HK1120255A - Diastereoisomers of 4-hydroxyisoleucine and uses thereof - Google Patents

Abstract

The invention relates to configurational isomers 4-hydroxyisoleucine, and to lactones, pharmaceutically acceptable salts, and prodrugs thereof, to processes for their preparation, and to pharmaceutical compositions comprising the same. The isomers of the invention exhibit insulinotropic activity and thus may be useful for the prevention and treatment of disorders of carbohydrate or lipid metabolism, including diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes, and Metabolic Syndrome.

Description

Diastereoisomers of4-hydroxyisoleucine and uses thereof

Background

a) Field of the invention

The present invention relates to isomers of4-hydroxyisoleucine and lactones, pharmaceutically acceptable salts and prodrugs thereof, to a preparation method thereof and a pharmaceutical composition comprising the same, and to uses thereof for preventing and treating disorders of carbohydrate metabolism or lipid metabolism, including diabetes (type 1 and type 2 diabetes), prediabetes and metabolic syndrome.

b) Brief description of the related art

Diabetes is a disease of carbohydrate metabolism and occurs when the body is unable to effectively control blood glucose levels. The disease is characterized by inadequate secretion or utilization of insulin, high glucose levels in blood and urine, and excessive thirst, hunger, weight loss, and urine production. It can lead to a number of serious complications, including cardiovascular disease, nephropathy, blindness, nerve damage and limb ischemia.

Diabetes is divided into two types, type 1 and type 2, with the latter accounting for about 90% of cases. In type 1 diabetes, the body destroys the insulin-producing beta cells of the pancreas, resulting in the body's inability to produce insulin. Type 1 diabetes commonly occurs in children or young adults and is generally managed by insulin administration, strict diet, and exercise. Type 1 diabetes is also found in older people after failure of treatment for type 2 diabetes. Type 2 diabetes is characterized by impaired insulin secretion (due to altered beta cell function) and a reduced ability of normal insulin-sensitive tissues (e.g., liver and muscle) to respond to insulin. Type 2 diabetes typically occurs in people over the age of 45, but is also recently found in younger people. The disease is associated with risk factors such as age, family history, obesity, lack of regular exercise, hypertension and hyperlipidemia. Treatments include strict dietary and exercise therapy, oral drug therapy (e.g., drug therapy that increases insulin secretion and/or insulin sensitivity), and in some cases insulin administration.

The importance of type 2 diabetes is rapidly increasing as a major public health concern in the western world. Although it was a relatively rare disease a hundred years ago, there are over 20000 million type 2 diabetic patients worldwide today, and it is estimated that this figure will increase to more than about 30000 million by 2025. This significant increase in the incidence of type 2 diabetes parallels the increase in obesity prevalence in western societies. Further, with more societies accepting western dietary habits, it is likely that type 2 diabetes will reach a worldwide epidemic proportion. Because of the severity of the complications associated with the disease and its rapidly increasing incidence, the development of effective treatments is a major concern in the medical field.

In 1973, Fowden et al, Phytochemistry 12: 1707-. Alcock et al (Phytochemistry 28: 1835-. This unusual material represents about 0.6% of the weight of the seed and greater than 85% of the free amino acids in the seed, and has two coexisting isomers: the (2S, 3R, 4S) isomer (. about.90%) and the (2R, 3R, 4S) isomer (. about.10%) (Sauvaire et al, Herbs, Botanicals and Teas (2000), eds. G.Mazza and BP.Oomah, pp. 107-). The (2S, 3R, 4S) isomer has been shown to have insulinotropic and insulin sensitizing activity (Broca et al, am. J. physiol.277: E617-E623, 1999; Broca et al, Eur. J. Pharmacol.390: 339-345, 2000; Broca et al, am. J. physiol.Endocrinol.Metah.287: E463-E471, 2004) and has since been developed for the treatment of diabetes (PCT publication Nos. WO 97/32577 and WO 01/15689). On the other hand, the (2R, 3R, 4S) isomer has been reported to have no or much less biological activity than the (2S, 3R, 4S) isomer (Broca et al, Eur. J. Pharmaco.390: 339-345, 2000).

Despite increasing evidence for the positive activity of4-hydroxyisoleucine for the treatment of diabetes, no one has demonstrated that configurational isomers of4-hydroxyisoleucine, other than the (2S, 3R, 4S) isomer, are useful in the prevention and/or treatment of metabolic diseases (e.g., diabetes).

In view of the above, there is a need for alternative and improved compounds for the prevention and treatment of diseases of carbohydrate and lipid metabolism, in particular diabetes.

There is also a need for pharmaceutical compositions and methods of treatment that stimulate glucose uptake and/or stimulate insulin secretion.

The present invention provides such compounds and methods for their use. Thus, the present invention fulfills the above-described needs, as well as other needs, which will become apparent to those skilled in the art upon review of the following specification.

Summary of The Invention

The present inventors have found that configurational isomers of (2S, 3R, 4S) -4-hydroxyisoleucine also exhibit insulinotropic activity in vitro assays: one was to monitor glucose-dependent stimulation of insulin secreting INS-1 cells, while the other monitored glucose uptake by differentiated3T3-L1 adipocytes.

Accordingly, the invention features the use of an isomer of4-hydroxyisoleucine (4-OH) as defined herein for therapeutic and/or prophylactic purposes. In a preferred embodiment, the isomer according to the invention is an isomer of 2S, 3R, 4S 4-hydroxyisoleucine selected from the group consisting of:

group (d) of (a).

Exemplary prodrugs include those compounds in which a carboxylic acid ester and a hydroxy group are condensed to form one of the following lactones:

another aspect of the invention features the following compounds:

or a lactone prodrug thereof:

in another aspect, the invention features pharmaceutical compositions that include one or more of such isomers and a pharmaceutically acceptable excipient.

In another aspect, the invention provides a method for treating a mammal suffering from a disorder of carbohydrate metabolism or lipid metabolism, which method comprises administering to said mammal one or more isomers of 4-OH as defined herein. Preferably, the disease is non-insulin dependent diabetes, more preferably type 2 diabetes. In one embodiment, the method may further comprise administering a second agent to the mammal, for example, wherein the second agent may be an anti-diabetic agent.

According to another aspect, the invention relates to a method of treatment of a disease in a mammal treatable by administration of a compound stimulating insulin secretion, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of at least one isomer of 4-OH according to the invention and a pharmaceutically acceptable carrier or excipient, alone or in combination with other pharmacologically active substances.

In another aspect, the invention relates to a method for stimulating glucose uptake in muscle cells and/or adipocytes, which comprises contacting said cells with an effective amount of an isoform according to the invention.

In another aspect, the invention relates to a method for stimulating the secretion of insulin from beta cells in the islets of langerhans, which method comprises contacting said cells with an effective amount of an isoform according to the invention.

In another aspect, the invention relates to pharmaceutical compositions, and in particular to the use of isomers according to the invention in the manufacture of medicaments for the treatment of diseases of carbohydrate metabolism or lipid metabolism where elevated circulating glucose levels are problematic, including but not limited to diabetes (type 1 and type 2 diabetes), prediabetes, metabolic syndrome, hyperglycemia, diabetic neuropathy and diabetic nephropathy.

In another aspect of the invention, there is provided a process for the preparation of an isomer according to the invention.

The present invention is advantageous in that it provides a novel and useful glucose uptake stimulant and insulin secretion stimulant. The present invention also provides compounds, compositions and methods for the unmet medical need for disorders of carbohydrate metabolism or lipid metabolism, particularly type 2 diabetes.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following description of non-limiting preferred embodiments (with reference to the accompanying drawings), which are exemplary and should not be construed as limiting the scope of the invention.

Brief Description of Drawings

FIG. 1 is a scheme showing the synthesis of each of the 8 configurational isomers of 4-hydroxyisoleucine.

FIG. 2 is a graph showing the stimulatory effect of configurational isomers of4-hydroxyisoleucine on glucose uptake by differentiated3T3-L1 adipocytes.

FIG. 3 is a graph showing glucose-dependent stimulation of insulin secretion by INS-1 cells by configurational isomers of 4-hydroxyisoleucine.

Detailed Description

The invention features compounds, pharmaceutical compositions, and methods that include isomers of (2S, 3R, 4S) -4-hydroxyisoleucine, which are compounds that have been shown to stimulate insulin secretion and reduce insulin resistance in a glucose-dependent manner (see, e.g., U.S. Pat. No. 5,470,879; WO 01/15689; Broca et al, am.J. physiol.277: E617-E623, 1999; and Broca et al, am.J. physiol.Endocrinol.Metab.287: E463-E471, 2004). More specifically, the invention features pharmaceutical compositions of (2S, 3S, 4S) -4-hydroxyisoleucine and one or more (2S, 3S, 4S), (2S, 3R, 4R), (2S, 3S, 4R), (2R, 3S, 4S), (2R, 3R, 4R), or (2R, 3R, 4S) configurational isomers including 4-hydroxyisoleucine and methods for treating a mammal having a disorder of carbohydrate metabolism or lipid metabolism.

