WO2011146845A1 - Modified macrocyclic ghrelin receptor modulators and methods of using the same - Google Patents

Modified macrocyclic ghrelin receptor modulators and methods of using the same Download PDF

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Publication number
WO2011146845A1
WO2011146845A1 PCT/US2011/037355 US2011037355W WO2011146845A1 WO 2011146845 A1 WO2011146845 A1 WO 2011146845A1 US 2011037355 W US2011037355 W US 2011037355W WO 2011146845 A1 WO2011146845 A1 WO 2011146845A1
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compound
compounds
disease
disorders
disorder
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PCT/US2011/037355
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French (fr)
Inventor
Éric MARSAULT
Helmut Thomas
Patrick BHÉRER
Lilian Clohs
Sophie Beauchemin
Martin Brassard
Felix Dupont
Serge Phoenix
Martin Vézina
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Tranzyme Pharma, Inc.
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Publication of WO2011146845A1 publication Critical patent/WO2011146845A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel conformationally-defined macrocyclic compounds that bind to and/or are functional modulators, in particular agonists, of the ghrelin (growth hormone secretagogue) receptor including GRLN (GHS-Rla) and subtypes, isoforms and/or variants thereof.
  • the present invention also relates to intermediates of these compounds, pharmaceutical compositions containing these compounds and methods of using the compounds.
  • GRS-Rla growth hormone secretagogue receptor
  • These novel macrocyclic compounds are useful as therapeutics for a range of disease indications.
  • these compounds are useful for treatment and prevention of gastrointestinal disorders including, but not limited to, postoperative ileus, gastroparesis, including diabetic and postsurgical gastroparesis, gastric stasis, opioid bowel dysfunction, chronic intestinal pseudo-obstruction, short bowel syndrome, constipation, functional gastrointestinal disorders and gastrointestinal dysmotility, such as that occurring in conjunction with other disease states, in critical care situations or as a result of treatment with pharmaceutical agents.
  • the pharmaceutical compositions have application to the treatment and prevention of metabolic and/or endocrine disorders, cardiovascular disorders, central nervous system disorders, bone disorders, inflammatory disorders, hyperproliferative disorders and genetic disorders.
  • Ghrelin is a recently characterized 28-amino acid peptide hormone isolated originally from the stomach of rats with the orthologue subsequently identified in humans, distinguished by an unusual n-octanoyl group modification on Ser 3 .
  • Kojima, ML Hosoda, H. et al. Nature 1999, 402, 656-660; Kojima, M. Regul.Pept. 2008, 145, 2-6.
  • the existence of this hormone in a wide range of other species suggests a conserved and important role in normal physiological function.
  • the ghrelin peptide has been demonstrated to be the endogenous ligand for a previously orphan G protein-coupled receptor (GPCR), type 1 growth hormone secretatogue receptor (GHS-Rla) (Howard, A.D.; Feighner, S.D.; et al. Science 1996, 273, 974-977; U.S. Pat. No. 6,242,199; Intl. Pat. Appl. Nos. WO 97/21730 and WO 97/22004).
  • GHS-Rla has recently been reclassified as the ghrelin receptor (GRLN) in recognition of its endogenous ligand (Davenport, A.P.; et al. Pharmacol Rev. 2005, 57, 541-546).
  • GRLN is found predominantly in the brain, in particular the arcuate nucleus and ventromedial nucleus in the hypothalamus, hippocampus and substantia nigra) and pituitary, but also is expressed in a number of other tissues and organs (Gnanapavan, S.; Kola, B.; Bustin, S.A.; et al. J Clin, Endocrinol Metab. 2002, 87, 2988-2991; Ueberberg, B.; Unger, N.; Saeger, W.; Mann, K.; Petersenn, S. Horm. Metab. Res. 2009, 41, 814-821).
  • the ghrelin peptide has been found to have a variety of endocrine and non-endocrine functions (Broglio, F.; Gottero, C; Arvat, E.; Ghigo, E. Horm. Res. 2003, 59, 109-117; Hosoda, H.; Kojima, M.; Kangawa, K. J. Pharmacol Sci. 2006, 100, 398-410; Gasco, V.; Beccuti, G.; Marotta, F.; Benso, A.; Granata, R.; Broglio, F.; Ghigo, E. Endocr. Dev.
  • Clinical trials have been initiated with certain of these agonists to take advantage of these effects.
  • GI gastrointestinal
  • the prokinetic effect of ghrelin in the gastrointestinal (GI) system makes ghrelin agonists useful for therapeutic purposes in disorders characterized by GI dysmotility or hypomotility.
  • Such disorders include, but are not limited to, postoperative ileus, paralytic ileus, gastroparesis, including diabetic and postsurgical gastroparesis, gastric stasis, including in enterally fed. patients, constipation, includmg chronic constipation, opioid bowel dysfunction, chronic intestinal pseudo -obstruction, short bowel syndrome, functional gastrointestinal disorders and gastrointestinal dysmotility occurring in conjunction with other disease states, such as in critical care situations or as a result of treatment with other pharmaceutical agents.
  • Ghrelin agonists also have application as therapeutics for the treatment of cardiovascular diseases (Nagaya, N.; Kangawa, K. Drugs 2006, 66, 439-448; Garcia, E.A.; Karbonits, M. Curr. Opin. Pharmacol 2006, 6, 142-147; Isgaard, J.; Barlind, A.; Johansson, I. Cardiovasc. Hematol. Disord. Drug Targets 2008, 8, 133-137; Zhang, G.; Yin, X.; Qi, Y.; Pendyala, L.; Chen, J.; Hou, D.; Tang, C. Curr. Cardiol Rev.
  • ghrelin plays a role in immune function and exhibits anti-inflammatory actions, hence ghrelin agonists can be applied to the treatment and prevention of inflammatory disorders.
  • ghrelin agonists can be applied to the treatment and prevention of inflammatory disorders.
  • a series of macrocyclic peptidomimetics recently has been described as modulators of the ghrelin receptor and their uses for the treatment and prevention of a range of medical conditions including metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, genetic disorders, hyperproliferative disorders and inflammatory disorders outlined (U.S. Pat. Nos. 7,452,862, 7,476,653, 7,491,695 and RE42013; Intl. Pat. Appl. Publ. Nos. WO 2006/009645, WO 2006/009674, WO 2006/046977, WO 2006/137974, WO 2008/130464, WO 2011/041369 and WO 2011/053821; U.S. Pat.
  • the compounds of the invention provide a number of advantages compared with the macrocyclic ghrelin modulators previously known.
  • the present invention provides novel conformationally-defmed macrocyclic compounds of formula (I) and pharmaceutically acceptable salts thereof. These compounds can function as modulators, in particular agonists, of the ghrelin (growth hormone secretagogue) receptor (GRLN GHS-Rla) and subtypes, isoforms and variants thereof.
  • GHLN GHS-Rla growth hormone secretagogue receptor
  • Ri is Ci-C 4 alkyl
  • R 2 is halogen or hydroxyl
  • W is hydrogen or hydroxyl
  • Xi, X 2 , X3 and X 4 are independently hydrogen or hydroxyl with the proviso that no more than two of X ls X 2 , X 3 and X 4 can be hydroxyl;
  • Yi and Y 2 are independently hydrogen or hydroxyl
  • compositions including a compound of formula (I) and a pharmaceutically acceptable carrier, excipient or diluent.
  • aspects of the present invention provide methods of treating a gastrointestinal disorder, a metabolic or endocrine disorder, a cardiovascular disorder, an inflammatory disorder, a bone disorder, a hyperproliferative disorder or a genetic disorder, including administering to a subject in need thereof an effective amount of a compound of formula I.
  • kits comprising one or more containers containing pharmaceutical dosage units comprising an effective amount of one or more compounds of the present invention packaged with optional instructions for the use thereof.
  • aspects of the present invention further provide methods of stimulating gastrointestinal motility, modulating GRLN (GHS- la) receptor activity in a mammal and/or treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a modulator that modulates a mammalian GRLN (GHS-Rla) receptor.
  • the modulator is a compound of formula (I).
  • Additional aspects of the present invention provide methods of diagnosing tumors and/or acromegaly, comprising administering compounds of the present invention and a radiolabeled metal binding agent and detecting the binding of the composition to a biological target, and treating tumors and/or acromegaly comprising administering a therapeutically effective amount of a composition comprising a compound of the present invention.
  • aspects of the invention provide methods of treating a patient suffering from reduced or dysfunctional gastrointestinal motility caused by a first medicament, pharmaceutical or pharmaceutical composition, wherein the first medicament, pharmaceutical or pharmaceutical composition is being employed to treat the patient for a metabolic, hyperproliferative or other disorder, with compounds of formula (I).
  • aspects of the present invention further relate to methods of preventing and/or treating disorders described herein, in particular, gastrointestinal disorders, including postoperative ileus, paralytic ileus, gastroparesis, such as diabetic and postsurgical gastroparesis, gastric stasis, opioid bowel dysfunction, chronic intestinal pseudoobstruction, short bowel syndrome, constipation, functional gastrointestinal disorders, gastrointestinal dysmotility, such as that occurring in conjunction with other disease states, in critical care situations or as a result of treatment with pharmaceutical agents, emesis such as caused by cancer chemotherapy, constipation such as associated with the hypomotility phase of irritable bowel syndrome (IBS), delayed gastric emptying associated with wasting conditions, gastroesophageal reflux disease (GERD), gastric ulcers, Crohn's disease, gastrointestinal disorders characterized by dysmotility or hypomotility, and other diseases and disorders of the gastrointestinal tract.
  • gastrointestinal disorders including postoperative ileus, paralytic ileus, gastroparesis, such as diabetic and postsurgical gastroparesis
  • the gastrointestinal disorder is postoperative ileus, paralytic ileus caused by surgery or other manipulations, gastroparesis, opioid-induced bowel dysfunction, gastric stasis or hypomotility caused by various diseases such as diabetes or by the administration of other pharmaceutical agents, or in enterally fed patients, chronic intestinal pseudo-obstruction, acute colonic pseudo-obstruction (Ogilvie's syndrome), enteric dysmotility, short bowel syndrome, emesis, constipation-predominant irritable bowel syndrome (IBS), chronic constipation, functional dyspepsia, cancer- associated dyspepsia syndrome, graft versus host disease, delayed gastric emptying, gastrointestinal dysfunction or delayed gastric emptying occurring in conjunction with other disease states, gastrointestinal dysmotility or delayed gastric emptying occurring in critical care situations, gastrointestinal dysfunction or delayed gastric emptying as a result of treatment with pharmaceutical agents, gastroesophageal reflux disease (GERD), gastric ulcers, gastroenteritis, colitis and Crohn's
  • the present invention also relates to compounds of formula (I) used for the preparation of a medicament for prevention and/or treatment of the disorders described herein.
  • Figure 1 shows a concentration-response graph for activation of the ghrelin receptor mediated signaling pathway with an exemplary compound of the present invention.
  • Figure 2 shows a concentration-response graph for activation of the ghrelin receptor mediated signaling pathway with another exemplary compound of the present invention.
  • Figure 3 shows a synthetic scheme for a representative tether building block of the present invention.
  • Figure 4 shows a synthetic scheme for another representative tether building block of the present invention.
  • Figure 5 shows a synthetic scheme for another representative tether building block of the present invention.
  • Figure 6 shows a synthetic scheme for a representative amino acid building block of the present invention.
  • Figure 7 shows a synthetic scheme for another representative amino acid building block of the present invention.
  • Figure 8 shows a synthetic scheme for a representative compound of the present invention.
  • alkyl refers to straight or branched chain saturated or partially unsaturated hydrocarbon groups having from 1 to 20 carbon atoms, in some instances 1 to 8 carbon atoms.
  • lower alkyl refers to alkyl groups containing 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, isopropyl, tert-butyl, 3-hexenyI, and 2-butynyl.
  • unsaturated is meant the presence of 1, 2 or 3 double or triple bonds, or a combination of the two. Such alkyl groups may also be optionally substituted as described below.
  • C 2 -C 4 alkyl indicates an alkyl group with 2, 3 or 4 carbon atoms.
  • cycloalkyl refers to saturated or partially unsaturated cyclic hydrocarbon groups having from 3 to 15 carbon atoms in the ring, in some instances 3 to 7, and to alkyl groups containing said cyclic hydrocarbon groups.
  • examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopropylmethyl, cyclopentyl, 2-(cyclohexyl)ethyl, cycloheptyl, and cyclohexenyl.
  • Cycloalkyl as defined herein also includes groups with multiple carbon rings, each of which may be saturated or partially unsaturated, for example decalinyl, [2.2.1]-bicycloheptanyl or adamantanyl. All such cycloalkyl groups may also be optionally substituted as described below.
  • aromatic refers to an unsaturated cyclic hydrocarbon group having a conjugated pi electron system that contains 4n+2 electrons where n is an integer greater than or equal to 1.
  • Aromatic molecules are typically stable and are depicted as a planar ring of atoms with resonance structures that consist of alternating double and single bonds, for example benzene or naphthalene.
  • aryl refers to an aromatic group in a single or fused carbocyclic ring system having from 6 to 15 ring atoms, in some instances 6 to 10, and to alkyl groups containing said aromatic groups.
  • aryl groups include, but are not limited to, phenyl, 1 -naphthyl, 2-naphthyl and benzyl.
  • Aryl as defined herein also includes groups with multiple aryl rings which may be fused, as in naphthyl and anthracenyt, or unfused, as in 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, indanyl or tetrahydronaphthyl (tetralinyl). All such aryl groups may also be optionally substituted as described below.
  • heterocycle refers to saturated or partially unsaturated monocyclic, bicyclic or tricyclic groups having from 3 to 15 atoms, in some instances 3 to 7, with at least one heteroatom in at least one of the rings, said heteroatom being selected from O, S or N.
  • Each ring of the heterocyclic group can contain one or two O atoms, one or two S atoms, 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 fused rings completing the bicyclic or tricyclic heterocyclic groups may contain only carbon atoms and may be saturated or partially unsaturated.
  • heterocyclic also refers to alkyl groups containing said monocyclic, bicyclic or tricyclic heterocyclic groups. Examples of heterocyclic rings include, but are not limited to, 2- or 3-piperidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl. All such heterocyclic groups may also be optionally substituted as described below
  • heteroaryl refers to an aromatic group in a single or fused ring system having from 5 to 15 ring atoms, in some instances 5 to 10, which have at least one heteroatom in at least one of the rings, said heteroatom being selected from O, S or N.
  • Each ring of the heteroaryl group can contain one or two O atoms, one or two S atoms, 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 fused rings completing the bicyclic or tricyclic groups may contain only carbon atoms and may be saturated, partially unsaturated or aromatic.
  • the N atoms may optionally be quaternized or oxidized to the N-oxide.
  • Heteroaryl also refers to alkyl groups containing said cyclic groups.
  • 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, pyridazinyl, and triazinyl.
  • bicyclic heteroaryl groups include, but are not limited to indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl.
  • tricyclic heteroaryl groups include, but are not limited to carbazolyl, benzindolyl, phenanthrollinyl, acridinyl, phenanthridinyl, and xanthenyl. All such heteroaryl groups may also be optionally substituted as described below.
  • hydroxy or "hydroxyl” refers to the group -OH.
  • alkoxy refers to the group -OR a , wherein R a is alkyl, cycloalkyl or heterocyclic. Examples include, but are not limited to methoxy, ethoxy, tert- butoxy, cyclohexyloxy and tetrahydropyranyloxy.
  • aryloxy refers to the group -03 ⁇ 4 wherein 3 ⁇ 4 is aryl or heteroaryl. Examples include, but are not limited to phenoxy, benzyloxy and 2-naphthyloxy.
  • amino acyl indicates an acyl group that is derived from an amino acid.
  • amino refers to an -NR d R e group wherein Rj and Re are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl.
  • R d and R e together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfmyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
  • R f and R g together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from 0, S or N.
  • Rj and R j together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
  • the term "carboxy” refers to the group -C0 2 H.
  • Carboxyalkyl refers to the group -CC ⁇ R k , wherein 3 ⁇ 4 is alkyl, cycioalkyl or heterocyclic.
  • Carboxyaryl refers to the group -CC ⁇ Rm, wherein R ra is aryl or heteroaryl.
  • cyano refers to the group -CN.
  • halo refers to fluoro, fluorine or fluoride, chloro, chlorine or chloride, bromo, bromine or bromide, and iodo, iodine or iodide, respectively.
  • mercapto refers to the group -SR dilemma wherein R réelle is hydrogen, alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl.
  • nitro refers to the group -N0 .
  • trifluoromethyl refers to the group -CF 3 .
  • R r and R s together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycioalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from 0, S or N.
  • R x and R y together form a heterocyclic ring or 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
  • R aa and R b together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
  • optionally substituted is intended to expressly indicate that the specified group is unsubstituted or substituted by one or more suitable substituents, unless the optional substituents are expressly specified, in which case the term indicates that the group is unsubstituted or substituted with the specified substituents.
  • various groups may be unsubstituted or substituted (i.e., they are optionally substituted) unless indicated otherwise herein (e.g., by indicating that the specified group is unsubstituted).
  • Ru, 3 ⁇ 4, Rmm, R PP , Rqq and R rr are independently selected from hydrogen, unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl or unsubstituted heteroaryl; and wherein Rkk and R nn are independently selected from unsubstituted alkyl, unsubstituted cycloalkyi, unsubstituted heterocyclic, unsubstituted aryl or unsubstituted heteroaryl.
  • R gg and Rhh, Rj and kk or R pp and R qq together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyi, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
  • substituted for aryl and heteroaryl groups includes as an option having one of the hydrogen atoms of the group replaced by cyano, nitro or trifluoromethyl,
  • substitution is made provided that any atom's normal valency is not exceeded and that the substitution results in a stable compound.
  • such substituted group is preferably not further substituted or, if substituted, the substituent comprises only a limited number of substituted groups, in some instances 1, 2, 3 or 4 such substituents.
  • stable compound or “stable structure” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity and formulation into an efficacious therapeutic agent.
  • amino acid refers to the common natural (genetically encoded) or synthetic amino acids and common derivatives thereof, known to those skilled in the art.
  • standard or “proteinogenic” refers to the genetically encoded 20 amino acids in their natural configuration.
  • unnatural or “unusual” refers to the wide selection of non-natural, rare or synthetic amino acids such as those described by Hunt, S. in Chemistry and Biochemistry of the Amino Acids, Barrett, G.C., Ed., Chapman and Hall: New York, 1985.
  • residue with reference to an amino acid or amino acid derivative refers to a group of the formula:
  • RAA is an amino acid side chain
  • n 0, 1 or 2 in this instance.
  • fragment with respect to a dipeptide, tripeptide or higher order peptide derivative indicates a group that contains two, three or more, respectively, amino acid residues.
  • amino acid side chain refers to any side chain from a standard or unnatural amino acid, and is denoted RAA-
  • RAA- amino acid side chain
  • the side chain of alanine is methyl
  • the side chain of valine is isopropyl
  • the side chain of tryptophan is 3-indolyImethyl.
  • agonist refers to a compound that duplicates at least some of the effect of the endogenous ligand of a protein, receptor, enzyme or the like.
  • antagonist refers to a compound that inhibits at least some of the effect of the endogenous ligand of a protein, receptor, enzyme or the like.
  • growth hormone secretagogue refers to any exogenously administered compound or agent that directly or indirectly stimulates or increases the endogenous release of growth hormone, growth hormone-releasing hormone, or somatostatin in an animal, in particular, a human.
  • a GHS may be peptidic or non-peptidic in nature, in some instances, with an agent that can be administered orally. In some instances, the agent can induce a pulsatile response.
  • modulator refers to a compound that imparts an effect on a biological or chemical process or mechanism.
  • a modulator may increase, facilitate, upregulate, activate, inhibit, decrease, block, prevent, delay, desensitize, deactivate, down regulate, or the like, a biological or chemical process or mechanism.
  • a modulator can be an "agonist” or an "antagonist.”
  • Exemplary biological processes or mechanisms affected by a modulator include, but are not limited to, receptor binding and hormone release or secretion.
  • Exemplary chemical processes or mechanisms affected by a modulator include, but are not limited to, catalysis and hydrolysis.
  • variable when applied to a receptor is meant to include dimers, trimers, tetramers, pentamers and other biological complexes containing multiple components. These components can be the same or different.
  • peptide refers to a chemical compound comprised of two or more amino acids covalently bonded together.
  • peptidomimetic refers to a chemical compound designed to mimic a peptide, but which contains structural differences through the addition or replacement of one of more functional groups of the peptide in order to modulate its activity or other properties, such as solubility, metabolic stability, oral bioavailability, lipophilicity, permeability, etc. This can include replacement of the peptide bond, side chain modifications, truncations, additions of functional groups, etc.
  • non- peptide peptidomimetic When the chemical structure is not derived from the peptide, but mimics its activity, it is often referred to as a "non- peptide peptidomimetic.”
  • protecting group refers to any chemical compound that may be used to prevent a potentially reactive functional group, such as an amine, a hydroxyl or a carboxyl, on a molecule from undergoing a chemical reaction while chemical change occurs elsewhere in the molecule.
