WO2016149628A1 - Tgr5 agonists - Google Patents

Tgr5 agonists Download PDF

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Publication number
WO2016149628A1
WO2016149628A1 PCT/US2016/023165 US2016023165W WO2016149628A1 WO 2016149628 A1 WO2016149628 A1 WO 2016149628A1 US 2016023165 W US2016023165 W US 2016023165W WO 2016149628 A1 WO2016149628 A1 WO 2016149628A1
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Prior art keywords
oxy
diazepan
fluorobenzyl
compound
methoxybenzyl
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PCT/US2016/023165
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French (fr)
Inventor
Alexander KASATKIN
Evgeny KURDYAVKA
Sergei SVIRIDOV
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Chembridge Corporation
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Publication of WO2016149628A1 publication Critical patent/WO2016149628A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D243/00—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms
    • C07D243/06—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4
    • C07D243/08—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4 not condensed with other rings

Definitions

  • TGR5 also known as G-protein coupled bile acid receptor 1 (GPBAR1) or GPR131
  • GPCR Gs protein-coupled receptor
  • TGR5 expression has also been characterized in macrophages (Kawamata et al., J. Biol. Chem., 278, 9435-9440, 2003; Keitel et al., Biochem. Biophys. Res. Commun., 372, 78- 84, 2008), where its activation inhibits pro-inflammatory cytokine production and prevents the development of LPS-induced liver inflammation (Wang et al., Hepatology, 54, 1421- 1432, 2011) and atherosclerosis in mice (Pols et al., Cell metabolism, 14, ⁇ - ⁇ 5 ⁇ , 2011).
  • TGR5 may be an effective target for pharmacologic treatment of metabolic disorders, such as obesity and diabetes, and inflammatory diseases, such as atherosclerosis.
  • TGR5 agonists [0004] Identification and characterization of TGR5 agonists have been reported (Budzik et al., Bioorg. Med. Chem. Lett., 20, 1363-1367, 2010; Dehmlow et al., Bioorg. Med. Chem. Lett, 23, 4627-4632, 2013; Duan et al., J. Med. Chem. , 55, 10475-10489, 2012).
  • TGR5 agonists with improved potency, efficacy, selectivity, metabolic stability and bioavailability, while lowering the toxicity and adverse effects of these compounds for long-term usage.
  • a TGR5 agonist of formula (I) is its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
  • the present invention features a composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, for example, a compound of formula (I) as described herein.
  • the present invention features a pharmaceutical composition comprising a TGR5 agonist of formula (I) and at least one pharmaceutically acceptable carrier.
  • the present invention provides a method of treating, preventing, suppressing, reducing the severity of, or reducing the risk of an inflammatory disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
  • a compound of the invention for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
  • the present invention provides a method for inducing increased GLP- 1 secretion in a cell, the method comprising contacting the cell with an inducing effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
  • a compound of the invention for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
  • the present invention provides a compound of formula (I), comprising a TGR5 agonist and showing selectivity for a heterogeneous population of TGR5 receptors.
  • the present invention identifies heterogeneity of the TGR5 receptor that can be exploited to separate the anti-inflammatory and metabolic effects of TRG5 agonists.
  • the present invention provides a compound of formula (I), comprising a TGR5 agonist and showing selectivity for heterogeneous activation of TGR5 receptors.
  • the present invention identifies heterogeneity of activating a TGR5 receptor that can be exploited to separate the anti-inflammatory and metabolic effects of TRG5 agonists.
  • Figures la-lf depict the characterization of synthetic TGR5 agonists.
  • Figure la A process of selecting the TGR5 agonists from over 20,000 compounds from the ChemBridge compound library (> 900,000 compounds).
  • a robotic primary screen which consisted of co-transfecting CHO cells with a hTGR5 expression construct and a luciferase reporter under the control of a cAMP response element (CRE), identified 621 hits out of these 20,000 compounds. These 621 primary hits were then re-tested in duplicate with the same reporter assay, identifying 313 confirmed hits. Out of these 313 hits, the 10 best candidates were selected according to their score and their EC 50 , and the microsomal stability of these 10 was tested.
  • CRE cAMP response element
  • Figures 2a-2d depict characterization of three compounds of the present invention.
  • Figure 2a Addition of compounds (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one), (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one), or 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l- (4-methoxybenzyl)-l,4-diazepan-2-one), each at a concentration of 30 ⁇ , significantly decreased the transcript levels for Tnfa, 116 and Mcpl in the RAW 264.7 murine macrophage cell line stimulated for 4 hours with lipopoly saccharide (LPS, 10 ng/mL).
  • LPS lipopoly sacchar
  • Figure 3d Oxygen consumption (V0 2 ) and locomotor activity (LA) measurements over a 24-hr period in male C57BL/6J mice after 7 weeks of HFD (solid circles) or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open squares) (at 30 mg/kg/d).
  • FIGS 5a-5h depict that administration of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-
  • Oxygen consumption and locomotor activity were comparable between Tgr5 ⁇ ' ⁇ mice treated with HFD (solid circles) or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open squares) during the active period (7PM-7AM - dark phase).
  • FIG. 5f Relative mRNA expression levels of Ucp-1, Dio-2, and Cpt-1 in brown adipose tissue of male 7gr5 _/ ⁇ mice showed no significant difference whether the animals were fed for 13 weeks with HFD (solid bars) or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) (open bars) (30 mg/kg/d).
  • FIG. 5h Oral glucose tolerance test (OGTT) in male Tgr5 ⁇ ' ⁇ mice fed for 9 weeks with HFD or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (30 mg/kg/d).
  • Data represent mean + SEM. * denotes p ⁇ 0.05.
  • FIG. 6a Body weight evolution in male LDLr-null (LDLr ⁇ ' ⁇ ) mice fed for 13 weeks with a high cholesterol diet (HCD) or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) at 30 mg/kg/d given as food admix.
  • Figure 6f Quantification of plasma cholesterol and triglyceride levels in LDLr 1' mice fed with HCD (solid bars) or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- 1 ,4-diazepan-2-one) (open bars) for various times.
  • Mice exposed to HCD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) showed a significant decrease of plasma cholesterol after 9 weeks of treatment, and this decrease persisted through the termination of the study at 12 weeks.
  • Figure 6g Quantification of Mcpl and 116 mRNA in aortas of LDLr' mice fed for 13 weeks with HCD (solid bars) or HCD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open bars) (upper panel). Quantification of ⁇ and KC/GRO in the plasma of LDLr ' mice fed for 13 weeks with HCD or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan- 2-one) (lower panel).
  • Figures 7a-7e depict functional differences at TGR5 between (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) enantiomers, thereby defining heterogeneity of the TGR5 receptor or TGR5 activiation.
  • Figure 7a The stereo enantiomers (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one)) and ( ⁇ 55)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one),) at 30 ⁇ , each similarly decreased mRNA levels of Tnfa, 116 and Mcpl in a murine macrophage cell line (RAW 264.7) stimulated for 4 hours with lipopoly saccharide (LPS, 10 ng/niL).
  • Rnfa murine macrophage cell line
  • LPS lipopoly saccharide
  • Figure 7b The enantiomer ( ⁇ 5R)-(6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one),) but not ( ⁇ 55)-(6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one),) at 30 ⁇ , significantly increased GLP-1 secretion from the murine entero-endocrine GLUTag cells.
  • R2 is Ci_6 alkyl, 0-Ci_ 6 alkyl, -NR a R b , C3_8 cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, each optionally mono- or polysubstituted with substituents selected from Ci_ 6 alkyl, halo, C1-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, nitro, and -NR a Rt > ;
  • R a and R b each are independently H or C1-3 alkyl.
  • R 3 is F, CI, Br, I, or 0-Ci_ 6 alkyl
  • n 1-5;
  • Ri is C 3 -8 cycloalkyl. In other embodiments, Ri is cyclobutyl. In certain embodiments, Ri is cyclohexyl or cyclopentyl.
  • the compound of formula (I) is represented by formula (IV)
  • R2 is O-C1-5 alkyl, -NR a Rb, C3-8 cycloalkyl, 2-furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, and O-C1-3 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro.
  • R2 is O-C1-5 alkyl or -NR a Rt > .
  • R2 is OCH2CH2CH3 or -NCH(CH 3 ) 2 .
  • R2 is C 3 -8 cycloalkyl.
  • the compound is 4-( ⁇ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy ⁇ methyl)benzonitrile ;
  • a compound of formula (I) is its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
  • the compound is the R enantiomer.
  • the compound is the S enantiomer.
  • the compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound is (65)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound is 4-(cyclopropylcarbonyl)-6-[(2- fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one
  • the compound is 4-acetyl-l-[2-(dimethylamino)ethyl]-6- [(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
  • This approach can be also used for parallel synthesis for obtaining libraries with a high diversity of substituent R 3 .
  • Scheme 3 shows a modification of the main approach of Scheme 1 to change the order of the introduction of substituents R2 and R 3 .
  • Both TBS and Boc groups are removed upon deprotection with HC1 to lead to an aminoalcohol.
  • HC1 HC1
  • Using HBTU as a condensing agent enables acylating selectively the aminoalcohol at the nitrogen atom.
  • the resulting compound is alkylated.
  • stable refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
  • haloalkyl refers to an alkyl group having one or more halogen substituents.
  • Example haloalkyl groups include CF 3 , C 2 F 5 , CHF 2 , CCI 3 , CHCI 2 , C 2 CI 5 , and the like.
  • aryl refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like. In some embodiments, an aryl group has from 6 to about 20 carbon atoms.
  • cycloalkyl refers to non-aromatic carbocycles including cyclized alkyl, alkenyl, and alkynyl groups.
  • Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems, including spirocycles.
  • cycloalkyl groups can have from 3 to about 20 carbon atoms, 3 to about 14 carbon atoms, 3 to about 10 carbon atoms, or 3 to 7 carbon atoms. Cycloalkyl groups can further have 0, 1 , 2, or 3 double bonds and/or 0, 1 , or 2 triple bonds.
  • Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like.
  • heteroaryl refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen.
  • Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Any ring- forming N atom in a heteroaryl group can also be oxidized to form an N-oxo moiety.
  • heteroaryl groups include without limitation, pyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like.
  • the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms.
  • cycloheteroalkyl or “heterocycloalkyl” refers to a non- aromatic heterocycle where one or more of the ring-forming atoms are a heteroatom such as an O, N, or S atom.
  • Cycloheteroalkyl or heterocycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well as spirocycles.
  • Example cycloheteroalkyl or heterocycloalkyl groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-l,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like.
  • cycloheteroalkyl or heterocycloalkyl moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles.
  • a cycloheteroalkyl or heterocycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion.
  • moieties where one or more ring-forming atoms are substituted by 1 or 2 oxo or sulfido groups.
  • halo or “halogen” includes fluoro, chloro, bromo, and iodo.
  • the term "substituted” refers to the replacement of a hydrogen moiety with a non-hydrogen moiety in a molecule or group.
  • the term "mono- substituted” or “poly-substituted” means substituted with one or more than one substituent up to the valence of the substituted group.
  • a mono- substituted group can be substituted with 1 substituent
  • a poly-substituted group can be substituted with 2, 3, 4, or 5 substituents.
  • the substituents can be independently selected from that group.
  • the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
  • a compound of this invneiton is the R- enantiomer of a compound of formula (I).
  • a compound of this invention is the 5-enantiomer of a compound of formula (I).
  • Compounds of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis.
  • R- and 5-enantiomers may have the same activity.
  • the R-enantiomer of the compound of formula (I) shows selective activity.
  • the 5-enantiomer of the compound of formula (I) shows selective activity.
  • an R-enatiomer of a compound of formula (I) selectively increases GLP-1 secretion while the 5-enantiomer of the compound does not increase GLP-1 secretion.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Compounds of the invention can also include all isotopes of atoms occurring in the intermediates or final compounds.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium.
  • the compound of the invention is substantially isolated.
  • substantially isolated is meant that the compound is at least partially or substantially separated from the environment in which it is formed or detected.
  • Partial separation can include, for example, a composition enriched in the compound of the invention.
  • Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the invention, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
  • a “therapeutically effective amount” or “inducing effective amount” as used herein refers to the amount which provides a therapeutic effect or inducing effect for a given condition and administration regimen.
  • compound as used herein is meant to include all stereoisomers, geometric iosomers, tautomers, and isotopes of the structures depicted.
  • the present invention also includes pharmaceutically acceptable salts of the compounds described herein.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
  • the physiologically acceptable salts may be obtained by neutralizing the bases with inorganic or organic acids or by neutralizing the acids with inorganic or organic bases.
  • suitable inorganic acids are hydrochloric acid, sulphuric acid, phosphoric acid, or hydrobromic acid
  • suitable organic acids are carboxylic acid, sulpho acid, or sulphonic acid, such as acetic acid, tartaric acid, lactic acid, propionic acid, glycolic acid, malonic acid, maleic acid, fumaric acid, tannic acid, succinic acid, alginic acid, benzoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, citric acid, maleic acid, salicylic acid, 3 -aminosalicylic acid, ascorbic acid, embonic acid, nicotinic acid, isonicotinic acid, oxalic acid, gluconic acid, amino acids, methanesulph
  • suitable inorganic bases are sodium hydroxide, potassium hydroxide and ammonia
  • suitable organic bases are amines, e.g., tertiary amines, such as trimethylamine, triethylamine, pyridine, N,N-dimethylaniline, quinoline, isoquinoline, a- picoline, ⁇ -picoline, ⁇ -picoline, quinaldine, or pyrimidine.
  • physiologically acceptable salts of the compounds according to formula (I) can be obtained by converting derivatives which possess tertiary amino groups into the corresponding quaternary ammonium salts in a manner known per se using quaternizing agents.
  • suitable quaternizing agents are alkyl halides, such as methyl iodide, ethyl bromide, and n-propyl chloride, and also arylalkyl halides, such as benzyl chloride or 2-phenylethyl bromide.
  • isomer includes, but is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like.
  • Compounds of the present invention can also be in the form of a hydrate, which means that the compound further includes a stoichiometric or non- stoichiometric amount of water bound by non-covalent intermolecular forces.
  • Compounds of of the present invention may exist in the form of one or more of the possible tautomers and depending on the particular conditions it may be possible to separate some or all of the tautomers into individual and distinct entities. It is to be understood that all of the possible tautomers, including all additional enol and keto tautomers and/or isomers are hereby covered.
  • the present invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), and at least one pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises a TGR5 receptor agonist compound, and at least one pharmaceutically acceptable carrier.
  • a therapeutically effective dose of the compound according to the invention is used, in addition to physiologically acceptable carriers, diluents and/or adjuvants for producing a pharmaceutical composition.
  • the dose of the active compound can vary depending on the route of administration, the age and weight of the patient, the nature and severity of the diseases to be treated, and similar factors.
  • the daily dose can be given as a single dose, which is to be administered once, or be subdivided into two or more daily doses, and is as a rule 0.001-2000 mg. In some embodiments, the daily dose is 0.1-500 mg, e.g. 0.1- 100 mg.
  • Suitable administration forms are oral, parenteral, intravenous, transdermal, topical, inhalative, intranasal and sublingual preparations.
  • administration forms are oral, parenteral, e.g. intravenous or intramuscular, intranasal, e.g. dry powder or sublingual preparations of the compounds according to the invention.
  • the customary galenic preparation forms such as tablets, sugar-coated tablets, capsules, dispersible powders, granulates, aqueous solutions, alcohol-containing aqueous solutions, aqueous or oily suspensions, syrups, juices or drops, are used.
  • Solid medicinal forms can comprise inert components and carrier substances, such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher molecular weight fatty acids, (such as stearic acid), gelatine, agar agar or vegetable or animal fats and oils, or solid high molecular weight polymers (such as polyethylene glycol), preparations which are suitable for oral administration can comprise additional flavorings and/or sweetening agents, if desired.
  • carrier substances such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher mole
  • Liquid medicinal forms can be sterilized and/or, where appropriate, comprise auxiliary substances, such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.
  • auxiliary substances such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.
  • additives are tartrate and citrate buffers, ethanol and sequestering agents (such as ethylenediaminetetraacetic acid and its nontoxic salts).
  • High molecular weight polymers such as liquid polyethylene oxides, microcrystalline celluloses, carboxymethyl celluloses, polyvinylpyrrolidones, dextrans or gelatine, are suitable for regulating the viscosity.
  • solid carrier substances are starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids (such as stearic acid), gelatine, agar agar, calcium phosphate, magnesium stearate, animal and vegetable fats, and solid high molecular weight polymers, such as polyethylene glycol.
  • Oily suspensions for parenteral or topical applications can be vegetable synthetic or semisynthetic oils, such as liquid fatty acid esters having in each case from 8 to 22 C atoms in the fatty acid chains, for example palmitic acid, lauric acid, tridecanoic acid, margaric acid, stearic acid, arachidic acid, myristic acid, behenic acid, pentadecanoic acid, linoleic acid, elaidic acid, brasidic acid, erucic acid or oleic acid, which are esterified with monohydric to trihydric alcohols having from 1 to 6 C atoms, such as methanol, ethanol, propanol, butanol, pentanol or their isomers, glycol or glycerol.
  • vegetable synthetic or semisynthetic oils such as liquid fatty acid esters having in each case from 8 to 22 C atoms in the fatty acid chains, for example palmitic acid, lauric acid, tride
  • fatty acid esters are commercially available miglyols, isopropyl myristate, isopropyl palmitate, isopropyl stearate, PEG 6-capric acid, caprylic/capric acid esters of saturated fatty alcohols, polyoxyethylene glycerol trioleates, ethyl oleate, waxy fatty acid esters, such as artificial ducktail gland fat, coconut fatty acid isopropyl ester, oleyl oleate, decyl oleate, ethyl lactate, dibutyl phthalate, diisopropyl adipate, polyol fatty acid esters, inter alia.
  • Silicone oils of differing viscosity are also suitable. It is furthermore possible to use vegetable oils, such as castor oil, almond oil, olive oil, sesame oil, cotton seed oil, groundnut oil or soybean oil.
  • Suitable solvents, gelatinizing agents and solubilizers are water or watermiscible solvents.
  • suitable substances are alcohols, such as ethanol or isopropyl alcohol, benzyl alcohol, 2-octyldodecanol, polyethylene glycols, phthalates, adipates, propylene glycol, glycerol, di- or tripropylene glycol, waxes, methyl cellosolve, cellosolve, esters, morpholines, dioxane, dimethyl sulphoxide, dimethylformamide, tetrahydrofuran, cyclohexanone, etc.
  • gelatinizing agents and film-forming agents are also perfectly possible.
  • ionic macromolecules such as sodium carboxymethyl cellulose, polyacrylic acid, polymethacrylic acid and their salts, sodium amylopectin semiglycolate, alginic acid or propylene glycol alginate as the sodium salt, gum arabic, xanthan gum, guar gum or carrageenan.
  • Stabilizers such as montmorillonites or colloidal silicic acids, for stabilizing emulsions or preventing the breakdown of active substances such as antioxidants, for example tocopherols or butylhydroxyanisole, or preservatives, such as p-hydroxybenzoic acid esters, can likewise be used for preparing the desired formulations.
  • Preparations for parenteral administration can be present in separate dose unit forms, such as ampoules or vials.
  • use is made of solutions of the active compound, for example, aqueous solution and, in particular, isotonic solutions and also suspensions.
  • solutions of the active compound for example, aqueous solution and, in particular, isotonic solutions and also suspensions.
  • injection forms can be made available as ready-to-use preparations or only be prepared directly before use, by mixing the active compound, for example the lyophilisate, where appropriate containing other solid carrier substances, with the desired solvent or suspending agent.
  • inhalable preparations can present as powders, solutions or suspensions.
  • inhalable preparations are in the form of powders, e.g. as a mixture of the active ingredient with a suitable formulation aid such as lactose.
  • the compound of the invention may be administered as a combination therapy with further active agents, e.g. therapeutically active compounds useful in the treatment of metabolic disorders, inflammatory diseases, atherosclerosis and associated conditions and dyslipidemias.
  • the active ingredients may be formulated as compositions containing several active ingredients in a single dose form and/or as kits containing individual active ingredients in separate dose forms.
  • the active ingredients used in combination therapy may be coadministered or administered separately.
  • the compounds of the present invention are TGR5 agonists which are useful for treating, preventing suppressing, reducing the severity of, or reducing the risk of conditions where stimulation of a TGR5 receptor can have a positive therapeutic effect in a subject in need thereof.
  • the therapeutic effect may be selective.
  • the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of an inflammatory disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof.
  • a compound of the invention for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof.
  • the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of an inflammatory disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a TGR5 receptor agonist compound.
  • the compound of the invention for the treatment or prevention of an inflammatory disease is 6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one, or 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4- methoxybenzyl)-l,4-diazepan-2-one, or 4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one.
  • methods of this invention for treating, preventing suppressing, reducing the severity of, or reducing the risk of an inflammatory disease comprise administering a compound of formula (I), wherein said compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound is (6S)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines.
  • the compound of formula (I) does not induce increased secretion of GLP- 1.
  • the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of a metabolic disease or disorder in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof.
  • the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of a metabolic disease or disorder in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a TGR5 receptor agonist compound.
  • the compound of the invention for the treatment or prevention of a metabolic disease or disorder is 6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one.
  • the compound of the invention for the treatment or prevention of a metabolic disease or disorder is racemic.
  • the compound of the invention for the treatment or prevention of a metabolic disease or disorder comprises an R- or an S- enantiomer of the compound.
  • the compound of the invention for the treatment or prevention of a metabolic disease or disorder comprises an S- enantiomer of the compound.
  • the compound of the invention for the treatment or prevention of a metabolic disease or disorder comprises an R- enantiomer of the compound.
  • the compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the invention of the present application features a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of atherosclerosis in a subject in need thereof the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof.
  • a compound of the invention for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof.
  • the compound of formula (I) has significantly reduced adverse effects on cardiac and gallbladder function relative to other TGR5 agonists known in the art, for example, the 13.4-benzofuranyloxynicotinamide, 4- phenoxynicotinamide and 4-phenoxypyrimidine-5-carboxamide derivatives.
  • the compound of the invention for the treatment or prevention of atherosclerosis is racemic. In some embodiments, the compound of the invention for the treatment or prevention of atherosclerosis is an R- or an S- enantiomer of the compound of formula (I). In some embodiments, the compound of the invention for the treatment or prevention of atherosclerosis is an R- enantiomer of the compound of formula (I). In some embodiments, the compound of the invention for the treatment or prevention of atherosclerosis is an S- enantiomer of the compound of formula (I).
  • methods of this invention for treating, preventing suppressing, reducing the severity of, or reducing the risk of atherosclerosis in a subject comprise administering a compound of formula (I), wherein said compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one.
  • the compound is (6S)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines.
  • the compound of formula (I) does not induce increased secretion of GLP- 1.
  • the invention of the present application provides a method of reducing plasma triglyceride and LDL-cholesterol levels in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof
  • a compound of the invention for reducing plasma triglyceride and LDL- cholesterol levels is racemic.
  • the compound for reducing plasma triglyceride and LDL-cholesterol levels is an R- or an S- enantiomer of the compound of formula (I).
  • the compound for reducing plasma triglyceride and LDL- cholesterol levels is an S- enantiomer of the compound of formula (I). In some embodiments, the compound for reducing plasma triglyceride and LDL-cholesterol levels is an R- enantiomer of the compound of formula (I). In some embodiments, the compound is 6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound is 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4- methoxybenzyl)-l,4-diazepan-2-one.
  • the compound is 4-acetyl-l- [2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one.
  • methods of this invention for reducing plasma triglyceride and LDL- cholesterol levels in a subject comprise administering a compound of formula (I), wherein said compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one.
  • the compound is (6S)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines.
  • the compound of formula (I) does not induce increased secretion of GLP-1.
  • the invention of the present application provides a method of inducing increased GLP- 1 secretion in a cell, the method comprising contacting the cell with an effective amount of the invention, e.g., a compound of formula (I), as described herein, or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof.
