, -NR^aR^b, hydroxy, cyano, aryl, or heteroaryl, wherein said -(Ci-C₆)alkyl, said aryl, or said heteroaryl is optionally substituted independently with one to three -COOH, -C(O)(C_rC₆)alkoxy, -C(O)(Ci-C₆)alkyl, -C(O)NR^aR^b, cyano, halogen, nitro, trifluoromethyl, -(Ci-C₆)alkyl, -(d-C₆)alkoxy, -(C₃-C₆)cycloalkyl, or phenyl, wherein: R^a and R^bare, independently, hydrogen, -(Ci-C₆)alkyl, aryl, or heteroaryl, or

R^a and R^b, taken together with the nitrogen atom to which they are attached, form a four- to six- membered heterocyclic ring, wherein said ring optionally incorporates an additional one or two nitrogen, oxygen, or sulfur ring heteroatoms;

R² and R³ are, independently, hydrogen, halogen, -(C_rC₆)alkyl, or -(C₃-C₈)cycloalkyl; R⁴ is (i) hydrogen, (ii) -COOH, (iii) -C(O) (C₁ -C₆)alkoxy, (iv) -C(O)(C_rC₆)alkyl, (v) -C(O)NR^aR^b, (vi) cyano, or (vii) -(Ci-C₆)alkyl, optionally substituted with one to six halogen atoms, -(Ci-C₆)alkoxy, cyano, hydroxy, or -NR^aR^b, (viii) -(C₃-C₆)cycloalkyl, (ix) -(Ci-C₆)arylalkyl, (x) aryl, or (xi) heteroaryl;

Q is a covalent bond, -C(O)-, or -SO₂-;

HET is a heterocycloalkyl ring moiety, optionally substituted independently with: (A) one to four -(Gr C₆)alkyl, optionally substituted with one to six halogen atoms, -(CrC₆)alkoxy, cyano, halogen, hydroxy, or -NR^aR^b, or (B) -(C_rC₆)arylalkyl, optionally substituted with one to six halogen atoms, -(CrC₆)alkoxy, cyano, halogen, hydroxy, or -NR^aR^b; - A -

n is zero or one;

X is hydrogen or cyano; and

Y is -CH₂-, -S-, -S(O)-, or -SO₂-; provided that R², R³ and R⁴ are not all hydrogen.

Generally preferred stereoisomeric configurations of the compounds of formula (I), the prodrugs thereof, and the pharmaceutically acceptable salts of the compounds and prodrugs, comprise the (2S) and (2S.4S) configurations, depicted in formulae (Ia) and (Ib) respectively, hereinbelow.

A generally preferred subgroup of formula (Ib) compounds comprises those compounds wherein: R¹ is aryl or heteroaryl, wherein said aryl or heteroaryl is optionally substituted independently with one to three cyano, halogen, nitro, trifluoromethyl, -(CrC₆)alkyl, -(CrC₆)alkoxy, -(C₃-C₆)cycloalkyl, or phenyl;

R², R³ and R⁴ are, independently, hydrogen or -(Ci-C₆)alkyl;

Q is a covalent bond, -C(O)-, or -SO₂-;

HET is azetidinyl, piperazinyl, piperidinyl, or pyrrolidinyl; n is zero or one;

X is hydrogen or cyano; and

Y is -CH₂- or -S-.

An especially preferred subgroup of the compounds of formula (Ib) comprises those compounds wherein: R¹ is pyrazinyl, pyridyl, pyrimidyl, or quinolyl, wherein said pyrazinyl, pyridyl, pyrimidyl, or quinoyl is optionally substituted independently with one to three cyano, halogen, nitro, trifluoromethyl, -(CτC₆)aIkyl,

-(CrC₆)alkoxy, -(C₃-C₆)cycloalkyl, or phenyl;

R² is -(C_rC₆)alkyI;

R³ and R⁴ are hydrogen; Q is a covalent bond;

HET is piperazinyl; n is one;

X is hydrogen or cyano; and

Y is -CH₂- or -S-. Even more preferably, for compounds of formula (Ib), R² is methyl; and

Y is -CH₂- .

The compounds and intermediates of the present invention may be named according to either the IUPAC (International Union for Pure and Applied Chemistry) or CAS (Chemical Abstracts Service) nomenclature systems. The carbon atom content of the various hydrocarbon-containing moieties may be indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix "- (C_a-C_b)alkyl" indicates an alkyl moiety of the integer "a" to "b" carbon atoms, inclusive.

The term "alkoxy" denotes a straight or branched, monovalent, saturated chains of carbon atoms bonded to an oxygen atom, wherein the alkoxy group optionally incorporates one or more double or triple bonds, or a combination of double bonds and triple bonds. Examples of alkoxy groups include methoxy, ethoxy, propoxy, /so-butoxy, terf-butoxy, and the like.

The term "alkyl" denotes a straight or branched chain of carbon atoms, wherein the alkyl group optionally incorporates one or more double or triple bonds, or a combination of double bonds and triple bonds. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, vinyl, allyl, 2- methylpropenyl, 2-butenyl, 1 ,3-butadienyl, ethynyl, propargyl, and the like.

The term "aryl" denotes a monocyclic or polycyclic aromatic hydrocarbon group, for example, anthracenyl, fluorenyl, naphthyl, phenanthrenyl, phenyl, and the like.

The term "arylalkyl" means an alkyl group, as defined hereinabove, wherein at least one of the hydrogen atoms thereof has been substituted with an aryl group, also as defined hereinabove. Examples of arylalkyl groups include, inter alia, benzyl groups.

The term "halogen" means chloro, bromo, fluoro, or iodo.