  • a potentially reactive functional group such as an amine, a hydroxyl or a carboxyl
  • a number of such protecting groups are known to those skilled in the art and examples can be found in "Protective Groups in Organic Synthesis," Theodora W. Greene and Peter G. Wuts, editors, John Wiley & Sons, New York, 3 rd edition, 1999 [ISBN 0471160199].
  • amino protectmg groups include, but are not limited to, phthalimido, trichloroacetyl, benzyloxycarbonyl, fert-butoxycarbonyl, and adamantyloxycarbonyl.
  • amino protecting groups are carbamate amino protecting groups, which are defined as an amino protecting group that when bound to an amino group forms a carbamate.
  • amino carbamate protecting groups are allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc) and ⁇ , ⁇ -dimethyl- 3,5-dimethoxybenzyloxycarbonyl (Ddz).
  • hydroxyl protecting groups include, but are not limited to, acetyl, ier/-butyldimethylsilyl (TBDMS), trityl (Trt), fert-butyl, and tetrahydropyranyl (THP).
  • carboxyl protecting groups include, but are not limited to methyl ester, tert-bv y ⁇ ester, benzyl ester, trimethylsilylefhyl ester, and 2,2,2-trichloroethyl ester.
  • solid phase chemistry refers to the conduct of chemical reactions where one component of the reaction is covalently bonded to a polymeric material (solid support as defined below). Reaction methods for performing chemistry on solid phase have become more widely known and established outside the traditional fields of peptide and oligonucleotide chemistry.
  • solid support refers to a mechanically and chemically stable polymeric matrix utilized to conduct solid phase
  • polystyrene polyethylene, polyethylene glycol, polyethylene glycol grafted or covalently bonded to polystyrene (also termed PEG-polystyrene, TentaGelTM, Rapp, W.; Zhang, L,; Bayer, E. In Innovations and Persepctives in Solid Phase Synthesis. Peptides, Polypeptides and Oligonucleotides; Epton, R. ?
  • polyacrylate polyacrylamide
  • polyurethane PEGA [polyethyleneglycol poly(N,N-dimethylacrylamide) co-polymer, Meldal, M. Tetrahedron Lett. 1992, 33, 3077- 3080], cellulose, etc.
  • PEGA polyethyleneglycol poly(N,N-dimethylacrylamide) co-polymer, Meldal, M. Tetrahedron Lett. 1992, 33, 3077- 3080]
  • cellulose etc.
  • These materials can optionally contain additional chemical agents to form cross-linked bonds to mechanically stabilize the structure, for example polystyrene cross-linked with divinylbenezene (DVB, usually 0.1-5%, preferably 0.5-2%).
  • DVD divinylbenezene
  • This solid support can include as non-limiting examples aminomethyl polystyrene, hydroxymethyl polystyrene, benzhydrylamine polystyrene (BHA), methylbenzhydrylamine (MBHA) polystyrene, and other polymeric backbones containing free chemical functional groups, most typically, -NH 2 or -OH, for further derivatization or reaction.
  • BHA benzhydrylamine polystyrene
  • MBHA methylbenzhydrylamine
  • the term is also meant to include "Ultraresins" with a high proportion ("loading") of these functional groups such as those prepared from polyethyleneimines and cross-linking molecules (Barth, M.; Rademann, J. J. Comb. Chem. 2004, 6, 340-349).
  • resins are typically discarded, although they have been shown to be able to be reused such as in Frechet, J.M.J.; Haque, K.E. Tetrahedron Lett. 1975, 16, 30
  • the materials used as resins are insoluble polymers, but certain polymers have differential solubility depending on solvent and can also be employed for solid phase chemistry.
  • polyethylene glycol can be utilized in this manner since it is soluble in many organic solvents in which chemical reactions can be conducted, but it is insoluble in others, such as diethyl ether.
  • reactions can be conducted homogeneously in solution, then the product on the polymer precipitated through the addition of diethyl ether and processed as a solid. This has been termed "liquid-phase" chemistry.
  • linker when used in reference to solid phase chemistry refers to a chemical group that is bonded covalently to a solid support and is attached between the support and the substrate typically in order to permit the release (cleavage) of the substrate from the solid support. However, it can also be used to impart stability to the bond to the solid support or merely as a spacer element. Many solid supports are available commercially with linkers already attached.
  • the term "effective amount” or “effective” is intended to designate a dose that causes a relief of symptoms of a disease or disorder as noted through clinical testing and evaluation, patient observation, and/or the like, and/or a dose that causes a detectable change in biological or chemical activity.
  • the detectable changes may be detected and/or further quantified by one skilled in the art for the relevant mechanism or process.
  • the dosage will vary depending on the administration routes, symptoms and body weight of the patient but also depending upon the compound being administered.
  • Administration of two or more compounds "in combination” means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other.
  • the two compounds can be administered simultaneously (concurrently) or sequentially. Simultaneous administration can be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration.
  • the phrases "concurrent administration”, “administration in combination”, “simultaneous administration” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time or immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.
  • pharmaceutically active metabolite is intended to mean a pharmacologically active product produced through metabolism in the body of a specified compound.
  • solvate is intended to mean a pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound.
  • examples of solvates include compounds of the invention in combination with water (termed hydrates), isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
  • Novel macrocyclic compounds of the present invention include macrocyclic compounds comprising a building block structure including a tether component that undergoes cyclization to form the macrocyclic compound.
  • the building block structure can comprise amino acids (standard and unnatural), hydroxy acids, hydrazino acids, aza-amino acids, specialized moieties such as those that play a role in the introduction of peptide surrogates and isosteres, and a tether component as described herein.
  • the present invention includes isolated compounds.
  • An isolated compound refers to a compound that, in some embodiements, comprises at least 10%, at least 25%, at least 50% or at least 70% of the compounds of a mixture.
  • the compound, pharmaceutically acceptable salt thereof or pharmaceutical composition containing the compound exhibits a statistically significant binding and/or antagonist activity when tested in biological assays at the human ghrelin receptor.
  • the compounds disclosed herein may have asymmetric centers.
  • the inventive compounds may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the scope of the present invention. In particular embodiments, however, the inventive compounds are used in optically pure form.
  • the terms "S” and "R” configuration as used herein are as defined by the IUPAC 1974 Recommendations for Section E, Fundamentals of Stereochemistry (Pure Appl Chem. 1976, 45, 13-30).
  • the compounds may be prepared as a single stereoisomer or a mixture of stereoisomers.
  • the non-racemic forms may be obtained by either synthesis or resolution.
  • the compounds may, for example, be resolved into the component enantiomers by standard techniques, for example formation of diastereomeric pairs via salt formation.
  • the compounds also may be resolved by covalently bonding to a chiral moiety.
  • the diastereomers can then be resolved by chromatographic separation and/or crystal lographic separation. In the case of a chiral auxiliary moiety, it can then be removed.
  • the compounds can be resolved through the use of chiral chromatography. Enzymatic methods of resolution could also be used in certain cases.
  • an “optically pure” compound is one that contains only a single enantiomer.
  • the term “optically active” is intended to mean a compound comprising at least a sufficient excess of one enantiomer over the other such that the mixture rotates plane polarized light.
  • Optically active compounds have the ability to rotate the plane of polarized light. The excess of one enantiomer over another is typically expressed as enantiomeric excess (e.e.).
  • the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s).
  • the prefixes "d” and “1" or (+) and (-) are used to denote the optical rotation of the compound (i.e., the direction in which a plane of polarized light is rotated by the optically active compound).
  • the "1" or (-) prefix indicates that the compound is levorotatory (i.e., rotates the plane of polarized light to the left or counterclockwise) while the "d” or (+) prefix means that the compound is dextrarotatory (i.e., rotates the plane of polarized light to the right or clockwise).
  • the sign of optical rotation, (-) and (+) is not related to the absolute configuration of the molecule, R and S.
  • a compound of the invention having the desired pharmacological properties will be optically active and, can be comprised of at least 90% (80% e.e.), at least 95% (90% e.e.), at least 97.5% (95% e.e.) or at least 99% (98% e.e.) of a single isomer.
  • Embodiments of the present invention further provide intermediate compounds formed through the synthetic methods described herein to provide the compounds of formula I and/or II.
  • the intermediate compounds may possess utility as a therapeutic agent for the range of indications described herein and/or a reagent for further synthesis methods and reactions.
  • the compounds of the present invention can be synthesized using traditional solution synthesis techniques or solid phase chemistry methods. In either, the construction involves four phases: first, synthesis of the building blocks comprising recognition elements for the biological target receptor, plus one tether moiety, primarily for control and definition of conformation. These building blocks are assembled together, typically in a sequential fashion, in a second phase employing standard chemical transformations. The precursors from the assembly are then cyclized in the third stage to provide the macrocyclic structures. Finally, the post-cyclization processing fourth stage involving removal of protecting groups, if necessary, and optional purification provides the desired final compounds. Synthetic methods for this general type of macrocyclic structure are described in Intl. Pat. Appls.
  • the macrocyclic compounds may be synthesized using solid phase chemistry on a soluble or insoluble polymer matrix as previously defined.
  • solid phase chemistry a preliminary stage involving the attachment of the first building block, also termed "loading," to the resin must be performed.
  • the resin utilized for the present invention preferentially has attached to it a linker moiety. These linkers are attached to an appropriate free chemical functionality, usually an alcohol or amine, although others are also possible, on the base resin through standard reaction methods known in the art, such as any of the large number of reaction conditions developed for the formation of ester or amide bonds.
  • linker moieties such as the thioester linker
  • Some linker moieties, such as the thioester linker, for the present invention are designed to allow for simultaneous cleavage from the resin with formation of the macrocycle in a process generally termed "cyclization-release.”
  • the thioester strategy proceeds through a modified route where the tether component is actually assembled during the cyclization step.
  • assembly of the building blocks proceeds sequentially, followed by cyclization (and release from the resin if solid phase).
  • An additional post-cyclization processing step is required to remove particular byproducts of the RCM reaction, but the remaining subsequent processing is done in the same manner as for the thioester or analogous base-mediated cyclization strategy.
  • steps including the methods provided herein may be performed independently or at least two steps may be combined. Additionally, steps including the methods provided herein, when performed independently or combined, may be performed at the same temperature or at different temperatures without departing from the teachings of the present invention.
  • Novel macro cyclic compounds of the present invention include those formed by a novel process including cyclization of a building block structure to form a macrocyclic compound comprising a tether component described herein. Accordingly, the present invention provides methods of manufactuiing the compounds of the present invention comprising (a) assembling building block structures, (b) chemically transforming the building block structures, (c) cyclizing the building block structures including a tether component, (d) removing protecting groups from the building block structures, and (e) optionally purifiying the product obtained from step (d). In some embodiments, assembly of the building block structures may be sequential. In further embodiments, the synthesis methods are carried out using traditional solution synthesis techniques or solid phase chemistry techniques.
  • Amino acids, Boc- and Fmoc-protected amino acids and side chain protected derivatives, including those of N-methyl and unnatural amino acids were obtained from commercial suppliers [for example Advanced ChemTech (part of CreoSalus, Louisville, KY, USA), Astatech (Bristol, PA, USA), Bachem (Bubendorf, Switzerland), Chemlmpex (Wood Dale, IL, USA), Novabiochem (subsidiary of Merck KGaA, Darmstadt, Germany), PepTech (Burlington, MA, USA), Synthetech (Albany, OR, USA)] or synthesized through standard methodologies known to those in the art.
  • Ddz-amino acids were either obtained commercially from Orpegen (Heidelberg, Germany) or Advanced ChemTech (part of CreoSalus, Louisville, Y, USA) or synthesized using standard methods utilizing Ddz-OPh or Ddz-N 3 .
  • Bts-amino acids were synthesized by known methods.
  • N-Alkyl amino acids in particular N-methyl amino acids, are commercially available from multiple vendors (Bachem, Novabiochem, Advanced ChemTech, Chemlmpex).
  • N-alkyl amino acid derivatives were accessed via literature methods.
  • the ⁇ - hydroxy derivatives of 4-fluorophenylalanine can be synthesized as described in the Examples or in the same manner as described for ⁇ -hydroxyphenylalanine stereoisomers.
  • Tethers were obtained from the methods previously described in Intl. Pat. Appl.
  • Tethers useful for the compounds of the present invention can include suitable tethers as described in the references cited above as well as known and/or novel tethers described herein.
  • Exemplary tethers include, but are not limited to, the following:
  • PGj is an amine protecting group and PG 2 and PG 3 are the same or different hydroxyl protecting groups.
  • the table following provides information on the building blocks used for the synthesis of representative compounds of the present invention using the standard methods.
  • a suitable protecting group(s) for the building block is typically indicated. These are directly applicable to solution phase synthesis.
  • modified protection strategies from that illustrated are typically employed to permit the use of a convergent approach. Additional synthetic details for the solution phase construction of representative macrocyclic compounds of the invention are presented in the Examples.
  • the compounds of the present invention were evaluated for their ability to interact at the human ghrelin receptor.
  • a competitive radioligand binding assay, fluorescence assay or Aequorin functional assay can be employed. Such methods can be conducted in a high throughput manner to permit the simultaneous evaluation of many compounds.
  • a competitive binding assay at the human growth hormone secretagogue receptor can be carried out analogously to assays described in the literature.
  • GRLN human growth hormone secretagogue receptor
  • GRLN GRLN
  • the functional activity of compounds of the invention found to bind to the GRLN (GHS-Rla) receptor can be determined using the methods described in the literature, which can also be used as a primary screen for ghrelin receptor activity in a high throughput fashion. See also U.S. Patent Nos. 7,491,695 and 7,476,653.
  • HRS-Rla GRLN
  • the in-vitro functional potency of compounds of the invention as activators of the ghrelin receptor mediated signaling pathway can also be determined using HEK-293 cells stably expressing the human GRLN (GHS-Rla).
  • IP3 a downstream metabolite of IP3, accumulates in cells following Gq receptor activation and is stable in the presence of LiCl, making it an ideal read out of receptor activation.
  • the IP-One assay is a competitive immunoassay that uses cryptate-labeled anti-IP 1 MAb and d2-labeled IPl. In the absence of endogenous IPl, cryptate Mab and IP-d2 interact and produce a quantifiable FRET (fluorescence energy transfer) signal.
  • FRET fluorescence energy transfer
  • HEK-293 cells stably expressing human GRLN receptor.
  • the cells were produced with the plasmid expressing the GRLN from Missouri University of Science and Technology (cat# GHSR0A0000).
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • a solution containing 100 units of penicillin G sodium, 100 ⁇ g ml of streptomycin sulfate, 292 ⁇ / ⁇ L-glutamine and 100 ⁇ sodium citrate was added to the culture medium (Gibco cat# 1789).
  • a solution of 0.25% Trypsin- EDTA was used to detach the cells (Gibco cat# 25200).
  • Phosphate buffered saline (PBS) (Gibco cat# 10010).
  • IP-One HTRF® assay (CisBio, Bedford, MA, USA, cat # 62P1APEC) quantifies myo -Inositol- 1 phosphate (IPl).
  • the Petri dish was washed with 10 ml PBS, treated with a 0,25% trypsin-EDTA solution for 5 min at 37°C.
  • the detached cells were resuspended in 20 ml of DMEM, 10% FBS, pen/strep/glut.
  • Ghrelin stock solution 1 mM in 3 ⁇ 40.
  • Results are calculated from the 665 nm/620 nm ratio and expressed in Delta F. Delta F calculation was performed according to the manufacturer as follows:
  • Ratio (Fluorescence 665 nm / Fluorescence 620 nm) X 10 000.
  • Test articles dosed at various concentrations, typically 1, 3, 10, 30 mg kg
  • vehicle and positive control metaloclopramide, a 5-HT ligand and prokinetic agent currently prescribed for the treatment of GI disorders, including gastroparesis
  • time 0
  • Opioid analgesics such as morphine
  • OBD opioid bowel dysfunction
  • Rats male, Sprague-Dawley, 250-300 g are implanted with jugular vein catheters to accommodate dosing of test articles.
  • Radioactivity ( 9m Tc) in each tissue isolate is measured as a means of measuring the transit of the meal.
  • cyclodextrin (CD) was added to solubilize the material.
  • Four to five (4-5) rats were tested per group, except in the case of the cyclodextrin control in which ten (10) rats comprise the group.
  • Kalff. Kalff, J.C.; Schraut, W.H.; Simmons, R.L.; Bauer, AJ. Ann. Surg. 1998, 228, 652-663.
  • Other known models can also be used to study the effect of compounds of the invention. (Trudel, L.; Bouin, M.; Tomasetto, C; Eberling, P.; St-Pierre, S.; Bannon, P.; L'Heureux, M.C.; Poitras, P.
  • Intestines and caecum are eviscerated and kept moist with saline.
  • Intestines are gently replaced into the abdomen and the abdominal wound was stitched closed under sterile conditions.
  • Rat are allowed to recover from isofluorane anaesthesia.
  • Test compounds are administered intravenously via previously
  • Geometric mean ⁇ (%totai radioactivity X number of segment)/100 I. Growth Hormone Response to Test Compounds
  • the compounds of the invention likewise can be tested in a number of animal models for their effect on GH release.
  • rats Boers, C.Y.; Momany, F.; Reynolds, G.A.; Chang, D.; Hong, A.; Chang, K. Endocrinology 1980, 106, 663-667
  • dogs Hickey, G.; Jacks, T.; Judith, F.; Taylor, J.; Schoen, W.R.; rupa, D.; Cunningham, P.; Clark, J.; Smith, R.G.
  • the following method is employed to determine the temporal pattern and magnitude of the growth hormone (GH) response to test compounds, administered either systemically or centrally. Analogous methods can be used for other appropriate animal models, such as dogs and cynomolgus monkeys.
  • Rats 225-300 g are purchased from Charles River Canada (St. Constant, Canada) and individually housed on a 12-h light, 12-h dark cycle (lights on, time: 0600-1800) in a temperature (22 + 1 ° C)- and humidity-controlled room.
  • Purina rat chow Rosina Co., St. Louis, MO
  • tap water are freely available.
  • chronic mtracerebroventricular (icv) and intracardiac venous cannulas are implanted under sodium pentobarbital (50 mg kg, ip) anesthesia using known techniques.
  • icv cannula The placement of the icv cannula are verified by both a positive drinking response to icv carbachol (100 ng/10 ⁇ ) injection on the day after surgery and methylene blue dye at the time of sacrifice.
  • the rats are placed directly in isolation test chambers with food and water freely available until body weight returned to preoperative levels (usually within 5-7 d). During this time, the rats are handled daily to minimize any stress associated with handling on the day of the experiment.
  • food is removed 1.5 h before the start of sampling and is returned at the end.
  • Test samples at various dosing levels or normal saline were administered either intravenously or orally at two different time points during a 6-h sampling period.
  • the times 1100 and 1300 are chosen because they reflect typical peak and trough periods of GH secretion, as previously documented.
  • the human ghrelin peptide (5 g, Phoenix Pharmaceuticals, Inc., Belmont, CA) is used as a positive control in the experiments and was diluted in normal saline just before use.
  • a 10- fold lower dose of the test sample or normal saline is administered icv at the same time points, 1100 and 1300.
  • Blood samples (0.35 mL) is withdrawn every 15 min over the 6-h sampling period (time: 1000-1600) from all animals.
  • an additional blood sample is obtained 5 min after each injection.
  • Plasma GH concentrations are measured in duplicate by double antibody RIA using materials supplied by the NIDDK Hormone Distribution Program (Bethesda, MD). The averaged plasma GH values for 5-6 rats per group are reported in terms of the rat GH reference preparation. All samples with values above the range of interest are reassayed at dilutions ranging from 1 :2 to 1:10. J. Mouse Model of Cancer Cachexia
  • Tumor cachexia is considered the major reason for mortality, rapidly declining quality of life and limitation of therapy in advanced tumor patients. Since agonism of the ghrelin receptor has been associated with increased food intake and the generation of a positive overall energy balance, the compounds of the present invention have applications to the treatment of this disorder.
  • the following method was designed to investigate the effects of test compounds as compared to ghrelin peptide on tumor cachexia in the G361 melanoma model grown as a subcutaneous xenograft in BALB/c nufnu mice. (Mori M, Yamaguchi K, Hyundai S, et al: Cancer Res, 1991, 51, 6656-6659.) Additional models are known in the art. (Emery, P.W. Nutrition 1999, 15, 600-603.)
  • tumour-bearing mice are randomised 12 days post-inoculation into two sets of 5 groups containing 6 animals each.
  • the average body weight loss of Set 1 and Set 2 animals relative to the initial average body weight is determined.
  • Treatment of Set 1 and 2 animals commences on Days 12 and 16, after tumor inoculation, respectively.
  • Groups 1 and 6 receive vehicle i.v. s.c. or oral (depending on the mode of administration of the test compound) bid alone, while Groups 5 and 10 were administered rat ghrelin peptide s.c. (1 mg/kg; bid, 6 h apart) as a positive control.
  • Test compounds are administered i.v., s.c.
  • mice are culled during the study according to predetermined criteria including >15% initial body weight loss and/or tumor volume in excess of 2000 mm 3 and/or display of severe clinical signs.