  • the cell is in vitro.
  • the cell is in vivo.
  • the compound of the invention for inducing increased GLP-1 secretion in a cell is racemic.
  • the compound of the invention for inducing increased GLP- 1 secretion in a cell is an R- or an S- enantiomer of the compound of formula (I). In some embodiments, the compound of the invention for inducing increased GLP- 1 secretion in a cell is an R- enantiomer of the compound of formula (I). In some embodiments, the compound inducing increased GLP-1 secretion in a cell is an S- enantiomer of the compound of formula (I).
  • the compound of the invention for inducing GLP-1 secretion in a cell is 6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one, 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4- methoxybenzyl)-l,4-diazepan-2-one, or 4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one.
  • the compound is (6R)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • the compound of the invention for inducing GLP-1 is selective, for example, the compound does not effect expression or elaboration of pro-inflammatory cytokines.
  • the present invention provides a method for decreasing expression or elaboration of pro-inflammatory cytokines in a cell, the method comprising contacting the cell with an effective amount of the invention, e.g., a compound of formula (I), as described herein, or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof.
  • the decreased expression or elaboration of pro- inflammatory cytokines is selective since the compound does not effect secretion of GLP-1.
  • the compound of the invention has diminished adverse effects on cardiac and gallbladder function.
  • the cell is in vitro. In other embodiments, the cell is in vivo.
  • the compound of the invention for decreasing expression or elaboration of pro-inflammatory cytokines is racemic. In some embodiments, the compound of the invention for decreasing expression or elaboration of proinflammatory cytokines comprises an R- or an S- enantiomer of the compound. In some embodiments, the compound of the invention for decreasing expression or elaboration of proinflammatory cytokines comprises an R- enantiomer of the compound. In some embodiments, the compound of the invention for decreasing expression or elaboration of pro-inflammatory cytokines comprises an S- enantiomer of the compound.
  • the compound comprises (65)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one. In other embodiments, the compound is ( ⁇ 5R)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)- 1 -(3-methylbenzyl)- 1 ,4-diazepan-2-one.
  • the present invention identifies heterogeneity of the TGR5 receptor that can be exploited to separate the anti-inflammatory and metabolic effects of TRG5 agonists.
  • the compounds of formula (I) are selective TGR5 agonists.
  • a TGR5 agonist compound of formula (I) selectively activates a TGR5 anti-inflammatory response.
  • a TGR5 agonist compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines.
  • a TGR5 agonist compound of formula (I) provides selective metabolic effects.
  • a TGR5 agonist comopound of formula (I) selectively increases GLP-1 secretion.
  • a TGR5 agonist compound of formula (I) activates a TGR5 anti-inflammatory response but does not increase GLP-1 secretion. In some embodiments, a TGR5 agonist compound of formula (I), activates a TGR5 decreased expression or eleaboration of pre-inflammaotry cytokines but does not increase GLP-1 secretion. In some embodiments, the TGR5 agonist that selectively increases GLP-1 secretion is a compound of formula (I). In other embodiments, the TGR5 agonist that selectively increases GLP-1 secretion is the S-enantiomer of a compound of formula (I).
  • the TGR5 agonist that selectively increases GLP-1 secretion is the R- enantiomer of a compound of formula (I). In certain embodiments, the TGR5 agonist that selectively increases GLP-1 secretion is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one.
  • the TGR5 agonist that selectively activiates a TGR5 anti-inflammatory effect and does not increase GLP-1 secretion is (65)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • a TGR5 agonist compound of this invention comprises any ligand, peptide, or antibody capable of binding the TGR5 receptor and producing a TGR5 receptor agonistic effect.
  • a compound may be a ligand, which in one embodiment refers to any molecule that provides an enhanced affinity for a TGR5 receptor, wherein said affinity results in a TGR5 receptor agonistic activity.
  • a TGR5 agonist compound of this invention is an antibody.
  • the term "antibody” refers to intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of specifcially interacting with a desired target as described herein, for example, binding to phagocytic cells.
  • the antibody fragments may be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • E. coli or mammalian cells e.g. Chinese hamster ovary cell culture or other protein expression systems
  • Antibody fragments can, in some embodiments, be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al , Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991 ; Bird et al , Science 242:423-426, 1988; Pack et al , Bio/Technology 11: 1271-77, 1993; and Ladner et al , U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al , Nature, 321:522-525 (1986); Riechmann et al , Nature 332:323-327 (1988); Verhoeyen et al , Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al , J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al , J. Immunol., 147(l):86-95 (1991)].
  • human can be made by introducing of human immunoglobulin loci into transgenic animals, e.g. mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • a TGR5 agonist compound provides a selective effect.
  • a ligand TGR5 agonist selectively binds the TGR5 receptor.
  • a peptide TGR5 agonist compound selectively binds the TGR5 receptor.
  • an antibody TGR5 agonist compound selectively binds the TGR5 receptor.
  • the present invention provides that different enantiomers of the compound of formula (I) induce different biologic effects at the TGR5 receptor, showing, for the first time, that there is heterogeneity of the TGR5 receptor or heterogeneity of activation of the TGR5 receptor, and that different compounds that are agonists at a TGR5 receptor can exploit this heterogeneity to have selective biological effects.
  • the compounds of this invention are selective TGR5 agonists able to provide an anti-inflammatory effect but not a metabolic effect in vivo.
  • the compounds of this invention are selective TGR5 agonists able to provide a metabolic effect but not an anti-inflammatory effect in vivo.
  • the present invention provides that different agonist compounds induce different biologic effects at the TGR5 receptor, showing heterogeneity of the TGR5 receptor binding or heterogeneity of activation of the TGR5 receptor.
  • Compounds that are agonists at a TGR5 receptor may be used to exploit the heterogeneity of the TGR5 receptor in order to provide selective biological effects.
  • the compounds of this invention are selective TGR5 agonists able to provide an anti- inflammatory effect but not a metabolic effect in vivo.
  • TLC thin layer chromatography
  • Carbon-13 nuclear magnetic resonance ( 13 C NMR) spectra were reported in parts per million on the ⁇ scale, and are referenced from the carbon resonances of the solvent (CDCI 3 : ⁇ 77.00, CD 2 CI 2 : ⁇ 54.00, DMSO- ⁇ : ⁇ 39.51). Peak assignments of intermediates are based on analyses of gradient COSY experiments and chemical shifts of individual protons. High resolution mass spectra (HRMS) were recorded on using either an electrospray (ESI) or direct analysis in real time (DART) ionization source.
  • ESI electrospray
  • DART direct analysis in real time
  • Example 1 6-[(3-Fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • Step 1 fert-Butyl Allylcarbamate.
  • allylamine 29.9 g, 0.525 mol
  • triethylamine 53.0 g, 0.525 mol
  • Boc-anhydride 114 g, 0.525 mol
  • dry Et 2 0 150 ml
  • the reaction mixture was stirred at room temperature overnight and concentrated under a reduced pressure.
  • the oily residue was dried in a vacuum (1-2 mm Hg) to afford the title compound which was used further without an additional purification.
  • Step 3 tert-Butyl ⁇ 2-Hydroxy-3-[(3- methylbenzyl)amino]propyl ⁇ carbamate.
  • 3-methylbenzyl)amine 54.2 g, 0.448 mol was added by one portion upon stirring and cooling in a cold-water-bath.
  • the reaction mixture was stirred at 50 °C (oil bath) for 4 h, cooled to room temperature, and concentrated under a reduced pressure.
  • the oily residue was re-evaporated with toluene (2 x 200 ml) and dried in a vacuum (1-2 mm Hg) for 3 h to afford the titled compound which was used in the next step without purification.
  • the reaction mixture was stirred at room temperature overnight and concentrated under a reduced pressure (-10-15 mm Hg) at -50-60 °C.
  • the residue was distributed between 10% aq. K 2 C0 3 (180 ml) and Et 2 0 (180 ml).
  • the organic layer was washed with water (2 x 100 ml), dried over MgS0 4 , and filtered.
  • 4M aq. HC1 (11.4 ml) was added dropwise, the formed precipitate was filtered, washed with abs. Et 2 0, and dried on air.
  • the obtained hydrochloride was shaken in a separating funnel with 20% aq.
  • Step 6 fert-Butyl 6- ⁇ [tert-Butyl(dimethyl)silyl]oxy ⁇ -4-(3-methylbenzyl)-3-oxo- 1,4-diazepane-l-carboxylate.
  • Step 7 fert-Butyl 6-Hydroxy-4-(3-methylbenzyl)-3-oxo-l,4-diazepane-l- carboxylate .
  • Step 9 6-[(3-Fluorobenzyl)oxy]-l-(3-methylbenzyl)-l,4-diazepan-2-one.
  • i ⁇ ?ri-butyl-6- [(3 -fluorobenzyl)oxy] -4-(3 -methylbenzyl)-3 -oxo- 1 ,4-diazepane- 1 - carboxylate (43.7 g, 0.0989 mol) in EtOAc (500 ml)
  • 4M solution of HC1 in EtOAc 198 ml, 0.791 mol
  • the reaction mixture was stirred at room temperature for 48 h.
  • Step 10 6-[(3-Fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one.
  • 2-furoyl chloride (16.8 g, 0.129 mol) in dry CH2CI2 (30 ml) was added dropwise upon stirring and cooling in an ice bath.
  • Step 1 tert-Butyl (Oxiran-2-ylmethyl)carbamate. Allylamine (17.5 g, 306.5 mmol, 23 ml) was dissolved in dichloromethane (150 ml). A solution of Boc-anhydride (70.8 g, 325 mmol) in dichloromethane (150 ml) was added in portions under cooling with a water bath and stirring. The reaction mixture was stirred at room temperature for 15 h. The obtained solution of feri-butyl allylcarbamate was cooled again in an ice bath, m- Chloroperoxybenzoic acid (92 g 70%, 370 mmol) was added under stirring for 30 min.
  • Step 2 tert-Butyi ⁇ 2-Hydroxy-3-[(4-methoxybenzyl)amino]propyl ⁇ carbamate.
  • i ⁇ ?ri-Butyl (oxiran-2-ylmethyl)carbamate 7 g, 41 mmol
  • isopropanol 40 ml
  • p-Methoxybenzylamine 6.7 g, 49 mmol
  • the obtained solution was kept under heating and stirring for 12 h.
  • the reaction mixture was evaporated.
  • the residue was purified by chromatograpny on silica gel in chloroform-isopropanol mixture (up to 10% of isopropanol).
  • Step 3 Target tert-Butyl ⁇ 2- ⁇ [tert-Butyl(dimethyl)silyl]oxy ⁇ -3-[(4- methoxybenzyl)-amino]propyl ⁇ carbamate. i ⁇ ?ri-Butyl ⁇ 2-hydroxy-3 - [(4- methoxybenzyl)amino]propyl ⁇ carbamate (8 g, 26 mmol) was dissolved in 1,2-dichloroethane (60 ml). Triethylamine (7.2 ml, 52 mmol) and feri-butyl dimethylsilyl chloride (5.4 g, 36 mmol) were added.
  • Step 4 tert-Butyi ⁇ 2- ⁇ [tert-Butyl(dimethyl)silyl]oxy ⁇ -3-[(chloroacetyl)(4- methoxy-benzyl)amino]propyl ⁇ carbamate.
  • Step 6 fert-Butyl 6-Hydroxy-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l- carboxylate. i ⁇ ?ri-Butyl 6- ⁇ [1 ⁇ 2ri-butyl(dimethyl)silyl]oxy ⁇ -4-(4-methoxybenzyl)-3-oxo-l,4- diazepane-l-carboxy-late (6.3 g, 13.8 mmol) and acetic acid (900 mg, 13.8 mmol) were dissolved in methanol (150 ml). Potassium fluoride (2.5 g, 42 mmol) was added.
  • Step 8. 6-[(2-Fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one Hydro-chloride.
  • feri-Butyl 6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4- diazepane-l-carboxylate (4.6 g, 10 mmol) was dissolved in dioxane (30 ml), and 4M HCl in dioxane (30 ml) was added. The resulting solution was stirred at room temperature for 12 h until compound (9) disappeared, and then evaporated in vacuum.
  • Example 3 (6/?)-6-[(2-Fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(3-thienylcarbonyl)- l,4-diazepan-2-one [000161]
  • Step 1. fert-Butyl [(2/?)-2,3-dihydroxypropyl]carbamate.
  • Freshly distilled S(- glycidol (10 g, 135 mmol) was dissolved in methanol (200 ml) containing 10% of water.
  • Sodium azide (44 g, 675 mmol) and ammonium chloride (16 g, 300 mmol) were added. The resulting mass was refluxed for 8 h and evaporated.
  • Target compound was isolated from the residue by column chromatography in chloroform-methanol mixture (up to 4% of methanol).
  • Compound (3) (15.5 g, 60%) was obtained.
  • Step 2 (2/?)-3-[(tert-Butoxycarbonyl)amino]-2-hydroxypropyl 4- methylbenzenesulfo-nate.
  • feri-Butyl [(2R)-2,3-dihydroxypropyl]carbamate (15.5 g, 81 mmol) was dissolved in absolute pyridine (50 ml).
  • Tosyl chloride (17 g, 90 mmol) was added in portions under cooling in an ice bath. The obtained solution was stirred for 4 h at room temperature.
  • the reaction mixture was poured into ethyl acetate (500 ml), washed with 1M citric acid (2 x 100 ml), with an aqueous solution of copper sulfate (100 ml), and dried with anhydrous sodium sulfate.
  • the target compound was isolated from the obtained solution by column chromatography in chloroform-methanol mixture (up to 2% of methanol) and obtained (16.8 g, 60%).
  • Step 3 tert-Butyl ⁇ (25)-2-hydroxy-3-[(4- methoxybenzyl)amino]propyl ⁇ carbamate.
  • (2R)-3-[(1 ⁇ 2ri-Butoxycarbonyl)amino]-2- hydroxypropyl 4-methylbenzenesulfonate (16.8 g, 48 mmol) was dissolved in acetonitrile (150 ml), and then p-methoxybenzylamine (8.4 g, 60 mmol), potash (16 g, 120 mmol), and potassium iodide (1.7 g, 10 mmol) were added. The resulting mixture was vigorously stirred at 60°C for 24 h until compound (4) disappeared.
  • Steps 4 - 9 Subsequent 6 steps of the transformation of feri-butyl ⁇ (25)-2- hydroxy-3-[(4-methoxybenzyl)amino]propyl ⁇ carbamate into the hydrochloride of (6R)-6-[(2- fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one are completely analogous to steps 3-8 of the Example 2
  • Step 10 (6/?)-6-[(2-Fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(3- thienylcarbonyl)-l,4-diazepan-2-one.
  • HBTU 160 mg, 0.42 mmol
  • thiophene-3-carboxylic acid 54 mg, 0.42 mmol
  • DIPEA 54 mg, 0.42 mmol
  • Steps 1-5 Identical to those described in Example 2.
  • Step 6 6-Hydroxy-l-(4-methoxybenzyl)-l,4-diazepan-2-one Hydrochloride. i ⁇ ?ri-Butyl 6- ⁇ [1 ⁇ 2ri-butyl(dimethyl)silyl]oxy ⁇ -4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l- carboxy-late (3.0 g, 6.5 mmol) was dissolved in dioxane (30 ml). Then 4M HCI in dioxane (30 ml) was added. The resulting solution was stirred at room temperature for 12 h until the deprotection was complete, and then evaporated in vacuum.
  • Step 7 4-(Cyclopropylcarbonyl)-6-hydroxy-l-(4-methoxybenzyl)-l,4- diazepan-2-one.
  • the product obtained at step 6 was dissolved in dichloromethane (50 ml), and DIPEA (1.3 g, 10 mmol) was added.
  • the resulting solution was cooled in an ice bath, and cyclopropanecarbonyl chloride (0.81 g, 7.8 mmol) was added dropwise.
  • the reaction mixture was stirred for 2 h at room temperature.
  • Dichloromethane (100 ml) and a 1M solution of citric acid (50 ml) were added.
  • the organic layer was separated and dried with anhydrous sodium sulfate.
  • the target product was isolated from the obtained solution by column chromatography in chloroform-methanol mixture. A crystalline solid (1.4 g, 70% for 2 steps) was obtained.
  • Step 8 HTC.
  • the intermediate (Example 4, step 7) was dissolved in THF-DMF (9:1) mixture (0.8 ml). A halide (1.2 - 1.3 eq) was added. Then a suspension of NaH (1.5 eq, preliminarily washed with hexane) in THF (0.3 ml) was added. The reaction mixture was stirred for 4 h at room temperature and neutralized with a solution of acetic acid in methanol to pH 7. After evaporation, the product was isolated by HPLC.
  • Steps 1-7 Identical to those described in Example 2.
  • Step 8 tert-Butyl 6-[(2-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate.
  • i ⁇ ?ri-Butyl 6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxylate (4.4 g, 9.6 mmol) was dissolved in a mixture of acetonitrile (80 ml) and water (20 ml).
  • Sodium acetate trihydrate (5.9 g, 43.2 mmol) and acetic acid (2.6 g, 43.2 mmol) were added.
  • Step 9 6-[(2-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride.
  • feri-Butyl 6-[(2-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate (1.95 g, 5.8 mmol) was dissolved in dioxane (25 ml), and 4M HC1 in dioxane (25 ml) was added. The resulting solution was stirred at room temperature for 12 h until the deprotection was complete, and then evaporated in vacuum. In order to remove excessive HC1 the residue was subjected to coevaporation with acetonitrile (2 x 100). The obtained oil of the hydrochloride was dried at 0.5 torr / 40°C to give an amorphous solid. The product was used without further purification.
  • Step 10 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one.
  • a solution of the crude 6-[(2-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride (5.8 mmol, step 9) and triethylamine (1 ml, 7.2 mmol) in dry CH 2 CI 2 (15 ml) a solution of 2-furoyl chloride (0.9 g, 7 mmol) in dry CH 2 CI 2 (5 ml) was added dropwise upon stirring and cooling in an ice bath.
  • Step 11 HTC.
  • the intermediate (Example 5, step 10) was dissolved in THF- DMF mixture (9: 1, 0.8 ml). A halide (1.2 - 1.3 eq) was added. Then a suspension of NaH (1.5 eq, preliminarily washed with hexane) in THF (0.3 ml) was added. The reaction mixture was stirred for 4 h at room temperature and neutralized with a solution of acetic acid in methanol to pH 7. After evaporation, the product was isolated by HPLC.
  • Steps 1-7 are similar to those described in Example 2, but allylamine was used as an amine, and 3-fluorobenzyl bromide as a halide.
  • Step 8 fert-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-4-(2-oxoethyl)-l,4-diazepane- 1-carboxylate.
  • Step 9 fert-Butyl 4-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-3-oxo- 1,4-diaze-pane-l-carboxylate. i ⁇ ?ri-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-4-(2-oxoethyl)- 1,4-diazepane-l-carboxylate (1.7 g, 4.5 mmol) was dissolved in 1 ,2-dichloroethane (40 ml).
  • Step 10 l-[2-(Dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2- one dihydrochloride. i ⁇ ?ri-Butyl 4-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-3-oxo- 1,4-diazepane-l-carboxylate (1.5 g, 3.7 mmol) was dissolved in dioxane (30 ml). Then 4M HCI in dioxane (30 ml) was added. The obtained solution was stirred at room temperature for 12 h until the starting compound disappeared, and then evaporated in vacuum.
  • Step 11 4-Acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4- diazepan-2-one.
  • DIPEA 1. mmol
  • acetic anhydride 1.5 g, 14.8 mmol
  • Steps 1-7 are similar to those described in Example 2, but 3-fluorobenzyl bromide was used as a halide.
  • Step 8 fert-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate.
  • i ⁇ ?ri-Butyl 6-[(3-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxylate (4.84 g, 10.6 mmol) was dissolved in a mixture of acetonitrile (90 ml) and water (25 ml). Sodium acetate trihydrate (6.5 g, 47.5 mmol) and acetic acid (2.9 g, 47.5 mmol) were added.
  • Step 9 6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride.
  • feri-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate (2.1 g, 6.2 mmol) was dissolved in dioxane (30 ml), and 4M HC1 in dioxane (30 ml) was added. The resulting solution was stirred at room temperature for 12 h until the deprotection was complete, and then evaporated in vacuum. In order to remove excessive HC1 the residue was subjected to coevaporation with acetonitrile (2 x 100).
  • Step 10 4-acetyl-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one.
  • a solution of the crude 6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride (6.2 mmol, step 9) and triethylamine (2 ml, 15 mmol) in dry CH 2 CI 2 (20 ml) a solution of acetyl chloride (0.6 g, 7.4 mmol) in dry CH 2 CI 2 (5 ml) was added dropwise upon stirring and cooling in an ice bath.
  • a library of 20,000 compounds were submitted to an automated high-throughput primary screening assay performed on a CHO cell line expressing hTGR5 together with a lucif erase reporter that was driven by a cAMP response element (CRE-luc) ( Figure la). All compounds were dissolved in DMSO and tested in 96 well plates at a single dose (lOuM), and a score was given to each potential hit, in accordance with its ability to activate the lucif erase reporter. The 621 compounds with the highest score were selected from this primary screening campaign. To assess the validity and specificity of the results, the screen was repeated and the 621 hits re-tested in CHO cells transfected with either an empty control vector or with hTGR5.
  • CRE-luc cAMP response element
  • these 10 hits were shown to be devoid of activity on the nuclear bile acid-activated receptor, farnesoid X receptor (FXR), as evaluated in Cos-7 cells transiently co-transfected with hFXR, mRXR and an FXR-RE-driven luciferase reporter (Houten et al., Mol Endocrinol, 21, 1312-1323, 2007) (data not shown).
  • FXR farnesoid X receptor
  • a mouse macrophage cell line (RAW 264.7) was treated with LPS, in the presence or absence of the three lead TGR5 agonists at a concentration of 30uM. These compounds' activity was compared to that of INT-777 as a benchmark positive control.
  • LPS stimulation induced the expression of the mRNAs of multiple inflammatory cytokines, including IL6, MCP-1 and TNFa.
  • TGR5 agonist (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) is due not only to its anti-inflammatory properties, but also to its rather profound effects on plasma lipid concentrations, effects that are not seen with INT-777 a semi- synthetic bile acid with TGR5 agonist activity (Pols et al., Cell metabolism, 4, 747-757, 2011).
  • Example 13 Effects on Metabolic Parameters both in vitro and, by oral administration, in vivo
  • TGR5 regulates glucose homeostasis by promoting the secretion of the insulinotropic incretin GLP-1 from enteroendocrine L-cells (Thomas et al., cell metabolism 10, 161 -111, 2009).
  • Body weight ( Figure 3a) and fat mass ( Figure 3b) were significantly reduced by (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) administration (compare to control mice given HFD alone), while lean mass ( Figure 3b) and water and food intake (Figure 3c) were comparable between the HFD+(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) and the HFD groups.
  • Results are expressed as a percentage of the activity of the reference agonist or antagonist for each receptor. Note that there was significant activity only at TGR5, where the reference agonist was INT-777. "Ago” - agonist; "Antago” - antagonist.
  • Example 15 In Vivo Effects of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) Show Specificity for the TGR5 Receptor
  • TGR5 agonists of the present invention are not only dependent on good efficacy and potency of the compounds, but also requires a lack of serious adverse effects.
  • Adverse effects on cardiac and gallbladder function have been reported for most known TGR5 agonists, for instance, 13.4-benzofuranyloxynicotinamide derivatives (Zou et al., Eur. J. Med. Chem. , 82, 1-15, 2014) and both 4-phenoxynicotinamide and 4- phenoxypyrimidine-5-carboxamide derivatives (Duan et al., J. Med. Chem., 55, 10475- 10489, 2012).
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • LDH lactate dehydrogenase
  • mice (6- [(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one)-treated mice were protected from the negative impact of HFD on liver function (Table 5), suggesting a hepatoprotective effect against the development of a fatty liver.
  • Serum amylase and creatine kinase levels were normal, indicating that (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) also had no adverse effect on exocrine pancreatic function, heart muscle or skeletal muscle (Table 5).
