# Patent

## Title
Glucagon antagonists

## Abstract

Provided herein are compounds, including enantiomerically pure forms thereof, and pharmaceutically acceptable salts or co-crystals and prodrugs thereof which have glucagon receptor antagonist or inverse agonist activity. Further, provided herein are pharmaceutical compositions comprising the same as well as methods of treating, preventing, delaying the time to onset or reducing the risk for the development or progression of a disease or condition for which one or more glucagon receptor antagonist is indicated, including Type I and II diabetes, insulin resistance and hyperglycemia. Moreover, provided herein are methods of making or manufacturing compounds disclosed herein, including enantiomerically pure forms thereof, and pharmaceutically acceptable salts or Co-crystals and prodrugs thereof. Formula I

## Background
RELATED APPLICATIONS
The present application is a national stage application of International Application No. PCT/US2009/053795, filed Aug. 13, 2009, and claims the benefit of the filing date of U.S. Provisional Application No. 61/088,697, filed Aug. 13, 2008, the contents of which are herein incorporated by reference in its entirety.


FIELD
Provided are antagonists of glucagon receptors. In particular, provided are compounds and compositions for use in treatment, prevention or amelioration of one or more symptoms, causes, or effects of a glucoregulatory or glucagon receptor-mediated disease or disorder.
BACKGROUND
Glucagon is a 29-amino acid pancreatic hormone which is secreted from the pancreatic a cells into the portal blood supply in response to hypoglycemia. It acts as a counterregulatory hormone to insulin. Most of the physiological effects of glucagon are mediated by its interaction with glucagon receptors in the liver, followed by activation of adenylate cyclase to increase intracellular cAMP levels. The result is an increase in glycogenolysis and gluconeogenesis, while attenuating the ability of insulin to inhibit these metabolic processes (Johnson et al., J. Biol. Chem. 1972, 247, 3229-3235). As such, overall rates of hepatic glucose synthesis and glycogen metabolism are controlled by the systemic ratio of insulin and glucagon (Roden et al., J. Clin. Invest. 1996, 97, 642-648; Brand et al., Diabetologia 1994, 37, 985-993).
Diabetes is a disease characterized by elevated levels of plasma glucose. Uncontrolled hyperglycemia is associated with an increased risk for microvascular and macrovascular diseases, including nephropathy, retinopathy, hypertension, stroke, and heart disease. Control of glucose homeostasis is a major approach to the treatment of diabetes. It has been demonstrated in healthy animals as well as in animal models of types I and II diabetes that removal of circulating glucagon with selective and specific antibodies results in reduction of the glycemic level (Brand et al., Diabetologia 1994, 37, 985-993; Brand et al., Diabetes 1994, 43(Suppl. 1), 172A). Therefore, one of the potential treatments for diabetes and other diseases involving impaired glycemia is to block a glucagon receptor with a glucagon receptor antagonist to improve insulin responsiveness, to decrease the rate of gluconeogenesis, and/or to lower plasma glucose levels by reducing the rate of hepatic glucose output in a patient.
Glucagon antagonists are known, e.g., UA20040014789, UA20040152750A1, WO04002480A1, U.S. Pat. No. 6,881,746B2, WO03053938A1, UA20030212119, UA20030236292, WO03048109A1, WO03048109A1, WO00069810A1, WO02040444A1, U.S. Pat. No. 6,875,760B2, UA20070015757A, WO04050039A2, UA20060116366A1, WO04069158A2, WO05121097A2, WO05121097A2, WO07015999A2, UA20070203186A 1, UA20080108620A1, UA20060084681A1, WO04100875A2, WO05065680A1, UA20070105930A1. U.S. Pat. No. 7,301,036B2, UA20080085926A1, WO08042223A1. WO07047177A1, UA20070088071A 1, WO07111864A2, WO06102067A1, WO07136577A2, WO06104826A2, WO05118542A1, WO05123668A1, WO06086488, WO07106181A2, WO07114855A2 UA20070249688A1, WO07123581A1, WO06086488A2, WO07120270A2, WO07120284A2, and UA20080125468A1, although at this time none are commercially available as therapeutics. Not all compounds that are glucagon antagonists have characteristics affording the best potential to become useful therapeutics. Some of these characteristics include high affinity at the glucagon receptor, duration of receptor activation, oral bioavailability, and stability (e.g., ability to formulate or crystallize, shelf life). Such characteristics can lead to improved safety, tolerability, efficacy, therapeutic index, patient compliance, cost efficiency, manufacturing ease, etc. It has been unexpectedly discovered that specific stereochemistry and functional groups of the compounds of the present invention exhibit one or more of these desired characteristics, including markedly improved receptor binding properties, oral bioavailability, and/or other advantageous features that enhance their suitability for therapeutic use.
All documents referred to herein are incorporated by reference into the present application as though fully set forth herein.
BRIEF SUMMARY
Provided herein are compounds, including enantiomerically pure forms thereof, and pharmaceutically acceptable salts or co-crystals and prodrugs thereof which have glucagon receptor antagonist or inverse agonist activity. Further, provided herein are pharmaceutical compositions comprising the same, as well as methods of treating, preventing, delaying the time to onset or reducing the risk for the development or progression of a disease or condition for which one or more glucagon receptor antagonist is indicated, including without limitation Type I and II diabetes, insulin resistance and hyperglycemia. Moreover, provided herein are methods of making or manufacturing compounds disclosed herein, including enantiomerically pure forms thereof, and pharmaceutically acceptable salts or co-crystals and prodrugs thereof.
By resolution of a synthetic intermediate into pure enantiomeric forms, a more active enantiomeric series was identified and was characterized as having the R configuration. Methods for synthesis of these enantiomers are described herein. No crystal structure was obtained, however, so while the depictions herein represent the R enantiomer, the compounds of the invention are those that represent the most active enantiomer and were obtained by the synthetic methods described herein. The most active enantiomers (compounds of the invention) are those that display improved characteristics, such as surprisingly increased ratio activity vs. the S enantiomer at the cellular level as compared to activity in the receptor displacement assay.
In one aspect, a compound of Formula I is provided:




wherein:

R44 is H, CH3 or CH3CH2;
R45 is C1-6-alkyl, alkenyl, alkoxy, C3-6-cycloalkyl, C4-8-cycloalkenyl, C4-8-bicycloalkenyl, aryl or heteroaryl, any of which can be optionally substituted with one or more substituents selected from C1-6alkyl, CF3, F, CN or OCF3;
L is phenyl, indenyl, benzoxazol-2-yl, C3-6-cycloalkyl, C4-8-cycloalkenyl or C4-8-bicycloalkenyl, any of which can be optionally substituted with one or more substituents selected from F, Cl, CH3, CF3, OCF3 or CN; and
R46 represents one or more substituents selected from H, F, Cl, CH3, CF3, OCF3 or CN;
or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
In another aspect, a compound of Formula II is provided:




wherein:

R44 is H;
R45 is cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl, 4,4-diethylcyclohexyl, 4,4-dipropylcyclohexyl, 4,4-diethylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl, (S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl, 4,4-dipropylcyclohex-1-enyl, 4-t-butylphenyl, (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl or (1R,4R)-1,7,7-trimethyl bicyclo[2.2.1]-2-hept-2-enyl;
L is phenyl, benzoxazol-2-yl, 4-alkyl-cyclohex-1-enyl, 4,4-dialkylcyclohex-1-enyl, 4-alkyl-cyclohexyl, 4,4-dialkylcyclohexyl or 4,4-dimethylcyclohexenyl, any of which can be optionally substituted with one or more substituents selected from F, Cl, CH3, CF3, OCF3 or CN; and
R46 is H;
or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
In another aspect, pharmaceutical compositions are provided comprising a compound provided herein, e.g., a compound of Formula I or Formula II, including a single enantiomer, a mixture of enantiomers, or a mixture of diastereomers thereof; or a pharmaceutically acceptable salt, solvate, or prodrug thereof in combination with one or more pharmaceutically acceptable carriers.
Further provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease associated with a glucagon receptor, comprising administering to a subject having or being suspected to have such a condition, disorder, or disease, a therapeutically effective amount of a compound provided herein, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
Additionally, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease responsive to the modulation of a glucagon receptor, comprising administering to a subject having or being suspected to have such a condition, disorder, or disorder, a therapeutically effective amount of a compound provided herein, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; or a pharmaceutical composition thereof.
Provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a GCGR-mediated condition, disorder, or disease, comprising administering to a subject having or being suspected to have such a condition, disorder, or disease, a therapeutically effective amount of a compound provided herein, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof or a pharmaceutical composition thereof.
Provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease responsive to a decrease in the hepatic glucose production or in the blood glucose level of a subject, comprising administering to the subject a therapeutically effective amount of a compound provided herein, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof or a pharmaceutical composition thereof.
Provided herein is a method of modulating the biological activity of a glucagon receptor, comprising contacting the receptor with one or more of the compounds provided herein, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; or a pharmaceutical composition thereof
These and other aspects of the invention will be more clearly understood with reference to the following preferred embodiments and detailed description.


DETAILED DESCRIPTION
a. Definitions
To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.
Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject.
The terms “treat,” “treating,” and “treatment” are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.
The terms “prevent,” “preventing,” and “prevention” are meant to include a method of delaying and/or precluding the onset of a disorder, disease, or condition, and/or its attendant symptom(s); barring a subject from acquiring a disease; or reducing a subject's risk of acquiring a disorder, disease, or condition.
The term “therapeutically effective amount” are meant to include the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. The term “therapeutically effective amount” also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.
The term “IC50” refers an amount, concentration, or dosage of a compound that is required for 50% inhibition of a maximal response in an assay that measures such response.
The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 5th Edition, Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition, Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004.
The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.
The terms “active ingredient” and “active substance” refer to a compound, which is administered, alone or in combination with one or more pharmaceutically acceptable excipients, to a subject for treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease. As used herein, “active ingredient” and “active substance” may be an optically active isomer of a compound described herein.
The terms “drug,” “therapeutic agent,” and “chemotherapeutic agent” refer to a compound, or a pharmaceutical composition thereof, which is administered to a subject for treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease.
The term “naturally occurring” or “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man. Similarly, “non-naturally occurring” or “non-native” refers to a material that is not found in nature or that has been structurally modified or synthesized by man.
The term “glucagon receptor” or “GCGR” refers to a glucagon receptor protein or variant thereof, which is capable of mediating a cellular response to glucagon in vitro or in vivo. GCGR variants include proteins substantially homologous to a native GCGR, i.e., proteins having one or more naturally or non-naturally occurring amino acid deletions, insertions, or substitutions (e.g., GCGR derivatives, homologs, and fragments), as compared to the amino acid sequence of a native GCGR. In certain embodiments, the amino acid sequence of a GCGR variant is at least about 80% identical, at least about 90% identical, or at least about 95% identical to a native GCGR. In certain embodiments, the GCGR is a human glucagon receptor.
The term “glucagon receptor antagonist” or “GCGR antagonist” refers to a compound that, e.g., partially or completely blocks, decreases, prevents, inhibits, or downregulates GCGR activity. These terms also refer to a compound that binds to, delays the activation of, inactivates, or desensitizes GCGR. A GCGR antagonist may act by interfering with the interaction of glucagon with GCGR.
The term “GCGR-mediated condition, disorder, or disease” refers to a condition, disorder, or disease characterized by inappropriate, e.g., less than or greater than normal, GCGR activity. Inappropriate GCGR functional activity might arise as the result of an increase in glucagon concentration, GCGR expression in cells which normally do not express GCGR, increased GCGR expression or degree of intracellular activation, leading to, e.g., abnormal plasma glucose levels; or decreased GCGR expression. A GCGR-mediated condition, disorder or disease may be completely or partially mediated by inappropriate GCGR activity. In particularly, a GCGR-mediated condition, disorder or disease is one in which modulation of GCGR results in some effect on the underlying symptom, condition, disorder, or disease, e.g., a GCGR antagonist results in some improvement in at least some of patients being treated.
The term “alkyl” and the prefix “alk” refers to a linear or branched saturated monovalent hydrocarbon radical, wherein the alkyl may optionally be substituted with one or more substituents. The term “alkyl” also encompasses linear, branched, and cyclic alkyl, unless otherwise specified. In certain embodiments, the alkyl is a linear saturated monovalent hydrocarbon radical that has 1 to 20 (C1-20), 1 to 15 (C1-15), 1 to 12 (C1-12), 1 to 10 (C1-10), or 1 to 6 (C1-6) carbon atoms, or branched saturated monovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 12 (C3-12), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. As used herein, linear C1-6 and branched C3-6 alkyl groups are also referred as “lower alkyl.” Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including all isomeric forms), n-propyl, isopropyl, butyl (including all isomeric forms), n-butyl, isobutyl, t-butyl, pentyl (including all isomeric forms), and hexyl (including all isomeric forms). For example, C1-6 alkyl refers to a linear saturated monovalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated monovalent hydrocarbon radical of 3 to 6 carbon atoms. Cycloalkyl also includes monocyclic rings fused to an aryl group in which the point of attachment is on the non-aromatic portion. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl and the like.
The term “alkenyl” refers to a linear or branched monovalent hydrocarbon radical, which contains one or more, in one embodiment, one to five, carbon-carbon double bonds. The alkenyl may be optionally substituted with one or more substituents. The term “alkenyl” also embraces radicals having “cis” and “trans” configurations, or alternatively, “E” and “Z” configurations, as appreciated by those of ordinary skill in the art. As used herein, the term “alkenyl” encompasses both linear and branched alkenyl, unless otherwise specified. For example, C2-6 alkenyl refers to a linear unsaturated monovalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated monovalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the alkenyl is a linear monovalent hydrocarbon radical of 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 12 (C2-12), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched monovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 12 (C3-12), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, isopropenyl, pentenyl, hexenyl, heptenyl, ethenyl, propen-1-yl, propen-2-yl, allyl, butenyl, 2-butenyl, 2-methyl-2-butenyl, 4-methylbutenyl, and the like.
The term “alkynyl” refers to a linear or branched monovalent hydrocarbon radical, which contains one or more, in one embodiment, one to five, carbon-carbon triple bonds. The alkynyl may be optionally substituted with one or more substituents. The term “alkynyl” also encompasses both linear and branched alkynyl, unless otherwise specified. In certain embodiments, the alkynyl is a linear monovalent hydrocarbon radical of 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 12 (C2-12), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched monovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 12 (C3-12), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl (—C≡CH), propargyl (—CH2C≡CH), 3-methyl-1-pentynyl, 2-heptynyl, and the like. For example, C2-6 alkynyl refers to a linear unsaturated monovalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated monovalent hydrocarbon radical of 3 to 6 carbon atoms.
The term “cycloalkyl” refers to a cyclic saturated bridged and/or non-bridged monovalent hydrocarbon radical, which may be optionally substituted with one or more substituents. In certain embodiments, the cycloalkyl has from 3 to 20 (C3-20), from 3 to 15 (C3-15), from 3 to 12 (C3-12), from 3 to 10 (C3-10), or from 3 to 7 (C3-7) carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl, and adamantyl.
The term “cycloalkenyl” refers to a cyclic unsaturated bridged and/or non-bridged monovalent hydrocarbon radical, which contains one or more double bonds in its ring. The cycloalkenyl may be optionally substituted with one or more substituents. In certain embodiments, the cycloalkenyl has from 3 to 20 (C3-20), from 3 to 15 (C3-15), from 3 to 12 (C3-12), from 3 to 10 (C3-10), or from 3 to 7 (C3-7) carbon atoms.
The term “aryl” (Ar) refers to a monocyclic aromatic group and/or multicyclic monovalent aromatic group that contain at least one aromatic hydrocarbon ring. In certain embodiments, the aryl has from 6 to 20 (C6-20), from 6 to 15 (C6-15), or from 6 to 10 (C6-10) ring atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to bicyclic or tricyclic carbon rings, where one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). In certain embodiments, aryl may also be optionally substituted with one or more substituents.
The term “aralkyl” or “aryl-alkyl” refers to a monovalent alkyl group substituted with aryl. In certain embodiments, both alkyl and aryl may be optionally substituted with one or more substituents.
The term “heteroaryl” (HAR) refers to a monocyclic aromatic group and/or multicyclic aromatic group that contain at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. In some embodiments, each ring contains 5 to 6 atoms. Each ring of a heteroaryl group can contain one or two O atoms, one or two S atoms, and/or one to four N atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. The heteroaryl may be attached to the main structure at any heteroatom or carbon atom which results in the creation of a stable compound. In certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyramidyl, pyridazinyl, triazolyl, tetrazolyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, benzothiophenyl, furo(2,3-b) pyridyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrollinyl, acridinyl, phenanthridinyl, and xanthenyl. In certain embodiments, heteroaryl may also be optionally substituted with one or more substituents. Heteroaryl also includes aromatic heterocyclic groups fused to heterocycles that are non-aromatic or partially aromatic, and aromatic heterocyclic groups fused to cycloalkyl rings. Heteroaryl also includes such groups in charged form, e.g., pyridinium.
The term “heterocyclyl” (Hetcy) or “heterocyclic” refers to a monocyclic non-aromatic ring system and/or multicyclic ring system that contains at least one non-aromatic ring, wherein one or more of the non-aromatic ring atoms are heteroatoms independently selected from O, S, or N; and the remaining ring atoms are carbon atoms. In certain embodiments, the heterocyclyl or heterocyclic group has from 3 to 20, from 3 to 15, from 3 to 10, from 3 to 8, from 4 to 7, or from 5 to 6 ring atoms. In certain embodiments, the heterocyclyl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused or bridged ring system, and in which the nitrogen or sulfur atoms may be optionally oxidized, the nitrogen atoms may be optionally quaternized, and some rings may be partially or fully saturated, or aromatic. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom which results in the creation of a stable compound. Examples of such heterocyclic radicals include, but are not limited to benzoxazinyl, benzodioxanyl, benzodioxolyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl, chromanyl, chromonyl, coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, 2,3-dihydrofuro(2,3-b)pyridyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dioxolanyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrazolyl, dihydropyrimidinyl, dihydropyrrolyl, 1,4-dithianyl, imidazolidinyl, imidazolinyl, indolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl, oxazolidinyl, oxiranyl, quinuclidinyl, tetrahydrofuryl, tetrahydrofuranyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, and 1,3,5-trithianyl. Heterocyclyl/heterocyclic also includes partially unsaturated monocyclic rings that are not aromatic, such as 2- or 4-pyridones attached through the nitrogen or N-substituted-(1H,3H)-pyrimidine-2,4-diones (N-substituted uracils). Heterocyclyl/heterocyclic also includes such moieties in charged form, e.g., piperidinium. In certain embodiments, heterocyclyl/heterocyclic may also be optionally substituted with one or more substituents.
The term “alkoxy” refers to an —OR radical, wherein R is, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl, each as defined herein. When R is aryl, it is also known as aryloxy. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, n-propoxy, 2-propoxy, n-butoxy, isobutoxy, tert-butoxy, cyclohexyloxy, phenoxy, benzoxy, and 2-naphthyloxy. In certain embodiments, alkoxy may also be optionally substituted with one or more substituents.
The term “acyl” refers to a —C(O)R radical, wherein R is, for example, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl, each as defined herein. Examples of acyl groups include, but are not limited to, formyl, acetyl, propionyl, butanoyl, isobutanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, docosanoyl, myristoleoyl, palmitoleoyl, oleoyl, linoleoyl, arachidonoyl, benzoyl, pyridinylcarbonyl, and furoyl. In certain embodiments, acyl may also be optionally substituted with one or more substituents.
The term “halogen”, “halide” or “halo” (Halo) refers to fluorine, chlorine, bromine, and/or iodine.
The term “optionally substituted” is intended to mean that a group, including alkyl, alkoxy, acyl, alkyl-cycloalkyl, hydroxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, aryl, aryloxy, aralkyl, aryl-alkenyl, aryl-alkynyl, heteroaryl, heteroarylalkyl, heteroaryl-alkenyl, heteroaryl-alkynyl, and heterocyclyl, or acyl, may be substituted with one or more substituents, in one embodiment, one, two, three, four substituents, where in some embodiments each substituent is independently selected from the group consisting of cyano, halo, oxo, nitro, C1-6 alkyl, C2-6 alkenyl, alkynyl, C3-7 cycloalkyl, C6-14 aryl, heteroaryl, heterocyclyl, —C(O)Re, —C(O)ORe, —C(O)NRfRg, —C(NRe)NRfRg, —ORe, —OC(O)Re, —OC(O)ORe, —OC(O)NRfRg, —OC(═NRe)NRfRg, —OS(O)Re, —OS(O)2Re, —OS(O)NRfRg, —OS(O)2NRfRg, —NRfRg, —NReC(O)Rf, —NReC(O)ORf, —NReC(O)NRfRg, —NReC(═NRh)NRfRg, —NReS(O)Rf, —NReS(O)2Rf, —NReS(O)NRfRg, —NReS(O)2NRfRg, —SRe, —S(O)Re, —S(O)2Re, and —S(O)2NRfRg, wherein each Re, Rf, Rg, and Rh is independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 cycloalkyl, C6-14 aryl, heteroaryl, or heterocyclyl; or Rf and Rg together with the N atom to which they are attached form heterocyclyl.
The term “optically active” refers to a collection of molecules, which has an enantiomeric excess of no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 91%, no less than about 92%, no less than about 93%, no less than about 94%, no less than about 95%, no less than about 96%, no less than about 97%, no less than about 98%, no less than about 99%, no less than about 99.5%, or no less than about 99.8%.
In describing an optically active compound, the prefixes R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The (+) and (−) are used to denote the optical rotation of the compound, that is, the direction in which a plane of polarized light is rotated by the optically active compound. The (−) prefix indicates that the compound is levorotatory, that is, the compound rotates the plane of polarized light to the left or counterclockwise. The (+) prefix indicates that the compound is dextrorotatory, that is, the compound rotates the plane of polarized light to the right or clockwise. However, the sign of optical rotation, (+) and (−), is not related to the absolute configuration of the molecule, R and S.
The term “solvate” refers to a compound provided herein or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.
“Binding” means the specific association of the compound of interest to the target of interest, e.g., a receptor.
The term “crystalline” and related terms used herein, when used to describe a substance, component or product, means that the substance, component or product is crystalline as determined by X-ray diffraction. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa., 173 (1990); The United States Pharmacopeia, 23rd ed., 1843-1844 (1995).
“Co-crystal” as used herein means a crystalline material comprised of two or more unique solids at room temperature that are H-bonded.
“Diabetes” refers to a heterogeneous group of disorders that share impaired glucose tolerance in common. Its diagnosis and characterization, including pre-diabetes, type I and type II diabetes, and a variety of syndromes characterized by impaired glucose tolerance, impaired fasting glucose, and abnormal glycosylated hemoglobin, are well known in the art. It may be characterized by hyperglycemia, glycosuria, ketoacidosis, neuropathy or nephropathy, increased hepatic glucose production, insulin resistance in various tissues, insufficient insulin secretion and enhanced or poorly controlled glucagon secretion from the pancreas.
The term “drug” refers to a compound, or a pharmaceutical composition thereof, which is administered to a subject for treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease.
The term “EC50” refers an amount, concentration, or dosage of a compound at which 50% of a maximal response is observed in an assay that measures such response.
The term “percent enantiomeric excess (% ee)” refers to optical purity. It is obtained by using the following formula:




  
    
      
        
          [
          R
          ]
        
        -
        
          [
          S
          ]
        
      
      
        
          [
          R
          ]
        
        +
        
          [
          S
          ]
        
      
    
    ×
    100
  
  =
  
    
      %
      ⁢
      
        
      
      ⁢
      R
    
    -
    
      %
      ⁢
      
        
      
      ⁢
      S
    
  




where [R] is the amount of the R isomer and [S] is the amount of the S isomer. This formula provides the % ee when R is the dominant isomer.
The term “enantiomerically pure” refers to a compound which comprises at least about 80% by weight of the designated enantiomer and at most about 20% by weight of the other enantiomer or other stereoisomer(s), at least about 90% by weight of the designated enantiomer and at most about 10% by weight of the other enantiomer or other stereoisomer(s), at least about 95% by weight of the designated enantiomer and at most about 5% by weight of the other enantiomer or other stereoisomer(s), at least about 96.6% by weight of the designated enantiomer and at most about 3.4% by weight of the other enantiomer or other stereoisomer(s), at least about 97% by weight of the designated enantiomer and at most about 3% by weight of the other enantiomer or other stereoisomer(s), at least about 99% by weight of the designated enantiomer and at most about 1% by weight of the other enantiomer or other stereoisomer(s), or at least about 99.9% by weight of the designated enantiomer and at most about 0.1% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the weights are based upon total weight of the compound.
As used in connection with the compounds of Formula I and II disclosed herein, the terms “R-isomer” and “R-enantiomer” refer to the configuration R of the aliphatic carbon which is alpha to the —C(O)NH— group. Formula I below shows the R-stereochemistry.




The term “chiral” as used herein includes a compound that has the property that it is not superimposable on its mirror image.
“Insulin resistance” is defined clinically as the impaired ability of a known quantity of exogenous or endogenous insulin to increase whole body glucose uptake and utilization.
“Impaired glucose tolerance (IGT)” refers to a condition known to precede the development of overt Type 2 diabetes. It is characterized by abnormal blood glucose excursions following a meal. The criteria for diagnosing and characterizing impaired glucose tolerance and related syndromes are well known in the art.
“Lower” referred to herein in connection with organic radicals or compounds respectively defines such radicals or compounds as containing up to and including 6 carbon atoms. One aspect provides organic radicals or compounds as containing up to and including 4 carbon atoms. Yet another aspect provides organic radicals or compounds that contain one to three carbon atoms. Such groups may be straight chain, branched, or cyclic.
“Metabolic disease” includes diseases and conditions such as obesity, diabetes and lipid disorders such as hypercholesterolemia, hyperlipidemia, hypertriglyceridemia as well as disorders that are associated with abnormal levels of lipoproteins, lipids, carbohydrates and insulin such as metabolic syndrome X, diabetes, impaired glucose tolerance, atherosclerosis, coronary artery disease, cardiovascular disease. The criteria for diagnosing and characterizing these conditions and related syndromes are well known in the art.
“Prodrug” as used herein refers to any compound that when administered to a biological system generates a biologically active compound as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), and/or metabolic chemical reaction(s), or a combination of each. Standard prodrugs are formed using groups attached to functionality, e.g., HO—, HS—, HOOC—, —NHR, associated with the drug, that cleave in vivo. Standard prodrugs include but are not limited to carboxylate esters where the group is alkyl, aryl, aralkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and amines where the group attached is an acyl group, an alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. The groups illustrated are exemplary, not exhaustive, and one skilled in the art could prepare other known varieties of prodrugs. Such prodrugs of the compounds of Formula I or II disclosed herein fall within this scope. Prodrugs must undergo some form of a chemical transformation to produce the compound that is biologically active or is a precursor of the biologically active compound. In some cases, the prodrug is biologically active, usually less than the drug itself, and serves to improve drug efficacy or safety through improved oral bioavailability, and/or pharmacodynamic half-life, etc. Prodrug forms of compounds may be utilized, for example, to improve bioavailability, improve subject acceptability such as by masking or reducing unpleasant characteristics such as bitter taste or gastrointestinal irritability, alter solubility such as for intravenous use, provide for prolonged or sustained release or delivery, improve ease of formulation, or provide site-specific delivery of the compound. Prodrugs are described in The Organic Chemistry of Drug Design and Drug Action, by Richard B. Silverman, Academic Press, San Diego, 1992. Chapter 8: “Prodrugs and Drug delivery Systems” pp. 352-401; Design of Prodrugs, edited by H. Bundgaard, Elsevier Science, Amsterdam, 1985; Design of Biopharmaceutical Properties through Prodrugs and Analogs, Ed. by E. B. Roche, American Pharmaceutical Association, Washington, 1977; and Drug Delivery Systems, ed. by R. L. Juliano, Oxford Univ. Press, Oxford, 1980.
b. Compounds
One aspect provides for compounds of Formula




wherein:

R44 is H, CH3 or CH3CH2;
R45 is C1-6-alkyl, alkenyl, alkoxy, C3-6-cycloalkyl, C4-8-cycloalkenyl, C4-8-bicycloalkenyl, aryl or heteroaryl, any of which can be optionally substituted with one or more substituents selected from C1-6alkyl, CF3, F, CN or OCF3;
L is phenyl, indenyl, benzoxazol-2-yl or 4,4-dimethylcyclohexenyl, any of which can be optionally substituted with one or more substituents selected from F, Cl, CH3, CF3, OCF3 or CN; and
R46 represents one or more substituents selected from H, F, Cl, CH3, CF3, OCF3 or CN.
In certain embodiments according to Formula I, the configuration of the aliphatic carbon which is alpha to the —C(O)NH— group is R.
In other embodiments according to Formula I, L is substituted with one or more substituents independently selected from F, Cl, CH3, CF3, OCF3 or CN. In another embodiment, L is phenyl, benzoxazol-2-yl or 4,4-dimethylcyclohexenyl, any of which can be optionally substituted with one or more substituents. In another embodiment, L is 4-chloro-2-methylphenyl, 4-methyl-2-benzoxazolyl, 2,4,6-trimethylphenyl, benzoxazol-2-yl, 4-chloro-3-methylphenyl or 4,4-dimethylcyclohexenyl.
In another embodiment, R44 is H or CH3. In another embodiment, R44 is H.
In certain embodiments, R45 is attached to the 3 (meta) or 4 (para) position. In another embodiment, R45 is attached to the 4 (para) position. In another embodiment, R45 is alkenyl, C3-6-cycloalkyl, C4-8-cycloalkenyl, C4-8-bicycloalkenyl or phenyl, any of which can be optionally substituted with one or more substituents. In yet other embodiments according to Formula I, R45 is selected from (CH3)3CCH═CH—, t-butyl-cycloalkyl-, dimethyl-cycloalkyl-, t-butyl-cycloalkenyl-, dimethyl-cycloalkenyl-, bicycloalkenyl or phenyl-.
In certain embodiments according to Formula I, R45 is substituted with one or more substituents independently selected from CH3 and (CH3)3C—.
In certain embodiments according to Formula I, R45 is trans-t-butylvinyl, cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl, cyclohex-1-enyl, (S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl, 4,4-diethylcyclohex-1-enyl, 4,4-diethylcyclohexyl, 4,4-dipropylcyclohex-1-enyl, 4,4-dipropylcyclohexyl, 4,4-dimethylcyclohexa-1,5-dienyl, (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]3-heptyl-2-ene, (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]2-heptyl-2-ene, 2-methyl-4-chloro-phenyl, 2,4,6-trimethylphenyl or 4-t-butylphenyl.
In certain embodiments according to Formula I, R45 is trans-t-butylvinyl, cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl, (S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl, (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]2-heptyl-2-ene or 4-t-butyl phenyl.
In another embodiment, R46 is H or CH3. In another embodiment, R46 is H.
In certain embodiments, the compound of Formula I is one presented below in Table I:







TABLE 1





Certain Compounds of Formula I













 








 








 








 








 








 








 








 








 








 








 








 















Another aspect provides for compounds, prodrugs thereof, and compositions comprising the compounds or prodrugs thereof wherein the compound is a structure of Formula II:




wherein:

R44 is H;
R45 is cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl, 4,4-diethylcyclohexyl, 4,4-dipropylcyclohexyl, 4,4-diethylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl, (S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl, 4,4-dipropylcyclohex-1-enyl, 4-t-butylphenyl, (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl or (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]-2-hept-2-enyl;
L is phenyl, benzoxazol-2-yl, 4-alkyl-cyclohex-1-enyl, 4,4-dialkylcyclohex-1-enyl, 4-alkyl-cyclohexyl, 4,4-dialkylcyclohexyl or 4,4-dimethylcyclohexenyl, any of which can be optionally substituted with one or more substituents selected from F, Cl, CH3, CF3, OCF3 or CN; and
R46 is H.
In certain embodiments according to Formula II, L is substituted with one or more substituents independently selected from Cl or CH3. In another embodiment, L is phenyl, benzoxazol-2-yl or 4,4-dimethylcyclohexenyl, any of which can be optionally substituted with one or more substituents selected from Cl or CH3.
In certain embodiments according to Formula II, R45 is attached to the 3 (meta) or 4 (para) position. In another embodiment, R45 is attached to the 4 (para) position.
In another embodiment, R45 is cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl, (S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl, 4,4-dipropylcyclohex-1-enyl, 4-t-butylphenyl, (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl or (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]-2-kept-2-enyl. In other embodiments, R45 is cis-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl, (S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl, 4-t-butylphenyl or (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl.
In certain embodiments the compound of Formula II is also a compound of Formula 1.
In certain embodiments according to Formula II, the configuration of the aliphatic carbon which is alpha to the —C(O)NH— group is R.
In certain embodiments, the compounds of Formula II are a racemic mixture.
Another aspect provides for enantiomerically pure compounds of Formula I or II. In certain embodiments, a single enantiomer is >60%, >70%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98% or >99% as compared to the total percentage of all other enantiomers of the same compound or other diastereomers present in the composition.
Another aspect provides enantiomerically pure compounds of Formula I or II. In certain embodiments, the compound comprises at least about 80% by weight of the designated enantiomer and at most about 20% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the compound comprises at least about 90% by weight of the designated enantiomer and at most about 10% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the compound comprises at least about 95% by weight of the designated enantiomer and at most about 5% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the compound comprises at least about 96.6% by weight of the designated enantiomer and at most about 3.4% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the compound comprises at least about 97% by weight of the designated enantiomer and at most about 3% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the compound comprises at least about 99% by weight of the designated enantiomer and at most about 1% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the compound comprises at least about 99.9% by weight of the designated enantiomer and at most about 0.1% by weight of the other enantiomer or other stereoisomer(s). In certain embodiments, the weights are based upon total weight of the compound.
Another aspect provides for salts, including pharmaceutically acceptable salts, of compounds of Formula I or II and pharmaceutical compositions comprising a pharmaceutically acceptable salt of compounds of Formula I or II. Salts of compounds of Formula I or II include an inorganic base addition salt such as sodium, potassium, lithium, calcium, magnesium, ammonium, aluminum salts or organic base addition salts.
Another aspect provides for anhydrates, hydrates and solvates of compounds of Formula I or II and pharmaceutical compositions comprising a pharmaceutically acceptable anhydrates, hydrates and solvates of compounds of Formula I or II. Included are an anhydrate, hydrate or solvate of a free form or salt of a compound of Formula I or II. Hydrates include, for example, a hemihydrate, monohydrate, dihydrate, trihydrate, quadrahydrate, pentahydrate, sesquihydrate.
In certain embodiments, the compounds of Formula I or II are able to displace radiolabeled glucagon from the human glucagon receptor by at least 15% at 1000 nM. In one embodiment, the compounds of Formula I or II are able to displace at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of radiolabeled glucagon from the human glucagon receptor as described in Example A.
Alternatively, the activities of the compounds of Formula I or II can be described in terms of the concentrations of compounds required for displacement of 50% of the radiolabeled glucagon from the human glucagon receptor (the IC50 values) according to the methods of Example A. In one embodiment, the IC50 values for the compounds of Formula I are less than <10,000 nM, 9,000 nM, 8,000 nM, 7.000 nM, 6,000 nM, 5,000 nM. 4,000 nM, 3,000 nM, 2,000 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM or 5 nM.
In another alternative, the activities of the compounds of Formula I or II can be described in terms of the concentrations of compounds required for functional antagonism of glucagon in hepatocytes from various species. The EC50 is determined using the method of Example B. In one embodiment, the EC50 values for the compounds of Formula I or II are less than <10,000 nM. 9,000 nM, 8,000 nM, 7,000 nM, 6,000 nM, 5,000 nM. 4,000 nM, 3,000 nM, 2,000 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM or 5 nM.
The compounds of Formula I or II disclosed herein also exhibit the ability to reduce blood glucose in an animal. In certain aspects, circulating blood glucose in fasting or non-fasting (freely-feeding) animals can be reduced between 10% and 100%. A reduction of 100% refers to complete normalization of blood glucose levels, not 0% blood glucose levels. Normal blood glucose in rats, for example, is approximately 80 mg/dl (fasted) and approximately 120 mg/dl (fed). Thus, contemplated herein is a method for reducing excessive circulating blood glucose levels in fasting or freely fed animals (e.g. rat), by administered 10 mg/kg of a compound of Formula I, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
c. Administration
Provided herein are pharmaceutical compositions including a compound provided herein as an active ingredient, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; in combination with a pharmaceutically acceptable vehicle, carrier, diluent, excipient, or a mixture thereof.
The pharmaceutical compositions may be formulated in various dosage forms, including, but limited to, the dosage forms for oral, parenteral, subcutaneous, intramuscular, transmucosal, inhaled, or topical/transdermal administration. The pharmaceutical compositions may also be formulated as modified release dosage forms, including, but not limited to, delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Deliver Technology, Rathbone et al., Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, N.Y., 2003; Vol. 126).
The pharmaceutical compositions provided herein may be provided in a unit- or multiple-dosage form. A unit-dosage form, as used herein, refers to a physically discrete unit suitable for administration to a subject as is known in the art. Examples of a unit-dosage form include an ampoule, syringe, and individually packaged tablet and capsule. A unit-dosage form may be administered in fractions or multiples thereof.
The pharmaceutical compositions provided herein may be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the pharmaceutical compositions provided herein.
Exemplary pharmaceutical compositions and components for use therewith are described in U.S. Provisional Application No. 61/088,697, the contents of which are herein incorporated by reference.
d. Methods of Use
In one embodiment, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease associated with impaired glucose tolerance, a metabolic syndrome, or a glucagon receptor, comprising administering to a subject having or being suspected to have such a condition, disorder, or disease, a therapeutically effective amount of a compound provided herein, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; or a pharmaceutical composition thereof. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.
In another embodiment, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a condition, disorder, or disease responsive to a decrease in the hepatic glucose production or in the blood glucose level of a subject, comprising administering to the subject having or being suspected to have such a condition, disorder, or disease, a therapeutically effective amount of a compound provided herein, e.g., a compound of Formula I or II, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; or a pharmaceutical composition thereof. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.
The conditions and diseases treatable with the methods provided herein include, but are not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, ketoacidosis, nonketotic hyperosmolar coma (nonketotic hyperglycemia), impaired glucose tolerance (IGT), insulin resistance syndromes, syndrome X, low HDL levels, high LDL levels, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia, hyperlipoproteinemia, hypercholesterolemia, dyslipidemia, arteriosclerosis, atherosclerosis, glucagonomas, acute pancreatitis, cardiovascular diseases, hypertension, cardiac hypertrophy, gastrointestinal disorders, obesity, vascular resenosis, pancreatitis, neurodegenerative disease, retinopathy, nephropathy, neuropathy, accelerated gluconeogenesis, excessive (greater than normal levels) hepatic glucose output, and lipid disorders.
Provided herein are also methods of delaying the time to onset or reducing the risk of the development or progression of a disease or condition responsive to decreased hepatic glucose production or responsive to lowered blood glucose levels.
Depending on the condition, disorder, or disease to be treated and the subject's condition, a compound provided herein may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or intraarterial (e.g., via catheter), ICV, intracistemal injection or infusion, subcutaneous injection, or implant), inhalation, nasal, vaginal, rectal, sublingual, and/or topical (e.g., transdermal or local) routes of administration, and may be formulated alone or together in suitable dosage unit with a pharmaceutically acceptable vehicle, carrier, diluent, excipient, or a mixture thereof, appropriate for each route of administration.
The dose may be in the form of one, two, three, four, five, six, or more sub-doses that are administered at appropriate intervals per day. The dose or sub-doses can be administered in the form of dosage units containing from about from about 0.01 to 2500 mg, from 0.1 mg to about 1,000 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 500 mg, from about 0.1 mg to about 500 mg, from about 0.1 mg to about 100 mg, from about 0.5 mg about to about 100 mg, from about 1 mg to about 100 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 500 mg, or from about 10 mg to about 100 mg of active ingredient(s) per dosage unit. For example, the dose or subdoses can be administered in the form of dosage units containing about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. If the condition of the patient requires, the dose can, by way of alternative, be administered as a continuous infusion.
In certain embodiments, an appropriate dosage level is about 0.01 to about 100 mg per kg patient body weight per day (mg/kg per day), about 0.01 to about 50 mg/kg per day, about 0.01 to about 25 mg/kg per day, or about 0.05 to about 10 mg/kg per day, which may be administered in single or multiple doses. A suitable dosage level may be about 0.01 to about 100 mg/kg per day, about 0.05 to about 50 mg/kg per day, or about 0.1 to about 10 mg/kg per day. Within this range, the dosage may be about 0.01 to about 0.1, about 0.1 to about 1.0, about 1.0 to about 10, or about 10 to about 50 mg/kg per day.
For oral administration, the pharmaceutical compositions can be provided in the form of tablets containing 1.0 to 1,000 mg of the active ingredient, particularly about 1, about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 750, about 800, about 900, and about 1,000 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compositions may be administered on a regimen of 1 to 4 times per day, including once, twice, three times, and four times per day. In various embodiments, the compositions may be administered before a meal, after a meal, in the morning hours, after awakening, in the evening hours, and/or at bedtime.
It will be understood, however, that the specific dose level, frequency, and timing of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
In still another embodiment, provided herein is a method of modulating the biological activity of a glucagon receptor, comprising contacting the receptor with one or more of the compounds provided herein, e.g., a compound of Formulas I or II, including a single enantiomer, a mixture of enantiomers, or a mixture of diastereomers thereof, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; or a pharmaceutical composition thereof. In one embodiment, the glucagon receptor is expressed by a cell.
The compounds provided herein may also be combined or used in combination with other therapeutic agents useful in the treatment, prevention, or amelioration of one or more symptoms of the conditions, disorders, or diseases for which the compounds provided herein are useful. As used herein, the term “in combination” includes the use of more than one therapeutic agents. However, the use of the term “in combination” does not restrict the order in which therapeutic agents are administered to a subject with a condition, disorder, or disorder. A first therapeutic agent (e.g., a therapeutic agent such as a compound provided herein) can be administered prior to (e.g., 5 min, 15 min, 30 min, 45 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, 96 hrs, 1 wk, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 8 wks, or 12 wks before), concomitantly with, or subsequent to (e.g., 5 min, 15 min, 30 min, 45 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, 96 hrs, 1 wk, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 8 wks, or 12 wks after) the administration of a second therapeutic agent to a subject to be treated.
When a compound provided herein is used contemporaneously with one or more therapeutic agents, a pharmaceutical composition containing such other agents in addition to the compound provided herein may be utilized, but is not required. Accordingly, the pharmaceutical compositions provided herein include those that also contain one or more other therapeutic agents, in addition to a compound provided herein.
In one embodiment, the other therapeutic agent is an antidiabetic agent. Suitable antidiabetic agents include, but are not limited to, insulin sensitizers, biguanides (e.g., buformin, metformin, and phenformin), PPAR agonists (e.g., troglitazone, pioglitazone, and rosiglitazone), insulin and insulin mimetics, somatostatin, α-glucosidase inhibitors (e.g., voglibose, miglitol, and acarbose), dipeptidyl peptidase-4 inhibitors, liver X receptor modulators, insulin secretagogues (e.g., acetohexamide, carbutamide, chlorpropamide, glibomuride, gliclazide, glimerpiride, glipizide, gliquidine, glisoxepid, glyburide, glyhexamide, glypinamide, phenbutamide, sulfonylureas, tolazamide, tolbutamide, tolcyclamide, nateglinide, and repaglinide), other glucagon receptor antagonists, GLP-1, GLP-1 mimetics (e.g., exenatide, liraglutide, DPPIV inhibitors), GLP-1 receptor agonists, GIP, GIP mimetics, GIP receptor agonists, PACAP, PACAP mimetics, PACAP receptor 3 agonists, cholesterol lowering agents, HMG-CoA reductase inhibitors (e.g. statins, such as lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, itavastatin, rivastatin, NK-104 (a.k.a. itavastatin, nisvastatin, and nisbastatin), and ZD-4522 (also known as rosuvastatin, atavastatin, and visastatin)), a cholesterol absorption inhibitor (e.g., ezetimibe), sequestrants, nicotinyl alcohol, nicotinic acid and salts thereof, PPAR α agonists, PPAR α/γ dual agonists, inhibitors of cholesterol absorption, acyl CoA:cholesterol acyltransferase inhibitors, anti-oxidants, PPAR δ agonists, antiobesity compounds, ileal bile acid transporter inhibitors, anti-inflammatory agents, and protein tyrosine phosphatase-1B (PTP-1B) inhibitors.
The dosages given will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
The weight ratio of a compound provided herein to the second active ingredient depends upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound provided herein is combined with a PPAR agonist the weight ratio of the compound provided herein to the PPAR agonist will generally range from about 1000:1 to about 1:1000 or about 200:1 to about 1:200. Combinations of a compound provided herein and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
Synthesis of Compounds
Compounds of Formula I and II can be prepared according to the methodology outlined in the following general synthetic schemes or with modifications of these schemes that will be evident to persons skilled in the art, or by other methods readily known to those of skill in the art.
In the following sections, the following abbreviations have the following meanings: THF: Tetrahydrofuran; DME: 1,2-Dimethoxyethane; DMF: N,N-Dimethylformamide; DCC: N,N′-Dicyclohexylcarbodiimide; EDCI or EDC: 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride; LiHMDS: Lithium hexamethyldisilyl azide; HOBt: 1-Hydroxybenzotriazole; EtOAc: Ethyl acetate; EtOH: Ethanol; IPA: iso-Propanol; ACN: Acetonitrile; DIPEA: N,N-Diisopropyl-ethyl amine; and MTBE: Methyl-tert-butyl ether.
a. Synthesis of Various Building Blocks:
The carboxylic acids 3 can be generated using standard methods. As shown in Scheme 1, a carboxylic ester or acid 1 can be alkylated by reaction with a base (such as lithium diisopropylamide or lithium hexamethyldisilylamide) in a suitable solvent (such as THF or DME) followed by reaction with an aralkyl halide to generate intermediates 2. In one embodiment, when Ra is not hydrogen, then the Ra and Rb groups are adequately chosen so that liberation of the carboxylic acid to generate 3 can take place selectively when Ra is H, 2 and 3 represent the same intermediate). For example, if Ra is a methyl or ethyl group, an Rb group can be a benzyl, t-butyl, 2-trimethylsilylethyl group or other groups that can be selectively removed under conditions where the ester group Ra would remain intact such as hydrogenolysis for the benzyl group, mild acid such as trifluoroacetic acid for the t-butyl group or a fluoride source for the 2-trimethylsilylethyl group such as a tetraalkyl ammonium fluoride (e.g. tetrabutyl ammonium fluoride).




An alternative route for the synthesis of this particular building block, shown in Scheme 2, involves the condensation of an acetic acid derivative 1 with an aldehyde or a ketone leading to the α,β-unsaturated ester intermediate 4. The esters 4 can be hydrogenated under conditions that are well-documented in the literature (for example, hydrogen atmosphere and palladium on carbon as a catalyst in a solvent such as ethanol) to generate the carboxylate esters 3.




Alternatively, if R44 in 4 is H (compound 5), 1,4-addition of an alkyl group can take place by reaction with a suitable carbon nucleophile (e.g. copper mediated reaction of alkyl lithium or alkyl Grignard reagents) to yield compounds 3 where R44 is alkyl (Scheme 3).




Scheme 4 shows an alternative route to precursors 5, involving the palladium catalyzed reaction of a vinylic halide 7 with an organometallic reagent such as an aryl boronic acid or an aryl stannane. These vinylic halides 7 (where Hal represents bromide or iodide), can be generated from the corresponding benzaldehydes and a halogenated Horner-Emmons reagent (RO)2P(O)CH(Hal)CO2Ra (Toke et al, Tetrahedron 51, 9167 (1995); Vanderwal et al, J. Am. Chem. Soc., 125 (18), 5393-5407 (2003)) in the presence of base or by the reaction of the same starting aldehyde with [Ph3P═C(IPh)CO2Ra](+)[BF4](−) in dichloromethane in the presence of a halide source such as tetra-n-butyl ammonium bromide or tetra-n-butyl ammonium iodide (Huang et al, J. Org. Chem. 67, 8261 (2002))




It is recognized that the carbon atom alpha to the central carbonyl group is an asymmetric center. The synthesis of compounds provided herein in enantiomerically pure form can be achieved by utilization of the methods described above if the starting material 8 exists in enantiomerically pure form. An optically pure precursor 8* or 8**, can be generated by resolution of racemic 8 or by use of synthetic methods that generate the asymmetric center in an enantioselective manner.
Resolution methods include the generation of a diastereomeric mixture of carboxylate salts with an optically active amine, which may be separated by fractional crystallization. Acidification of the individual diastereomeric salts and isolation of the carboxylic acid affords the individual enantiomers of the carboxylic acid (D. Kozma: ‘CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation’ CRC Press, 2001). Alternatively, diastereomeric mixtures of ester or amide derivatives may be prepared by condensation of the racemic carboxylic acid with an optically active alcohol or amine, respectively; these diastereomers may be separated by chromatographic methods and/or fractional crystallization. The pure enantiomers are then generated from the individual diastereomers by reconversion to the carboxylic acid, using methods that are well established in the literature (Scheme 5).