The term "heterocycloalkyl", as employed with reference to HET hereinabove, refers to a saturated four- to eight-membered heterocyclic ring system, optionally fused to a five- or six-membered aromatic or heteroaromatic ring system. Examplary, non-limiting heterocycloalkyl groups comprise homopiperazinyl, piperazinyl, piperidinyl, pyrrolidinyl, azetidinyl, 2-aza-bicyclo[2.2.1]heptanyl, 3-aza- bicyclo[3.1.0]hexanyl, 2,5-diaza-bicyclo[2.2.1]heptanyl, 5,6,7,8-tetrahydro-2H-imidazo[1 ,2-a]pyrazinyl, 5,6,7,8-tetrahydro[1 ,2,4]triazolo[4,3-a]pyrazinyl, 3,4,5,6-tetrahydro-2H-[1 ,2']bipyrazinyl, 4,5,6,7- tetrahydropyrazolo[1 ,5-a]pyrazinyl, 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl, 5,6,7,8- tetrahydropyrido[4,3-d]pyrimidinyl, octahydropyrrolo[3,4-b]pyrrolyl, octahydropyrrolo[3,4-c]pyrrolyl, 6- azabicyclo[3.2.1]octanyl, 3,8-diazabicyclo[3.2.1]octanyl, 2,3-dihydrospiro[indene-1 ,4'-piperidinyl], spiro[indene-1 ,4'-piperidinyl], 1 -oxa-8-azaspiro[4.5]decanyl, 8-azabicyclo[3.2.1]octanyl, 2,3,4,5- tetrahydrobenzo[f][1 ,4]oxazepinyl, hexahydro-2H-pyrrolo[3,4-d]isothiazolyl-1 ,1 -dioxide, 2,7- diazaspiro[4.4]nonanyl, 6,7,8,9-tetrahydro-5H-[1 ,2,4]triazolo[4,3-g][1 ,4]diazepinyl, 5,6-dihydro-8H- imidazo[1 ,2-a]pyrazinyl, 5,6-dihydro-8H-[1 ,2,4]triazolo[4,3-a]pyrazinyl, 7,8-dihydro-5H-pyrido[4,3- a]pyrimidinyl, and the like.

The term "heteroaryl" denotes a monocyclic or polycyclic aromatic heterocyclic ring system. Examples of heteroaryl groups comprise benzoisothiazolyl, benzisoxazolyl, benzooxazolyl, furyl, imidazolyl, indolyl, isothiazolyl, isoxadiazolyl, isoxazolyl, oxazolopyridyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidyl, pyrrolyl, quinolyl, quinoxalinyl, thiazolyl, thiadiazolyl, thienyl, triazinyl, 1 ,1- dioxo-1 H-1 ,2-benzoisothiazolyI, oxazolo[4,5-c]pyridyl, and the like.

The term "mammal" means animals including, for example, dogs, cats, cows, sheep, horses, and humans. Preferred mammals include humans of either gender.

The term "pharmaceutically acceptable" indicates that the designated carrier, vehicle, or diluent, and/or salt must be chemically and/or physically compatible with the other ingredients comprising the formulation and physiologically compatible with the recipient thereof. The term "prodrug" refers to a compound that is a drug precursor which, following administration, releases the drug in vivo via a chemical or physiological process (e.g., upon being brought to physiological pH or through enzymatic activity). A discussion of the preparation and use of prodrugs is provided by T. Higuchi and W. Stella, "Prodrugs as Novel Delivery Systems", Vol.14 of the ACS Symposium Series and in "Bioreversible Carriers in Drug Design", Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

The term "salts" refers to both organic and inorganic salts of a compound, prodrug, or stereoisomer of formula (I). Such salts can be prepared in situ during the final isolation and purification of a compound, or by separately reacting a compound, prodrug, or stereoisomer of formula (I) with a suitable organic or inorganic acid or base and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, besylate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts, and the like. These may also include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. For additional examples see, for example, Berge, et al., J. Pharm. ScL, 66, 1-19 (1977).

The term "substituted" means that a hydrogen atom on a molecule has been replaced with a different atom or functional group. The atom or functional group replacing the hydrogen atom is denoted as a "substituent."

The symbol "-" represents a covalent bond.

The phrase "reaction-inert solvent" or "inert solvent" refers to a solvent, or mixture of solvents, that does not interact with starting materials, reagents, intermediates, or products in a manner that adversely affects their desired properties.

The terms "treating", "treated", or "treatment" as employed herein includes preventative (e.g., prophylactic), palliative, or curative use or result.

The compounds and prodrugs of formula (I) may contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds and prodrugs of formula (I) as well as mixtures thereof, including racemic mixtures, form part of the present invention. In addition, the present invention embraces all geometric and positional isomers. For example, if a compound or prodrug of formula (I) incorporates a double bond, both the cis- and trans- forms, as well as mixtures thereof, are embraced within the scope of the invention.

Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well-known to those of ordinary skill in the art, such as by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydroiyzing) the individual diastereomers to the corresponding pure enantiomers. Also, some of the compounds of formula (I) may be atropisomers (e.g., substituted biaryls) and are also considered as part of the invention. The compounds, prodrugs, and stereoisomers of formula (I) 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.

It is also possible that the compounds, prodrugs, and stereoisomers of formula (I) may exist as tautomeric isomers in equilibrium, and all such forms are embraced within the scope of the invention. The present invention also embraces isotopically-labeled compounds of formula (I), 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 formula (I) include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸0, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶CI, respectively. The compounds of formula (I), the prodrugs, and stereoisomers thereof, and the pharmaceutically acceptable salts thereof, that contain the aforementioned isotopes and/or other isotopes of the other atoms are intended to be within the scope of the instant invention. Certain isotopically-labeled compounds of formula (I), for example those compounds into which radioactive isotopes such as ³H and ¹⁴C are incorporated, 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 relative ease of preparation and facile detection. Furthermore, 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. The isotopically-labeled compounds of formula (I) can generally be prepared by carrying out procedures analogous to those disclosed in the Schemes and/or Examples set forth hereinbelow, by substituting an isotopically-labeled reagent for a non-isotopically-labeled reagent. In another aspect, the invention is directed to pharmaceutical compositions comprising an amount of a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of the compound, prodrug, or stereoisomer, and a pharmaceutically acceptable carrier, vehicle, or diluent.