  • the compound had no adverse impact on serum triglycerides or total-, HDL- or LDL-cholesterol levels (Table 5) and, in fact, seemed to slightly decrease the levels of LDL-cholesterol.
  • an in vivo GLP-1 secretion assay was conducted by challenging diet-induced obese C57BL/6J mice with an oral test meal, 30 minutes after the administration of saline, (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one) or (65)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one) at a concentration of 30mg/kg of body weight.

Abstract

The invention relates to novel 1,4-diazepan-2-one compounds, which are TGR5 agonists and useful for the treatment of metabolic disorders, inflammatory diseases, atherosclerosis and dyslipidemias.

Description

TGR5 AGONISTS
FIELD OF THE INVENTION
[0001] The invention relates to novel 1 ,4-diazepan-2-one compounds, which are TGR5 agonists and useful for the treatment of metabolic disorders, inflammatory diseases, atherosclerosis and associated conditions, and dyslipidemias.
BACKGROUND OF THE INVENTION
[0002] TGR5, also known as G-protein coupled bile acid receptor 1 (GPBAR1) or GPR131, is a Gs protein-coupled receptor (GPCR) which, upon binding to bile acids (BAs), stimulates downstream adenylate cyclase/cAMP signaling in a wide array of tissues, including gallbladder, adipose tissue, intestine, spleen, placenta and ovaries (Kawamata et al., J Biol Chem 278, 9435-9440, 2003; Maruyama et al., Biochem. Biophys. Res. Commun., 298, 714-719, 2002; Pols et al, Cell metabolism, 14, 747-757, 2011). Functional activation of TGR5 by BAs is crucial for the regulation of several physiological processes. TGR5 modulates chloride and fluid secretion from the gallbladder (Keitel et al., Hepatology, 50, 861-870, 2009; Lavoie et al. Journal of physiology, 588, 3295-3305, 2010; Li et al., Molecular endocrinology, 25, 1066-1071, 2011). In brown adipose tissue and skeletal muscle, TGR5 activation increases energy expenditure, and a diet containing 0.5% of cholic acid (CA), which efficiently activates TGR5, attenuates diet-induced obesity (DIO) in mice (Watanabe et al., Nature, 439, 484-489, 2006). TGR5 also controls the secretion of the insulinotropic incretin GLP-1 (glucagon-like peptide- 1) from enteroendocrine L cells, both in vitro (Katsuma et al., Biochem. Biophys. Res. Commun., 329, 386-390, 2005) and in vivo (Harach et al., Scientific reports, 2, 430, 2012; Thomas et al., Cell metabolism, 10, 167-177, 2009). The therapeutic relevance of increasing GLP-1 levels in plasma is currently well- established, since enhancement of the half-life of this incretin upon treatment with dipeptidyl peptidase 4 inhibitors (DPP4i) or activation of the GLP-1 receptor by exendin-4 (Ex-4) derivatives has proven to be efficacious in the treatment of type 2 diabetes (Drucker, Cell metabolism, 3, 153-165, 2006; Drucker and Nauck, Lancet, 368, 1696-1705, 2006). Therefore, the direct stimulation of intestinal GLP-1 release upon TGR5 activation could constitute a new therapeutic approach to the treatment of metabolic diseases.
[0003] TGR5 expression has also been characterized in macrophages (Kawamata et al., J. Biol. Chem., 278, 9435-9440, 2003; Keitel et al., Biochem. Biophys. Res. Commun., 372, 78- 84, 2008), where its activation inhibits pro-inflammatory cytokine production and prevents the development of LPS-induced liver inflammation (Wang et al., Hepatology, 54, 1421- 1432, 2011) and atherosclerosis in mice (Pols et al., Cell metabolism, 14, ΊΑΊ-Ί5Ί, 2011). These observations suggest that TGR5 may be an effective target for pharmacologic treatment of metabolic disorders, such as obesity and diabetes, and inflammatory diseases, such as atherosclerosis.
[0004] Identification and characterization of TGR5 agonists have been reported (Budzik et al., Bioorg. Med. Chem. Lett., 20, 1363-1367, 2010; Dehmlow et al., Bioorg. Med. Chem. Lett, 23, 4627-4632, 2013; Duan et al., J. Med. Chem. , 55, 10475-10489, 2012). There is still an on-going need for TGR5 agonists with improved potency, efficacy, selectivity, metabolic stability and bioavailability, while lowering the toxicity and adverse effects of these compounds for long-term usage. The present invention provides a novel class of small molecule TGR5 agonists with beneficial in vitro and in vivo effects on metabolism (improved glucose homeostasis and cholesterol lowering), inflammation and atherosclerosis, while greatly diminishing adverse effects on cardiac and gallbladder function.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides TGR5 agonists of formula (I)
Figure imgf000003_0001
(I)
wherein
Ri is aryl, heteroaryl, C3-8 cycloalkyl, cycloheteroalkyl, Ci_6 alkyl, C2-6 alkenyl, C2-6 alkynyl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, aryl, halo, Ci_3 haloalkyl, OH, 0-Ci_6 alkyl, cyano, nitro, and -NRaRt,;
R2 is Ci-6 alkyl, O-Ci-6 alkyl, -NRaRt>, C3-8 cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, nitro, and -NRaRt>;
R3 is F, CI, Br, I, or 0-Ci_6 alkyl;
n is 0-5 ; and
Ra and Rb each are independently H or C1-3 alkyl.
[0006] In another aspect, the present invention features TGR5 agonists of formula (I)
Figure imgf000004_0001
(I)
wherein
Ri is aryl, -CH2CH2-aryl, C3-8 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, or -CH2NRa b, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro;
R2 is Ci-6 alkyl, O-Ci-6 alkyl, -NRaRt>, C3-8 cycloalkyl, 2-furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, Ci-3 haloalkyl, OH, and 0-Ci_6 alkyl, cyano, amino, Ci_3 alkylamino, di-Ci-3 alkylamino, and nitro;
R3 is F, CI, Br, I, or 0-Ci_6 alkyl;
n is 1-5; and
Ra and Rb each are independently Ci_6 alkyl.
[0007] In another aspect, a TGR5 agonist of formula (I) is its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
[0008] In another aspect, the present invention features a composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, for example, a compound of formula (I) as described herein. In another aspect, the present invention features a pharmaceutical composition comprising a TGR5 agonist of formula (I) and at least one pharmaceutically acceptable carrier.
[0009] In yet another aspect, the present invention provides a method of treating, preventing, suppressing, reducing the severity of, or reducing the risk of a metabolic disease or disorder in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the present invention, for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof. In some embodiments, the metabolic disease is diabetes mellitus, obesity, dyslipidemia, insulin resistance, or insulin sensitivity, any any combination thereof.
[00010] In another aspect, the present invention provides a method of treating, preventing, suppressing, reducing the severity of, or reducing the risk of an inflammatory disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
[00011] In yet another aspect, the present invention provides a method of treating, preventing, suppressing, reducing the severity of, or reducing the risk of atherosclerosis in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
[00012] In yet another aspect, the present invention provides a method of reducing plasma triglyceride and LDL-cholesterol levels in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
[00013] In yet another aspect, the present invention provides a method for inducing increased GLP- 1 secretion in a cell, the method comprising contacting the cell with an inducing effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof.
[00014] In yet another aspect, the present invention provides a method for selectively increasing GLP-1 secretion in a cell, the method comprising contacting the cell with effective amount of a compound of formula (I). In a further aspect, the present invention provides a method for selectively increasing GLP-1 secretion in a cell, the method comprising contacting the cell with effective amount of a compound of (6R)-6-[(3-fluorobenzyl)oxy]-4- (2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
[00015] In yet another aspect, the present invention provides a method for decreasing expression or elaboration of pro-inflammatory cytokines in a cell, the method comprising contacting the cell with an effective amount of a compound of the invention as described herein, or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof. In another embodiment, a method of this invention comprising contacting the cell with an effective amount of a compound of the invention does not induce an increase in GLP- 1 secretion in the cell. [00016] In yet another aspect, the present invention provides a method of selectively binding a subpopulation of TGR5 receptors, said method comprising the step of contacting the TGR5 with a compound of the invention, e.g., a compound of formula (I), or its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof, in an amount effective to bind the compound to the TGR5 receptor.
[00017] In another aspect, the present invention provides a compound of formula (I), comprising a TGR5 agonist and showing selectivity for a heterogeneous population of TGR5 receptors. In yet another aspect, the present invention identifies heterogeneity of the TGR5 receptor that can be exploited to separate the anti-inflammatory and metabolic effects of TRG5 agonists. In another aspect, the present invention provides a compound of formula (I), comprising a TGR5 agonist and showing selectivity for heterogeneous activation of TGR5 receptors. In yet another aspect, the present invention identifies heterogeneity of activating a TGR5 receptor that can be exploited to separate the anti-inflammatory and metabolic effects of TRG5 agonists.
[00018] In another aspect, the compound of this invention is a selective TGR5 agonist. In some embodiments, a TGR5 agonist compound of this invention activates a TGR5 antiinflammatory response. In other embodiments, a TGR5 agonist compound of this invention decreases expression or elaboration of pro-inflammatory cytokines. In some embodiments, a TGR5 agonist compound of this invention provides metabolic effects. In other embodiments, a TGR5 agonist compound of this invention increases GLP-1 secretion. In some embodiments, a TGR5 agonist compound of this invention provides selective TGR5 activation. In certain embodiments, a TGR5 agonist compound of formula (I) is an R- enantiomer. In certain embodiments, a TGR5 agonist compound of formula (I) is an S- enantiomer. In certain embodiments, a TGR5 agonist compound of formula (I) is (6R)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In certain embodiment, a TGR5 agonist comound of formula (I) is (65)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
[00019] In yet another aspect, in contrast to other known TGR5 agonists, the compounds of the present inventon show diminished adverse effects upon the heart or the gall bladder.
[00020] In yet another aspect, the present invention provides that different enantiomers of the TGR5 agonist of formula (I) induce selective biologic effects at the TGR5 receptor, showing, for the first time, that there is heterogeneity of the TGR5 receptor or heterogeneity of activation of the TGR5 receptor, and that enantiomers of TGR5 agonist compounds can exploit this heterogeneity resulting in different biological effects. [00021] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[00022] Figures la-lf depict the characterization of synthetic TGR5 agonists. Figure la: A process of selecting the TGR5 agonists from over 20,000 compounds from the ChemBridge compound library (> 900,000 compounds). A robotic primary screen, which consisted of co-transfecting CHO cells with a hTGR5 expression construct and a luciferase reporter under the control of a cAMP response element (CRE), identified 621 hits out of these 20,000 compounds. These 621 primary hits were then re-tested in duplicate with the same reporter assay, identifying 313 confirmed hits. Out of these 313 hits, the 10 best candidates were selected according to their score and their EC50, and the microsomal stability of these 10 was tested. The 3 best candidates were selected for detailed characterization using in vitro/in vivo assays. Figure lb: Representation of the scores in the primary screens; 313 hits were confirmed out of the 621 primary hits. Each point represents the average score of the hit. The score was calculated by plate. The relative luciferase activity value of DMSO represented a score of 0, and Lithocholic acid (LCA) at 10 μΜ represented a score of 1. In blue are the hits that were not confirmed and in red the confirmed hits. Figure lc: EC50 determination of the 3 best compounds using the CRE-luciferase/hTGR5 co-transfection assay in CHO cells. Relative luciferase activity of Lithocholic acid (LCA) at 10μΜ was used as 100% efficacy. Figure Id: Intrinsic clearance rate of the 3 best compounds using human microsomes. Figure le: Chemical structure of the 3 selected compounds: (4- acetyl- 1- [2- (dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one), (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) and 4-
(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one). Figure If: Intrinsic clearance rate of the 3 best compounds using mouse microsomes.
[00023] Figures 2a-2d depict characterization of three compounds of the present invention. Figure 2a: Addition of compounds (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one), (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one), or 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l- (4-methoxybenzyl)-l,4-diazepan-2-one), each at a concentration of 30μΜ, significantly decreased the transcript levels for Tnfa, 116 and Mcpl in the RAW 264.7 murine macrophage cell line stimulated for 4 hours with lipopoly saccharide (LPS, 10 ng/mL). The semi-synthetic bile acid INT-777 (at 30μΜ), a known selective TGR5 agonist, was used as a positive control. Figure 2b: Acute effect of INT-777, (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one), (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one), and 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l- (4-methoxybenzyl)-l,4-diazepan-2-one) (30μΜ) on GLP-1 secretion from cultured murine entero-endocrine GLUTag cells. All compounds increased GLP-1 release above vehicle- treated cells. Graphs represent the mean + SEM of at least three independent experiments. * denotes p<0.05. Figure 2c: Acute effect of INT-777, (4-acetyl-l-[2-(dimethylamino)ethyl]- 6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one), (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) and 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l- (4-methoxybenzyl)-l,4-diazepan-2-one) (30μΜ) on GLP-1 secretion from cultured human entero-endocrine NCI-H716 cells. All the tested compounds increased GLP-1 release above vehicle-treated cells. Graphs represent the mean + SEM of at least three independent experiments. *denotes p<0.05. Figure 2d: Analysis of plasma GLP-1 levels in diet-induced obese C57BL/6J mice after a test meal challenge (Ensure Plus at 10 mL/kg) given 30 min after a single oral administration of INT-777, (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one), 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l- (4-methoxybenzyl)-l,4-diazepan-2-one), or (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) at 30mg/kg of body weight (n = 5 mice per group). GLP- 1 secretion was significantly increased in mice treated with INT-777 and (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one). Data represent mean + SEM. *p<0.05.
[00024] Figures 3a-3h depict that the activation of TGR5 signaling by (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) stimulates energy expenditure and GLP-1 secretion in vivo. Figure 3a: Body weight evolution in male wild type C57BL/6J mice (Tgr5+/+ mice) fed for 13 weeks with a high-fat diet (HFD) or HFD+((6- [(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) at 30 mg/kg/d mixed with the food. Mice treated with HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) gained significantly less weight than mice fed with HFD alone (n = 10 mice per group). Figure 3b: Body composition was assessed by EchoMRI after 7 weeks of treatment as described in (a). Mice fed with HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) had a significant decrease in body fat mass compared to the mice fed with HFD alone (n = 10 mice per group). Figure 3c: Food and water intake measurements during 24 hours in the mice on HFD or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) revealed no significant differences between the treatment groups (n = 8 mice per group). Figure 3d: Oxygen consumption (V02) and locomotor activity (LA) measurements over a 24-hr period in male C57BL/6J mice after 7 weeks of HFD (solid circles) or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open squares) (at 30 mg/kg/d). Mice treated with HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) had a significant increase in O2 consumption, but did not move more, during the active period (7PM-7AM - dark phase) (n = 8 mice per group). The insets represent the average area under the curve (AUC) during the dark phase. Figure 3e: Cold test in male C57BL/6J mice fed for 10 weeks with HFD or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (at 30 mg/kg/d). Mice were exposed to a cold temperature (6°C), and their rectal temperature was monitored during 6 hours. The inset represents the average area under the temperature curve (AUC). Mice treated with HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one) maintained their core body temperature better than did control mice on HFD (n = 10 mice per group). Figure 3f: Relative mRNA expression levels of Ucp-1, Dio-2, and Cpt-1 in brown adipose tissue of male C57BL/6J mice fed for 13 weeks with HFD (solid bars) or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one) (open bars) (at 30 mg/kg/d). Mice exposed to HFD+((6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) showed a significant increase in the expression of these genes. Figure 3g: Analysis of plasma GLP-1 levels after a test meal (Ensure Plus at 10 mL/kg) in diet-induced obese C57BL/6J mice after 8 weeks of treatment with HFD plus vehicle or HFD plus (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) at 30mg/kg/day given as a food admix (n = 10 mice per group). GLP-1 secretion was significantly increased by (6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) administration. Figure 3h: Oral glucose tolerance test (OGTT) in male C57BL/6J mice fed for 9 weeks with either HFD or HFD+((6- [(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one). Mice treated with HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) had significant lower glucose levels after a glucose challenge (n = 10 mice per group). Data represent mean + SEM. * denotes p<0.05.
[00025] Figures 4a-4d depict the absence of adverse effects on the heart and gallbladder in mice chronically exposed to (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one). Figure 4a: Echocardiography and non-invasive blood pressure measurement in male C57BL/6J mice after 12 weeks on HFD or on HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) at 30 mg/kg/d. M- mode echocardiographic images and recorded parameters, such as posterior wall thickness (PWT), septum thickness (ST), left ventricular fractional shortening fraction (LVFS), and left ventricular ejection fraction (LVEF), were not different between HFD (solid bars) and HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open bars) treated mice (n = 5 mice per group). Similarly, heart rate (HR) and systolic blood pressure (SBP), which were measured in non-anesthesized mice, were also indistinguishable between the two treatment groups. Figure 4b: Macroscopic morphology (left), echographic evaluation (middle) and immunohistochemistry (right) of the gallbladder of male C57BL/6J mice after 13 weeks on HFD or on HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) at 30 mg/kg/d. No differences were detected between the treatment groups. Figure 4c: The area of the gallbladder, as measured by echographic analyses, was similar in mice treated with HFD (solid bar) or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open bar) after an overnight fast, when the gallbladder is filled (n = 5 mice per group). Figure 4d: Plasma concentration of total bile acids (BAs) after 13 weeks of HFD or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (n = 10 mice per group).
[00026] Figures 5a-5h depict that administration of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-
1- (3-methylbenzyl)-l,4-diazepan-2-one) admixed with HFD does not alter metabolic parameters in 7gr5-null mice, in support of the fact that the compound acts specifically at TGR5. Figure 5a: Body weight evolution in male 7gr5-null (Tgr5~'~) mice fed for 13 weeks with HFD or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-
2- one) at 30 mg/kg/d mixed with the food. The weight gain in Tgr5~'~ mice treated with HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) was comparable to that of Tgr5~'~ mice fed with HFD alone, indicating that the effect of (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) is dependent on the presence of TGR5 (n = 10 mice per group). Figure 5b: Body composition was assessed by EchoMRI after 7 weeks of HFD or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) at 30 mg/kg/d. Lean and fat mass were not altered in Tgr5~'~ mice treated with HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) compared to Tgr5~'~ mice that received only HFD (n = 10 mice per group). Figure 5c: Food and water intake during 24 hours was similar in the Tgr5~'~ mice whether on HFD or on HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one) (n = 8 mice per group). Figure 5d: Oxygen consumption (V02) and locomotor activity (LA) over a 24-hr period in male Tgr5~'~ mice after 7 weeks of HFD or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (30 mg/kg/d) (n = 8 mice per group). Oxygen consumption and locomotor activity were comparable between Tgr5~'~ mice treated with HFD (solid circles) or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open squares) during the active period (7PM-7AM - dark phase). In fact, if anything, the HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) group showed a statistically-insignificant decrease in oxygen consumption, a result in sharp contrast to that seen in normal mice (Figure 3d). Insets represent the average area under the curve (AUC) during the dark phase. Figure 5e: Cold test in male Tgr5~'~ mice fed for 10 weeks with HFD or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (30 mg/kg/d). Mice were exposed to cold temperature (6°C), and the rectal temperature was monitored over 6 hours. The inset represents the average area under the curve (AUC). Core body temperature was similar in Tgr5~'~ mice treated with HFD or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (n = 10 mice per group). Figure 5f: Relative mRNA expression levels of Ucp-1, Dio-2, and Cpt-1 in brown adipose tissue of male 7gr5_/~mice showed no significant difference whether the animals were fed for 13 weeks with HFD (solid bars) or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) (open bars) (30 mg/kg/d). Figure 5g: Absence of a difference in plasma GLP-1 levels after a test meal challenge (Ensure Plus at lOmL/kg) in diet- induced obese Tgr5~'~ mice after 8 weeks of treatment with HFD or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) given at 30mg/kg/day admixed with the food (n = 10 mice per group). Figure 5h: Oral glucose tolerance test (OGTT) in male Tgr5~'~ mice fed for 9 weeks with HFD or HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (30 mg/kg/d). Glucose levels after a glucose challenge in Tgr5~'~ mice treated with HFD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) were identical to those that received HFD alone (n = 10 mice per group). Data represent mean + SEM. * denotes p<0.05.
[00027] Figures 6a-6g depict that activation of TGR5 by (6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) protects against the development and progression of atherosclerosis in LDLr-null mice fed with a high cholesterol diet. Figure 6a: Body weight evolution in male LDLr-null (LDLr ~'~) mice fed for 13 weeks with a high cholesterol diet (HCD) or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) at 30 mg/kg/d given as food admix. Mice treated with HCD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) gained significantly less weight than mice fed HCD alone (n = 12 mice per group). Figure 6b: Body composition was assessed by EchoMRI in LDLr ~'~ mice after 7 weeks of HCD or HCD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) at 30 mg/kg/d. Body fat mass of LDLr ~'~ mice treated with HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l- (3-methylbenzyl)-l,4-diazepan-2-one) was significantly decreased compared to that of LDLr ~ '~ mice fed with HCD alone (n = 12 mice per group). Figure 6c: Food intake during 24 hours in the LDLr ' mice on either HCD or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) (n = 12 mice per group) was the same. ASAT plasma level in LDLr ~'~ mice fed for 13 weeks with eitherHCD or HCD+((6-[(3-fluorobenzyl)oxy]-4- (2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) was also the same. Figure 6d: Representative en-face images of aortas of LDLr ~'~ mice fed for 13 weeks with HCD or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) were stained with Oil-Red O. Figure 6e: Quantification of the surface area of the aortic plaque in LDLr ~'~ mice fed HCD showed a significant decrease in the plaque area in animals that received HCD plus (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan- 2-one) (open squares) compared to the plaque area in animals that received HCD alone (solid circles) (n = 12 mice per group). Data are expressed as the mean + SEM. *p<0.05 determined by Student's t tests or by Mann- Whitney U for the aortic plaque quantification. Figure 6f: Quantification of plasma cholesterol and triglyceride levels in LDLr 1' mice fed with HCD (solid bars) or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- 1 ,4-diazepan-2-one) (open bars) for various times. Mice exposed to HCD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) showed a significant decrease of plasma cholesterol after 9 weeks of treatment, and this decrease persisted through the termination of the study at 12 weeks. Figure 6g: Quantification of Mcpl and 116 mRNA in aortas of LDLr' mice fed for 13 weeks with HCD (solid bars) or HCD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (open bars) (upper panel). Quantification of ΙΡΝγ and KC/GRO in the plasma of LDLr ' mice fed for 13 weeks with HCD or HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan- 2-one) (lower panel). In all cases (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) significantly decreased the expression or elaboration of proinflammatory cytokines.
[00028] Figures 7a-7e depict functional differences at TGR5 between (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) enantiomers, thereby defining heterogeneity of the TGR5 receptor or TGR5 activiation. Figure 7a: The stereo enantiomers (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one)) and (<55)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one),) at 30μΜ, each similarly decreased mRNA levels of Tnfa, 116 and Mcpl in a murine macrophage cell line (RAW 264.7) stimulated for 4 hours with lipopoly saccharide (LPS, 10 ng/niL). The TGR5 agonist INT-777 (30μΜ), used as a positive control, decreased only the 116 and Mcpl transcripts. Figure 7b: The enantiomer (<5R)-(6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one),) but not (<55)-(6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one),) at 30μΜ, significantly increased GLP-1 secretion from the murine entero-endocrine GLUTag cells. Bar graphs represent the mean + SEM of three independent experiments. *p<0.05. Figure 7c: (<5R)-(6-[(3-fluorobenzyl)oxy]- 4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one)) significantly increased GLP-1 release from GLUTag cells secretion at concentrations of 10 and 30μΜ, while INT-777 showed such activity at 30 and 60μΜ. Bar graphs represent the mean + SEM of three independent experiments. *p<0.05. Figures 7d and 7e: analysis of plasma GLP-1 levels after a test meal challenge (Ensure Plus at lOmL/kg), given 30 min after the oral administration to diet- induced obese C57BL/6J mice of either (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)) (Figure 7d) or (65)-(6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one)) (Figure 7e). Compounds were given at 30mg/kg of body weight (n = 5 mice per group). GLP-1 secretion was significantly increased only in the mice treated with (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one).) Data expressed as the mean + SEM. * denotes a p<0.05.
