US005998.463A United States Patent (19) 11 Patent Number: 5,998,463 Hulin et al. (45) Date of Patent: Dec. 7, 1999

54 GLYCOGEN PHOSPHORYLASE 96.39384 12/1996 WIPO ...... CO7D 209/42 INHIBITORS 96.39385 12/1996 WIPO ...... CO7D 209/42 75 Inventors: Bernard Hulin, Essex; Reinhard Sarges, Mystic, both of Conn. Primary Examiner Johann Richter ASSistant Examiner-Dominic Keating 73 Assignee: Pfizer Inc, New York, N.Y. Attorney, Agent, or Firm-Peter C. Richardson; Gregg C. Benson; Martha A. Gammill 21 Appl. No.: 09/251,141 57 ABSTRACT 22 Filed: Feb. 16, 1999 This invention relates to certain 5-acyl-2-oxo-indole-3- carboxamides useful as inhibitors of glycogen Related U.S. Application Data phosphorylase, methods of treating glycogen phosphorylase 60 Provisional application No. 60/076,132, Feb. 27, 1998. dependent diseases or conditions with Such compounds and 51) Int. Cl...... A01N 43/38; CO7D 209/34 pharmaceutical compositions comprising Such compounds. 52 U.S. Cl...... 514/418; 548/486 This invention also relates to pharmaceutical compositions 58 Field of Search ...... 548/486, 514/418 comprising those 5-acyl-2-oxo-indole-3-carboxamides in combination with antidiabetes agents and methods of treat 56) References Cited ing glycogen phosphorylase dependent diseases or condi tions with Such compositions. FOREIGN PATENT DOCUMENTS 96/39384 12/1996 WIPO. 23 Claims, No Drawings 5,998,463 1 2 GLYCOGEN PHOSPHORYLASE disease (CVD) due to atherosclerosis. In recent years, lead INHIBITORS ers of the medical profession have placed renewed emphasis on lowering plasma cholesterol levels, and low density This application is a continuation of U.S. Provisional lipoprotein cholesterol in particular, as an essential Step in Application No. 60/076,132 filed Feb. 27, 1998. prevention of CVD. The upper limits of “normal” are now known to be significantly lower than heretofore appreciated. BACKGROUND OF THE INVENTION AS a result, large Segments of Western populations are now This invention relates to glycogen phosphorylase realized to be at particular high risk. Other independent risk inhibitors, pharmaceutical compositions containing Such factors include glucose intolerance, left ventricular inhibitors and the use of Such inhibitors to treat diabetes, hypertrophy, hypertension, and being of the male SeX. Car hyperglycemia, hypercholesterolemia, hypertension, diovascular disease is especially prevalent among diabetic hyperinsulinemia, hyperlipidemia, atherosclerosis, myocar Subjects, at least in part because of the existence of multiple dial ischemia and other tissue ischemias in animals, particu independent risk factors in this population. Successful treat larly mammals. In particular, this invention relates to certain ment of hyperlipidemia in the general population, and in 5-acyl-2-oxo-indole-3-carboxamides having Such glycogen 15 diabetic Subjects in particular, is therefore of exceptional phosphorylase inhibitor activity. medical importance. In spite of the early discovery of and its Subse Hypertension (or high blood pressure) is a condition quent widespread use in the treatment of diabetes, and the which occurs in the human population as a Secondary later discovery of and use of (e.g., Chlorpro Symptom to various other disorderS Such as renal artery pamide TM Pfizer), TM (Upjohn), Acetohexam Stenosis, pheochromocytoma or endocrine disorders. ide TM (E. I. Lilly), TM (Upjohn)) and However, hypertension is also evidenced in many Subjects in (e.g., PhenforminTM (Ciba Geigy), MetforminTM (G. D. whom the causative agent or disorder is unknown. While Searle)), alpha-glucosidase inhibitors (e.g., glucophage) and Such “essential” hypertension is often associated with dis insulin Sensitizing agents (e.g., ) as oral orderS Such as obesity, diabetes and hypertriglyceridemia, hypoglycemic agents, there is still a need for improved 25 the relationship between these disorderS has not been elu diabetes therapies. The use of insulin, necessary in about cidated. Additionally, many Subjects display the Symptoms 10% of diabetic subjects in which synthetic hypoglycemic agents are not effective (Type I diabetes, insulin dependent of high blood pressure in the complete absence of any other diabetes mellitus), requires multiple daily doses, usually by Signs of disease or disorder. Self injection. Determination of the proper dosage of insulin It is known that hypertension can directly lead to heart requires frequent estimations of the Sugar in urine or blood. failure, renal failure and stroke (brain hemorrhaging). These The administration of an exceSS dose of insulin causes conditions are capable of causing rapid death in a Subject. hypoglycemia, with effects ranging from mild abnormalities Hypertension can also contribute to the development of atherosclerosis and coronary disease. These conditions in blood glucose to coma, or even death. Treatment of gradually weaken a Subject, leading to eventual death due to non-insulin dependent diabetes mellitus (Type 2 diabetes, 35 NIDDM) usually consists of a combination of diet, exercise, cardiovascular failure. oral agents, e.g. Sulfonylureas, and in more Severe cases, The exact cause of essential hypertension is unknown, insulin. However, the clinically available hypoglycemics though a number of factors are believed to contribute to the can have other side effects which limit their use. In any onset of the disease. Among Such factors are StreSS, uncon event, where one of these agents may fail in an individual 40 trolled emotions, unregulated hormone release (the renin, case, another may Succeed. A continuing need for hypogly angiotensin, aldosterone System), excessive Salt and water cemic agents, which may have fewer Side effects or Succeed due to kidney malfunction, wall thickening and hypertrophy where others fail, is clearly evident. of the vasculature resulting in constricted blood vessels and AtherOSclerosis, a disease of the arteries, is recognized to genetic factors. be the leading cause of death in the United States and 45 The treatment of essential hypertension has been under Western Europe. The pathological Sequence leading to ath taken bearing the foregoing factors in mind. Thus, a broad erosclerosis and occlusive heart disease is well known. The range of beta-blockers, vasoconstrictors, angiotensin con earliest Stage in this Sequence is the formation of "fatty Verting enzyme inhibitors and the like have been developed Streaks” in the carotid, coronary and cerebral arteries and in and marketed as antihypertensives. The treatment of hyper the aorta. These lesions are yellow in color due to the 50 tension utilizing these compounds has proven beneficial in presence of lipid deposits found principally within Smooth the prevention of deaths resulting from little or no periods of muscle cells and in macrophages of the intima layer of the illness Such as heart failure, renal failure and brain hemor arteries and aorta. Further, it is postulated that most of the rhaging. However, the development of atherOSclerosis or cholesterol found within the fatty Streaks, in turn, give rise heart disease due to hypertension over a long period of time to development of the “fibrous plaque', which consists of 55 remains a problem. This implies that although high blood accumulated intimal Smooth muscle cells laden with lipid preSSure is being reduced, the underlying cause of essential and Surrounded by extracellular lipid, collagen, elastin and hypertension is not responding to this treatment. proteoglycans. The cells plus matrix form a fibrous cap that Hypertension has been associated with elevated blood covers a deeper deposit of cell debris and more extracellular insulin levels, a condition known as hyperinsulinemia. lipid. The lipid is primarily free and esterified cholesterol. 60 Insulin, a peptide hormone whose primary actions are to The fibrous plaque forms slowly, and is likely in time to promote glucose utilization, protein Synthesis and the for become calcified and necrotic, advancing to the “compli mation and Storage of neutral lipids, also acts to promote cated lesion” which accounts for the arterial occlusion and vascular cell growth and increase renal Sodium retention, tendency toward mural thrombosis and arterial muscle among other things. These latter functions can be accom Spasm that characterize advanced atherosclerosis. 65 plished without affecting glucose levels and are known Epidemiological evidence has firmly established hyper causes of hypertension. Peripheral vasculature growth, for lipidemia as a primary risk factor in causing cardiovascular example, can cause constriction of peripheral capillaries, 5,998,463 3 4 while Sodium retention increases blood volume. Thus, the agents. WO96/39384 discloses certain substituted lowering of insulin levels in hyperinsulinemics can prevent N-(indole-2-carbonyl)-glycinamides and derivatives as abnormal vascular growth and renal Sodium retention caused antidiabetic agents. by high insulin levels and thereby alleviate hypertension. The mechanism(s) responsible for the myocardial or other Cardiac hypertrophy is a Significant risk factor in the tissue injury observed after ischemia and reperfusion is not development of Sudden death, myocardial infarction, and congestive heart failure. These cardiac events are due, at fully understood. It has been reported (M. F. Allard, et al. least in part, to increased Susceptibility to myocardial injury Am. J. Physiol. 267, H66–H74, 1994) that “pre ischemic after ischemia and reperfusion which can occur in out glycogen reduction . . . is associated with improved post Subject as well as perioperative Settings. There is an unmet ischemic left ventricular functional recovery in hypertro medical need to prevent or minimize adverse myocardial phied rat hearts”. perioperative outcomes, particularly perioperative myocar Thus, although there are a variety of hyperglycemia, dial infarction. Both non-cardiac and cardiac Surgery are hypercholesterolemia, hypertension, hyperlipidemia, ath asSociated with Substantial risks for myocardial infarction or erosclerosis and myocardial ischemia therapies, there is a death. Some 7 million Subjects undergoing non-cardiac continuing need and a continuing Search in this field of art Surgery are considered to be at risk, with incidences of 15 for alternative therapies. perioperative death and Serious cardiac complications as high as 20-25% in some series. In addition, of the 400,000 SUMMARY OF THE INVENTION Subjects undergoing coronary by-pass Surgery annually, This invention is directed to glycogen phosphorylase perioperative myocardial infarction is estimated to occur in inhibitor compounds of Formula I which are useful for the 5% and death in 1-2%. There is currently a need for treatment of diabetes, hyperglycemia, hypercholesterolemia, improved drug therapy which reduces damage to cardiac hypertension, hyperinsulinemia, hyperlipidemia, atheroscle tissue from perioperative myocardial ischemia or enhances rosis and myocardial or other tissue ischemia. cardiac resistance to ischemic episodes. If Such a therapy This invention is particularly directed to compounds of were found, it would be expected to reduce hospitalizations, Formula I enhance quality and length of life and reduce overall health 25 care costs of high risk Subjects.