Methods that generate the chiral center in an enantioselective manner include, but are not limited to, the alkylation of precursors containing a chiral auxiliary Xc. This may generate two diastereomers, which may be separated by fractional crystallization or chromatography (Scheme 6). After the separation of the diastereomers, they can be converted into the enantiomerically pure acid 3 and its enantiomer 3* by known procedures and further elaborated into the compounds provided herein as described in the Examples below.




Asymmetric centers may be present in other positions of the molecule. As an example, substitution on a cyclohexenyl group generates a new chiral center in the compound of Example 1. This center can be established in an appropriately functionalized precursor prior to construction of the target molecule. A potential route to this chiral precursor involves the desymmetrization of a racemic ketone as illustrated in Scheme 7. The reaction of 4-t-butylcyclohexanone with a chiral amide base has been reported to generate the corresponding chiral enolate in an enantioselective manner [Busch-Petersen and Corey, Tetrahedron Letters 41, 6941 (2000), Lyapkalo et al, Synlett 1292 (2001)]. Conversion of the enolate into a trifluoromethanesulfonate or a nonafluorobutanesulfonate [Busch-Petersen and Corey, Tetrahedron Letters 41, 6941 (2000), Lyapkalo et al. Synlett 1292 (2001)], leads to a chiral precursor that may be used in subsequent steps (A specific enantiomer is shown below, but it should be understood that either enantiomer can be synthesized by modifications of this method). The precursor 9 so obtained can then be elaborated into the single enantiomer as described above.




In a related manner, the two enantiomers of a 4-substituted cyclohex-1-enyl system can be obtained through resolution of the corresponding racemic alkene. For example, (enantiospecific or enantioselective) reaction of an alkene 10 (wherein R50 and R51 are different groups) generates a mixture of diastereomers which upon separation provides 11 and 12. Regeneration of the alkene provides the two enantiomers 13 and 13* (Scheme 8).




In addition to the methods described above, optically pure compounds can be obtained from their racemic parent compounds through chromatographic methods that utilize a chiral stationary phase.
A method that can be used to synthesize compounds of formula I is exemplified below (Scheme 9). The carboxylic acids 8 are converted to the corresponding amides by methods known for amide bond formation reactions. As an example, generation of an acid chloride 14 from 8 takes place under standard conditions (e.g. thionyl chloride in toluene or oxalyl chloride and catalytic DMF in dichloromethane). Treatment of acid chloride 14 with amines or anilines generates the amides 15. Alternatively, amines can be directly coupled with the carboxylic acid 8 by use of an activating agent (for example, DCC or EDCI with or without a catalyst such as DMAP or HOBT) to directly generate the amides 15. When L is a halo group such as bromo or iodo, aryl amides 15 can be further functionalized through metal-mediated (e.g. Palladium) C—C bond coupling reactions to give further L-functionalized amides 15. Hydrolysis of the ester group of 15 (e.g. Rb═—CH3 or —C(CH3)3) results in a carboxylic acid 15 (wherein Rb═H), which can then be coupled with taurine derivatives using standard amide bond forming reactions to generate the targeted compounds 16.
The amide bond in the last step can also be formed by other reported methods known for amide bond formation, for example, reaction of an N-hydroxysuccinimidyl ester of 15 (Rb═O-succinimidyl) and taurine gives the target taurine amide derivative 16. Other activated esters (e.g. pentafluorophenyl esters) can also be used to effect the amide bond formation.




The following examples are provided so that this disclosure can be more fully understood. They should not be construed as limiting the disclosure in any way.
EXAMPLES
Biological Examples
Example A
Human Glucagon Receptor Affinity
Compounds provided herein are dissolved in a suitable solvent (e.g., dimethlysulfoxide) at a concentration of 10 mM and then diluted in buffer (e.g., 50 mM Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, and 0.2% BSA) to concentrations ranging from 1 nM to 100 μM. Compounds (20 μL/well) and [125I]glucagon at the final concentration of 0.125 nM (20 μl/well) (Perkin Elmer) are added to and mixed in wells of a 96-well plate (Costar, Corning) containing 120 μL of buffer. Next, an appropriate aliquot of a membrane preparation containing the human glucagon receptor (isolated from human liver samples or obtained from a recombinant cell line) is added to the wells. The binding mixtures are incubated for 2 hrs at room temperature. In the meantime, a MultiScreen 96-well filter plate (Millipore) is treated with 200 μL of the buffer, which is vacuumed through the filter just before the binding mixtures are transferred to the plate. At the end of incubation, binding mixtures are transferred to the wells of the MultiScreen 96-well filter plate and filtered through by applying vacuum. The plate is washed once with 200 μL per well of the buffer, and the filters are dried and counted using a gamma counter.
Compounds provided herein have been shown to have high affinity for the glucagon receptor. Examples of some compounds are provided in the Table below. The Table below displays the results of testing the compounds shown in the human glucagon receptor binding assay. Also shown are data from the human hepatocyte assay, oral bioavailability in the rat, and glucose lowering in the db/db mouse (see Examples below for assay descriptions). The column marked “stereo” indicates whether the tested compound tested was racemic or the R-isomer (rac=racemic).
















 





















stereo
Hu  IC50 (nM)
Hu Hepatocyte EC50 (nM)
OBAV in rat
% Glu lowering  in db/db mouse @ 30 mg/kg
























R
5
4
73
57


 










R
10
12
52
45


 










R
9
3
54
53


 










rac
18
27
—
—


 










R
6
31
62
33


 










R
8
36
69
29


 










rac
18
412
—
—


 










rac
10
194
—
45


 










rac
12
41
—
58


 










rac
15
132
—
—


 










rac
15
329
—
—


 










rac
11
560
—
—


 










rac
15
215
—
—


 










rac
11
30
—
20


 










rac
19
—
—
—


 










rac
18
60
—
—


 










rac
12
661
—
—


 










rac
10
—
—
—


 










rac
16
—
—
—


 










rac
8
116
—
—


 










rac
18
161
—
—


 










rac
12
30
—
—


 










rac
17
49
48
42


 










rac
16
—
—
—


 










rac
16
78
—
—


 










rac
15
137
—
—


 










rac
20
143
—
—


 










rac
13
62
—
39


 










rac
9
26
38
56


 










rac
14
32
—
—


 










rac
19
108
—
—


 










rac
15
30
—
28


 










rac
18
61
—
—


 










rac
12
241
—
—


 










rac
8
167
—
—


 










rac
12
—
—
—


 










rac
18
—
—
—


 










rac
16
—
—
25


 










rac
12
—
—
—


 










rac
18
—
—
—


 










rac
10
—
—
—


 










rac
14
—
—
—


 










rac
10
192
—
—


 










rac
17
65
—
30


 










rac
10
—
—
—









The compounds of Formula I provided herein which have been tested displace radiolabeled glucagon from the human glucagon receptor with an IC50 of <15 nM.
For certain compounds, the R-enantiomer displays up to 5-fold higher affinity for the human glucagon receptor than the S-enantiomer.
The Table below displays the relative potency in the human glucagon receptor binding assay of the R- vs. S-isomers of the compounds shown. (The stereocenter being changed is marked with an asterisk; the R-isomer is shown in the drawing.)
















 

















Hu 1050 (nM)












R
S





















14
61


 










6
20


 










7
19


 










11
37


 










10
43


 










10
49









The compounds in the above table display nanomolar affinity for the human glucagon receptor. For certain compounds, the R-enantiomer displays up to 5-fold higher affinity for the human glucagon receptor than the S-enantiomer.
Example B
Functional Antagonism in Hepatocytes from Various Species
Primary human, monkey, dog, rat, or mouse hepatocytes are seeded onto collagen-coated 24-well plates in Williams E medium (supplemented with 10% fetal bovine serum) and incubated at 37° C. overnight in M199 medium (supplemented with 15 mM glucose and 10 nM human insulin). The following day cells are washed twice with a glucose-free Kreb-bicarbonate buffer, pH 7.4, containing 0.1% BSA. Cells are then incubated at 37° C. with the aforementioned buffer containing 1 nM glucagon and varying concentrations of a glucagon antagonist (0-100 microM). Control wells without glucagon or antagonist are also included. After 1 hour, an aliquot of the medium is removed and analyzed for glucose content by means of the glucose oxidase method. The background glucose levels observed in the control wells are subtracted from the glucagon and antagonist containing wells. A graph of % glucose concentration vs drug concentration is plotted and an EC50 value for inhibition of glucose production generated using Sigmaplot software (SAS, Cary, N.C.). Alternatively, intracellular cAMP levels are measured using standard kits and EC50 values determined by plotting these levels against drug concentration. Antagonists of the glucagon receptor inhibit glucagon-induced cAMP production.
The R-enantiomer compounds of Formula I provided herein which have been tested show functional antagonism of glucose production in human hepatocytes with an EC50 of <40 nM.
The compounds disclosed herein display significant functional antagonism of glucose production in human hepatocytes. For certain compounds, the R-enantiomer displays up to 50-fold greater functional antagonism in human hepatocytes than the S-enantiomer.
The Table below displays the relative potency in the human hepatocyte functional assay of the R- vs. S-isomers of the compounds shown. (The stereocenter being changed is marked with an asterisk; the R-isomer is shown in the drawing.)
















 

















Hu cell EC50 (nM)












R
S





















37
1177


 










7
350


 










6
165


 










33
213









Example C
Glucose Lowering in Diabetic Animals
The effects of compounds provided herein on blood glucose levels are assessed in animal models of type 1 or 2 diabetes such as, but not limited to, the db/db mouse, the Zucker fatty (ZF) rat, the Zucker diabetic (ZDF) rat, the glucagon-challenged dog, the alloxan- or streptozotocin-treated mouse or rat, the NOD mouse or the BB rat.
Compounds are dissolved in an appropriate vehicle such as polyethylene glycol-400 or cyclodextrin and administered to animals at doses of 0.1 to 100 mg/kg either by intraperitoneal injection, intravenous injection, or oral gavage. Animal models used in this evaluation [e.g., the db/db mouse, the ZF rat, the ZDF rat, the glucagon-challenged (0.3-5 μg/kg) dog, the alloxan- or streptozotocin-treated mouse or rat, the NOD mouse, or BB rat] are either freely-feeding or fasted from 3 to 24 hours prior to compound administration. In some instances, animals may be subjected to a glucose tolerance test following compound administration by intravenous or oral administration of up to 2 g/kg of glucose. Blood glucose levels are assessed in blood samples obtained by tail bleed or by sampling an appropriate blood vessel by means of a syringe or catheter. Glucose is measured using a portable glucometer such as the OneTouch or HemoCue meters at regular time intervals for up to 24 hours. The extent of blood glucose lowering elicited by the compounds of Formula I or II is determined by comparison to those in control animals administered only the vehicle. The percentage of blood glucose lowering attained is calculated relative to blood glucose levels in vehicle-treated nondiabetic or non-glucagon-challenged control animals.
Example D
Glucose Lowering in db/db Mice
To assess the effects of compounds provided herein on blood glucose levels in the db/db mouse, an animal model of type 2 diabetes, compounds are dissolved in polyethylene glycol-400 and administered by oral gavage to db/db mice in the freely-feeding state at doses of 30 and/or 100 mg/kg. Blood glucose levels are assessed in blood samples obtained by tail bleed at baseline (prior to drug administration) and at regular time intervals over 24 hrs using a portable glucometer such as the OneTouch or HemoCue meters. The magnitude of blood glucose lowering elicited by the compounds provided herein is determined by comparison to those in db/db mice administered only the vehicle. The percentage glucose lowering is calculated by factoring in the blood glucose levels of vehicle-treated lean db/+ (heterozygote) mice, with 100% representing the normalization of blood glucose levels from the hyperglycemic state (vehicle-treated db/db mice) to the normoglycemic state (vehicle-treated db/+ mice).
The compounds disclosed herein which have been tested lowered blood glucose of db/db mice in the freely-feeding state. In particular, the percentage blood glucose lowering achieved ranged from 36 to 57% relative to lean control animals.
The compounds disclosed herein have pronounced antihyperglycemic activity in animal models of type 2 diabetes.
EXAMPLES
Chemical Synthetic Examples
Example 1
Sodium; 2-{4-[2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate
Step 1: 4-[2-(4-Bromophenyl)-2-carboxy-ethyl]-benzoic acid methyl ester




In a 3-neck flask, a solution of 4-bromophenyl acetic acid (26.91 g) in THF (485 mL) was cooled to <10° C. under a nitrogen atmosphere. A solution of LiHMDS in THF (263 mL, 1.0 M) was added dropwise, ensuring that the internal temperature remained at <10° C. After the addition was complete, the mixture was stirred at 0° C. for about 15 min. The cooling bath was the removed and the reaction mixture was allowed to warm up to 20° C.
The reaction mixture was then cooled to <−60° C. From an addition funnel, a solution of 4-bromomethyl methylbenzoate (29.53 g) in THF (270 mL) was added dropwise, ensuring that the temperature did not rise above −60° C. After the addition was complete, the mixture was stirred at −60° C. for about 15 min, and poured over 300 mL of cold 1M aqueous HCl (saturated with sodium chloride). The organic layer was washed with 1M aqueous HCl (saturated with sodium chloride). The combined aqueous layers were extracted with toluene (50 mL). The combined organic phases were then dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was recrystallized from toluene to yield the carboxylic acid as a white solid. HNMR (300 MHz, DMSO-d6): 12.54 (1H, broad s), 7.82 (2H, d, J=6.4 Hz), 7.49 (2H, d, J=6.7 Hz), 7.32 (2H, d, J=8.2 Hz), 7.26 (2H, d, J=8.5 Hz), 3.95 (1H, t, J=7.9 Hz), 3.81 (3H, s), 3.3 (1H, m, overlaps with residual HOD), 3.03 (1H, m)
Step 2: 4-{2-(4-Bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid methyl ester




To a solution of 4-[2-(4-Bromophenyl)-2-carboxy-ethyl]-benzoic acid methyl ester (step 1 above, 0.6 g) in THF: ethanol: water (6 mL: 3 mL: 1.5 mL) added 4-t-butyl-1-cyclohexenyl-boronic acid (0.5 g), PdCl2(P(o-tolyl)3)2, and sodium carbonate (0.7 g). The resulting mixture was heated at 125° C. for a 1 h period. The reaction was then cooled to room temperature, treated with an excess of aqueous HCl (1M) and the resulting heterogeneous mixture was filtered through a celite pad. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The crude residue was dissolved in toluene (25 mL), treated with thionyl chloride (0.26 mL) and heated at 100° C. for a 1 h period. The toluene was removed under reduced pressure. The resulting acid chloride was redissolved in toluene (15 mL), treated with 4-bromoaniline (0.3 g) and diisopropyl ethyl amine (0.3 mL), and heated at 100° C. for a 1 h period. After cooling to room temperature, the mixture was partitioned between ethyl acetate and 1M aqueous HCl. The organic layer was washed (water, saturated sodium chloride), dried over magnesium sulfate and concentrated under reduced pressure. The product obtained was carried to the next step without further purification.
Step 3: Sodium; 2-(4-{2-(4-bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonate




A solution of 4-{2-(4-Bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid methyl ester (Step 2, 0.8 g) in THF: methanol: water (8 mL: 6 mL: 2 mL) was treated with lithium hydroxide (0.4 g) and stirred at room temperature for 16 h. Added an excess of aqueous HCl (1M) and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride in water and dried over sodium sulfate. The solvents were then removed under reduced pressure. The residue obtained was dissolved in DMF (10 mL), and treated with EDCI (0.4 g), HOBt-H2O (0.32 g), taurine (0.26 g) and diisopropyl ethyl amine (0.71 mL). The reaction mixture was then stirred at room temperature for a 16 h period. The solvent was removed under reduced pressure. The residue was partitioned between ethyl acetate and 1M aqueous HCl. The organic phase was washed with saturated sodium chloride, and concentrated. The residue in methanol was treated with an excess of sodium hydroxide and loaded on top of a C-18 reverse phase flash chromatography column. The column was eluted with an acetonitrile—water gradient to afford the sodium salt of the sulfonate as a white solid. LCMS m/z: 665.6 [C34H38N2O5BrS]− Step 4:




To a solution sodium; 2-(4-{2-(4-bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonate (Step 3 above, 96 mg) in DME: ethanol: water (2 mL: 1 mL: 0.5 mL) added 4-t-butyl-1-cyclohexenyl-boronic acid (0.5 g), PdCl2(P(o-tolyl)3))2, and sodium carbonate (0.7 g). The resulting mixture was heated at 125° C. for a 1 h period. The reaction was then cooled to room temperature, treated with an excess of aqueous HCl (1M) and the resulting heterogeneous mixture was filtered through a celite pad. The mixture was partitioned (ethyl acetate/water). The organic phase was washed with saturated sodium chloride, and concentrated. The residue in methanol was treated with an excess of sodium hydroxide and loaded on top of a C-18 reverse phase flash chromatography column. The column was eluted with an acetonitrile—water gradient to afford the sodium salt of the sulfonate as a white solid. LC-MS m/z=711.6 [C44H44N2O5ClS]−
Example 2
Sodium-2-(4-{2-(4′-chloro-2′-methyl biphenyl-4-ylcarbamoyl)-2-[3-(4,4-dimethyl-cyclohexyl)-phenyl]-ethyl}-benzoylamino-ethane sulfonic acid
Step 1: 4-Benzyl-3-[2-(3-bromo-phenyl)-acetyl]-oxozolidin-2-one




To a solution of (3-bromo-phenyl)-acetic acid (5.0 g, 23.2 mmol) in CH2Cl2 (50 mL) at room temperature was added oxalyl chloride (5.86 g, 46.5 mmol). The reaction mixture was stirred at room temperature for overnight and the solvent was removed under reduced pressure. The residue was dried under vacuum for 3-4 h and used without further purification.
In a separate flask, to a stirred solution of R-(+)-4-benzyl-oxazolidinone (4.34 g, 24.5 mmol) in THF (30 mL) at −78° C. was added n-BuLi (26.7 mL, 26.7 mmol, 1.0 M solution in hexane). The reaction mixture was stirred for 1 h, at −78° C., then the crude acid chloride (5.2 g, 22.3 mmol) in THF was added dropwise. The mixture was stirred for 2 h at −78° C. and allowed to warm to rt and stirred for another hour (monitored by TLC). The reaction was quenched with saturated NH4Cl solution (100 mL) and stirred for 10 min. The reaction mixture was extracted with ethyl acetate (2×250 mL) and the combined organic layers were washed with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with EtOAc-hexanes (5-25%) to afford 4-benzyl-3-[2-(3-bromo-phenyl)-acetyl]-oxozolidin-2-one. 1H NMR (300 MHz, CDCl3): 7.51 (d, J=2.1 Hz. 1 H), 7.44 (dd, J=3.9,4.8 Hz, 1 H), 7.32-7.20 (m, 6 H), 7.15(d, J=3.9 Hz, 1 H), 4.60 (m, 1 H), 4.30 (m, 2 H), 4.26-4.19 (m, 3 H), 3.27 (dd, J=1.8, 8.1 Hz, 1 H), 2.78 (dd, J=5.4, 7.8 Hz, 1 H); TLC conditions: Uniplate silica gel, 250 microns; mobile phase=ethyl acetate/hexanes (5:1); Rf=0.6.
Step 2: 4-{3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-[4-(3-bromophenyl)-3-oxo-propyl}-benzoic acid tert-butyl ester




To a stirred solution of 4-benzyl-3-[2-(3-bromo-phenyl)-acetyl]-oxozolidin-2-one (4.02 g, 10.7 mmol) in anhydrous THF (50 mL) was added LiHMDS (16.5 mL, 16.5 mmol, 1.0 M solution in THF) at −78° C. The reaction mixture was stirred for 1.5 h at −78° C., and then tert-butyl-4-bromo methyl benzoate (3.75 g, 11.8 mmol, in THF 10 mL) was added dropwise, stirred for 2 h at −78° C. and then allowed to warm to rt for 1 h. After completion of the reaction quenched with saturated NH4Cl solution (100 mL) and stirred for 10 min. The reaction mixture was extracted with ethyl acetate (2×250 mL) and the organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was precipitated from minimum amount of EtOAc and hexane at room temperature to afford 4-{3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-[4-(3-bromophenyl)-3-oxo-propyl}-benzoic acid tert-butyl ester as a yellow solid 1H NMR (300 MHz, CDCl3): δ 7.75 (d, J=6.3 Hz, 2 H), 7.44 (d, J=2.1 Hz, 2 H), 7.26-7.04 (m, 9 H), 6.79 (dd, J=1.8, 5.7 Hz, 2 H), 5.29 (dd, J=2.1, 6.3 Hz, 1 H), 3.89 (s, 3 H), 3.83 (d, J=7.5 Hz, 1 H), 3.41 (dd, J=8.4, 13.8 Hz, 1 H), 3.05 (dd, J=7.2, 13.5 Hz, 1 H); TLC conditions: Uniplate silica gel, 250 microns; mobile phase=ethyl acetate-hexanes (1:4); Rf=0.45.
Step 3: 4-[2-carboxy-2-(3-bromo-phenyl)-ethyl]-benzoic acid tert-butyl ester (5)




To a stirred solution of 4-{3-(4-benzyl-2-oxo-oxazolidin-3-yl)-2-[4-(3-bromophenyl)-3-oxo-propyl}-benzoic acid tert-butyl ester (2.3 g, 3.7 mmol) in THF/H2O (20 mL) (3:1) at room temperature were added H2O2 (1.25 g, 37.0 mmol 35% in H2O) followed by LiOH (0.62 g, 14.8 mmol). The reaction mixture was stirred for 3 h, at room temperature and quenched with 0.1 N HCl. The reaction mixture was extracted with ethyl acetate (100 mL) and dried over MgSO4, filtered and concentrated under reduced pressure to afford the crude acid. This crude product was purified by column chromatography on silica gel, eluting with CH2Cl2/MeOH 2%-15% to afford 4-[2-carboxy-2-(3-bromo-phenyl)-ethyl]-benzoic acid tert-butyl ester (1.5 g, 75%). 1H NMR (300 MHz, DMSO-d6): δ 12.52 (s, 1 H), 7.77 (d, J=8.4 Hz, 2 H). 7.48 (d, J=8.7 Hz, 2 H), 7.28 (d, J=8.4 Hz, 2 H), 7.22 (d, J=8.4 Hz, 2 H). 3.93 (t, J=7.8 Hz, 1 H), 3.30 (dd, J=8.4, 13.8 Hz, 1 H), 3.0 (dd, J=8.1, 13.8 Hz, 1 H), 1.49 (s, 9 H), TLC conditions: Uniplate silica gel, 250 microns; mobile phase ═CH2Cl2/MeOH (10%); Rf=0.4. Chiral HPLC conditions: Kromasil 100-5-TBB chiral column 250×4.6 cm, (5% hexane/2-propanol to 30%), 35 min, flow rate 1 mL/min, RT=12.41 min (enantiomeric excess: >96%)
Step 4: 4′-Chloro-2′-methyl-biphenyl-4-amine




A mixture of 4-iodo-aniline (25.0 g, 114.1 mmol), 2-methyl-4-chlorophenyl-boronic acid (29.17 g, 171.1 mmol), PdCl2(P(o-tolyl)3)2 (11.66 g, 14.8 mmol), and Na2CO3 (60.49 g, 570.7 mmol) in DME/EtOH/H2O (100/50/25 mL) was heated 125° C. for 2 h. The reaction mixture was cooled to room temperature, filtered and washed with EtOAc (200 mL). The solvent was removed under reduced pressure. The crude mixture was extracted with ethyl acetate (500 mL) and the organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The resulting crude was purified by column chromatography on silica gel, eluting with 5-30% Hexane/EtOAc to afford 4′-Chloro-2′-methyl biphenyl-4-ylamine. 1H NMR (300 MHz, CDCl3): δ 7.24-7.08 (m, 5 H), 6.72 (d. J=7.2 Hz, 2 H), 3.70 (bs, 2 H), 2.27 (s, 3 H).
Step 5: 4-[2-(3-Bromo-phenyl)-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid tert-butyl ester




To a stirred suspension of 4-[2-carboxy-2-(3-bromo-phenyl)-ethyl]-benzoic acid tert-butyl ester (2.41 g, 5.94 mmol) in anhydrous CH2Cl2 (20 mL), was added oxalylchloride (1.0 mL, 11.8 mmol) at room temperature The reaction mixture was stirred for 14 h, concentrated under reduced pressure and azeotroped with CH2Cl2 (2×10 mL). The crude acid chloride (2.2 g, 1.61 mmol) was treated with 4-chloro-2-methyl biphenyl-4-ylamine (1.24 g, 5.71 mmol) and N,N-diispropylethylamine (2.53 mL, 15.5 mmol) in CH2Cl2 (25 mL) at 0° C. The reaction mixture was stirred for 14 h at room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with CH2Cl2-hexanes (30%-100%) to give 4-[2-(3-bromo-phenyl)-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid tert-butyl ester as a brownish solid (2.4 g, 88%). 1H NMR (500 MHz, CDCl3): δ 7.74 (d, J=14.0 Hz, 2 H), 7.58-7.49 (m, 4 H), 7.39-7.22 (m, 4 H), 7.24-7.19 (m, 3 H), 7.14 (d, J=14.0 Hz, 2 H), 4.03 (t, J=11.0 Hz, 1 H), 3.42 (dd, J=15.5, 22.5 Hz, 1 H), 3.03 (dd, J=11.0, 22.5 Hz, 1 H), 2.17 (s, 3 H), 1.49 (s, 9 H); Chiral HPLC conditions: Chiralcel OD-H T=23° C.; mobile phase=5-25% hexane/IPA; flow rate=1.0 mL/min; detection=254, 280, 220 nm retention time in min: 16.66 min (enantiomeric excess: 97.3%).
Step 6: 4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid tert-butyl ester




To 4-[2-(3-bromo-phenyl)-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid tert-butyl ester (1.2 g, 1.98 mmol) in DME (30 mL), was added 4,4-dimethyl-cyclo-hex-1-enyl-boronic acid (0.76 g. 4.96 mmol), PdCl2(P(o-tolyl)3)2 (202 mg, 0.25 mmol), and diisopropylethylamine (1.0 mL, 5.94 mmol). The resulting mixture was heated at 85° C. for 2 h, allowed to cool to room temperature and filtered. Partitioned the filtrate between EtOAc (20 mL) and water. The organic phase was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The resulting crude was purified by column chromatography on silica gel, eluting with CH2C17:hexanes (20%-100%) to afford 4-{4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid tert-butyl ester as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 10.14 (s, 1 H), 7.76 (d. J=8.5 Hz. 2 H). 7.57 (d, J=8.4 Hz, 2 H), 7.48 (s, 1 H), 7.35-7.20 (m, 9 H), 7.13 (d, J=8.5 Hz, 1 H), 6.06 (bt, 1 H), 4.03 (t, J=6.5 Hz, 1 H), 3.48 (dd, J=4.5, 13.5 Hz, 1 H). 3.04 (dd. J=6.5, 14.0 Hz, 1 H), 2.37-2.34 (m, 2 H), 2.23 (t, J=7.0 Hz, 1 H), 2.18 (s, 3 H), 1.57 (t, J=6.5 Hz, 2 H), 1.50 (s, 9 H), 1.47 (t, J=6.5 Hz, 1 H), 0.93 (s, 6 H).
Step 7: 4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid




To a stirred solution of 4-{4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid tert-butyl ester (0.82 g, 1.29 mmol) in CH2Cl2 (30 mL) at room temperature, added trifluoroacetic acid (2.5 ml), and conc HCl (1.0 mL) The reaction mixture was stirred overnight. The organic solvents were removed under reduced pressure. The residue was extracted with ethyl acetate (2×100 mL), dried over MgSO4 and concentrated o afford 4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid as a solid. 1H NMR (500 MHz, DMSO-d6): 10.05 (s, 1 H), 7.73 (d, J=8.0 Hz, 2 H), 7.48 (d, J=8.5 Hz, 2 H), 7.40 (s, 1 H), 7.29-7.05 (m, 10 H), 5.99 (s, 1 H), 3.96 (t, J=10.5 Hz. 1 H), 2.95 (dd, J=5.0, 14.0 Hz, 1 H), 2.20-2.18 (m, 1 H), 2.14 (t, J=7.5 Hz, 1 H), 2.09 (s, 3 H), 1.89 (bs, 2 H), 1.49 (t, J=7.5 Hz, 1 H), 1.39 (t, J=6.0 Hz, 2 H), 0.84 (s, 6 H). Chiral HPLC conditions: Chiralcel OD-H T=23° C.; mobile phase=10-30% hexane/IPA; flow rate=1.0 mL/min; detection=254, 280, 220 nm retention time in min: 19.81 min (enantiomeric excess: 70.8%)
Step 8: 4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohexyl)-phenyl]-ethyl}-benzoic acid




A stirred solution of 4-{4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid (0.62 g, 1.07 mmol) in ethyl acetate (30 mL) at room temperature, was added Pd/C (100 mg). The mixture was stirred under 1 atm of H2 (gas) at room temperature for 4 h. The catalyst was removed by filtration through a Celite plug and washed with ethyl acetate (2×50 mL). Concentration of the filtrate afforded 4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid as a solid. 1H NMR (300 MHz, DMSO-d6): 12.69 (s, 1 H), 10.06 (s, 1 H), 7.72 (d, J=7.0 Hz, 2 H), 7.49 (d, J=7.0 Hz, 2 H), 7.27-7.03 (m, 11 H), 3.94 (t, J=5.5 Hz, 1 H), 3.39 (t, J=11.5 Hz, 1 H), 2.93 (dd, J=5.5, 13.0 Hz, 1 H), 2.16 (t, J=7.0 Hz, 1 H), 2.09 (s, 3 H), 1.48-1.43 (m, 4 H), 1.24-1.19 (m, 2 H), 0.87 (s, 3 H), 0.84 (s, 3 H).
Step 9: Sodium-2-(4-{2-(4′-chloro-2′-methyl biphenyl-4-ylcarbamoyl)-2-[3-(4,4-dimethyl-cyclohexyl)-phenyl]-ethyl}-benzoylamino-ethane sulfonic acid




To a mixture of 4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoic acid (0.6 g, 1.03 mmol) and EDCI (290 mg, 1.55 mmol) in DMF (7 mL), added HOBt (230 mg, 1.55 mmol), diispropylethylamine (0.4 g, 2.06 mmol), and taurine (250 mg, 0.5 mmol). The resulting mixture was stirred for 14 h. The reaction solvent was removed under reduced pressure. The residue mixture was dissolved in 0.1 N NaHCO3 and acetonitrile, purified by column chromatography on a C-18 silica gel flash chromatography column, eluting with an acetonitrile-water gradient. Sodium-2-(4-{2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino-ethane sulfonic acid was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6): 9.98 (s, 1 H), 8.25 (bs, 1H), 7.50 (d, J=3.0 Hz, 2 H), 7.43 (d, J=5.5 Hz, 2 H), 7.19-6.98 (m, 11 H), 3.85 (bs, 1 H), 3.32-3.18 (m, 3 H), 2.83 (d, J=14.0 Hz, 2 H), 2.47 (bs, 2 H), 2.23 (bs, 1 H), 2.03 (5, 3 H), 1.42-1.15 (m, 6 H), 0.81 (s, 3 H), 0.78 (s, 3 H); LC-MS m/z=685 [C39H42N2O5S]+; Anal Calcd: (MF:C398H42N2O5SNa+3.3 H2O) Calcd: C:60.94; H:6.37; N:3.64. Found: C:60.82; H:6.08; N:3.57. Chiral HPLC conditions: Regis-Whelk-01-786615, (S,S)10/100 250×10 mm T=23° C.; mobile phase=100% ACN/(5% NH4CO3,+H2O) flow rate=1.0 mL/min; detection=254 nm retention time in min: 12.39 min (enantiomeric excess: 95.1%)
Example 3
Ammonium, 2-(S)-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methyl biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate
Step 1: [4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester




To a mixture of 4-bromophenyl ethyl acetate (780 mg), 4-t-butyl-cyclohexen-1-yl boronic acid (897 mg), PdCl2(P(o-tolyl)3)2 (254 mg) in THF:ethanol:water (8 mL:4 mL:2 mL), added sodium carbonate (1.377 g). The sealed flask was heated at 140° C. for a 5 min period. The heterogeneous mixture was treated with an excess of 1M aqueous hydrochloric acid and filtered through a celite pad. The organic solvents were removed under reduced pressure and the residue partitioned between ethyl acetate and water. The organic phase was washed with water and saturated aqueous sodium chloride, dried over magnesium sulfate and chromatographed on silica gel using an ethyl acetate/hexanes gradient. The product was obtained as a yellow oil.
1H NMR (500 MHz, CDCl3): 7.32 (d, J=8 Hz, 2 H), 7.20 (d, J=8 Hz, 2 H), 6.10 (m, 1 H), 4.12 (q, J=7 Hz, 2 H), 3.57 (5, 2 H), 2.52-2.32 (m, 2 H), 2.26-2.18 (m, 1 H), 2-1.9 (m, 2 H), 1.38-1.2 (m, 5 H), 0.91 (s, 9 H).
Step 2: [4-((1R,2R,4S)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-acetic acid ethyl ester and [4-((1R,2R,4R)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-acetic acid ethyl ester