  • Body weights are measured, along with quantity of food and water consumption.
  • plasma levels of cholesterol, triglyceride, non-esterified fatty acids and blood glucose are determined during the course of treatment to provide further measures of the effects of the test compounds on the overall health of the animal.
  • This method is employed to evaluate the potency of compounds of the invention as a prokinetic agent by treatment of rat stomach fundus strips in an organ bath ex vivo in the presence or absence of electrical field stimulation (EFS) using ghrelin peptide as a reference, Methods
  • Fundus strips (approximately 0.4 x 1 cm) were cut from the stomach of adult male Wistar rats parallel to the circular muscle fibers. They were placed between two platinum ring electrodes, 1 cm apart ( adnoti, ADInstruments, USA) in 10 ml tissue baths containing Krebs solution bubbled with 5 % C0 2 in 0 2 and maintained at 37 °C. Tissues were suspended under 1.5 g resting tension. Changes of tension were measured isometrically with force transducers and recorded with a PowerLab 8/30 data acquisition system (ADInstruments, USA). Tissues were allowed to equilibrate for 60 min during which time bath solutions were changed every 15 min.
  • EFS was achieved by applying 0.5 ms pulses, 5 Hz frequency, at a maximally effective voltage of 70 V. EFS was applied for 30 sec at 3 min intervals for a 30 min initial period. This initial period was separated by a 5 min interval with wash out of the bath solution. Then, a second period of stimulation was started. After obtaining consistent EFS-evoked contractions (after three or four 30 sec stimulations), the effects of ghrelin, test compounds at various concentrations (for example 0.01-10 ⁇ ), L-NAME (300 ⁇ , as control) or their respective vehicles, applied non-cumulatively, on responses to EFS were studied over a 30 min period. Responses to the agents were measured and expressed as % of the mean of three or four pre-drug responses to EFS. All compounds were dissolved at 1 mM in distilled water or MeOH, as stock solutions.
  • the pharmacokinetic and pharmacodynamic properties of drugs are largely a function of the reversible binding of drugs to plasma or serum proteins such as albumin and oti-acid glycoprotein.
  • plasma or serum proteins such as albumin and oti-acid glycoprotein.
  • drugs with low plasma protein binding generally have large volumes of distribution and rapid clearance since only unbound drug is available for glomerular filtration and, in some cases, hepatic clearance.
  • the ideal range for plasma protein binding is in the range of 87-98% for most drug products.
  • Protein binding studies were performed using human plasma. Briefly, 96-well microplates were used to incubate various concentrations of the test article for 60 min at 37°C. Bound and unbound fractions are separated by equilibrium dialysis, where the concentration remaining in the unbound fraction is quantified by LC-MS or LC-MS-MS analysis. Drugs with known plasma protein binding values such as quinine (-35%), warfarin (-98%) and naproxen (-99.7%) were used as reference controls.
  • Cytochrome P450 enzymes are implicated in the phase I metabolism of drugs. The majority of drug-drug interactions are metabolism-based and, moreover, these interactions typically involve inhibition of cytochrome P450s. Six CYP450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) appear to be commonly responsible for the metabolism of most drugs and the associated drug-drug interactions. Assays to determine the binding of compounds of the invention to the various metabolically important isoforms of cytochrome P450 metabolizing enzymes are commercially available, for example NoAb BioDiscoveries (Mississaugua, ON, Canada) and Absorption Systems (Exton, PA, USA).
  • the Caco-2 cell line derived from a human colorectal carcinoma, has become an established in vitro model for the prediction of drug absorption across the human intestine.
  • Assays to determine the permeability of compounds of the invention utilizing Caco-2 cells are commercially available, for example NoAb BioDiscoveries (Mississaugua, ON, Canada) and Absorption Systems (Exton, PA, USA).
  • PAMPA parallel artificial membrane permeability assays
  • Permeability across the Caco-2 cell layer was determined by growing the cells on a membrane placed between two (donor and acceptor) chambers. Drug candidates are typically added to the apical (A) side of the cell layer and their appearance in the basolateral (B) side is measured over incubation time. Permeability in this direction represents intestinal absorption. Permeability may also be determined from the basolateral to the apical side of the Caco-2 cell. A higher apical to basolateral P app , compared to the basolateral to apical P app , is indicative of carrier-mediated transport. P-gp mediated transport is suggested when a higher basolateral to apical P app is observed relative to die apical to basolateral P app ,
  • Permeability (10 ⁇ ) for compounds of the invention in the apical to basolateral and basolateral to apical direction were tested in duplicate. Samples will be collected from the donor and acceptor chambers at the beginning (0 min) and following 60 min of incubation at 37°C and stored frozen at -70° C until bioanalysis. Samples for each test compound generated from the Caco-2 permeability assay were further analyzed by LC- MS-MS. The permeability of [ 3 H]-mannitol and [ 3 H] -propranolol were determined in parallel as controls.
  • dQ/dT represents the permeability rate
  • C denotes the initial concentration in the donor compartment
  • A represents the surface area of the filter.
  • Q is determined from the mean concentration of duplicate samples taken prior to addition to the donor compartment. Permeability rates were calculated by plotting the cumulative amount of compound measured in the acceptor compartment over time and determining the slope of the line by linear regression analysis. The duplicate and mean apical to basolateral and basolateral to apical P app 's were reported for each compound and standard.
  • Ghrelin, GHRP-6 and capromorelin were used as reference agonists
  • the macrocyclic compounds of the present invention or pharmacologically acceptable salts thereof according to the invention may be formulated into pharmaceutical compositions of various dosage forms.
  • one or more compounds, including optical isomers, enantiomers, diastereomers, racemates or stereochemical mixtures thereof, or pharmaceutically acceptable salts thereof as the active ingredient is intimately mixed with appropriate carriers and additives according to techniques known to those skilled in the art of pharmaceutical formulations.
  • a pharmaceutically acceptable salt refers to a salt form of the compounds of the present invention in order to permit their use or formulation as pharmaceuticals and which retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically or otherwise undesirable.
  • Examples of such salts are described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, ermuth, C.G. and Stahl, P.H. (eds.), Wiley- Verlag Helvetica Acta, Zurich, 2002 [ISBN 3-906390-26-8].
  • Examples of such salts include alkali metal salts and addition salts of free acids and bases.
  • Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenylacetates, phenylprop
  • a desired salt may be prepared by any suitable method known to those skilled in the art, including treatment of the free base with an inorganic acid, such as, without limitation, hydrochloric acid, hydrobromic acid, hydroiodic, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, including, without limitation, formic acid, acetic acid, propionic acid, maieic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, stearic acid, ascorbic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-to
  • an inventive compound is an acid
  • a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like.
  • an inorganic or organic base such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like.
  • suitable salts include organic salts derived from amino acids such as glycine, lysine and arginine; ammonia; primary, secondary, and tertiary amines such as ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine, choline, and procaine, and cyclic amines, such as piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
  • compositions for oral administration may be, for example, solid preparations such as tablets, sugar- coated tablets, hard capsules, soft capsules, granules, powders and the like, with suitable carriers and additives being starches, sugars, binders, diluents, granulating agents, lubricants, disintegrating agents and the like. Because of their ease of use and higher patient compliance, tablets and capsules represent the most advantageous oral dosage forms for many medical conditions.
  • compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, elixirs, and the like with suitable carriers and additives being water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like.
  • suitable carriers and additives being water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like.
  • Typical preparations for parenteral administration comprise the active ingredient with a carrier such as sterile water or parenterally acceptable oil including polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with other additives for aiding solubility or preservation may also be included.
  • a carrier such as sterile water or parenterally acceptable oil including polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with other additives for aiding solubility or preservation may
  • compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, topical (i.e., both skin and mucosal surfaces, including airway surfaces), transdermal administration and parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intrathecal, intracerebral, intracranially, intraarterial, or intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active agent which is being used.
  • compositions for injection will include the active ingredient together with suitable carriers including propylene glycol-alcohol-water, isotonic water, sterile water for injection (USP), emulPhorTM-alcohol-water, cremophor-ELTM or other suitable carriers known to those skilled in the art.
  • suitable carriers including propylene glycol-alcohol-water, isotonic water, sterile water for injection (USP), emulPhorTM-alcohol-water, cremophor-ELTM or other suitable carriers known to those skilled in the art.
  • carriers may be used alone or in combination with other conventional solubilizing agents such as ethanol, propylene glycol, or other agents known to those skilled in the art.
  • the compounds may be used by dissolving or suspending in any conventional diluent
  • the diluents may include, for example, physiological saline, Ringer's solution, an aqueous glucose solution, an aqueous dextrose solution, an alcohol, a fatty acid ester, glycerol, a glycol, an oil derived from plant or animal sources, a paraffin and the like. These preparations may be prepared according to any conventional method known to those skilled in the art.
  • compositions for nasal administration may be formulated as aerosols, drops, powders and gels.
  • Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a physiologically acceptable aqueous or non-aqueous solvent.
  • Such formulations are typically presented in single or multidose quantities in a sterile form in a sealed container.
  • the sealed container can be a cartridge or refill for use with an atomizing device.
  • the sealed container may be a unitary dispensing device such as a single use nasal inhaler, pump atomizer or an aerosol dispenser fitted with a metering valve set to deliver a therapeutically effective amount, which is intended for disposal once the contents have been completely used.
  • the dosage form comprises an aerosol dispenser, it will contain a propellant such as a compressed gas, air as an example, or an organic propellant including a fiuorochlorohydrocarbon or fluorohydrocarbon.
  • compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth or gelatin and glycerin.
  • a carrier such as sugar and acacia, tragacanth or gelatin and glycerin.
  • compositions for rectal administration include suppositories containing a conventional suppository base such as cocoa butter.
  • compositions suitable for transdermal administration include ointments, gels and patches.
  • compositions known to those skilled in the art can also be applied for percutaneous or subcutaneous administration, such as plasters.
  • compositions comprising the active ingredient or ingredients in admixture with components necessary for the formulation of the compositions
  • other conventional pharmacologically acceptable additives may be incorporated, for example, excipients, stabilizers, antiseptics, wetting agents, emulsifying agents, lubricants, sweetening agents, coloring agents, flavoring agents, isotonicity agents, buffering agents, antioxidants and the like.
  • additives there may be mentioned, for example, starch, sucrose, fructose, dextrose, lactose, glucose, mannitol, sorbitol, precipitated calcium carbonate, crystalline cellulose, carboxymethylcellulose, dextrin, gelatin, acacia, EDTA, magnesium stearate, talc, hydroxypropylmethylcellulose, sodium metabisulfite, and the like.
  • the composition is provided in a unit dosage form such as a tablet or capsule.
  • kits including one or more containers comprising pharmaceutical dosage units comprising an effective amount of one or more compounds of the present invention.
  • the present invention further provides prodrugs comprising the compounds described herein.
  • prodrug is intended to mean a compound that is converted under physiological conditions or by solvolysis or metabolically to a specified compound that is pharmaceutically active.
  • the "prodrug” can be a compound of the present invention that has been chemically derivatized such that, (i) it retains some, all or none of the bioactivity of its parent drug compound, and (ii) it is metabolized in a subject to yield the parent drug compound.
  • the prodrug of the present invention may also be a "partial prodrug" in that the compound has been chemically derivatized such that, (i) it retains some, all or none of the bioactivity of its parent drug compound, and (ii) it is metabolized in a subject to yield a biologically active derivative of the compound.
  • Known techniques for derivatizmg compounds to provide prodrugs can be employed. Such methods may utilize formation of a hydrolyzable coupling to the compound.
  • the present invention further provides that the compounds of the present invention may be administered in combination with a therapeutic agent used to prevent and/or treat metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, bone disorders, genetic disorders, hyperproiiferative disorders and inflammatory disorders.
  • a therapeutic agent used to prevent and/or treat metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, bone disorders, genetic disorders, hyperproiiferative disorders and inflammatory disorders.
  • agents include analgesics (including opioid analgesics), anesthetics, antifungals, antibiotics, antiinflammatories (including nonsteroidal anti-inflammatory agents), anthelmintics, antiemetics, antihistamines, antihypertensives, antipsychotics, antiarthritics, antitussives, antivirals, cardioactive drugs, cathartics, chemotherapeutic agents (such as DNA-interactive agents, antimetabolites, tubulin-interactive agents, hormonal agents, and agents such as asparaginase or hydroxyurea), corticoids (steroids), antidepressants, depressants, diuretics, hypnotics, minerals, nutritional supplements, parasympathomimetics, hormones (such as corticotrophin releasing hormone, adrenocorticotropin, growth hormone releasing hormone, growth hormone, thyrptropin- releasing hormone and thyroid stimulating hormone), sedatives, sulfonamides, stimulants, sympathomimetics,
  • Subjects suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects, and are preferably mammalian.
  • Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates, humans, and the like, and mammals in utero. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects are preferred.
  • Illustrative avians include chickens, ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) and domesticated birds (e.g., parrots and canaries), and birds in ovo.
  • the present invention is primarily concerned with the treatment of human subjects, but the invention can also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes, and for drug screening and drug development purposes.
  • the compounds of the present invention or an appropriate pharmaceutical composition thereof may be administered in an effective amount. Since the activity of the compounds and the degree of the therapeutic effect vary, the actual dosage administered will be determined based upon generally recognized factors such as age, condition of the subject, route of delivery and body weight of the subject.
  • the dosage can be from about 0.1 to about 100 mg/kg, administered orally 1-4 times per day.
  • compounds can be administered by injection at approximately 0.01 - 20 mg kg per dose, with administration 1-4 times per day. Treatment could continue for weeks, months or longer. Determination of optimal dosages for a particular situation is within the capabilities of those skilled in the art.
  • the compounds of the present invention can be used for the prevention and treatment of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, bone disorders, genetic disorders, hyperproliferative disorders, inflammatory disorders and combinations thereof where the disorder may be the result of multiple underlying maladies.
  • the disease or disorder is irritable bowel syndrome (IBS), non-ulcer dyspepsia, Crohn's disease, gastroesophogeal reflux disorders, constipation, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, carcinoid syndrome, malabsorption syndrome, diarrhea, diabetes including diabetes mellitus (type II diabetes), obesity, atrophic colitis, gastritis, gastric stasis, constipation, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, celiac disease, an eating disorder or obesity.
  • the disease or disorder is congestive heart failure, ischemic heart disease or chronic heart disease.
  • the disease or disorder is osteoporosis and/or frailty, congestive heart failure, accelerating bone fracture repair, metabolic syndrome, attenuating protein catabolic response, cachexia, protein loss, impaired or risk of impaired wound healing, impaired or risk of impaired recovery from burns, impaired or risk of impaired recovery from surgery, impaired or risk of impaired muscle strength, impaired or risk of impaired mobility, altered or risk of altered skin thickness, impaired or risk of impaired metabolic homeostasis or impaired or risk of impaired renal homeostasis.
  • the disease or disorder involves facilitating neonatal development, stimulating growth hormone release in humans, maintenance of muscle strength and function in humans, reversal or prevention of frailty in humans, prevention of catabolic side effects of glucocorticoids, treatment of osteoporosis, stimulation and increase in muscle mass and muscle strength, stimulation of the immune system, acceleration of wound healing, acceleration of bone fracture repair, treatment of renal failure or insufficiency resulting in growth retardation, treatment of short stature, treatment of obesity and growth retardation, accelerating the recovery and reducing hospitalization of burn patients, treatment of intrauterine growth retardation, treatment of skeletal dysplasia, treatment of hypercortisolism, treatment of Cushing's syndrome, induction of pulsatile growth hormone release, replacement of growth hormone in stressed patients, treatment of osteochondrodysplasias, treatment of Noonans syndrome, treatment of schizophrenia, treatment of depression, treatment of Alzheimer's disease, treatment of emesis, treatment of memory loss, treatment of reproduction disorders, treatment of delayed wound healing, treatment of
  • inventions provide for methods of treatment of inflammatory disorders, including ulcerative colitis, inflammatory bowel disease, Crohn's disease, pancreatitis, rheumatoid arthritis, osteoarthritis, asthma, vasculitis, psoriasis, allergic rhinitis, peptic ulcer disease, postoperative intra-abdominal sepsis, ischemia-reperfusion injury, pancreatic and liver damage, sepsis and septic shock, gastric damage caused by certain drugs, stress-induced gastric damage, gastric damage caused by H. pylori, inflammatory pain, chronic kidney disease and intestinal inflammation.
  • inflammatory disorders including ulcerative colitis, inflammatory bowel disease, Crohn's disease, pancreatitis, rheumatoid arthritis, osteoarthritis, asthma, vasculitis, psoriasis, allergic rhinitis, peptic ulcer disease, postoperative intra-abdominal sepsis, ischemia-reperfusion injury, pancreatic
  • a method for the treatment of postoperative ileus, cachexia (wasting syndrome), such as that caused by cancer, AIDS, cardiac disease and renal disease, gastroparesis, such as that resulting from type I or type II diabetes, other gastrointestinal disorders, growth hormone deficiency, bone loss, and other age-related disorders in a human or animal patient suffering therefrom comprises administering to said patient an effective amount of at least one member selected from the compounds disclosed herein having the ability to modulate the ghrelin receptor.
  • diseases and disorders treated by the compounds disclosed herein include short bowel syndrome, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, celiac disease, and hyperproliferative disorders such as tumors, cancers, and neoplastic disorders, as well as premalignant and non-neoplastic or non-malignant hyperproliferative disorders.
  • tumors, cancers, and neoplastic tissue that can be treated by the present invention include, but are not limited to, malignant disorders such as breast cancers, osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas, leukemias, lymphomas, sinus tumors, ovarian, uretal, bladder, prostate and other genitourinary cancers, colon, esophageal and stomach cancers and other gastrointestinal cancers, lung cancers, myelomas, pancreatic cancers, liver cancers, kidney cancers, endocrine cancers, skin cancers and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas.
  • malignant disorders such as breast cancers, osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas
  • leukemias lymphomas
  • sinus tumors ovarian, uretal, bladder, prostate
  • the macrocyclic compounds of the present invention can be used to treat postoperative ileus.
  • the compounds of the present invention can be used to treat gastroparesis.
  • the compounds of the present invention can be used to treat diabetic gastroparesis.
  • the compounds of the present invention can be used to treat gastric stasis.
  • the compounds of the present invention can be used to treat opioid-induced bowel dysfunction.
  • the compounds of the present invention can be used to treat chronic intestinal pseudoobstruction.
  • the compounds of the present invention can be used to treat postoperative ileus, paralytic ileus following surgical or other manipulation, gastroparesis, opioid-induced bowel dysfunction, chronic intestinal pseudo-obstruction, acute colonic pseudo-obstruction (Ogilvie's syndrome), enteric dysmotility, short bowel syndrome, emesis, constipation-predominant irritable bowel syndrome (IBS), chronic constipation, functional dyspepsia, cancer-associated dyspepsia syndrome, graft versus host disease, gastric stasis or hypomotility caused by various diseases such as diabetes or by the administration of other pharmaceutical agents, or in enterally fed patients, delayed gastric emptying, gastrointestinal dysfunction or delayed gastric emptying occurring in conjunction with other disease states, gastrointestinal dysmotility or delayed gastric emptying occurring in critical care situations, gastrointestinal dysfunction or delayed gastric emptying as a result of treatment with pharmaceutical agents, gastroesophageal reflux disease (GERD), gastric ulcers, gastroente
  • the present invention further provides methods of treating a horse or canine for a gastrointestinal disorder comprising administering a therapeutically effective amount of a modulator having the structure of formula I.
  • the gastrointestinal disorder is ileus or colic.
  • treatment is not necessarily meant to imply cure or complete abolition of the disorder or symptoms associated therewith.
  • the compounds of the present invention can further be utilized for the preparation of a medicament for the treatment of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, genetic disorders, bone disorders, hyperproliferative disorders and inflammatory disorders.
  • the table below presents binding activity at the human ghrelin receptor for representative compounds of the invention.
  • Kj values are expressed as follows: A ⁇ 10 nM, B ⁇ 100 nM, C ⁇ 500 nM, D > 500 nM.
  • This tether in a representative protected form was synthesized from the monobenzoate of resorcinol (205-1) using the multi-step process shown in Figure 3.
  • the desired compound was accessed from 2-hydroxybenzaldehyde (206-1) as outlined in Figure 4.
  • the presence of one fixed chiral center would permit the separation of the two diastereoisomers of the hydroxyl chiral center to be isolated by preparative HPLC.
  • the desired tether was constructed as shown below.
  • the protected tether T209 was prepared from an intermediate in the T207 synthesis as presented below.
  • the (R)-isomer of the ⁇ -hydroxy derivative of 4-fluorophenylalanine can be prepared as outlined in Figure 7 using 4-fluorobenzaldehyde (2-2) as the substrate for an Evans chiral aldol reaction (Evans, D. A. Aldrichimica Acta 1982, 15, 23) followed by a chiral azidation through the intermediate bromide.