Hepatic glucose production is an important target for Type 2 therapy. The liver is the major regulator of plasma glucose levels in the post absorptive (fasted) State, and the rate of hepatic glucose production in Type 2 diabetic SubjectS is Significantly elevated relative to normal individuals. NHR, Likewise, in the postprandial or fed State, where the liver has a proportionately Smaller role in the total plasma glucose Supply, hepatic glucose production is abnormally high in Type 2 diabetic subjects. 35 Glycogenolysis is an important target for interruption of hepatic glucose production. The liver produces glucose by prodrugs thereof and pharmaceutically acceptable Salts of glycogenolysis (breakdown of the glucose polymer Said compounds and Said prodrugs wherein: glycogen) and gluconeogenesis (Synthesis of glucose from 40 R" is (C-C)alkyl, (C-C)cycloalkyl, phenyl or phenyl 2- and 3-carbon precursors). Several lines of evidence independently Substituted with up to three (C-C) indicate that glycogenolysis may make an important contri alkyl, (C-C)alkoxy or halogen; bution to hepatic glucose output in Type 2 diabetes. First, in R is (C-C)alkyl optionally substituted with up to three normal post absorptive humans, up to 75% of hepatic fluoro atoms, and glucose production is estimated to result from glycogenoly 45 R is (C-C)cycloalkyl; phenyl; phenyl substituted at the Sis. Second, Subjects having liver glycogen Storage diseases, para position with (C-C)alkyl, halo or trifluorom including Hers disease (glycogen phosphorylase ethyl, phenyl Substituted at the meta position with deficiency), display episodic hypoglycemia. These observa fluoro; or phenyl substituted at the ortho position with tions Suggest that glycogenolysis may be a Significant pro fluoro. ceSS for hepatic glucose production. 50 A first group of preferred compounds of Formula I are the Glycogenolysis is catalyzed in liver, muscle, and brain by compounds of Formula I, prodrugs thereof and pharmaceu tissue-specific isoforms of the enzyme glycogen phospho tically acceptable Salts of Said compounds and Said prodrugs rylase. This enzyme cleaves the glycogen macromolecule to wherein R is phenyl; R is ethyl; and R is phenyl, phenyl release glucose-1-phosphate and a new shortened glycogen Substituted at the para position with (C-C)alkyl, halo or macromolecule. Two types of glycogen phosphorylase 55 trifluoromethyl, phenyl substituted at the meta position with inhibitors have been reported to date: glucose and glucose fluoro, or phenyl substituted at the ortho position with analogs Martin, J. L. et al. Biochemistry, 1991, 30, 10101 fluoro. and caffeine and other purine analogs Kasvinsky, P. J. et al. A Second group of preferred compounds of Formula I are J. Biol. Chem., 1978, 253, 3343–3351 and 9102-9106). the compounds of Formula I, prodrugs thereof and pharma These compounds, and glycogen phosphorylase inhibitors in 60 ceutically acceptable Salts of Said compounds and Said general, have been postulated to be of potential use for the prodrugs wherein R' is methyl and R is ethyl. treatment of Type 2 diabetes by decreasing hepatic glucose A more preferred group of compounds, hereinafter termed production and lowering glycemia. Blundell, T. B. et al. the third group of preferred compounds, within the Second Diabetologia, 1992, 35, Suppl. 2,569–576 and Martin et al. preferred group of compounds are those compounds of the Biochemistry 1991, 30, 10101). 65 Second group of preferred compounds of Formula I, pro WO96/39385 discloses certain substituted N-(indole-2- drugs thereof and pharmaceutically acceptable Salts of Said carbonyl)-B-alanimamides and derivatives as antidiabetic compounds and said prodrugs wherein R is phenyl, phenyl 5,998,463 S 6 Substituted at the para position with (C-C)alkyl, halo or compound, a prodrug thereof or a pharmaceutically accept trifluoromethyl, phenyl substituted at the meta position with able Salt of Said compound or Said prodrug. fluoro, or phenyl substituted at the ortho position with Yet another aspect of this invention is directed to a method fluoro. for treating hyperlipidemia in an animal, particularly a An especially preferred group of compounds within the mammal, by administering to Said animal Suffering from third preferred group of compounds are 5-acetyl-1-ethyl-2- hyperlipidemia a hyperlipidemia treating amount of a For oxo-2,3-dihydro-1H-indole-3-carboxylic acid (3-p- mula I compound, a prodrug thereof or a pharmaceutically tolylcarbamoyl-phenyl)-amide, 5-acetyl-1-ethyl-2-oxo-2,3- acceptable Salt of Said compound or said prodrug. Included dihydro-1H-ind ole-3-carboxylic acid (3-(4- in the treatment of hyperlipidemia is the lowering of trig bromophenylcarbamoyl-phenyl)-amide; and 5-acetyl-1- lycerides and free fatty acids. ethyl-2-oxo-2,3-dihydro-1H-indole-3-carboxylic acid Yet another aspect of this invention is directed to a method (3-phenylcarbamoyl-phenyl)-amide. for treating or preventing a tissue ischemic injury in an A particularly preferred compound within the third group animal, particularly a mammal, by administering to Said of compounds is the compound wherein R is p-tolyl. animal a tissue ischemic injury preventing amount of a Another particularly preferred compound within the third 15 Formula I compound, a prodrug thereof or a pharmaceuti group of compounds is the compound wherein R is cally acceptable Salt of Said compound or Said prodrug. 4-bromophenyl. Included within tissue ischemic injury is ischemic injury due Yet another particularly preferred compound within the to organ transplant. third group of compounds is the compound wherein R is Yet another aspect of this invention is directed to a method phenyl. for treating or preventing a myocardial ischemic injury in an Yet another aspect of this invention is directed to a method animal, particularly a mammal, by administering to Said for treating a glycogen phosphorylase dependent disease or animal at risk for myocardial ischemic injury a myocardial condition in an animal, particularly a mammal, by admin ischemic injury treating or preventing amount of a Formula istering to Said animal Suffering from a glycogen phospho I compound, a prodrug thereof or a pharmaceutically accept rylase dependent disease or condition a glycogen phospho 25 able Salt of Said compound or Said prodrug. rylase dependent disease or condition treating amount of a Yet another aspect of this invention is directed to a method Formula I compound, a prodrug thereof or a pharmaceuti for treating or preventing a perioperative myocardial cally acceptable Salt of Said compound or said prodrug. ischemic injury in an animal, particularly a mammal, by Yet another aspect of this invention is directed to a method administering to Said animal at risk for perioperative myo for treating hyperglycemia in an animal, particularly a cardial ischemic injury a perioperative myocardial ischemic mammal, by administering to Said animal Suffering from injury treating or preventing effective amount of a Formula hyperglycemia a hyperglycemia treating amount of a For I compound, a prodrug thereof or a pharmaceutically accept mula I compound, a prodrug thereof or a pharmaceutically able Salt of Said compound or said prodrug. acceptable Salt of Said compound or said prodrug. This invention is also directed to pharmaceutical compo Yet another aspect of this invention is directed to a method 35 Sitions which comprise a therapeutically effective amount of for treating diabetes in an animal, particularly a mammal, by a compound of Formula I, a prodrug thereof or a pharma administering to Said animal Suffering from diabetes a ceutically acceptable Salt of Said compound or Said prodrug diabetes treating amount of a Formula I compound, a and a pharmaceutically acceptable carrier or diluent. prodrug thereof or a pharmaceutically acceptable Salt of Said Preferred compositions include pharmaceutical composi compound or Said prodrug. Included in the treatment of 40 tions for the treatment of glycogen phosphorylase dependent diabetes is the prevention or attenuation of long term com diseases or conditions in animals, particularly mammals, plications Such as, but not limited to, neuropathy, which comprise a glycogen phosphorylase dependent dis nephropathy, retinopathy or cataracts. ease or condition treating amount of a compound of Formula Yet another aspect of this invention is directed to a method I, a prodrug thereof or a pharmaceutically acceptable Salt of for treating hypercholesterolemia in an animal, particularly 45 Said compound or said prodrug and a pharmaceutically a mammal, by administering to Said animal Suffering from acceptable carrier or diluent. hypercholesterolemia a hypercholesterolemia treating Preferred pharmaceutical compositions within the imme amount of a Formula I compound, a prodrug thereof or a diately preceding group are those compositions wherein the pharmaceutically acceptable Salt of Said compound or said glycogen phosphorylase inhibitor is a compound of Formula prodrug. 50 I. Yet another aspect of this invention is directed to a method Another aspect of this invention is directed to pharma for treating atherOSclerosis in an animal, particularly a ceutical compositions for the treatment of diabetes which mammal, by administering to Said animal Suffering from or comprise a therapeutically effective amount of a glycogen Susceptible to atherOSclerosis an atherosclerosis treating phosphorylase inhibitor of Formula I; amount of a Formula I compound, a prodrug thereof or a 55 one or more antidiabetic agents Such as insulin and insulin pharmaceutically acceptable Salt of Said compound or said analogs (e.g. LysPro insulin); GLP-1 (7-37) prodrug. (insulinotropin) and GLP-1 (7-36)-NH; Sulfonylureas Yet another aspect of this invention is directed to a method and analogS Such as , , for treating hyperinsulinemia in an animal, particularly a tolbutamide, tolaZamide, , , mammal, by administering to Said animal Suffering from 60 , and ; biguanides hyperinsulinemia a hyperinsulinemia treating amount of a Such as mefformin, and ; Formula I compound, a prodrug thereof or a pharmaceuti C-antagonists and imidazolines Such as midaglizole, cally acceptable Salt of Said compound or said prodrug. isaglidole, deriglidole, idaZOXan, efaroXan and flupar Yet another aspect of this invention is directed to a method OXan; other insulin Secretagogues Such as linogliride for treating hypertension in an animal, particularly a 65 and A-4166; glitaZones Such as ciglita Zone, mammal, by administering to Said animal Suffering from , , troglitaZone, dargilitaZone hypertension a hypertension treating amount of a Formula I and BRL49653; aldose reductase inhibitors Such as 5,998,463 7 8 , , tolrestat and Zopolrestat; Sorbitol drugs thereof or pharmaceutically acceptable Salts of dehydrogenase inhibitors, fatty acid oxidation inhibi Said antidiabetic agents or said prodrugs and a phar torS Such as clomoxir and etomoxir; C-glucosidase maceutically acceptable carrier or diluent in a Second inhibitorS Such as , , emiglitate, unit dosage form; and , MDL-25,637, camiglibose and MDL-73, c. a container. 945; f-agonists Such as (4-(2-(2-(6-aminopyridin-3- Another aspect of this invention is directed to a compound yl)-2(R)-hydroxyethylamino)ethoxy)phenyl)acetic of formula II, acid and BRL 35135, BRL 37344, Ro 16-8714, ICI D7114 and CL 316,243; phosphodiesterase inhibitors II

Such as L-386,398; lipid-lowering agents Such as ben fluorex, antiobesity agents Such as fenfiluramine, Sibutramine and ; Vanadate and Vana dium complexes (e.g. naglivan) and peroxovanadium complexes, antagonists; glucagon antagonists; NHR, growth hormone Secretagogues; gluconeogenesis 15 inhibitorS Such as glucose-6-phosphatase inhibitors, Somatostatin analogs, antilipolytic agents Such as nico tinic acid, acipimox and WAG994; prodrugs thereof or pharmaceutically acceptable Salts of Said antidiabetic agents or Said prodrugs and a pharmaceutically accept wherein: able carrier or diluent. R" is (C-C)alkyl, (C-C)cycloalkyl, phenyl or phenyl Another aspect of this invention is directed to a method independently Substituted with up to three (C-C) for treating glycogen phosphorylase dependent diseases or alkyl, (C-C)alkoxy or halogen; conditions in an animal, particularly a mammal, which R is (C-C)alkyl optionally substituted with up to three comprises administering to Said animal Suffering from a 25 fluoro atoms, and glycogen phosphorylase dependent disease or condition a R is (C-C)cycloalkyl; phenyl; phenyl substituted at the glycogen phosphorylase dependent disease or condition para position with (C-C)alkyl, halo or trifluorom treating amount of a composition Set forth in the immedi ethyl, phenyl Substituted at the meta position with ately preceding paragraph. fluoro; or phenyl substituted at the ortho position with Another aspect of this invention is a method of treating fluoro. diabetes in an animal, particularly a mammal, with the above Glycogen phosphorylase dependent diseases or condi described combination compositions. tions refers to disorders which are mediated, initiated or Another aspect of this invention is directed to a kit maintained, in whole or in part, by the cleavage of the comprising glycogen macromolecule by glycogen phosphorylase a. an amount of a compound of Formula I, a prodrug 35 enzymes to release glucose-1-phosphate and a new short thereof or a pharmaceutically acceptable Salt of Said ened glycogen molecule. These disorders are ameliorated by compound or said Salt in a first unit dosage form; reduction of or characterized by an elevation of glycogen b. an amount of an antidiabetic agent Such as insulin and phosphorylase activity. Examples include diabetes, insulin analogs (e.g. LysPro insulin); GLP-1 (7-37) hyperglycemia, hypercholesterolemia, hypertension, (insulinotropin) and GLP-1 (7-36)-NH; Sulfonylureas 40 hyperinsulinemia, hyperlipidemia, atherOSclerosis and myo and analogS Such as chlorpropamide, glibenclamide, cardial ischemia. tolbutamide, tolaZamide, acetohexamide, glipizide, The term glycogen phosphorylase inhibitor refers to any glimepiride, repaglinide and meglitinide; biguanides Substance or agent or any combination of Substances and/or Such as , phenformin and buformin; agents which reduces, retards, or restrains the enzymatic C2-antagonists and imidazolines Such as midaglizole, 45 action of glycogen phosphorylase. The currently known isaglidole, deriglidole, idaZOXan, efaroXan and flupar enzymatic action of glycogen phosphorylase is the degra OXan; other insulin Secretagogues Such as linogliride dation of glycogen by catalysis of the reversible reaction of and A4166; glita Zone S Such as ciglita Zone, a glycogen macromolecule and inorganic phosphate to pioglitaZone, englitaZone, troglitaZone, dargilitaZone glucose-1-phosphate and a glycogen macromolecule which and BRLA9653; aldose reductase inhibitors Such as 50 is one glucosyl residue shorter than the original glycogen epalrestat, Sorbinil, tolrestat and Zopolrestat; fatty acid macromolecule (forward direction of glycogenolysis). oxidation inhibitorS Such as clomoxir and etomoxir; The term “treating as used herein includes preventative C-glucosidase inhibitorS Such as acarbose, miglitol, (e.g., prophylactic), palliative and curative treatment. emiglitate, voglibose, MDL-25,637, camiglibose and By halo is meant chloro, bromo, iodo, or fluoro. MDL-73,945; B-agonists such as (4-(2-(2-(6- 55 By alkyl is meant Straight chain or branched Saturated aminopyridin-3-yl)- 20R)-hydroxyethylamino)ethoxy) hydrocarbon. Exemplary of Such alkyl groups (assuming the phenyl)acetic acid and BRL 35135, BRL 37344, Ro designated length encompasses the particular example) are 16-8714, ICI D7114 and CL 316,243; phosphodi methyl, ethyl, propyl, isopropyl, butyl, Sec-butyl, tertiary esterase inhibitors such as L-386,398; lipid-lowering butyl, pentyl, isopentyl, hexyl and isohexyl. agents Such as , antiobesity agents Such as 60 By alkoxy is meant Straight chain or branched Saturated fenfluramine, Sibutramine and bromocriptine; Vanadate alkyl bonded through an Oxy. Exemplary of Such alkoxy and Vanadium complexes (e.g. naglivan) and peroxo groups (assuming the designated length encompasses the Vanadium complexes; amylin antagonists; glucagon particular example) are methoxy, ethoxy, propoxy, antagonists, growth hormone Secretagogues, gluconeo isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, genesis inhibitorS Such as glucose-6-phosphatase 65 isopentoxy, heXOXy and isohexoxy. inhibitors, Somatostatin analogs, antilipolytic agents The expression "prodrug” refers to compounds that are Such as nicotinic acid, acipimox and WAG 994; pro drug precursors, which, following administration, release 5,998,463 10 the drug in Vivo via a chemical or physiological proceSS Other features and advantages will be apparent from the (e.g., a prodrug on being brought to the physiological pH is Specification and claims which describe the invention. converted to the desired drug form). AS used herein, the expressions “reaction-inert Solvent' DETAILED DESCRIPTION OF THE and “inert Solvent” refers to a solvent or mixture of Solvents 5 which does not interact with Starting materials, reagents, INVENTION intermediates or products in a manner which adversely affects the yield of the desired product. In general the compounds of Formula I can be made by The chemist of ordinary skill will recognize that certain processes which include processes known in the chemical compounds of this invention will contain one or more atoms arts, particularly in light of the description contained herein. which may be in a particular Stereochemical or geometric Certain processes for the manufacture of Formula I com configuration, giving rise to Stereoisomers and configura pounds are provided as further features of the invention and tional isomers. All Such isomers and mixtures thereof are are illustrated by the following reaction Schemes.