In another aspect, the invention is directed to pharmaceutical compositions comprising amounts of: a) a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of the compound, prodrug, or stereoisomer; b) a second compound selected from an antidiabetic agent, a prodrug or pharmaceutically acceptable salt thereof, insulin or an analog thereof, insulinotropin, a biguanide, an α₂-antagonist, an imidazoline, a glitazone, an aldose reductase inhibitor, a glycogen phosphorylase inhibitor, a sorbitol dehydrogenase inhibitor, a fatty acid oxidation inhibitor, an α-glucosidase inhibitor, a β-agonist, a phosphodiesterase inhibitor, a lipid-lowering agent, an antiobesity agent, a vanadate, a vanadium complex, a peroxovanadium complex, an amylin antagonist, a glucagon antagonist, a growth hormone secretagogue, a gluconeogenesis inhibitor, a somatostatin analog, an antilipolytic agent, or an inhibitor of renal glucose; and c) a pharmaceutically acceptable carrier, vehicle, or diluent. In another aspect, the invention is directed to methods of inhibiting DPP-IV which methods comprise administering to a mammal in need of such inhibition a DPP-IV inhibiting amount of a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of the compound, prodrug or stereoisomer; a DPP-IV inhibiting amount of a pharmaceutical composition comprising a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer; and a pharmaceutically acceptable vehicle, carrier, or diluent; or a DPP-IV inhibiting amount of the pharmaceutical composition described above comprising a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer, the second compound, and a pharmaceutically acceptable carrier, vehicle, or diluent.

In another aspect, the invention is directed to methods of treating conditions mediated by DPP- IV in mammals in need of such treatment which methods comprise administering to the mammal a therapeutically effective amount of a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of the compound, prodrug or stereoisomer; a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer; and a pharmaceutically acceptable vehicle, carrier, or diluent; or a therapeutically effective amount of the pharmaceutical composition described above comprising a compound of formula (I), a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer, the second compound, and a pharmaceutically acceptable carrier, vehicle, or diluent.

Conditions treatable according to the instant methods are Type 2 diabetes, progression of disease in Type 2 diabetes, metabolic syndrome (Syndrome X and/or IRS), hyperglycemia, impaired glucose tolerance, glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, Type 1 diabetes, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, short stature due to bone growth deficiency, acute coronary syndrome, infertility due to polycystic ovary syndrome, short bowel syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, eating disorders, chronic pain, alcohol addiction, diseases associated with intestinal motility, ulcers, irritable bowel syndrome, or inflammatory bowel syndrome. Preferably, the condition is Type 2 diabetes.

The compounds of formula (I), the prodrugs and stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds, prodrugs, and stereoisomers, may be administered to mammals at dosage levels in the range of 0.01 mg/kg/day to 30 mg/kg/day, preferably 0.01 mg/kg/day to 1 mg/kg/day, in single or divided doses. Some variations in dosage will necessarily occur, however, depending on the condition of the subject being treated. The individual responsible for dosing will, in any event, determine the appropriate dose for the individual subject. Preferably, a single dose is administered orally.

The compounds of formula (I) may be administered to a subject in need of treatment by a variety of conventional routes of administration, including orally and parenterally, {e.g., intravenously, subcutaneously or intramedullary). Further, the pharmaceutical compositions of this invention may be administered intranasally, as a suppository, or using a "flash" formulation, i.e., allowing the medication to dissolve in the mouth without the need to use water. The appropriate dosage regimen, the amount of each dose administered and the intervals between doses of the compound will depend upon the compound of formula (I), or the prodrug or stereoisomer being used, the type of pharmaceutical compositions being used, the characteristics of the subject being treated, and/or the severity of the conditions being treated. Administration may be in single (e.g., once daily) or multiple doses or via constant infusion. The compounds may also be administered alone or, preferably, in combination with pharmaceutically acceptable carriers, vehicles, or diluents, in either single or multiple doses. Suitable pharmaceutical carriers, vehicles, and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. The pharmaceutical compositions formed by combining the compounds of this invention and the pharmaceutically acceptable carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like.

Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and/or calcium phosphate may be employed along with various disintegrants such as starch, alginic acid and/or certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and/or acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Generally preferred materials for oral include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired, the active pharmaceutical agent therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and/or combinations thereof. For parenteral administration, solutions in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solutions may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.

For intranasal administration or administration by inhalation, the compounds of formula (I) are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of a compound of this invention. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound or compounds of the invention and a suitable powder base such as lactose or starch. Methods of preparing pharmaceutical compositions with amounts of active ingredients are known, or will be apparent in light of this disclosure, to those skilled in this art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995).

The compounds of formula (I), the prodrugs and stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds, prodrugs, and stereoisomers, may be prepared according to the exemplary routes disclosed in the Schemes and Examples below, as well as by other conventional preparative procedures known, or apparent in light of the instant disclosure, to one of ordinary skill in the art. The methods disclosed in the instant Schemes and Examples are intended for purposes of exemplifying the instant invention, and are not to be construed in any manner as limitations thereon. In the discussions below, the following abbreviations are used: BOC (te/t-butoxycarbonyl), Cbz

(benzyloxycarbonyl), DMF (N,N-dimethylformamide), NMP (N-methyl-2-pyrrolidinone), DMAC (N, N- dimethylacetamide), DME (dimethoxyethane), DMSO (dimethylsulfoxide), TFA (trifluoroacetic acid), TEA (triethylamine), THF (tetrahydrofuran), DIPEA (diisopropylethylamine), EDC (1-(3-dimethylaminopropyl)- 3-carbodiimide)), DCC (dicyclohexylcarbodiimide), CDI (1 ,1 '-carbonyldiimidazole), HATU (O-(7- azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HOAT (1 -hydroxy-7- azabenzotriazole), HOBT (N-hydroxybenzotriazole), and EEDQ (2-ethoxy-1 -ethoxycarbonyl-1 ,2- dihydroquinoline).