  • Evans chiral aldol reaction Evans, D.A.; Britton, T.C.; Ellman, J.A.; Dorow, R.L, J. Am. Chem. Soc. 1990, 112, 4011-4030; Evans, D.A.; Ellman, J.A.; Dorow, R.L. Tet. Lett. 1987, 28, 1123-1126.
  • Subsequent hydrolytic cleavage and Staudinger reaction provided the free amino acid, which can be protected using standard methods.

Abstract

The present invention provides novel conformationally-defined macrocyclic compounds that have been demonstrated to be selective modulators of the ghrelin receptor (growth hormone secretagogue receptor, GRLN, GHS-R1a and subtypes, isoforms and variants thereof). Methods of synthesizing the novel compounds are also described herein. These compounds are useful as agonists of the ghrelin receptor and as medicaments for treatment and prevention of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, bone disorders, genetic disorders, hyperproliferative disorders and inflammatory disorders.

Description

Modified Macrocyclic Ghrelin Receptor Modulators and
Methods of Using the Same
Related Application Data
This application claims priority to U.S. Provisional Application serial number 61/347,166, filed May 21, 2010. The disclosure of which is incorporated herein by reference in its entirety.
Field of the Invention
The present invention relates to novel conformationally-defined macrocyclic compounds that bind to and/or are functional modulators, in particular agonists, of the ghrelin (growth hormone secretagogue) receptor including GRLN (GHS-Rla) and subtypes, isoforms and/or variants thereof. The present invention also relates to intermediates of these compounds, pharmaceutical compositions containing these compounds and methods of using the compounds. These novel macrocyclic compounds are useful as therapeutics for a range of disease indications. In particular, these compounds are useful for treatment and prevention of gastrointestinal disorders including, but not limited to, postoperative ileus, gastroparesis, including diabetic and postsurgical gastroparesis, gastric stasis, opioid bowel dysfunction, chronic intestinal pseudo-obstruction, short bowel syndrome, constipation, functional gastrointestinal disorders and gastrointestinal dysmotility, such as that occurring in conjunction with other disease states, in critical care situations or as a result of treatment with pharmaceutical agents. Additionally, the pharmaceutical compositions have application to the treatment and prevention of metabolic and/or endocrine disorders, cardiovascular disorders, central nervous system disorders, bone disorders, inflammatory disorders, hyperproliferative disorders and genetic disorders.
Background of the Invention
Ghrelin is a recently characterized 28-amino acid peptide hormone isolated originally from the stomach of rats with the orthologue subsequently identified in humans, distinguished by an unusual n-octanoyl group modification on Ser3. (Kojima, ML; Hosoda, H. et al. Nature 1999, 402, 656-660; Kojima, M. Regul.Pept. 2008, 145, 2-6.) The existence of this hormone in a wide range of other species suggests a conserved and important role in normal physiological function. The ghrelin peptide has been demonstrated to be the endogenous ligand for a previously orphan G protein-coupled receptor (GPCR), type 1 growth hormone secretatogue receptor (GHS-Rla) (Howard, A.D.; Feighner, S.D.; et al. Science 1996, 273, 974-977; U.S. Pat. No. 6,242,199; Intl. Pat. Appl. Nos. WO 97/21730 and WO 97/22004). GHS-Rla has recently been reclassified as the ghrelin receptor (GRLN) in recognition of its endogenous ligand (Davenport, A.P.; et al. Pharmacol Rev. 2005, 57, 541-546). GRLN is found predominantly in the brain, in particular the arcuate nucleus and ventromedial nucleus in the hypothalamus, hippocampus and substantia nigra) and pituitary, but also is expressed in a number of other tissues and organs (Gnanapavan, S.; Kola, B.; Bustin, S.A.; et al. J Clin, Endocrinol Metab. 2002, 87, 2988-2991; Ueberberg, B.; Unger, N.; Saeger, W.; Mann, K.; Petersenn, S. Horm. Metab. Res. 2009, 41, 814-821).
The ghrelin peptide has been found to have a variety of endocrine and non-endocrine functions (Broglio, F.; Gottero, C; Arvat, E.; Ghigo, E. Horm. Res. 2003, 59, 109-117; Hosoda, H.; Kojima, M.; Kangawa, K. J. Pharmacol Sci. 2006, 100, 398-410; Gasco, V.; Beccuti, G.; Marotta, F.; Benso, A.; Granata, R.; Broglio, F.; Ghigo, E. Endocr. Dev. 2010, 17, 86-95) and this range of actions has led to the pursuit of modulators of the ghrelin receptor for a number of therapeutic purposes. (Kojima, M.; Kangawa, K. Nat. Clin. Pract. Endocrinol. Metab. 2006, 2, 80-88; Akamizu, T.; Kangawa, K. Endocr. J. 2006, 53, 585- 591; Leite-Moreira, A.F.; Soares, J.-B. Drug Disc. Today 2007, 12, 276-288; Katergari, S.A.; Milousis, A.; Pagonopoulou, O.; Asimakopoulos, B.; Nikolettos, N.K. Endocr. J.
2008, 55, 439-453; Angelidis, G.; Valotassiou, V.; Georgoulias, P. J Endocrinol Invest. 2010, 33, 823-838; Nass, R.; Gaylinn, B.D.; Thomer, M.O. Mol. Cell. Endocrinol. 2011, doi: 10.1016/j.mce.201 1.02.010) For example, ghrelin and ghrelin agonists have been demonstrated to have positive effects in wasting syndromes, such as cachexia. (Kamiji, M.M.; Inui, A. Curr. Opin. Clin. Nutr. Metab. Care 2008, 11, 443-451; DeBoer, M.D. Nutrition 2008, 24, 806-814; Ashitani, J.; Matsumoto, N.; Nakazato, M. Peptides 2009, 30, 1951-1956; Akamizu, T.; Kangawa, K. J. Cachex. Sarcopenia Muscle 2010, 1, 169-176; Ueno, H.; Shiiya, T.; Nakazato, M. Ann. NY Acad. Sci. 2010, 1200, 120-127.) Clinical trials have been initiated with certain of these agonists to take advantage of these effects. (Garcia, J.M.; Polvino, W.J. The Oncologist 2007, 12, 594-600; Strasser, F.; Lutz, T.A.; Maeder, M.T. Br. J. Cancer 2008, 98, 300-308; Garcia, JM.; Polvino, W.J. Growth Horm. IGF Res.
2009, 19, 267-273.) These agents also have been investigated as intervention agents in aging. (Smith, .G.; Sun, Y.; Jiang, H.; Albarran-Zeckler, R.; Timchenko, N. Ann. N. Y. Acad. Sci. 2007, 1119, 147-164; Nass, R.; Pezzoli, S.S.; Oliveri, M.C.; et al. Ann. Intern. Med. 2008, 7^, 601-611.)
As another example, the prokinetic effect of ghrelin in the gastrointestinal (GI) system makes ghrelin agonists useful for therapeutic purposes in disorders characterized by GI dysmotility or hypomotility. (Peeters, T.L, Curr. Opin. Pharmacol. 2006, 6, 553-558; Sanger, G.J. Drug Disc. Today 2008, 13, 234-239; Venkova, K.; Greenwood-Van Meerveld, B. Curr. Opin. Invest. Drugs 2008, 9, 1103-1107; DeSmet, B.; Mitselos, A.; Depoortere, I. Pharmacol. Ther. 2009, 123, 207-223; Camilleri, M.; Papathanasopoulos, A.; Odunsi, S.T. Nat. Rev. Gastroenterol. Hepatol 2009, 6, 343-352; El-Salhy, M. Int. J. Mol. Med. 2009, 24, 727-732; Deane, A.M.; Fraser, R.J.; Chapman, MJ. Crit. Care Resusc. 2009, 11, 132-143; Jeffery, P.; McDonald, V.; Tippett, E.; McGuckin, M. Mol. Cell. Endocrinol. 2011, doi: 10.1016/j.mce.2011.03.002; Greenwood-Van Meerveld, B.; Kriegsman, M.; Nelson, R. Peptides 2011, doi: 10.1016/j.peptides.2011.03.014.) Such disorders include, but are not limited to, postoperative ileus, paralytic ileus, gastroparesis, including diabetic and postsurgical gastroparesis, gastric stasis, including in enterally fed. patients, constipation, includmg chronic constipation, opioid bowel dysfunction, chronic intestinal pseudo -obstruction, short bowel syndrome, functional gastrointestinal disorders and gastrointestinal dysmotility occurring in conjunction with other disease states, such as in critical care situations or as a result of treatment with other pharmaceutical agents. Ghrelin agonists also have application as therapeutics for the treatment of cardiovascular diseases (Nagaya, N.; Kangawa, K. Drugs 2006, 66, 439-448; Garcia, E.A.; Karbonits, M. Curr. Opin. Pharmacol 2006, 6, 142-147; Isgaard, J.; Barlind, A.; Johansson, I. Cardiovasc. Hematol. Disord. Drug Targets 2008, 8, 133-137; Zhang, G.; Yin, X.; Qi, Y.; Pendyala, L.; Chen, J.; Hou, D.; Tang, C. Curr. Cardiol Rev. 2010, 6, 62-70;Isgaard, J.; Granata, R. Mol Cell Endocrinol. 2011, doi: 10.1016/j.mce.2011.03.006), such as chronic heart failure, since ghrelin has been shown to be a powerful vasodilator, the treatment of bone disorders, such as osteoporosis (Svensson, J.; Lall, S.; Dickson, S.L. et al. Endocrine 2001, 14, 63-66; van der Velde, M.; Delhanty, P.; et al. Vitamins and Hormones 2007, 77, 239-258; Wong, LP.; Baldock, P.A.; Herzog, H. Curr. Opin. Endocrinol. Diabetes Obes. 2010, 17, 44-50; Nikolopoulos, D.; Theocharis, S.; Kouraklis, G. Med. Sci. Monit. 2010, 16, RA147-RA162; Napoli, N.; Pedone, C; Pozzilli, P.; Lauretani, F.; Bandinelli, S.; Ferrucci, L.; Incalzi, R.A. Bone 2011, doi: 10.1016/j.bone.2011.03.772), as anti-angiogenic agents for hyperproliferative disorders such as cancer (Baiguera, S.; Concord, M.T.; Guidolin, D.; et al. Int. J. Mol. Med. 2004, 14, 849-854; Conconi, M.T.; Nico, B.; Guidolin, D.; et al. Peptides 2004, 25, 2179-2185; Nikolopoulos, D.; Theocharis, S.; Kouraklis, G. Regul Pept.
2010, 163, 7-17), for regulating reproduction (Tena-Sempere, M,; Nat. Clin. Pract. Endocrinol Metab. 2008, 4, 666-674; Mitchell, G.A. Int. J. Obes. 2009, 33, S41-S47;
Unniappan, S. Gen. Comp. Endocrinol. 2010, 167, 340-343) and for preventing, modulating or ameliorating conditions involving the CNS, including anxiety, depression, stress, reward and motivation, cognitive enhancement and sleep regulation. (Seoane, L.M.; Al-Massadi, O.; Lage, M.; Dieguez, C; Casanueva, F.F. Pediatr. Endocrinol. Rev. 2004, 1, 432-437; McNay, E.C. Curr. Opin. Pharmacol. 2007, 7, 628-632; Olszewski, P.K.; Schioth, H.B.; Levine, A.S. Brain Res. Rev. 2008, 58, 160-170; Ferrini, F.; Salio, C; Lossi, L.; Merighi, A. Curr. Neuropharmacol 2009, 7, 37-49; Atcha, Z.; Chen, W.S.; Ong, A.B.; et al. Psychopharmacol. 2009, 206, 415-427; Andrews, Z.B. Trends Neurosci. 2011, 34, 31-40; Steiger, A.; Dresler, M.; Schussler, P.; Kluge, M. Mol. Cell. Endocrinol. 2011, doi; 10.1016/j.mce.201 1.02.013; Dickson, S.L.; Egeciogiu, E.; Landgren, S.; Skibicka, K.P.; Engel, J.A.; Jerlhag, E. Mol. Cell. Endocrinol. 2011, doi: 10.1016/j.mce.2011.02.017) Lastly, ghrelin plays a role in immune function and exhibits anti-inflammatory actions, hence ghrelin agonists can be applied to the treatment and prevention of inflammatory disorders. (Vixit, V.D.; Taub, D.D. Exp. Gerontol. 2005, 40, 900-910; Taub, D.D. Vitamins and. Hormones 2007, 77, 325-346; Baatar, D.; Patel, K.; Taub, D.D. Mol. Cell. Endocrinol.
2011, doi: 10.1016/j.mce.2011.04.019.)
A series of macrocyclic peptidomimetics recently has been described as modulators of the ghrelin receptor and their uses for the treatment and prevention of a range of medical conditions including metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, genetic disorders, hyperproliferative disorders and inflammatory disorders outlined (U.S. Pat. Nos. 7,452,862, 7,476,653, 7,491,695 and RE42013; Intl. Pat. Appl. Publ. Nos. WO 2006/009645, WO 2006/009674, WO 2006/046977, WO 2006/137974, WO 2008/130464, WO 2011/041369 and WO 2011/053821; U.S. Pat. Appl. Publ. Nos. 2006/025566, 2007/021331, 2008/051383 and 2008/194672). The activity of one of these macrocycles, compound 298, in a rat model of POI has been reported. (Venkova, K.; Fraser, G.; Hoveyda, H.R.; Greenwood-Van Meerveld, B. Dig. Dis. Set 2007, 52, 2241-2248; Fraser, G.L.; Venkova, K.; Hoveyda, H.R.; Thomas, H.; Greenwood- Van Meerveld, B. Eur. J Pharmacol. 2009, 604, 132-137.) In contrast to other types of ghrelin agonists, compound 298 did not stimulate concurrent GH secretion in these animal models. (Fraser, G.L.; Hoveyda, H.R.; Tannenbaum, G.S. Endocrinology 2008, 149, 6280-6288.) Compound 298 has exhibited appropriate safety and pharmacokinetic properties in humans (Lasseter, K.C.; Shaughnessy, L.; Cummings, D.; et al. J. Clin. Pharmacol. 2008, 48, 193-202) and also has proven to have GI prokinetic actions in patients suffering from postoperative ileus (POI, Popescu, I.; Fleshner,P.R.; Pezzullo, J.C.; Charlton, P. A.; Kosutic, G.; Senagore, A.J. Dis. Colon Rectum 2010, 53, 126-134) and gastroparesis (Ejskjaer, N.; Vestergaard, E.T.; Hellstrom, P.M.; Gormsen, L.C.; Madsbad, S.; Madsen, J.L.; Jensen, T.A.; Pezzullo, J.C.; Christiansen, J.S.; Shaughnessy, L.; Kosutic, G. Aliment. Pharmacol. Ther. 2009, 29, 1179- 1187; Wargin, W.; Thomas, H.; Clohs, L,; St-Louis, C; Ejskjaer, N.; Gutierrez, M.; Shaughnessy, L.; Kosutic, G. Clin. Drug Investig. 2009, 29, 409-418; Ejskjaer, N.; Dimcevski, G.; Wo, J.; et al. Nenrogastro. Motil. 2010, 22, 1069-e281; Wo, J.M.; Ejskjaer, N.; Hellstrom, P.M.; et al. Aliment. Pharmacol. Ther. 2011, 33, 679-688).
Figure imgf000007_0001
(Compound 298)
Similar structures to compound 298 have been reported as antagonists of the motilin receptor and their uses for the treatment of a variety of GI disorders and to modulate the migrating motor complex summarized. (U.S. Pat. Nos. 7,452,862 and 7,521,420; Eur. Pat.
Nos. 1 633 774 and 1 648 922; Intl. Pat. Appl. Publ. WO 2004/111077; U.S. Pat Appl. Publ. 2005/054562; Marsault, E.; Hoveyda, H.R.; Peterson, M.L.; Saint-Louis, C; Landry,
A.; Vezina, M.; Ouellet, L.; Wang, Z.; Ramaseshan, M.; Beaubien, S.; Benakli, K.;
Beauchemin, S.; Deziel, R.; Peeters, T.; Fraser, G.L. J. Med. Chem. 2006, 49, 7190-7197;
Marsault, E.; Benakli, K.; Beaubein, S.; Saint-Louis, C; Deziel, R.; Fraser, G. Bioorg Med.
Chem. Lett. 2007, 17, 4187-4190.) In this series, introduction of a hydroxyl group in these structures resulted in approximately a 100-fold weaker activity. The motilin receptor is the most homologous receptor to the ghrelin receptor, sharing 52% homology, 86% in the key transmembrane domains. (Kojima, M.; Kangawa, K. Physiol. Rev. 2005, 85, 495-522.)
Compounds of the invention were found surprisingly to possess very potent ghrelin receptor binding activity in contrast to the guidance of the prior art. These compounds, generally exhibit beneficial physico chemical properties and can also permit access to alternative structures and derivatives not possible from the unsubstituted macrocycle.
Hence, the compounds of the invention provide a number of advantages compared with the macrocyclic ghrelin modulators previously known.
Summary of the Invention
The present invention provides novel conformationally-defmed macrocyclic compounds of formula (I) and pharmaceutically acceptable salts thereof. These compounds can function as modulators, in particular agonists, of the ghrelin (growth hormone secretagogue) receptor (GRLN GHS-Rla) and subtypes, isoforms and variants thereof.
Figure imgf000008_0001
wherein:
Ri is Ci-C4 alkyl;
R2 is halogen or hydroxyl;
W is hydrogen or hydroxyl;
Xi, X2, X3 and X4 are independently hydrogen or hydroxyl with the proviso that no more than two of Xls X2, X3 and X4 can be hydroxyl;
Yi and Y2 are independently hydrogen or hydroxyl; and
i and Z2 are independently hydrogen or hydroxyl with the proviso that both are not hydroxyl. Aspects of the present invention further provide pharmaceutical compositions including a compound of formula (I) and a pharmaceutically acceptable carrier, excipient or diluent.
Other aspects provide specific compounds of formula (I).
Aspects of the present invention provide methods of treating a gastrointestinal disorder, a metabolic or endocrine disorder, a cardiovascular disorder, an inflammatory disorder, a bone disorder, a hyperproliferative disorder or a genetic disorder, including administering to a subject in need thereof an effective amount of a compound of formula I.
Additional aspects of the present invention provide kits comprising one or more containers containing pharmaceutical dosage units comprising an effective amount of one or more compounds of the present invention packaged with optional instructions for the use thereof.
Aspects of the present invention further provide methods of stimulating gastrointestinal motility, modulating GRLN (GHS- la) receptor activity in a mammal and/or treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a modulator that modulates a mammalian GRLN (GHS-Rla) receptor. In still other embodiments, the modulator is a compound of formula (I).
Additional aspects of the present invention provide methods of diagnosing tumors and/or acromegaly, comprising administering compounds of the present invention and a radiolabeled metal binding agent and detecting the binding of the composition to a biological target, and treating tumors and/or acromegaly comprising administering a therapeutically effective amount of a composition comprising a compound of the present invention.
Other aspects of the invention provide methods of treating a patient suffering from reduced or dysfunctional gastrointestinal motility caused by a first medicament, pharmaceutical or pharmaceutical composition, wherein the first medicament, pharmaceutical or pharmaceutical composition is being employed to treat the patient for a metabolic, hyperproliferative or other disorder, with compounds of formula (I).
Further aspects of the present invention relate to methods of making the compounds of formula (I).
Aspects of the present invention further relate to methods of preventing and/or treating disorders described herein, in particular, gastrointestinal disorders, including postoperative ileus, paralytic ileus, gastroparesis, such as diabetic and postsurgical gastroparesis, gastric stasis, opioid bowel dysfunction, chronic intestinal pseudoobstruction, short bowel syndrome, constipation, functional gastrointestinal disorders, gastrointestinal dysmotility, such as that occurring in conjunction with other disease states, in critical care situations or as a result of treatment with pharmaceutical agents, emesis such as caused by cancer chemotherapy, constipation such as associated with the hypomotility phase of irritable bowel syndrome (IBS), delayed gastric emptying associated with wasting conditions, gastroesophageal reflux disease (GERD), gastric ulcers, Crohn's disease, gastrointestinal disorders characterized by dysmotility or hypomotility, and other diseases and disorders of the gastrointestinal tract.
In particular embodiments, the gastrointestinal disorder is postoperative ileus, paralytic ileus caused by surgery or other manipulations, gastroparesis, opioid-induced bowel dysfunction, gastric stasis or hypomotility caused by various diseases such as diabetes or by the administration of other pharmaceutical agents, or in enterally fed patients, chronic intestinal pseudo-obstruction, acute colonic pseudo-obstruction (Ogilvie's syndrome), enteric dysmotility, short bowel syndrome, emesis, constipation-predominant irritable bowel syndrome (IBS), chronic constipation, functional dyspepsia, cancer- associated dyspepsia syndrome, graft versus host disease, delayed gastric emptying, gastrointestinal dysfunction or delayed gastric emptying occurring in conjunction with other disease states, gastrointestinal dysmotility or delayed gastric emptying occurring in critical care situations, gastrointestinal dysfunction or delayed gastric emptying as a result of treatment with pharmaceutical agents, gastroesophageal reflux disease (GERD), gastric ulcers, gastroenteritis, colitis and Crohn's disease.