DETAILED DESCRIPTION OF THE INVENTION
[00029] The present invention provides a compound of formula (I)
Figure imgf000013_0001
(I)
wherein
Ri is aryl, heteroaryl, C3-8 cycloalkyl, cycloheteroalkyl, Ci_6 alkyl, C2-6 alkenyl, C2-6 alkynyl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, aryl, halo, C1-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, nitro, and -NRaRt>;
R2 is Ci_6 alkyl, 0-Ci_6 alkyl, -NRaRb, C3_8 cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, nitro, and -NRaRt>;
R3 is F, CI, Br, I, or 0-Ci_6 alkyl;
n is 0-5 ; and
Ra and Rb each are independently H or C1-3 alkyl.
[00030] The present invention further provides a compound of formula (I)
Figure imgf000014_0001
(I)
wherein
R! is aryl, -CH2CH2-aryl, C3_8 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, or -CH2NRaRb, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro;
R2 is Ci-6 alkyl, O-Ci-6 alkyl, -NRaRb, C3-8 cycloalkyl, 2-furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, and O-Ci-6 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro;
R3 is F, CI, Br, I, or 0-Ci_6 alkyl;
n is 1-5; and
[00031] Ra and Rb each are independently Ci_6 alkyl.In some embodiments, R! is aryl, optionally mono- or polysubstituted with substituents selected from Ci_5 alkyl, halo, Ci-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro. In some embodiments, Ri is phenyl, optionally mono- or polysubstituted with substituents selected from Ci-5 alkyl, halo, C1-3 haloalkyl, OH, O-C1-3 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro. In other embodiments, Ri is phenyl, optionally mono-substituted with methyl, methoxy, halo, or cyano. In certain embodiments, Ri is phenyl, optionally mono-substituted with methyl, methoxy, flouro, chloro, or cyano.
[00032] In some embodiments, Ri is C3-8 cycloalkyl. In other embodiments, Ri is cyclobutyl. In certain embodiments, Ri is cyclohexyl or cyclopentyl.
[00033] In some embodiments, Ri is -CH2NRaRt>. In certain embodiments, Ri is - CH2N(CH3)2. In other embodiments, R is -CH2N(C2H5)2.
[00034] In some embodiments, R! is CH2CH2-aryl, optionally mono- or polysubstituted with substituents selected from Ci-5 alkyl, halo, C1-3 haloalkyl, OH, O-C1-3 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro. In some embodiments, Ri is CH2CH2- phenyl, optionally mono-substituted with methyl, methoxy, flouro, chloro, or cyano.
[00035] In some embodiments, the compound of formula (I) is represented by formula (II)
Figure imgf000015_0001
(II).
wherein R4 is F, CI, Br, I, O-C1-5 alkyl, Ci-5 alkyl, or cyano; and m is 1-5.
[00036] In some embodiments, the compound of formula (I) is represented by formula
(III)
Figure imgf000015_0002
(III).
[00037] In some embodiments, the compound of formula (I) is represented by formula (IV)
Figure imgf000015_0003
(IV).
[00038] In some embodiments, R2 is O-C1-5 alkyl, -NRaRb, C3-8 cycloalkyl, 2-furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, and O-C1-3 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro.
[00039] In some embodiments, R2 is O-C1-5 alkyl or -NRaRt>. In certain embodiments, R2 is OCH2CH2CH3 or -NCH(CH3)2.
[00040] In some embodiments, R2 is C3-8 cycloalkyl.
[00041] In some embodiments, R2 is 2-furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono-substituted with methyl, methoxy, flouro, chloro, or cyano. In other embodiments, R2 is thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl. In other embodiments, R2 is thienyl. In certain embodiments, R2 is 2-furanyl.
[00042] In some embodiments, R3 is F, CI, Br, or I. In some embodiments, R3 is F. In other embodiments, R3 is CI. In some embodiments, R3 is 2-F or 3-F.
[00043] In some embodiments, n is 1.
[00044] In some embodiments, the compound of formula (I) is represented by formula (V)
Figure imgf000016_0001
(V)
wherein Ri, R2, and R3 are defined as above.
[00045] In some embodiments, the compound of formula (I) is represented by formula
(VI)
Figure imgf000016_0002
(VI)
wherein Ri, R2, and R3 are defined as above. [00046] In some embodiments, the compound of formula (I) is represented by formula (VII)
Figure imgf000017_0001
(VII) wherein Ri, ]¾, and R3 are defined as above.
[00047] In some embodiments, the compound of formula (I) is
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] -N-isopropyl-4-(4-methoxybenzyl)-3 -oxo- 1 ,4-diazepane- 1 - carboxamide;
6- [(2-fluorobenzyl)oxy] -4-glycoloyl- 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one (MH
416);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(tetrahydrofuran-2-ylcarbonyl)-l,4- diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(lH-pyrrol-2-ylcarbonyl)-l,4- diazepan-2-one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4- [(5-methyl- 1 H-pyrazol-3 - yl)carbonyl]-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(5-methylisoxazol-3-yl)carbonyl]- 1 ,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4- [(2-methyl- 1 ,3-thiazol-4- yl)carbonyl]-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(isoxazol-5-ylcarbonyl)-l-(4-methoxybenzyl)-l,4- diazepan-2-one;
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan- 2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(5-methyl-l,3-oxazol-4- yl)carbonyl]-l,4-diazepan-2-one;
propyl 6- [(2-fluorobenzyl)oxy] -4-(4-methoxybenzyl)-3 -oxo- 1 ,4-diazepane- 1 - carboxylate;
4-butyryl-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4-( 1 H-pyrazol-3 -ylcarbonyl)- 1 ,4- diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(lH-imidazol-4-ylcarbonyl)-l-(4-methoxybenzyl)-l,4- diazepan-2-one;
N-ethyl-6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l- carboxamide;
6-[(2-fluorobenzyl)oxy]-4-(isoxazol-3-ylcarbonyl)-l-(4-methoxybenzyl)-l,4- diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(lH-imidazol-2-ylcarbonyl)-l-(4-methoxybenzyl)-l,4- diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-isobutyryl-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-propionyl-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(methoxyacetyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-(cyclobutylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-
2-one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4- [( 1 -methyl- 1 H-pyrazol-3 - yl)carbonyl]-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] -4- [2-furyl(oxo)acetyl] - 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2- one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(l,3-thiazol-4-ylcarbonyl)-l,4- diazepan-2-one; and
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(pyridin-2-ylcarbonyl)-l,4-diazepan-
2-one.
[00048] In some embodiments, the compound is 4-({[4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzonitrile ;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(4-methoxybenzyl)oxy]-l,4- diazepan-2-one;
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-
2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-3-ylmethoxy)-l,4-diazepan-
2-one;
4-(cyclopropylcarbonyl)-6-[(4-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-
2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-2-ylmethoxy)-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-6-[(3-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(2-methoxybenzyl)oxy]-l,4-diazepan-2- one;
6-[(2-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzoic acid ;
4-(cyclopropylcarbonyl)- 1 -(4-methoxybenzyl)-6-(pyridin-4-ylmethoxy)- 1 ,4-diazepan-
2-one;
2- ({[4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzonitrile ;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(4-methylbenzyl)oxy]-l,4- diazepan-2-one;
6-(benzyloxy)-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one; 6-[(4-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-
2-one;
3- ({[4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzonitrile ;
4- (cyclopropylcarbonyl)-6-[(3,5-dimethylisoxazol-4-yl)methoxy]-l-(4- methoxybenzyl)- 1 ,4-diazepan-2-one; 3- ({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl]oxy}methyl)benzoic acid;
4- (cyclopropylcarbonyl)- 1 -(4-methoxybenzyl)-6- [(2-methylbenzyl)oxy]- 1 ,4- diazepan-2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(3-methylbenzyl)oxy]-l,4- diazepan-2-one;
4-(cyclopropylcarbonyl)- 1 -(4-methoxybenzyl)-6- [(3-methoxybenzyl)oxy]- 1 ,4- diazepan-2-one; or
6-[(3-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan- 2-one.
[00049] In some embodiments, the compound of formula (I) is
4-{ [6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile; l-benzyl-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
l-(2-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-phenylethyl)-l,4-diazepan-2-one;
l-butyl-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-2-ylmethyl)-l,4-diazepan-2-one;
l-(cyclobutylmethyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(l,3-thiazol-4-ylmethyl)-l,4-diazepan-2-one;
l-(4-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] -4-(2-furoyl)- 1 - [2-( IH-pyrazol- 1 -yl)ethyl] - 1 ,4-diazepan-2-one; l-(cyclopropylmethyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
1 - [(3 ,5 -dimethylisoxazol-4-yl)methyl] -6- [(2-fluorobenzyl)oxy] -4-(2-furoyl)- 1 ,4-diazepan-2- one;
l-(2-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(2-methyl-l,3-oxazol-4-yl)methyl]-l,4-diazepan-2- one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-phenylpropyl)-l,4-diazepan-2-one; 1- (4-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
2- {[6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile; 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbut-2-en-l-yl)-l,4-diazepan-2-one;
3- {[6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methylbenzyl)-l,4-diazepan-2-one;
6 (2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(2-methyl-l,3-thiazol-4-yl)methyl]-l,4-diazepan-2- one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(3-methylisoxazol-5-yl)methyl]-l,4-diazepan-2-one; l-(3-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-4-ylmethyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-3-ylmethyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-methylbenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methoxybenzyl)-l,4-diazepan-2-one;
l-(3-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methoxyethyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(4-methylbenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-[(l-methyl-lH-imidazol-5-yl)methyl]-l,4- diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(l,3-thiazol-4-ylmethyl)-l,4-diazepan-2-one; 4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methylbenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(3-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(3-methylbenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-[(5-methyl-l,2,4-oxadiazol-3-yl)methyl]-l,4- diazepan-2-one;
4-acetyl-l-(cyclopropylmethyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
2- { 4-acetyl-6- [(3-fluorobenzyl)oxy] -2-oxo- 1 ,4-diazepan- 1 -yl } -N,N- dimethylacetamide ;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-[(3-methylisoxazol-5-yl)methyl]-l,4-diazepan-2- one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(pyridin-2-ylmethyl)-l,4-diazepan-2-one;
4-acetyl-l-(4-fluorobenzyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(4-methoxypyridin-2-yl)methyl] - 1 ,4-diazepan-2- one;
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(2-methyl- 1 ,3 -oxazol-4-yl)methyl] - 1 ,4-diazepan-
2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-phenylethyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(pyridin-4-ylmethyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(l,3-thiazol-2-ylmethyl)-l,4-diazepan-2-one; 4-acetyl-l-benzyl-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-l-(cyclohexylmethyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl- 1 -(3 -fluorobenzyl)-6- [(3 -fluorobenzyl)oxy] - 1 ,4-diazepan-2-one;
4-acetyl-l-(2-fluorobenzyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(pyridin-3-ylmethyl)-l,4-diazepan-2-one;
4-acetyl- 1 -(cyclobutylmethyl)-6- [(3 -fluorobenzyl)oxy] - 1 ,4-diazepan-2-one ; or 4-acetyl- l-[(3,5-dimethylisoxazol-4-yl)methyl]-6-[(3-fluorobenzyl)oxy]-l, 4- diazepan-2-one.
[00050] In one embodiment, a compound of formula (I) is its isomer, pharmaceutically acceptable salt, or tautomer, or any combination thereof. In some embodiments, the compound is the R enantiomer. In some embodiments, the compound is the S enantiomer.
[00051] In some embodiments, the compound is 6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)- l-(3-methylbenzyl)-l,4-diazepan-2-one
Figure imgf000022_0001
[00052] In some embodiments, the compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In other embodiments, the compound is (65)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. [00053] In some embodiments, the compound is 4-(cyclopropylcarbonyl)-6-[(2- fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one
Figure imgf000023_0001
[00054] In some embodiments, the compound is 4-acetyl-l-[2-(dimethylamino)ethyl]-6- [(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
Figure imgf000023_0002
[00055] A compound of the present invention can be prepared as shown in the schemes herein.
Scheme 1 ^J ^ ^A
Figure imgf000023_0003
[00056] Scheme 1 illustrates the main approach to the prepration of the compounds of the present invention. Nucleophilic ring opening of the epoxide with an amine enables obtaining an unsymmetrically substituted l,3-diaminopropan-2-ol. To create the 7- membered ring, the TBS protecting group has been found to be appropriate to protect the hydroxyl group without affecting the secondary amine. The secondary amine is acylated with chloroacetyl chloride. The obtained product is cyclized under the action of sodium hydride. If these two steps are performed without product isolation, the yield would be considerably lower, and purification would be more difficult. The next step of the synthesis is TBS deprotection, followed by alkylation of the hydroxyl group. After Boc deprotection an acyl substituent is introduced.
[00057] This approach can be also used for parallel synthesis for obtaining libraries with a high diversity of substituent R3.
Scheme 2
Figure imgf000024_0001
[00058] Scheme 2 shows an approach for obtaining chiral products. In this approach, readily available R- and 5-glycidyl alcohols have been used. The epoxide ring opening with an azide followed by reduction and protection enables obtaining a chiral diol. The less hindered alcohol group of the diol is selectively transformed into a sulphonate. This product can be used for obtaining a chiral epoxide via the approach as described in Scheme 1. Alternatively, it can be used directly for alkylating the amine. This gives the chiral intermediate, which is identical in all other respects to that described in Scheme 1. Accordingly, other compounds of the present invention using the same intermediate have been prepared in the similar method.
Scheme 3
o
Figure imgf000024_0002
[00059] Scheme 3 shows a modification of the main approach of Scheme 1 to change the order of the introduction of substituents R2 and R3. Both TBS and Boc groups are removed upon deprotection with HC1 to lead to an aminoalcohol. Using HBTU as a condensing agent enables acylating selectively the aminoalcohol at the nitrogen atom. The resulting compound is alkylated.
[00060] This approach has been used for obtaining libraries with a high diversity of substituent R2. Further, it can be useful for obtaining products with substituents R2 labile in acidic media.
Scheme 4
Figure imgf000025_0001
[00061] Scheme 4 shows a modification of the approach as described in Scheme 1 to change the order of the introduction of substituents Ri, R2 and R3. It includes synthesizing the final product with Ri = PMB by the method shown in the Scheme 1. This protecting group PMB can be removed in a moderate yield upon treatment with CAN. As a result, a new required substituent R! is introduced.
[00062] This approach has been used for obtaining libraries with a high diversity of substituent Further, it can be useful for obtaining products with substituents R! labile in acidic media. In this case substituents R2 and R3 of Scheme 4 must be rather stable for using CAN.
[00063] For example, thist approach has been used for the preparation ofcompounds of the invention, including compound 2 (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one) of Table 1.
Definitions
[00064] At various places in the present specification, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the members of such groups and ranges. For example, the term "Ci_6 alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.
[00065] It is further intended that the compounds of the invention are stable. As used herein "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
[00066] It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.
[00067] In some embodiments, the term "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t- butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.
[00068] In some embodiments, "haloalkyl" refers to an alkyl group having one or more halogen substituents. Example haloalkyl groups include CF3, C2F5, CHF2, CCI3, CHCI2, C2CI5, and the like.
[00069] In some embodiments, "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like. In some embodiments, an aryl group has from 6 to about 20 carbon atoms.
[00070] In some embodiments, "cycloalkyl" refers to non-aromatic carbocycles including cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems, including spirocycles. In some embodiments, cycloalkyl groups can have from 3 to about 20 carbon atoms, 3 to about 14 carbon atoms, 3 to about 10 carbon atoms, or 3 to 7 carbon atoms. Cycloalkyl groups can further have 0, 1 , 2, or 3 double bonds and/or 0, 1 , or 2 triple bonds. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo derivatives of cyclopentane, cyclopentene, cyclohexane, and the like. A cycloalkyl group having one or more fused aromatic rings can be attached through either the aromatic or non-aromatic portion. One or more ring-forming carbon atoms of a cycloalkyl group can be oxidized, for example, having an oxo or sulfido substituent. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like.
[00071] In some embodiments, "heteroaryl" refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Any ring- forming N atom in a heteroaryl group can also be oxidized to form an N-oxo moiety. Examples of heteroaryl groups include without limitation, pyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms.
[00072] In some embodiments, "cycloheteroalkyl" or "heterocycloalkyl" refers to a non- aromatic heterocycle where one or more of the ring-forming atoms are a heteroatom such as an O, N, or S atom. Cycloheteroalkyl or heterocycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well as spirocycles. Example cycloheteroalkyl or heterocycloalkyl groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-l,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Also included in the definition of cycloheteroalkyl or heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles. A cycloheteroalkyl or heterocycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. Also included in the definition of cycloheteroalkyl or heterocycloalkyl are moieties where one or more ring-forming atoms are substituted by 1 or 2 oxo or sulfido groups. In some embodiments, the cycloheteroalkyl or heterocycloalkyl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the cycloheteroalkyl or heterocycloalkyl group contains 3 to about 20, 3 to about 14, 3 to about 7, or 5 to 6 ring- forming atoms. In some embodiments, the cycloheteroalkyl or heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, the cycloheteroalkyl or heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the cycloheteroalkyl or heterocycloalkyl group contains 0 to 2 triple bonds.
[00073] In some embodiments, "halo" or "halogen" includes fluoro, chloro, bromo, and iodo.
[00074] In some embodiments, the term "substituted" refers to the replacement of a hydrogen moiety with a non-hydrogen moiety in a molecule or group. The term "mono- substituted" or "poly-substituted" means substituted with one or more than one substituent up to the valence of the substituted group. For example, a mono- substituted group can be substituted with 1 substituent, and a poly-substituted group can be substituted with 2, 3, 4, or 5 substituents. Generally when a list of possible substituents is provided, the substituents can be independently selected from that group.
[00075] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. In some embodiments, a compound of this invneiton is the R- enantiomer of a compound of formula (I). In other embodiments, a compound of this invention is the 5-enantiomer of a compound of formula (I). Compounds of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. In some embodiments, R- and 5-enantiomers may have the same activity. In other embodiments, the R-enantiomer of the compound of formula (I) shows selective activity. In some embodiments, the 5-enantiomer of the compound of formula (I) shows selective activity. In some embodiments, an R-enatiomer of a compound of formula (I) selectively increases GLP-1 secretion while the 5-enantiomer of the compound does not increase GLP-1 secretion.
[00076] Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
[00077] Compounds of the invention can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.
[00078] In some embodiments, the compound of the invention is substantially isolated. By "substantially isolated" is meant that the compound is at least partially or substantially separated from the environment in which it is formed or detected. Partial separation can include, for example, a composition enriched in the compound of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the invention, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
[00079]
[00080] In some embodiments, a "therapeutically effective amount" or "inducing effective amount" as used herein refers to the amount which provides a therapeutic effect or inducing effect for a given condition and administration regimen.
[00081] The term "compound" as used herein is meant to include all stereoisomers, geometric iosomers, tautomers, and isotopes of the structures depicted.
[00082] All compounds, and pharmaceuticaly acceptable salts thereof, are also meant to include solvated or hydrated forms.
[00083] The present invention also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
[00084] The physiologically acceptable salts may be obtained by neutralizing the bases with inorganic or organic acids or by neutralizing the acids with inorganic or organic bases. Examples of suitable inorganic acids are hydrochloric acid, sulphuric acid, phosphoric acid, or hydrobromic acid, while examples of suitable organic acids are carboxylic acid, sulpho acid, or sulphonic acid, such as acetic acid, tartaric acid, lactic acid, propionic acid, glycolic acid, malonic acid, maleic acid, fumaric acid, tannic acid, succinic acid, alginic acid, benzoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, citric acid, maleic acid, salicylic acid, 3 -aminosalicylic acid, ascorbic acid, embonic acid, nicotinic acid, isonicotinic acid, oxalic acid, gluconic acid, amino acids, methanesulphonic acid, ethanesulphonic acid, 2-hydroxyethanesulphonic acid, ethane- 1,2- disulphonic acid, benzenesulphonic acid, 4-methylbenzenesulphonic acid or naphthalene-2-sulphonic acid. Examples of suitable inorganic bases are sodium hydroxide, potassium hydroxide and ammonia, while examples of suitable organic bases are amines, e.g., tertiary amines, such as trimethylamine, triethylamine, pyridine, N,N-dimethylaniline, quinoline, isoquinoline, a- picoline, β-picoline, γ-picoline, quinaldine, or pyrimidine.
[00085] In addition, physiologically acceptable salts of the compounds according to formula (I) can be obtained by converting derivatives which possess tertiary amino groups into the corresponding quaternary ammonium salts in a manner known per se using quaternizing agents. Examples of suitable quaternizing agents are alkyl halides, such as methyl iodide, ethyl bromide, and n-propyl chloride, and also arylalkyl halides, such as benzyl chloride or 2-phenylethyl bromide.
[00086] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[00087] The term "isomer" includes, but is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like.
[00088] Compounds of the present invention can also be in the form of a hydrate, which means that the compound further includes a stoichiometric or non- stoichiometric amount of water bound by non-covalent intermolecular forces. [00089] Compounds of of the present invention may exist in the form of one or more of the possible tautomers and depending on the particular conditions it may be possible to separate some or all of the tautomers into individual and distinct entities. It is to be understood that all of the possible tautomers, including all additional enol and keto tautomers and/or isomers are hereby covered.
Compositions and Administration
[00090] In one embodiment, the present invention features a pharmaceutical composition comprising a compound of formula (I), and at least one pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises a TGR5 receptor agonist compound, and at least one pharmaceutically acceptable carrier.
[00091] A therapeutically effective dose of the compound according to the invention is used, in addition to physiologically acceptable carriers, diluents and/or adjuvants for producing a pharmaceutical composition. The dose of the active compound can vary depending on the route of administration, the age and weight of the patient, the nature and severity of the diseases to be treated, and similar factors. The daily dose can be given as a single dose, which is to be administered once, or be subdivided into two or more daily doses, and is as a rule 0.001-2000 mg. In some embodiments, the daily dose is 0.1-500 mg, e.g. 0.1- 100 mg.
[00092] Suitable administration forms are oral, parenteral, intravenous, transdermal, topical, inhalative, intranasal and sublingual preparations. In some embodiments, administration forms are oral, parenteral, e.g. intravenous or intramuscular, intranasal, e.g. dry powder or sublingual preparations of the compounds according to the invention. The customary galenic preparation forms, such as tablets, sugar-coated tablets, capsules, dispersible powders, granulates, aqueous solutions, alcohol-containing aqueous solutions, aqueous or oily suspensions, syrups, juices or drops, are used.
[00093] Solid medicinal forms can comprise inert components and carrier substances, such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher molecular weight fatty acids, (such as stearic acid), gelatine, agar agar or vegetable or animal fats and oils, or solid high molecular weight polymers (such as polyethylene glycol), preparations which are suitable for oral administration can comprise additional flavorings and/or sweetening agents, if desired.
[00094] Liquid medicinal forms can be sterilized and/or, where appropriate, comprise auxiliary substances, such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators. Examples of such additives are tartrate and citrate buffers, ethanol and sequestering agents (such as ethylenediaminetetraacetic acid and its nontoxic salts). High molecular weight polymers, such as liquid polyethylene oxides, microcrystalline celluloses, carboxymethyl celluloses, polyvinylpyrrolidones, dextrans or gelatine, are suitable for regulating the viscosity. Examples of solid carrier substances are starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids (such as stearic acid), gelatine, agar agar, calcium phosphate, magnesium stearate, animal and vegetable fats, and solid high molecular weight polymers, such as polyethylene glycol.