The compounds of formula (I), the prodrugs and stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds, prodrugs, and stereoisomers, may be prepared according to the exemplary synthetic routes set forth in the Schemes and Examples hereinbelow, as well as by other conventional organic preparative methods known, or apparent in light of the instant disclosure, to one of ordinary skill in the relevant art. It is to be understood that the methods disclosed in the instant Schemes are intended for purposes of exemplifying the instant invention, and are not to be construed in any manner as limitations thereon. A generalized method for preparing the compounds of formula (I) is depicted in Scheme 1 hereinbelow.

Scheme 1

In Scheme 1 , a compound of formula (II), prepared as described in Scheme 2, wherein P represents a nitrogen-protecting group, is deprotected according to known methods. If P represents BOC, deprotection is typically effected by first treating (II), dissolved in an inert solvent such as ethyl acetate (EtOAc), ether, or dioxane, with cooling at a suitable temperature, such as about 0^°C, with gaseous acid (e.g., hydrogen chloride) for a suitable time, such as about 5 minutes to about an hour. The solution is allowed to warm to room temperature (RT), followed by stirring for an additional amount of time, typically an additional 30 minutes to about 16 hours. Preferably, the reaction mixture is stirred about 15 minutes, allowed to reach room temperature, then stirred an additional 30 minutes. Alternatively, (II) is dissolved in TFA and, after a suitable time (e.g., about 30 min to about 24 hours), excess TFA is removed in vacuo, and the residual product is triturated with a solvent such as ether. If P represents Cbz, deprotection may be performed by hydrogenolysis in the presence of catalyst, such as 10% palladium or palladium hydroxide, in a suitable solvent such as ethanol (EtOH) or EtOAc, at a pressure of about 30 psi to about 60 psi, for a sufficient period of time, usually overnight, at a temperature of between about 20° C and about 80° C. Preferably, hydrogenolysis is effected at a pressure of about 45 psi at RT.

The compounds of formula (II) may be prepared by coupling an appropriately-substituted carboxylic acid derivative (III) with an appropriately-substituted amine derivative (IV) as depicted hereinbelow in Scheme 2.

Scheme 2

The coupling is typically accomplished by combining (III) and (IV) in a reaction-inert solvent, preferably an aprotic solvent such as acetonitrile, dichloromethane, DMF, or chloroform. A coupling agent, such as EDC, HATU, DCC, EEDQ, CDI, or diethylphosphorylcyanide is then added, optionally in the presence of a base, such as TEA or pyridine, and an optional adjuvant, such as HOBT or HOAT. The coupling is typically effected at a temperature of between about 0° C and about 50° C, for a suitable time, such as from about one hour and about 24 hours, for example about 16 hours. For a discussion of other conditions useful for coupling carboxylic acids see Houben-Weyl, Vol. XV, Part II, E. Wunsch, Ed., G. Theime Verlag, (1974), Stuttgart; M. Bodansky, "Principles of Peptide Synthesis", Springer-Verlag Berlin (1984); and "The Peptides: Analysis, Synthesis and Biology" (ed. E. Gross and J. Meienhofer), VoIs. 1 -5 (Academic Press NY 1979-1983). The compounds of formulae (III) may be prepared by known methods or, alternatively, according to the exemplary preparative procedures described hereinbelow. The formula (IV) amines are commercially available or may be prepared according to known methods.

Alternatively, the compounds of formula (II) may be prepared as described below in Scheme 3.

Scheme 3

In Scheme 3, the compounds of formula (II) where R⁴ is hydrogen, are prepared by reductive amination of a protected ketone (V), prepared as described hereinbelow in Scheme 4, with an appropriately-substituted heterocycloalkylamine (Vl). Such amination reactions are well-known to one skilled in the art. See, for example, A.F. Abdel-Magid, et al., J. Org. Chem., 61, 3849 (1996); R. F. Borch, et al., J. Am. Chem. Soc, 93, 2897 (1971); and S. Bhattacharyya, et al., Synlett, 1079 (1995). The formula (Vl) amines are well-known in the relevant art and may be obtained commercially or prepared by known methods. See, for example, D.A. Horton, et al., Chem. Rev., 103, 893-930 (2003). Typically, (V) and (Vl) are condensed in the presence of a reducing agent such as sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, tetramethylammonium triacetoxyborohydride, or hydrogen in the presence of a catalyst (10% Pd/C, platinum oxide, etc.), optionally in the presence of an acid (e.g. acetic acid (AcOH), hydrochloric acid, etc.). The coupling is normally effected in a reaction-inert solvent, such as 1 ,2-dichloroethane, THF, DMF, EtOH, or methanol (MeOH). The reaction is performed at a suitable temperature, such as 0 to 50⁰C, for a suitable period of time, such as between about one to about 24 hours, for example, about 16 hours.

The compounds of formula (II), where R⁴ is other than hydrogen, may be generally prepared by treating (V) and (Vl) first with a cyanide source, followed by an organometallic reagent. The reaction is effected by first forming an enamine or iminium species by reacting ketone (V) with an amine (Vl) in the presence of a dehydrating agent, such as titanium (IV) isopropoxide or molecular sieves, followed by treatment with the cyanide source. Examples of suitable cyanide sources include (C₂H₅)₂AICN, acetone cyanohydrin, and trimethylsilylcyanide (TMSCN). The intermediate α-aminonitrile can be converted into (II), where R⁴ is other than hydrogen, by subsequent hydrolysis to a carboxylic acid and conversion thereof to an ester, amide, or ketone by known methods or by treatment with an organometallic reagent. Examples of suitable organometallic reagents include Grignard reagents such as methyl magnesium bromide or methyl magnesium chloride. Such transformations are well known to one skilled in the art. See, for example, E. Coderc, et al., Eur. J. Med. Chem., 30, 463 (1995) and A. Palani, et al., J. Med. Chem., 45, 3143 (2002).

The compounds of formula (V) may be prepared as described hereinbelow in Scheme 4, beginning with, as appropriate, commercially available carboxylic acid (VII), ketocarboxylic acid (IX), or ketoester (X).

Scheme 4

In Scheme 4, Step 1 , protected acid (VII) is coupled with amine (IV) as described hereinabove in Scheme 2 to afford alcohol (VIII).