To provide a clearer and more consistent understanding of the specification and claims, including the scope to be given such terms herein, the following definitions are provided:

A) definition of

The term "administration" or "administering" refers to a method of administering a dosage of a pharmaceutical composition to a mammal (e.g., a human), for example, wherein the method is oral, subcutaneous, topical, intravenous, intraperitoneal, or intramuscular. The preferred method of administration may vary depending on various factors such as the components of the pharmaceutical composition, the site of the underlying or actual disease, and the severity of the disease.

"disorders of carbohydrate metabolism" means metabolic disorders in which a patient suffering from the disorder is unable to metabolize sugars properly. Examples of such diseases include, for example, diabetes (type 1 and type 2), prediabetes, hyperglycemia, impaired glucose tolerance, metabolic syndrome, glucosuria, diabetic neuropathy and diabetic nephropathy, obesity and eating disorders.

"lipid metabolism disorder" means a metabolic disorder in which a patient suffering from the disorder is unable to properly metabolize, partition and/or store fat. Examples of such diseases include, but are not limited to, type 2 diabetes, prediabetes, and metabolic syndrome.

"effective amount" means the amount of a compound required for the treatment or prevention of a disease of carbohydrate metabolism or a disease of lipid metabolism (e.g., diabetes and metabolic syndrome). The effective amount of active compound for use in the practice of the present invention for treating or preventing conditions caused by or contributed to by disorders of carbohydrate metabolism or disorders of lipid metabolism will vary depending on the mode of administration and the age, weight and general health of the patient. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. An effective amount may also be one that provides some relief from one or more symptoms of the condition or disease, or reduces the likelihood of the condition developing.

Compounds having the same molecular formula but differing in the nature or order of bonding of their atoms or the spatial arrangement of their atoms are referred to as "isomers". Isomers in which the connectivity between atoms is the same but the arrangement of atoms in space is different are referred to as "stereoisomers". Stereoisomers that are not mirror images of each other are referred to as "diastereomers", and stereoisomers that are non-superimposable mirror images of each other are referred to as "enantiomers". When a compound has an asymmetric center, for example, it is bound to four different groups, then a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and are described and designated as dextrorotatory or levorotatory (i.e., the (+) or (-) -isomers, respectively) by the R-and S-cis rules of Cahn, Ingold and Prelog, or by the manner in which molecules rotate the plane of polarized light. The chiral compounds may exist as individual enantiomers or as mixtures thereof. Mixtures containing enantiomers in the same proportions are referred to as "racemic mixtures".

The compounds of the invention may present asymmetric or chiral centers. Unless otherwise indicated, the description or naming of a particular compound in the specification and claims is intended to include all individual enantiomers and racemic or otherwise mixtures thereof. Methods for determining stereochemistry and separating stereoisomers are well known in the art (see discussion in chapter 4 of "Advanced Organic Chemistry", 4 th edition j. march, John Wiley and Sons, New York, 1992). The individual stereoisomers of the compounds of the invention are prepared synthetically from commercially available starting materials containing asymmetric or chiral centers, or by preparing mixtures of enantiomeric compounds, followed by resolution as is well known to those skilled in the art. These resolution methods are exemplified by (1) attaching a racemic mixture of enantiomers (designated (+/-) to a chiral auxiliary, separating the resulting diastereomers by recrystallization or chromatography, and liberating the optically pure product from the auxiliary, or (2) directly separating a mixture of optical enantiomers on a chiral chromatography column. Enantiomers are designated herein by the symbol "R" or "S" depending on the configuration of the substituents around the chiral carbon atom, or are drawn in a conventional manner using bold lines (defining substituents above the plane of the paper in three-dimensional space) and random or hatched lines (defining substituents below the plane of the printed paper in three-dimensional space).

As is generally understood by those skilled in the art, an optically pure compound is an enantiomerically pure compound. As used herein, the term "optically pure" is intended to mean a compound that includes at least a sufficient amount of a single enantiomer to produce a compound having the desired pharmacological activity. Preferably, "optically pure" is intended to mean a compound comprising at least 90% of a single isomer (80% enantiomeric excess, i.e., "e.e"), preferably at least 95% (90% e.e.), more preferably at least 97.5% (95% e.e.), and most preferably at least 99% (98% e.e.). Preferably, the compounds of the present invention are optically pure.

The terms "isomer of 4-hydroxyisoleucine", "isomer of 4-OH", "isomer of the present invention", or "compound of the present invention" as used herein refer to the diastereoisomer of (2S, 3R, 4S) -4-hydroxyisoleucine as defined herein and includes pharmaceutically acceptable lactones, salts, crystalline forms, metabolites, solvates, esters and prodrugs thereof.

The term "pharmaceutically acceptable salts" as used herein, means those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are known in the art. For example, s.m.berge et al, in j.pharmaceutical Sciences 66: pharmaceutically acceptable salts are described in detail in 1-19, 1977. The salts may be prepared in situ during the final isolation and purification of the compounds of the invention or isolated by reacting the free base with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbic acid, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, heptonate (glucoheptonate), glycerophosphate, hemisulfate, heptonate (heptanate), hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectin (pectate), persulfate, 3-phenylpropionate, salicylate, and mixtures thereof, Phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, tosylates, undecanoates, valerates, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

The term "pharmaceutically acceptable ester" as used herein represents an ester that hydrolyzes in vivo, including esters that readily decompose in the human body to leave the parent compound or salt thereof. For example, suitable ester groups include esters derived from pharmaceutically acceptable aliphatic carboxylic acids (particularly alkanoic, alkenoic, naphthenic and alkanedioic acids), wherein each alkyl or alkenyl group preferably has not more than 6 carbon atoms. Examples of specific esters include formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.

The term "prodrug" as used herein represents a compound that is rapidly converted in vivo, for example by hydrolysis in blood, to the parent compound of the above formula. A full discussion is provided in t.higuchi and v.stella, Pro-drugs as Novel Delivery Systems, volume 14 of a.c.s.symposium Series; edited by Edward b.roche, Bioreversible Carriers in Drug Design (Bioreversible Carriers in Drug Design), american pharmaceutical Association and Pergamon Press, 1987; and Judkins et al, Synthetic Communications 26 (23): 4351-4367, 1996, each of which is incorporated herein by reference.

Prodrugs of isomers according to the invention are prepared by modifying functional groups in such a way that the modification can be cleaved in vivo to release the parent isomer. Prodrugs include modified isomers in which the hydroxy or amino group, respectively, of any of the isomers is bonded to any group that can be cleaved in vivo to regenerate the free hydroxy or amino group. Examples of prodrugs include, but are not limited to, esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N-dimethylaminocarbonyl), and the like of hydroxy functional groups in compounds of formula (I), (II), or (III).

The term "pharmaceutically acceptable prodrug" as used herein means those prodrugs of the compounds of the present invention, as well as the zwitterionic forms of the compounds of the present invention (when possible), which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.

"pharmaceutically acceptable active metabolite" is intended to mean the pharmacologically active product produced by metabolism of the 4-OH isomer in vivo.

"pharmaceutically acceptable solvate" is intended to mean a solvate that retains the biological effects and properties of the biologically active components of the isomers according to the invention. Examples of pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

B) Compounds according to the invention

As will be described in detail later, the present inventors have prepared a series of isomers of 4-hydroxyisoleucine. According to a preferred embodiment of the present invention, these isoforms are effective for stimulating glucose uptake and/or stimulating insulin secretion in mammals, and thus are useful for preventing and/or treating diseases in which elevated glucose levels are an issue. Thus, providing such isomers is expected not only for the treatment of diabetes, but also for the treatment of other diseases of carbohydrate metabolism or lipid metabolism.

According to a first aspect, the invention features isomers of 2S, 3R, 4S 4-hydroxyisoleucine and pharmaceutically acceptable lactones, salts, crystalline forms, prodrugs, esters, metabolites or solvates thereof. In certain embodiments, the isomers of the present invention are selected from:

group (d) of (a).

Exemplary prodrugs include those compounds in which a carboxylic acid ester and a hydroxy group are condensed to form one of the following lactones:

in a preferred embodiment, the isomer of the present invention is selected from:

group (d) of (a).

According to a related aspect, the invention features the following compounds:

and pharmaceutically acceptable lactones, salts, crystalline forms, prodrugs, esters, metabolites or solvates thereof. In a preferred embodiment, the lactone is a lactone prodrug having the structure:

the isomers and compositions of the present invention (see below) are prepared using readily available starting materials by employing techniques available in the art. For example, processes for preparing (2S, 3R, 4S) -4-hydroxyisoleucine have been described, for example, see U.S. patent application publication nos. US 2003/0219880; Rolland-Fulcrand et al, Eur.J.org.chem.873-877, 2004; and Wang et al, Eur.J.org.chem.834-839, 2002. In addition, the compounds can be isolated from fenugreek (Trigonella foenum-graecum) seeds. Methods for preparing other isomers of4-hydroxyisoleucine or prodrugs thereof have been described in PCT/FR2005/02805(WO 2006/__, published in May __ 2006), filed on 11/10/2005, which is incorporated herein by reference.