[00095] Oily suspensions for parenteral or topical applications can be vegetable synthetic or semisynthetic oils, such as liquid fatty acid esters having in each case from 8 to 22 C atoms in the fatty acid chains, for example palmitic acid, lauric acid, tridecanoic acid, margaric acid, stearic acid, arachidic acid, myristic acid, behenic acid, pentadecanoic acid, linoleic acid, elaidic acid, brasidic acid, erucic acid or oleic acid, which are esterified with monohydric to trihydric alcohols having from 1 to 6 C atoms, such as methanol, ethanol, propanol, butanol, pentanol or their isomers, glycol or glycerol. Examples of such fatty acid esters are commercially available miglyols, isopropyl myristate, isopropyl palmitate, isopropyl stearate, PEG 6-capric acid, caprylic/capric acid esters of saturated fatty alcohols, polyoxyethylene glycerol trioleates, ethyl oleate, waxy fatty acid esters, such as artificial ducktail gland fat, coconut fatty acid isopropyl ester, oleyl oleate, decyl oleate, ethyl lactate, dibutyl phthalate, diisopropyl adipate, polyol fatty acid esters, inter alia. Silicone oils of differing viscosity, or fatty alcohols, such as isotridecyl alcohol, 2-octyldodecanol, cetylstearyl alcohol or oleyl alcohol, or fatty acids, such as oleic acid, are also suitable. It is furthermore possible to use vegetable oils, such as castor oil, almond oil, olive oil, sesame oil, cotton seed oil, groundnut oil or soybean oil.
[00096] Suitable solvents, gelatinizing agents and solubilizers are water or watermiscible solvents. Examples of suitable substances are alcohols, such as ethanol or isopropyl alcohol, benzyl alcohol, 2-octyldodecanol, polyethylene glycols, phthalates, adipates, propylene glycol, glycerol, di- or tripropylene glycol, waxes, methyl cellosolve, cellosolve, esters, morpholines, dioxane, dimethyl sulphoxide, dimethylformamide, tetrahydrofuran, cyclohexanone, etc.
[00097] Mixtures of gelatinizing agents and film-forming agents are also perfectly possible. In this case, use is made, in particular, of ionic macromolecules such as sodium carboxymethyl cellulose, polyacrylic acid, polymethacrylic acid and their salts, sodium amylopectin semiglycolate, alginic acid or propylene glycol alginate as the sodium salt, gum arabic, xanthan gum, guar gum or carrageenan. The following can be used as additional formulation aids: glycerol, paraffin of differing viscosity, triethanolamine, collagen, allantoin and novantisolic acid. Use of surfactants, emulsifiers or wetting agents, for example of Na lauryl sulphate, fatty alcohol ether sulphates, di-Na-N-lauryl- -iminodipropionate, polyethoxylated castor oil or sorbitan monooleate, sorbitan monostearate, polysorbates (e.g. Tween), cetyl alcohol, lecithin, glycerol monostearate, polyoxyethylene stearate, alkylphenol poly glycol ethers, cetyltrimethylammonium chloride or mono-/dialkylpoly glycol ether orthophosphoric acid monoethanolamine salts can also be required for the formulation. Stabilizers, such as montmorillonites or colloidal silicic acids, for stabilizing emulsions or preventing the breakdown of active substances such as antioxidants, for example tocopherols or butylhydroxyanisole, or preservatives, such as p-hydroxybenzoic acid esters, can likewise be used for preparing the desired formulations.
[00098] Preparations for parenteral administration can be present in separate dose unit forms, such as ampoules or vials. In some embodiments, use is made of solutions of the active compound, for example, aqueous solution and, in particular, isotonic solutions and also suspensions. These injection forms can be made available as ready-to-use preparations or only be prepared directly before use, by mixing the active compound, for example the lyophilisate, where appropriate containing other solid carrier substances, with the desired solvent or suspending agent.
[00099] Intranasal preparations can be present as aqueous or oily solutions or as aqueous or oily suspensions. They can also be present as lyophilisates which are prepared before use using the suitable solvent or suspending agent.
[000100] Inhalable preparations can present as powders, solutions or suspensions. Preferably, inhalable preparations are in the form of powders, e.g. as a mixture of the active ingredient with a suitable formulation aid such as lactose.
[000101] The preparations are produced, aliquoted and sealed under the customary antimicrobial and aseptic conditions.
[000102] As indicated above, the compound of the invention may be administered as a combination therapy with further active agents, e.g. therapeutically active compounds useful in the treatment of metabolic disorders, inflammatory diseases, atherosclerosis and associated conditions and dyslipidemias. For a combination therapy, the active ingredients may be formulated as compositions containing several active ingredients in a single dose form and/or as kits containing individual active ingredients in separate dose forms. The active ingredients used in combination therapy may be coadministered or administered separately. Pharmaceutical Methods
[000103] The compounds of the present invention are TGR5 agonists which are useful for treating, preventing suppressing, reducing the severity of, or reducing the risk of conditions where stimulation of a TGR5 receptor can have a positive therapeutic effect in a subject in need thereof. In some embodiment, the therapeutic effect may be selective.
[000104] In one aspect, the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of an inflammatory disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof. In another aspect, the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of an inflammatory disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a TGR5 receptor agonist compound.
[000105] Exemplary inflammatory diseases that can be treated according to the present invention include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, atherosclerosis, coronary artery disease, stroke, allergy, osteoarthritis, appendicitis, bronchial asthma, pancreatitis, and psoriasis. In some embodiments, the inflammatory disease is rheumatoid arthritis. In other embodiments, the inflammatory disease is allergy.
[000106] In some embodiments, a compound of formula (I) has diminished adverse effects on cardiac and gallbladder function compared with other known TGR5 agonists.
[000107] In some embodiments, the compound of the invention for the treatment or prevention of an inflammatory disease is 6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one, or 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4- methoxybenzyl)-l,4-diazepan-2-one, or 4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one. In some embodiments, the compound of the invention for the treatment or prevention of an inflammatory disease comprises an R- or an S- enantiomer of the compound. In some embodiments, the compound of the invention for the treatment or prevention of an inflammatory disease comprises an R-enantiomer of the compound. In some embodiments, the compound of the invention for the treatment or prevention of an inflammatory disease comprises an 5-enantiomer of the compound. In some embodiments, methods of this invention for treating, preventing suppressing, reducing the severity of, or reducing the risk of an inflammatory disease comprise administering a compound of formula (I), wherein said compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In other embodiments, the compound is (6S)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In some embodiments, the compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines. In some embodiments, the compound of formula (I) does not induce increased secretion of GLP- 1.
[000108] In another aspect, the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of a metabolic disease or disorder in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof. In another aspect, the present invention provides a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of a metabolic disease or disorder in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a TGR5 receptor agonist compound.
[000109] Metabolic diseases that can be treated according to the invention include, but are not limited to, dyslipidemia, obesity, insulin resistance/sensitivity, and diabetes mellitus. In some embodiments, the metabolic disease is obesity. In other embodiments, the metabolic diseas is diabetes, e.g., Type 2 diabetes mellitus. In certain embodiments, the metabolic disease is insulin sensitivity.
[000110] In some embodiments, the compound of formula (I) has significantly diminished adverse effects on cardiac and gallbladder function relative to other known TGR5 agonists known in the art, for example, the 13.4-benzofuranyloxynicotinamide, 4- phenoxynicotinamide and 4-phenoxypyrimidine-5-carboxamide derivatives.
[000111] In some embodiments, the compound of the invention for the treatment or prevention of a metabolic disease or disorder is 6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one. In some embodiments, the compound of the invention for the treatment or prevention of a metabolic disease or disorder is racemic. In some embodiments, the compound of the invention for the treatment or prevention of a metabolic disease or disorder comprises an R- or an S- enantiomer of the compound. In some embodiments, the compound of the invention for the treatment or prevention of a metabolic disease or disorder comprises an S- enantiomer of the compound. In some embodiments, the compound of the invention for the treatment or prevention of a metabolic disease or disorder comprises an R- enantiomer of the compound. In certain embodiments, the compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
[000112] In another aspect, the invention of the present application features a method of treating, preventing suppressing, reducing the severity of, or reducing the risk of atherosclerosis in a subject in need thereof the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I), or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof. In some embodiments, the compound of formula (I) has significantly reduced adverse effects on cardiac and gallbladder function relative to other TGR5 agonists known in the art, for example, the 13.4-benzofuranyloxynicotinamide, 4- phenoxynicotinamide and 4-phenoxypyrimidine-5-carboxamide derivatives. In some embodiments, the compound of the invention for the treatment or prevention of atherosclerosis is racemic. In some embodiments, the compound of the invention for the treatment or prevention of atherosclerosis is an R- or an S- enantiomer of the compound of formula (I). In some embodiments, the compound of the invention for the treatment or prevention of atherosclerosis is an R- enantiomer of the compound of formula (I). In some embodiments, the compound of the invention for the treatment or prevention of atherosclerosis is an S- enantiomer of the compound of formula (I). In some embodiments, methods of this invention for treating, preventing suppressing, reducing the severity of, or reducing the risk of atherosclerosis in a subject comprise administering a compound of formula (I), wherein said compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one. In other embodiments, the compound is (6S)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In some embodiments, the compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines. In some embodiments, the compound of formula (I) does not induce increased secretion of GLP- 1.
[000113] In another aspect, the invention of the present application provides a method of reducing plasma triglyceride and LDL-cholesterol levels in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of the invention, for example, a compound of formula (I) as described herein, or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof some embodiments, the compound of the invention for reducing plasma triglyceride and LDL- cholesterol levels is racemic. In some embodiments, the compound for reducing plasma triglyceride and LDL-cholesterol levels is an R- or an S- enantiomer of the compound of formula (I). In some embodiments, the compound for reducing plasma triglyceride and LDL- cholesterol levels is an S- enantiomer of the compound of formula (I). In some embodiments, the compound for reducing plasma triglyceride and LDL-cholesterol levels is an R- enantiomer of the compound of formula (I). In some embodiments, the compound is 6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In other embodiments, the compound is 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4- methoxybenzyl)-l,4-diazepan-2-one. In certain embodiments, the compound is 4-acetyl-l- [2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one. In some embodiments, methods of this invention for reducing plasma triglyceride and LDL- cholesterol levels in a subject comprise administering a compound of formula (I), wherein said compound is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one. In other embodiments, the compound is (6S)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In some embodiments, the compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines. In some embodiments, the compound of formula (I) does not induce increased secretion of GLP-1.
[000114] In another aspect, the invention of the present application provides a method of inducing increased GLP- 1 secretion in a cell, the method comprising contacting the cell with an effective amount of the invention, e.g., a compound of formula (I), as described herein, or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof. In some embodiments, the cell is in vitro. In other embodiments, the cell is in vivo. In some embodiments, the compound of the invention for inducing increased GLP-1 secretion in a cell is racemic. In some embodiments, the compound of the invention for inducing increased GLP- 1 secretion in a cell is an R- or an S- enantiomer of the compound of formula (I). In some embodiments, the compound of the invention for inducing increased GLP- 1 secretion in a cell is an R- enantiomer of the compound of formula (I). In some embodiments, the compound inducing increased GLP-1 secretion in a cell is an S- enantiomer of the compound of formula (I). In some embodiments, the compound of the invention for inducing GLP-1 secretion in a cell is 6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one, 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4- methoxybenzyl)-l,4-diazepan-2-one, or 4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one. In certain embodiments, the compound is (6R)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one. In some embodiments, the compound of the invention for inducing GLP-1 is selective, for example, the compound does not effect expression or elaboration of pro-inflammatory cytokines.
[000115] In another aspect, the present invention provides a method for decreasing expression or elaboration of pro-inflammatory cytokines in a cell, the method comprising contacting the cell with an effective amount of the invention, e.g., a compound of formula (I), as described herein, or an isomer, a pharmaceutically acceptable salt or a tautomer, or any combination thereof. In some embodiments, the decreased expression or elaboration of pro- inflammatory cytokines is selective since the compound does not effect secretion of GLP-1. In some embodiments, the compound of the invention has diminished adverse effects on cardiac and gallbladder function. In some embodiments, the cell is in vitro. In other embodiments, the cell is in vivo. In some embodiments, the compound of the invention for decreasing expression or elaboration of pro-inflammatory cytokines is racemic. In some embodiments, the compound of the invention for decreasing expression or elaboration of proinflammatory cytokines comprises an R- or an S- enantiomer of the compound. In some embodiments, the compound of the invention for decreasing expression or elaboration of proinflammatory cytokines comprises an R- enantiomer of the compound. In some embodiments, the compound of the invention for decreasing expression or elaboration of pro-inflammatory cytokines comprises an S- enantiomer of the compound. In some embodiments, the compound comprises (65)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one. In other embodiments, the compound is (<5R)-6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)- 1 -(3-methylbenzyl)- 1 ,4-diazepan-2-one.
[000116] In yet another aspect, the present invention identifies heterogeneity of the TGR5 receptor that can be exploited to separate the anti-inflammatory and metabolic effects of TRG5 agonists. In some embodiments, the compounds of formula (I) are selective TGR5 agonists. In other embodiments, a TGR5 agonist compound of formula (I) selectively activates a TGR5 anti-inflammatory response. In some embodiments, a TGR5 agonist compound of formula (I) selectively decreases expression or elaboration of pro-inflammatory cytokines. In some embodiments, a TGR5 agonist compound of formula (I) provides selective metabolic effects. In other embodiments, a TGR5 agonist comopound of formula (I) selectively increases GLP-1 secretion. In some embodiments, a TGR5 agonist compound of formula (I), activates a TGR5 anti-inflammatory response but does not increase GLP-1 secretion. In some embodiments, a TGR5 agonist compound of formula (I), activates a TGR5 decreased expression or eleaboration of pre-inflammaotry cytokines but does not increase GLP-1 secretion. In some embodiments, the TGR5 agonist that selectively increases GLP-1 secretion is a compound of formula (I). In other embodiments, the TGR5 agonist that selectively increases GLP-1 secretion is the S-enantiomer of a compound of formula (I). In some embodiments, the TGR5 agonist that selectively increases GLP-1 secretion is the R- enantiomer of a compound of formula (I). In certain embodiments, the TGR5 agonist that selectively increases GLP-1 secretion is (6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one. In certain embodiments, the TGR5 agonist that selectively activiates a TGR5 anti-inflammatory effect and does not increase GLP-1 secretion is (65)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
[000117] In some embodiments, a TGR5 agonist compound of formula (I) of this invention provides selective TGR5 activation. In other embodiments, in contrast to other known TGR5 agonists, the compounds of the present inventon show diminished adverse effects upon the heart or the gall bladder. Other known TGR5 agonists in the art include but are not limited to 13.4-benzofuranyloxynicotinamide, 4-phenoxynicotinamide and 4-phenoxypyrimidine-5- carboxamide derivatives. In some embodiments, a TGR5 agonist of formula (I) is an R- enantiomer. In other embodiments, a TGR5 agonist of formula (I) is an 5-enantiomer.
[000118] In some embodiments, a TGR5 agonist compound of this invention comprises any ligand, peptide, or antibody capable of binding the TGR5 receptor and producing a TGR5 receptor agonistic effect. For example, a compound may be a ligand, which in one embodiment refers to any molecule that provides an enhanced affinity for a TGR5 receptor, wherein said affinity results in a TGR5 receptor agonistic activity.
[000119] In another embodiment, a TGR5 agonist compound is a peptide. As used herein, the term "peptide" includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into bacterial cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=0, 0=C-NH, CH2-0, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
[000120] Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-0-0-C(R)-N-), ketomethylen bonds (-CO-CH2-), OC-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)- CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.
[000121] These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
[000122] Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
[000123] In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
[000124] In another embodiment, a TGR5 agonist compound of this invention is an antibody. In some embodiments, the term "antibody" refers to intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of specifcially interacting with a desired target as described herein, for example, binding to phagocytic cells.
In some embodiments, the antibody fragments comprise:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
(2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
[000125] Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
[000126] In some embodiments, the antibody fragments may be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
[000127] Antibody fragments can, in some embodiments, be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
[000128] Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al , Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991 ; Bird et al , Science 242:423-426, 1988; Pack et al , Bio/Technology 11: 1271-77, 1993; and Ladner et al , U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
[000129] Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
[000130] In some embodiments, the antibodies or fragments as described herein may comprise "humanized forms" of antibodies. In some embodiments, the term "humanized forms of antibodies" refers to non-human (e.g. murine) antibodies, which are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al , Nature, 321 :522-525 (1986); Riechmann et al , Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
[000131] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al , Nature, 321:522-525 (1986); Riechmann et al , Nature 332:323-327 (1988); Verhoeyen et al , Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
[000132] Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al , J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al , J. Immunol., 147(l):86-95 (1991)]. Similarly, human can be made by introducing of human immunoglobulin loci into transgenic animals, e.g. mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al , Bio/Technology 10, 779- 783 (1992); Lonberg et al , Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al , Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[000133] In another embodiment, a TGR5 agonist compound provides a selective effect. In one embodiment, a ligand TGR5 agonist selectively binds the TGR5 receptor. In another embodiment, a peptide TGR5 agonist compound selectively binds the TGR5 receptor. In still another embodiment, an antibody TGR5 agonist compound selectively binds the TGR5 receptor.
[000134] In yet another embodiment, the present invention provides that different enantiomers of the compound of formula (I) induce different biologic effects at the TGR5 receptor, showing, for the first time, that there is heterogeneity of the TGR5 receptor or heterogeneity of activation of the TGR5 receptor, and that different compounds that are agonists at a TGR5 receptor can exploit this heterogeneity to have selective biological effects. In some embodiments, the compounds of this invention are selective TGR5 agonists able to provide an anti-inflammatory effect but not a metabolic effect in vivo. In other embodiments, the compounds of this invention are selective TGR5 agonists able to provide a metabolic effect but not an anti-inflammatory effect in vivo.
[000135] In another embodiment, the present invention provides that different agonist compounds induce different biologic effects at the TGR5 receptor, showing heterogeneity of the TGR5 receptor binding or heterogeneity of activation of the TGR5 receptor. Compounds that are agonists at a TGR5 receptor may be used to exploit the heterogeneity of the TGR5 receptor in order to provide selective biological effects. In some embodiments, the compounds of this invention are selective TGR5 agonists able to provide an anti- inflammatory effect but not a metabolic effect in vivo. TGR5 agonist compounds able to provide an anti-inflammatory effect but not a metabolic effect, may for example, decrease exression or elaboration of pro-inflammatory cytokines while not increasing GLP-1 secretion upon binding to the TGR5 receptor. In other embodiments, the compounds of this invention are selective TGR5 agonists able to provide a metabolic effect but not an anti-inflammatory effect in vivo. TGR5 agonist able to provide a metabolic effect but not an anti-inflammatory effect, may for example, increase secretion of GLP-1 while not decreasing exression or elaboration of pro-inflammatory cytokines upon binding to the TGR5 receptor.
[000136] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLES
General Procedures
[000137] All reactions were performed in oven-dried or flame-dried round-bottom flasks. The flasks were fitted with rubber septa and reactions were conducted under a positive pressure of argon. Gas-tight syringes with stainless steel needles or cannulae were used to transfer air- and moisture-sensitive liquids. Flash column chromatography was performed as described by Still et al. using granular silica gel (60-A pore size, 40-63 μιη, 4-6% ]¾0 content, Zeochem) (Still, et al. J. Org. Chem. 1978, 43, 2923). Analytical thin layer chromatography (TLC) was performed using glass plates pre-coated with 0.25 mm 230^400 mesh silica gel impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to short wave ultraviolet light (254 nm) and a solution of p- Anisaldehyde stain (PAA), Potassium permanganate (ΚΜη04), eerie ammonium molybdate (CAM), or vanillin stain, followed by heating on a hot plate (-250 °C). Organic solutions were concentrated at 29-30 °C on rotary evaporators capable of achieving a minimum pressure of ~2 torr.
Materials
[000138] Commercial reagents and solvents were used as received with the following exceptions: dichloromethane, benzene, and toluene were distilled over calcium hydride; acetonitrile was pre-dried over calcium hydride then was distilled over calcium hydride; acetone was distilled over calcium sulfate; tetrahydrofuran was distilled from sodium benzophenone ketyl. Pyridine, triethylamine, and Hiinig's base were dried over potassium hydroxide pellets for at least 48 hours before use.
Instrumentation
[000139] Proton nuclear magnetic resonance ( H NMR) spectra are reported in parts per million on the δ scale, and are referenced from the residual proton in the NMR solvent (CDC13: δ 7.26 (CHC13), CD2C12: δ 5.32 (CHDC12), DMSO-d6: δ 2.50 (DMSO-d5)). Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, sp = septet, m = multiplet), coupling constant(s) in Hertz, integration, assignment]. Carbon-13 nuclear magnetic resonance (13C NMR) spectra were reported in parts per million on the δ scale, and are referenced from the carbon resonances of the solvent (CDCI3: δ 77.00, CD2CI2: δ 54.00, DMSO-^: δ 39.51). Peak assignments of intermediates are based on analyses of gradient COSY experiments and chemical shifts of individual protons. High resolution mass spectra (HRMS) were recorded on using either an electrospray (ESI) or direct analysis in real time (DART) ionization source.
Experimental
Example 1: 6-[(3-Fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
Figure imgf000045_0001
[000140] Step 1. fert-Butyl Allylcarbamate. To a solution of allylamine (29.9 g, 0.525 mol) and triethylamine (53.0 g, 0.525 mol) in dry Et20 (300 ml), a solution of Boc-anhydride (114 g, 0.525 mol) in dry Et20 (150 ml) was added portionwise upon stirring and cooling in a cold-water-bath. The reaction mixture was stirred at room temperature overnight and concentrated under a reduced pressure. The oily residue was dried in a vacuum (1-2 mm Hg) to afford the title compound which was used further without an additional purification. The yield: 82.4 g (100%); the product contains traces of B0C2O and i-BuOH according to H NMR data. !H NMR (δ, DMSO-d6): 1.45 (s, 9H), 3.50-3.56 (m, 2H), 4.99-5.04 (m, 1H), 5.05-5.11 (m, 1H), 5.71-5.81 (m, 1H), 6.98 (br. s, 1H).
Figure imgf000046_0001
[000141] Step 2. tert-Butyl (Oxiran-2-ylmethyl)carbamate. To a solution of the compound i<?ri-Butyl Allylcarbamate (82.4 g, 0.525 mol) in CH2CI2 (1300 ml), commercial (75% purity) m-chloroperbenzoic acid (157 g, 0.683 mol) was added portionwise upon stirring and cooling in an ice-bath. The reaction mixture was stirred at -5-7 °C for 1 h, then overnight at room temperature. The white precipitate was filtered, the mother liquid was concentrated under a reduced pressure. The residue was dissolved in Et20 (1000 ml); the resulted solution was washed with 10% aq. Na2S03 (500 ml), sat. aq. NaHC03 (4 x 500 ml), dried over MgS04, filtered, and concentrated under a reduced pressure. The resulted oil was dried in a vacuum (1-2 mm Hg) to afford the titled compound which was used further without an additional purification. The yield: 70.4 g (77.5%); the product contains -5-7% of m- chlorobenzoic acid according to lH NMR data. lH NMR (δ, DMSO-d6): 1.38 (s, 9H), 2.45 (dd, Jj = 5.1 Hz, J2 = 2.7 Hz, 1H), 2.65-2.70 (m, 1H), 2.91-2.97 (m, 1H), 2.99-3.15 (m, 1H), 6.96-7.03 (m, 1H).
Figure imgf000046_0002
[000142] Step 3. tert-Butyl {2-Hydroxy-3-[(3- methylbenzyl)amino]propyl}carbamate. To a solution of the feri-Butyl (Oxiran-2- ylmethyl)carbamate (70.4 g, 0.407 mol) in dry isopropanol (700 ml), (3-methylbenzyl)amine (54.2 g, 0.448 mol) was added by one portion upon stirring and cooling in a cold-water-bath. The reaction mixture was stirred at 50 °C (oil bath) for 4 h, cooled to room temperature, and concentrated under a reduced pressure. The oily residue was re-evaporated with toluene (2 x 200 ml) and dried in a vacuum (1-2 mm Hg) for 3 h to afford the titled compound which was used in the next step without purification.