In Scheme 4, Step 2, alcohol (VIII) is oxidized to ketone (Va) by treating (VIII) with an oxidizing agent in a reaction-inert solvent. Examples of appropriate oxidizing agents comprise pyridine/sulfur trioxide in DMSO; aqueous sodium hypochlorite in the presence of sodium bromide and TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) free radical catalyst; chromium-based reagents, such as chromium trioxide, pyridinium dichromate, or pyridinium chlorochromate; and oxalyl chloride in DMSO in the presence of a tertiary amine. Examples of reaction-inert solvents comprise dichloromethane, EtOAc, toluene, or pyridine. The oxidation is typically conducted at a temperature of between about -78⁰C and about 5O⁰C, for between about one and about 24 hours, for example, about 16 hours. Such oxidations are well known to one skilled in the art. See, for example, M. Tamaki, et al., J. Org. Chem., 66, 3593 (2001) and X-I. Qiu, et al., J. Org. Chem., 67, 7162 (2002).

In Scheme 4, Step 3, protected ketocarboxylic acid (IX) is first coupled with amine (IV), as described hereinabove in Scheme 2, to afford (Va), which is then alkylated (Step 4) to afford ketone (V). The alkylation is typically effected by first forming an enamine by reacting ketone (Va) with a secondary amine, for example, pyrrolidine, piperidine or morpholine, followed by treatment with an alkylating agent, optionally in the presence of a base, such as potassium carbonate. Typically, the reaction is effected in a solvent such as benzene, toluene, acetonitrile, or dioxane. Such conversions are well-known to one skilled in the art. See, for example, G. Stork, et al., J. Am. Chem. Soc, 85, 207 (1963); M.W. Holladay, et al., J. Med. Chem., 34, 455 (1991); and P. Barraclough, et al., Tetrahedron, 51_, 4195 (1995).

In Scheme 4, Step 5, protected ketoester (X), wherein R represents an alkyl or arylaikyl moiety, is alkylated under the conditions previously described in Step 4 to afford ketoester (Xl). In Scheme 4, Step 6, ketoester (Xl) is saponified to yield the corresponding carboxylic acid which, in Step 7, is coupled with an appropriately-substituted amine (IV), as previously described hereinabove in Scheme 2. The saponification step is typically accomplished by dissolving (Xl) in a water- miscible solvent, such as MeOH or EtOH, and water in the presence of a base, such as lithium hydroxide or sodium hydroxide. The saponification is effected at suitable temperature, such as between about 0 °C and about 100 ⁰C, preferably RT, for a suitable time, such as between about one and about 24 hours, for example, about 16 hours.

PREPARATIVE EXPERIMENTAL

EXAMPLES

The following Preparations and Examples are provided for purposes of exemplification and not limitation. Unless otherwise noted, all reagents employed were obtained commercially.

El - electron ionization

HPLC - high performance liquid chromatography hr - hour(s) min - minute(s)

MS - mass spectrometry

Preparation 1 ferf-butyl (2S)-4-Oxo-2-(pyrrolidin-1 -yl-carbonvπpyrrolidine-1 -carboxylate

1-(fert-Butoxycarbonyl)-4-oxo-L-proline (1.15 g, 5.0 mmol), HOBt (1.01 g, 7.5 mmol), EDC (1.15 g, 6.0 mmol), and pyrrolidine (0.42 mL, 5.0 mmol) were dissolved in 10 mL of dichloromethane. After stirring the reaction overnight, 15 mL of saturated sodium bicarbonate and 15 mL of dichloromethane were added. The aqueous layer was extracted with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by chromatography (Biotage^® Flash 4OM; A Dynax Corp.; Charlottesville, VA) eluting with EtOAc to afford the title compound 0.82 g (70% yield). MS (El) m/z= 283 (MH⁺). Preparation 2 te/t-butyl (2S)-4-Cvano-4-(4-phenyl-piperazin-1 -vD-2-(pyrrolidin-1 -ylcarbonvDpyrrolidine-1 -carboxylate To an ether solution of the title compound of Preparation 1 (0.50 g, 1.77 mmol) was added

TMSCN, followed by the addition of a catalytic amount of zinc iodide (~ 1 mg). After 15 min., a solution of phenylpiperazine (0.29 g, 1.77 mmol) in 6 mL of MeOH was added. The solution was refluxed for three hrs and then stirred at RT for 12 hr. The solution was concentrated to dryness and the product recrystallized from EtOAc/ether/hexanes to afford 630 mg (78.5% yield) of the title compound. MS (El) m/z= 454 (MH⁺).

The compounds of formula (I), the stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds and stereoisomers, may be prepared as described in the following Examples. Example 1 r(2S,4S)-4-Methyl-4-(4-phenyl-piperazin-1-yl)-pyrro]idin-2-yl1-pyrrolidin-1 -yl-methanone dihvdrochloride Step i ferf-butyl (2S.4S)-4-Methyl-4-(4-phenylpiperazin-1 -yl)-2-(pyrrolidin-1 -ylcarbonyl)pyrrolidine-1 -carboxylate Methyl magnesium bromide (0.5 ml_, 1.5 mmol., 1.4 M in THF/toluene) was added to a solution of the title compound of Preparation 2 (227 mg, 0.5 mmol) in 2 ml_ of toluene. After stirring the reaction mixture at RT for 18 hrs, the reaction was quenched with saturated ammonium chloride, extracted with EtOAc, and concentrated. The crude material was purified on a Shimadzu preparative HPLC (Shimadzu Corp.; Kyoto, Japan) (gradient 35% acetonitrile/water to 70% acetonitrile/water in 15 min at a flow rate of 100 mL/min.; 100 x 50 mm column; Waters Instrument Co., Milford, MA; λ = 254 nm) to afford 20 mg (9% yield) of the title compound. MS (El) m/z= 443 (MH⁺). Step 2

The product of Step 1 (21 mg, 0.047 mmol) was treated with 4N HCI in dioxane (2 ml_). After stirring overnight at RT, the mixture was concentrated to dryness to afford 17 mg of the title compound as a white solid. MS (El) m/z= 343 (MH⁺).