SCHEMEI O O AlCl3 1. NaH, RI N C O 2. H. Pd-C O RCOCI HCO N 4 N R2 AlCl V V R2 H 1. 2 3 O / \ HOCH2CH2OH O O 1. TSOH 1. R O (when R* = Me, primary - R O N or secondarwalkwly alkyl) N

4 Ye 5 Ye (when R = 4 4 NaH, R“OCOOR 4 4 tertiary alkyl (R' Me or Et) NaH, R“OCOOR or aryl) We O (R = Me or Et) O COOR4 / \ O O COOR R1 O R1 O N N R2 V 7 R2 6

O O HN HN 1O 1O HNYR3 HNYR3

HCI

included in this invention. Hydrates and Solvates of the compounds of this invention, of the produgs of the com 65 pounds of this invention, and pharmaceutically acceptable Salts of Said hydrates and Solvates are also included. 5,998,463 12 reaction is most readily accomplished by reacting a carboxy lic acid ester derivative of formula 6 or formula 7 with the SCHEME II appropriate aniline derivative of formula 10. Typically, a Y compound of formula 6 or formula 7 is dissolved in a reaction inert Solvent and a compound of formula 10 is R1 added. Molecular sieves (4 A) are added and the reaction O -- mixture is generally heated at the reflux temperature of the chosen Solvent until the Starting materials are no longer N present as determined by thin layer chromatography or other Ye analytical technique well known to those skilled in the art. Y is FO or OCHCHO The coupled product of formula 9 or Formula I is then 4 or 5 isolated according to methods well known to those skilled in O the art. Alternatively, the compounds of Formula I may be pre pared according to the procedure Set forth in Scheme II. In n N N YR3 H this procedure, a compound of formula 5 wherein Y is as Set Ph 11 O forth in Scheme II is reacted with an activated amide Such as a N,N-diphenylureido derivative in the presence of a base to form the compound of Formula I. Typically, the com s pound of formula 5 wherein Y is =O (when R is tertiary H alkyl or aryl) or -OCH2CH2O- (when R is primary or N-R3 secondary alkyl) and R' and R are as described above is dissolved in a Suitable solvent and is treated with a base O which is strong enough to deprotonate the carbon atom alpha