Another aspect of the invention relates to a novel process for the synthesis of isomers according to the invention. Examples some new and exemplary methods of preparing the compounds of the present invention have been described in the examples section. Such methods are within the scope of the present invention.

C) Methods for stimulating glucose uptake and methods for stimulating insulin secretion

The isoforms of the present invention preferably stimulate glucose uptake in muscle tissue or adipose tissue and/or stimulate insulin secretion by pancreatic beta cells. The biological activity of the isomers of the invention can be measured by any method available to those skilled in the art, including in vivo and in vitro assays. Some examples of suitable assays for such measurements are described in the examples section herein. Other examples of suitable art-recognized assays for such measurements are well known.

Accordingly, a related aspect of the invention provides a method of stimulating glucose uptake in muscle and or adipose tissue, the method comprising

-providing at least one isomer according to the invention as defined herein;

-providing an in vitro cell-based functional assay wherein stimulation of glucose uptake is evaluable; and

-introducing an effective amount of said isomer into said assay for stimulating glucose uptake activity.

In one embodiment, the in vitro cell-based assay comprises 3T3-L1 adipocytes and at about 10. mu.M 2-deoxy-D-glucose and about 16. mu.M³In the presence of H-deoxy-D-glucose.

Accordingly, a related aspect of the invention provides a method of stimulating insulin secretion from a beta cell, the method comprising:

-providing at least one isomer according to the invention as defined herein;

-providing an in vitro cell-based functional assay wherein stimulation of insulin secretion is evaluable; and

-introducing an effective amount of said isomer into said assay for stimulating insulin secretion.

In one embodiment, the in vitro cell-based assay comprises INS-1 cells and is performed in the presence of a glucose concentration of about 2mM to about 10 mM.

D) Pharmaceutical compositions and therapeutic applications

Without wishing to be bound by any particular theory, the inventors have demonstrated that the isoforms of the present invention are suitable for use in stimulating glucose uptake and/or stimulating insulin secretion. Accordingly, the present invention pertains to methods of using isomers of 4-OH and pharmaceutical compositions thereof for therapeutic or prophylactic purposes. In a preferred embodiment, the method comprises administering any of the individual isomers described herein or any combination thereof.

According to a preferred embodiment of the invention, the mammal is a human patient in need of treatment by the method and/or the isomer of the invention and is selected for treatment based on this need. Persons in need of treatment, particularly in the case of type 2 diabetes, are well known in the art and include patients who have been identified as having abnormally high blood glucose levels, impaired glucose tolerance, dyslipidemias, and may suffer from excess body weight (e.g., the patient may be obese). The person in need of treatment can also be a person at risk for such a disease or condition and is expected to benefit from treatment (e.g., cure, heal, prevent, alleviate, relieve, alter, correct, ameliorate, improve, or affect the disease or condition, a symptom of the disease or condition, or a risk of the disease or condition) based on diagnosis (e.g., medical diagnosis).

Accordingly, a related aspect of the invention relates to the use of the isomers of the invention as active ingredients in pharmaceutical compositions for therapeutic or prophylactic purposes. As used herein, "treatment" or "treating" is intended to mean the alleviation of a disease condition associated with at least a disease of carbohydrate metabolism or a disease of lipid metabolism, and in particular type 2 diabetes in a mammal (e.g., a human) (which type 2 diabetes is alleviated by stimulation of insulin secretion and/or by stimulation of glucose uptake), and includes the total or partial cure, healing, inhibition (e.g., arresting or reducing the development of, relieving from, ameliorating and/or relieving the disease condition (e.g., causing regression of the disease and its clinical symptoms).

As used herein, "prevent" or "prevention" is intended to mean a reduction in the likelihood of a disease condition associated with at least a disease of carbohydrate metabolism or a disease of lipid metabolism, and in particular type 2 diabetes in humans. Predisposing factors for type 2 diabetes identified or suggested in the scientific literature include, but are not limited to: (i) a genetic predisposition that will suffer from the disease condition but has not been diagnosed as having it, (ii) obesity, (iii) having a dyslipidemic disorder and/or (iv) having a sedentary lifestyle. For example, type 2 diabetes can be prevented or treated by administering the isomer of the present invention or a composition comprising the same when the human is pre-diabetic, when the human is overweight, when the human shows abnormally high blood glucose levels, and/or when the human shows reduced glucose tolerance.

The patient may be a woman or a man, and it may be a child, adolescent or adult.

According to a particular aspect, the invention features a method for treating a mammal (e.g., a human) having diabetes (type 1 or type 2 diabetes), prediabetes, or metabolic syndrome, the method including administering to the mammal an amount of an isomer of the invention and/or a composition comprising the same sufficient to lower its circulating glucose level.

According to certain embodiments, the isomers, compositions, and methods of the present invention are administered at a therapeutically effective dose sufficient to reduce the glucose level in the plasma of a patient by about at least 5, 10, 15, 20, 25, 30, 40, 50, 75, or 100 percent (as compared to the original level prior to treatment).

According to certain embodiments, the isomers, compositions, and methods of the present invention are administered at a therapeutically effective dose sufficient to raise insulin levels in the plasma of a patient by about at least 5, 10, 15, 20, 25, 30, 40, 50, 75, or 100 percent (as compared to the original level prior to treatment).

Typically, the isoforms of the present invention are administered until the glucose and/or insulin levels return to normal. Due to the nature of the conditions and disorders to which the isomers of the invention are directed, long-term or lifetime administration may be required. In a preferred embodiment, the isomer and the pharmaceutical composition according to the present invention are administered 1 to 3 times per day.

The amount of glucose or insulin in a patient's blood or plasma can be assessed by using techniques and methods well known to those skilled in the art, including, but not limited to, hand-held blood glucose meters, enzyme assays (e.g., glucose oxidase or hexokinase substrate assays), enzyme-linked immunosorbent assays (ELISA), quantitative immunoblot assays, and radio-labeled immunoassays (RIA).

Accordingly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an isomer of 4-OH as described herein in combination with a pharmaceutically acceptable carrier or excipient. Suitable carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical compositions may be administered in any effective conventional manner, for example, including but not limited to: administration by the topical route, administration by the parenteral route, administration by the oral route, administration by the rectal route, administration by the vaginal route, administration by the intravenous route, administration by the intraperitoneal route, administration by the intramuscular route, administration by the intraocular route, administration by the subcutaneous route, administration by the intranasal route, administration by the intrabronchial route, or administration by the intradermal route.

Acceptable methods for preparing pharmaceutical compositions in suitable pharmaceutical forms are known to those skilled in the art. For example, pharmaceutical formulations can be prepared by conventional techniques of medicinal chemists to produce the desired products for various routes of administration, involving steps such as: mixing, granulating and compressing (as is necessary for tablets), or mixing, filling and dissolving the ingredients appropriately.

Toxicity and therapeutic efficacy of the isoforms according to the invention can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals. The therapeutic efficacy of the isoforms according to the invention can be evaluated in animal model systems, which can predict efficacy in human diseases. For example, animal models for evaluating the efficacy of glucose uptake include animal models for diabetes or other relevant animal models in which glucose infusion rates can be measured. Animal models for evaluating insulinotropic efficacy include models for diabetes or other relevant animal models in which insulin secretion can be measured. Examples of suitable animal models for diabetes include, but are not limited to, DIO mice, ob/ob mice, db/db mice, and Zucker fa/fa rats. Alternatively, the ability of isoforms may be assessed in vitro by testing the ability of the compound to stimulate glucose uptake using differentiated3T3-L1 adipocytes (see example 2) or using L6 myocytes, and by testing the ability of the compound to stimulate insulin secretion using INS-1 cells (see example 3) or using perfused pancreas. While substances exhibiting toxic side effects may be used, care should be taken to design a delivery system that directs such substances to the affected tissue site to minimize potential damage to the uninvolved cells and thereby reduce side effects whenever possible.

The isomers, compositions and methods of the present invention can be used with a wide range of drugs. Such agents may be selected from antidiabetic agents, antihypertensive agents, anti-inflammatory agents, antiobesity agents, and the like.