Using appropriate starting materials, the compounds of Examples 2 to 5, disclosed in Table 1 hereinbelow, were prepared in a manner analogous to that described in Example 1.

Table 1

Example 6

2-(4-r(3S.4R,5S)-4-Methyl-5-(pyrrolidine-1-carbonyl)-pyrrolidin-3-yll-piperazin-1-yl|-nicotinonitrile Step i (S)-(2,3.4.5,2',5'-Hexahvdro-1 '-H ,3'lbipyrroyl-5'-yl)-pyrrolidin-1 -yl-methanone

The title compound of Preparation 1 (5.6 g, 20 mmol) was dissolved in benzene (50 ml_) containing 4 A molecular sieves (7.9 g) and treated with pyrrolidine (2.0 ml_, 24 mmol). The solution was filtered and concentrated to dryness, leaving an orange foam (7.0 g, 100% yield). Step 2 (2S,3R)-3-Methyi-4-oxo-2-(pyrrolidine-1 -carbonyl)-pyrrolidine-1 -carboxylic acid ferf-butyl ester

A solution of the product of Step 1 (7.0 g, 20 mmol) in acetonitrile (100 ml_) was added to crushed potassium carbonate (5.2 g, 38 mmol) and treated with methyl iodide (1.5 ml_, 24 mmol). The mixture was heated to 90^s C for 16 hrs, cooled to RT, and concentrated. The residue was taken up in chloroform (150 mL) and a mixture of AcOH (5 ml_) and water (45 ml_) was added. After three hrs at RT, the layers were separated, the aqueous layer was extracted with chloroform (3 x 25 mL), and the combined organic phases were washed with saturated sodium bicarbonate (2 x 25 mL) and brine, dried over magnesium sulfate, and concentrated to a brown oil. The oil was dissolved in ether (75 ml_), filtered, and concentrated to a pale brown solid (0.97 g, 16% yield). Step 3

(2S,3R.4S)-4-[4-(3-Cvano-pyridin-2-vπ-piperazin-1 -yl1-3-methyl-2-(pyrrolidine-1 -carbonyl)-pyrrolidine-1- carboxylic acid terf-butyl ester

To a mixture of the product of Step 2 (74 mg, 0.25 mmol), AcOH (16 μl_), and sodium acetate (23 mg, 0.28 mmol) in MeOH (1 ml.) was added sodium cyanoborohydride (21 mg, 028 mmol). The mixture was stirred at RT for 65 hrs and then concentrated. The residue was taken up in EtOAc (20 ml.) and the solution was washed with 1 N sodium hydroxide (2 x 3ml_) and brine (5 ml_), dried over magnesium sulfate, and concentrated to dryness. The residue was purified by chromatography (Biotage^® Flash 4OM; 1% followed by 2% MeOH/dichloromethane) and the product was isolated as a yellow solid (13.5 mg, 11 % yield). Step 4

Hydrogen chloride (4M) in dioxane (0.5 ml_) was added to a solution of the product of Step 3 (13.5 mg, 0.029 mmol) in acetonitrile (1 ml_). After 16 hrs, the mixture was concentrated to dryness and the residue was triturated with ether (2 ml_). The title compound was obtained as a solid (12.5 mg, 97% yield). MS m/z 369 (MH⁺).

Using appropriate starting materials, the compounds of Examples 7 to 15, disclosed in Table 2 hereinbelow, were prepared in a manner analogous to that described in Example 6.

Table 2

BIOLOGICAL METHODOLOGIES

The utility of the compounds of formula (I), the prodrugs and stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds, prodrugs, and stereoisomers, in the treatment or prevention of the conditions enumerated hereinabove in mammals may be demonstrated in conventional assays known to one of ordinary skill in the relevant art, including the in vivo and in vitro assays described below. Such assays also provide a means by which the activities of the compounds of formula (I), the prodrugs, and stereoisomers thereof, and the pharmaceutically acceptable salts of the compounds, prodrugs, and stereoisomers, may be compared with the activities of other compounds.

In Vitro Assay for DPP-IV Inhibition DPP-IV inhibition may be demonstrated in vitro by the following assay, which is adapted from methods of Scharpe, et al., A. Clin. Chem., 2299 (1988) and Lodja, Z. Czechoslovak Medicine, 181

(1988). 150 μL of an enzyme-substrate solution is pipetted into microtiter wells of a polystyrene 96-well plate, and maintained at 4°C. The enzyme-substrate solution comprises 50 μM Gly-Pro-4-methoxy-β- naphthylamide hydrochloride in 5OmM Tris assay buffer pH 7.3 containing 0.1 M sodium chloride, 0.1% (v/v) Triton and 50 μU/mL DPP-IV (MP Biomedicals, Livermore, CA; DPP-IV 5 mU/mL stock). Five μL per well of the compound of formula (I) is added, bringing the final concentrations of the formula (I) compound to between three μM and 10 nM per well.

Controls. Enzyme is omitted from four wells, as a reagent blank. Five μL of 3 mM Diprotin A

(Bachem Bioscience, Inc.; King of Prussia, PA) is added to four wells as a positive quality control, providing a final Diprotin A concentration of 100 μM. To measure total enzyme activity {i.e., a negative control), without the influence of any compounds of formula (I), five μL of distilled water is added to four wells.

The entire assay is incubated overnight (between 14 and 18 hours) at 37 ⁰C. The reaction is quenched by adding 10 μL of Fast Blue B solution (0.5 mg/mL Fast Blue B in a buffer comprising 0.1 M sodium acetate pH 4.2 and 10% (v/v) Triton X-100 to each well, followed by shaking for approximately 5 min at room temperature. The plates may be analyzed on a Spectramax spectrophotometer (Molecular

Devices; Sunnyvale, CA), or equivalent equipment, (absorption maximum at 525 nm). IC₅₀ data for compounds may be obtained by measuring the activity of DPP-IV over a range of compound concentrations from 1 OnM to 3μM.