The present invention also relates to compounds of formula (I) used for the preparation of a medicament for prevention and/or treatment of the disorders described herein.
The foregoing and other aspects of the present invention are explained in greater detail in the specification set forth below.
Brief Description of the Drawings
Figure 1 shows a concentration-response graph for activation of the ghrelin receptor mediated signaling pathway with an exemplary compound of the present invention.
Figure 2 shows a concentration-response graph for activation of the ghrelin receptor mediated signaling pathway with another exemplary compound of the present invention. Figure 3 shows a synthetic scheme for a representative tether building block of the present invention.
Figure 4 shows a synthetic scheme for another representative tether building block of the present invention.
Figure 5 shows a synthetic scheme for another representative tether building block of the present invention.
Figure 6 shows a synthetic scheme for a representative amino acid building block of the present invention.
Figure 7 shows a synthetic scheme for another representative amino acid building block of the present invention.
Figure 8 shows a synthetic scheme for a representative compound of the present invention.
Detailed Description
The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
All publications, U.S. patent applications, U.S. patents and other references cited herein are incorporated by reference in their entireties.
The term "alkyl" refers to straight or branched chain saturated or partially unsaturated hydrocarbon groups having from 1 to 20 carbon atoms, in some instances 1 to 8 carbon atoms. The term "lower alkyl" refers to alkyl groups containing 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, isopropyl, tert-butyl, 3-hexenyI, and 2-butynyl. By "unsaturated" is meant the presence of 1, 2 or 3 double or triple bonds, or a combination of the two. Such alkyl groups may also be optionally substituted as described below.
When a subscript is used with reference to an alkyl or other hydrocarbon group defined herein, the subscript refers to the number of carbon atoms that the group may contain. For example, C2-C4 alkyl indicates an alkyl group with 2, 3 or 4 carbon atoms.
The term "cycloalkyl" refers to saturated or partially unsaturated cyclic hydrocarbon groups having from 3 to 15 carbon atoms in the ring, in some instances 3 to 7, and to alkyl groups containing said cyclic hydrocarbon groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopropylmethyl, cyclopentyl, 2-(cyclohexyl)ethyl, cycloheptyl, and cyclohexenyl. Cycloalkyl as defined herein also includes groups with multiple carbon rings, each of which may be saturated or partially unsaturated, for example decalinyl, [2.2.1]-bicycloheptanyl or adamantanyl. All such cycloalkyl groups may also be optionally substituted as described below.
The term "aromatic" refers to an unsaturated cyclic hydrocarbon group having a conjugated pi electron system that contains 4n+2 electrons where n is an integer greater than or equal to 1. Aromatic molecules are typically stable and are depicted as a planar ring of atoms with resonance structures that consist of alternating double and single bonds, for example benzene or naphthalene.
The term "aryl" refers to an aromatic group in a single or fused carbocyclic ring system having from 6 to 15 ring atoms, in some instances 6 to 10, and to alkyl groups containing said aromatic groups. Examples of aryl groups include, but are not limited to, phenyl, 1 -naphthyl, 2-naphthyl and benzyl. Aryl as defined herein also includes groups with multiple aryl rings which may be fused, as in naphthyl and anthracenyt, or unfused, as in 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, indanyl or tetrahydronaphthyl (tetralinyl). All such aryl groups may also be optionally substituted as described below.
The term "heterocycle" or "heterocyclic" refers to saturated or partially unsaturated monocyclic, bicyclic or tricyclic groups having from 3 to 15 atoms, in some instances 3 to 7, with at least one heteroatom in at least one of the rings, said heteroatom being selected from O, S or N. Each ring of the heterocyclic group can contain one or two O atoms, one or two S atoms, 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 fused rings completing the bicyclic or tricyclic heterocyclic groups may contain only carbon atoms and may be saturated or partially unsaturated. The N and S atoms may optionally be oxidized and the N atoms may optionally be quaternized. Heterocyclic also refers to alkyl groups containing said monocyclic, bicyclic or tricyclic heterocyclic groups. Examples of heterocyclic rings include, but are not limited to, 2- or 3-piperidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl. All such heterocyclic groups may also be optionally substituted as described below
The term "heteroaryl" refers to an aromatic group in a single or fused ring system having from 5 to 15 ring atoms, in some instances 5 to 10, which have at least one heteroatom in at least one of the rings, said heteroatom being selected from O, S or N. Each ring of the heteroaryl group can contain one or two O atoms, one or two S atoms, 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 fused rings completing the bicyclic or tricyclic groups may contain only carbon atoms and may be saturated, partially unsaturated or aromatic. In structures where the lone pair of electrons of a nitrogen atom is not involved in completing the aromatic pi electron system, the N atoms may optionally be quaternized or oxidized to the N-oxide. Heteroaryl also refers to alkyl groups containing said cyclic groups. 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, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, benzopyranyl, 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. All such heteroaryl groups may also be optionally substituted as described below.
The term "hydroxy" or "hydroxyl" refers to the group -OH.
The term "alkoxy" or "alkoxyl" refers to the group -ORa, wherein Ra is alkyl, cycloalkyl or heterocyclic. Examples include, but are not limited to methoxy, ethoxy, tert- butoxy, cyclohexyloxy and tetrahydropyranyloxy.
The term "aryloxy" refers to the group -0¾ wherein ¾ is aryl or heteroaryl. Examples include, but are not limited to phenoxy, benzyloxy and 2-naphthyloxy.
The term "acyl" refers to the group -C(=0)-Rc wherein Rc is alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl. Examples include, but are not limited to, acetyl, benzoyl and furoyl.
The term "amino acyl" indicates an acyl group that is derived from an amino acid. The term "amino" refers to an -NRdRe group wherein Rj and Re are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl. Alternatively, Rd and Re together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfmyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term "amido" refers to the group -C(=0)-NRfRg wherein and Rg are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aiyl and heteroaryl. Alternatively, Rf and Rg together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from 0, S or N.
The term "amidino" refers to the group -C(=NRh)NRiRj wherein Rh is selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl; and Rj and Rj are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl. Alternatively, Rj and Rj together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N. The term "carboxy" refers to the group -C02H.
The term "carboxyalkyl" refers to the group -CC^Rk, wherein ¾ is alkyl, cycioalkyl or heterocyclic.
The term "carboxyaryl" refers to the group -CC^Rm, wherein Rra is aryl or heteroaryl.
The term "cyano" refers to the group -CN.
The term "formyl" refers to the group -C(=0)H, also denoted -CHO,
The term "halo," "halogen" or "halide" refers to fluoro, fluorine or fluoride, chloro, chlorine or chloride, bromo, bromine or bromide, and iodo, iodine or iodide, respectively.
The term "oxo" refers to the bivalent group =0, which is substituted in place of two hydrogen atoms on the same carbon to form a carbonyl group.
The term "mercapto" refers to the group -SR„ wherein R„ is hydrogen, alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl.
The term "nitro" refers to the group -N0 .
The term "trifluoromethyl" refers to the group -CF3.
The term "sulfinyl" refers to the group -S(;=0)Rp wherein Rp is alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl.
The term "sulfonyl" refers to the group -S(=0)2-Rql wherein Rqi is alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl.
The term "aminosulfonyl" refers to the group -NRq2-S(=0)2-Rq3 wherein Rq2 is hydrogen, alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl; and Rq3 is alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl.
The term "sulfonamido" refers to the group -S(=0)2-NRrRs wherein Rr and Rs are independently selected from the group consisting of hydrogen, alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl. Alternatively, Rr and Rs together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycioalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from 0, S or N.
The term "carbamoyl" refers to a group of the formula -N(Rt)-C(=0)-ORu wherein Rt is selected from hydrogen, alkyl, cycioalkyl, heterocyclic, aryl or heteroaryl; and Ru is selected from alkyl, cycioalkyl, heterocylic, aryl or heteroaryl. The term "guanidino" refers to a group of the formula -N(Rv)-C(=NRw)-NRxRy wherein Rv, Rw, Rx and Ry are independently selected from hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl. Alternatively, Rx and Ry together form a heterocyclic ring or 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term "ureido" refers to a group of the formula -N(Rz)-C(=0)-NRaaRbb wherein Rz, Raa and Rb are independently selected from hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl. Alternatively, Raa and R b together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term "optionally substituted" is intended to expressly indicate that the specified group is unsubstituted or substituted by one or more suitable substituents, unless the optional substituents are expressly specified, in which case the term indicates that the group is unsubstituted or substituted with the specified substituents. As defined above, various groups may be unsubstituted or substituted (i.e., they are optionally substituted) unless indicated otherwise herein (e.g., by indicating that the specified group is unsubstituted).
The term "substituted" when used with the terms alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl refers to an alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl group having one or more of the hydrogen atoms of the group replaced by substituents independently selected from unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, halo, oxo, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino, ureido and groups of the formulas -NRccC(=0)Rdd, -NReeC(=NRff)Rgg, -OC(=0)NRhhRiij -OC(=0)Rjj, -OC(=0)ORkk, -NRmmS02Rnn, or -NRPpS02NRqqRrr wherein R^, dd, Ree, Rff, Rgg, Rhh! Ru, ¾, Rmm, RPP, Rqq and Rrr are independently selected from hydrogen, unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl or unsubstituted heteroaryl; and wherein Rkk and Rnn are independently selected from unsubstituted alkyl, unsubstituted cycloalkyi, unsubstituted heterocyclic, unsubstituted aryl or unsubstituted heteroaryl. Alternatively, Rgg and Rhh, Rj and kk or Rpp and Rqq together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyi, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N. hi addition, the term "substituted" for aryl and heteroaryl groups includes as an option having one of the hydrogen atoms of the group replaced by cyano, nitro or trifluoromethyl,
A substitution is made provided that any atom's normal valency is not exceeded and that the substitution results in a stable compound. Generally, when a substituted form of a group is present, such substituted group is preferably not further substituted or, if substituted, the substituent comprises only a limited number of substituted groups, in some instances 1, 2, 3 or 4 such substituents.
When any variable occurs more than one time in any constituent or in any formula herein, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
A "stable compound" or "stable structure" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity and formulation into an efficacious therapeutic agent.
The term "amino acid" refers to the common natural (genetically encoded) or synthetic amino acids and common derivatives thereof, known to those skilled in the art. When applied to amino acids, "standard" or "proteinogenic" refers to the genetically encoded 20 amino acids in their natural configuration. Similarly, when applied to amino acids, "unnatural" or "unusual" refers to the wide selection of non-natural, rare or synthetic amino acids such as those described by Hunt, S. in Chemistry and Biochemistry of the Amino Acids, Barrett, G.C., Ed., Chapman and Hall: New York, 1985.
The term "residue" with reference to an amino acid or amino acid derivative refers to a group of the formula:
Figure imgf000018_0001
wherein RAA is an amino acid side chain, and n = 0, 1 or 2 in this instance.
The term "fragment" with respect to a dipeptide, tripeptide or higher order peptide derivative indicates a group that contains two, three or more, respectively, amino acid residues.
The term "amino acid side chain" refers to any side chain from a standard or unnatural amino acid, and is denoted RAA- For example, the side chain of alanine is methyl, the side chain of valine is isopropyl and the side chain of tryptophan is 3-indolyImethyl.
The term "agonist" refers to a compound that duplicates at least some of the effect of the endogenous ligand of a protein, receptor, enzyme or the like.
The term "antagonist" refers to a compound that inhibits at least some of the effect of the endogenous ligand of a protein, receptor, enzyme or the like.
The term "growth hormone secretagogue" (GHS) refers to any exogenously administered compound or agent that directly or indirectly stimulates or increases the endogenous release of growth hormone, growth hormone-releasing hormone, or somatostatin in an animal, in particular, a human. A GHS may be peptidic or non-peptidic in nature, in some instances, with an agent that can be administered orally. In some instances, the agent can induce a pulsatile response.
The term "modulator" refers to a compound that imparts an effect on a biological or chemical process or mechanism. For example, a modulator may increase, facilitate, upregulate, activate, inhibit, decrease, block, prevent, delay, desensitize, deactivate, down regulate, or the like, a biological or chemical process or mechanism. Accordingly, a modulator can be an "agonist" or an "antagonist." Exemplary biological processes or mechanisms affected by a modulator include, but are not limited to, receptor binding and hormone release or secretion. Exemplary chemical processes or mechanisms affected by a modulator include, but are not limited to, catalysis and hydrolysis.
The term "variant" when applied to a receptor is meant to include dimers, trimers, tetramers, pentamers and other biological complexes containing multiple components. These components can be the same or different. The term "peptide" refers to a chemical compound comprised of two or more amino acids covalently bonded together.
The term "peptidomimetic" refers to a chemical compound designed to mimic a peptide, but which contains structural differences through the addition or replacement of one of more functional groups of the peptide in order to modulate its activity or other properties, such as solubility, metabolic stability, oral bioavailability, lipophilicity, permeability, etc. This can include replacement of the peptide bond, side chain modifications, truncations, additions of functional groups, etc. When the chemical structure is not derived from the peptide, but mimics its activity, it is often referred to as a "non- peptide peptidomimetic."
The term "peptide bond" refers to the amide [-C(=0)-NH-] functionality with which individual amino acids are typically covalently bonded to each other in a peptide.
The term "protecting group" refers to any chemical compound that may be used to prevent a potentially reactive functional group, such as an amine, a hydroxyl or a carboxyl, on a molecule from undergoing a chemical reaction while chemical change occurs elsewhere in the molecule. A number of such protecting groups are known to those skilled in the art and examples can be found in "Protective Groups in Organic Synthesis," Theodora W. Greene and Peter G. Wuts, editors, John Wiley & Sons, New York, 3rd edition, 1999 [ISBN 0471160199]. Examples of amino protectmg groups include, but are not limited to, phthalimido, trichloroacetyl, benzyloxycarbonyl, fert-butoxycarbonyl, and adamantyloxycarbonyl. In some embodiments, amino protecting groups are carbamate amino protecting groups, which are defined as an amino protecting group that when bound to an amino group forms a carbamate. In other embodiments, amino carbamate protecting groups are allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc) and α,α-dimethyl- 3,5-dimethoxybenzyloxycarbonyl (Ddz). For a recent discussion of newer nitrogen protecting groups: Theodoridis, G. Tetrahedron 2000, 56, 2339-2358. Examples of hydroxyl protecting groups include, but are not limited to, acetyl, ier/-butyldimethylsilyl (TBDMS), trityl (Trt), fert-butyl, and tetrahydropyranyl (THP). Examples of carboxyl protecting groups include, but are not limited to methyl ester, tert-bv y\ ester, benzyl ester, trimethylsilylefhyl ester, and 2,2,2-trichloroethyl ester.
The term "solid phase chemistry" refers to the conduct of chemical reactions where one component of the reaction is covalently bonded to a polymeric material (solid support as defined below). Reaction methods for performing chemistry on solid phase have become more widely known and established outside the traditional fields of peptide and oligonucleotide chemistry.
The term "solid support," "solid phase" or "resin" refers to a mechanically and chemically stable polymeric matrix utilized to conduct solid phase
chemistry. This is denoted by "Resin," "P-" or the following symbol:
Figure imgf000020_0001
Examples of appropriate polymer materials include, but are not limited to, polystyrene, polyethylene, polyethylene glycol, polyethylene glycol grafted or covalently bonded to polystyrene (also termed PEG-polystyrene, TentaGel™, Rapp, W.; Zhang, L,; Bayer, E. In Innovations and Persepctives in Solid Phase Synthesis. Peptides, Polypeptides and Oligonucleotides; Epton, R.? Ed.; SPCC Ltd.: Birmingham, UK; p 205), polyacrylate (CLEAR™), polyacrylamide, polyurethane, PEGA [polyethyleneglycol poly(N,N-dimethylacrylamide) co-polymer, Meldal, M. Tetrahedron Lett. 1992, 33, 3077- 3080], cellulose, etc. These materials can optionally contain additional chemical agents to form cross-linked bonds to mechanically stabilize the structure, for example polystyrene cross-linked with divinylbenezene (DVB, usually 0.1-5%, preferably 0.5-2%). This solid support can include as non-limiting examples aminomethyl polystyrene, hydroxymethyl polystyrene, benzhydrylamine polystyrene (BHA), methylbenzhydrylamine (MBHA) polystyrene, and other polymeric backbones containing free chemical functional groups, most typically, -NH2 or -OH, for further derivatization or reaction. The term is also meant to include "Ultraresins" with a high proportion ("loading") of these functional groups such as those prepared from polyethyleneimines and cross-linking molecules (Barth, M.; Rademann, J. J. Comb. Chem. 2004, 6, 340-349). At the conclusion of the synthesis, resins are typically discarded, although they have been shown to be able to be reused such as in Frechet, J.M.J.; Haque, K.E. Tetrahedron Lett. 1975, 16, 3055.
In general, the materials used as resins are insoluble polymers, but certain polymers have differential solubility depending on solvent and can also be employed for solid phase chemistry. For example, polyethylene glycol can be utilized in this manner since it is soluble in many organic solvents in which chemical reactions can be conducted, but it is insoluble in others, such as diethyl ether. Hence, reactions can be conducted homogeneously in solution, then the product on the polymer precipitated through the addition of diethyl ether and processed as a solid. This has been termed "liquid-phase" chemistry. The term "linker" when used in reference to solid phase chemistry refers to a chemical group that is bonded covalently to a solid support and is attached between the support and the substrate typically in order to permit the release (cleavage) of the substrate from the solid support. However, it can also be used to impart stability to the bond to the solid support or merely as a spacer element. Many solid supports are available commercially with linkers already attached.
Abbreviations used for amino acids and designation of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972, 247, 977-983. This document has been updated: Biochem. J, 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; Internal J. Pept. Prot. Res., 1984, 24, following p 84; J Biol Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; and in Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pp 39-67. Extensions to the rules were published in the JCBN/NC-IUB Newsletter 1985, 1986, 1989; see Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pp 68-69.
The term "effective amount" or "effective" is intended to designate a dose that causes a relief of symptoms of a disease or disorder as noted through clinical testing and evaluation, patient observation, and/or the like, and/or a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and/or further quantified by one skilled in the art for the relevant mechanism or process. As is generally understood in the art, the dosage will vary depending on the administration routes, symptoms and body weight of the patient but also depending upon the compound being administered.
Administration of two or more compounds "in combination" means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two compounds can be administered simultaneously (concurrently) or sequentially. Simultaneous administration can be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration. The phrases "concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.
The term "pharmaceutically active metabolite" is intended to mean a pharmacologically active product produced through metabolism in the body of a specified compound.
The term "solvate" is intended to mean a pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates, without limitation, include compounds of the invention in combination with water (termed hydrates), isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
1. Compounds
Novel macrocyclic compounds of the present invention include macrocyclic compounds comprising a building block structure including a tether component that undergoes cyclization to form the macrocyclic compound. The building block structure can comprise amino acids (standard and unnatural), hydroxy acids, hydrazino acids, aza-amino acids, specialized moieties such as those that play a role in the introduction of peptide surrogates and isosteres, and a tether component as described herein.
The present invention includes isolated compounds. An isolated compound refers to a compound that, in some embodiements, comprises at least 10%, at least 25%, at least 50% or at least 70% of the compounds of a mixture. In some embodiments, the compound, pharmaceutically acceptable salt thereof or pharmaceutical composition containing the compound exhibits a statistically significant binding and/or antagonist activity when tested in biological assays at the human ghrelin receptor.
In the case of compounds, salts, or solvates that are solids, it is understood by those skilled in the art that the inventive compounds, salts, and solvates may exist in different crystal or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulas.
The compounds disclosed herein may have asymmetric centers. The inventive compounds may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the scope of the present invention. In particular embodiments, however, the inventive compounds are used in optically pure form. The terms "S" and "R" configuration as used herein are as defined by the IUPAC 1974 Recommendations for Section E, Fundamentals of Stereochemistry (Pure Appl Chem. 1976, 45, 13-30).
Unless otherwise depicted to be a specific orientation, the present invention accounts for all stereoisomeric forms. The compounds may be prepared as a single stereoisomer or a mixture of stereoisomers. The non-racemic forms may be obtained by either synthesis or resolution. The compounds may, for example, be resolved into the component enantiomers by standard techniques, for example formation of diastereomeric pairs via salt formation. The compounds also may be resolved by covalently bonding to a chiral moiety. The diastereomers can then be resolved by chromatographic separation and/or crystal lographic separation. In the case of a chiral auxiliary moiety, it can then be removed. As an alternative, the compounds can be resolved through the use of chiral chromatography. Enzymatic methods of resolution could also be used in certain cases.
As generally understood by those skilled in the art, an "optically pure" compound is one that contains only a single enantiomer. As used herein, the term "optically active" is intended to mean a compound comprising at least a sufficient excess of one enantiomer over the other such that the mixture rotates plane polarized light. Optically active compounds have the ability to rotate the plane of polarized light. The excess of one enantiomer over another is typically expressed as enantiomeric excess (e.e.). In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes "d" and "1" or (+) and (-) are used to denote the optical rotation of the compound (i.e., the direction in which a plane of polarized light is rotated by the optically active compound). The "1" or (-) prefix indicates that the compound is levorotatory (i.e., rotates the plane of polarized light to the left or counterclockwise) while the "d" or (+) prefix means that the compound is dextrarotatory (i.e., rotates the plane of polarized light to the right or clockwise). The sign of optical rotation, (-) and (+), is not related to the absolute configuration of the molecule, R and S.