to the carbonyl. The anion thus formed is treated with the activated amide compound and the reaction mixture is stirred for 16 hours to 7 days. Typically, the reaction is complete after Stirring for three days. The reaction mixture is then acidifed to form the compound of Formula I directly. 3O The compounds of formulas 6 and 7 in Scheme I are prepared by Standard acylation chemistry. For example, the compounds of formula 4 are acylated directly when R is tertiary alkyl or aryl by reacting the compound of formula 4 under Standard acylation conditions, e.g., base and acylating According to Scheme I, the compounds of Formula I 35 agent, to obtain the compound of formula 6. When R' is wherein R, R and Rare as defined above may be prepared primary or Secondary alkyl, the ketone moiety attached to by either of two general procedures. The procedures involve the 5-position of the OXindole ring must be protected using coupling a carboxylic acid or carboxylic acid ester deriva Standard ketone protecting groups as Set forth in Greene and tive of formula 6 or 7 with an appropriate 3-substituted Wuts, Supra. The protected compound of formula 5 is then aniline derivative of formula 10. When the coupling is 40 acylated in the same manner as the compound of formula 4 performed using a compound of formula 7, the immediate to obtain the compound of formula 7. The acylation reaction product is a compound of Formula I. When the coupling is described in this paragraph is readily carried out using performed using a compound of formula 6, which com procedures well known to those skilled in the art or by using pound contains a protected ketone moiety, the intermediate methods analogous to those set forth in Melvin, U.S. Pat. result is a compound of formula 9 which is converted to a 45 No. 4,686,224, the teachings of which are incorporated compound of Formula I by a Subsequent deprotection Step. herein by reference. Deprotection, if required, is performed Typically, the deprotection of the formula 9 compounds using methods analogous to those described in Greene and may be carried out using methods well known to those Wuts, supra. skilled in the art, for example, methods described in “Pro The compounds of formulas 1, 2, 3, 4 and 10 are prepared tecting Groups in Organic Synthesis,” Second Edition, T. W. 50 according to methods well known to those skilled in the art. Greene and P. G. M. Wuts, John Wiley and Sons, Inc., 1991. Further, the Starting materials and reagents for the above Generally, the compound of formula 9 is dissolved in a described reaction Schemes are also readily available from reaction inert Solvent Such as tetrahydrofuran and Strong commercial Sources or can be readily Synthesized by those aqueous acid is added. The temperature of the reaction may skilled in the art using conventional methods of organic be varied from 0° C. to 50° C. Generally, however, the 55 Synthesis. reaction is performed at room temperature. The reaction is The compounds of Formula I have an asymmetric carbon Stirred until all or most of the Starting material has reacted atom and therefore are racemic mixtures of enantiomers as determined by thin layer chromatography or other ana when prepared from nonoptically active intermediates and lytical technique well known to those skilled in the art. reagents. Enantiomers can be separated by reacting the Ordinarily, the reaction is stirred for about fifteen minutes to 60 enantiomeric mixture with an appropriate optically active twenty four hours and preferably for one hour. The resulting compound (e.g.,amine) to form a mixture of diastereomeric compound of Formula I is then isolated according to meth Salts of the compound of Formula I and Separating the ods well known to those skilled in the art. diastereomers by crystallization or other method well known The coupling reaction referred to above is used to gen to those skilled in the art. It will be recognized by those erate the compounds of Formula I directly from the com 65 skilled in the art that racemization of the optically active pounds of formula 7 or to generate the compounds of center may occur upon removal of the ammonium counte formula 9 from the compounds of formula 6. The coupling rion. Therefore, when resolving the compounds of Formula 5,998,463 13 14 I using optically active amine compounds, it is particularly (Pharmacia Biotech. Inc., Piscataway, N.J.) in E. coli strain advantageous to use pharmaceutically acceptable optically XL-1 Blue (Stratagene Cloning Systems, LaJolla, Calif.). active amines Such as naturally occurring amino acids The strain is inoculated into LB medium (consisting of 10 g protected as carboxylic acid esters or other pharmaceutically tryptone, 5 g yeast extract, 5 g NaCl, and 1 ml 1N NaOH per acceptable protected amino acids. Other physical resolution liter) plus 100 mg/L ampicillin, 100 mg/L pyridoxine and techniques Such as chromatography are well known to those 600 mg/L MnCl and grown at 37° C. to a cell density of skilled in the art and these techniques may also be used to ODsso-1.0. At this point, the cells are induced with 1 mM resolve the enantiomers of Formula I. All Such diastere isopropyl-1-thio-f-D-galactoside (IPTG). Three hours after omers and enantiomers and mixtures thereof are considered induction the cells are harvested by centrifugation and cell as part of this invention. pellets are frozen at -70° C. until needed for purification. It will be recognized that the compounds of this invention The HBGP cDNA can be expressed by several are acidic and they form a Salt with a pharmaceutically methodologies, for example, by the method described by acceptable cation. All Such Salts are within the Scope of this Crerar, et al. (J. Biol. Chem. 270: 13748–13756). The invention and they can be prepared by conventional meth method described by Crerar, et al. (J. Biol. Chem. ods. Typical bases used to form Such cationic Salts are 15 270:13748–13756) for the expression of HBGP is as fol Sodium hydroxide, Sodium methoxide, Sodium ethoxide, lows: the HBGP cl)NA can be expressed from plasmid Sodium hydride, potassium methoxide, magnesium pTACTAC in E. coli strain 25A6. The strain is inoculated hydroxide, calcium hydroxide, benzathine, choline, into LB medium (consisting of 10 g tryptone, 5 g yeast diethanolamine, piperazine and tromethamine. For example, extract, 5 g NaCl, and 1 ml 1N NaOH per liter) plus 50 mg/L the cationic Salts can be prepared simply by contacting the ampicillin and grown overnight, then resuspended in fresh acidic and basic entities, usually in a Stoichiometric ratio, in LB medium plus 50 mg/L amplicillin, and reinoculated into either an aqueous, non-aqueous or partially aqueous a 40x volume of LB/amp media containing 250 uM medium, as appropriate. The Salts are recovered either by isopropyl-1-thio-f-D-galactoside (IPTG), 0.5 mM pyridox filtration, by precipitation with a non-solvent followed by ine and 3 mM MnCl and grown at 22°C. for 48-50 hours. filtration, by evaporation of the Solvent, or, in the case of The cells can then be harvested by centrifugation and cell aqueous Solutions, by lyophilization, as appropriate. 25 pellets are frozen at -70° C. until needed for purification. In addition, when the compounds of this invention form The HLGP cl)NA is expressed from plasmid pBlueBac III hydrates or Solvates they are also within the Scope of the (Invitrogen Corp., San Diego, Calif.) which is cotransfected invention. with BaculoGold Linear Viral DNA (Pharmingen, San The utility of the compounds of the present invention as Diego, Calif.) into Sf9 cells. Recombinant virus is subse medical agents in the treatment of metabolic diseases (Such quently plaque-purified. For production of protein, Sf9 cells as are detailed herein) in animals, particularly mammals grown in serum-free medium (Sf-900 II serum free medium, (e.g. humans) is demonstrated by the activity of the com Gibco BRL, Life Technologies, Grand Island, N.Y.) are pounds of this invention in conventional assays and the in infected at an moi of 0.5 and at a cell density of 2x10° Vitro and in Vivo assays described below. Such assays also 35 cells/ml. After growth for 72 hours at 27 C., cells are provide a means whereby the activities of the compounds of centrifuged, and the cell pellets frozen at -70° C. until this invention can be compared with the activities of other needed for purification. known compounds. The results of these comparisons are Purification of Glycoen Phosphorylase Expressed in E. coli useful for determining dosage levels in animals, particularly The E. coli cells in pellets described above are resus mammals, including humans, for the treatment of Such 40 pended in 25 mM f-glycerophosphate (pH 7.0) with 0.2 mM diseases. DTT, 1 mM MgCl, plus the following protease inhibitors: The three different purified glycogen phosphorylase (GP) 0.7 tug/mL Pepstatin A isoenzymes, wherein glycogen phosphorylase is in the acti 0.5 lug/mL Leupeptin vated “a” state (referred to as glycogen phosphorylase a, or 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and the abbreviation GPa), and referred to here as human liver 45 0.5 mM EDTA, glycogen phosphorylase a (HLGPa), human muscle glyco lysed by pretreatment with 200 tug/mL lysozyme and 3 gen phosphorylase a (HMGPa), and human brain glycogen lug/mL DNAase followed by Sonication in 250 mL batches phosphorylase a (HBGPa), can be obtained by the following for 5x1.5 minutes on ice using a Branson Model 450 procedures. ultrasonic cell disrupter (Branson Sonic Power Co., Danbury Expression and Fermentation 50 Conn.). The E. coli cell lysates are then cleared by centrifu The HLGP clDNAS (obtained as described in Newgard et gation at 35,000xg for one hour followed by filtration al., Proc. Natl. Acad. Sci., 1986, 83,8132-8136; and New through 0.45 micron filters. GP in the soluble fraction of the gard et al., Proc. Natl. Acad. Sci., 1988, 263, 3850-3857, lysates (estimated to be less than 1% of the total protein) is respectively) and HMGP clDNAS (obtained by screening a purified by monitoring the enzyme activity (as described in Stratagene (Stratagene Cloning Systems, La Jolla, Calif.) 55 GPa Activity Assay section, below) from a series of chro human muscle cDNA library with a polymerase chain reac matographic Steps detailed below. tion (PCR)-generated cDNA fragment based on information Immobilized Metal Affinity Chromatography (IMAC) and methodology reported for isolation of the human skel This step is based on the method of Luong et al (Luong etal muscle glycogen phosphorylase gene and partial cDNA et al. Journal of Chromatography (1992) 584, 77–84). 500 sequence by Kubisch et al., Center for Molecular 60 mL of the filtered soluble fraction of cell lysates (prepared Neurobiology, University of Hamburg, Martinistrasse 85, from approximately 160-250 g of original cell pellet) are Hamburg, 20246 Germany. Genbank (National Center for loaded onto a 130 mL column of IMAC Chelating Biotechnology Information, National Institutes of Health, Sepharose (Pharmacia LKB Biotechnology, Piscataway, USA) Accession Numbers U94774, U94775, U94776 and N.J.) which has been charged with 50 mM CuCl and 25 U94777, Submitted Mar. 20, 1997; Burke et al., Proteins, 65 mM f-glycerophosphate, 250 mM NaCl and 1 mM imida 1987, 2:177-187; and Hwang et al., Eur. J. Biochem., 1985, zole at pH 7 (equilibration buffer). The column is washed 152:267-274) are expressed from plasmid pKK233-2 with equilibration buffer until the Aso returns to baseline. 5,998,463 15 16 The sample is then eluted from the column with the same Anion Exchange Chromatography buffer containing 100 mM imidazole to remove the bound GP and other bound proteins. Fractions containing the GP Following activation of the IMAC purified GPb to GPa by activity are pooled (approximately 600 mL), and ethylene reaction with the immobilized phosphorylase kinase, as diaminetetraacetic acid (EDTA), DL-dithiothreitol (DTT), described below, the pooled GPa fractions are dialyzed phenylmethylsulfonyl fluoride (PMSF), leupeptin and pep against 25 mM Tris-HCl, pH 7.5, containing 0.5 mM DTT, statin A are added to obtain 0.3 mM, 0.2 mM, 0.2 mM, 0.5 0.2 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride tug/mL and 0.7 ug/mL concentrations respectively. The (PMSF), 1.0 ug/mL leupeptin and 1.0 ug/mL pepstatin A. pooled GP is desalted over a Sephadex G-25 column (Sigma The fraction is then loaded onto a MonoO Anion Exchange Chemical Co., St. Louis, Mo.) equilibrated with 25 mM Chromatography column (Pharmacia Biotech. Inc., Tris-HCl (pH 7.3), 3 mM DTT buffer (Buffer A) to remove imidazole and is Stored on ice and Subjected to a Second Piscataway, N.J.). The column is washed with equilibration chromatographic step (below) if necessary. buffer until the Aso returns to baseline. The sample is then 5'-AMP-Sepharose Chromatography eluted from the column with a linear gradient of 0-0.25 M The desalted pooled GPsample (approximately 600 mL) NaCl to remove the bound GP and other bound proteins. is next mixed with 70 mL of 5'-AMP Sepharose (Pharmacia 15 GP-containing fractions elute between 0.1-0.2 M NaCl LKB Biotechnology, Piscataway, N.J.) which has been range, as detected by monitoring the eluant for peak protein equilibrated with Buffer A (see above). The mixture is gently absorbance at Aso. The GP protein is then identified by agitated for one hour at 22 C. then packed into a column visualizing the M approximately 97 kdal GP protein band and washed with Buffer Auntil the Aso returns to baseline. by Sodium dodecyl Sulfate polyacrylamide gel electrophore GP and other proteins are eluted from the column with 25 sis (SDS-PAGE) followed by silver staining (2D-silver Stain mM Tris-HCl, 0.2 mM DTT and 10 mM adenosine II “Daiichi Kit”, Daiichi Pure Chemicals Co., LTD., Tokyo, 5'-monophosphate (AMP) at pH 7.3 (Buffer B). Japan) and then pooled. The pooled GP is dialyzed into 25 GP-containing fractions are pooled following identification mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid by determining enzyme activity described below and Visu 25 (BES), 1.0 mM DTT, 0.5 mM EDTA, 5 mM NaCl, pH 6.8 alizing the M approximately 97 kdal GP protein band by buffer and Stored on ice until use. Sodium dodecyl Sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (2D-silver Stain II Determination of GP Enzyme Activity “Daiichi Kit', Daiichi Pure Chemicals Co., LTD., Tokyo, A) Activation of GP: Conversion of GPb to GPa. Japan) and then pooled. The pooled GP is dialyzed into 25 Prior to the determination of GP enzyme activity, the mM B-glycerophosphate, 0.2 mM DTT, 0.3 mM EDTA, 200 enzyme is converted from the inactive form as expressed in mM NaCl, pH 7.0 buffer (Buffer C) and stored on ice until E.coli strain XL-1 Blue (designated GPb) (Stragene Cloning Sc. Systems, La Jolla, Calif.), to the active form (designated Prior to use of the GP enzyme, the enzyme is converted GPa) by phosphorylation of GP using phosphorylase kinase from the inactive form as expressed in E.coli strain XL-1 35 as follows. The fraction of inactive enzyme as expressed in Blue (designated GPb) (Stragene Cloning Systems, LaJolla, Sf9 cells (designated GPb) is also converted to the active Calif.), to the active form (designated GPa) by the procedure form (designated GPa) by the follow procedure. described in Section (A) Activation of GP below. Purification of Glycogen Phosphorylase Expressed in Sf9 GP Reaction with Immobilized Phosphorylase Kinase Cells Phosphorylase kinase (Sigma Chemical Company, St. The Sf9 cells in pellets described above are resuspended 40 Louis, Mo.) is immobilized on Affi-Gel(R) 10 (BioPad Corp., in 25 mM B-glycerophosphate (pH 7.0) with 0.2 mM DTT, Melvile, N.Y.) as per the manufacturer's instructions. In 1 mM MgCl2, plus the following protease inhibitors: brief, the phosphorylase kinase enzyme (10 mg) is incubated 0.7 tug/mL Pepstatin A with washed Affi-Gel(B) beads (1 mL) in 2.5 mL of 100 mM 0.5 lug/mL Leupeptin 45 HEPES and 80 mM CaCl at pH 7.4 for 4 hours at 4°C. The 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and Affi-Gel(E) beads are then washed once with the same buffer 0.5 mM EDTA, prior to blocking with 50 mM HEPES and 1 M glycine lysed by pretreatment with 3 tug/mL DNAase followed by methyl ester at pH 8.0 for one hour at room temperature. Sonication in batches for 3x1 minutes on ice using a Branson Blocking buffer is removed and replaced with 50 mM Model 450 ultrasonic cell disrupter (Branson Sonic Power 50 HEPES (pH 7.4), 1 mM f-mercaptoethanol and 0.2% NaN Co., Danbury Conn.). The Sf9 cell lysates are then cleared for storage. Prior to use to convert GPb to GPa, the Affi by centrifugation at 35,000xg for one hour followed by Gelf immobilized phosphorylase kinase beads are equili filtration through 0.45 micron filters. GP in the soluble brated by washing in the buffer used to perform the kinase fraction of the lysates (estimated to be 1.5% of the total reaction, consisting of 25 mM f-glycerophosphate, 0.3 mM protein) is purified by monitoring the enzyme activity (as 55 DTT, and 0.3 mM EDTA at pH 7.8 (kinase assay buffer). described in GPa Activity Assay section, below) from a Series of chromatographic Steps detailed below. The partially purified, inactive GPb obtained from Immobilized Metal Affinity Chromatography (IMAC) 5'-AMP-Sepharose chromatography above (from E. coli) or Immobilized Metal Affinity Chromatography is per the mixture of GPa and GPb obtained from IMAC above formed as described in the section above. The pooled, 60 (from Sf9 cells) is diluted 1:10 with the kinase assay buffer desalted GP is then stored on ice until further processed. then mixed with the aforementioned phosphorylase kinase Activation of GP enzyme immobilized on the Affi-Gel(R) beads. NaATP is Before further chromatography, the fraction of inactive added to 5 mM and MgCl2 to 6 mM. The resulting mixture enzyme as expressed in Sf9 cells (designated GPb) is is mixed gently at 25 C. for 30 to 60 minutes. The activated converted to the active form (designated GPa) by the fol 65 Sample is removed from the beads and the percent activation lowing procedure described in Section (A) Activation of GP of GPb by conversion to GPa is estimated by determining below. GP enzyme activity in the presence and absence of 3.3 mM 5,998,463 17 18 AMP. The percentage of total GP enzyme activity due to Shechosky, S. and Madsen, N. B. (1970) Can. J. Biochem. GPa enzyme activity (AMP-independent) is then calculated 48,746–754 modified as follows: 1 to 100 ug GPa is diluted as follows: to 1 mL in Buffer E (pH 7.2, 50 mM HEPES, 100 mM KC1, 2.5 mM EGTA, 2.5 mM MgCl, and 0.5 mM dithiothreitol). HLGP activity - AMP 20 ul of this stock is added to 80 ul of Buffer E with 1.25 % of total HLGPa= mg/mL glycogen, 9.4 mM glucose, and 0.63 mM glucose HLGP activity + AMP 1-phosphate. The compound to be tested is added as 5 till of solution in 14% DMSO prior to the addition of the enzyme. Alternately, the conversion of GPb to GPa can be moni The basal rate of GPa enzyme activity in the absence of tored by isoelectric focusing, based on the shift in electro added inhibitors, e.g., a compound of this invention, is phoretic mobility that is noted following conversion of GPb determined by adding 5 till of 14% DMSO and a fully to GPa. GP Samples are analyzed by isoelectric focusing inhibited rate of GPa enzyme activity is obtained by adding (IEF) utilizing the Pharmacia PfastGel System (Pharmacia 20 u, of 50 mM caffeine. This mixture is incubated at room Biotech. Inc., Piscataway, N.J.) using precast gels (p range temperature for 1 hour and the inorganic phosphate released 4-6.5) and the manufacturer's recommended method. The 15 from the glucose-1-phosphate is measured by the general resolved GPa and GPb bands are then visualized on the gels method of Lanzetta et al. Lanzetta, P. A., Alvarez, L. J., by silver staining (2D-silver Stain II “Daiichi Kit”, Daiichi Reinach, P. S. and Candia, O.A. (1979) Anal. Biochem. 