A non-limiting list of useful antidiabetic agents that may be used in combination with the isomers of the present invention includes: insulin, biguanides such as metformin (Glucophage ®, Bristol-Myers Squibb company, U.S.; Stagid ®, Lipa Sante, Europe); sulfonylureas, such as gliclazide (Diamicron ®), glyburide, glipizide (gluconrol ® and gluconrol XL ®, Pfizer), glimepiride (amyl ®, Aventis), chlorpropamide (e.g., diabines ®, Pfizer), tolbutamide, and glibenclamide (e.g., micron ®, Glynase ®, and Diabeta ®); glynoids, for example repaglinide (Prandin ® or Novo Norm ®; Novo Nordisk), ormitiglilide, nateglinide (Starlix)®), lenaliena, and BTS-67582; insulin sensitisers, for example glitazones (glitazones), thiazolidinediones such as rosiglitazone maleate (Avandia ®, Glaxo Smith Kline), pioglitazone (Actos ®, EIi Lilly, Takeda), troglitazone, ciglitazone, issagltazone (isaglitazone), daglipzone, englitazone, CS-011/CI-1037, T174, GI 262570, YM-440, MCC-555, JTT-501, AR-H920342, KRP-297, GW-409544, CRE-16336, AR-H049020, LY 5109292929, MBX-102, CLX-0940, GW-501516 and in WO 97/41097(DRF 23456565631), WO 354117, WO97/41120, WO 98/45292, WO 99/19313 (NN/DRF-368295), WO 00/23415, WO 27223, WO 4934, WO 493 00/23417, WO 4934, WO 3531, KRP-297, GW-409544, CRE-1634, Compounds described in WO 00/23445, WO 00/23451, WO 00/41121, WO 00/50414, WO 00/63153, WO 00/63189, WO 00/63190, WO 00/63191, WO 00/63192, WO 00/63193, WO 00/63196 and WO 00/63209; glucagon-like peptide 1(GLP-1) receptor agonists, e.g. Exendin-4(1-39) (Ex-4), Byetta^TM(Amylin Pharmaceuticals Inc.), CJC-1131(ConjuchemInc.), NN-2211(Scios Inc.), and those GLP-1 agonists described in WO 98/08871 and WO 00/42026; substances that slow the absorption of carbohydrates, such as alpha-glucosidase inhibitors (e.g., acarbose, miglitol, voglibose, and emiglitate); substances that inhibit gastric emptying, such as glucagon-like peptide 1, cholecystokinin (cholecystokinin), dextrin and pramlintide; glucagon antagonists, e.g. quinoxaline derivatives (e.g. 2-styryl-3- [3- (dimethylamino) propylmethylamino)]-6, 7-dichloroquinoxaline; collins et al, Bioorganic and medicinal chemistry Letters 2 (9): 915-918, 1992), celestine and celestine analogs (e.g., those described in WO 94/14426), 1-phenylpyrazole derivatives (e.g., those described in U.S. Pat. No. 4,359,474), substituted disilylcyclohexanes (e.g., those described in U.S. Pat. No. 4,374,130), substituted pyridines and biphenyls (e.g., those described in WO 98/04528), substituted pyridylpyrroles (e.g., those described in U.S. Pat. No. 5,776,954), 2, 4-diaryl-5-pyridylimidazoles (e.g., those described in WO 98/21957, WO98/22108, WO 98/22109)And those described in U.S. patent No. 5,880,139), 2, 5-substituted arylpyrroles (e.g., those described in WO 97/16442 and U.S. patent No. 5,837,719), substituted pyrimidinones, pyridones, and pyrimidine compounds (e.g., those described in WO 98/24780, WO 98/24782, WO 99/24404, and WO 99/32448), 2- (benzimidazol-2-ylthio) -1- (3, 4-dihydroxyphenyl) -1-ethanone (see Madsen et al, j.med.chem.41: 5151-5157, 1998), alkylene hydrazines (such as those described in WO 99/01423 and WO 00/39088) and other compounds (such as those described in WO 00/69810, WO 02/00612, WO02/40444, WO 02/40445 and WO 02/40446); and glucokinase activators such as those described in WO 00/58293, WO 01/44216, WO 01/83465, WO 01/83478, WO 01/85706, and WO 01/85707.

Other examples of antidiabetic agents that may be used in combination with one or more isomers according to the present invention include imidazolines (e.g., efaroxan, idazoxan, phentolamine, and 1-phenyl-2- (imidazolin-2-yl) benzimidazole); glycogen phosphorylase inhibitors (see, e.g., WO 97/09040); oxadiazolidinediones, dipeptidyl peptidase-IV (DPP-IV) inhibitors, protein tyrosine phosphatase (PTP enzyme) inhibitors, liver enzyme inhibitors involved in the stimulation of gluconeogenesis and/or glycogenosis, glucose uptake modulators, glycogen synthase kinase-3 (GSK-3) inhibitors, compounds that modulate lipid metabolism (e.g., antihyperlipidemic and antilipemic agents), peroxisome proliferator-activated receptor (PPAR) agonists or antagonists (in general), Retinoid X Receptor (RXR) agonists (e.g., ALRT-268, LG-1268, and LG-1069), and antihyperlipidemic or antilipemic agents (e.g., cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, and dextrothyroxine). Other suitable antidiabetic agents are listed in table 1 provided elsewhere herein.

Examples of antihypertensive agents that can be used with the isomers of the present invention include β -blockers (e.g., alprenolol, atenolol, timolol, pindolol, propranolol, and metoprolol), Angiotensin Converting Enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril, and ramipril), calcium channel blockers (e.g., nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem, and verapamil), and α -blockers (e.g., doxazosin, urapidil, prazosin, and terazosin).

Examples of anti-inflammatory agents that may be used with the isomers of the present invention include antihistamines and anti-TNF α.

Examples of anti-obesity agents that can be used with the isomers of the present invention include Xenicacal^TM(Roche)、Meridia^TM(Abbott)、Acomplia^TM(Sanofi-Aventis), pramlintide (Amylin) and sympathomimetic phentermine.

The isomers, compositions, AND methods OF the present invention may also be used with 4-OH ANALOGS, such as described in PCT application entitled "4-HYDROXYISOLEUCINE ANALOGS AND USES THEREOF (ANALOGS OF 4-hydroxyisoline AND USES THEREOF), which claims priority from U.S. provisional patent application 60/654,342, filed on 18/2/2005.

Accordingly, another aspect relates to a pharmaceutical kit or pharmaceutical composition comprising any of the isomers of 4-OH described herein or a combination thereof and a second antidiabetic agent. The pharmaceutical kit or composition may include the isomer of4-hydroxyisoleucine and a second antidiabetic agent formulated as a single composition, such as a tablet or capsule. The invention also provides methods of treating diabetes (type 1 diabetes or type 2 diabetes), prediabetes, or metabolic syndrome in a patient comprising administering to the patient one or more isomers of4-hydroxyisoleucine (such as those described herein) and one or more antidiabetic agents. The combination of substances may be administered simultaneously or at different times to each other.

The inventive combination offers several advantages. For example, because the drug combinations described herein may be used to achieve an enhanced (e.g., increased or synergistic) effect, it is possible to consider administering fewer individual drugs, resulting in a reduction in the overall exposure of the patient to the drug as well as any adverse side effects of any drug. In addition, greater disease control can be achieved because the drug can fight the disease through different mechanisms.

Administration of drugs

With respect to the treatment methods of the present invention, it is contemplated that administration of the compounds to the mammal is limited to a particular mode of administration, dosage, frequency of administration; the present invention encompasses all modes of administration, including oral, intraperitoneal, intramuscular, intravenous, intra-articular, intralesional, subcutaneous, inhalation, or any other route sufficient to provide a dose sufficient to prevent or treat diabetes (type 1 diabetes or type 2 diabetes) and other diseases of carbohydrate metabolism or lipid metabolism, such as those described herein. The one or more compounds may be administered to the mammal in a single dose or in multiple doses. When multiple doses are administered, the doses may be spaced apart from each other, for example, by a few hours, a day, or a week. It will be understood that for any particular patient, the specific dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering or instructing the administration of the composition. Exemplary mammals that can be treated using the isomers, compositions and methods of the invention include humans, primates (e.g., monkeys), farming-related animals (e.g., cows, pigs, sheep, goats, buffalos and horses), and domesticated pets (e.g., dogs and cats). The isomers and compositions of the invention can also be administered to rodents (e.g., mice, rats, gerbils, hamsters, guinea pigs, and rabbits) for therapeutic purposes and/or for experimental purposes (e.g., to study the mechanism of action of the compounds, to screen and test the efficacy of the isomers, structural design, etc.).