Figure imgf000046_0003
[000143] Ste 4.tert-Butyl-{2-{[tert-Butyl(dimethyl)silyl]oxy}-3-[(3- methylbenzyl)amino]propyl}-carbamate. To a solution of the crude i<?ri-Butyl {2- Hydroxy-3-[(3-methylbenzyl)amino]propyl}-carbamate (0.407 mol) and triethylamine (181 g, 1.79 mol) and dry DMF (750 ml), i-butyldimethylsilyl chloride (135 g, 0.895 mol) was added portionwise upon stirring and cooling in an ice bath. The reaction mixture was stirred at room temperature overnight and concentrated under a reduced pressure (-10-15 mm Hg) at -50-60 °C. The residue was distributed between 10% aq. K2C03 (180 ml) and Et20 (180 ml). The organic layer was washed with water (2 x 100 ml), dried over MgS04, and filtered. To the resulted solution, 4M aq. HC1 (11.4 ml) was added dropwise, the formed precipitate was filtered, washed with abs. Et20, and dried on air. The obtained hydrochloride was shaken in a separating funnel with 20% aq. K2C03 (120 ml) and EtOAc (120 ml) until two homogeneous layers were formed. The organic layer was separated, dried over MgS04, filtered, and concentrated under a reduced pressure. The residue was purified by column chromatography on silica gel (CHCI3 - MeOH, 10: 1) to afford the titled compound (oil). The yield: 73.4 g (44% from 3). H NMR (δ, DMSO-d6): 0.01 (s, 3H), 0.04 (s, 3H), 0.82 (s, 9H), 1.35 (s, 9H), 2.28 (s, 3H), 2.41 (dd, = 12.0 Hz, J2 = 5.6 Hz, 1H,), 2.90-3.05 (m, 2H), 3.64 (br.s, 2H), 3.72-3.79 (m, 1H), 6.70 (t, J = 5.9 Hz, 1H,), 7.02 (d, J = 7.6 Hz, 1H,), 7.07 (d, J = 7.6 Hz, 1H), 7.10 (s, 1H), 7.17 (dd, Ji = J2 = 7.6 Hz, 1H).
Figure imgf000047_0001
[000144] Step 5. tert-Butyl {2-{[tert-Butyl(dimethyl)silyl]oxy}-3-[(chloroacetyl)(3- methylbenzyl)-amino]propyl}carbamate. To a solution of i<?ri-Butyl {2-{ [tert- Butyl(dimethyl)-silyl]oxy}-3-[(3-methylbenzyl)amino]propyl}-carbamate (73.4 g, 0.180 mol) and triethylamine (36.4 g, 0.360 mol) in dry CH2C12 (600 ml), a solution of chloroacetyl chloride (26.4 g, 0.234 mol) in dry CH2C12 (50 ml) was added dropwise upon stirring and cooling in an ice bath. The reaction mixture was stirred overnight at room temperature. EtOAc (1500 ml) and brine (1200 ml) were added; the organic layer was washed with brine (1200 ml), sat. aq. NaHCC>3 (2 x 600 ml), dried over MgS04, filtered, and concentrated under a reduced pressure. The residue was purified by column chromatography on silica gel (hexane - EtOAc, 4: 1 to 3: 1) to afford the titled compound (oil). The yield: 73.7 g (85%).
!H NMR (δ, DMSO-d6): -0.05, 0.00 (s, 3H), 0.05, 0.09 (s, 3H), 0.83 (s, 9H), 1.35 (s, 9H), 2.28, 2.30 (s, 3H), 2.35-2.45, 2.48-3.08 (m, 2H), 3.13 (d, J = 14.2 Hz), 3.32-3.45 (m), 3.92- 4.00 (m, 1H), 4.12 (d, J = 15.2 Hz), 4.38-4.42 (m), 4.56 (d, J = 16.9 Hz), 4.63 (d, J = 13.5 Hz), 4.73 (d, J = 16.9 Hz), 4.86 (d, J = 15.2 Hz), 6.67, 6.93 (t, J = 6.6 Hz, 1H), 6.96-7.03 (m, 2H), 7.07, 7.12 (d, J = 7.6 Hz, 1H), 7.21, 7.27 (dd, = = 7.6 Hz, 1H).
Figure imgf000048_0001
[000145] Step 6. fert-Butyl 6-{[tert-Butyl(dimethyl)silyl]oxy}-4-(3-methylbenzyl)-3-oxo- 1,4-diazepane-l-carboxylate. To a solution of i<?ri-butyl {2-{ [tert- butyl(dimethyl)silyl]oxy}-3-[(chloroacetyl)(3-methylbenzyl)-amino]propyl}carbamate (73.7 g, 0.152 mol) in abs. DMF (950 ml), sodium hydride (7.92 g of -60% suspension in mineral oil, 0.198 mol) was added portionwise upon stirring and cooling in a cold- water-bath. The reaction mixture was stirred at room temperature for 6 h. Water (20 ml) was added, and the mixture was stirred for 30 min and diluted with water (2600 ml) and EtOAc (1400 ml). The organic layer was washed with brine (3 x 1200 ml), dried over MgS04, filtered, and concentrated under a reduced pressure. The residue was dried in a vacuum (1-2 mm Hg) at room temperature to afford the crude product (oil) which was used in the next step without an additional purification. LCMS(ES): 393 (MH-56)+, 349 (MH-100)+.
Figure imgf000048_0002
[000146] Step 7. fert-Butyl 6-Hydroxy-4-(3-methylbenzyl)-3-oxo-l,4-diazepane-l- carboxylate . To a solution of the crude i<?ri-Butyl 6-{ [½ri-Butyl(dimethyl)silyl]oxy}-4-(3- methylbenzyl)-3-oxo-l,4-diazepane-l-carboxylate (0.152 mol) in dry THF (400 ml), tetrabutylammonium fluoride (198 ml of 1.0M solution in THF, 0.198 mol) was added by one portion. The reaction mixture was stirred overnight at room temperature and concentrated under a reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc - hexane, 2: 1 to EtOAc neat) to afford the titled compound (oil). The yield: 42.4 g (83.5% from 6). H NMR (δ, DMSO-d6): 1.39 (s, 9H), 2.28 (s, 3H), 2.77-3.02 (m, 1H), 3.13- 3.24 (m, 1H), 3.31-3.52 (m, 2H), 3.76-4.06 (m, 2H), 4.08-4.23 (m, 2H), 4.70-4.79 (m, 1H), 5.14 (br. s, 1H), 6.96-7.04 (m, 2H), 7.07 (d, J = 7.6 Hz, 1H), 7.19 (dd, Ji = J2 = 7.6 Hz, 1H). LCMS(ES): 335 (MH+), 279 (MH-56)+, 235 (MH-100)
Figure imgf000049_0001
[000147] Step 8. fert-Butyl 6-[(3-Fluorobenzyl)oxy]-4-(3-methylbenzyl)-3-oxo-l,4- diazepane-l-carboxylate. To a solution of i<?ri-Butyl 6-Hydroxy-4-(3-methylbenzyl)-3-oxo- 1,4-diazepane-l-carboxylate (40.7 g, 0.122 mol) and 3-fluorobenzyl bromide (30.1 g, 0.159 mol) in abs. DMF (550 ml), sodium hydride (7.32 g of -60% suspension in mineral oil, 0.183 mol) was added portionwise upon stirring and cooling in an ice bath. The reaction mixture was stirred overnight at room temperature. Water (15 ml) was added, and the mixture was stirred for 30 min, poured into water (1500 ml), and extracted with EtOAc (2 x 1100 ml). The organic layer was washed with brine (2 x 1000 ml), dried over MgS04, filtered, and concentrated under a reduced pressure. The residue was purified by column chromatography on silica gel (CHC13 - MeOH, 30: 1 to 20: 1) to afford the titled compound (oil). The yield: 43.7 g (81%). !H NMR (δ, DMSO-d6): 1.31, 1.38 (s, 9H), 2.22 (s, 3H), 3.31-3.70 (m, 5H), 4.02-4.13 (m, 3H), 4.35-4.48 (m, 2H), 4.78-4.90 (m, 1H), 6.97-7.20 (m, 7H), 7.32-7.40 (m, 1H). LCMS(ES): 387 (MH-56)+, 343 (MH-100)+.
Figure imgf000049_0002
[000148] Step 9. 6-[(3-Fluorobenzyl)oxy]-l-(3-methylbenzyl)-l,4-diazepan-2-one. To a solution of i<?ri-butyl-6- [(3 -fluorobenzyl)oxy] -4-(3 -methylbenzyl)-3 -oxo- 1 ,4-diazepane- 1 - carboxylate (43.7 g, 0.0989 mol) in EtOAc (500 ml), 4M solution of HC1 in EtOAc (198 ml, 0.791 mol) was added portionwise upon stirring and cooling in a cold-water-bath. The reaction mixture was stirred at room temperature for 48 h. To the resulted solution, a solution of K2CO3 (156 g, 1.13 mol) in water (620 ml) was carefully added portionwise upon stirring (gas evolution was observed); the mixture was stirred for 30 min at room temperature. The organic layer was separated, and the aqueous layer was extracted with EtOAc (600 ml). The combined organic layer was dried over MgS04, filtered, and concentrated under a reduced pressure. The residue was dried in a vacuum (1-2 mm Hg) at room temperature to afford the crude product which was used in the next step without an additional purification. LCMS(ES): 343 (MH+).
Figure imgf000050_0001
[000149] Step 10. 6-[(3-Fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one. To a solution of the crude 6-[(3-Fluorobenzyl)oxy]-l-(3-methylbenzyl)-l,4- diazepan-2-one (0.0989 mol) and triethylamine (14.9 g, 0.148 mol) in dry CH2C12 (650 ml), a solution of 2-furoyl chloride (16.8 g, 0.129 mol) in dry CH2CI2 (30 ml) was added dropwise upon stirring and cooling in an ice bath. The reaction mixture was stirred overnight at room temperature, diluted with EtOAc (1300 ml), washed with brine (2 x 1000 ml), sat. aq. NaHCC>3 (2 x 1 L), dried over MgS04, filtered, and concentrated under a reduced pressure. The residue was purified by column chromatography on silica gel (CHCI3 - MeOH, 20: 1 to 10: 1) to afford the titled compound (oil). The yield: 41.0 g (95% from step 8). H NMR (δ, DMSO-d6): 2.21 (s, 3H), 3.50-3.70 (m, 3H), 3.80-4.05 (m, 2H), 4.11-4.21 (m, 1H), 4.23- 4.68 (m, 4H), 4.77-4.88 (m, 1H), 6.55-6.68 (m, 1H), 6.85-7.25 (m, 8H), 7.30-7.40 (m, 1H), 7.75-7.90 (m, 1H). LCMS(ES): 437 (MH+).
Example 2: 6-[(2-Fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one Hydrochloride
Figure imgf000050_0002
[000150] Step 1. tert-Butyl (Oxiran-2-ylmethyl)carbamate. Allylamine (17.5 g, 306.5 mmol, 23 ml) was dissolved in dichloromethane (150 ml). A solution of Boc-anhydride (70.8 g, 325 mmol) in dichloromethane (150 ml) was added in portions under cooling with a water bath and stirring. The reaction mixture was stirred at room temperature for 15 h. The obtained solution of feri-butyl allylcarbamate was cooled again in an ice bath, m- Chloroperoxybenzoic acid (92 g 70%, 370 mmol) was added under stirring for 30 min. The reaction mixture was allowed to heat up slowly in the bath to room temperature for 24 h. The formed precipitate was filtered off and washed with cold dichloromethane (2 x 100 ml). The filtrate was evaporated to half volume, washed with a 10% aqueous solution of potash (300 ml), and dried with anhydrous sodium sulfate. Target i<?ri-butyl (oxiran-2- ylmethyl)carbamate was isolated from the obtained solution by column chromatography in dichloromethane to give a fluid oil (39.4 g, 75%). H-NMR (400 MHz, DMSO-d6): 1.4 (s, 9H), 2.46 (m, IH), 2.67 (t, IH), 2.95 (m, IH), 3.08 (m, 2H), 6.95 (t, IH).
Figure imgf000051_0001
[000151] Step 2. tert-Butyi {2-Hydroxy-3-[(4-methoxybenzyl)amino]propyl}carbamate. i<?ri-Butyl (oxiran-2-ylmethyl)carbamate (7 g, 41 mmol) was dissolved in isopropanol (40 ml). p-Methoxybenzylamine (6.7 g, 49 mmol) was added. The obtained solution was kept under heating and stirring for 12 h. The reaction mixture was evaporated. The residue was purified by chromatograpny on silica gel in chloroform-isopropanol mixture (up to 10% of isopropanol). i<?ri-Butyl {2-hydroxy-3-[(4-methoxybenzyl)amino]propyl}carbamate (8.15 g, 65%) was obtained as oil of purity sufficient for the next step. (An impurity of the corresponding benzylamine is typical.) LCMS(ES): 311 (MH+). H-NMR (400 MHz, DMSO-d6): 1.4(s, 9H), 2.0 (br s, IH), 2.4 (m, 2H), 2.9 (m, 2H), 3.55 (m, IH), 3.6 (s, 2H), 3.72 (s, 3H), 4.63 (br s, IH), 6.63(t, IH), 6.85 (d, 2H), 7.2(d, 2H).
Figure imgf000051_0002
80%
[000152] Step 3. Target tert-Butyl {2-{[tert-Butyl(dimethyl)silyl]oxy}-3-[(4- methoxybenzyl)-amino]propyl}carbamate. i<?ri-Butyl { 2-hydroxy-3 - [(4- methoxybenzyl)amino]propyl}carbamate (8 g, 26 mmol) was dissolved in 1,2-dichloroethane (60 ml). Triethylamine (7.2 ml, 52 mmol) and feri-butyl dimethylsilyl chloride (5.4 g, 36 mmol) were added. The reaction mixture was kept at 60°C for 24 h until the conversion was complete, and then poured into a mixture of chloroform (200 ml) and saturated aqueous K2CO3 (80 ml). The organic layer was separated and dried with anhydrous sodium sulfate. Target compound was isolated from the obtained solution by column chromatography in chloroform-ethanol mixture. Target compound (8.7 g, 80%) was obtained as oil. LCMS(ES): 425 (MH+). !H-NMR (400 MHz, DMSO-d6): 0 (d, 6H), 0.8 (s, 9H), 1.38 (s, 9H), 1.8 (br s, IH), 2.45 (m, 2H), 2.98 (m, 2H), 3.62 (s, 2H), 3.72 (s, 3H), 3.75 m, IH), 6.66 (t, IH), 6.85 (d, 2H), 7.2 (d, 2H).
Figure imgf000051_0003
[000153] Step 4. tert-Butyi {2-{[tert-Butyl(dimethyl)silyl]oxy}-3-[(chloroacetyl)(4- methoxy-benzyl)amino]propyl}carbamate. i<?ri-Butyl {2-{ [½ri-butyl(dimethyl)silyl]oxy }- 3-[(4-methoxy-benzyl)amino]propyl}carbamate (8.7 g, 21 rnmol) and DIPEA (7.3 ml, 42 mmol) were dissolved in dichloromethane (200 ml). Chloroacetyl chloride (2.2 ml, 27.3 mmol) was added dropwise under cooling in an ice bath and stirring. When starting compound (5) disappeared (1 h), the reaction mixture was poured into a mixture of dichloromethane (200 ml) and 1M citric acid (200 ml). The organic layer was separated and dried with anhydrous sodium sulfate. Target compound was isolated from the obtained solution by column chromatography in chloroform-methanol mixture. Compound (6) (9.2 g, 90%) was obtained as oil. LCMS(ES): 502 (MH+), 402 (MH-Boc)+. ^-NMR (400 MHz, DMSO-d6) - very rotameric, several forms.
Figure imgf000052_0001
[000154] Step 5. tert-Butyi 6-{[tert-Butyl(dimethyl)silyl]oxy}-4-(4-methoxybenzyl)-3- oxo-l,4-diazepane-l-carboxylate. i<?ri-Butyl {2-{ [½ri-butyl(dimethyl)silyl]oxy}-3- [(chloroacetyl)-(4-methoxybenzyl)amino]propyl} carbamate (9.2 g, 19 mmol) was dissolved in DMFA (40 ml). Sodium hydride (960 mg, 60% in mineral oil, 24 mmol) was added in three portions under stirring and slight cooling. The resulting suspension was vigorously stirred at room temperature for 12 h until compound (6) disappeared. The reaction mixture was poured into a mixture of ethyl acetate (300 ml) and 1M citric acid (200 ml). The organic layer was separated, washed with water (2 x 150 ml), and dried with anhydrous sodium sulfate. Target compound was isolated from the obtained solution by column chromatography in chloroform-methanol mixture. Compound (6.3 g, 75%) was obtained as oil. LCMS(ES): 465 (MH+). ^-NMR (400 MHz, DMSO-d6): 0 (d, 6H), 0.8 (s, 9H), 1.4 (s, 9H), 2.95 (m, IH), 3.1 (t, IH), 3.5 (m, IH), 3.7 (s, 3H), 3.7^.25 (m, 5H), 5.0 (m, IH), 6.85 (d, 2H), 7.1 (d, 2H).
Figure imgf000052_0002
[000155] Step 6. fert-Butyl 6-Hydroxy-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l- carboxylate. i<?ri-Butyl 6-{ [½ri-butyl(dimethyl)silyl]oxy}-4-(4-methoxybenzyl)-3-oxo-l,4- diazepane-l-carboxy-late (6.3 g, 13.8 mmol) and acetic acid (900 mg, 13.8 mmol) were dissolved in methanol (150 ml). Potassium fluoride (2.5 g, 42 mmol) was added. The reaction mixture was refluxed for 24 h and evaporated. The residue was partitioned in a mixture of dichloromethane (200 ml) and saturated aqueous potash (50 ml). The organic layer was separated and dried with anhydrous sodium sulfate. Target compound was isolated from the obtained solution by column chromatography in chloroform-methanol mixture to give 4.2 g (90%) of the product . LCMS(ES): 251 (MH-100)+. ^-NMR (400 MHz, DMSO- d6): 1.4 (s, 9H), 2.95 (m, 1H), 3.1 (t, 1H), 3.5 (m, 1H), 3.7 (s, 3H), 3.7-4.25 (m, 5H), 4.8 (m, 1H), 5.15 (d, 1H), 6.85 (d, 2H), 7.1 (d, 2H).
Figure imgf000053_0001
[000156] Step 7. fert-butyl 6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4- diazepane-l-carboxylate. i<?ri-Butyl 6-hydroxy-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane- 1-carboxylate (4.2 g, 12.2 mmol) was dissolved in DMFA (50 ml). Sodium hydride (600 mg, 60% in mineral oil, 15 mmol) was added under slight cooling and stirring. The resulting suspension was vigorously stirred at room temperature for 20 min. l-(Bromomethyl)-2- fluorobenzene (2.8 g, 15 mmol) was added dropwise. The reaction mixture was stirred for 12 h at room temperature. The resulting solution was poured into a mixture of ethyl acetate (300 ml) and 1M citric acid (200 ml). The organic layer was separated, washed with water (2x150 ml), and dried with anhydrous sodium sulfate. Target compound was isolated from the obtained solution by column chromatography in chloroform-methanol mixture. Compound (9) (4.6 g, 85%) was obtained as oil. LCMS(ES): 359 (MH-100)+.
Figure imgf000053_0002
[000157] Step 8. 6-[(2-Fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one Hydro-chloride. feri-Butyl 6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4- diazepane-l-carboxylate (4.6 g, 10 mmol) was dissolved in dioxane (30 ml), and 4M HCl in dioxane (30 ml) was added. The resulting solution was stirred at room temperature for 12 h until compound (9) disappeared, and then evaporated in vacuum. The residue was coevaporated with acetonitrile (2 x 100 ml). The residual oil was dried at 0.5 torr/40°C to give an amorphous solid. The hydrochloride of (10) (3.9 g, 99%) was obtained. LCMS(ES): 359 (MH+).
Figure imgf000054_0001
[000158] Step 9 (HTC). Amides. Solutions of BB's-acids (1.1 eq; 220 μιηοΐ) in DMF (500 μΐ) were placed into 6-ml glass tubes standing in a 48-well reaction rack (6 x 8). A solution of HBTU (1.1 eq; 220 μιηοΐ) in DMF (400 μΐ) and NMM (50 μΐ) were added. The reaction mixtures were stirred with the use of Vortex and then solution of the intermediate (Example 2, Step 8) (1 eq; 200 μιηοΐ) in DMF (500 μΐ) was added. The reaction mixtures were stirred with the use of Vortex again and left for 24 h at room temperature. The reaction DMF was evaporated to dryness on a Savant evaporator at 60°C for 6 h. The products were handed over for HPLC purification.
[000159] Ureas, Urethanes and Amides. A solution of the intermediate (1 eq; 250 μιηοΐ) in dichloromethane (500 μΐ) was mixed with trietylethylamine (75 μΐ), and solution sulfonyl chloride (or isocyanate) (1.3 eq; 325 μιηοΐ) in dichloromethane (500 μΐ) was added. The reaction mixtures were stirred with use of Vortex and kept for 16 h at room temperature. The reaction solvent was evaporated to dryness and the products were handed over for HPLC purification.
[000160] The following compounds of the present invention were synthesized by this method:
6-[(2-fluorobenzyl)oxy]-N-isopropyl-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l- carboxamide, (MH+ 444);
6- [(2-fluorobenzyl)oxy] -4-glycoloyl- 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one, (MH+ 416); 6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(tetrahydrofuran-2-ylcarbonyl)-l,4- diazepan-2-one, (MH+ 456);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(lH-pyrrol-2-ylcarbonyl)-l,4-diazepan-2- one, (MH+ 451); 6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(5-methyl-lH-pyrazol-3-yl)carbonyl]-l,4- diazepan-2-one, (MH+ 466);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(5-methylisoxazol-3-yl)carbonyl]-l,4- diazepan-2-one, (MH+ 467);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(2-methyl-l,3-thiazol-4-yl)carbonyl]-l,4- diazepan-2-one, (MH+ 483);
6-[(2-fluorobenzyl)oxy]-4-(isoxazol-5-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2- one, (MH+ 453);
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 426);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(5-methyl-l,3-oxazol-4-yl)carbonyl]-l,4- diazepan-2-one, (MH+ 467);
propyl 6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxylate, (MH+ 444);
4-butyryl-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 428);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(lH-pyrazol-3-ylcarbonyl)-l,4-diazepan-2- one, (MH+ 452);
6-[(2-fluorobenzyl)oxy]-4-(lH-imidazol-4-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan- 2-one, (MH+ 452);
N-ethyl-6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxamide, (MH+ 429);
6-[(2-fluorobenzyl)oxy]-4-(isoxazol-3-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2- one, (MH+ 453);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 452);
6-[(2-fluorobenzyl)oxy]-4-(lH-imidazol-2-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan- 2-one, (MH+ 452);
6-[(2-fluorobenzyl)oxy]-4-isobutyryl-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 428);
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4-propionyl- 1 ,4-diazepan-2-one, (MH+ 414);
6-[(2-fluorobenzyl)oxy]-4-(methoxyacetyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 430); 4-acetyl-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH 400);
4-(cyclobutylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 440);
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4- [( 1 -methyl- lH-pyrazol-3 -yl)carbonyl] -1,4- diazepan-2-one, (MH+ 466);
6-[(2-fluorobenzyl)oxy]-4-[2-furyl(oxo)acetyl]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 480);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(l,3-thiazol-4-ylcarbonyl)-l,4-diazepan-2- one, (MH+ 469); and
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(pyridin-2-ylcarbonyl)-l,4-diazepan-2-one, (MH+ 463).