A compound of the invention having the desired pharmacological properties will be optically active and, can be comprised of at least 90% (80% e.e.), at least 95% (90% e.e.), at least 97.5% (95% e.e.) or at least 99% (98% e.e.) of a single isomer.
Likewise, many geometric isomers of double bonds and the like can also be present in the compounds disclosed herein, and all such stable isomers are included within the present invention unless otherwise specified. Also included in the invention are tautomers and ro tamers of the compounds.
The use of the following symbols at the right refers to NH) substitution of one or more hydrogen atoms of the indicated ring
Figure imgf000024_0001
with the defined substituent .
The use of the following symbol indicates a single bond or an optional double bond:
Embodiments of the present invention further provide intermediate compounds formed through the synthetic methods described herein to provide the compounds of formula I and/or II. The intermediate compounds may possess utility as a therapeutic agent for the range of indications described herein and/or a reagent for further synthesis methods and reactions.
2. Synthetic Methods
The compounds of the present invention can be synthesized using traditional solution synthesis techniques or solid phase chemistry methods. In either, the construction involves four phases: first, synthesis of the building blocks comprising recognition elements for the biological target receptor, plus one tether moiety, primarily for control and definition of conformation. These building blocks are assembled together, typically in a sequential fashion, in a second phase employing standard chemical transformations. The precursors from the assembly are then cyclized in the third stage to provide the macrocyclic structures. Finally, the post-cyclization processing fourth stage involving removal of protecting groups, if necessary, and optional purification provides the desired final compounds. Synthetic methods for this general type of macrocyclic structure are described in Intl. Pat. Appls. WO 01/25257, WO 2004/111077, WO 2005/012331, WO 2005/012332, WO 2006/009645, WO 2006/009674, WO 2008/033328, WO 2008/130464, WO 2011/050270 and WO 2011/050276, including purification procedures described in WO 2004/111077 and WO 2005/012331. Solution phase synthesis routes, including methods amenable to larger scale manufacture, were described in U.S. Patent Appl. Publ. Nos. 2006/025566, US 2007/0021331 and WO 2011/053821.
In some embodiments of the present invention, the macrocyclic compounds may be synthesized using solid phase chemistry on a soluble or insoluble polymer matrix as previously defined. For solid phase chemistry, a preliminary stage involving the attachment of the first building block, also termed "loading," to the resin must be performed. The resin utilized for the present invention preferentially has attached to it a linker moiety. These linkers are attached to an appropriate free chemical functionality, usually an alcohol or amine, although others are also possible, on the base resin through standard reaction methods known in the art, such as any of the large number of reaction conditions developed for the formation of ester or amide bonds. Some linker moieties, such as the thioester linker, for the present invention are designed to allow for simultaneous cleavage from the resin with formation of the macrocycle in a process generally termed "cyclization-release." (van Maarseveen, J.H. Solid phase synthesis of heterocycles by cyclization/cleavage methodologies. Comb. Chem. High Throughput Screen. 1998, 1, 185-214; Ian W. James, Linkers for solid phase organic synthesis. Tetrahedron 1999, 55, 4855-4946; Eggenweiler, H.-M. Linkers for solid-phase synthesis of small molecules: coupling and cleavage techniques. Drug Discovery Today 1998, 3, 552-560; Backes, B.J.; Ellman, J.A. Solid support linker strategies. Curr. Opin. Chem. Biol. 1997, 1, 86-93. Of particular utility in this regard for compounds of the invention is the 3-thiopropionic acid linker. (Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117; Zhang, L.; Tarn, J. J. Am. Chem. Soc. 1999, 121, 3311-3320.)
Such a process provides material of higher purity as only cyclic products are released from the solid support and no contamination with the linear precursor occurs as would happen in solution phase. After sequential assembly of all the building blocks and tether into the linear precursor using known or standard reaction chemistry, base-mediated intramolecular attack on the carbonyl attached to this linker by an appropriate nucleophilic functionality that is part of the tether building block results in formation of the amide or ester bond that completes the cyclic structure as shown (Scheme 1). An analogous methodology adapted to solution phase can also be applied as would likely be preferable for larger scale applications.
Scheme 1. Cyclization-release Strategy
Figure imgf000025_0001
Although this description accurately represents the pathway for one of the methods of the present invention, the thioester strategy, another method of the present invention, that of ring-closing metathesis ( CM), proceeds through a modified route where the tether component is actually assembled during the cyclization step. However, in the RCM methodology as well, assembly of the building blocks proceeds sequentially, followed by cyclization (and release from the resin if solid phase). An additional post-cyclization processing step is required to remove particular byproducts of the RCM reaction, but the remaining subsequent processing is done in the same manner as for the thioester or analogous base-mediated cyclization strategy.
Moreover, it will be understood that steps including the methods provided herein may be performed independently or at least two steps may be combined. Additionally, steps including the methods provided herein, when performed independently or combined, may be performed at the same temperature or at different temperatures without departing from the teachings of the present invention.
Novel macro cyclic compounds of the present invention include those formed by a novel process including cyclization of a building block structure to form a macrocyclic compound comprising a tether component described herein. Accordingly, the present invention provides methods of manufactuiing the compounds of the present invention comprising (a) assembling building block structures, (b) chemically transforming the building block structures, (c) cyclizing the building block structures including a tether component, (d) removing protecting groups from the building block structures, and (e) optionally purifiying the product obtained from step (d). In some embodiments, assembly of the building block structures may be sequential. In further embodiments, the synthesis methods are carried out using traditional solution synthesis techniques or solid phase chemistry techniques.
A. Amino acids
Amino acids, Boc- and Fmoc-protected amino acids and side chain protected derivatives, including those of N-methyl and unnatural amino acids, were obtained from commercial suppliers [for example Advanced ChemTech (part of CreoSalus, Louisville, KY, USA), Astatech (Bristol, PA, USA), Bachem (Bubendorf, Switzerland), Chemlmpex (Wood Dale, IL, USA), Novabiochem (subsidiary of Merck KGaA, Darmstadt, Germany), PepTech (Burlington, MA, USA), Synthetech (Albany, OR, USA)] or synthesized through standard methodologies known to those in the art. Ddz-amino acids were either obtained commercially from Orpegen (Heidelberg, Germany) or Advanced ChemTech (part of CreoSalus, Louisville, Y, USA) or synthesized using standard methods utilizing Ddz-OPh or Ddz-N3. (Birr, C; Lochinger, W.; Stahnke, G.; Lang, P. Justus Lie bigs Ann. Chem. 1972, 763, 162-172.) Bts-amino acids were synthesized by known methods. (Vedejs, E.; Lin, S.; Klapara, A.; Wang, J. J. Am. Chem. Soc. 1996, 118, 9796-9797, also WO 01/25257, WO 2004/1 1 1077) N-Alkyl amino acids, in particular N-methyl amino acids, are commercially available from multiple vendors (Bachem, Novabiochem, Advanced ChemTech, Chemlmpex). In addition, N-alkyl amino acid derivatives were accessed via literature methods. (Hansen, D. W., Jr.; Pilipauskas, D. J. Org. Chem. 1985, 50, 945-950.) The β- hydroxy derivatives of 4-fluorophenylalanine can be synthesized as described in the Examples or in the same manner as described for β-hydroxyphenylalanine stereoisomers. (Jung, M.E.; Jung, Y.H. Tet. Lett. 1989, 30, 6637 - 6640.)
B. Tethers
Tethers were obtained from the methods previously described in Intl. Pat. Appl.
Nos. WO 01/25257, WO 2004/111077, WO 2005/012331, WO 2006/009645, WO 2006/009674, WO 2008/130464 and WO 2011/053821. Procedures for the synthesis of representative tethers as described herein are also presented in the Examples below. Tethers useful for the compounds of the present invention can include suitable tethers as described in the references cited above as well as known and/or novel tethers described herein. Exemplary tethers include, but are not limited to, the following:
Figure imgf000028_0001
where PGj is an amine protecting group and PG2 and PG3 are the same or different hydroxyl protecting groups.
C. Solid and Solution Phase Techniques
Synthetic methods for this general type of macrocyclic structure are described in Intl. Pat. Appls. WO 01/25257, WO 2004/111077, WO 2005/012331, WO 2005/012332, WO 2006/009645, WO 2006/009674, WO 2008/033328, WO 2008/130464, WO 2011/050270 and WO 2011/050276 including purification procedures described in WO 2004/111077 and WO 2005/012331. Solution phase synthesis routes, including methods amenable to larger scale manufacture, were described in U.S. Patent Appl. Publ. Nos. 2006/025566, US 2007/0021331 and Intl. Pat. Appl. WO 2011/053821.
The table following provides information on the building blocks used for the synthesis of representative compounds of the present invention using the standard methods. A suitable protecting group(s) for the building block is typically indicated. These are directly applicable to solution phase synthesis. For solid phase syntheses, modified protection strategies from that illustrated are typically employed to permit the use of a convergent approach. Additional synthetic details for the solution phase construction of representative macrocyclic compounds of the invention are presented in the Examples.
Synthesis of Representative Compounds of the Invention
Figure imgf000030_0001
The table below presents analytical data for representative compounds of the present invention.
Analytical Data for Representative Compounds of the Invention
Figure imgf000031_0001
11 C30H37FN4O5 552.6 553 525 (-CO), 507
(-CO-H20), 440,
422, 383, 258
Notes
1. Molecular formulas and molecular weights are calculated automatically from the structure via ChemBioUltra software (CambridgeSoft, Cambridge, MA, USA).
2. M+H obtained from LC-MS analysis using standard methods.
3. All analyses conducted on material after preparative purification.
3. Biological Methods
The compounds of the present invention were evaluated for their ability to interact at the human ghrelin receptor. A competitive radioligand binding assay, fluorescence assay or Aequorin functional assay can be employed. Such methods can be conducted in a high throughput manner to permit the simultaneous evaluation of many compounds.
Specific assay methods for the human (GRLN, hGHS-Rla), swine and rat GHS- receptors (U.S. Pat. No. 6,242,199, Intl. Pat. Appl. Nos. WO 97/21730 and 97/22004), as well as the canine GHS-receptor (U.S. Pat. No. 6,645,726), and their use in generally identifying agonists and antagonists thereof are known.
Appropriate methods for determining the functional and in vivo activity of compounds of the present invention that interact at the human ghrelin receptor are also described below. In addition, methods established in the art can be used to determine other parameters important for use as pharmaceutical agents, such as pharmacokinetics, Caco-2 permeability, plasma protein binding.
A. Competitive Radioligand Binding Assay (Ghrelin Receptor)
A competitive binding assay at the human growth hormone secretagogue receptor (GRLN, hGHS-Rla) can be carried out analogously to assays described in the literature. (Bednarek, M.A.; et al J. Med. Chem, 2000, 43, 4370-4376; Palucld, B.L.; et ai. Bioorg. Med. Chem. Lett. 2002, 11, 1955-1957.) See also U.S. Patent Nos. 7,491,695 and 7,476,653.
B. Aequorin Functional Assay (Ghrelin Receptor)
The functional activity of compounds of the invention found to bind to the GRLN (GHS-Rla) receptor can be determined using the methods described in the literature, which can also be used as a primary screen for ghrelin receptor activity in a high throughput fashion. See also U.S. Patent Nos. 7,491,695 and 7,476,653. (LePoul, E,; et al. J. Biomol Screen. 2002, 7, 57-65; Bednarek, M.A.; et al. J. Med. Chem. 2000, 43, 4370- 4376; Palucki, B,L.; et al. Bioorg. Med. Chem. Lett. 2001, 11, 1955-1957.)
C. IPl Functional Assay (Ghrelin Receptor)
The in-vitro functional potency of compounds of the invention as activators of the ghrelin receptor mediated signaling pathway can also be determined using HEK-293 cells stably expressing the human GRLN (GHS-Rla).
Summary
Several metabolites in the inositol phosphates pathway, including IP3, have extremely short half lives, making them difficult to quantify accurately. IPl, a downstream metabolite of IP3, accumulates in cells following Gq receptor activation and is stable in the presence of LiCl, making it an ideal read out of receptor activation. The IP-One assay is a competitive immunoassay that uses cryptate-labeled anti-IP 1 MAb and d2-labeled IPl. In the absence of endogenous IPl, cryptate Mab and IP-d2 interact and produce a quantifiable FRET (fluorescence energy transfer) signal.
Materials
HEK-293 cells stably expressing human GRLN receptor. The cells were produced with the plasmid expressing the GRLN from Missouri University of Science and Technology (cat# GHSR0A0000).
96-well microtiter plates: white, flat-bottom from Falcon (cat # 353296).
Culture medium and reagents: DMEM from Gibco (cat# 11995) containing 10% fetal bovine serum (FBS) (Gibco cat#12483). A solution containing 100 units of penicillin G sodium, 100 μg ml of streptomycin sulfate, 292 μ§/πύ L-glutamine and 100 μΜ sodium citrate was added to the culture medium (Gibco cat# 1789). A solution of 0.25% Trypsin- EDTA was used to detach the cells (Gibco cat# 25200). Phosphate buffered saline (PBS) (Gibco cat# 10010).
Detection kit: IP-One HTRF® assay (CisBio, Bedford, MA, USA, cat # 62P1APEC) quantifies myo -Inositol- 1 phosphate (IPl). Cell preparation
Cells were plated in 96-weli plates the day before the experiment and incubated in a C02 incubator (5% C02) at 37°C.
The cell contents of one confluent 10 cm Petri dish were split into one 96-well plate (approx 100,000 cells/well) as follows:
• The Petri dish was washed with 10 ml PBS, treated with a 0,25% trypsin-EDTA solution for 5 min at 37°C. The detached cells were resuspended in 20 ml of DMEM, 10% FBS, pen/strep/glut.
* 200 μΐ of resuspended cells were distributed in each well.
Methods
All compounds were tested in duplicate each in three independent experiments typically at the following concentrations: 0 nM , 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μΜ, 10 μΜ
Ghrelin dilution:
1. Ghrelin stock solution: 1 mM in ¾0.
2. 1st dilution: 1/ 0 in H20 (final concentration: 0.1 mM).
3. Serial dilutions were made in the 96-well plate.
Test compound dilution:
1. All compounds were prepared as stock solutions at 1 mM in 100 % DMSO.
2. 1st dilution: 10 μΐ of stock solution in 90 μΐ ¾0 (100 μΜ final, 10% DMSO).
3. 2nd dilution: 10 μΐ of 100 μΜ solution in 90 μΐ H20 (10 μΜ final).
4. Serial dilutions were performed in stimulation buffer to obtain the final test concentrations.
Stimulation buffer, pH 7.4:
• HEPES 10 mM
• CaCl2 1 mM
• MgCl2 0.5 mM
• C1 4.2 mM
• NaCl 146 mM
• Glucose 5.5 mM
• LiCl 50 mM Assay Protocol:
1. Remove plates from the C02 incubator
2. Discard culture medium from the plate by inversion
3. Remove traces of culture medium with a micropipette
4. Add 70 μΐ of compound in stimulation buffer/well at 10 sec interval
5. Incubate in a water bath for 30 min at 37°C
6. Add ^l of cAMP-d2/well
7. Add 15 μΐ of anti-cAMP-cryptate/well
8. Incubate 1 h at room temp on an orbital shaker at 100 RPM
Read the fluorescence of wells with the GeniosPro (Tecan) fluorescence reader or similar instrument
Data Analysis
Results are calculated from the 665 nm/620 nm ratio and expressed in Delta F. Delta F calculation was performed according to the manufacturer as follows:
1. Ratio = (Fluorescence 665 nm / Fluorescence 620 nm) X 10 000.
2. Delta F = (Sample ratio - Neg. control ratio *) / Neg. control ratio X 100
* Neg. control = fluorescence measurement of wells where IPl-d2 was absent. Data were analyzed with the curve-fitting program XLFit4 (IDBS)
D. Pharmacokinetic Analysis of Representative Compounds of the Invention
The pharmacokinetic behavior of compounds of the invention can be ascertained by methods well known to those skilled in the art. (Wilkinson, G. R. "Pharmacokinetics: The Dynamics of Drug Absorption, Distribution, and Elimination" in Goodman & Oilman's The Pharmacological Basis of Therapeutics, Tenth Edition, Hardman, J.G.; Limbird, L.E., Eds., McGraw Hill, Columbus, OH, 2001, Chapter 1.) The following method was used to investigate the pharmacokinetic parameters (elimination half-life, total plasma clearance, etc.) for intravenous, subcutaneous and oral administration of compounds of the present invention. See also U.S. Patent Nos. 7,491,695 and 7,476,653and Intl. Pat. Appl. No. WO 2008/033328. E. Gastric Emptying in Fasted Rat Model
To examine the effects of compounds of the invention in a model for gastroparesis, compounds were evaluated for possible effects on gastric emptying in fasted rats. This model is used to determine the potential of compounds of the invention to promote motility in fasted rats.
Methods
1. Overnight-fasted rats (male, Wistar, 200g, n = 5/group) were given a meal (2 mL) of methylcellulose (2%) by intragastric gavage. The meal was labeled with phenol red (0.05%).
2. Test articles (dosed at various concentrations, typically 1, 3, 10, 30 mg kg), vehicle and positive control (metoclopramide, a 5-HT ligand and prokinetic agent currently prescribed for the treatment of GI disorders, including gastroparesis) were administered by oral gavage immediately after the meal (time = 0).
3. Animals were sacrificed 15 minutes later; the stomach was immediately removed and homogenized in 0.1 N NaOH and centrifuged.
4. Total phenol red remaining in the stomach was quantified by a colorimetric method at 560 nin.
5. A 30% or more increase in gastric emptying (as compared to the vehicle control) was considered significant.
6. One-way ANOVA, Dunnet's post-hoc statistical test was applied.
F. Opioid-Delayed Gastric Emptying
The following protocol is used for the study of the effects of representative compounds of the invention on Gastric emptying and GI transit in a rat model of opioid- delayed gastric emptying.
Opioid analgesics, such as morphine, are well known to delay gastrointestinal transit which is an important side-effect for this class of drugs. The clinical term for this syndrome is opioid bowel dysfunction (OBD). Importantly, patients recovering from abdominal surgery experience post-operative ileus that is further exacerbated by concomitant opioid therapy for post-surgical pain. The objective of this procedure is to determine whether compounds of the invention may have therapeutic utility in the treatment of OBD. Methods
1. Rats (male, Sprague-Dawley, 250-300 g) are implanted with jugular vein catheters to accommodate dosing of test articles.
2. Overnight-fasted rats are administered morphine (3 mg/kg s.c).
3. After 30 min, rats are to be dosed with vehicle or test compound at appropriate dose levels (for example 300 or 1000 μg/kg, i.v., n = 4-to-6/gp) followed by intragastric gavage of 99mTc methylcellulose (2%) meal
4. After 15 min, the rats are euthanized and the stomach and consecutive 10 cm
segments of the intestine are isolated. Radioactivity ( 9mTc) in each tissue isolate is measured as a means of measuring the transit of the meal.
G. Gastroparesis Animal Model
High caloric meals are well known to impede gastric emptying. This observation has recently been exploited by Megens, A.A.; et al. (unpublished) to develop a rat model for delayed gastric emptying as experienced in gastroparesis.
Materials
1. Wistar rats, male, 200-250 g
2. Chocolate test meal: 2 mL Clinutren ISO® (1.0 kcal/mL, Nestle SA, Vevey
Switzerland)
Method
The test meal is given to the subjects by oral gavage at time = 0. After 60 min, the subjects are sacrificed, the stomachs excised and the contents weighed.
Test compounds are administered intravenously as aqueous solutions, or solutions in normal saline, at time = 0 typically at three dose levels (for example 0.08 mg/kg; 0.30- 0.31 mg/kg, 1.25 mg/kg). When necessary, cyclodextrin (CD) was added to solubilize the material. Test compounds to be examined utilizing subcutaneous injection are administered at time = -30 min. Four to five (4-5) rats were tested per group, except in the case of the cyclodextrin control in which ten (10) rats comprise the group.
Results are reported as percentage relative to the stomach weight for injection only of solvent as a control to illustrate the gastric emptying capability of the compounds of the present invention. H. Gastric Emptying and Intestinal Transit in Rat Model of Postoperative Ileus
This clinically relevant model for POI is adapted from that of Kalff. (Kalff, J.C.; Schraut, W.H.; Simmons, R.L.; Bauer, AJ. Ann. Surg. 1998, 228, 652-663.) Other known models can also be used to study the effect of compounds of the invention. (Trudel, L.; Bouin, M.; Tomasetto, C; Eberling, P.; St-Pierre, S.; Bannon, P.; L'Heureux, M.C.; Poitras, P. Peptides 2003, 24, 531-534; (b) Trudel, L.; Tomasetto, C; Rio, M.C.; Bouin, M.; Plourde, V.; Eberling, P.; Poitras, P. Am. J. Physiol. 2002, 282, G948-G952.)