100, Pure Chemicals Co., LTD., Tokyo, Japan). Identification of 95-97 modified as follows: 150 till of 10 mg/mL ammo GPa and GPb is made by comparison to E. coli derived GPa. nium molybdate, 0.38 mg/mL malachite green in 1 NHCl is and GPb Standards that are run in parallel on the same gels added to 100 ul of the enzyme mix. After a 20 minute as the experimental Samples. incubation at room temperature, the absorbance is measured B) GPa Activity Assay at 620 nm. The disease/condition treating/preventing activities The above assays carried out with a range of concentra described herein of the compounds of this invention can be tions of test compound allows the determination of an ICso indirectly determined by assessing the effect of the com 25 value (concentration of test compound required for 50% pounds of this invention on the activity of the activated form inhibition) for the in vitro inhibition of GPa enzyme activity of glycogen phosphorylase (GPa) by one of two methods; by that test compound. glycogen phosphorylase a activity is measured in the for The compounds of this invention are readily adapted to ward direction by monitoring the production of glucose-1- clinical use as hypoglycemic agents. The hypoglycemic phosphate from glycogen or by following the reverse activity of the compounds of this invention can be deter reaction, measuring glycogen Synthesis from glucose-1- mined by the amount of test compound that reduces glucose phosphate by the release of inorganic phosphate. All reac levels relative to a vehicle without test compound in male tions are run in triplicate in 96-well microtiter plates and the ob/ob mice. The test also allows the determination of an change in absorbance due to formation of the reaction approximate minimal effective dose (MED) value for the in product is measured at the wavelength specified below in a 35 Vivo reduction of plasma glucose concentration in Such mice MCC/340 MKII Elisa Reader (Lab Systems, Finland), con for Such test compounds. nected to a Titertech Microplate Stacker (ICN Biomedical Since the concentration of glucose in blood is closely Co, Huntsville, Ala.). related to the development of diabetic disorders, the com To measure the GPa enzyme activity in the forward pounds of this invention, by Virtue of their hypoglycemic direction, the production of glucose-1-phosphate from gly 40 action, prevent, arrest and/or regreSS diabetic disorders. cogen is monitored by the multienzyme coupled general Five to eight week old male C57BL/6J-ob/ob mice method of Pesce et al. Pesce, M. A., Bodourian, S. H., (obtained from Jackson Laboratory, Bar Harbor, Me.) are Harris, R. C. and Nicholson, J. F. (1977) Clinical Chemistry housed five per cage under Standard animal care practices. 23, 1711–1717 modified as follows: 1 to 100 lug GPa, 10 After a one week acclimation period, the animals are units phosphoglucomutase and 15 units glucose-6- 45 weighed and 25 microliters of blood are collected from the phosphate dehydrogenase (Boehringer Mannheim retro-orbital Sinus prior to any treatment. The blood Sample Biochemicals, Indianapolis, Ind.) are diluted to 1 mL in is immediately diluted 1:5 with saline containing 0.025% Buffer D (pH 7.2, 50 mM HEPES, 100 mM KC1, 2.5 mM Sodium heparin, and held on ice for metabolite analysis. ethyleneglycoltetraacetic acid (EGTA), 2.5 mM MgCl, 3.5 Animals are assigned to treatment groups. So that each group mM KHPO and 0.5 mM dithiothreitol). 20 ul of this stock 50 has a Similar mean for plasma glucose concentration. After is added to 80 ul of Buffer D containing 0.47 mg/mL group assignment, animals are dosed orally each day for four glycogen, 9.4 mM glucose, 0.63 mM of the oxidized form of days with the vehicle consisting of either: (1) 0.25% w/v nicotinamide adenine dinucleotide phosphate (NADP+). methyl cellulose in water without pH adjustment; or (2) The compound to be tested is added as 5 lull of solution in 0.1% Pluronic(R) P105 Block Copolymer Surfactant (BASF 14% dimethylsulfoxide (DMSO) prior to the addition of the 55 Corporation, Parsippany, N.J.) in 0.1% saline without pH enzymes. The basal rate of GPa enzyme activity in the adjustment. On day 5, the animals are weighed again and absence of inhibitors, e.g., a compound of this invention, is then dosed orally with a test compound or the vehicle alone. determined by adding 5 till of 14% DMSO and a fully All compounds are administered in vehicle consisting of inhibited rate of GPa enzyme activity is obtained by adding either: (1) 0.25% w/v methyl cellulose in water without pH 20 ul of 50 mM of the positive control test substance, 60 adjustment; or (2) 10% DMSO/0.1% Pluronic(R) in 0.1% caffeine. The reaction is followed at room temperature by saline without pH adjustment. The animals are then bled measuring the conversion of oxidized NADP+ to reduced from the retro-orbital sinus three hours later for determina NADPH at 340 nm. tion of blood metabolite levels. The freshly collected To measure the GPa enzyme activity in the reverse Samples are centrifuged for two minutes at 10,000xg at room direction, the conversion of glucose-1-phosphate into gly 65 temperature. The Supernatant is analyzed for glucose, for cogen plus inorganic phosphate is measured by the general example, by the Abbott VPTM (Abbott Laboratories, Diag method described by Engers et al. Engers, H. D., nostics Division, Irving, Tex.) and VP Super System(R) 5,998,463 19 20 Autoanalyzer (Abbott Laboratories, Irving, Tex.), using the out pH adjustment. Single daily dosing (S.i.d.) or twice daily A-GentTMGlucose-UV Test reagent system (Abbott dosing (b.i.d.) is maintained for 1 to, for example, 15 days. Laboratories, Irving, Tex.) (a modification of the method of Control mice receive the 10% DMSO/O.1% Pluronic(R) P105 Richterich and Dauwalder, Schweizerische Medizinische in 0.1% saline without pH adjustment or the 0.25% w/v Wochenschrift, 101,860 (1971)) (hexokinase method) using methylcellulose in water without pH adjustment only. a 100 mg/dL Standard. Plasma glucose is then calculated by Three hours after the last dose is administered, the ani the equation: mals are Sacrificed by decapitation and trunk blood is Plasma glucose (mg/dL)=Sample valuex8.14 collected into 0.5 mL Serum Separator tubes containing 3.6 mg of a 1:1 weight/weight Sodium fluoride: potassium where 8.14 is the dilution factor, adjusted for plasma oxalate mixture. The freshly collected Samples are centri hematocrit (assuming the hematocrit is 44%). fuged for two minutes at 10,000xg at room temperature, and The animals dosed with vehicle maintain substantially the Serum Supernatant is transferred and diluted 1:1 Volume/ unchanged hyperglycemic glucose levels (e.g., greater than volume with a 1 TIU/mL aprotinin solution in 0.1% saline or equal to 250 mg/dL), animals treated with compounds without pH adjustment. having hypoglycemic activity at Suitable doses have signifi 15 The diluted serum samples are then stored at -80 C. until cantly depressed glucose levels. Hypoglycemic activity of analysis. The thawed, diluted Serum Samples are analyzed the test compounds is determined by Statistical analysis for insulin, triglycerides, free fatty acids and cholesterol (unpaired t-test) of the mean plasma glucose concentration levels. Serum insulin concentration is determined using between the test compound group and vehicle-treated group Equate(R) RIA INSULIN kits (double antibody method; as on day 5. The above assay carried out with a range of doses Specified by the manufacturer) purchased from Binax, South of a test compound allows the determination of an approxi Portland, Me. The inter assay coefficient of variation is mate minimal effective dose (MED) value for the in vivo s 10%. Serum triglycerides are determined using the Abbott reduction of plasma glucose concentration. VPTM and VP Super System(R Autoanalyzer (Abbott The compounds of this invention are readily adapted to Laboratories, Irving, Tex.), or the Abbott Spectrum CCXTM clinical use as hyperinsulinemia reversing agents, triglycer 25 (Abbott Laboratories, Irving, Tex.) using the A-GentTM ide lowering agents and hypocholesterolemic agents. Such Triglycerides Test reagent System (Abbott Laboratories, activity can be determined by the amount of test compound Diagnostics Division, Irving, Tex.) (lipase-coupled enzyme that reduces insulin, triglycerides or cholesterol levels rela method; a modification of the method of Sampson, et al., tive to a control vehicle without test compound in male Clinical Chemistry 21, 1983 (1975)). Serum total cholesterol ob/ob mice. levels are determined using the Abbott VPTM and VP Super Since the concentration of cholesterol in blood is closely System(R) Autoanalyzer (Abbott Laboratories, Irving, Tex.), related to the development of cardiovascular, cerebral vas and A-GentTM Cholesterol Test reagent system (cholesterol cular or peripheral vascular disorders, the compounds of this esterase-coupled enzyme method; a modification of the invention, by virtue of their hypocholesterolemic action, method of Allain, et al. Clinical Chemistry 20, 470 (1974)) prevent, arrest and/or regreSS atherosclerosis. 35 using 100 and 300 mg/dL standards. Serum free fatty acid Since the concentration of insulin in blood is related to the concentration is determined utilizing a kit from Amano promotion of vascular cell growth and increased renal International Enzyme Co., Inc., as adapted for use with the Sodium retention, (in addition to the other actions, e.g., Abbott VPTM and VP Super System(E) Autoanalyzer (Abbott promotion of glucose utilization) and these functions are Laboratories, Irving, Tex.), or the Abbott Spectrum CCXTM known causes of hypertension, the compounds of this 40 (Abbott Laboratories, Irving, Tex.). Serum insulin, invention, by Virtue of their hypoinSulinemic action, triglycerides, free fatty acids and total cholesterol levels are prevent, arrest and/or regreSS hypertension. then calculated by the equations, Since the concentration of triglycerides in blood contrib utes to the overall levels of blood lipids, the compounds of Serum insulin (ul J/mL)=Sample valuex2 this invention, by virtue of their triglyceride lowering and/or 45 free fatty acid lowering activity prevent, arrest and/or Serum triglycerides (mg/dL)=Sample valuex2 regreSS hyperlipidemia. Serum total cholesterol (mg/dL)=Sample valuex2 Free fatty acids contribute to the overall level of blood lipids and independently have been negatively correlated Serum free fatty acid (uEq/L)=Sample valuex2 with insulin Sensitivity in a variety of physiologic and 50 where 2 is the dilution factor. pathologic States. The animals dosed with vehicle maintain substantially Five to eight week old male C57BL/6J-ob/ob mice unchanged, elevated Serum insulin (e.g., 275 uU/mL), Serum (obtained from Jackson Laboratory, Bar Harbor, Me.) are triglycerides (e.g., 235 mg/dl), serum free fatty acid (1500 housed five per cage under Standard animal care practices mEq/mL) and Serum total cholesterol (e.g., 190 mg/dL) and fed standard rodent diet ad libitum. After a one week 55 levels, while animals treated with compounds of this inven acclimation period, the animals are weighed and 25 micro tion generally display reduced Serum insulin, triglycerides, liters of blood are collected from the retro-orbital sinus prior free fatty acid and total cholesterol levels. The serum insulin, to any treatment. The blood sample is immediately diluted triglycerides, free fatty acid and total cholesterol lowering 1:5 with Saline containing 0.025% sodium heparin, and held activity of the test compounds are determined by Statistical on ice for plasma glucose analysis. Animals are assigned to 60 analysis (unpaired t-test) of the mean Serum insulin, treatment groups. So that each group has a similar mean for triglycerides, or total cholesterol concentration between the plasma glucose concentration. The compound to be tested is test compound group and the vehicle-treated control group. administered by oral gavage as an about 0.02% to 2.0% Activity in providing protection from ischemia (e.g., solution (weight/volume (w/v)) in either (1) 10% DMSO/ damage to heart tissue) for the compounds of this invention 0.1% Pluronic(R) P105 Block Copolymer Surfactant (BASF 65 can be demonstrated in Vitro according to procedures Set Corporation, Parsippany, N.J.) in 0.1% saline without pH forth in Butwell et al., Am. J. Physiol., 264, H1884–H1889, adjustment or (2) 0.25% w/v methylcellulose in water with 1993 and Allard et al., Am. J. Physio., 1994, 267, H66-H74. 5,998,463 21 22 Experiments are performed using an isoVolumic isolated rat hearts are then exposed through a left thoracotomy and a heart preparation, essentially as described in the above Snare (00 silk) placed around a prominent branch of the left referenced article. Normal male Sprague-Dawley rats, male coronary artery. lSchemia is induced by pulling the Snare Sprague-Dawley rats treated to possess cardiac hypertrophy tight and clamping it in place. Releasing the Snare allowed by an aortic banding operation, acutely diabetic male BB/W the affected area to reperfuse. Myocardial ischemia is evi rats, or non-diabetic BB/W age matched control rats are denced by regional cyanosis, reperfusion was evidenced by pretreated with heparin (1000 U, i.p.), followed by pento reactive hyperemia. barbital (65 mg/kg, i.p.). After deep anesthesia is achieved as Protocol: Once arterial pressure and heart rate has been determined by the absence of a foot reflex, the heart is stable for at least 30 minutes the experiment is started. rapidly excised and placed into iced Saline. The heart is Ischemic preconditioning is induced by twice occluding the retrogradely perfused through the aorta within 2 minutes. coronary artery for 5 min followed by a 10 min reperfusion. Heart rate and Ventricular pressure are determined using a Pharmacological preconditioning is induced by twice infus latex balloon in the left ventricle with high pressure tubing ing a test compound over, for example, 5 minutes and connected to a pressure transducer. The heart is perfused allowing 10 minutes before further intervention or by infus with a perfusate solution consisting of (mM) NaCl 118, KCl 15 ing the adenosine agonist, PIA (0.25 mg/kg). Following 4.7, CaCl 1.2, MgCl, 1.2, NaHCO25 and glucose 11. The ischemic preconditioning, pharmacological preconditioning perfusion apparatus is tightly temperature-controlled with or no conditioning (unconditioned, vehicle control) the heated baths used for the perfusate and for the water artery is occluded for 30 minutes and then reperfused for two jacketing around the perfusion tubing to maintain heart hours to induce myocardial infarction. The test compound temperature at 37 C. Oxygenation of the perfusate is and PIA are dissolved in Saline or other Suitable vehicle and provided by a pediatric hollow fiber oxygenator (Capiax, delivered at 1 to 5 ml/kg, respectively. Staining (Liu et al., Terumo Corp., Tokyo, Japan) immediately proximal to the Circulation 84:350–356, 1991): At the end of the 2 hour heart. Hearts are exposed to perfusion Solution with and reperfusion period, the hearts are quickly removed, hung on without test compound for about 10 minutes or more, a Langendorff apparatus, and flushed for 1 minute with followed by 20 minutes of global ischemia and 60 minutes 25 normal saline heated to body temperature (38 C.). The silk of reperfusion in the absence of the test compound. The heart Suture used as the Snare is then tied tightly to reocclude the beats of the control and test compound treated hearts are artery and a 0.5% suspension of fluorescent particles (1-10 compared in the period following ischemia. The left ven lum in diameter, obtained from Duke Scientific Corp., Palo tricular pressure of the control and test compound treated Alto, Calif.) is infused with the perfusate to stain all of the hearts are compared in the period following ischemia. At the myocardium except the area at risk (nonfluorescent end of the experiment, hearts are also perfused and Stained ventricle). The hearts are then quickly frozen and stored to determine the ratio of infarct area relative to the area at overnight at -20°C. On the following day, the hearts are cut risk (% IA/AAR) as described below. into 2 mm slices and Stained with 1% triphenyl tetrazolium The therapeutic effects of the compounds of this invention chloride (TTC). Since TTC reacts with living tissue, this in preventing heart tissue damage otherwise resulting from 35 Stain differentiates between living (red Stained) tissue, and an ischemic insult can also be demonstrated in Vivo accord dead tissue (unstained infarcted tissue). The infarcted area ing to procedures Set forth in Liu et al., Circulation, Vol. 84, (no stain) and the area at risk (no fluorescent particles) are No. 1, (July 1991), as described specifically herein. The in calculated for each Slice of left Ventricle using a pre Vivo assay tests the cardioprotection of a test compound calibrated image analyzer. To normalize the ischemic injury relative to the control group which receives Saline vehicle. 