For clinical use in therapy or as a prophylactic agent, the isomer or composition of the present invention may be generally administered, for example, orally, subcutaneously, parenterally, intravenously, intramuscularly, colonically, nasally, intraperitoneally, rectally, by inhalation, or buccally. Compositions suitable for use in human or veterinary medicine containing at least one isomer of4-hydroxyisoleucine according to the invention may be in a form which allows administration by an appropriate route. These compositions may be prepared according to conventional methods, using one or more pharmaceutically acceptable carriers or excipients. Such carriers include, but are not limited to, diluents, sterile aqueous media, and various non-toxic organic solvents. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington: "Science and Practice of Pharmacy" (The Science and Practice of Pharmacy) "(20 th edition), editions A.R. Gennaro, Lippincott Williams & Wilkins, 2000, Philadelphia and" Encyclopedia of Pharmaceutical Technology ", editions J.Swarbrick and J.C. Boylan, 1988-. The composition may be in the form of tablets, pills, granules, powders, aqueous solutions or suspensions, injections, elixirs or syrups, and may optionally contain one or more substances selected from the group consisting of sweetening agents, flavouring agents, colouring agents and stabilising agents in order to obtain a pharmaceutically acceptable formulation.

The choice of carrier and the amount of active substance in the carrier will generally be determined by the solubility and chemical properties of the product, the particular mode of administration and the conditions found in pharmaceutical practice. For example, excipients (e.g., sodium citrate, calcium carbonate, and dicalcium phosphate) and disintegrants (e.g., starch, alginic acid, and certain complex silicic acids in combination with lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc)) can be used to prepare tablets. For the preparation of capsules, it is advantageous to use high molecular weight polyethylene glycols. When an aqueous suspension is used, it may contain an emulsifier to facilitate suspension. Diluents such as ethanol, polyethylene glycol, propylene glycol, glycerol, chloroform or mixtures thereof may also be used. In addition, low-calorie sweeteners such as isomalt, sorbitol, xylitol may be used in the formulations of the present invention.

For parenteral administration, emulsions, suspensions or solutions of the compositions of the invention in a vegetable oil (e.g., sesame, peanut or olive oil), an aqueous organic solution (e.g., water and propylene glycol), an injectable organic ester (e.g., ethyl oleate) or a pharmaceutically acceptable salt in sterile aqueous solution may be used. The salt solutions of the compositions of the present invention are particularly useful for administration by intramuscular or subcutaneous injection. An aqueous solution of pure distilled water containing the salt solution may be used for intravenous administration, provided that (i) its pH is appropriately adjusted, (ii) it is appropriately buffered and made isotonic with a sufficient amount of sodium chloride, and (iii) it is sterilized by heating, irradiation, or microfiltration. Suitable compositions containing the isomer of the invention may be dissolved or suspended in a suitable carrier for aerosols or suspensions or solution-type aerosols, or may be absorbed or adsorbed on a suitable solid carrier for dry powder inhalers. Solid compositions for rectal administration include suppositories formulated according to known methods. It will be appreciated that the appropriate dosage and concentration of the agent (i.e., the isomer of4-hydroxyisoleucine alone and/or in combination with other drugs) in the formulation will vary depending upon a number of factors, including: the dosage of the agent to be administered, the route of administration, the nature of the agent, the frequency and manner of administration, the desired therapy, the form in which the agent is administered, the potency of the agent, the sex, age, weight and general condition of the patient to be treated, the nature and severity of the condition being treated, any complications to be treated, and other factors that will be apparent to those skilled in the art. The dosage of the pharmaceutical composition contains at least a therapeutically effective amount of the isomer according to the present invention and preferably consists of one or more pharmaceutical dosage units. The selected dosage can be administered to a human patient in need of treatment. "therapeutically effective amount" is intended to mean the amount of an isomer of the present invention that has a therapeutic effect on the treatment of a patient. The therapeutic effect may be objective (i.e., measurable by some test or marker (e.g., insulin or glucose level)) or subjective (i.e., an indication or sensory effect produced by the patient).

The dosage of the pharmaceutical composition contains at least a therapeutically effective amount of the isomer according to the present invention and preferably consists of one or more pharmaceutical dosage units. The selected dosage can be administered to a mammal, e.g., a human, in need of treatment. As stated above, a "therapeutically effective amount" is intended to mean that amount of an isomer of the present invention which, when administered to a patient to treat a disease, has a therapeutic effect on the patient being treated. The therapeutic effect may be objective (i.e., measurable by some test or marker (e.g., insulin or glucose level)) or subjective (i.e., an indication or sensory effect produced by the patient). For example, in one embodiment involving type 2 diabetes, a "therapeutically effective" amount will increase glucose uptake by muscle tissue and/or adipose tissue, and/or it will stimulate insulin secretion by pancreatic beta cells. For example, in another embodiment involving type 2 diabetes, a "therapeutically effective" amount results in a decrease in glucose levels in the blood of the patient and/or an increase in insulin levels by at least about 20%, or at least about 40%, or even at least about 60% or at least about 80%, relative to an untreated patient.

The amount corresponding to a "therapeutically effective amount" will vary depending on, for example, the following factors: the particular compound, route of administration, use of excipients, disease condition and severity thereof, identification of the patient in need thereof, body weight of the patient to be treated, etc., and the possibility of co-use with other substances used to treat the disease. However, the therapeutically effective amount can be readily determined by one skilled in the art.

For administration to mammals and, in particular, humans, it is contemplated that dosages of from about 0.1mg to about 50mg (e.g., about 5mg to about 100mg, about 1mg to about 50mg, or about 5mg to about 25mg) of each active compound per kg body weight per day may be used in adult human therapy. For example, a typical oral dose may be administered in a range of about 50mg to about 5g (e.g., about 100mg to about 4g, 250mg to 3g, or 500mg to 2g) per day in one or more doses (e.g., 1 to 3 doses). The dosage can be increased or decreased as desired, as can be readily determined by one skilled in the art. For example, if determined to be appropriate, the amount of a particular substance may be reduced when used in combination with another substance. In addition, reference may be made to the standard amounts and methods mentioned herein for administering the substances.

Table 1 below provides examples of dosages for the antidiabetic agents mentioned herein. The antidiabetic agent may be used in these dosages when combined with the configurational isomer of4-hydroxyisoleucine, which is generally administered in an amount ranging, for example, from 250 mg/day to 1 g/day (e.g., from 350 mg/day to 900 mg/day, from 450 mg/day to 800 mg/day, or from 550 mg/day to 700 mg/day). Alternatively, due to the potential increased or synergistic effect obtained when using the pharmaceutical combinations of the present invention, the amounts in table 1 and/or the amount of the administered isomer may be reduced (e.g., by about 10% to 70%, 20% to 60%, 30% to 50%, or 35% to 45%) as determined to be appropriate by one of skill in the art.

In any event, the physician will determine the actual dosage which will be most suitable for the individual. The above dosages are exemplary averages. Of course, there may be individual cases where higher or lower dosage ranges are contemplated and these are within the scope of the present invention.

With respect to administration, it is understood that: the duration of treatment with any compound or composition of the invention will vary depending on several factors, such as those listed previously herein for dosing. However, one skilled in the art can readily determine the appropriate duration of administration. According to certain embodiments, the compounds of the present invention are administered daily, weekly, or continuously.

Table 1: list of known antidiabetic agents

Antidiabetic agents	Recommended dosage and/or administration
		Insulin	400IU per bottle-40 IU per day (average)
Gliclazide (Diamicron)	80 mg/tablet-1 to 4 tablets per day
		Glibenclamide (Danil) or glibenclamide (Micronase, Glynase, Diabeta)	5 mg/tablet-1 to 3 tablets per day (glibenclamide); 1.25mg to 6 mg/tablet-1 to 2 tablets per day (you Jiang Tang)
Glipizide (Glucotrol, Glibenese)	5 mg/tablet-1 to 4 tablets per day
		Glimepiride (Amamyl, Amarel)	1mg to 5mg per tablet-up to 6mg per day
Chloropropyl urea (Diabinese)	250 mg/tablet-125 mg to 1000mg per day
		Tolbutamide	500 mg/tablet-1 to 4 tablets per day
Repaglinide (Prandin)	0.5mg to 16mg per day
		Nateglinide, lenalide (Starlix)	60mg to 120 mg/tablet-3 tablets per day
Tulasulfenuron	100mg to 500 mg/tablet
		Rosiglitazone	2mg to 8mg per tablet-up to 8mg per day
Pyrrolidinones	15mg to 45mg per tablet15mg to 45mg per day
		Troglitazone	200mg to 400mg per tablet-200 mg to 600mg per day
Ciglitazone	0.1 mg/tablet
		Exenatide(Amylin)	0.09mg to 0.270mg per day
Acarbose	50mg to 100mg per tablet-150 mg to 600mg per day
		Miglitol	50mg to 100 mg/tablet-150 mg to 300mg per day
Voglibose	0.1mg to 0.9mg per day
		Phentolamine	50 mg-4 times per day-6 times per day
Cholestyramine (Colestipol)	4 g/unit-12 g to 16g per day
		Clofibrate ester	500 mg/capsule-1 to 4 capsules per day
Gemfibrozil (Lipur)	450 mg/tablet-2 tablets per day
		Lovastatin	10 mg/tablet and 20 mg/tablet
Pravastatin	20 mg/tablet-10 mg to 40mg per day
		Simvastatin (Zocor, Lodales)	5 mg/tablet and 20 mg/tablet-5 mg to 40mg per day
Probucol	250 mg/tablet-1 g per day
		Dextrothyroxine	2mg to 6mg per day
Alprenolol	5 mg/tablet-4 to 8 tablets per day
		Atenolol	50mg to 100 mg/tablet-100 mg to 200mg per day
Timolol	10 mg/tablet-10 mg to 20mg per day
		Indolol	5 mg/tablet and 15 mg/tablet-5 mg to 60mg per day
Propranolol (Propranolol)	40 mg/tablet-80 mg to 160mg per day
		Metoprolol	100 mg/tablet and 200 mg/tablet-50 mg to 200mg per day
Captopril	25 mg/tablet and 50 mg/tablet-12.5 mg to 150mg per day
		Enalapril	5 mg/tablet and 20 mg/tablet-5 mg to 40mg per day
Nifedipine	10 mg/capsule-30 mg to 60mg per day
		Diltiazem	60 mg/tablet-3 to 6 tablets per day
Verapamil	120 mg/capsule-240 mg to 360mg per day
		Doxazosin	2mg to 8mg per day
Prazozin	2.5 mg/tablet and 5 mg/tablet-2.5 mg to 20mg per day