Example 3: (6/?)-6-[(2-Fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(3-thienylcarbonyl)- l,4-diazepan-2-one
Figure imgf000056_0001
[000161] Step 1. fert-Butyl [(2/?)-2,3-dihydroxypropyl]carbamate. Freshly distilled S(- glycidol (10 g, 135 mmol) was dissolved in methanol (200 ml) containing 10% of water. Sodium azide (44 g, 675 mmol) and ammonium chloride (16 g, 300 mmol) were added. The resulting mass was refluxed for 8 h and evaporated. The residue was coevaporated with acetonitrile (200 ml) and treated with ether-methanol mixture 1 : 1 (300 ml). The inorganic solid was filtered off. The filtrate was evaporated to give crude (2R)-3-azidopropane-l,2- diol. The latter was dissolved in methanol (300 ml) without additional purification. Then 10% Pd/C (1 g) and Boc-anhydride (29 g, 135 mmol) were added. The obtained mass was hydrogenated in a Parr apparatus at a hydrogen pressure of 3 atm and room temperature for 20 h. The reaction mixture was filtered to separate the catalyst. Target compound was isolated from the residue by column chromatography in chloroform-methanol mixture (up to 4% of methanol). Compound (3) (15.5 g, 60%) was obtained. ^-NMR (400 MHz, DMSO- d6): 1.4 (s, 9H), 2.85 (m, 1H), 3.05 (m, 1H), 3.28 (t, 2H), 3.45 (m, 1H), 4.43 (t, 1H), 4.59 (d, 1H), 6.56 (br t, 1H).
Figure imgf000057_0001
[000162] Step 2. (2/?)-3-[(tert-Butoxycarbonyl)amino]-2-hydroxypropyl 4- methylbenzenesulfo-nate. feri-Butyl [(2R)-2,3-dihydroxypropyl]carbamate (15.5 g, 81 mmol) was dissolved in absolute pyridine (50 ml). Tosyl chloride (17 g, 90 mmol) was added in portions under cooling in an ice bath. The obtained solution was stirred for 4 h at room temperature. The reaction mixture was poured into ethyl acetate (500 ml), washed with 1M citric acid (2 x 100 ml), with an aqueous solution of copper sulfate (100 ml), and dried with anhydrous sodium sulfate. The target compound was isolated from the obtained solution by column chromatography in chloroform-methanol mixture (up to 2% of methanol) and obtained (16.8 g, 60%). LCMS (ES): 246 (MH-100)+. ^-NMR (400 MHz, DMSO-d6): 1.4 (s, 9H), 2.4 (s, 3H), 2.9 (t, 2H), 3.65 (m, 1H), 3.77 (m, 1H), 3.95 (m, 1H), OH, 6.75 (br t, 1H), 7.48 (d, 2H), 7.78 (d, 2H).
Figure imgf000057_0002
[000163] Step 3. tert-Butyl {(25)-2-hydroxy-3-[(4- methoxybenzyl)amino]propyl}carbamate. (2R)-3-[(½ri-Butoxycarbonyl)amino]-2- hydroxypropyl 4-methylbenzenesulfonate (16.8 g, 48 mmol) was dissolved in acetonitrile (150 ml), and then p-methoxybenzylamine (8.4 g, 60 mmol), potash (16 g, 120 mmol), and potassium iodide (1.7 g, 10 mmol) were added. The resulting mixture was vigorously stirred at 60°C for 24 h until compound (4) disappeared. The reaction mixture was evaporated. The residue was treated with dichloromethane. The organic layer was evaporated. The residue was purified by column chromatography in chloroform-isopropanol mixture (up to 10% of isopropanol) to give target compound (7.6 g, 50%) as oil of purity sufficient for the next step. (An impurity of the corresponding benzylamine is typical.). Analytical data: see Example 2.
[000164] Steps 4 - 9. Subsequent 6 steps of the transformation of feri-butyl {(25)-2- hydroxy-3-[(4-methoxybenzyl)amino]propyl}carbamate into the hydrochloride of (6R)-6-[(2- fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one are completely analogous to steps 3-8 of the Example 2
Figure imgf000058_0001
[000165] Step 10. (6/?)-6-[(2-Fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(3- thienylcarbonyl)-l,4-diazepan-2-one. HBTU (160 mg, 0.42 mmol), thiophene-3-carboxylic acid (54 mg, 0.42 mmol), and DIPEA (54 mg, 0.42 mmol) were dissolved in absolute dioxane (5 ml). The solution was stirred for 5 min at room temperature. Then a second solution containing the hydrochloride of (6R)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4- diazepan-2-one (139 mg, 0.35 mmol) and DIPEA (130 mg, 1 mmol) in DMFA (2 ml) was added. After 24 h the reaction mixture was evaporated in vacuum at 1 torr. The residue was partitioned in a mixture of ethyl acetate (100 ml) and 1M citric acid (50 ml) and washed with an aqueous solution of sodium hydrocarbonate. The organic layer was separated and dried with anhydrous sodium sulfate. The target compound was isolated from the obtained solution by column chromatography in chloroform-methanol mixture. The target compound (140 mg, 85%) was obtained. LCMS (ES): 469 (MH+). ^-NMR (400 MHz, DMSO-d6): 3.5-3.8 (m, 8H including s, 3H at 3.7), 3.9-4.5 (m, 5H), 4.8 (d, 1H), 6.9 (d, 2H), 7.1-8.0 (m, 9H).
Example 4
[000166] Steps 1-5: Identical to those described in Example 2.
Figure imgf000058_0002
[000167] Step 6. 6-Hydroxy-l-(4-methoxybenzyl)-l,4-diazepan-2-one Hydrochloride. i<?ri-Butyl 6-{ [½ri-butyl(dimethyl)silyl]oxy}-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l- carboxy-late (3.0 g, 6.5 mmol) was dissolved in dioxane (30 ml). Then 4M HCI in dioxane (30 ml) was added. The resulting solution was stirred at room temperature for 12 h until the deprotection was complete, and then evaporated in vacuum. Excessive HCI was removed by coevaporation with acetonitrile (2 x 100). The obtained oil of the hydrochloride was dried at 0.5 torr / 40°C to give an amorphous solid. The product was used without further purification.
Figure imgf000059_0001
[000168] Step 7. 4-(Cyclopropylcarbonyl)-6-hydroxy-l-(4-methoxybenzyl)-l,4- diazepan-2-one. The product obtained at step 6 was dissolved in dichloromethane (50 ml), and DIPEA (1.3 g, 10 mmol) was added. The resulting solution was cooled in an ice bath, and cyclopropanecarbonyl chloride (0.81 g, 7.8 mmol) was added dropwise. The reaction mixture was stirred for 2 h at room temperature. Dichloromethane (100 ml) and a 1M solution of citric acid (50 ml) were added. The organic layer was separated and dried with anhydrous sodium sulfate. The target product was isolated from the obtained solution by column chromatography in chloroform-methanol mixture. A crystalline solid (1.4 g, 70% for 2 steps) was obtained. LCMS(ES): 319 (MH+).
Figure imgf000059_0002
[000169] Step 8 (HTC). The intermediate (Example 4, step 7) was dissolved in THF-DMF (9:1) mixture (0.8 ml). A halide (1.2 - 1.3 eq) was added. Then a suspension of NaH (1.5 eq, preliminarily washed with hexane) in THF (0.3 ml) was added. The reaction mixture was stirred for 4 h at room temperature and neutralized with a solution of acetic acid in methanol to pH 7. After evaporation, the product was isolated by HPLC.
[000170] The following compunds of the invention were obtained by this method:
4-({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl]oxy}methyl)benzonitrile, (MH+ 434);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(4-methoxybenzyl)oxy]-l,4-diazepan-2- one, (MH+ 439);
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 427);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-3-ylmethoxy)-l,4-diazepan-2-one, (MH+ 410); 4-(cyclopropylcarbonyl)-6-[(4-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 427);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-2-ylmethoxy)-l,4-diazepan-2-one, (MH+ 410);
4-(cyclopropylcarbonyl)-6-[(3-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 427);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(2-methoxybenzyl)oxy]-l,4-diazepan-2- one, (MH+ 439);
6-[(2-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 443, 445);
4-({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl]oxy}methyl)benzoic acid, (MH+ 453);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-4-ylmethoxy)-l,4-diazepan-2-one, (MH+ 410);
2-({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl]oxy}methyl)benzonitrile, (MH+ 434);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(4-methylbenzyl)oxy]-l,4-diazepan-2-one, (MH+ 423);
6-(benzyloxy)-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 409);
6-[(4-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 443, 445);
3-({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl]oxy}methyl)benzonitrile, (MH+ 434);
4-(cyclopropylcarbonyl)-6-[(3,5-dimethylisoxazol-4-yl)methoxy]-l-(4-methoxybenzyl)-l,4- diazepan-2-one, (MH+ 428);
3- ({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl]oxy}methyl)benzoic acid, (MH+ 453);
4- (cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(2-methylbenzyl)oxy]-l,4-diazepan-2-one, (MH+ 423);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(3-methylbenzyl)oxy]-l,4-diazepan-2-one, (MH 423);
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(3-methoxybenzyl)oxy]-l,4-diazepan-2- one, (MH+ 439); and
6-[(3-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 443, 445).
Example 5
[000171] Steps 1-7: Identical to those described in Example 2.
Figure imgf000061_0001
[000172] Step 8. tert-Butyl 6-[(2-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate. i<?ri-Butyl 6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxylate (4.4 g, 9.6 mmol) was dissolved in a mixture of acetonitrile (80 ml) and water (20 ml). Sodium acetate trihydrate (5.9 g, 43.2 mmol) and acetic acid (2.6 g, 43.2 mmol) were added. Then CAN (15.8 g, 29 mmol) was added under vigorous stirring. The reaction mixture was stirred at room temperature until the starting compound disappeared (about 4 h) and diluted with ethyl acetate (200 ml). The inorganic solid was filtered off and washed with a saturated aqueous solution of sodium hydrocarbonate (100 ml). The filtrate was dried with anhydrous sodium sulfate and evaporated to dryness. Target i<?ri-butyl 6-[(2-fluorobenzyl)oxy]-3-oxo- 1,4-diazepane-l-carboxylate was isolated by column chromatography in chloroform- methanol mixture. The target compound (1.95 g, 60%) was obtained. LCMS (ES): 339 (MH+).
Figure imgf000061_0002
[000173] Step 9. 6-[(2-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride. feri-Butyl 6-[(2-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate (1.95 g, 5.8 mmol) was dissolved in dioxane (25 ml), and 4M HC1 in dioxane (25 ml) was added. The resulting solution was stirred at room temperature for 12 h until the deprotection was complete, and then evaporated in vacuum. In order to remove excessive HC1 the residue was subjected to coevaporation with acetonitrile (2 x 100). The obtained oil of the hydrochloride was dried at 0.5 torr / 40°C to give an amorphous solid. The product was used without further purification.
Figure imgf000062_0001
[000174] Step 10. 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one. To a solution of the crude 6-[(2-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride (5.8 mmol, step 9) and triethylamine (1 ml, 7.2 mmol) in dry CH2CI2 (15 ml), a solution of 2-furoyl chloride (0.9 g, 7 mmol) in dry CH2CI2 (5 ml) was added dropwise upon stirring and cooling in an ice bath. The reaction mixture was stirred overnight at room temperature, diluted with EtOAc (100 ml), washed with brine (2 x 30 ml), sat. aq. NaHC03 (2 x 30 ml), dried over Na2S04, filtered, and concentrated under a reduced pressure. The residue was purified by column chromatography on silica gel (CHCI3 - MeOH, 20: 1 to 10: 1) to afford the titled compound (oil). The yield: 1.6 g (85% from step 8).
Figure imgf000062_0002
[000175] Step 11 (HTC). The intermediate (Example 5, step 10) was dissolved in THF- DMF mixture (9: 1, 0.8 ml). A halide (1.2 - 1.3 eq) was added. Then a suspension of NaH (1.5 eq, preliminarily washed with hexane) in THF (0.3 ml) was added. The reaction mixture was stirred for 4 h at room temperature and neutralized with a solution of acetic acid in methanol to pH 7. After evaporation, the product was isolated by HPLC.
[000176] The following compounds of the invention were obtained by this method:
4-{ [6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile, (MH+ 448);
l-benzyl-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 423);
l-(2-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+457, 459);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-phenylethyl)-l,4-diazepan-2-one, (MH+ 437); l-butyl-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 389);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one, (MH+ 437); 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-2-ylmethyl)-l,4-diazepan-2-one, MH 424); l-(cyclobutylmethyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 401);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(l,3-thiazol-4-ylmethyl)-l,4-diazepan-2-one, (MH+ 430);
l-(4-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 457, 459);
6- [(2-fluorobenzyl)oxy] -4-(2-furoyl)- 1 - [2-( 1 H-pyrazol- 1 -yl)ethyl] - 1 ,4-diazepan-2-one, (MH+ 427);
l-(cyclopropylmethyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 387) l-[(3,5-dimethylisoxazol-4-yl)methyl]-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2- one, (MH+ 442);
l-(2-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 441);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(2-methyl-l,3-oxazol-4-yl)methyl]-l,4-diazepan-2- one, (MH+ 428);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-phenylpropyl)-l,4-diazepan-2-one, (MH+ 451);
1- (4-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 441);
2- { [6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile, (MH+ 448);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbut-2-en-l-yl)-l,4-diazepan-2-one, (MH+ 401);
3- { [6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile, (MH+ 448);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 453);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methylbenzyl)-l,4-diazepan-2-one, (MH+ 437); 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(2-methyl-l,3-thiazol-4-yl)methyl]-l,4-diazepan-2- one, (MH+ 444);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(3-methylisoxazol-5-yl)methyl]-l,4-diazepan-2-one, (MH+ 428);
l-(3-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 457, 459); 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-4-ylmethyl)-l,4-diazepan-2-one, (MH 424);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-3-ylmethyl)-l,4-diazepan-2-one, (MH+ 424);
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-methylbenzyl)-l,4-diazepan-2-one, (MH+ 437);
6- [(2-fluorobenzyl)oxy] -4-(2-furoyl)- 1 -(3 -methoxybenzyl)- 1 ,4-diazepan-2-one, (MH+ 453) ; l-(3-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one, (MH+ 441); and
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-methoxybenzyl)-l,4-diazepan-2-one, (MH+ 453). Example 6: 4-Acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan- 2-one
[000177] Steps 1-7: Steps 1-7 are similar to those described in Example 2, but allylamine was used as an amine, and 3-fluorobenzyl bromide as a halide.
Figure imgf000064_0001
[000178] Step 8. fert-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-4-(2-oxoethyl)-l,4-diazepane- 1-carboxylate. feri-Butyl 4-allyl-6-[(3-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l- carboxylate (2.87 g, 7.6 mmol) was dissolved in a mixture of THF (20 ml) and water (20 ml). A solution of Os04 in CC14 (8 mg, or 30 μιηοΐ of pure osmium tetraoxide) and NaI04 (3.9 g, 18.2 mmol) was added. The reaction mixture was vigorously stirred at room temperature for 1 h until the starting alkene disappeared. Ethyl acetate (200 ml) was added. The organic layer was separated, dried with anhydrous sodium sulfate, and evaporated. The residue was purified by column chromatography (eluent: dichloromethane-ethyl acetate 3:2 - ethyl acetate). The target product (1.7 g, 60%) was obtained as oil. LCMS (ES): 381 (MH+). 1H- NMR (400 MHz, CDC13): 1.45 (s, 9H), 4.0-3.4 (m, 6H), 4.7-4.1 (m, 5H), 7.0 (m, 3H), 7.3 (m, 1H), 9.55 (s, 1H).
Figure imgf000065_0001
[000179] Step 9. fert-Butyl 4-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-3-oxo- 1,4-diaze-pane-l-carboxylate. i<?ri-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-4-(2-oxoethyl)- 1,4-diazepane-l-carboxylate (1.7 g, 4.5 mmol) was dissolved in 1 ,2-dichloroethane (40 ml). STAB (2.9 g, 13.4 mmol) and a 2M solution of dimethylamine in methanol (9 ml of the solution, 18 mmol) were added. The reaction mixture was vigorously stirred at room temperature for 16 h, and then partitioned between saturated aqueous potash (50 ml) and dichloromethane (200 ml). The organic layer was separated, dried with anhydrous sodium sulfate, and evaporated. The target product was isolated by column chromatography (eluent: dichloromethane - dichloromethane-methanol 4: 1). The target product (1.5 g, 85%) was obtained as oil. LCMS (ES): 410 (MH+). H-NMR (400 MHz, DMSO-d6): 1.4 (d, 9H), 2.1(s, 6H), 2.3 (m, 2H), 3.2 (m, 1H), 3.5-3.7 (m, 6H), 4.0 (m, 2H), 4.5-4.7 (m, 2H), 7.05-7.2 (m, 3H), 7.4 (m, 1H).
Figure imgf000065_0002
[000180] Step 10. l-[2-(Dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2- one dihydrochloride. i<?ri-Butyl 4-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-3-oxo- 1,4-diazepane-l-carboxylate (1.5 g, 3.7 mmol) was dissolved in dioxane (30 ml). Then 4M HCI in dioxane (30 ml) was added. The obtained solution was stirred at room temperature for 12 h until the starting compound disappeared, and then evaporated in vacuum. The residue was subjected to coevaporation with acetonitrile (2x100 ml). The obtained oil was dried at 0.5 torr / 40°C to give an amorphous solid. The target product (1.4 g, quantitative yield) was obtained. LCMS (ES): 310 (MH+). H-NMR (400 MHz, DMSO-d6): 2.5-4.1 (br m, 17H), 4.7 (m, 2H), 7.1-7.5 (m, 4H), 9.8-10.4 (m, 3H).
Figure imgf000066_0001
[000181] Step 11. 4-Acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4- diazepan-2-one. 1 - [2-(Dimethylamino)ethyl] -6- [(3 -fluorobenzyl)oxy] - 1 ,4-diazepan-2-one dihydro-chloride (1.4 g, 3.7 mmol) was dissolved in acetonitrile (30 ml). DIPEA (1.4 g, 11.1 mmol) and acetic anhydride (1.5 g, 14.8 mmol) were added. The reaction mixture was refluxed for 2 h until the starting amine disappeared according to LCMS. The obtained mass was evaporated, and the residue was partitioned between saturated aqueous potash (20 ml) and ethyl acetate (200 ml). The organic layer was separated, dried with anhydrous sodium sulfate, and evaporated. The residue was lklpurified by column chromatography (eluent: dichloromethane-methanol). The target compound (1.1 g, 90%) was obtained as oil. LCMS (ES): 352 (MH+). ^-NMR (400 MHz, DMSO-d6): 2.0 (2s-rotamers, 3H), 2.65 (2s-rotamers, 6H), 3.1 (br s, 2H), 3.5-4.3 (m, 9H), 4.5-4.7 (m, 2H), 7.15 (m, 3H), 7.4 (m, 1H).
Example 7:
[000182] Steps 1-7: are similar to those described in Example 2, but 3-fluorobenzyl bromide was used as a halide.
Figure imgf000066_0002
[000183] Step 8. fert-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate. i<?ri-Butyl 6-[(3-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxylate (4.84 g, 10.6 mmol) was dissolved in a mixture of acetonitrile (90 ml) and water (25 ml). Sodium acetate trihydrate (6.5 g, 47.5 mmol) and acetic acid (2.9 g, 47.5 mmol) were added. Then CAN (17.4 g, 32 mmol) was added under vigorous stirring. The reaction mixture was stirred at room temperature until the starting compound disappeared (about 4 h) and diluted with ethyl acetate (250 ml). The inorganic solid was filtered off and washed with a saturated aqueous solution of sodium hydrocarbonate (100 ml). The filtrate was dried with anhydrous sodium sulfate and evaporated to dryness. Target i<?ri-butyl 6-[(3-fluorobenzyl)oxy]-3-oxo- 1,4-diazepane-l-carboxylate was isolated by column chromatography in chloroform- methanol mixture. The target compound (2.1 g, 60%) was obtained. LCMS (ES): 339 (MH+). 1H-NMR(400 MHz, DMSO-d6): 1.4(double s, 9H), 3.3(m, 2H), 3.5-3.7(m, 3H), 3.9(m, 2H) , 4.6(m, 2H), 7.05-7.2(m, 3H), 7.4(m, 2H).
Figure imgf000067_0001
[000184] Step 9. 6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride. feri-Butyl 6-[(3-fluorobenzyl)oxy]-3-oxo-l,4-diazepane-l-carboxylate (2.1 g, 6.2 mmol) was dissolved in dioxane (30 ml), and 4M HC1 in dioxane (30 ml) was added. The resulting solution was stirred at room temperature for 12 h until the deprotection was complete, and then evaporated in vacuum. In order to remove excessive HC1 the residue was subjected to coevaporation with acetonitrile (2 x 100). The obtained oil of the hydrochloride was dried at 0.5 torr / 40°C to give an amorphous solid. The product was used without further purification. LCMS (ES): 239 (MH+). 1H-NMR(400 MHz, DMSO-d6): 3.3-3.8(m, 6H ), 4.5(m, 1H), 4.6(q, 2H) , 7.15( m, 1H), 7.2(d, 1H), 7.25 (d, 1H), 7.4(q, 1H), 8.1 (tr, 1H), 9.0-9.6 (br d, 2H).
[000185]
Figure imgf000067_0002
[000186] Step 10. 4-acetyl-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one. To a solution of the crude 6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one hydrochloride (6.2 mmol, step 9) and triethylamine (2 ml, 15 mmol) in dry CH2CI2 (20 ml), a solution of acetyl chloride (0.6 g, 7.4 mmol) in dry CH2CI2 (5 ml) was added dropwise upon stirring and cooling in an ice bath. The reaction mixture was stirred overnight at room temperature, diluted with EtOAc (100 ml), washed with brine (2 x 30 ml), sat. aq. NaHC03 (2 x 30 ml), dried over Na2S04, filtered, and concentrated under a reduced pressure. The residue was purified by column chromatography on silica gel (CHC13 - MeOH, 20:1 to 10: 1) to afford the titled compound (oil). The yield: 1.5 g (85% from step 8). LCMS (ES): 281 (MH+). 1H-NMR (400 MHz, DMSO-d6): 2.1
(double s, 3H), 3.3(m, 2H), 3.5-3.7(m, 3H), 3.9(m, 2H), 4.6(m, 2H), 7.05-7.2(m, 3H), 2H).
Figure imgf000068_0001
[000187] Step 11 (HTC). The intermediate (Example 7, step 10) was dissolved in THF- DMF mixture (9: 1, 0.8 ml). A halide (1.2 - 1.3 eq) was added. Then a suspension of NaH (1.5 eq, preliminarily washed with hexane) in THF (0.3 ml) was added. The reaction mixture was stirred for 4 h at room temperature and neutralized with a solution of acetic acid in methanol to pH 7. After evaporation, the product was isolated by HPLC.
[000188] The following compounds of the invention were obtained by this method:
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methoxyethyl)-l,4-diazepan-2-one (MH+ 339);
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(4-methylbenzyl)-l,4-diazepan-2-one (MH+ 385); 4-acetyl-6-[(3-fluorobenzyl)oxy]-l-[(l-methyl-lH-imidazol-5-yl)methyl]-l,4- diazepan-2-one (MH+ 375);
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 -( 1 ,3 -thiazol-4-ylmethyl)- 1 ,4-diazepan-2-one (MH+ 378);
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one (MH+ 401);
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methylbenzyl)-l,4-diazepan-2-one (MH+ 385); 4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(3-methoxybenzyl)-l,4-diazepan-2-one (MH+
401);
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 -(3 -methylbenzyl)- 1 ,4-diazepan-2-one (MH+ 385); 4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(5 -methyl- 1 ,2,4-oxadiazol-3 -yl)methyl] -1,4- diazepan-2-one (MH+ 377);
4-acetyl- l-(cyclopropylmethyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one (MH+
335);
2- { 4-acetyl-6- [(3 -fluorobenzyl)oxy] -2-oxo- 1 ,4-diazepan- 1 -yl } -N,N- dimethylacetamide (MH+ 366) ;
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(3 -methylisoxazol-5-yl)methyl] - 1 ,4-diazepan-2- one (MH+ 376);
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 -(pyridin-2-ylmethyl)- 1 ,4-diazepan-2-one (MH+
372);
4-acetyl-l-(4-fluorobenzyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one (MH+ 389);
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(4-methoxypyridin-2-yl)methyl] - 1 ,4-diazepan-2- one (MH+ 402);
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(2-methyl- 1 ,3 -oxazol-4-yl)methyl] - 1 ,4-diazepan- 2-one (MH+ 376);
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-phenylethyl)-l,4-diazepan-2-one (MH+ 385); 4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 -(pyridin-4-ylmethyl)- 1 ,4-diazepan-2-one (MH+
372);
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 -( 1 ,3 -thiazol-2-ylmethyl)- 1 ,4-diazepan-2-one (MH+ 378);
4-acetyl-l-benzyl-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one (MH+ 371);
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methoxybenzyl)-l,4-diazepan-2-one (MH+
401);
4-acetyl-l-(cyclohexylmethyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one (MH+
377);
4-acetyl- 1 -(3 -fluorobenzyl)-6- [(3 -fluorobenzyl)oxy] - 1 ,4-diazepan-2-one (MH+ 389) ; 4-acetyl- 1 -(2-fluorobenzyl)-6- [(3 -fluorobenzyl)oxy] - 1 ,4-diazepan-2-one (MH+ 389) ; 4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 -(pyridin-3 -ylmethyl)- 1 ,4-diazepan-2-one (MH+
372);
4-acetyl- l-(cyclobutylmethyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one (MH+ 349); and
4-acetyl- l-[(3,5-dimethylisoxazol-4-yl)methyl]-6-[(3-fluorobenzyl)oxy]-l, 4- diazepan-2-one (MH+ 390).