40 for differences in the area at risk between hearts, the data is AS background information, it is noted that brief periods of expressed as the ratio of infarct area VS. area at risk (% myocardial ischemia followed by coronary artery reperfu IA/AAR). All data are expressed as Meani-SEM and com Sion protects the heart from Subsequent Severe myocardial pared Statistically using Single factor ANOVA or unpaired ischemia (Murry et al., Circulation 74:1124-1136, 1986). t-test. Significance is considered as p-0.05. Cardioprotection, as indicated by a reduction in infarcted 45 The compounds of this invention can be tested for its myocardium, can be induced pharmacologically using intra utility in reducing or preventing ischemic injury in non venously administered adenosine receptor agonists in intact, cardiac tissues, for example, the brain, or the liver, utilizing anesthetized rabbits Studied as an in Situ model of myocar procedures reported in the Scientific literature. The com dial ischemic preconditioning (Liu et al., Circulation pounds of this invention can be administered by the pre 84:350–356, 1991). The in vivo assay tests whether a 50 ferred route and vehicle of administration and at the pre compound can pharmacologically induce cardioprotection, ferred time of administration either prior to the ischemic i.e., reduced myocardial infarct size, when parenterally episode, during the ischemic episode, following the administered to intact, anesthetized rabbits. The effects of ischemic episode (reperfusion period) if included, or during the compounds of this invention can be compared to any of the below-mentioned experimental Stages. ischemic preconditioning using the A1 adenosine agonist, 55 The benefit of the compounds of this invention to reduce N-1-(phenyl-2R-isopropyl) adenosine (PIA) that has been ischemic brain damage can be demonstrated, for example, in shown to pharmacologically induce cardioprotection in mammals using the method of Park, et al (Ann. Neurol. intact anesthetized rabbits studied in situ (Liu et al., Circu 1988:24:543–551). In brief, in the procedure of Park, et al. lation 84:350–356, 1991). The exact methodology is adult male Sprague Dawley rats are anesthetized initially described below. 60 with 2% halothane, and thereafter by mechanical ventilation Surgery: New Zealand White male rabbits (3–4 kg) are with a nitrous oxide-oxygen mixture (70%:30%) containing anesthetized with Sodium pentobarbital (30 mg/kg, i.v.). A 0.5–1% halothane. A tracheostomy is then performed. The tracheotomy is performed via a Ventral midline cervical Stroke Volume of the ventilator is adjusted to maintain incision and the rabbits are ventilated with 100% oxygen arterial carbon dioxide tension at approximately 35 mm Hg using a positive pressure ventilator. Catheters are placed in 65 and adequate arterial oxygenation (PaC)>90 mm Hg). Body the left jugular vein for compound administration and in the temperature can be monitored by a rectal thermometer, and left carotid artery for blood preSSure measurements. The the animals can be maintained normothermic, if necessary, 5,998,463 23 24 by external heating. The animals next undergo Subtemporal example, by the reflectometry method using an automatic craniectomy to expose the main trunk of the left middle Kodak Ektachem 500 analyzer reported by Nakano, et al. cerebral artery (MCA) under an operating microscope, and (Hepatology 1995:22:539-545). The ability of the com the exposed artery is occluded with microbipolar coagula pounds of this invention to reduce ischemic liver damage tion to generate large ischemic lesions in the cerebral cortex 5 induced by occlusion is noted based on a reduction in the and basal ganglia. After three hours of MCA occlusion, the release of hepatocellular enzymes immediately following rats are deeply anesthetized with 2% halothane and a tho the occlusive period and/or during the postischemic racotomy is performed to infuse heparinized Saline into the reperfusion period in the perfused livers from the rats in the left ventricle. The effluent is collected via an incision of the treatment group compared to perfused livers from rats in a right atrium. The Saline washout is followed by approxi placebo-treated control group. mately 200 ml of a fixative, e.g., 40% formaldehyde, glacial Other methods and parameters which could alternatively acetic acid and absolute methanol solution (FAM; 1:1:8, be utilized to demonstrate the benefit of the compounds of V/v/v), then the animals are decapitated and the head is this invention to reduce ischemic liver damage include those stored in the fixative for 24 hours. The brain is then described by Nakano, et al. (Hepatology 1995:22:539-545). removed, dissected, processed, embedded in paraffin wax, 15 Administration of the compounds of this invention can be and Sectioned (approximately 100 Sections per brain). The via any method which delivers a compound of this invention Sections are then Stained with hematoxylin-eosin or with a preferentially to the liver and/or cardiac tissues. These combination of cresyl violet and Luxol(R) (registered to Dow methods include oral routes, parenteral, intraduodenal Chemical Co. and obtainable from Sigma Chemical Co., St. routes, etc. Generally, the compounds of the present inven Louis, Mo.) fast blue, and examined by light microscopy to tion are administered in Single (e.g., once daily) or multiple identify and quantitate the ischemic damage using an image doses. analyzer (e.g. the Quantimet 720 Image Analyzer, Leica, However, the amount and timing of compound(s) admin Inc., Deerfield, Ill.). The ischemic volumes and areas are istered will, of course, be dependent on the particular expressed in absolute units (mm and mm) and as a disease/condition being treated, the Subject being treated, on percentage of the total region examined. The effect of the 25 the Severity of the affliction, on the manner of administration invention to reduce ischemic brain damage induced by MCA and on the judgment of the prescribing physician. Thus, occlusion is noted based on a reduction in the area or Volume because of Subject to Subject variability, the dosages given of relative or absolute ischemic damage in the brain Sections below are a guideline and the physician may titrate doses of from the rats in the treatment group compared to brain the drug to achieve the activity (e.g., glucose lowering Sections from rats in a placebo-treated control group. activity) that the physician considers appropriate for the Other methods which could alternatively be utilized to Subject. In considering the degree of activity desired, the demonstrate the benefit of the compounds of this invention physician must balance a variety of factorS Such as Starting to reduce ischemic brain damage include those described by level, other risk (cardiovascular) factors, presence of preex Nakayama, et al. in Neurology 1988;38:1667-1673, isting disease, and age of the Subject and the Subject's Memezawa, et al. in Stroke 1992:23:552–559, Folbergrova, 35 motivation. et al. in Proc. Nati. Acad. Sci 1995;92:5057-5059, or Gofti, In general an effective dosage for the compounds of this et al. in Brain Res. 1990;522:290–307. invention, for example in the blood glucose, triglycerides, The benefit of the compounds of this invention to reduce free fatty acid and cholesterol lowering activities and hyper ischemic liver damage can be demonstrated, for example, in insulinemia reversing activities of the compounds of this mammals using the method of Yokoyama, et al. (Am. J. 40 invention is in the range of 0.005 to 50 mg/kg/day, prefer Physiol. 1990;258:G564–G570). In brief, in the procedure ably 0.01 to 25 mg/kg/day and most preferably 0.1 to 15 of Yokoyama, et al., fasted adult male Sprague Dawley rats mg/kg/day. are anesthetized with Sodium pentobarbital (40 mg/kg i.p.), Generally, the compounds of this invention are adminis then the animals are tracheotomized and mechanically ven tered orally, but parenteral administration (e.g., intravenous, tilated with room air. The liver is extirpated and placed in an 45 intramuscular, Subcutaneous or intramedullary) may be environmental chamber maintained at constant temperature utilized, for example, where oral administration is inappro (37° C), then perfused through the portal vein at a constant priate for the instant target or where the Subject is unable to pressure of 15 cm H2O with a modified, hemoglobin-free ingest the drug. Topical administration may also be Krebs-Henseleit buffer (in mM: 118 NaCl, 4.7 KC1, 27 indicated, for example, where the Subject is Suffering from NaHCO, 2.5 CaCl, 1.2 MgSO, 1.2 KHPO, 0.05 EDTA, 50 gastrointestinal disorders or whenever the is best and 11 mM glucose, plus 300 U heparin). The pH of the applied to the Surface of a tissue or organ as determined by perfusate is maintained at 7.4 by gassing the buffer with 95% the attending physician. O-5% CO. Each liver is perfused at a flow rate of 20 The compounds of the present invention are generally ml/min in a single-pass manner for a 30 min Washout and administered in the form of a pharmaceutical composition equilibration period (preischemic period), followed by a 2 55 comprising at least one of the compounds of this invention hour period of global ischemia, and then a 2 hour period of together with a pharmaceutically acceptable carrier or dilu reperfusion under conditions identical to the preischemic ent. Thus, the compounds of this invention can be admin period. Aliquots (20 ml) of the perfusate are collected during istered individually or together in any conventional oral, the preischemic period, immediately after the occlusive parenteral or transdermal dosage form. ischemic period, and every 30 min of the 2 hour reperfusion 60 For oral administration, a pharmaceutical composition period. The perfusate Samples are assayed for the appear can take the form of Solutions, Suspensions, tablets, pills, ance of hepatocellular enzymes, for example, aspartate capsules, powders, and the like. Tablets containing various amino-transferase (AST), alanine amino-transferase (ALT), excipients Such as Sodium citrate, calcium carbonate and and lactate dehydrogenase (LDH), which are taken to quan calcium phosphate are employed along with various disin titatively reflect the degree of ischemic liver tissue damage 65 tegrants Such as Starch and preferably potato or tapioca during the procedure. AST, ALT, and LDH activities in the Starch and certain complex Silicates, together with binding perfusate can be determined by Several methods, for agents Such as polyvinylpyrrollidone, Sucrose, gelatin and 5,998,463 25 26 acacia. Additionally, lubricating agents Such as magnesium whereby the numbers correspond with the days of the Stearate, Sodium lauryl Sulfate and talc are often very useful regimen which the dosage form So Specified should be for tabletting purposes. Solid compositions of a similar type ingested. Another example of Such a memory aid is a are also employed as fillers in Soft and hard-filled gelatin capsules, preferred materials in this connection also include calendar printed on the card, e.g., as follows "First Week, lactose or milk Sugar as well as high molecular weight Monday, Tuesday, . . . etc . . . Second Week, Monday, polyethylene glycols. When aqueous Suspensions and/or Tuesday, ... '' etc. Other variations of memory aids will be elixirs are desired for oral administration, the compounds of readily apparent. A "daily dose” can be a Single tablet or this invention can be combined with various Sweetening capsule or Several tablets or capsules to be taken on a given agents, flavoring agents, coloring agents, emulsifying agents day. Also, a daily dose of a Formula I compound, a prodrug and/or Suspending agents, as well as Such diluents as water, ethanol, propylene glycol, glycerin and various like combi thereof or a pharmaceutically acceptable Salt of Said com nations thereof. pound or said prodrug can consist of one tablet or capsule For purposes of parenteral administration, Solutions in while a daily dose of the Second compound can consist of Sesame or peanut oil or in aqueous propylene glycol can be Several tablets or capsules and Vice versa. The memory aid employed, as well as Sterile aqueous Solutions of the corre 15 should reflect this. sponding water-Soluble Salts. Such aqueous Solutions may be suitably buffered, if necessary, and the liquid diluent first In another specific embodiment of the invention, a dis rendered isotonic with Sufficient Saline or glucose. These penser designed to dispense the daily doses one at a time in aqueous Solutions are especially Suitable for intravenous, the order of their intended use is provided. Preferably, the intramuscular, Subcutaneous and intraperitoneal injection dispenser is equipped with a memory-aid, So as to further purposes. In this connection, the Sterile aqueous media facilitate compliance with the regimen. An example of Such employed are all readily obtainable by Standard techniques a memory-aid is a mechanical counter which indicates the well-known to those skilled in the art. number of daily doses that has been dispensed. Another For purposes of transdermal (e.g.,topical) administration, example of Such a memory-aid is a battery-powered micro dilute sterile, aqueous or partially acqueous Solutions (usually 25 chip memory coupled with a liquid crystal readout or in about 0.1% to 5% concentration), otherwise similar to the audible reminder Signal which, for example, reads out the above parenteral Solutions, are prepared. date that the last daily dose has been taken and/or reminds Since the present invention has an aspect that relates to a one when the next dose is to be taken. combination of active ingredients which may be adminis tered Separately, the invention also relates to combining Methods of preparing various pharmaceutical composi Separate pharmaceutical compositions in kit form. The kit tions with a certain amount of active ingredient are known, comprises two separate pharmaceutical compositions: a or will be apparent in light of this disclosure, to those skilled compound of Formula I, a prodrug thereof or a pharmaceu in this art. For examples, see Remington's Pharmaceutical tically acceptable Salt of Said compound or said prodrug and Sciences, Mack Publishing Company, Easton, Pa., 19th a Second compound as described above. The kit comprises 35 Edition (1990). container means for containing the Separate compositions Pharmaceutical compositions according to the invention such as a divided bottle or a divided foil packet, however, the may contain 0.1%-95% of the compound(s) of this Separate compositions may also be contained within a invention, preferably 1%–70%. In any event, the composi Single, undivided container. Typically, the kit comprises tion or formulation to be administered will contain a quan directions for the administration of the Separate components. 40 The kit form is particularly advantageous when the Separate tity of a compound(s) according to the invention in an components are preferably administered in different dosage amount effective to treat the disease/condition of the Subject forms (e.g., oral and parenteral), are administered at differ being treated, i.e., a glycogen phosphorylase dependent ent dosage intervals, or when titration of the individual disease/condition. components of the combination is desired by the prescribing 45 physician. TECHNIOUES An example of Such a kit is a So-called blister pack. Blister packs are well known in the packaging industry and are NMR spectra were recorded on a Varian XL-300 (Varian being widely used for the packaging of pharmaceutical unit Co., Palo Alto, Calif.) or Bruker AM-300 spectrometer dosage forms (tablets, capsules, and the like). Blister packs 50 (Bruker Co., Billerica, Mass.) at about 23° C. at 300 MHz generally consist of a sheet of relatively Stiff material for proton and 75.4 mHz for carbon nuclei. Chemical shifts covered with a foil of a preferably transparent plastic mate are expressed in parts per million downfield from trimeth rial. During the packaging proceSS recesses are formed in the ylsilane. plastic foil. The recesses have the size and shape of the Column chromatography was performed with Amicon tablets or capsules to be packed. Next, the tablets or capsules 55 silica gel (30 uM, 60A pore size) (Amicon D Vision, W.R. are placed in the recesses and the sheet of relatively Stiff Grace & Co., Beverly, Mass.) in glass columns under low material is Sealed against the plastic foil at the face of the foil nitrogen preSSure. Unless otherwise Specified, reagents were which is opposite from the direction in which the recesses used as obtained from commercial Source S. were formed. As a result, the tablets or capsules are Sealed Dimethylformamide, 2-propanol, tetrahydrofuran, and in the recesses between the plastic foil and the sheet. 60 Preferably the strength of the sheet is such that the tablets or dichloromethane used as reaction Solvents were the anhy capsules can be removed from the blister pack by manually drous grade Supplied by Aldrich Chemical Company applying pressure on the recesses whereby an opening is (Milwaukee, Wis.). Microanalyses were performed by formed in the sheet at the place of the recess. The tablet or Schwarzkopf Microanalytical Laboratory, Woodside, N.Y. capsule can then be removed via Said opening. 65 The terms “concentrated” and “coevaporated” refer to It may be desirable to provide a memory aid on the kit, removal of Solvent at water aspirator pressure on a rotary e.g., in the form of numbers next to the tablets or capsules evaporator with a bath temperature of less than 45 C. 5,998,463 27 28 EXAMPLE 1. Prepared as in Example 1 from 1-ethyl-5-(2-methyl-1,3 dioxolan-2-yl)-2-oxo-2,3-dihydro-1H-indole-3-carboxylic 5-Acetyl-1-ethyl-2-oxo-2,3-dihydro-1H-indole-3- acid methyl ester and 3-amino-N-cyclohexyl-benzamide. carboxylic acid (3-phenylcarbamoyl-phenyl)-amide Purified by trituration in ethyl acetate. mp 180° C. dec. 5 EXAMPLE 3