The isomers and compositions of the present invention are believed to be primarily effective in the treatment of disorders of carbohydrate metabolism, particularly type 2 diabetes. However, it is contemplated that the isomers and compositions according to the present invention may also be useful in lipodystrophy diseases, including but not limited to lipodystrophy associated with HIV and lipemia, as it may affect fat distribution.

It is also contemplated to use the isomers of the present invention for other related or unrelated uses. For example, it may be used to provide internal devices for improving cardiovascular function, such as catheters coated with the isoforms of the present invention.

Examples

The examples provided herein below provide exemplary syntheses of the compounds of the invention. Also provided are methods for determining the activity of the compounds of the invention as glucose uptake stimulators and as insulin secretion stimulators. These examples are given to enable those skilled in the art to more fully understand and practice the present invention, and are not intended to limit or restrict the scope thereof.

Example 1: general procedure for the preparation of4-hydroxyisoleucine isomers

A) General Experimental procedures

FIG. 1 shows the synthesis scheme of 8 different configurational isomers of4-hydroxyisoleucine (SRS, SRR, SSS, SSR, RSR, RSS, RRR and RRS). Imine intermediate 1(Cordova et al, a highly enantioselective amino acid catalysed route to functionalized alpha amino acids, J.Am.chem.Soc.124: 1842-43, 2002) was prepared from p-anisidine and ethyl glyoxylate. The reaction of imine 1 with 2-butanone in the presence of L-proline as catalyst (followed by silica gel chromatography) produced 2S, 3S isomer 2. Epimerization at C-3 was achieved using 1, 5-diazabicyclo [4.3.0] non-5-ene (DBN) to produce 2S, 3R isomer 3. The (2S, 3R, 4S), (2S, 3R, 4R), (2S, 3S, 4S) and (2S, 3S, 4R) isomers of4-hydroxyisoleucine were obtained from 2 or 3 as follows:

deprotection of the amine moiety of 3 (removal of the p-methoxyphenyl group) using Ceric Ammonium Nitrate (CAN) and subsequent use of KBH₄Reduction in water and concomitant cyclization provided lactone 4, which was followed by basic hydrolysis using lithium hydroxide and recrystallization from absolute ethanol to yield pure (2S, 3R, 4S) -4-hydroxyisoleucine 5. Alternatively, deprotection of the amine moiety of 3 using CAN is followed by isolation of amine intermediate 6, which is subsequently reduced using potassium borohydride in methanol to yield lactone intermediate 7, which is followed by basic hydrolysis using lithium hydroxide and recrystallization from ethanol to yield (2S, 3R, 4)R) -4-hydroxyisoleucine (Compound 8). Further purification of compound 8 was performed using preparative HPLC.

Similar reactions starting from compound 2 using sodium borohydride instead of potassium borohydride for the preparation of lactone 12 from aminoketone 11 resulted in the isolation of (2S, 3S, 4S) 4-hydroxyisoleucine (compound 10) and (2S, 3S, 4R) 4-hydroxyisoleucine (compound 13).

When compound 1 is reacted with 2-butanone in the presence of a catalytic amount of D-proline, compound 14 is formed, which is the enantiomer of compound 2. As above, epimerization of C-3 of compound 14 was achieved using 1, 5-diazabicyclo [4.3.0] non-5-ene (DBN) to produce 2R, 3S isomer 15. The (2R, 3S, 4R), (2R, 3S, 4S), (2R, 3R, 4R), and (2R, 3R, 4S) isomers (compounds 17, 20, 22, and 25, respectively) were obtained from compounds 14 and 15 by the same reaction sequence as used for the preparation of compounds 5,8, 10, and 13.

Specific reaction conditions used in the preparation of compounds 1 to 25 are as follows.¹H and¹³c NMR spectrum was D₂Of O solution and using methanol (C)¹H is delta 3.34, and¹³c is δ 49.50) as an internal standard in ppm to indicate chemical shift.

B) Specific experimental procedures

Synthesis of Compound 1

To a stirred toluene solution (400mL) of p-anisidine (50g, 406mmol) in a 1-liter round-bottom flask was added sodium sulfate (200g, 2.5 eq). Ethyl glyoxylate (82mL, 50% in toluene, 406mmol) was slowly added to the above reaction mixture and the mixture was stirred for 30 minutes. After this time, the sodium sulfate was filtered off using celite (celite), and the toluene was removed under reduced pressure. After drying, Compound 1(80g, 95%) was isolated and used for the next reaction.

Synthesis of Compound 2

Chamber under nitrogen in dry DMF (600mL)A mixture of 2-butanone (800mL, 22eq) and L-proline (15.8g, 0.35eq) was stirred warm. To the reaction mixture was slowly added dry DMF (200mL) and Et of Compound 1₃N (22.4ml, 0.40eq) solution. After stirring the reaction mixture at room temperature for 8 hours, the L-proline was filtered off, the excess 2-butanone was removed under reduced pressure and the DMF was removed in vacuo at 500 ℃. By column chromatography (SiO)₂85: 15 Hexane/EtOAc) was purified.

Synthesis of Compound 3

Compound 2 was dissolved in t-BuOMe (15mL) and 1, 5-diazabicyclo [4.3.0] non-5-ene (DBN) (1mL, 0.04eq) was added to the stirred reaction mixture. The reaction mixture was stirred under nitrogen for 2 h. After evaporation of the solvent at room temperature overnight a solid mass was obtained, which was followed by recrystallization from hot ethanol to yield compound 3(48g, 43% yield).

Synthesis of (2S, 3R, 4S) -4-hydroxyisoleucine (Compound 5)

To compound 3(11.6g, 40mmol) in CH was added with stirring at 0 deg.C₃To CN aqueous solution (20mL) was added cerium (IV) ammonium nitrate (CAN) (65.6g, 3eq) aqueous solution (120 mL). After CAN addition, the color gradually changed from blue to green. The reaction mixture was stirred for 2.5 hours and the progress of the reaction was followed by TLC analysis. After completion, the reaction mixture was extracted with EtOAc (4X 150mL) and the aqueous phase was used for the next step.

The aqueous phase is saturated Na₂CO₃Neutralized to pH7 and cooled to-15 ℃ and stirred. After cooling for 30 minutes, KBH was added to the reaction mixture₄(3.2g, 60mmol, 1.5 eq). The reaction mixture was heated to 0 ℃ for 45 minutes and followed by TLC. Then 2N Na was used₂CO₃The reaction mixture was made basic to pH8-9 and treated with CH₂Cl₂(5X 400 mL). The organic phase is washed with water and Na₂SO₄Dried and evaporated under reduced pressure to give a 90: 10 mixture of lactones (compound 4(3S, 4R, 5S) and compound 7(3S, 4R, 5R); 3.73g, 62.6%).