A mixture of AD-mix-beta (6.512 g, J. Org. Chem. 57, 2768 (1992)) and methanesulfonamide (443 mg, 4.66 mmol) in tert-butanol (23 mL) and water (28 mL) was cooled to 2-4° C. To this mixture, [4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester (1.4 g, 4.66 mmol) in tert-Butanol (5 mL) was added slowly while making sure that the temperature remained in the 2-4° C. range. The mixture was stirred at the same temperature for a period of 4 days then quenched by adding sodium sulfite (1.5 g/mmol starting material) in water (20 mL). After allowing to warm to room temperature, the reaction mixture was stirred an additional 1 h before being partitioned between ethyl acetate and water. The organic phase was washed with brine then concentrated under reduced pressure to afford crude material which was purified by chromatography on silica gel, eluting with an ethyl acetate/hexanes gradient. Two products were obtained. In agreement with the report from Hamon et al (Tetrahedron 57, 9499 (2001)) they were assigned as follows:
First eluting product: [4-((1R,2R,4R)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-15 phenyl]-acetic acid ethyl ester 1H NMR (500 MHz, CDCl3): 7.46 (m, 2H), 7.28 (m, 2 H), 4.16 (q, J=7 Hz, 2H), 3.99 (m, 1H), 3.61 (s, 2 H), 2.59 (m, 1 H), 1.92 (m, 2 H), 1.64-1.46 (m, 5 H), 1.27 (t, J=7 Hz, 3H), 0.93 (s, 9 H).
Second eluting product: [4-((1R,2R,4S)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-acetic acid ethyl ester. 1H NMR (500 MHz, CDCl3): 7.51 (m, 2H), 7.30 (m, 2 H), 4.36 (m, 1H), 4.16 (q, J=7 Hz, 2H), 3.62 (s, 2 H), 2.74 (m, 1 H), 2.26 (m, 1H), 2.20 (m, 1H). 2.07 (m, 1H), 1.92 (m, 1 H), 1.75 (m, 1 H), 1.27 (t, J=7 Hz, 3H), 1.17 (m, 2H), 0.80 (s, 9 H).
Step 3:[4-(3R,6R,7R)-6-tert-Butyl-2-thioxo-tetrahydro-benzo[1,3]dioxol-3-yl)-phenyl]-acetic acid ethyl ester




A solution of [4-((1R,2R,4R)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-acetic acid ethyl ester (319 mg, 0.95 mmol) and thiocarbonyl diimidazole (309 mg, 1.91 mmol) in THF (15 mL) was refluxed under N, overnight. The reaction mixture was partitioned between ethyl acetate and brine, dried over Na2SO4 and concentrated under reduced pressure. Purification of the residue by chromatography on silica gel using an ethyl acetate/hexanes gradient afforded 297 mg of product.
1H NMR (500 MHz, CDCl3): 7.31 (m, 4 H), 4.97 (dd, J=9 Hz, J=7 Hz, 1 H), 4.13 (q, J=7 Hz, 2 H). 3.59 (s, 2 H), 2.53-2.48 (m, 1 H), 2.40-2.31 (m, 1 H), 1.90-1.72 (in. 2 H), 1.40-1.15 (m, 6 H), 0.91 (s, 9 H).
Step 4:[4-((R)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester




A solution of [4-((3aR,6S,7aR)-6-tert-Butyl-2-thioxo-tetrahydro-benzo[1,3]dioxol-3a-yl)-phenyl]-acetic acid ethyl ester (297 mg, 0.80 mmol) in triethylphosphite (3 mL) was slowly added to a solution of triethylphosphite (10 mL) heated to reflux—rate of addition was such that the reaction temperature exceeded 150. After refluxing overnight, the solvent was removed under vacuum and the crude reaction mixture was loaded on top of a silica column and eluted with an ethyl acetate/hexanes gradient to afford 145 mg of the title compound. 1H NMR (500 MHz, CDCl3): 7.32 (d, J=8 Hz, 2 H), 7.20 (d, J=8 Hz, 2 H), 6.10 (m, 1 H), 4.12 (q, J=7 Hz, 2 H), 3.57 (s, 2 H), 2.52-2.32 (m, 2 H), 2.26-2.18 (m, 1 H), 2-1.9 (m, 2 H), 1.38-1.2 (m, 5 H), 0.91 (s, 9 H).
Determination of the enantiomeric excess: A sample of the product was treated with an excess of aqueous 1M NaOH:ethanol:water (1:2:3 ratio by volume) and heated at 125° C. for a 5 min period. The organic solvents were removed under reduced pressure and the residue was partitioned between ethyl acetate and 1M aqueous HCl. The organic phase was washed with water and a saturated sodium chloride solution and then dried over magnesium sulfate. The enantiomeric excess of the product was determined to be >99% by chiral HPLC utilizing a Chiral Technologies ChiralPak AD-H 250 mm×4.6 mm column, eluting at a 1.0 mL/min flow rate using a mixture of hexanes: isopropanol: methanesulfonic acid in a 95:5:0.1 ratio. The sample was dissolved at 1 mg/mL in ethanol prior to injection. The retention time observed was 6.2 min.
Step 5: 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-ethoxylcarbonyl-ethyl}-benzoic acid tert-butyl ester




To [4-((R)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester (95 mg. 0.32 mmol) in anhydrous THF (5 mL), chilled to −78° C. was added 380 uL (0.38 mmol) of 1M lithium hexamethyldisilazane in THF. The resulting solution was stirred for 1 hr before 4-Bromomethylbenzoic acid tert-butyl ester (94 mg, 0.35 mmol) was added. The reaction mixture was allowed to warm to rt overnight then quenched with saturated NH4Cl solution. After partitioning between ethyl acetate and brine the organic portion was dried over Na2SO4 and concentrated under reduced pressure. Purification of the crude by prep TLC (Analtech, 2 mm silica plates) using an hexane/ethyl acetate (10:1) gave 63 mg of product. 1H NMR (500 MHz, CDCl3): 7.83 (d, J=8.5 Hz, 2 H), 7.31 (d, J=8.5 Hz, 2 H), 7.20 (d, J=8 Hz, 2 H), 7.14 (d, J=8 Hz, 2 H), 6.12 (m, 1H), 4.03 (q, J=7 Hz 2 H), 3.80 (t, J=8.5 Hz, 1H), 3.41 (dd, =14, 8.5 Hz, 1 H), 3.03 (dd, J=14, 7 Hz, 1 H), 2.56-2.4 (m, 2H), 2.35-2.2 (m, 1H). 2.05-1.9 (m, 2H), 1.56 (s, 9 H), 1.36-1.2 (m, 2 H), 1.11 (t, J=7 Hz, 3 H), 0.89 (s, 9H).
Step 6: 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoic acid




To 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-ethoxylcarbonyl-ethyl}-benzoic acid tert-butyl ester (63 mg, 0.13 mmol) dissolved in a solution of THF (3 mL), MeOH (1 mL) and water (1 mL) was added lithium hydroxide (27 mg, 0.64 mmol). The solution was stirred at rt for 5 hrs then neutralized with 3M KH2PO4 and extracted with ethyl acetate. The organic portion was washed with brine, dried over Na2SO4 and concentrated under vacuum to afford crude material which was used without further purification.
Step 7: 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid tert-butyl ester




To 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoic acid tert-butyl ester (697 mg, 1.51 mmol) in anhydrous dichloromethane (30 mL) was added oxalyl chloride (650 uL, 7.53 mmol) and 3 drops of DMF. The resulting solution was stirred at rt for 1 hr before being concentrated under vacuum. The residue was co-evaporated with toluene (1×5 mL) then dissolved in toluene again (20 mL). To the mixture was added 4-chloro-2-methylbiphenyl-4-ylamine (361 mg, 1.66 mmol) and DIPEA (1.3 mL, 7.53 mmol). The resulting mixture was refluxed for 90 min, diluted with ethyl acetate and washed with saturated NaHCO3. The organic portion was dried over Na2SO4 and concentrated under vacuum to afford crude material which was crystallized from MeOH to afford a white solid (590 mg). Removing the solvent and purification of the residue by chromatography on silica gel using an ethyl acetate/hexanes gradient afforded an additional 137 mg of product. 1H NMR (500 MHz, DMSO-d6): 10.16 (s, 1H), 7.78 (d, J=8 Hz, 2 H), 7.59 (d, J=8.5 Hz, 2 H), 7.4-7.32 (m, 7 H), 7.21 (d, J=8 Hz, 2 H), 7.15 (d, J=8 Hz, 2 H), 6.14 (m, 1H), 4.03 (dd, J=9, 7 Hz, 1 H), 3.48 (dd, J=13.5, 9.5 Hz, 1 H), 3.41 (dd, J=13.5, 6.5 Hz, 1 H), 2.4-2.3 (m, 1H), 2.2-2.15 (m, 4H), 1.96-1.9 (m, 2H), 1.52 (s, 9 H), 1.32-1.2 (m, 2 H), 0.89 (s, 9H).
Step 8: 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid




To 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid tert-butyl ester (727 mg, 1.1 mmol) was added 4N HCl/dioxane (30 mL), water (5 mL) and conc. HCl (1 mL). The resulting solution was stirred at rt overnight. The excess solvent was removed under vacuum and the residue co-evaporated with toluene to afford the desired crude product as a gummy oil. The crude material was used the next step without further purification.
Step 9: Sodium, 2-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate




To crude 4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methyl

biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid (assumed 1.1 mmol) in DMF (25 mL) was added EDC (316 mg, 1.65 mmol), HOBt (252 mg, 1.65 mmol), taurine (206 mg, 1.65 mmol) and DIPEA (550 uL, 3.29 mmol). The resulting mixture was stirred at rt overnight. The excess solvent was removed under vacuum and to the oily residue was added excess 1N HCl. After decanting off the excess 1N HCl, the residue was dissolved in acetonitirile/MeOH, made basic with saturated NaHCO3 and purified using reverse phase flash chromatography and eluting with an acetonitrile/water gradient. The sodium salt was obtained as a white solid. LCMS: 711.6 [M-H]−
Step 10: Ammonium, 2-(S)-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate




Sodium, 2-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate (obtained on Step 8 above), was dissolved in DMF. The product was subjected to preparative HPLC on a Pirkle Covalent (S,S)-Whelk-01 column (250 mm×10 mm), eluting at 10 mL/min with a gradient of acetonitrile and 5 mM ammonium bicarbonate. The title compound was the first of the two diastereomers to elute. Conditions for the determination of the enantiomeric excess by HPLC: Regis-Whelk-01-786615, (S,S)10/100 250×10 mm T=23° C.; mobile phase=100% ACN/(5% Phosphate pH=7.0, ACN) flow rate=1.0 mL/min; detection=254 nm. Retention time in min: 18.22 min (enantiomeric excess: 99.1%). 1H NMR (500 MHz, CD3OD): 7.71 (d, J=8.5 Hz, 2 H), 7.51 (d, J=8.5 Hz, 2 H), 7.4-7.25 (m, 7 H), 7.20 (d, J=8.5 Hz, 2 H), 7.13 (d, J=8.5 Hz, 2 H), 3.97 (dd, J=9.5, 6.5 Hz, 1 H), 3.78 (t, J=6.5 Hz, 2H), 3.52 (dd, J=13.5, 9.5 Hz, 1 H), 3.10 (dd, J=13.5, 6.5 Hz, 1 H), 3.07 (t, J=6.5 Hz, 2H), 2.5-2.3 (m, 2H), 2.3-2.20 (m, 4H), 2.1-1.95 (m, 2H), 1.38 (s, 10 H), 0.94 (s, 9H).
Example 4
Ammonium, 2-(R)-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate




The title compound was the second compound eluting from the chiral chromatography reported in Example 3, Step 9. Conditions for the determination of the enantiomeric excess by HPLC: Regis-Whelk-01-786615, (S,S)10/100 250×10 mm T=23° C.; mobile phase=100% ACN/(5% Phosphate pH=7.0, ACN) flow rate=1.0 mL/min; detection
=254 nm. Retention time in min: 23.55 min (enantiomeric excess: 99.5%). 1H NMR (500 MHz, CD3OD): 7.71 (d, J=8.5 Hz, 2 H). 7.51 (d, J=8.5 Hz, 2 H). 7.4-7.25 (m, 7 H), 7.20 (d, J=8.5 Hz, 2 H), 7.13 (d, J=8.5 Hz, 2 H), 3.97 (dd, J=9.5, 6.5 Hz, 1 H), 3.78 (t, J=6.5 Hz, 2H), 3.52 (dd, J=13.5, 9.5 Hz, 1 H), 3.10 (dd, J=13.5, 6.5 Hz, 1 H), 3.07 (t, J=6.5 Hz, 2H), 2.5-2.3 (m, 2H), 2.3-2.20 (m, 4H), 2.1-1.95 (m, 2H), 1.38 (s, 10 H), 0.94 (s, 9H).
Example 5
Ammonium, 2-(S)-{4-[2-[4-(4-(S)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methyl biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate
Step 1: [4-((S)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester




Utilizing the second eluting diol in Example 3, Step 2, the chiral alkene shown above was obtained after utilizing the methods described in Example 3, Steps 3 and 4. 1H NMR (500 MHz, CDCl3): 7.32 (d, J=8 Hz, 2 H), 7.20 (d, J=8 Hz, 2 H), 6.10 (m, 1 H), 4.12 (q, J=7 Hz, 2 H), 3.57 (s, 2 H), 2.52-2.32 (m, 2 H), 2.26-2.18 (m, 1 H), 2-1.9 (m, 2 H), 1.38-1.2 (m, 5 H), 0.91 (s, 9 H).
Determination of the enantiomeric excess: A sample of the product was treated with an excess of aqueous 1M NaOH:ethanol:water (1:2:3 ratio by volume) and heated at 125° C. for a 5 min period. The organic solvents were removed under reduced pressure and the residue was partitioned between ethyl acteate and 1M aqueous HCl. The organic phase was washed with water and a saturated sodium chloride solution and then dried over magnesium sulfate. The enantiomeric excess of the product was determined to be >99% by chiral HPLC utilizing a Chiral Technologies ChiralPak AD-H 250 mm×4.6 mm column, eluting at a 1.0 mL/min flow rate using a mixture of hexanes: isopropanol: methane sulfonic acid in a 95:5:0.1 ratio. The sample was dissolved at 1 mg/mL in ethanol prior to injection. The retention time observed was 5.6 min.
Step 2: Ammonium, 2-(S)-{4-[2-[4-(4-(S)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate




[4-((S)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester (Step 1, above), was utilized to yield the title compound through the sequence illustrated in Example 3, Steps 5-10. The title compound eluted first among the two possible diastereomers after preparative HPLC on a Pirkle Covalent (S,S)-Whelk-01 column (250 mm×10 mm), eluting at 10 mL/min with a gradient of acetonitrile and 5 mM ammonium bicarbonate.
Conditions for the determination of the enantiomeric excess by HPLC: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 65% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM potassium phosphate monobasic (pH 6.8). The product was detected by UV at 254 nm. Retention time in min: 18.23 min (enantiomeric excess: >99.5%). 1H NMR (500 MHz, CD3OD): 7.71 (d, J=8.5 Hz, 2 H), 7.51 (d, J=8.5 Hz, 2 H), 7.4-7.25 (m, 7 H), 7.20 (d, J=8.5 Hz, 2 H), 7.13 (d, J=8.5 Hz, 2 H), 3.97 (dd, J=9.5, 6.5 Hz, 1 H), 3.78 (t, J=6.5 Hz, 2H), 3.52 (dd, J=13.5, 9.5 Hz, 1 H), 3.10 (dd, J=13.5, 6.5 Hz, 1 H), 3.07 (t, J=6.5 Hz, 2H), 2.5-2.3 (m, 2H), 2.3-2.20 (m, 4H), 2.1-1.95 (m, 2H), 1.38 (s, 10 H), 0.94 (s, 9H).
Example 6
Ammonium, 2-(R)-{4-[2-[4-(4-(S)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate




The title compound was the second compound eluting from the chiral chromatography reported in Example 5, Step 2. The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615. (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 65% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM potassium phosphate monobasic (pH 6.8). The product was detected by UV at 254 nm. Retention time in min:23.41 min (enantiomeric excess: >99.5%). 1H NMR (500 MHz, CD3OD): 7.71 (d, J=8.5 Hz, 2 H), 7.51 (d, J=8.5 Hz, 2 H), 7.4-7.25 (m, 7 H), 7.20 (d, J=8.5 Hz, 2 H), 7.13 (d, J=8.5 Hz, 2 H), 3.97 (dd, J=9.5, 6.5 Hz, 1 H), 3.78 (t, J=6.5 Hz, 2H)), 3.52 (dd, J=13.5, 9.5 Hz, 1 H), 3.10 (dd, J=13.5, 6.5 Hz, 1 H), 3.07 (t, J=6.5 Hz, 2H), 2.5-2.3 (m, 2H), 2.3-2.20 (m, 4H), 2.1-1.95 (m, 2H), 1.38 (s, 10 H), 0.94 (s, 9H).
Using the methods described in Examples 1-6, the following compounds were synthesized.
Example 7
Sodium-2-(R)-4-[2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(4-(3,3-dimethyl-but-1-enyl)-phenyl]-ethyl}-benzoylamino ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): 10.14 (s, 1 H). 8.39 (t, J=9.8 Hz, 1 H), 7.61 (d, J=8.7 Hz, 2 H), 7.55 (d, J=8.7 Hz, 2 H), 7.33-7.12 (m, 11 H), 6.25 (dd, J=16.2, 3.6 Hz, 2 H), 3.98 (t, J=8.4 Hz, 1 H), 3.49-3.38 (m, 3 H), 3.0 (dd, J=6.3, 7.8 Hz, 1 H), 2.61 (t, J=7.8 Hz, 2 H), 2.17 (s, 3 H), 1.06 (s, 3 H); LC-MS m/z=657 [C37H38N2O5SClNa+H]+; HPLC conditions: Waters Atlantis C-18 OBD 4.6×150 mm; mobile phase=ACN/(H2O:0.1TFA) flow rate=1.0 mL/min; detection=UV@254, 220 nm retention time in min: 12.33; Anal Calcd: (MF: C37H38N7O5SClNa+1.2H2O) Calcd: C:63.23, H:5.79, N:3.99. Found: C:63.02, H:5.89, N:4.15.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 65% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM potassium phosphate monobasic (pH 6.8). The product was detected by UV at 254 nm. Retention time in min: 14.08 min (enantiomeric excess: >97.06%)
Example 8
Sodium-2-(S)-4-[2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-(4-(3,3-dimethyl-but-1-enyl)-phenyl]-ethyl}-benzoylamino ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): 10.14 (s, 1 H), 8.39 (t, J=9.8 Hz, 1 H), 7.61 (d, J=8.7 Hz, 2 H), 7.56 (d, J=6.6 Hz, 2 H), 7.33-7.12 (m, 11 H), 6.26 (dd, J=16.2, 3.6 Hz, 2 H), 3.98 (t, J=9.9 Hz, 1 H), 3.49-3.39 (m, 3 H), 3.0 (dd, J=6.3, 7.8 Hz, 1 H), 2.61 (t, J=7.2 Hz, 2 H), 2.17 (s, 3 H), 1.05 (s, 3 H); LC-MS m/z=657 [C37H38N2O5SClNa+H]+; HPLC conditions: Waters Atlantis C-18 OBD 4.6×150 mm; mobile phase=ACN/(H2O:0.1TFA) flow rate=1.0 mL/min; detection=UV@254, 220 nm retention time in min: 12.33; Anal Calcd: (MF: C37H38N2O5SClNa+1.5H2O) Calcd: C:62.75, H:5.83, N:3.96. Found: C:62.65, H:5.81, N:4.13.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 65% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM potassium phosphate monobasic (pH 6.8). The product was detected by UV at 254 nm. Retention time in min: 17.69 min (enantiomeric excess: >97.4%).
Example 9
2-(4-{(R)-2-(4′-Chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[4-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 10.14 (s, 1 H), 8.40 (t, J=2.8 Hz, 1 H), 7.64 (d, J=8.4 Hz, 2 H). 7.57 (d, J=8.9 Hz. 2 H), 7.37-7.14 (m, 11 H), 6.07 (bs, 1 H), 4.0 (t, J=5.5 Hz, 1 H), 3.47-3.45 (m, 3 H), 3.0-2.85 (m, 1 H), 2.62 (t, J=4.2 Hz, 2 H), 2.35 (t, J=1.2 Hz, 2 H), 2.18 (s, 3 H), 1.95 (t, J=2.8 Hz, 2 H), 1.46 (t, J=2.8 Hz, 2 H), 0.91 (s, 6H). LC-MS m/z=685 [C39H40N2O4SCl+H]+.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min: 17.92 min (enantiomeric excess: 99.5%).
Example 10
2-(4-{(S)-2-(4′-Chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[4-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 10.15 (s, 1 H), 8.40 (t, J=2.8 Hz, 1 H), 7.64 (d, J=8.4 Hz, 2 H), 7.57 (d, J=8.9 Hz, 2 H), 7.37-7.11 (m, 11 H), 6.07 (bs, 1 H), 4.0 (t, J=5.5 Hz, 1 H), 3.47-3.45 (m, 3 H), 3.0-2.85 (m, 1 H), 2.62 (t, J=8.4 Hz, 2 H), 2.35 (t, J=1.2 Hz, 2 H), 2.18 (s, 3 H), 1.95 (t, J=2.8 Hz, 2 H), 1.46 (t, J=2.8 Hz, 2 H), 0.91 (s, 6H). LC-MS m/z=684 [C39H40N2O4SCl]+.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min: 13.87 min (enantiomeric excess: 97.6%).
Example 11
Sodium-2-[4-(S)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(4′-chloro-2′-methyl-phenyl-carbamoyl)-ethyl}-benzoylamino-ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 7.70 (d, J=6.0 Hz, 2 H), 7.57-7.47 (m, 11 H), 7.33 (d, J=8.4 Hz, 2 H), 7.21-7.13 (m, 4 H), 4.0 (t, J=6.0 Hz, 1 H), 3.74 (t, J=13.5 Hz, 2 H), 3.53 (dd, J=6.3, 3.0 Hz, 1 H), 3.15 (dd, J=6.6, 2.8 Hz, 1 H), 3.06 (t, J=6.9 Hz, 2 H), 2.20 (s, 3 H), 1.34 (s, 9 H). LC-MS m/z=709 [C35H36N2O5SClNa]+.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min: 23.87 min (enantiomeric excess: 99.5%).
Example 12
Sodium-2-[4-(R)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(4′-chloro-2′-methyl-phenyl-carbamoyl)-ethyl}-benzoylamino-ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 7.70 (d, J=6.0 Hz, 2 H), 7.57-7.47 (m, 11 H), 7.33 (d, J=8.4 Hz, 2 H), 7.21-7.13 (m, 4 H), 4.0 (t, J=6.0 Hz, 1 H), 3.74 (t, J=13.5 Hz, 2 H), 3.53 (dd, J=6.3, 3.0 Hz, 1 H), 3.15 (dd, J=6.6, 2.8 Hz, 1 H), 3.06 (t, J=6.9 Hz, 2 H), 2.20 (s, 3 H), 1.34 (s, 9 H). LC-MS m/z=707 [C35H36N2O5SClNa-2]+.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min: 23.86 min (enantiomeric excess: >96.9%).
Example 13
Sodium-2-[4-(R)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(2′′,4′,6′-trimethyl-biphenyl-4-ylcarbamoyl)-ethyl}-benzoylamino-ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 7.65 (d, J=7.5 Hz, 2 H), 7.51 (d, J=8.0 Hz, 2 H), 7.48-7.43 (m, 11 H), 7.38 (d, J=8.4 Hz, 2 H). 7.28 (d, J=8.4 Hz, 2 H). 6.92 (d, J=8.5 Hz, 2 H), 6.80 (s, 1 H), 3.97 (t, J=6.5 Hz, 1 H), 3.71 (t, J=7.0 Hz, 2 H), 3.50 (dd, J=9.0, 13.5 Hz. 1 H), 3.08 (dd, J=6.6, 13.5 Hz, 1 H), 2.99 (t, J=6.5 Hz, 2 H), 2.19 (s, 3 H), 1.86 (s, 6 H), 1.28 (s, 9 H). LC-MS m/z=702 [C43H45N2O5SCl]+; Anal Calcd: (MF: C43H45N2O5SClNa+1.2H2O+0.1 NaHCO3) Calcd: C:68.57; H:6.34; N:3.71. Found: C:68.24; H:5.98; N:3.58.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min: 28.908 min (enantiomeric excess: >98.79%).
Example 14
Ammonium-2-(R)-(4-{2-[4′-tert-butyl-biphenyl-4-yl)-2-[3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl-4-ylcarbamoyl]-ethyl}-benzoylamino-ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 10.07 (s, 1 H), 8.39 (t, J=5.0 Hz, 1 H), 7.64 (d, J=8.0 Hz. 2 H), 7.57 (d, J=8.0 Hz. 2 H), 7.54 (d, J=7.5 Hz. 2 H), 7.50-7.44 (m, 6 H), 7.32-7.29 (m, 4 H), 6.0 (bs, 1 H), 4.05 (dd, J=6.0, 8.5 Hz, 1 H), 3.48 (dd, J=7.0, 12.5 Hz, 3 H), 3.05 (dd, J=6.0, 13.5 Hz, 1 H), 2.63 (t, J=7.0 Hz, 2 H), 2.18 (bs, 3 H), 1.94 (bs, 2 H), 1.44 (t, J=12.0 Hz, 2 H), 1.29 (s, 9 H), 0.90 (s, 6 H). LC-MS m/z=692 [C42H48N2O5S]+; Anal Calcd: (MF: C42H48N2O5S+2.8H2O+0.6 NH3) Calcd: C:66.94, H:7.41, N:4.83 Found: C:66.90, H:7.20, N:4.44.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min:13.87 min (enantiomeric excess: >99.0%).
Example 15
2-(4-[2-(4-Benzooxazol-2-yl-phenylcarbamoyl)-2-4-(1R,4R)-1,7,7-trimethyl-bicyclo[2,2,1]hept-2-en-2-yl)-phenyl]ethyl-benzoylamino)-ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 8.13 (d, J=8.5 Hz, 2 H), 7.74-7.65 (m, 4 H), 7.40-7.38 (m, 6 H), 7.33 (d. J=8.0 Hz, 2 H), 7.21 (d, J=8.0 Hz. 2 H), 5.97 (d, J=3.5 Hz, 2 H), 4.05 (t, J=6.0, Hz, 1 H), 3.77 (t, J=6.5, Hz, 1 H), 3.56-3.46 (m. 1 H), 3.14 (dd, J=6.0, 14.0 Hz, 1 H), 3.06 (t, J=6.5 Hz. 2 H). 2.37 (t, J=3.5 Hz, 2 H), 1.99-1.93 (m, 2 H), 1.72-1.67 (m, 2 H), 1.33-1.29 (m, 2 H), 1.15-1.09 (m, 4 H), 0.90 (s, 3 H), 0.88 (s, 3 H). LC-MS m/z=703 [C41H41N3O6S]+. HPLC conditions: 250×10 mm T=23° C.; mobile phase=100% ACN/(H2O/CAN+0.1 TFA) flow rate=1.0 mL/min; detection=254, 280, 220 nm retention time in min: 7.39 min (95.0%).
Example 16
Sodium-2-[4-(R)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(4′-chloro-3′-methyl-phenyl-carbamoyl)-ethyl}-benzoylamino-ethane sulfonic acid




1H NMR (500 MHz, DMSO-d6): δ 7.71 (d, J=8.0 Hz, 2 H), 7.59-7.44 (m, 13 H). 7.36-7.33 (m, 4 H), 4.0 (dd, J=6.0, 9.0 Hz, 1 H), 3.77 (t, J=7.0 Hz, 2 H), 3.56 (dd, J=9.0, 13.0 Hz, 1 H), 3.14 (dd, J=6.0, 13.0 Hz, 1 H), 3.06 (t, J=6.5 Hz, 2 H), 2.41 (s, 3 H), 1.34 (s, 9 H). LC-MS m/z=731 [C35H36N2O5SClNa]+.
Example 17
Ammonium-2-(R)-(4-{2-[4′-tert-butyl-biphenyl-4-yl)-2-(4-methyl-benzooxazol-2-yl)phenylcarbamoyl]-ethyl}-benzoylamino)-ethane sulfonic acid
Step 1: 4-(4-Methyl-benzooxazol-2-yl)-phenylamine




To a suspension of 4-amino-benzoic acid (2.0 g, 14.5 mmol) in PPA ( ̃85 g) was added 2-amino-m-cresol (1.8 g, 15.3 mmol). The reaction was heated to 160° C. for 14 h, then carefully quenched in aqueous sodium carbonate ( ̃50% saturated) at room temperature. Ethyl acetate was added, and the organic layer was washed with water and brine, and dried over sodium sulfate. The crude product was obtained was subsequently purified by flash column chromatography on silica gel eluting with ethyl acetate in hexanes to afford the desired product, 4-(4-methyl-benzooxazol-2-yl)-phenylamine as a light pink solid, 1.8 g (56%). LC-MS m/z=225 [C14H12N2O+H]+.
Step 2: The Methods Described in Examples 1-6 were Used to Generate the Title Compound from 4-(4-Methyl-benzooxazol-2-yl)-phenylamine




1H NMR (500 MHz, DMSO-d6): δ 10.48 (s, 1 H), 8.41 (t, J=3.0 Hz, 1 H), 8.12 (d, J=5.4 Hz, 2 H), 7.77 (d, J=5.1 Hz, 2 H), 7.67-7.46 (m, 11 H), 7.34 (d, J=5.1 Hz, 1 H), 7.25 (dd, J=4.5 Hz, 1 H), 7.17 (d, J=4.8 Hz, 1 H), 4.13 (t, J=6.0 Hz, 1 H), 3.53-3.41 (m, 3 H), 3.11 (dd, J=3.0, 6.5 Hz, 1 H), 2.62 (t, J=3.9 Hz, 2 H), 2.55 (s, 3 H), 1.29 (s, 9 H); LC-MS m/z=717[C42H41N3O6S]+.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min: 26.69 min (enantiomeric excess: >99.5%).
Example 18
Ammonium-2-(S)-(4-{2-[4′-tert-butyl-biphenyl-4-yl)-2-(4-methyl-benzooxazol-2-yl)phenylcarbamoyl]-ethyl}-benzoylamino)-ethanesulfonate




This compound was generated using the methods described in Example 19. 1H NMR (500 MHz, DMSO-d6): δ 10.48 (s, 1 H), 8.41 (t, J=3.3 Hz, 1 H), 8.11 (d, J=5.1 Hz, 2 H), 7.78 (d, J=2.1 Hz, 2 H), 7.67-7.46 (m, 11 H), 7.34 (d, J=5.1 Hz, 1 H), 7.25 (dd, J=4.8 Hz, 1 H), 7.19 (d, J=4.8 Hz, 1 H), 4.13 (t, J=6.0 Hz, 1 H), 3.51-3.37 (m. 3 H), 3.09 (dd, J=3.9, 4.5 Hz, 1 H), 2.62 (t, J=1.2 Hz, 2 H), 2.60 (s, 3 H), 1.29 (s, 9 H); LC-MS m/z=717[C47H41N3O6S]+.
Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mL concentration. It was injected into an HPLC system equipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with a gradient of two solvents A and B for a 30 min period. The composition of the gradient ranged from 40% A to 70% A over this period (with the balance of the solvent being B). Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrile and 95% water containing 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO2). The product was detected by UV at 254 nm. Retention time in min: 26.48 min (enantiomeric excess: >99.8%).
Example 19
2-{4-[(R)-2-[4-(4-(cis)-tert-Butylcyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonic acid




Step 1: 4-Chloromethyl benzoic acid tert-butyl ester




Oxalyl chloride (101 mL) was added dropwise over a 30 min period to a slurry of 4-chloromethyl benzoic acid (181.8 g) in dichloromethane (1.2 L) containing 5 mL of DMF. After the addition was complete the reaction mixture was stirred at room temperature for 24 h, concentrated under reduced pressure and then co-evaporated with toluene. To the residue was added 908 mL of MTBE and the mixture was cooled to −5° C. A solution of potassium tert-butoxide in THF (1.0 M, 1172 mL) was added dropwise ensuring that the internal temperature remained below 10° C. After the addition was complete, the reaction mixture was stirred for an additional 1 hour then treated with 500 mL of saturated sodium bicarbonate solution. After stirring 5 minutes, then settling, the organic phase was separated, washed with saturated sodium chloride solution and dried over magnesium sulfate. Concentration yielded 241.7 g (86% yield) as a dark oil. HNMR: CDCl3, 1.59 ppm (s, 9H), 4.61 (s, 2H), 7.45 (d, 2H), 7.99 (d, 2H)
Step 2: 4-Iodomethyl benzoic acid tert-butyl ester