NH 1O

NH

15 Step A. 1-Ethyl-5-(2-methyl-1,3dioxolan-2-yl)-2-oxo-2,3- Cl dihydro-1H-indole-3-carboxylic acid (3-phenylcarbamoyl phenyl)-amide. 1-Ethyl-5-(2-methyl-1,3dioxolan-2-yl)-2- 5-Acetyl-1-ethyl-2-oxo-2,3-dihydro-1H-indole-3- oxo-2,3-dihydro-1H-indole-3-carboxylic acid methyl ester carboxylic acid 3-(4-chlorophenylcarbamoyl)-phenyl (3.6 g., 11.8 mmol) and 3-amino-N-phenyl-benzamide (5.0 g, amide. 24 mmol) were combined in benzene (160 mL) in a flask Prepared as in Example 1 from 1-ethyl-5-(2-methyl-1,3 fitted with a Soxhlet containing activated 4 A molecular dioxolan-2-yl)-2-oxo-2,3-dihydro-1H-indole-3-carboxylic sieves. The mixture was heated to reflux for 30 min, then acid methyl ester and 3-amino-N-(4-chloro-phenyl)- cooled, washed with 0.1 NHCl (2x30 mL), water and brine, 25 benzamide. Recrystallized from ethyl acetate-hexanes. mp dried over magnesium Sulfate and concentrated in vacuo to 119-121 C. a dark orange foam which was purified by flash chromatography (chloroform/methanol, 20:1) to give a EXAMPLE 4 slightly orange foamy solid (3.75 g. 66%) Step B. 5-Acetyl-1-ethyl-2-oxo-2,3-dihydro-1H-indole-3- carboxylic acid (3-phenylcarbamoyl-phenyl)-amide. To a solution of 1-ethyl-5-(2-methyl-1,3dioxolan-2-yl)-2-oxo O O 2,3-dihydro-1H-ind ole-3-carboxylic acid O (3-phenylcarbamoyl-phenyl)-amide (the title compound of 35 NH Example 1, Step A, 3.75 g, 7.8 mmol) in THF (70 mL) was Me added 2 NHCl (20 mL). The mixture was stirred at room O temperature for 1 hour then poured into water (200 mL) and N extracted with ethyl acetate. The extracts were washed with V water and brine, dried over magnesium Sulfate and concen Et Me trated in vacuo to afford an orange foam. The foam was 40 dissolved in a Small amount of ethyl acetate and precipitated 5-Acetyl-1-ethyl-2-oxo-2,3-dihydro-1H-indole-3- with hexane to yield a tan Solid, which was dried on high carboxylic acid (3-p-tolylcarbamoylphenyl)-amide. vacuum (mp 173–175° C., 2.5 g., 78%). Calculated for Prepared as in Example 1 from 1-ethyl-5-(2-methyl-1,3 CHNO: C 70.74; H 5.25; N 9.52; found C70.91; H dioxolan-2-yl)-2-oxo-2,3-dihydro-1H-indole-3-carboxylic 5.56; N 9.25. 45 acid methyl ester and 3-amino-N-(p-tolyl)-benzamide. Puri EXAMPLES 2-15 fied by trituration in ethyl acetate hexanes. mp 168 dec. Examples 2 to 15 were prepared from the appropriate Starting materials in a manner analogous to the method of EXAMPLE 5 Example 1, with variations in reaction time, temperature, 50 and reagents as noted.

EXAMPLE 2

55

60 5-Acetyl-1-ethyl-2-oxo-2,3-dihydro-1H-indole-3- carboxylic acid 3-(4-fluorophenylcarbamoyl)-phenyl amide. 65 Prepared as in Example 1 from 1-ethyl-5-(2-methyl-1,3 5-Acetyl-1-ethyl-2-oxo-2,3-dihydro-1H-indole-3- dioxolan-2-yl)-2-oxo-2,3-dihydro-1H-indole-3-carboxylic carboxylic acid (3-cyclohexylcarbamoyl-phenyl)-amide. acid methyl ester and 3-amino-N-(4-fluoro-phenyl)-

5,998,463 31 32 (hexanes/acetone, 1:1) followed by trituration in ethyl acid methyl ester and 3-amino-N-phenyl-benzamide. Puri acetate/hexanes. mp 139-142 C. fied by flash-chromatography (chloroform/methanol, 20:1) EXAMPLE 12 followed by trituration in hexanes/ethyl acetate. mp 179-180° C. Calculated for C2HNO: C 71.19; H 5.53; N 9.22; found C 70.79; H 5.73; N 8.79.

EXAMPLE 1.5 1O

O O N O H 15 NH 5-Acetyl-1-methyl-2-oxo-2,3-dihydro-1H-indole-3- carboxylic acid (3-phenylcarbamoyl-phenyl)-amide. Prepared as in Example 1 from 1-methyl-5-(2-methyl-1, N O O 3dioxolan-2-yl)-2-oxo-2,3-dihydro-1H-indole-3- carboxylic acid methyl ester and 3-amino-N-phenyl N. benzamide. Purified by trituration in hexanes/ethyl acetate. mp 178–179° C. 5-Cyclopentanecarbonyl-1-ethyl-2-oxo-2,3-dihydro-1H Calculated for CHNO: C 70.25; H 4.95; N 9.83; indole-3-carboxylic acid (3-phenylcarbamoyl-phenyl)- found C 69.89; H 4.79; N 9.69. 25 amide. EXAMPLE 13 Prepared as in Example 1 from 5-(2-cyclopentyl-1,3 dioxolan-2-yl)-1-ethyl-2-oxo-2,3-dihydro-1H-indole-3- carboxylic acid ethyl ester and 3-amino-N-phenyl

benzamide. Purified by flash-chromatography (hexanes/ acetone, 1:1) followed by trituration in ethyl acetate. mp 208-213 C. NH EXAMPLE 16 35

Br

5-Acetyl-1-methyl-2-oxo-2,3-dihydro-1H-indole-3- 40 carboxylic acid 3-(4-bromophenylcarbamoyl)-phenyl NH amide. Prepared as in Example 1 from 1-methyl-5-(2-methyl-1, 3dioxolan-2-yl)-2-oxo-2,3-dihydro-1H-indole-3- carboxylic acid methyl ester and 3-amino-N-(4-bromo phenyl)-benzamide. Purified by flash-chromatography 45 (hexanes/acetone, 1:1). CF mp 234-236 C. Calculated for CH-BrNNO: C 59.30; H 3.98; N 8.30; found C 59.12; H 4.15; N 8.08. 5-Acetyl-2-oxo-1-(2,2,2-trifluoro-ethyl)-2,3-dihydro-1H EXAMPLE 1.4 50 indole-3-carboxylic acid (3-phenylcarbamoyl-phenyl)- amide. Sodium hydride (0.13 g of a 60% suspension, 3.3 mmol) was added to a solution of 5-(2-methyl-1,3dioxolan-2-yl)-

55 1-(2,2,2-trifluoroethyl)-1,3-dihydro-indol-2-one (0.5g, 1.66 mmol) in HMPA (6 mL) and after 10 min 3-(3,3-diphenyl NH ureido)-N-phenyl-benzamide (0.74g, 1.8 mmol) was added. The reaction mixture was stirred for three days, acidified with 1 NHCl, poured into water and extracted with ethyl 60 acetate. The combined extracts were washed with water and brine, dried over magnesium Sulfate and concentrated in vacuo to a dark foam. 1-Ethyl-2-oxo-5-propionyl-2,3-dihydro-1H-indole-3- Trituration in ethyl acetate/hexanes gave the title com carboxylic acid (3-phenylcarbamoyl-phenyl)-amide. 65 pound as a colorless solid (124 mg, 15%, mp 197-203 C). Prepared as in Example 1 from 1-ethyl-5-(2-ethyl-1,3 Calculated for CHFNO: C 63.03; H 4.07; N 8.48; dioxolan-2-yl)-2-oxo-2,3-dihydro-1H-indole-3-carboxylic found C 62.83; H 4.32; N 8.47. 5,998,463 33 34 Preparation 1 (100 mL) was added a solution of 3-nitrobenzoyl chloride

(30 g, 160 mmol) in dichloromethane (60 mL). The mixture was stirred for 15 min at 0° C. then overnight at room temperature. It was then poured into Saturated Sodium bicar bonate (1L) and stirred vigorously for 15 min. The precipi tate was collected, washed with water and dried (40 g, 100%). Preparation 6

1-Ethyl-5-(2-methyl-1,3dioxolan-2-yl)-2-oxo-2,3- dihydro-1H-indole-3-carboxylic acid methyl ester. 15 Sodium (2.68 g, 112 mmol) was added to methanol (110 mL) in a 3-neck flask fitted with a reflux condenser, while controlling the temperature with an ice-bath. After dissolu tion dimethyl carbonate (9.4 mL, 112 mmol) was added followed by 1-ethyl-5-(2-methyl-1,3dioxolan-2-yl)-1,3- dihydro-indol-2-one (U.S. Pat. No. 4,686.224, 9.2 g, 37 3-Amino-N-phenyl-benzamide. mmol). The mixture was heated to reflux for 70 hours, then cooled, concentrated to about 25 mL, diluted with water, 10% Palladium on carbon (2.0 g) was added to a solution acidified to pH 7 with acetic acid and extracted twice with of 3-nitro-N-phenyl-benzamide (20g, 83 mmol) in ethanol ethyl acetate. The combined extracts were washed with 25 (225 mL) and the mixture was hydrogenated at 45 psi for 2 brine, dried over magnesium Sulfate and concentrated in hours. The mixture was filtered through diatomaceous earth vacuo to an oily Solid which was triturated in isopropyl and concentrated to give a colorless solid (mp 118-120° C., ether, collected and dried (7.9 g, 70%). 16 g, 90%). Preparations 2-4 The compounds of Preparations 2 to 4 were prepared from Preparations 7-15 the appropriate Starting materials in a manner analogous to The compounds of Preparations 7 to 15 were prepared from the method of Preparation 1. the appropriate commercially available starting materials in Preparation 2 a manner analogous to the methods of Preparations 5 and 6 1-Methyl-5-(2-methyl-1,3dioxolan-2-yl)-2-oxo-2,3- 35 performed Sequentially. dihydro-1H-indole-3-carboxylic acid methyl ester. Prepared from 1-methyl-5-(2-methyl-1,3dioxolan-2-yl)- Preparation 7 2,3-dihydro-indol-2-one, which was prepared as disclosed in 3-Amino-N-cyclohexyl-benzamide. U.S. Pat. No. 4,686,224. 40 Preparation 3 Preparation 8 1-Ethyl-5-(2-ethyl-1,3dioxolan-2-yl)-2-oxo-2,3-dihydro 3-Amino-N-(4-chloro-phenyl)-benzamide. 1H-indole-3-carboxylic acid methyl ester. Prepared from 1-ethyl-5-(2-ethyl-1,3dioxolan-2-yl)-2,3- Preparation 9 dihydro-indol-2-one, the title compound of Preparation 22. 45 3-Amino-N-(p-tolyl)-benzamide. Preparation 4 5-(2-Cyclopentyl-1,3dioxolan-2-yl)-1-ethyl-2-oxo-2,3- Preparation 10 dihydro-1H-indole-3-carboxylic acid ethyl ester. 3-Amino-N-(4-fluoro-phenyl)-benzamide. Preparaed from 5-(2-cyclopentyl-1,3dioxolan-2-yl)-1- 50 ethyl-2,3-dihydro-indol-2-one, the title compound of Prepa ration 24. Preparation 11 3-Amino-N-(4-ethyl-phenyl)-benzamide. Preparation 5 55 Preparation 12 3-Amino-N-(3-fluoro-phenyl)-benzamide. Preparation 13 60 3-Amino-N-(4-bromo-phenyl)-benzamide.