Animals
I. Rat, Sprague-Dawley, male, -300 g.
2. Fasted O/N prior to study.
Induction of post-operative ileus (POI)
1. Isofluorane anaesthesia under sterile conditions.
2. Midline abdominal incision.
3. Intestines and caecum are eviscerated and kept moist with saline.
4. The intestines and caecum are manipulated along its entire length with moist
cotton applicators analogous to the 'running of the bowel' in the clinical setting. This procedure was timed to last for 10 min.
5. Intestines are gently replaced into the abdomen and the abdominal wound was stitched closed under sterile conditions.
Dosing
1. Rat are allowed to recover from isofluorane anaesthesia.
2. Test compounds (or vehicle) are administered intravenously via previously
implanted jugular catheter.
3. Immediate intragastric gavage of methylcellulose (2%) labeled with radioactive 99mTc, t = 0.
Experimental
1. At t = 15 min, animal are euthanized with C02.
2. Stomach and 10 cm sections along the small intestine are to be immediately
ligated, cut and placed in tubes for measuring of 99mTc in gamma counter.
3. Stomach emptying and small intestinal transit are measured by calculation of the geometric mean.
Geometric mean =∑(%totai radioactivity X number of segment)/100 I. Growth Hormone Response to Test Compounds
The compounds of the invention likewise can be tested in a number of animal models for their effect on GH release. For example, rats (Bowers, C.Y.; Momany, F.; Reynolds, G.A.; Chang, D.; Hong, A.; Chang, K. Endocrinology 1980, 106, 663-667), dogs (Hickey, G.; Jacks, T.; Judith, F.; Taylor, J.; Schoen, W.R.; rupa, D.; Cunningham, P.; Clark, J.; Smith, R.G. Endocrinology 1994, 134, 695-701; Jacks, T.; Hickey, G.; Judith, F.; Taylor, J.; Chen, H.; Krupa, D.; Feeney, W.; Schoen, W.R.; Ok, D.; Fisher, M,; Wyvratt, M.; Smith, R. J Endocrinology 1994, 143, 399-406; Hickey, G.J.; Jacks, T.M.; Schleim, K.D.; Frazier, E.; Chen, H.Y.; Krupa, D.; Feeney, .; Nargund, R.P.; Patchett, A.A.; Smith, R.G. J. Endocrinol. 1997, 152, 183-192), and pigs (Chang, C.H.; Rickes, E.L.; Marsilio, F,; McGuire, L.; Cosgrove, S.; Taylor, J.; Chen, H.Y.; Feighner, S.; Clark, J.N.; Devita, R.; Schoen, W.R.; Wyvratt, M.; Fisher, M.; Smith, R.G.; Hickey, G. Endocrinology 1995, 136, 1065-1071; (b) Peschke, B.; Hanse, B.S. Bioorg. Med. Chem. Lett. 1999, 9, 1295-1298) have all been successfully utilized for the in vivo study of the effects of GHS and would likewise be applicable for investigation of the effect of ghrelin agonists on GH levels. The measurement of ghrelin of GH levels in plasma after appropriate administration of compounds of the invention can be performed using radioimmunoassay via standard methods known to those in the art. (Deghenghi, R.; et al. Life Sciences 1994, 54, 1321-1328.) Binding to tissue can be studied using whole body autoradiography after dosing of an animal with test substance containing a radioactive label. (Ahnfelt-Rianne, I.; Nowak, J.; Olsen, U.B. Do growth hormone-releasing peptides act as ghrelin secretagogues? Endocrine 2001, 14, 133-135.)
The following method is employed to determine the temporal pattern and magnitude of the growth hormone (GH) response to test compounds, administered either systemically or centrally. Analogous methods can be used for other appropriate animal models, such as dogs and cynomolgus monkeys.
Dosing and sampling procedures for in vivo studies of GH release
Adult male Sprague Dawley rats (225-300 g) are purchased from Charles River Canada (St. Constant, Canada) and individually housed on a 12-h light, 12-h dark cycle (lights on, time: 0600-1800) in a temperature (22 + 1 ° C)- and humidity-controlled room. Purina rat chow (Ralston Purina Co., St. Louis, MO) and tap water are freely available. For these studies, chronic mtracerebroventricular (icv) and intracardiac venous cannulas are implanted under sodium pentobarbital (50 mg kg, ip) anesthesia using known techniques. The placement of the icv cannula are verified by both a positive drinking response to icv carbachol (100 ng/10 μΐ) injection on the day after surgery and methylene blue dye at the time of sacrifice. After surgery, the rats are placed directly in isolation test chambers with food and water freely available until body weight returned to preoperative levels (usually within 5-7 d). During this time, the rats are handled daily to minimize any stress associated with handling on the day of the experiment. On the test day, food is removed 1.5 h before the start of sampling and is returned at the end. Test samples at various dosing levels or normal saline were administered either intravenously or orally at two different time points during a 6-h sampling period. The times 1100 and 1300 are chosen because they reflect typical peak and trough periods of GH secretion, as previously documented. The human ghrelin peptide (5 g, Phoenix Pharmaceuticals, Inc., Belmont, CA) is used as a positive control in the experiments and was diluted in normal saline just before use. To assess the central actions of test compounds on pulsatile GH release, a 10- fold lower dose of the test sample or normal saline is administered icv at the same time points, 1100 and 1300. Blood samples (0.35 mL) is withdrawn every 15 min over the 6-h sampling period (time: 1000-1600) from all animals. To document the rapidity of the GH response to the test compound, an additional blood sample is obtained 5 min after each injection. All blood samples are immediately centrifuged, and plasma is separated and stored at -20° C for subsequent GH assay. To avoid hemodynamic disturbance, the red blood cells are resuspended in normal saline and returned to the animal after removal of the next blood sample. All animal studies are conducted under procedures approved by an animal care oversight committee.
GH assay method
Plasma GH concentrations are measured in duplicate by double antibody RIA using materials supplied by the NIDDK Hormone Distribution Program (Bethesda, MD). The averaged plasma GH values for 5-6 rats per group are reported in terms of the rat GH reference preparation. All samples with values above the range of interest are reassayed at dilutions ranging from 1 :2 to 1:10. J. Mouse Model of Cancer Cachexia
Tumor cachexia is considered the major reason for mortality, rapidly declining quality of life and limitation of therapy in advanced tumor patients. Since agonism of the ghrelin receptor has been associated with increased food intake and the generation of a positive overall energy balance, the compounds of the present invention have applications to the treatment of this disorder. The following method was designed to investigate the effects of test compounds as compared to ghrelin peptide on tumor cachexia in the G361 melanoma model grown as a subcutaneous xenograft in BALB/c nufnu mice. (Mori M, Yamaguchi K, Honda S, et al: Cancer Res, 1991, 51, 6656-6659.) Additional models are known in the art. (Emery, P.W. Nutrition 1999, 15, 600-603.)
For the method, 60 tumour-bearing mice are randomised 12 days post-inoculation into two sets of 5 groups containing 6 animals each. At initiation of treatment, the average body weight loss of Set 1 and Set 2 animals relative to the initial average body weight is determined. Treatment of Set 1 and 2 animals commences on Days 12 and 16, after tumor inoculation, respectively. Groups 1 and 6 receive vehicle i.v. s.c. or oral (depending on the mode of administration of the test compound) bid alone, while Groups 5 and 10 were administered rat ghrelin peptide s.c. (1 mg/kg; bid, 6 h apart) as a positive control. Test compounds are administered i.v., s.c. or oral twice daily, 6 h apart, at three dose levels (for example 3, 10, 30 mg/kg) for 20-40 consecutive days. Mice are culled during the study according to predetermined criteria including >15% initial body weight loss and/or tumor volume in excess of 2000 mm3 and/or display of severe clinical signs.
Body weights are measured, along with quantity of food and water consumption. In addition, plasma levels of cholesterol, triglyceride, non-esterified fatty acids and blood glucose are determined during the course of treatment to provide further measures of the effects of the test compounds on the overall health of the animal.
K. Ex-vivo Potency Evaluation on the Rat Stomach Fundus
This method is employed to evaluate the potency of compounds of the invention as a prokinetic agent by treatment of rat stomach fundus strips in an organ bath ex vivo in the presence or absence of electrical field stimulation (EFS) using ghrelin peptide as a reference, Methods
Fundus strips (approximately 0.4 x 1 cm) were cut from the stomach of adult male Wistar rats parallel to the circular muscle fibers. They were placed between two platinum ring electrodes, 1 cm apart ( adnoti, ADInstruments, USA) in 10 ml tissue baths containing Krebs solution bubbled with 5 % C02 in 02 and maintained at 37 °C. Tissues were suspended under 1.5 g resting tension. Changes of tension were measured isometrically with force transducers and recorded with a PowerLab 8/30 data acquisition system (ADInstruments, USA). Tissues were allowed to equilibrate for 60 min during which time bath solutions were changed every 15 min.
EFS was achieved by applying 0.5 ms pulses, 5 Hz frequency, at a maximally effective voltage of 70 V. EFS was applied for 30 sec at 3 min intervals for a 30 min initial period. This initial period was separated by a 5 min interval with wash out of the bath solution. Then, a second period of stimulation was started. After obtaining consistent EFS-evoked contractions (after three or four 30 sec stimulations), the effects of ghrelin, test compounds at various concentrations (for example 0.01-10 μΜ), L-NAME (300 μΜ, as control) or their respective vehicles, applied non-cumulatively, on responses to EFS were studied over a 30 min period. Responses to the agents were measured and expressed as % of the mean of three or four pre-drug responses to EFS. All compounds were dissolved at 1 mM in distilled water or MeOH, as stock solutions.
L. Plasma Protein Binding
The pharmacokinetic and pharmacodynamic properties of drugs are largely a function of the reversible binding of drugs to plasma or serum proteins such as albumin and oti-acid glycoprotein. In general, only unbound drug is available for diffusion or transport across cell membranes, and for interaction at the pharmacological target. On the other hand, drugs with low plasma protein binding generally have large volumes of distribution and rapid clearance since only unbound drug is available for glomerular filtration and, in some cases, hepatic clearance. Thus, the extent of plasma protein binding can influence efficacy, distribution and elimination. The ideal range for plasma protein binding is in the range of 87-98% for most drug products.
Protein binding studies were performed using human plasma. Briefly, 96-well microplates were used to incubate various concentrations of the test article for 60 min at 37°C. Bound and unbound fractions are separated by equilibrium dialysis, where the concentration remaining in the unbound fraction is quantified by LC-MS or LC-MS-MS analysis. Drugs with known plasma protein binding values such as quinine (-35%), warfarin (-98%) and naproxen (-99.7%) were used as reference controls.
M. Assay for Cytochrome P450 Inhibition
Cytochrome P450 enzymes are implicated in the phase I metabolism of drugs. The majority of drug-drug interactions are metabolism-based and, moreover, these interactions typically involve inhibition of cytochrome P450s. Six CYP450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) appear to be commonly responsible for the metabolism of most drugs and the associated drug-drug interactions. Assays to determine the binding of compounds of the invention to the various metabolically important isoforms of cytochrome P450 metabolizing enzymes are commercially available, for example NoAb BioDiscoveries (Mississaugua, ON, Canada) and Absorption Systems (Exton, PA, USA). As well, a number of appropriate methods have been described or reviewed in the literature. (White, R.E. Ann. Rev. Pharmacol. Toxicol. 2000, 40, 133-157; Li, A.P. Drug. Disc. Today 2001, 6, 357-366; Turpeinen, M.; orhonen, L.E. Tolonen, A.; et al. Eur. J. Pharm. Sci. 2006, 29, 130-138.)
Methods
1. Assay was performed on microsomes (Supersomes®, BD Gentest, Becton-Dickinson) prepared from insect cells expressing individual human CYP-450 subtypes, specifically:
- CYP subtypes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4
- Two substrates are typically tested for CYP-3A4 as this enzyme exhibits complex inhibition kinetics
2. Assays monitored, via fluorescence detection, the formation of a fluorescent metabolite following incubation of the microsomes with a specific CYP substrate.
3. Compounds of the present invention were tested in duplicate samples at eight test concentrations using 3-fold serial dilutions (concentration range of 0.0457 to 100 μΜ).
4. For each CYP-450 enzyme, a specific inhibitor was tested in duplicate at eight concentrations as a positive control.
5. The concentration of the inhibitor or test compound that inhibited metabolite formation by 50% (IC50) was calculated by non-linear regression analysis of the % inhibition vs. log concentration (M) curve. N. Determination of Caco-2 Permeability
The Caco-2 cell line, derived from a human colorectal carcinoma, has become an established in vitro model for the prediction of drug absorption across the human intestine. (Sun, D.; Yu, L.X.; Hussain, M.A.; Wall, D.A.; Smith, R.L.; Amidon, G.L. Curr. Opin. Drug Discov. Devel. 2004, 7, 75-85; Bergstrom, C.A. Basic Clin. Pharmacol Toxicol. 2005, 96, 156-61 ; Balimane, P.V.; Han, Y.H.; Chong, S. A APS J. 2006, 8, El-13; Shah, P.; Jogani, V.; Bagchi, T.; Misra, A. Biotechnol. Prog. 2006, 22, 186-198.) When cultured on semi-permeable membranes, Caco-2 cells differentiate into a highly functionalized epithelial barrier with remarkable morphological and biochemical similarity to the small intestinal columnar epithelium. Fully differentiated cell monolayers can be used to assess the membrane transport properties of novel compounds. In addition, the apparent permeability coefficients (Papp) obtained from Caco-2 cell transport studies have been shown to reasonably correlate with human intestinal absorption.
Assays to determine the permeability of compounds of the invention utilizing Caco-2 cells are commercially available, for example NoAb BioDiscoveries (Mississaugua, ON, Canada) and Absorption Systems (Exton, PA, USA).
Alternatively, parallel artificial membrane permeability assays (PAMPA) can be utilized to assess intestinal permeability. (Avdeef, A. Expert Opin. Drug. Metab. Toxicol. 2005, 1, 325-42.)
Methods
Permeability across the Caco-2 cell layer was determined by growing the cells on a membrane placed between two (donor and acceptor) chambers. Drug candidates are typically added to the apical (A) side of the cell layer and their appearance in the basolateral (B) side is measured over incubation time. Permeability in this direction represents intestinal absorption. Permeability may also be determined from the basolateral to the apical side of the Caco-2 cell. A higher apical to basolateral Papp, compared to the basolateral to apical Papp, is indicative of carrier-mediated transport. P-gp mediated transport is suggested when a higher basolateral to apical Papp is observed relative to die apical to basolateral Papp,
Permeability (10 μΜ) for compounds of the invention in the apical to basolateral and basolateral to apical direction were tested in duplicate. Samples will be collected from the donor and acceptor chambers at the beginning (0 min) and following 60 min of incubation at 37°C and stored frozen at -70° C until bioanalysis. Samples for each test compound generated from the Caco-2 permeability assay were further analyzed by LC- MS-MS. The permeability of [3H]-mannitol and [3H] -propranolol were determined in parallel as controls.
The permeability coefficient (PapP) of each compound and radiolabeled standard was determined using the following equation:
Figure imgf000045_0001
dT
where dQ/dT represents the permeability rate, C denotes the initial concentration in the donor compartment, and A represents the surface area of the filter. Q is determined from the mean concentration of duplicate samples taken prior to addition to the donor compartment. Permeability rates were calculated by plotting the cumulative amount of compound measured in the acceptor compartment over time and determining the slope of the line by linear regression analysis. The duplicate and mean apical to basolateral and basolateral to apical Papp's were reported for each compound and standard.
0. Activation-Desensitization Profile
It is well-known that agonists of G-protein coupled receptors can induce desensitization or tachyphylaxis, thereby limiting the potential of agents acting at the receptor as therapeutics for chronic use. (Luttrell, L.M. Methods Mol, Biol. 2006, 332, 3- 49; Kenakin, T. Ann. Rev. Pharmacol Toxicol. 2002, 42, 349-379; enakin, T. Nat. Rev. Drug Discov. 2002, 1, 103-110; Ferguson, S.S. Pharmacol. Rev. 2001, 53, 1-24.) This method is used to assess the receptor activation- desensitization profile of compounds of the present invention relative to reference agonists using HEK293 cells stably expressing hGHS-Rla.
Methods
1. Ghrelin, GHRP-6 and capromorelin were used as reference agonists
2. Agonist-induced Ca+2 fluxes were measured after loading with Ca+2 indicator Fluo-4- AM.
3. The negative logarithm of the agonist concentration causing 50% maximal stimulation of MjHS-Rla was calculated (pEC50)
4. The negative logarithm of the pre-incubation concentration reducing the maximum response to ghrelin to 50% of its control value was calculated (pDC5o) 5. To compare the relative activation-desensitization profile of ghrelin agonists, the difference between pEC5o and pDC5o values for individual compounds were calculated, with the higher positive numbers expected to have less desensitization potential and hence suitable for chronic applications as therapeutics.
4. Pharmaceutical Compositions
The macrocyclic compounds of the present invention or pharmacologically acceptable salts thereof according to the invention may be formulated into pharmaceutical compositions of various dosage forms. To prepare the pharmaceutical compositions of the invention, one or more compounds, including optical isomers, enantiomers, diastereomers, racemates or stereochemical mixtures thereof, or pharmaceutically acceptable salts thereof as the active ingredient is intimately mixed with appropriate carriers and additives according to techniques known to those skilled in the art of pharmaceutical formulations.
A pharmaceutically acceptable salt refers to a salt form of the compounds of the present invention in order to permit their use or formulation as pharmaceuticals and which retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically or otherwise undesirable. Examples of such salts are described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, ermuth, C.G. and Stahl, P.H. (eds.), Wiley- Verlag Helvetica Acta, Zurich, 2002 [ISBN 3-906390-26-8]. Examples of such salts include alkali metal salts and addition salts of free acids and bases. Examples of pharmaceutically acceptable salts, without limitation, include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates, methanesulfonates, ethane sulfonates, propanesulfonates, toluenesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, and mandelates.
If an inventive compound is a base, a desired salt may be prepared by any suitable method known to those skilled in the art, including treatment of the free base with an inorganic acid, such as, without limitation, hydrochloric acid, hydrobromic acid, hydroiodic, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, including, without limitation, formic acid, acetic acid, propionic acid, maieic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, stearic acid, ascorbic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, cyclohexyl- aminosulfonic acid or the like.
If an inventive compound is an acid, a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like. Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine, lysine and arginine; ammonia; primary, secondary, and tertiary amines such as ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine, choline, and procaine, and cyclic amines, such as piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
The carriers and additives used for such pharmaceutical compositions can take a variety of forms depending on the anticipated mode of administration. Thus, compositions for oral administration may be, for example, solid preparations such as tablets, sugar- coated tablets, hard capsules, soft capsules, granules, powders and the like, with suitable carriers and additives being starches, sugars, binders, diluents, granulating agents, lubricants, disintegrating agents and the like. Because of their ease of use and higher patient compliance, tablets and capsules represent the most advantageous oral dosage forms for many medical conditions.
Similarly, compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, elixirs, and the like with suitable carriers and additives being water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like. Typical preparations for parenteral administration comprise the active ingredient with a carrier such as sterile water or parenterally acceptable oil including polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with other additives for aiding solubility or preservation may also be included. In the case of a solution, it can be lyophilized to a powder and then reconstituted immediately prior to use. For dispersions and suspensions, appropriate carriers and additives include aqueous gums, celluloses, silicates or oils.
The pharmaceutical compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, topical (i.e., both skin and mucosal surfaces, including airway surfaces), transdermal administration and parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intrathecal, intracerebral, intracranially, intraarterial, or intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active agent which is being used.
Compositions for injection will include the active ingredient together with suitable carriers including propylene glycol-alcohol-water, isotonic water, sterile water for injection (USP), emulPhor™-alcohol-water, cremophor-EL™ or other suitable carriers known to those skilled in the art. These carriers may be used alone or in combination with other conventional solubilizing agents such as ethanol, propylene glycol, or other agents known to those skilled in the art.
Where the macrocyclic compounds of the present invention are to be applied in the form of solutions or injections, the compounds may be used by dissolving or suspending in any conventional diluent The diluents may include, for example, physiological saline, Ringer's solution, an aqueous glucose solution, an aqueous dextrose solution, an alcohol, a fatty acid ester, glycerol, a glycol, an oil derived from plant or animal sources, a paraffin and the like. These preparations may be prepared according to any conventional method known to those skilled in the art.
Compositions for nasal administration may be formulated as aerosols, drops, powders and gels. Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a physiologically acceptable aqueous or non-aqueous solvent. Such formulations are typically presented in single or multidose quantities in a sterile form in a sealed container. The sealed container can be a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single use nasal inhaler, pump atomizer or an aerosol dispenser fitted with a metering valve set to deliver a therapeutically effective amount, which is intended for disposal once the contents have been completely used. When the dosage form comprises an aerosol dispenser, it will contain a propellant such as a compressed gas, air as an example, or an organic propellant including a fiuorochlorohydrocarbon or fluorohydrocarbon.
Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth or gelatin and glycerin.
Compositions for rectal administration include suppositories containing a conventional suppository base such as cocoa butter.
Compositions suitable for transdermal administration include ointments, gels and patches.
Other compositions known to those skilled in the art can also be applied for percutaneous or subcutaneous administration, such as plasters.
Further, in preparing such pharmaceutical compositions comprising the active ingredient or ingredients in admixture with components necessary for the formulation of the compositions, other conventional pharmacologically acceptable additives may be incorporated, for example, excipients, stabilizers, antiseptics, wetting agents, emulsifying agents, lubricants, sweetening agents, coloring agents, flavoring agents, isotonicity agents, buffering agents, antioxidants and the like. As the additives, there may be mentioned, for example, starch, sucrose, fructose, dextrose, lactose, glucose, mannitol, sorbitol, precipitated calcium carbonate, crystalline cellulose, carboxymethylcellulose, dextrin, gelatin, acacia, EDTA, magnesium stearate, talc, hydroxypropylmethylcellulose, sodium metabisulfite, and the like.
In some embodiments, the composition is provided in a unit dosage form such as a tablet or capsule.
In further embodiments, the present invention provides kits including one or more containers comprising pharmaceutical dosage units comprising an effective amount of one or more compounds of the present invention.
The present invention further provides prodrugs comprising the compounds described herein. The term "prodrug" is intended to mean a compound that is converted under physiological conditions or by solvolysis or metabolically to a specified compound that is pharmaceutically active. The "prodrug" can be a compound of the present invention that has been chemically derivatized such that, (i) it retains some, all or none of the bioactivity of its parent drug compound, and (ii) it is metabolized in a subject to yield the parent drug compound. The prodrug of the present invention may also be a "partial prodrug" in that the compound has been chemically derivatized such that, (i) it retains some, all or none of the bioactivity of its parent drug compound, and (ii) it is metabolized in a subject to yield a biologically active derivative of the compound. Known techniques for derivatizmg compounds to provide prodrugs can be employed. Such methods may utilize formation of a hydrolyzable coupling to the compound.
The present invention further provides that the compounds of the present invention may be administered in combination with a therapeutic agent used to prevent and/or treat metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, bone disorders, genetic disorders, hyperproiiferative disorders and inflammatory disorders. Exemplary agents include analgesics (including opioid analgesics), anesthetics, antifungals, antibiotics, antiinflammatories (including nonsteroidal anti-inflammatory agents), anthelmintics, antiemetics, antihistamines, antihypertensives, antipsychotics, antiarthritics, antitussives, antivirals, cardioactive drugs, cathartics, chemotherapeutic agents (such as DNA-interactive agents, antimetabolites, tubulin-interactive agents, hormonal agents, and agents such as asparaginase or hydroxyurea), corticoids (steroids), antidepressants, depressants, diuretics, hypnotics, minerals, nutritional supplements, parasympathomimetics, hormones (such as corticotrophin releasing hormone, adrenocorticotropin, growth hormone releasing hormone, growth hormone, thyrptropin- releasing hormone and thyroid stimulating hormone), sedatives, sulfonamides, stimulants, sympathomimetics, tranquilizers, vasoconstrictors, vasodilators, vitamins and xanthine derivatives.
Subjects suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects, and are preferably mammalian. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates, humans, and the like, and mammals in utero. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects are preferred. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) can be treated according to the present invention, Illustrative avians according to the present invention include chickens, ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) and domesticated birds (e.g., parrots and canaries), and birds in ovo.
The present invention is primarily concerned with the treatment of human subjects, but the invention can also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes, and for drug screening and drug development purposes.
In therapeutic use for treatment of conditions in mammals (i.e. humans or animals) for which a modulator, such as an agonist, of the ghrelin receptor is effective, the compounds of the present invention or an appropriate pharmaceutical composition thereof may be administered in an effective amount. Since the activity of the compounds and the degree of the therapeutic effect vary, the actual dosage administered will be determined based upon generally recognized factors such as age, condition of the subject, route of delivery and body weight of the subject. The dosage can be from about 0.1 to about 100 mg/kg, administered orally 1-4 times per day. In addition, compounds can be administered by injection at approximately 0.01 - 20 mg kg per dose, with administration 1-4 times per day. Treatment could continue for weeks, months or longer. Determination of optimal dosages for a particular situation is within the capabilities of those skilled in the art.
5. Methods of Use
The compounds of the present invention can be used for the prevention and treatment of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, bone disorders, genetic disorders, hyperproliferative disorders, inflammatory disorders and combinations thereof where the disorder may be the result of multiple underlying maladies. In particular embodiments, the disease or disorder is irritable bowel syndrome (IBS), non-ulcer dyspepsia, Crohn's disease, gastroesophogeal reflux disorders, constipation, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, carcinoid syndrome, malabsorption syndrome, diarrhea, diabetes including diabetes mellitus (type II diabetes), obesity, atrophic colitis, gastritis, gastric stasis, constipation, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, celiac disease, an eating disorder or obesity. In other embodiments, the disease or disorder is congestive heart failure, ischemic heart disease or chronic heart disease. In still other embodiments, the disease or disorder is osteoporosis and/or frailty, congestive heart failure, accelerating bone fracture repair, metabolic syndrome, attenuating protein catabolic response, cachexia, protein loss, impaired or risk of impaired wound healing, impaired or risk of impaired recovery from burns, impaired or risk of impaired recovery from surgery, impaired or risk of impaired muscle strength, impaired or risk of impaired mobility, altered or risk of altered skin thickness, impaired or risk of impaired metabolic homeostasis or impaired or risk of impaired renal homeostasis. In other embodiments, the disease or disorder involves facilitating neonatal development, stimulating growth hormone release in humans, maintenance of muscle strength and function in humans, reversal or prevention of frailty in humans, prevention of catabolic side effects of glucocorticoids, treatment of osteoporosis, stimulation and increase in muscle mass and muscle strength, stimulation of the immune system, acceleration of wound healing, acceleration of bone fracture repair, treatment of renal failure or insufficiency resulting in growth retardation, treatment of short stature, treatment of obesity and growth retardation, accelerating the recovery and reducing hospitalization of burn patients, treatment of intrauterine growth retardation, treatment of skeletal dysplasia, treatment of hypercortisolism, treatment of Cushing's syndrome, induction of pulsatile growth hormone release, replacement of growth hormone in stressed patients, treatment of osteochondrodysplasias, treatment of Noonans syndrome, treatment of schizophrenia, treatment of depression, treatment of Alzheimer's disease, treatment of emesis, treatment of memory loss, treatment of reproduction disorders, treatment of delayed wound healing, treatment of psychosocial deprivation, treatment of pulmonary dysfunction, treatment of ventilator dependency; attenuation of protein catabolic response, reducing cachexia and protein loss, treatment of hyperinsulinemia, adjuvant treatment for ovulation induction, stimulation of thymic development, prevention of thymic function decline, treatment of immunosuppressed patients, improvement in muscle mobility, maintenance of skin thickness, metabolic homeostasis, renal homeostasis, stimulation of osteoblasts, stimulation of bone remodeling, stimulation of cartilage growth, stimulation of the immune system in companion animals, treatment of disorders of aging in companion animals, growth promotion in livestock, and/or stimulation of wool growth in sheep. Other embodiments provide for methods of treatment of inflammatory disorders, including ulcerative colitis, inflammatory bowel disease, Crohn's disease, pancreatitis, rheumatoid arthritis, osteoarthritis, asthma, vasculitis, psoriasis, allergic rhinitis, peptic ulcer disease, postoperative intra-abdominal sepsis, ischemia-reperfusion injury, pancreatic and liver damage, sepsis and septic shock, gastric damage caused by certain drugs, stress-induced gastric damage, gastric damage caused by H. pylori, inflammatory pain, chronic kidney disease and intestinal inflammation.
According to a further aspect of the invention, there is provided a method for the treatment of postoperative ileus, cachexia (wasting syndrome), such as that caused by cancer, AIDS, cardiac disease and renal disease, gastroparesis, such as that resulting from type I or type II diabetes, other gastrointestinal disorders, growth hormone deficiency, bone loss, and other age-related disorders in a human or animal patient suffering therefrom, which method comprises administering to said patient an effective amount of at least one member selected from the compounds disclosed herein having the ability to modulate the ghrelin receptor. Other diseases and disorders treated by the compounds disclosed herein include short bowel syndrome, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, celiac disease, and hyperproliferative disorders such as tumors, cancers, and neoplastic disorders, as well as premalignant and non-neoplastic or non-malignant hyperproliferative disorders. In particular, tumors, cancers, and neoplastic tissue that can be treated by the present invention include, but are not limited to, malignant disorders such as breast cancers, osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas, leukemias, lymphomas, sinus tumors, ovarian, uretal, bladder, prostate and other genitourinary cancers, colon, esophageal and stomach cancers and other gastrointestinal cancers, lung cancers, myelomas, pancreatic cancers, liver cancers, kidney cancers, endocrine cancers, skin cancers and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas.
In particular embodiments, the macrocyclic compounds of the present invention can be used to treat postoperative ileus. In other embodiments, the compounds of the present invention can be used to treat gastroparesis. In still other embodiments, the compounds of the present invention can be used to treat diabetic gastroparesis. In another embodiment, the compounds of the present invention can be used to treat gastric stasis. In yet another embodiment, the compounds of the present invention can be used to treat opioid-induced bowel dysfunction. In further embodiments, the compounds of the present invention can be used to treat chronic intestinal pseudoobstruction. In still further particular embodiments of the present invention, the compounds of the present invention can be used to treat postoperative ileus, paralytic ileus following surgical or other manipulation, gastroparesis, opioid-induced bowel dysfunction, chronic intestinal pseudo-obstruction, acute colonic pseudo-obstruction (Ogilvie's syndrome), enteric dysmotility, short bowel syndrome, emesis, constipation-predominant irritable bowel syndrome (IBS), chronic constipation, functional dyspepsia, cancer-associated dyspepsia syndrome, graft versus host disease, gastric stasis or hypomotility caused by various diseases such as diabetes or by the administration of other pharmaceutical agents, or in enterally fed patients, delayed gastric emptying, gastrointestinal dysfunction or delayed gastric emptying occurring in conjunction with other disease states, gastrointestinal dysmotility or delayed gastric emptying occurring in critical care situations, gastrointestinal dysfunction or delayed gastric emptying as a result of treatment with pharmaceutical agents, gastroesophageal reflux disease (GERD), gastric ulcers, gastroenteritis and Crohn's disease,
The present invention further provides methods of treating a horse or canine for a gastrointestinal disorder comprising administering a therapeutically effective amount of a modulator having the structure of formula I. In some embodiments, the gastrointestinal disorder is ileus or colic.
As used herein, "treatment" is not necessarily meant to imply cure or complete abolition of the disorder or symptoms associated therewith.
The compounds of the present invention can further be utilized for the preparation of a medicament for the treatment of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, genetic disorders, bone disorders, hyperproliferative disorders and inflammatory disorders.
Further embodiments of the present invention will now be described with reference to the following examples. It should be appreciated that these examples are for the purposes of illustrating embodiments of the present invention, and do not limit the scope of the invention. EXAMPLES
Example 1
Binding Activity
The table below presents binding activity at the human ghrelin receptor for representative compounds of the invention.
Figure imgf000055_0001
a Binding activity determined using standard method 3C, Kj values are expressed as follows: A < 10 nM, B < 100 nM, C < 500 nM, D > 500 nM.
Example 2
Drug-like Properties
A number of parameters have been found to be reasonably predictive of the ability of a compound to eventually be able to be developed as a pharmaceutical. These parameters can be calculated in silico. The following Table provides data for representative compounds of the invention, showing the differences with ulimorelin.
Figure imgf000055_0002
6 4.43 120.0
7 3.95 140.2
8 3.66 120.0
9 4.55 116.8
10 4.32 120.0
11 4.41 116.8
12 4.15 120.0
13 3.44 120.0
14 3.95 140.2
15 3.95 140.2
16 3.95 140.2
17 3.95 140.2
18 3.17 140.2
19 3.17 140.2
20 3.17 140.2
21 3.17 140.2
22 3.83 140.2
23 3.83 140.2
24 3.83 140.2
25 3.83 140.2 26 2.95 140.2
27 2.95 140.2
28 3.67 140.2
29 2.95 140.2
30 2.95 140.2
31 3.67 140.2
32. 3.67 140.2
33 3.67 140.2
34 3.29 140.2
35 2.58 140.2
36 2.80 140.2
37 3.46 140.2
38 1.80 140.2
39 2.46 140.2
40 2.52 140.2
41 3.18 140.2
42 3.31 140.2
Calculated using ChemBioDrawUltra v. 12.0; TPSA = total polar surface area Example 3
Synthesis of Tethers
A. Standard Procedure for the Synthesis of Tether T205
This tether in a representative protected form was synthesized from the monobenzoate of resorcinol (205-1) using the multi-step process shown in Figure 3.
B. Standard Procedure for the Synthesis of Tether T206
The desired compound was accessed from 2-hydroxybenzaldehyde (206-1) as outlined in Figure 4. The presence of one fixed chiral center would permit the separation of the two diastereoisomers of the hydroxyl chiral center to be isolated by preparative HPLC.
C. Standard Procedure for the Synthesis of Tether T207
The preparation of protected tether T207 originates from a previously reported tether, T165 (Intl. Pat. Appl. Publ. No. WO 201 1/053821) as presented in Figure 5. To synthesize the other stereoisomer at the hydroxyl chiral center, the reagent AD-mix-a (Aldrich Chemical, Milwaukee, WI, USA) can be employed for the asymmetric dihydroxylation. (Sharpless, .B.; Amberg, W.; Bennani, Y.L.; et al J Org. Chem. 1992, 57, 2768-2771; Kolb, H.C.; VanNieuwenhze, M.S.; Sharpless, K.B. Chem. Rev. 1994, 94, 2483-2547.)
D. Standard Procedure for the Synthesis of Tether T208
Starting from an intermediate from the T206 synthesis, the desired tether was constructed as shown below.
Dess-Martin Periodinane H20, DCM, rt, O/N
Figure imgf000058_0001
206-4 208-1
Figure imgf000058_0002
208-2 BOC-T208 E. Standard Procedure for the Synthesis of Tether T209
In an analogous manner to that described for tether T208, the protected tether T209 was prepared from an intermediate in the T207 synthesis as presented below.
Figure imgf000059_0001
207-4 209-1
Figure imgf000059_0002
BOC-T209
Example 4
Synthesis of Amino Acid Building Blocks
A. Standard Procedure for the Synthesis of Amino Acid AA1
Starting from Boc-Phe(4F)-OH, the (S)-isomer of the β -hydroxy derivative accessed as presented in Figure 6.
B. Standard Procedure for the Synthesis of Amino Acid AA2
The (R)-isomer of the β-hydroxy derivative of 4-fluorophenylalanine can be prepared as outlined in Figure 7 using 4-fluorobenzaldehyde (2-2) as the substrate for an Evans chiral aldol reaction (Evans, D. A. Aldrichimica Acta 1982, 15, 23) followed by a chiral azidation through the intermediate bromide. (Evans, D.A.; Britton, T.C.; Ellman, J.A.; Dorow, R.L, J. Am. Chem. Soc. 1990, 112, 4011-4030; Evans, D.A.; Ellman, J.A.; Dorow, R.L. Tet. Lett. 1987, 28, 1123-1126.) Subsequent hydrolytic cleavage and Staudinger reaction provided the free amino acid, which can be protected using standard methods. Example 5
Standard Procedure for the Synthesis of Macro cycles
The representative synthesis of compound 4 as outlined in Figure 8 demonstrates the chemistry strategy and procedures for construction of the macrocyclic compounds of the invention. Alternative methods for coupling and protection can be employed as well.
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

! Claimed is:
1. A compound of formula I):
Figure imgf000061_0001
and pharmaceutically acceptable salts thereof
wherein:
Ri is Ci-C4 alkyl;
R2 is halogen or hydroxyl;
W is hydrogen or hydroxyl;
Xlt X2, X3 and X4 are independently hydrogen or hydroxyl with the proviso that no more than two of X1} X2, X3 or X can be hydroxyl;
Yi and Y2 are independently hydrogen or hydroxyl;
Zi and Z2 are independently hydrogen or hydroxyl with the proviso that both are not hydroxyl.
2. The compound of formula (I) wherein Ri is methyl.
3. The compound of formula (I) wherein R2 is fluorine.
4. The compound of formula (I) having the following structure:
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
or an optical isomer, enantiomer or diastereomer thereof.
5. A pharmaceutical composition comprising:
(a) a compound of formula (I) of claim 1 ; and
(b) a pharmaceutically acceptable carrier, excipient or diluent.
Figure imgf000065_0001
or
wherein FGi is hydrogen or an amine protecting group; PG2 is hydrogen or a hydroxyl protecting group; and PG3 is hydrogen or a hydroxyl protecting group.
7. The compound of claim 6, wherein PGj is selected from the group consisting of tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), a,a-dimethyl-3 ,5-dimethoxybenzyloxycarbonyl (Ddz) and allyloxycarbonyl (Alloc).
8. The compound of claim 6, wherein PG2 is tetrahydropyranyl (THP).
9. The compound of claim 6, wherein PG is benzyl.
10. A method of using a compound of claim 6 to synthesize a compound of formula (I) as describd in claim 1.
11. A method of treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a compound of claim 1.
12. The method of claim 11, wherein the gastrointestinal disorder is characterized by gastrointestinal dysmotility or hypomotility.
The method of claim 11 , wherein the compound is administered orally.
14. The method of claim 11, wherein the compound is administered parenterally.
15. The method of claim 11 , wherein the subj ect is a mammal.
16. The method of claim 11 , wherein the subject is a human.
17. The method of claim 11 , wherein the subject is a horse.
18. The method of claim 11, wherein the compound is co-administered with an additional agent useful for stimulating gastrointestinal motility.
19. The method of claim 11, wherein the gastrointestinal disorder is selected from the group consisting of postoperative ileus, paralytic ileus, gastroparesis, opioid- induced bowel dysfunction, gastric stasis, clironic intestinal pseudo-obstruction, acute colonic pseudo-obstruction (Ogilvie's syndrome), enteric dysmotility, short bowel syndrome, emesis, constipation-predominant irritable bowel syndrome (IBS), chronic constipation, functional dyspepsia, cancer-associated dyspepsia syndrome, graft versus host disease, delayed gastric emptying, gastrointestinal dysfunction or delayed gastric emptying occurring in conjunction with other disease states, gastrointestinal dysmotility or delayed gastric emptying occurring in critical care situations, gastrointestinal dysfunction or delayed gastric emptying as a result of treatment with pharmaceutical agents, gastroesophageal reflux disease (GERD), gastric ulcers, gastroenteritis, colitis and Crohn's disease.
20. The method of claim 19, wherein the gastroparesis is diabetic gastroparesis or postsurgical gastroparesis syndrome.
21. The method of claim 19, wherein the gastric stasis is caused by disease, by the administration of other pharmaceutical agents, or occurs in enterally fed patients,
22. A kit comprising one or more containers containing pharmaceutical dosage units comprising an effective amount of one or more compounds of claim 1 or claim 4.
23. A method of treating a metabolic or endocrine disorder comprising administering to a subject in need thereof an effective amount of a compound of claim 1 :
24. The method of claim 23, wherein the metabolic or endocrine disorder is characterized by lack of appetite, decrease in food intake, reduced energy expenditure, or muscle wasting.
25. The method of claim 23, wherein the metabolic or endocrine disorder is cachexia.
26. A method of treating a cardiovascular disease comprising administering to a subject in need thereof an effective amount of a compound of claim 1.
27. The method of claim 26, wherein the cardiovascular disease is chronic heart failure.
28. A method of treating a central nervous system disorder comprising administering to a subject in need thereof an effective amount of a compound of claim 1.
29. The method of claim 28, wherein the central nervous system disorder is Alzheimer's disease, Parkinson's disease, anxiety, stress, insomnia, or is characterized by reduced cognitive function or by disruption of normal sleep patterns.
30. A method of treating an inflammatory disorder comprising administering to a subject in need thereof an effective amount of a compound of claim 1.
31. The method of claim 30, wherein the inflammatory disorder is ulcerative colitis, inflammatory bowel disease, Crohn's disease, pancreatitis, rheumatoid arthritis, osteoarthritis, asthma, vasculitis, psoriasis, allergic rhinitis, peptic ulcer disease, postoperative intra-abdominal sepsis, ischemia-reperfusion injury, pancreatic and liver damage, sepsis and septic shock, gastric damage caused by certain drugs, stress-induced gastric damage, gastric damage caused by H. pylori, radiation-induced damage, chemotherapy-induced damage, inflammatory pain, chronic kidney disease and intestinal inflammation.
32. A method of treating a hyperproliferative disorder comprising administering to a subject in need thereof an effective amount of a compound of claim 1.
33. The method of claim 32, wherein the hyperproliferative disorder is cancer.
34. A method of treating a bone disorder comprising administering to a subject in need thereof an effective amount of a compound of claim 1.
The method of claim 34, wherein the bone disorder is osteoporosis.
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