In Vivo Assay for Glucose Lowering

The glucose lowering effects of DPP-IV inhibitors, including the compounds of formula (I), may be exemplified in 4-6 week old KK/H1 J mice (Jackson Labs; Bar Harbor, ME) in the context of an oral glucose tolerance test. Oral glucose tolerance tests (OGTT) have been in use in humans since, at least, the 1930s, as described by Pincus, et al., Am. J. Med. Sci., 782 (1934), and are routinely used in the diagnosis of human diabetes, though not to evaluate the efficacy of therapeutic agents in patients.

KK mice have been used to evaluate (i) glitazones (Fujita etal. Diabetes, 804 (1983); Fujiwara, et al., Diabetes, 1549 (1988); and Izumi, et al., Biopharm Drug. Dispos., 247 (1997)); (ii) metformin (Reddi, et al., Diabet. Metabol., 44 (1993)); (iii) glucosidase inhibitors (Hamada, et al., Jap. Pharmacol.

Ther., 17 (1988) and Matsuo, et al., Am. J. Clin. Nutr., 314S (1992)), and (iv) extra-pancreatic effects of sulfonylureas (Kameda, et al., Arzneim. Forsch./Drug Res., 39044 (1982) and Muller et al., Horm.

Metabl. Res., 469 (1990)).

KK mice are derived from an inbred line first established and described by Kondo, et al., Bull. Exp. Anim., 107 (1957). These mice spontaneously develop a hereditary form of polygenic diabetes that progresses to cause renal, retinal, and neurological complications analogous to those seen in human diabetic subjects, however, they do not require insulin or other medication for survival.

Another aspect of the invention is directed to the use of KK mice to evaluate the effects of insulin secretagogue agents in the context of an oral glucose tolerance test. The mice are fasted overnight (about 14 to about 18 hr), but allowed free access to water. After fasting, (time "t" = 0), 25 μl_ of blood is drawn from the retro-orbital sinus and added to 0.025% heparinized saline (100 μL) on ice. The mice (10 per group) are then orally dosed with a solution of a compound of formula (I) in 0.5% methylcellulose (0.2 mL/mouse). Two controls groups receive only 0.5% methylcellulose. At t = 15 min, the mice are bled, as described above, and then dosed with 1 mg/kg glucose in distilled water (0.2 mL/mouse). The first control group is dosed with glucose. The second control group is dosed with water. At t = 45 min, the mice are again bled, as described above. The blood samples are centrifuged, the plasma collected and analyzed for glucose content on a Roche-Hitachi 912 glucose analyzer (Roche Diagnostics Corp.; Indianapolis, IN). The data may be expressed as percent (%) inhibition of glucose excursion relative to the two control groups (i.e., the glucose level in the animals receiving glucose but no test compound representing 0% inhibition and the glucose concentration in the animals receiving only water representing 100% inhibition).

The compounds of formula (I) generally exhibit inhibitory activity, expressed as IC₅₀ ¹S, against DPP-IV that are less than 3,000 nM. More specifically, the compounds of Examples 1-5, herein, were generally found to have IC₅₀ values of less than 3000 nM with the exception of the compound of Example 2 which exhibited an IC₅₀ value of more than 3000 nM. In addition, the compound [(2S,4S)-4- cyano-4-(4-phenyl-piperazin-1-yl)-pyrrolidin-2-yl]-pyrroIidin-1-yl-methanone dihydrochloride, which has a structure similar to those of the compounds of Examples 1 -5, was also prepared. This compound also exhibited an IC₅₀ value of more than 3000 nM.

When this assay was performed on the compounds of Examples 6-15, herein, said compounds were found to have IC₅₀ values of less than 100 nM with the exception of the compound of Example 6 that had an IC₅₀ value of 149 nM.

Generally preferred compounds have IC₅₀ ¹S of less thanlOO nM. For example, the compound of Example 15, {(2S,3R,4S)-4-[4-(3,5-dichloro-pyridin-4-yl)-piperazin-1 -yl]-3-methyl-pyrrolidin-2-yl}- pyrrolidin-1 -yl-methanone has an IC₅₀ of 6.13 nM.

Claims

We claim:

A compound of formula (I)

a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of the compound, prodrug, or stereoisomer, wherein:

R¹ is hydrogen, -(Ci-C₆)alkyl, -(C_rC₆)alkoxy, -(C_rC₆)arylalkyl, -NR^aR^b, hydroxy, cyano, aryl, or heteroaryl, wherein said -(d-C₆)alkyl, said aryl, or said heteroaryl is optionally substituted independently with one to three -COOH, -C(O)(C₁ -C₆)alkoxy, -C(O)(Ci-C₆)alkyl, -C(O)NR^aR^b, cyano, halogen, nitro, trifluoromethyl, -(C₁-C₆)alkyl, -(Ci-C₆)alkoxy, -(C₃-C₆)cycloalkyl, or phenyl, wherein:

R^a and R^b are, independently, hydrogen, -(d-C₆)alkyl, aryl, or heteroaryl, or

R^a and R^b, taken together with the nitrogen atom to which they are attached, form a four- to six-membered heterocyclic ring, wherein said ring optionally incorporates an additional one or two nitrogen, oxygen, or sulfur ring heteroatoms;

R² and R³ are, independently, hydrogen, halogen, -(Ci-C₆)alkyl, or -(C₃-C₈)cycloalkyl;

R⁴ is (i) hydrogen, (ii) -COOH, (iii) -C(O) (C₁ -C₆)alkoxy, (iv) -C(O)(C_rC₆)alkyl, (v) -C(O)NR^aR^b,

(vi) cyano, or (vii) -(CrC₆)alkyl, optionally substituted with one to six halogen atoms, -(C₁-

C₆)alkoxy, cyano, hydroxy, or -NR^aR^b, (viii) -(C₃-C₆)cycloalkyl, (ix) -(CrC₆)arylalkyl, (x) aryl, or

(xi) heteroaryl;

Q is a covalent bond, -C(O)-, or -SO₂-;

HET is a heterocycloalkyl ring moiety, optionally substituted independently with: (A) one to four -