NO2 Preparation 14 3-Amino-N-(4-iodo-phenyl)-benzamide. 3-Nitro-N-phenyl-benzamide. 65 To a cooled (0°C.) solution of aniline (17 g., 180 mmol) Preparation 15 and triethylamine (27 mL, 190 mmol) in dichloromethane 3-Amino-N-(3-methyl-phenyl)-benzamide. 5,998,463 35 36 Preparation 16 Preparation 19

O

O N N. N N CF 1-(2,2,2-Trifluoro-ethyl)-1H-indole-2,3-dione. 5-(2-Methyl-1,3dioxolan-2-yl)-1-(2,2,2-trifluoro-ethyl)-1, Sodium hydride (8.65 g of a 60% oil dispersion, 0.22 mol) 3-dihydro-indol-2-one. was washed with hexane and Suspended in hexamethylphos 15 A solution of 5-acetyl-1-(2,2,2-trifluoro-ethyl)-1,3- phoramide (200 mL). Isatin (29.4g, 0.20 mol)) was added dihydro-indol-2-one (1.04 g, 4.0 mmol), ethylene glycol carefully. After the gas evolution Subsided, 2,2,2- (1.37 mL, 24 mmol) and p-toluenesulfonic acid (10 mg) in trifluoroethyl iodide (46.2g, 0.22 mol) was added and the benzene (25 mL) was heated to reflux for 4 hours in a flask mixture was heated to 55° C. for 4 hours. The Solution was fitted with a Dean-Stark trap. The solution was diluted with cooled, diluted with water (1 L), and the precipitate was ethyl acetate, washed with Saturated Sodium bicarbonate, collected. The filtrate was acidified with 6 NHCl and a new water and brine, dried over magnesium Sulfate and concen precipitate formed and was collected. The combined Solids trated in vacuo to an oil which Solidified upon Standing (1.19 were recrystallized from 95% ethanol. Yield 22.5 g (49%). g, 98%, mp 87-89° C). mp 161-163° C. 25 Preparation 20 Preparation 17

l O N N NH N CF 35 c 1-(2,2,2-Trifluoro-ethyl)-1,3-dihydro-indol-2-one. A mixture of 1-(2,2,2-trifluoro-ethyl)-1H-indole-2,3- 3-(3,3-Diphenyl-ureido)-N-phenyl-benzamide. dione (9.2g, 40 mmol) and 10% palladium on carbon (2.4 A mixture of 3-amino-N-phenylbenzamide (2.0 g, 9.4 g) in acetic acid (100 mL) and 70% perchloric acid (6.4 mL, 40 mmol), diphenylcarbamoyl chloride (2.2g, 9.4 mmol) and 80 mmol) was hydrogenated in a Parr apparatus for 22 triethylamine (2.6 mL, 19 mmol) in ethanol (10 mL) was hours. The mixture was filtered through diatomaceous earth, heated to reflux for 4.5 hours. The mixture was cooled and diluted with water (1.5 L) and the precipitate was collected concentrated, water was added, the slurry was acidified with and dried. mp 155–162° C. Yield 7.5 g (87%). 1 N HCl and the Solid was collected, washed with water, 45 dried and recrystallized from acetone/hexanes, to give a Preparation 18 colorless solid (1.63 g, 42%). Preparation 21 1-Ethyl-5-Propionyl-2,3-dihydro-indol-2-one. Prepared from readily available starting materials 50 (N-ethyloxindole and propionyl chloride) in a manner analo gous to that set forth in Preparation 18. N Preparation 22 N 1-Ethyl-5-(2-ethyl-1,3dioxolan-2-yl)-2,3-dihydro-indol-2- CF 55 OC. Prepared from the title compound of Preparation 21 in a 5-Acetyl-1-(2,2,2-trifluoro-ethyl)-1,3-dihydro-indol-2-one. manner analogous to that Set forth in Preparation 19. To a solution of 1-(2,2,2-trifluoro-ethyl)-1,3-dihydro Preparation 23 indol-2-one (2.0 g, 9.3 mmol) and acetylchloride (0.86 mL, 60 5-Cyclopentanecarbonyl-1-ethyl-2,3-dihydro-indol-2-one. 12 mmol) in carbon disulfide (40 mL) was added aluminum Prepared from readily available starting materials trichloride (7.4g, 56 mmol) by portions. The mixture was (N-ethyloxindole and cyclopentanecarbonyl chloride) in a heated to reflux for 3 hours, then cooled. The liquid phase manner analogous to that Set forth in Preparation 18. was decanted and the residue was quenched carefully with ice, then water. The Solids were filtered, washed with water, 65 Preparation 24 dried and recrystallized from acetone/hexanes to give a pale 5-(2-cyclopentyl-1,3dioxolan-2-yl)-1-ethyl-2,3-dihydro pink solid, mp 170–171° C. Yield 1.23 g (51%). indol-2-one. 5,998,463 37 38 Prepared from the title compound of Preparation 23 in a 10. The method as recited in claim 9 wherein the glycogen manner analogous to that Set forth in Preparation 19. phorphorylase dependent disease or condition is hypergly It should be understood that the invention is not limited to cemia which comprises administering to Said animal Suffer the particular embodiments described herein, but that vari ing from hyperglycemia a hyperglycemia treating amount of ous changes and modifications may be made without depart Said compound, a prodrug thereof or a pharmaceutically ing from the Spirit and Scope of this novel concept as defined acceptable Salt of Said compound or said prodrug. by the following claims. 11. The method of claim 9 wherein the glycogen phos We claim: phorylase dependent disease or condition is diabetes in an 1. A compound of Formula I animal, which comprises administering to Said animal Suf fering from diabetes a diabetes treating amount of Said 1O compound, a prodrug thereof or a pharmaceutically accept

able Salt of Said compound or Said prodrug. 12. The method of claim 9 wherein the glycogen phos phorylase dependent disease or condition is hypercholester olemia in an animal, which comprises administering to Said 15 animal Suffering from hypercholesterolemia a hypercholes NHR3, terolemia treating amount of Said compound, a prodrug thereof or a pharmaceutically acceptable Salt of Said com pound or Said prodrug. 13. The method of claim 9 wherein the glycogen phos phorylase dependent disease or condition is atherOSclerosis a prodrug thereof or a pharmaceutically acceptable Salt of in an animal, which comprises administering to Said animal Said compound or Said prodrug wherein: Suffering from or Susceptible to atherosclerosis an athero R" is (C-C)alkyl, (C-C)cycloalkyl, phenyl or phenyl Sclerosis treating amount of Said compound, a prodrug independently Substituted with up to three (C-C) thereof or a pharmaceutically acceptable Salt of Said com alkyl, (C-C)alkoxy or halogen; 25 pound or Said prodrug. R’ is (C-C)alkyl optionally substituted with up to three 14. The method of claim 9 wherein the glycogen phos fluoro atoms, and phorylase dependent disease or condition is hyperinsuline R is (C-C)cycloalkyl; phenyl; phenyl substituted at the mia in an animal, which comprises administering to Said para position with (C-C)alkyl, halo or trifluorom animal Suffering from hyperinsulinemia a hyperinsulinemia ethyl, phenyl Substituted at the meta position with treating amount of Said compound, a prodrug thereof or a fluoro; or phenyl substituted at the ortho position with pharmaceutically acceptable Salt of Said compound or Said fluoro. prodrug. 2. A compound of claim 1, a prodrug thereof or a 15. The method of claim 9 wherein the glycogen phos pharmaceutically acceptable Salt of Said compound or said phorylase dependent disease or condition is hypertension in prodrug, wherein R' is phenyl, R is ethyl; and R is phenyl, 35 an animal, which comprises administering to Said animal phenyl Substituted at the para position with (C-C)alkyl, Suffering from hypertension a hypertension treating amount halo or trifluoromethyl, phenyl Substituted at the meta posi of Said compound, a prodrug thereof or a pharmaceutically tion with fluoro, or phenyl substituted at the ortho position acceptable Salt of Said compound or said prodrug. with fluoro. 16. The method of claim 9 wherein the glycogen phos 3. A compound of claim 1, a prodrug thereof or a 40 phorylase dependent disease or condition is hyperlipidemia pharmaceutically acceptable Salt of Said compound or said in an animal, which comprises administering to Said animal prodrug, wherein R' is methyl and R is ethyl. Suffering from hyperlipidemia a hyperlipidemia treating 4. A compound of claim 3, a prodrug thereof or a amount of Said compound, a prodrug thereof or a pharma pharmaceutically acceptable Salt of Said compound or said ceutically acceptable Salt of Said compound or Said prodrug. prodrug, wherein R is phenyl, phenyl Substituted at the para 45 17. The method of claim 9 wherein the glycogen phos position with (C-C)alkyl, halo or trifluoromethyl, phenyl phorylase dependent disease or condition is tissue ischemic Substituted at the meta position with fluoro, or phenyl injury in an animal, which comprises administering to Said substituted at the ortho position with fluoro. animal at risk for tissue ischemic injury a tissue ischemic 5. A compound of claim 4 selected from 5-acetyl-1-ethyl injury preventing amount of Said compound, a prodrug 2-oxo-2,3-dihydro-1H-indole-3-carboxylic acid (3-p- 50 thereof or a pharmaceutically acceptable Salt of Said com tolylcarbamoyl-phenyl)-amide, 5-acetyl-1-ethyl-2-oxo-2,3- pound or Said prodrug. dihydro-1H-in dole-3-carboxylic acid (3-( 18. The method of claim 9 wherein the glycogen phos 4-bromophenylcarbamoyl-phenyl)-amide; and 5-acetyl-1- phorylase dependent disease or condition is myocardial ethyl-2-oxo-2,3-dihydro-1H-indole-3-carboxylic acid ischemic injury in an animal, which comprises administer (3-phenylcarbamoyl-phenyl)-amide. 55 ing to Said animal at risk for myocardial ischemic injury a 6. The compound of claim 4 wherein R is p-tolyl. myocardial ischemic injury treating or preventing amount of 7. The compound of claim 4 wherein R is Said compound, a prodrug thereof or a pharmaceutically 4-bromophenyl. acceptable Salt of Said compound or said prodrug. 8. The compound of claim 4 wherein R is phenyl. 19. The method of claim 18 wherein said myocardial 9. A method for treating a glycogen phosphorylase depen 60 ischemic injury is perioperative myocardial ischemic injury. dent disease or condition in an animal, which comprises 20. A pharmaceutical composition which comprises a administering to Said animal Suffering from a glycogen therapeutically effective amount of a compound of claim 1, phosphorylase dependent disease or condition a glycogen a prodrug thereof or a pharmaceutically acceptable Salt of phosphorylase dependent disease or condition treating Said compound or said prodrug and a pharmaceutically amount of a compound of claim 1, a prodrug thereof or a 65 acceptable carrier of diluent. pharmaceutically acceptable Salt of Said compound or said 21. The pharmaceutical composition of claim 20 for the prodrug. treatment of glycogen phosphorylase dependent diseases or 5,998,463 39 40 conditions in animals, which comprises a glycogen phos agents, Vanadate and Vanadiumn complexes and per phorylase dependent disease or condition treating amount of OXOVanadium complexes, amylin antagonists; gluca Said compound, a prodrug thereof or a pharmaceutically gon antagonists, growth hormone Secretagogues; glu acceptable Salt of Said compound or said prodrug and a coneogenesis inhibitors, Somatostatin analogs, pharmaceutically acceptable carrier or diluent. 22. A pharmaceutical composition which comprises a antilipotic agents, prodrugs of Said antidiabetic agents therapeutically effective amount of and pharmaceutically acceptable Salts of Said antidia a) a glycogen phosphorylase inhibitor as recited in claim betic agents and Said prodrugs, and 1; c) a pharmaceutically acceptable carrier or diluent. b) an antidiabetic agent Selected from insulin and insulin 23. A method for treating a glycogen phosphorylase analogs, dependent disease or condition in an animal, particularly a insulinotropin; Sulfonylureas and analogs, biguanides, mammal, which comprises administering to Said animal C-antagonists and imidazolines; insulin Secreta Suffering from a glycogen phosphorylase dependent disease gogues, glitaZones, aldose reductase inhibitors, Sorbitol or condition a glycogen phosphorylase dependent disease or dehydrogenase inhibitors, fatty acid oxidation inhibi 15 condition treating amount of a composition of claim 1. tors, C-glucosidase inhibitors, B-agonists, phosphodi esterase inhibitors, lipid-lowering agents, antiobesity UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,998.463 Page 1 of 1 DATED : December 7, 1999 INVENTOR(S) : Bernard Hulin and Reinhard Sarges

it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 40 Line 1, “vanadiumn should read -- vanadium --, Line 5, "antilipotic' should read -- antilipolytic -: Line 11, “particularly a mammal' should be deleted; Line 15, “claim 1' should read -- claim 22 --.

Signed and Sealed this Twenty-eighth Day of August, 2001 Attest: 7c44, f abeel

NICHOLASP. GODC Attesting Officer Acting Director of the United States Patent and Trademark Office