To the 90: 10 aqueous lactone mixture (96mL, 0.3M) was added LiOH (1.1g, 43.3mmol, 1.5 eq.) and the mixture was stirred at room temperature for 2 h. After the reaction was complete, it was acidified by careful addition of AcOH (43.3mmol, 2.4 mL). The reaction mixture was concentrated under reduced pressure and the last traces of water were removed by repeated addition and removal of ethanol. The crude product was crystallized from anhydrous EtOH to yield 1.56g of 98% pure (2S, 3R, 4S) 4-hydroxyisoleucine (compound 5). Compound 5, which yields a white shiny powder, was further purified by preparative HPLC: mp 215-; [ alpha ] to]_D ^H2O+30.7(C，1)；¹H NMR(200MHz)δ3.90(m，¹H)，3.84(m，¹H)，1.91(m，¹H)，1.23(d，J＝5.6Hz，3H)0.95(d，J＝6.6Hz，3H)；¹³C NMR(75MHz)δ174.32，70.46，57.54，41.90，21.30，12.70。

Synthesis of (2S, 3R, 4R) -4-hydroxyisoleucine (Compound 8)

To compound 3(11.6g, 40mmol) in CH was added with stirring at 0 deg.C₃To CN aqueous solution (20mL) was added Cerium Ammonium Nitrate (CAN) (65.6g, 3eq) aqueous solution (120 mL). After CAN addition, the color gradually changed from blue to green. The reaction mixture was stirred for 45 minutes and the progress of the reaction was followed by TLC. After completion, the reaction mixture was extracted with EtOAc (4X 150mL) and the aqueous phase was taken with saturated Na₂CO₃The solution was carefully neutralized to a slightly alkaline pH (. about.8). Using CH₂Cl₂(4X 150mL) the aqueous phase was extracted and the organic extracts combined, washed with brine, anhydrous Na₂SO₄Dried and concentrated under reduced pressure to yield 5.52g (79.7%) of compound 6 as a brown oil.

To a solution of Compound 6 in methanol (15mL) cooled to 0 deg.C was added KBH quickly₄(2.58g, 47.8 mmol). The reaction mixture was stirred at ℃ for 45 minutes and then gradually warmed to room temperature. The solvent was removed in vacuo and the mixture was diluted with water. Using CH₂Cl₂The aqueous phase was extracted (4X 150 mL). The organic phase was washed with brine and anhydrous Na₂SO₄Drying and vacuumEvaporation was carried out to give a 75: 25 mixture of compound 7(3S, 4R, 5R) and compound 4(3S, 4R, 5S) (2.9g, 70.2%).

An aqueous solution of compound 7/compound 4 (100mL) was treated with LiOH (805mg, 33.7mmol) and stirred at room temperature for 1 h, after which it was carefully acidified with AcOH (1.91mL, 33.72 mmol). After concentration under reduced pressure, trace amounts of water were removed by repeated addition and removal of absolute ethanol. Crude off-white solid was obtained from a 90% cold ethanol solution. Further recrystallization from 90% ethanol yielded 1.4g of 75: 25 diastereomer ratio of compound 8 to compound 5. Repeated recrystallization increased the purity of compound 8 to 90% and further purified using preparative HPLC to yield pure (2S, 3R, 4R) 4-hydroxyisoleucine (compound 8) as a white lustrous material: mp 202-; [ alpha ] to]_D ^H2O-21.6(c，0.5)；¹H-NMR(300MHz)54.05(m，1H)，3.80(d，J＝4.2Hz，1H)，2.13(m，1H)1.20(d，J＝6.3Hz，3H)，1.05(d，J＝7.2Hz，3H)；¹³C NMR(75MHz)δ174.49，69.13，59.97，39.12，20.71，9.38。

Synthesis of (2S, 3S, 4S) -4-hydroxyisoleucine (Compound 10)

Compound 2(5.6g, 20mmol) was dissolved in acetonitrile (10mL), and an aqueous solution (60mL) of Cerium Ammonium Nitrate (CAN) (33g, 60mmol) was added with stirring at 0 ℃. After CAN addition, the color gradually changed from blue to green. The reaction mixture was stirred for 45 minutes and extracted with ethyl acetate (4X 150 mL). The water phase is saturated Na₂CO₃Neutralize and carefully adjust pH to 7. After cooling the reaction mixture to-15 ℃ for 90 minutes, KBH was added₄(1.6g, 60mmol, 1.5 eq). The reaction mixture was warmed to 0 ℃ for about 45 minutes, then 2N Na was used₂CO₃Treatment to pH8-9 followed by CH₂Cl₂(5X 400 mL). Washing the organic phase with water and anhydrous Na₂SO₄Dried and evaporated under reduced pressure to give 1.42g of a 75: 25 mixture of lactones (compound 9(3S, 4S, 5S) and compound 12(3S, 4S, 5R)).

To the lactone mixture in water (35mL) was added LiOH (395mg, 16.5mmol, 1.5eq) and the mixture was stirred at room temperature for 2 h. After this time, the reaction mixture was carefully acidified with AcOH (16.5mmol, 0.9 mL). The solvent was removed under vacuum, and repeated addition and removal of absolute ethanol resulted in complete removal of water. The crude product obtained was dissolved in 90% EtOH and left overnight. The isolated white solid was filtered, washed several times with EtOH, and recrystallized from 90% EtOH to obtain white crystals of (2S, 3S, 4S) -4-hydroxyisoleucine (compound 10, 500 mg). Further purification using preparative HPLC formed a pure lustrous material: mp 253-255 ℃; [ alpha ] to]_D ^H2O+28(c，0.25)；¹H NMR(300MHz)54.11(m，1H)，3.87(d，J＝2.7Hz，1H)，2.21(m，1H)，1.23(d，J＝6.3Hz，3H)，0.92(d，J＝7.5Hz，3H)；¹³C NMR(75MHz)δ174.64，71.39，60.39，38.97，21.11，6.19。

Synthesis of (2S, 3S, 4R) -4-hydroxyisoleucine (Compound 13)

To a solution of compound 2(11.6g, 40mmol) in acetonitrile (20mL) was added an aqueous solution of cerium (IV) ammonium nitrate (CAN) (65.6g, 120mmol) (120mL) with stirring at 0 ℃. After the addition of CAN, the reaction mixture gradually changed in color from blue to green. The reaction mixture was stirred for 45 minutes and extracted with ethyl acetate (4X 150 mL). The water phase is saturated Na₂CO₃The solution was carefully neutralized to pH8, followed by CH₂Cl₂(4X 150 mL). The combined organic extracts were washed with brine, anhydrous Na₂SO₄Dried above and concentrated under reduced pressure to yield 4g of compound 11 as a brown oil.

To a 0 ℃ solution of Compound 11 in MeOH (15mL) was added NaBH quickly₄(962mg, 1.1eq, 25.43 mmol). The reaction mixture was stirred vigorously at ℃ for 45 minutes and then gradually warmed to room temperature. The solvent was removed under reduced pressure and the residue was diluted with water and CH was used₂Cl₂The aqueous phase was extracted (4X 150 mL). The combined organic phases were washed with brine, anhydrous Na₂SO₄Dried and evaporated in vacuo to give 2g of a compoundA mixture of substance 12(3S, 4S, 5R) and compound 9(3S, 4S, 5S).

The mixture was dissolved in water (40mL) and LiOH (556.9mg, 18.6mmol) was added. The reaction mixture was stirred at room temperature for 1 hour and carefully acidified with AcOH (1.31 mL). The solvent was removed under vacuum. The crude product was dissolved in a minimal amount of water and the compound was loaded onto a bed using dowex 50 w.times.8 (H)⁺) Resin (50g) packed column. The column was first eluted with water (4X 50mL) and then 2M NH₄OH elution collects fractions. The isolated product was dissolved in 90% EtOH and left overnight. The isolated solid (250mg) was filtered, washed with cold EtOH, and recrystallized from 90% EtOH to obtain a mixture of diastereomers.

This diastereomeric mixture of compounds 10 and 13 was purified by preparative HPLC to yield (2S, 3S, 4R) 4-hydroxyisoleucine (compound 13) as a white, lustrous powder: mp173-175 ℃; [ alpha ] to]_D ^H2O+6.0(c，0.25)；¹H NMR(300MHz)δ4.02(d，J＝3Hz，1H)，3.81(m，1H)，2.12(m，1H)1.28(d，J＝6.6Hz，3H)，0.97(d，J＝7.2Hz，3H)；¹³C NMR(75MHz)δ174.93，70.18，56.34，40.46，21.24，12.15。

(2R, 3S, 4R) -4-hydroxyisoleucine (Compound 17), (2R, 3S, 4S) -4-hydroxyisoleucine (Compound 20), (2R, 3R, 4R) -4-hydroxyisoleucine (Compound 22), and (2R, 3R, 4S) -4-hydroxy Synthesis of isoleucine (Compound 25)

The procedures for the synthesis of compounds 17, 20, 22 and 25 are the same as those for compounds 5,8, 10 and 13 except that compound 1 is reacted with 2-butanone in the presence of D-proline to produce compound 14 (the enantiomer of compound 12). Physical and NMR data for compounds 17, 20, 22 and 25 are as follows:

(2R, 3S, 4R) -4-hydroxyisoleucine (Compound 17)：mp 217-225℃(subl.)；[α]_D ^H2O-31(c，1)；¹H NMR(200MHz)53.89(m，1H)，3.84(m，1H)，1.90(m，1H)1.23(d，J＝6.4Hz，3H)，0.95(d，J＝7Hz，3H)；¹³C NMR(50MHz)δ174.36，70.43，57.51，41.91，21.30，12.6。