(Ci-C₆)alkyl, optionally substituted with one to six halogen atoms, -(CrC₆)alkoxy, cyano, halogen, hydroxy, or -NR^aR^b, or (B) -(CrC₆)arylalkyl, optionally substituted with one to six halogen atoms, -(CrC₆)alkoxy, cyano, halogen, hydroxy, or -NR^aR^b; n is zero or one;

Xishydrogenorcyano;and

Yis-CH₂-, -S-, -S(O)-,or-SO₂-; provided that R², R³ and R⁴ are not all hydrogen. A compound of claim 1 , having the structure

wherein:

R¹ is aryl or heteroaryl, wherein said aryl or heteroaryl is optionally substituted independently with one to three cyano, halogen, nitro, trifluoromethyl, -(Ci-C₆)alkyl, -(d-C₆)alkoxy, -(C₃-

C₆)cycloalkyl, or phenyl;

R², R³ and R⁴ are, independently, hydrogen or -(C_rC₆)aikyl; Q is a covalent bond, -C(O)-, or -SO₂-; HET is azetidinyl, piperazinyl, piperidinyl, or pyrrolidinyl; n is zero or one;

X is hydrogen or cyano; and

Y is -CH₂- or -S-.

3. A compound of claim 2, wherein: R¹ is pyrazinyl, pyridyl, pyrimidyl, or quinolyl, wherein said pyrazinyl, pyridyl, pyrimidyl, or quinoyl is optionally substituted independently with one to three cyano, halogen, nitro, trifluoromethyl, -

(Ci-C₆)alkyl, -(CrC₆)alkoxy, -(C₃-C₆)cycloalkyl, or phenyl;

R² is -(C_rC₆)alkyl;

R³ and R⁴ are both hydrogen; Q is a covalent bond;

HET is piperazinyl; n is one;

X is hydrogen or cyano; and

Y is -CH₂- or -S-.

4. A compound of claim 3, wherein: R² is methyl; and

Y is -CH₂- .

5. {(2S,3R,4S)-4-[4-(3,5-Dichloro-pyridin-4-yl)-piperazin-1 -yl]-3-methyl-pyrrolidin-2-yl}-pyrrolidin-1 - yl-methanone;

{(2S,3R,4S)-3-methyl-4-[4-(3-trifluoromethyl-pyridin-2-yl)-piperazin-1-yl]- pyrrolidin-2-yl}- pyrrolidin-1 -yl-methanone;

4-[(3S,4R,5S)-4-methyl-5-(pyrrolidine-1 -carbonyl)-pyrrolidin-3-yl]-3,4,5,6-tetrahydro-2H- [1 ,2']bipyrazinyl-3'-carbonitrile; and {(2S,3R,4S)-3-methyl-4-[4-(2-trifluoromethyl-quinolin-4-yl)-piperazin-1 -yl]-pyrrolidin-2-yl}- pyrrolidin-1 -yl-methanone; a prodrug thereof, or a pharmaceutically acceptable salt of said prodrug.

6. A pharmaceutical composition comprising a compound of claims 1 -5, a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer; and a pharmaceutically acceptable vehicle, carrier, or diluent.

7. A method of inhibiting DPP-IV in a mammal in need of such inhibition which method comprises administering to said mammal a DPP-IV inhibiting amount of a compound of formula (I) of claims 1 -5, a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer.

8. A method of treating a condition mediated by DPP-IV in a mammal in need of such treatment which method comprises administering to said mammal a therapeutically effective amount of a compound of formula (I) of claims 1 -5, a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer; or a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (I) of claims 1 -5, a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer; and a pharmaceutically acceptable vehicle, carrier, or diluent.

9. The method of claim 8, wherein said condition is Type 2 diabetes, progression of disease in Type 2 diabetes, metabolic syndrome (Syndrome X and/or IRS), hyperglycemia, impaired glucose tolerance, glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, Type 1 diabetes, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, short stature due to bone growth deficiency, acute coronary syndrome, infertility due to polycystic ovary syndrome, short bowel syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, eating disorders, chronic pain, alcohol addiction, diseases associated with intestinal motility, ulcers, irritable bowel syndrome, or inflammatory bowel syndrome.

10. The method of claim 9, wherein said condition is Type 2 diabetes.

11. A pharmaceutical composition comprising: a) an amount of a compound of formula (I) of claims 1 -5, a prodrug or stereoisomer thereof, or a pharmaceutically acceptable salt of said compound, prodrug, or stereoisomer; b) an amount of a second compound selected from an antidiabetic agent, a prodrug or pharmaceutically acceptable salt thereof, insulin or an analog thereof, insulinotropin, a biguanide, an α₂-antagonist, an imidazoline, a glitazone, an aldose reductase inhibitor, a glycogen phosphorylase inhibitor, a sorbitol dehydrogenase inhibitor, a fatty acid oxidation inhibitor, an α-glucosidase inhibitor, a β-agonist, a phosphodiesterase inhibitor, a lipid-lowering agent, an antiobesity agent, a vanadate, a vanadium complex, a peroxovanadium complex, an amylin antagonist, a glucagon antagonist, a growth hormone secretagogue, a gluconeogenesis inhibitor, a somatostatin analog, an antilipolytic agent, or an inhibitor of renal glucose; and c) a pharmaceutically acceptable carrier, vehicle, or diluent.

12. A method of treating a condition mediated by DPP-IV in a mammal in need of such treatment which method comprises administering to said mammal a therapeutically effective amount of a pharmaceutical composition of claim 11.

13. The method of claim 12, wherein said condition is Type 2 diabetes, progression of disease in Type 2 diabetes, metabolic syndrome (Syndrome X and/or IRS), hyperglycemia, impaired glucose tolerance, glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, Type 1 diabetes, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, short stature due to bone growth deficiency, acute coronary syndrome, infertility due to polycystic ovary syndrome, short bowel syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, eating disorders, chronic pain, alcohol addiction, diseases associated with intestinal motility, ulcers, irritable bowel syndrome, or inflammatory bowel syndrome.

14. The method of claim 13, wherein said condition is Type 2 diabetes.