Sodium iodide (229.2 g) was added to a solution of 4-chloromethyl benzoic acid tert-butyl ester (315.2 g) in acetone (1.5 L). The reaction mixture was heated to reflux for about 2 h and then allowed to cool to room temperature. The precipitate was removed by filtration and the filtrate concentrated under reduced pressure. The residue was partitioned between water (500 mL) and MTBE (1500 mL). The organic phase was washed with saturated sodium bicarbonate and dried over magnesium sulfate. Concentration under reduced pressure afforded 442.2 g (97% yield) dark oil. HNMR: CDCl3, 1.59 ppm (s, 9H), 4.47 (s, 2H), 7.42 (d, 2H), 7.91 (d, 2H)




Step 3: 4-Bromophenyl acetic acid methyl ester
Sulfuric acid (56.5 mL) was very slowly added to a solution of 206.6 g of 4-bromophenyl acetic acid in methanol (800 mL). After completion of the addition, the mixture was heated to reflux for 2 h. The reflux condenser was replaced by a distillation head and 400 mL of methanol was atmospherically distilled. The temperature was the reduced to 50° C. and the reaction stirred for additional 16 h, when the mixture was then cooled to room temperature and partitioned between dichloromethane (1 L) and water (600 mL). The organic phase was washed with saturated sodium bicarbonate and dried over magnesium sulfate. Concentration under reduced pressure provided 220.1 g (98% yield) colorless oil. HNMR: CDCl3, 3.59 (s, 2H), 3.70 (s, 3H), 7.16 (d, 2H), 7.45 (d, 2H)
Step 4: 4-[2-(4-Bromo-phenyl)-2-methoxycarbonyl-ethyl]-benzoic acid tert-butyl ester




A solution of 246.63 g of methyl-4-bromophenyl acetate and 342.54 g of 4-Iodomethyl benzoic acid tert-butyl ester in THF (1233 mL) was cooled to −8° C. A solution of lithium hexamethyl disilazide in THF (1185 mL, 1.0 M) was added dropwise ensuring that the temperature remaine below −2° C. After the addition was complete, the reaction was allowed to proceed for  ̃45 min at the same temperature and then poured over a stirring mixture of ethyl acetate (2.46 L) and water (1.23 L). The organic phase was washed with saturated ammonium chloride and then with water. Dried over magnesium sulfate and concentrated under reduced pressure to obtain 450.5 g (100% yield) thick oil. HNMR: CDCl3, 1.41 (s, 9H), 2.88-2.90 (m, 1H), 3.24-3.28 (m, 1H), 3.45 (s, 3H), 3.63 (t, 1H). 6.96-6.99 (m, 4H), 7.25 (d, 2H), 7.68 (d, 2H)
Step 5: (R)-2-(4-Bromo-phenyl)-3-(4-tert-butoxycarbonyl-phenyl)-propionate (S)-2-hydroxymethyl pyrrolidinium




4-[2-(4-Bromo-phenyl)-2-methoxycarbonyl-ethyl]-benzoic acid tert-butyl ester (769 g) was dissolved in THF (5.38 L) and water (3.85 L) and treated with lithium hydroxide monohydrate (153.9 g). The reaction mixture was heated to 45° C. for approximately 1 hour. After allowing the reaction to cool to 32 the reaction was poured into a stirring mixture of 11.6 L of ethyl acetate and 3.9 L of 1M aqueous hydrochloric acid. The separated organic layer was washed with water, dried over magnesium sulfate and concentrated under reduced pressure. Added ethyl acetate (2.045 L) to the residue and warmed to 78° C. for  ̃5 min for dissolution. The mixture was allowed to cool to 68° C. and treated with (S)-(+)-prolinol (90.5 mL). The solid that precipitated after cooling to room temperature was filtered and rinsed with a cold mixture (7° C.) of 1:1 ethyl acetate: heptane (740 mL). The solid isolated (232.4 g, 35% yield) was shown to have 94% enantiomeric excess (R isomer) by chiral HPLC analysis. HNMR: CDCl3, 1.57 (s, 9H), 1.67-1.74, (m, 2H), 2.64-2.69 (m, 1H), 2.76-2.81 (m, 1H), 2.94-2.99 (m, 1H), 3.14-3.19 (m, 1H), 3.32-3.39 (m, 2H), 3.60-3.67 (m, 2H), 7.14-7.19 (m, 4H), 7.35 (d, 2H), 7.80 (d, 2H). Conditions for chiral HPLC analysis: Kromasil 100-5-TBB column, 250×4.6 mm, 1 mL/min, 15% (1% AcOH/MTBE)/85% hexanes, 230/240/250 nm.
To 228 g of the product above were added 684 mL of ethyl acetate. The mixture was warmed to reflux (additional 228 mL of ethyl acetate were added when the temperature reached 69° C. for mobility) where it was held for about 10 min. The suspension was then allowed to cool to room temperature and filtered. Vacuum dried at 50° C. Product is a white solid (224.6 g, 98% yield) “R’ enantiomer with enantiomeric excess of 96.9% by HPLC analysis as described above.
Step 6: 4-[(R)-2-(4-Bromo-phenyl)-2-carboxy-ethyl-]-benzoic acid tert-butyl ester




A stirring slurry of 216.8 g of (R)-2-(4-Bromo-phenyl)-3-(4-tert-butoxycarbonyl-phenyl)-propionate (S)-2-hydroxymethyl pyrrolidinium in 2168 mL of ethyl acetate at 21° C. was treated 1084 mL of 10% aqueous formic acid. After 20 minutes, the separated organic phase was washed with water and dried over magnesium sulfate. The ethyl acetate solution was atmospherically displaced into heptanes to yield the product as a granular solid. 166.3 g (96% yield) of white solid, enantiomeric excess (“R” enantiomer) of 96.9% by chiral HPLC analysis as described above. HNMR: CDCl3, 1.50 (s, 9H), 2.96-3.00 (m, 1H), 3.32-3.37 (m, 1H), 3.73 (t, 1H), 7.03-7.08 (m, 4H), 7.35 (d, 2H), 7.76 (d, 2H)
Step 7: 4-{(R)-2-[4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoic acid tert-butyl ester




A mixture of 3.1 g of 4-[(R)-2-(4-Bromo-phenyl)-2-carboxy-ethyl-]-benzoic acid tert-butyl ester (3.Step 2, above), 1.5 g of 4-t-butyl-cyclohex-1-enyl boronic acid, 644 mg of PdCl2(P(o-tolyl)3)2, and 2.21 g of sodium carbonate in 12 mL of DME and 6 mL of ethanol and 3 mL of water was heated to reflux for a 16 h period. The reaction mixture was quenched with an excess of aqueous ammonium chloride, added ethyl acetate and the heterogeneous mixture was filtered through a celite pad. The organic phase was washed (water, saturated sodium chloride), dried over magnesium sulfate and concentrated. The residue was chromatographed on silica gel using a methanol-dichloromethane gradient to yield the carboxylic acid. HNMR (300 MHz, CDCl3, partial): 6.14 (1H, m), 1.58 (9H, s), 0.92 (9H, s). LCMS m/z=407.9 [(C30H38O4+H)—C4H9]+.
Step 8: 4-{(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-carboxy-ethyl}-benzoic acid tert-butyl ester




To a solution of 4-{(R)-2-[4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoic acid tert-butyl ester (3.0 g) in ethyl acetate (100 mL) added 10% palladium on carbon (300 mg). The mixture was stirred under a balloon filled with hydrogen, until proton NMR indicated the disappearance of the olefinic signal. The reaction was filtered through a plug of celite, and the filtrate was concentrated under reduced pressure to give a mixture of cis/trans isomers (in a ratio of 1:1, based on 1H NMR). The cis1 and trans isomers were separated by reverse phase chromatography with the latter being cis (1.34 g, 2.9 mmol, 35%). 1H NMR (CDCl3): δ 0.95 (9H, s), 1.23-1.38 (4H, m), 1.58 (9H, s), 1.88-1.98 (4H, m), 2.39-2.56 (1H, m), 3.05-3.12 (1H, m), 3.19-3.50 (1H, m), 3.82-3.90 (1H, m), 7.19-7.22 (6H, m), 8.81 (2H, d).
Based on the following reference and references within the article, the cis was assigned as described above. Garbisch, E. W.; Patterson, D. B., J. Am. Chem. Soc., 1963, 85, 3228.
Step 9: 4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chlor-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid tert-butyl ester




To 4-{(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-carboxy-ethyl}-benzoic acid tert-butyl ester (300 mg) in dichloromethane (10 mL) was added a solution of oxalyl chloride in dichloromethane (2.0M, 0.54 mL) followed by 2 drops of DMF. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The residue was dissolved in dichloromethane (20 mL), and treated with 4′-Chloro-2′-methyl-biphenyl-4-amine (142 mg) and diisopropyl ethyl amine (0.120 mL). After stirring for 1 h at room temperature, the solvent was removed under reduced pressure and the residue treated with methanol. The white precipitated formed was washed with methanol, dried under vacuum and used without further purification in the following step.
Step 10: 4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chlor-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid




A solution of 431 mg of 4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chlor-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid tert-butyl ester in dichloromethane (10 mL) was treated with trifluoroacetic acid (2 mL) and concentrated aqueous hydrochloric acid (1 mL). The resulting mixture was stirred for 16 h at room temperature. The organic phase was separated, washed with water and dried over magnesium sulfate. Concentration left a residue that was used without further purification.
Step 11: 2-{4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexy)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonic acid




(R)-4-[2-(4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid (350 mg, 0.6 mmol), was taken up in 3 mL of DMF, followed by addition of HOBt (133 mg, 0.9 mmol), EDCI (132 mg, 0.7 mmol), taurine (86 mg, 0.7 mmol) and Hunig's base (374 mg, 2.9 mmol). The resulting reaction mixture was then stirred for 16 h at room temperature. The reaction solution was diluted with EtOAc (25 mL) and 10 mL of water, acidified with 2.4 N HCl. The layers were separated and the aqueous layer was extracted with EtOAc (2×20 mL). The organic extracts were combined, dried with Na2SO4, filtered through a frit and concentrated under reduced pressure to give a foam. The material was subjected to reverse phase HPLC purification to give the desired product as a white solid (150 mg, 36%). 1H NMR (CD3OD): δ 0.89 (9H, s), 1.10-1.60 (6H, m), 1.80-1.90 (4H, m), 2.38 (3H, s), 2.32-2.53 (1H, m), 3.05-3.10 (4H, m), 3.47-3.55 (1 h. dd), 3.77 (2H, t), 3.93-3.98 (1H, m), 7.09-7.47 (1H, m), 7.50 (2H, d), 7.70 (2H, d). Anal. Calcd. For C42H47ClN2O5S+NH3+1.2 H2O; C=65.22; H=7.13; N=5.57. Found C=65.31; H=7.00; N=5.57.
Step 12. (R)-2-{4-[2-[4-(4-(trans)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonic acid
The trans isomer isolated from step 8, (R)-4-{2-[4-(4-(trans)-tert-Butyl-cyclohexyl)-phenyl]-2-carboxy-ethyl]-benzoic acid tert-butyl ester, was subjected to the procedures of steps 9-11 to give (R)-2-{4-[2-[4-(4-(trans)-tert-butyl-cyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl-benzoylamino}-ethanesulfonic acid.




The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entireties as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.

## Claims

1. A compound of Formula I:



wherein
R44 is H, CH3 or CH3CH2;
R45 is C1-6-alkyl, alkenyl, alkoxy, C3-6-cycloalkyl, C4-8-cycloalkenyl, C4-8-bicycloalkenyl, aryl or heteroaryl, any of which can be optionally substituted with one or more substituents selected from C1-6alkyl, CF3, F, CN or OCF3;
L is phenyl, indenyl, benzoxazol-2-yl, C3-6-cycloalkyl, C4-8-cycloalkenyl or C4-8-bicycloalkenyl, any of which can be optionally substituted with one or more substituents selected from F, Cl, CH3, CF3, OCF3 or CN; and
R46 represents one or more substituents selected from H, F, Cl, CH3, CF3, OCF3 or CN;
or a pharmaceutically acceptable salt, solvate, or prodrug thereof having an optical purity of at least 95% of the R enantiomer.

2. The compound of claim 1, wherein:
R44 is H, CH3 or CH3CH2;
R45 is C1-6-alkyl, alkenyl, alkoxy, C3-6-cycloalkyl, C4-8-cycloalkenyl, C4-8-bicycloalkenyl, aryl or heteroaryl, any of which can be optionally substituted with one or more substituents selected from C1-6alkyl, CF3, F, CN or OCF3;
L is phenyl, indenyl, benzoxazol-2-yl or 4,4-dimethylcyclohexenyl, any of which can be optionally substituted with one or more substituents selected from F, Cl, CH3, CF3, OCF3 or CN; and
R46 is H, F, Cl, CH3, CF3, OCF3 or CN.

3. The compound of claim 2, wherein L is phenyl, benzoxazol-2-yl or 4,4-dimethylcyclohexenyl, any of which can be optionally substituted with one or more substituents selected from F, Cl, CH3, CF3, OCF3 or CN.

4. The compound of claim 2, wherein L is 4-chloro-2-methylphenyl, 4-methyl-2-benzoxazolyl, 2,4,6-trimethylphenyl, 2-benzoxazolyl, 4-chloro-3-methylphenyl or 4,4-dimethylcyclohexenyl.

5. The compound of claim 2, wherein R44 is H or CH3.

6. The compound of claim 2, wherein R45 is attached to the 3 (meta) or 4 (para) position.

7. The compound of claim 2, wherein R45 is alkenyl, C3-6-cycloalkyl, C4-8-cycloalkenyl, C4-8-bicycloalkenyl or phenyl, any of which can be optionally substituted with one or more substituents selected from C1-6alkyl or CF3.

8. The compound of claim 2, wherein R45 is substituted with one or more substituents independently selected from CH3 and (CH3)3C—.

9. The compound of claim 2, wherein R45 is selected from (CH3)3CCH═CH—, t-butyl-cycloalkyl-, dimethyl-cycloalkyl-, t-butyl-cycloalkenyl-, dimethyl-cycloalkenyl-, bicycloalkenyl-or t-butyl-phenyl-.

10. The compound of claim 2, wherein R45 is trans-t-butylvinyl, cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl, 4,4dimethylcyclohexyl, cyclohex-1-enyl, (S)-4-t-butylcyclohex-1 -enyl, (R)-4-t-butylcyclohex-1 -enyl, 4,4-dimethylcyclohex-1 -enyl, 4,4-diethylcyclohex -1 -enyl, 4,4-diethylcyclohexyl, 4,4 dipropylcyclohex-1 -enyl, 4,4-dipropylcyclohexyl, 4,4-dimethylcyclo hexa-1,5 -dienyl, (1R,4 S)1,7,7-trimethylbicyclo [2.2.1]3-heptyl -2-ene, (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]2-heptyl-2-ene, 2-methyl-4-chloro-phenyl, 2,4,6-trimethylphenyl or 4-t-butylphenyl.

11. The compound of claim 10, wherein is R45 is trans-t-butylvinyl, cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl, (S)-4-t-butylcyclohex-lenyl, (R)-4-t-butylcyclohex-1 -enyl, 4,4-dimethylcyclohex-1 -enyl, (1R,4R)-1,7,7-trimethylbicyclo [2.2.1]2-heptyl-2-ene or 4-t -butylphenyl.

12. The compound of claim 2, wherein R46 is H or CH3.

13. The compound of claim 1, wherein the compound is selected from the group










having an optical purity of at least 95% of the R enantiomer.

14. The compound of claim 1, wherein the compound is:




and wherein the compound has an optical of at least 95% of the R enantiomer.

15. A pharmaceutical composition comprising the compound of claim 1, and one or more pharmaceutically acceptable excipients or carriers.

16. The pharmaceutical composition of claim 15, further comprising a second therapeutic agent.

17. The pharmaceutical composition of claim 16, wherein the second therapeutic agent is an antidiabetic agent.

18. A method of treating one or more symptoms of a disease responsive to the modulation of a glucagon receptor in a subject, wherein the disease is diabetes, comprising administering to a subject in need thereof the compound of claim 1.

19. A method of treating one or more symptoms of a disease responsive to a decrease in the hepatic glucose production or in the blood glucose level in a subject, wherein the disease is diabetes, comprising administering to a subject in need thereof the compound of claim 1.