(2R, 3S, 4S) -4-hydroxyisoleucine (Compound 20)：mp 200-204℃(subl.)；[α]_D ^H2O+22(C，0.5)；¹H NMR(200MHz)54.04(m，1H)，3.80(m，1H)，2.12(m，1H)，1.19(d，J＝6.2Hz，3H)1.05(d，J＝7.2Hz，3H)；¹³C NMR(50MHz)δ174.55，69.12，59.97，39.12，20.73，9.40。

(2R, 3R, 4R) -4-hydroxyisoleucine (Compound 22)：mp 250-254℃；[α]_D ^H2O-30(c，0.25)；¹H-NMR(200MHz)54.10(m，1H)，3.87(d，J＝2.6Hz 1H)，2.23(m，1H)1.23(d，J＝6.6Hz，3H)，0.92(d，J＝7.2Hz，3H)；¹³C NMR(50MHz)δ174.64，71.29，60.35，38.96，21.12，6.22。

(2R, 3R, 4S) -4-hydroxyisoleucine (Compound 25)：mp 173℃；[α]_D ^H2O-5.6(c，0.25)；¹H NMR(300MHz)54.01(d，J＝2.7Hz，1H)，3.80(m，1H)，2.11(m，1H)1.27(d，J＝6.3Hz，3H)，0.97(d，J＝7.2Hz，3H)；¹³C NMR(75MHz)δ174.96，70.18，56.35，40.44，21.23，12.10。

Example 2: configurational isomer pair of (2S, 3R, 4S) -4-hydroxyisoleucine to differentiate 3T3-L1 fat Stimulation of glucose uptake by cells

3T3-L1 adipocytes (ATCC; CI-173) were cultured in 12-well tissue culture plates for 3 days to achieve confluency (Lakshmann et al, Analysis of insulin-stimulated glucose uptake in differentiated3T3-L1 adipocytes (Analysis of insulin-stimulated glucose uptake in differentiated3T3-L1 adipocytes). Diabetes mellitis: Methods and Protocols, Saire Ozena, eds., Humana Press Inc., Tonowa, New Jersey 97-103, 2003). The medium was removed and replaced with differentiation medium (Green and Meut)h, Cell 3: 127-; madsen et al, biochem.j.375: 539-549, 2003) and the cells are cultured for another 9 days. The differentiation state was confirmed by visual observation. Cell starvation was performed for 5 hours by replacing the differentiation medium with medium lacking fetal bovine serum. During the last 30 minutes of the starvation phase, cells were exposed to the respective configurational isomers of4-hydroxyisoleucine (compounds 5,8, 10, 13, 17, 20, 22 and 25) at a concentration of 0.5 mM. Cells exposed to insulin (0.0167U/mL; Sigma; Cat. No.15534) for the last 30 minutes of the starvation phase were used as positive controls, and cells exposed to 0.5mM isoleucine were used as controls for background uptake. All treatments were performed in four groups. Cells were washed and added with 16. mu.M³H-deoxy-D-glucose (0.5. mu. Ci/mL) and 10. mu.M of 2-deoxy-D-glucose in fresh medium, and cells were cultured for 10 minutes. Glucose uptake was stopped by washing the cells with ice-cold PBS. Cells were lysed and the relative phase in lysates determined³Specific activity of background uptake of H-deoxy-glucose. The results shown in figure 2 were normalized on a per well protein content basis.

As expected, insulin strongly increased glucose uptake, while isoleucine did not. All configurational isomers of (2S, 3R, 4S) -4-hydroxyisoleucine (compound 5) showed good stimulation of glucose uptake, and compounds 8, 13, 20 and 25 had the best activity. Thus, configurational isomers of (2S, 3R, 4S) -4-hydroxyisoleucine have the potential to be therapeutic agents for the treatment of diabetes and related disorders.

Example 3: configurational isomer of (2S, 3R, 4S) -4-hydroxyisoleucine to islets of INS-1 cells Glucose-dependent stimulation of hormone secretion

The insulinotropic effect of4-hydroxyisoleucine configurational isomers on INS-1 cells was tested in a blind manner. Briefly, cells were plated at 2X 10⁵Was plated in 12-well plates and cultured in RPMI containing 10% fetal bovine serum and 11mM glucose for 2 days. On day 3 after plating the medium was removed and replaced with RPMI containing 3mM glucose and 10% fetal bovine serum. CellsThe culture was continued for another 24 hours. On day 4 after plating, the medium was removed and replaced with Krebs-Ringer bicarbonate buffer containing 2mM glucose. The cells were cultured for 30 minutes, the buffer was removed and replaced with Krebs-Ringer bicarbonate buffer containing 4.5mM glucose and the optical isomer at a concentration of 0.5 mM. The cell culture was 1 hour. Basal insulin secretion was determined by culturing the cells in the presence of a buffer containing 2mM glucose. The presence of 4.5mM and 10mM glucose stimulated insulin secretion and served as reference and positive controls, respectively. The positive stimulation response of the configurational isomer of4-hydroxyisoleucine tested was taken as a response above that initiated by 4.5mM glucose. As shown in FIG. 3, all configurational isomers of4-hydroxyisoleucine except Compound 13 (the 2S, 3S, 4R isomer) exhibit insulinotropic activity, and Compounds 8, 10 and 20 are as effective as Compound 5 (the 2S, 3R, 4S isomer known to have insulinotropic activity, see Broca et al, 4-hydroxyisoleucine: the effect of synthetic and natural analogs on insulin secretion (effects of synthetic and natural analogs on insulin secretion), Eur.J.Pharmacol.390: 339-.

It should be understood that: the examples and embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims (22)

1. A compound having the structure:

or a pharmaceutically acceptable salt, lactone, or prodrug thereof.

2. The compound of claim 1, wherein the lactone is

3. Use of the compound of claim 1 and/or a pharmaceutically acceptable salt, lactone, or prodrug of said compound in the manufacture of a medicament for preventing or treating a disorder of carbohydrate metabolism or lipid metabolism in a human.

4. A pharmaceutical composition, comprising: (i) a compound according to claim 1, and/or a pharmaceutically acceptable salt, lactone or prodrug comprising said compound; and (ii) a pharmaceutically acceptable carrier or excipient.

5. A pharmaceutical composition, comprising: (i) a compound selected from the group consisting of:

and

and/or a pharmaceutically acceptable salt, lactone, or prodrug of said compound; and (ii) a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition of claim 5, wherein the lactone is selected from the group consisting of:

and

7. the pharmaceutical composition of claim 5, further comprising at least one antidiabetic agent selected from those listed in Table 1.

8. A pharmaceutical kit, comprising: (i) a compound selected from the group consisting of:

and

and/or a pharmaceutically acceptable salt, lactone, or prodrug of said compound; and (ii) instructions for using the compound to reduce circulating glucose levels in a human patient.

9. A compound selected from the group consisting of:

and

and/or a pharmaceutically acceptable salt, lactone or prodrug of said compound in the manufacture of a medicament for the prophylaxis or treatment of a disorder of carbohydrate metabolism or lipid metabolism in a human.

10. The use according to claim 9, wherein the disorder of carbohydrate metabolism is diabetes.

11. The use according to claim 9, wherein the disorder of carbohydrate metabolism is type 2 diabetes.

12. A compound selected from the group consisting of:

and

and/or a pharmaceutically acceptable salt, lactone or prodrug of said compound for use in the manufacture of a medicament for the treatment of type 2 diabetes in a human.

13. A method for stimulating glucose uptake in muscle cells and/or adipocytes, which comprises administering an effective amount of a compound selected from the group consisting of:

and

and/or an effective amount of a pharmaceutically acceptable salt, lactone, or prodrug of said compound.

14. A method for stimulating insulin secretion from pancreatic beta cells, the method comprising administering an effective amount of a compound selected from the group consisting of:

and

and/or contacting the cell with an effective amount of a pharmaceutically acceptable salt, lactone, or prodrug of the compound.

15. A method for treating a mammal having a disorder of carbohydrate metabolism or a disorder of lipid metabolism, the method comprising administering to the mammal a compound selected from the group consisting of:

and

and/or administering to the mammal a pharmaceutically acceptable salt, lactone, or prodrug of the compound, wherein the compound, salt, lactone, or prodrug is administered in an amount sufficient to lower circulating glucose levels in the mammal.

16. The method of claim 15, wherein the mammal is selected from the group consisting of primates, animals involved in farming and pasturing, rodents, and domesticated companion animals.

17. The method of claim 15, wherein the mammal is a human.

18. The method of claim 15, wherein the disorder of carbohydrate metabolism is diabetes.

19. The method of claim 15, wherein the disorder of carbohydrate metabolism is type 2 diabetes.

20. The method of claim 15, wherein the disorder of carbohydrate metabolism is metabolic syndrome.

21. The method of claim 15, wherein the disorder of carbohydrate metabolism is pre-diabetes.

22. The method of claim 15, wherein the disorder of lipid metabolism is type 2 diabetes.