Example 8: (R)-6-[(3-Fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one and (S)-6-[(3-Fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one
[000189] The pure enantiomers (R- and S- of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)) of the compound of Example 1 were obtained by the same methods with the exception of steps 1-2 that were analogous to those shown in Example 3. The spectral characteristics of the products are identical to those of the racemic compound. The chiral purity of the obtained products was tested by HPLC in 10% iPrOH/heptane in a Chiralcel OJ-H column. The purity of the obtained products was -98%.
Example 9: Identification of TGR5 agonists in a Pre-clinical Assay
[000190] A library of 20,000 compounds were submitted to an automated high-throughput primary screening assay performed on a CHO cell line expressing hTGR5 together with a lucif erase reporter that was driven by a cAMP response element (CRE-luc) (Figure la). All compounds were dissolved in DMSO and tested in 96 well plates at a single dose (lOuM), and a score was given to each potential hit, in accordance with its ability to activate the lucif erase reporter. The 621 compounds with the highest score were selected from this primary screening campaign. To assess the validity and specificity of the results, the screen was repeated and the 621 hits re-tested in CHO cells transfected with either an empty control vector or with hTGR5. Through this exercise, 313 of our primary hits were confirmed to be specific TGR5 agonists (Figure lb). Each of the 313 compounds was then examined in a dose-response study to determine its efficacy and EC50. Lithocholic acid (LCA), a well- established natural ligand of TGR5 (Maruyama et al., Biochem. Biophys. Res. Commun. 298, 714-719, 2002), was used as a positive control, and its efficacy was set at 100%. From these studies, 10 hits were selected on the basis of their efficacy (%Eff > 65%) and potency (EC50≤ 0.1 μΜ) (Table 1 and Figure lc). Importantly, these 10 hits were shown to be devoid of activity on the nuclear bile acid-activated receptor, farnesoid X receptor (FXR), as evaluated in Cos-7 cells transiently co-transfected with hFXR, mRXR and an FXR-RE-driven luciferase reporter (Houten et al., Mol Endocrinol, 21, 1312-1323, 2007) (data not shown).
Table 1. Potency and efficacy of selected compounds of the present invention with the 10 lead com ounds listed first in the table
Figure imgf000070_0001
Figure imgf000071_0001
l,4-diazepan-2-one c ra
Figure imgf000072_0001
Figure imgf000073_0001
l-butyl-6-((3- fluorobenzyl)oxy)-4-
388.43 0.05 62.5 (furan-2-carbonyl)-l,4- diazepan-2-one
(R)-4-(3-(lH-pyrazol-l- yl)propanoyl)-l- isobutyl-6-((3- 428.52 0.05 99.2 methoxybenzyl)oxy)- l,4-diazepan-2-one
4-acetyl-l- (cyclobutylmethyl)-6-
348.41 0.05 73 ((2-fluorobenzyl)oxy)- l,4-diazepan-2-one
4-((6-((3- fluorobenzyl)oxy)-4- (furan-2-carbonyl)-2- 447.46 0.06 87.9 oxo- 1 ,4-diazepan- 1 - yl)methyl)benzonitrile
6-((3-fluorobenzyl)oxy)- 4- (f uran-2-carbonyl)- 1 -
400.44 0.06 57.1 (3-methylbut-2-en-l-yl)- l,4-diazepan-2-one l-benzyl-6-((3- fluorobenzyl)oxy)-4-
422.45 0.06 61.5 (furan-2-carbonyl)-l,4- diazepan-2-one
6- [(2-fluorobenzy l)oxy] - %
4-(2-furoyl)-l-(4- ¾
468.54 0.06 56.6 methoxybenzyl) - 1 ,4- diazepan-2-one
l-benzyl-6-((3- fluorobenzyl)oxy)-4-
438.51 0.06 101.9 (thiophene-2-carbonyl) - l,4-diazepan-2-one
Figure imgf000075_0001
l-(3-fluorobenzyl)-6-((3- fluorobenzyl)oxy)-4-
440.44 0.07 62.4 (furan-2-carbonyl)-l,4- diazepan-2-one
6- [(2-fluorobenzy l)oxy] - N-isopropyl-4- (4- ft
methoxybenzyl)-3-oxo- 443.51 0.07 69.5
1,4-diazepane-l- "
carboxamide
4-acetyl-l- «
(cyclohexy lmethyl) -6- "A* J. )
f < 376.47 0.08 76.1 [(3-fluorobenzyl)oxyl- l,4-diazepan-2-one
6-((2-fluorobenzyl)oxy)- l-(4-methoxybenzyl)-4-
468.54 0.08 74.3 (thiophene-3-carbonyl)- l,4-diazepan-2-one
4-acetyl-6-((2- fluorobenzyl)oxy)-l-(3-
398.47 0.08 72.7 phenylpropyl)-l,4- diazepan-2-one
6- [(2-fluorobenzy l)oxy] - . i! V *
4-(2-furoyl)-l-(3- r
88.2 methy lbenzy 1) - 1 ,4 - VV_/ 436.48 0.08
diazepan-2-one
6-((3-fluorobenzyl)oxy)- 4- (f uran-2-carbonyl)- 1 -
450.5 0.08 97.8 (3-phenylpropyl)-l,4- diazepan-2-one
Figure imgf000077_0001
Example 10: Metabolic Stability
[000191] 10 compounds obtained in Example 9 were tested for metabolic stability using human microsomes (Figure Id). After calculation of the half-life (T1/2) and of the intrinsic clearance (CLint), the 10 hits were ranked in relation to their stability (Table 2), efficacy, EC50 and intrinsic clearance. 3 compounds, (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3- fluorobenzyl)oxy]-l,4-diazepan-2-one), (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) and 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l- (4-methoxybenzyl)-l,4-diazepan-2-one), were prepared for more detailed study (Figure le). These 3 compounds also showed good stability in a murine microsomal assay (Figure If and Table 3).
Table 2. Human microsomal stability of the 10 best compounds based on their intrinsic clearance and their half-life
Figure imgf000078_0001
Table 3: Mouse microsomal stability of the 3 best compounds based on their intrinsic clearance and their half-life
Figure imgf000079_0001
Example 11: In Vitro Anti-inflammatory Activity
[000192] To assess the anti-inflammatory activity of the compounds, a mouse macrophage cell line (RAW 264.7) was treated with LPS, in the presence or absence of the three lead TGR5 agonists at a concentration of 30uM. These compounds' activity was compared to that of INT-777 as a benchmark positive control. In this experimental setting, LPS stimulation induced the expression of the mRNAs of multiple inflammatory cytokines, including IL6, MCP-1 and TNFa. Two of the selected compounds, (4-acetyl- l-[2-(dimethylamino)ethyl] -6- [(3-fluorobenzyl)oxy]-l,4-diazepan-2-one) and (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one), were able to reduce the LPS-mediated increase in the mRNA levels of all three of these cytokines (Figure 2a). INT-777 and 4- (cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one), however, were unable to reduce TNFa mRNA levels under these conditions but were able to reduce the LPS-mediated increase of IL6 and MCP-1 mRNA (Figure 2a).
Example 12: In Vivo Anti-inflammatory and Anti-atherogenic Activity of (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one)
[000193] The in vivo anti-inflammatory and anti-atherogenic activity of (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) was studied in atherosclerosis-susceptible LDLr- null (LDLr ~'~) mice fed for 13 weeks with a high- cholesterol atherogenic diet (HCD) with or without the administration of (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one). Body weight (Figure 6a) and fat mass (Figure 6b) were significantly reduced by (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one), while lean mass (Figure 6b) and food intake (Figure 6c) were unaffected. Importantly, (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) showed no evidence of hepatotoxicity, as demonstrated by the normal ASAT levels in the plasma of LDLr i- mice fed for 13 weeks with HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) (Figure 6c). The key studies were plaque analyses of the thoraco-abdominal aorta, which demonstrated that LDLf'~ mice fed for 13 weeks with HCD+((6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) developed significantly fewer and smaller atherosclerotic plaques than did control mice fed HCD alone (Figures 6d and 6e). This decrease in plaque formation was paralleled by a time-dependent reduction in total plasma cholesterol and triglyceride content in HCD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)-fed LDLf'~ mice (Figure 6f). It was noted that LDL- cholesterol was also significantly reduced by chronic (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)- l-(3-methylbenzyl)-l,4-diazepan-2-one) administration (HCD 22.87±0.9621 vs. HCD+((6- [(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) 19.02±1.206, p=0.02), while HDL-cholesterol was unchanged (HCD 8.681±0.3025 vs. HCD+((6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) 8.549±0.3142, p=0.7656). In addition, chronic (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) administration reduced the pro-inflammatory action of the HCD, as demonstrated by the very substantial reduction of Mcpl and 116 mRNA expression in the aorta and of ΙΡΝγ and KC/GRO in the plasma (Figure 6f). These data suggest that the antiatherogenic effect of the TGR5 agonist (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) is due not only to its anti-inflammatory properties, but also to its rather profound effects on plasma lipid concentrations, effects that are not seen with INT-777 a semi- synthetic bile acid with TGR5 agonist activity (Pols et al., Cell metabolism, 4, 747-757, 2011).
Example 13: Effects on Metabolic Parameters both in vitro and, by oral administration, in vivo
[000194] In addition to the anti-inflammatory effect, activation of TGR5 regulates glucose homeostasis by promoting the secretion of the insulinotropic incretin GLP-1 from enteroendocrine L-cells (Thomas et al., cell metabolism 10, 161 -111, 2009). Both the mouse enteroendocrine-L cell line, GLUTag, and the human enteroendocrine cell line, NCI-H716, were used to evaluate the activity of the selected compounds on GLP-1 release. After lh of exposure, all three compounds were able to induce GLP-1 secretion in both mouse and human systems (Figures 2b and 2c). [000195] The ability of these compounds to stimulate GLP-1 secretion in vivo was tested. For this purpose, diet-induced obese C57BL/6J mice were challenged with an oral test meal, and their plasma was assayed for GLP-1 secretion. Mice were either given saline, INT-777, (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one), 4- (cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one) or (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) at a concentration of 30mg/kg of body weight, 30 minutes prior to the administration of a test meal. Pre-administration of (4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]- l,4-diazepan-2-one) or 4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4- methoxybenzyl)-l,4-diazepan-2-one) did not enhance GLP-1 release, while INT-777 and (6- [(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) significantly stimulated both GLP-1 and insulin secretion (Figure 2c). These data indicated that compound (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) did stimulate GLP-1 release in vivo.
[000196] To examine the long-term effects of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) administration on metabolism, (6-[(3-fluorobenzyl)oxy]- 4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) was admixed with the food and studied in mice that were fed a high fat diet (HFD). Body weight (Figure 3a) and fat mass (Figure 3b) were significantly reduced by (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) administration (compare to control mice given HFD alone), while lean mass (Figure 3b) and water and food intake (Figure 3c) were comparable between the HFD+(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) and the HFD groups. In indirect calorimetry studies, HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l- (3-methylbenzyl)-l,4-diazepan-2-one)-treated mice displayed increased oxygen consumption, and this increase was not due to any difference in locomotor activity (Figure 3d).
[000197] The (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one) -dependent decrease in body weight and increase in energy expenditure is best explained by an effect on brown adipose tissue (BAT). Indeed, it was reported that TGR5 activation in vivo is associated with a stimulation of BAT function (Watanabe et al., 2006). Consistent with an effect on brown adipose tissue, HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)-treated mice exposed to cold temperature (6°C) maintained their core body temperature better than did control mice fed HFD alone (Figure 3e). Furthermore, (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one) led to higher mRNA levels of Ucp-1, Dio-2 and Cpt-1, genes involved in fatty acid metabolism and energy expenditure in brown adipose tissue (Figure 3f). Importantly, the effect of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) on GLP-1 secretion was not merely short-lived, as shown by the lasting effect of chronic administration of the compound to HFD-fed C57BL/6J male mice (Figure 3g). This increase in GLP-1 release was paralleled by the improved glucose clearance seen with (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) in an oral glucose tolerance test (OGTT) (Figure 3h).
Example 14: TGR5 Specificity of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)
[000198] In order to ascertain whether (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) was working through the selective activation of TGR5 rather than by the activation and/or inhibition of other GPCRs, the activity of this compound in vitro was tested on a comprehensive panel of GPCRs both in the agonist (Ago) and antagonist (Antago) mode. In this study, a reference agonist for each GPCR (used at the concentration where it elicited a maximal response) was included and the ability of (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) to further activate or inhibit the receptor was tested (Table 4). (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) was highly selective for TGR5 and did not activate or inhibit any of the other 166 receptors in the panel. These studies are furthermore aligned with the in vivo studies (discussed later - Figure 5) that also showed the strict dependence of the in vivo effects of our compounds on the presence of TGR5.
Table 4. Human GPCR profiling (Full Panel) with (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)- l-(3-methylbenzyl)-l,4-diazepan-2-one)
Figure imgf000083_0001
Tab e 4 legend: Results are expressed as a percentage of the activity of the reference agonist or antagonist for each receptor. Note that there was significant activity only at TGR5, where the reference agonist was INT-777. "Ago" - agonist; "Antago" - antagonist.
Example 15: In Vivo Effects of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one) Show Specificity for the TGR5 Receptor
[000199] To affirm that the biological effects of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) were due to the activation of TGR5 in vivo, the effect of this compound was tested in 7gr5-null (Tgr5~'~) knock-out mice mice that were fed either HFD or HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one) (Figures 5a-5h). Unlike in normal mice, (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) had no effect on body weight (Figure 5a), body composition (Figure 5b), food and water intake (Figure 5c), energy expenditure (Figure 5d), cold exposure (Figure 5e), thermogenic gene expression (Figure 5f), GLP-1 secretion (Figure 5g) or glucose tolerance (Figure 5h). These data demonstrate unequivocally that the metabolic effects of (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan- 2-one) in the C57BL/6J mice (Figures 2 and 3) are due to its activity as an agonist at the TGR5 receptor.
Example 16: Diminished Adverse Effects
[000200] Clinical effectiveness of the TGR5 agonists of the present invention is not only dependent on good efficacy and potency of the compounds, but also requires a lack of serious adverse effects. Adverse effects on cardiac and gallbladder function have been reported for most known TGR5 agonists, for instance, 13.4-benzofuranyloxynicotinamide derivatives (Zou et al., Eur. J. Med. Chem. , 82, 1-15, 2014) and both 4-phenoxynicotinamide and 4- phenoxypyrimidine-5-carboxamide derivatives (Duan et al., J. Med. Chem., 55, 10475- 10489, 2012). Thus, the possible side effects of chronic administration of (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) in vivo were assessed. Cardiac function and morphology were not influenced by chronic (6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) administration. Indeed, echocardiography showed normal heart morphology and function, as evidenced by absence of changes in posterior wall thickness (PVT), septum thickness (ST), left ventricular (LV) fractional shortening (LVFS), and LV ejection fraction (LVEF) (Figure 4a). Furthermore, there was no difference between (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)-treated and control mice in heart rate (HR) and noninvasive systolic (SBP) and diastolic blood pressure measurements in basal (non- anaesthetized) mice (Figure 4a and data not shown). [000201] Gallbladder volume and morphology also were normal. In fact, both in vivo echographic and post-mortem gross morphological and histological analyses (Figure 4b and 4c) of the gallbladder failed to show an effect of chronic (6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) dosing. Total plasma bile acid (BA) content was comparable between HFD and HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)-fed mice (Figure 4d), demonstrating that chronic TGR5 activation by (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) did not impact gallbladder function. The lack of hepatotoxicity or renal toxicity after chronic exposure to (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) was shown by normal plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatinine (Table 5). In fact, (6- [(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one)-treated mice were protected from the negative impact of HFD on liver function (Table 5), suggesting a hepatoprotective effect against the development of a fatty liver. Serum amylase and creatine kinase levels were normal, indicating that (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one) also had no adverse effect on exocrine pancreatic function, heart muscle or skeletal muscle (Table 5). Furthermore, the compound had no adverse impact on serum triglycerides or total-, HDL- or LDL-cholesterol levels (Table 5) and, in fact, seemed to slightly decrease the levels of LDL-cholesterol.
Table 5. Plasma analyte levels in 10 male C57BL/6J mice after 13 weeks on a high fat diet (HFD) or an HFD supplemented with 30 mg/kg/d of (6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one).
Figure imgf000085_0001
Mice were fasted overnight. *p<0.05 HFD+((6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l- (3-methylbenzyl)-l,4-diazepan-2-one) vs. HFD alone. Example 17: Selective activation of GLP-1 secretion
[000202] As (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one) is a random mixture of (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l,4-diazepan-2-one)) and (<55)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one)) enantiomers, the two enantionmers' respective activities in vitro were analyzed. First, the ability of the two enantiomers, (<5R)-(6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) and (<55)-(6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one),) to reduce the LPS-dependent increase of inflammatory cytokine expression in the mouse macrophage cell line RAW 264.7 was tested. Both enantiomers, after 4 hours' treatment at a concentration of 30uM, reduced the mRNA level of TNFoc, IL6 and MCP1 similarly to the positive control INT-777 (Figure 7a). Then, the effects of the two enantiomers on GLP-1 secretion in the entero-endocrine GLUTag cell line were investigated. After 1 hour of treatment, only INT-777 and the (<5R)-(6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one)) enantiomer significantly increased GLP-1 secretion (Figure 7b). A dose-response study of the active enantiomer (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2- one)) and INT-777 was then performed. Both (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l- (3-methylbenzyl)-l,4-diazepan-2-one)) and INT-777 showed a significant dose-dependent increase of GLP-1 secretion, but (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)) was about three-fold more potent than was INT-777 (Figure 7c). These data suggest that only the R enantiomer of (6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one) is active on GLP-1 secretion.
[000203] To corroborate these data, an in vivo GLP-1 secretion assay was conducted by challenging diet-induced obese C57BL/6J mice with an oral test meal, 30 minutes after the administration of saline, (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one) or (65)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4- diazepan-2-one) at a concentration of 30mg/kg of body weight. Pre-administration of (65)- (6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one)) did not increase GLP-1 release, while (6R)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3- methylbenzyl)-l,4-diazepan-2-one)) significantly enhanced GLP-1 secretion (Figures 7c and 7d). These data suggest structural and/or conformational heterogeneity of the TGR5 receptor and that this heterogeneity can be exploited to improve the pharmacologic specificity of compounds active at TGR5. [000204] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A compound of formula (I)
Figure imgf000088_0001
(I)
wherein
Ri is aryl, -C]¾CH2-aryl, C3-8 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, or -CH2NRaRb, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, O-Ci-6 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro;
R2 is Ci-6 alkyl, O-Ci-6 alkyl, -NRaRb, C3-8 cycloalkyl, 2-furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono- or polysubstituted with substituents selected from Ci-6 alkyl, halo, C1-3 haloalkyl, OH, and O-Ci-6 alkyl, cyano, amino, C1-3 alkylamino, di- C1-3 alkylamino, and nitro;
R3 is F, CI, Br, I, or 0-Ci_6 alkyl;
n is 1-5; and
Ra and Rb each are independently Ci_6 alkyl.
2. The compound of claim 1, wherein R! is aryl, optionally mono- or polysubstituted with substituents selected from Ci_5 alkyl, halo, Ci_3 haloalkyl, OH, 0-Ci_6 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro.
3. The compound of claim 1, wherein R! is phenyl, optionally mono- or polysubstituted with substituents selected from Ci-5 alkyl, halo, C1-3 haloalkyl, OH, O-C1-3 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro.
4. The compound of claim 1, wherein Ri is phenyl, optionally mono-substituted with methyl, methoxy, halo, or cyano.
5. The compound of claim 1, wherein Ri is phenyl, optionally mono-substituted with methyl, methoxy, flouro, chloro, or cyano.
6. The compound of claim 1, wherein Ri is C3-8 cycloalkyl.
7. The compound of claim 1, wherein Ri is cyclobutyl.
8. The compound of claim 1, wherein Ri is -CH2NRaRt>.
9. The compound of claim 1, wherein Ri is -0¾Ν(0¾)2.
10. The compound of claim 1, wherein Ri is CH2CH2-aryl, optionally mono- or polysubstituted with substituents selected from Ci_5 alkyl, halo, Ci_3 haloalkyl, OH, 0-Ci_3 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro.
11. The compound of claim 1, wherein R! is CH2CH2-phenyl, optionally mono- substituted with methyl, methoxy, flouro, chloro, or cyano.
12. The compound of claim 1, wherein said compound is represented by formula (II)
Figure imgf000089_0001
(Π) wherein R4 is F, CI, Br, I, 0-Ci_5 alkyl, Ci_5 alkyl, or cyano; and m is 1-5.
13. The compound of claim 1 , wherein the compound is represented by formula (III)
Figure imgf000089_0002
(III).
14. The compound of claim 1 , wherein the compound is represented by formula (IV)
Figure imgf000090_0001
(IV).
15. The compound of claim 1, wherein R2 is O-C1-5 alkyl, -NRaRb, C3-8 cycloalkyl, 2- furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono- or polysubstituted with substituents selected from Ci_6 alkyl, halo, C1-3 haloalkyl, OH, and 0-Ci_ 3 alkyl, cyano, amino, C1-3 alkylamino, di-Ci-3 alkylamino, and nitro.
16. The compound of claim 1, wherein R2 is O-C1-5 alkyl or -NRaRt>.
17. The compound of claim 1 , wherein R2 is OCH2CH2CH3 or -NCH(CH3)2.
18. The compound of claim 1, R2 is C3-8 cycloalkyl.
19. The compound of claim 1, wherein R2 is 2-furanyl, thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl, each optionally mono- substituted with methyl, methoxy, flouro, chloro, or cyano.
20. The compound of claim 1, wherein R2 is thienyl, thiazolyl, pyrazoly, pyrrolyl, or isoxazolyl.
21. The compound of claim 1 , wherein R2 is thienyl.
22. The compound of claim 1, wherein R2 is 2-furanyl.
23. The compound of claim 1, wherein R3 is F, CI, Br, or I.
24. The compound of claim 1, wherein R3 is F.
25. The compound of claim 1, wherein said compound is represented by formula (V)
Figure imgf000090_0002
26. The compound of claim 1 , wherein said compound is represented by formula (VI)
Figure imgf000091_0001
(VI).
27. The compound of claim 1, wherein said compound is represented by formula (VII)
Figure imgf000091_0002
(VII).
28. The compound of claim 1, wherein R3 is F or CI.
29. The compound of claim 1, wherein R3 is 2-F or 3-F.
30. The compound of claim 1, wherein n is 1.
31. The compound of claim 1 , wherein the compound is
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-N-isopropyl-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l- carboxamide;
6- [(2-fluorobenzyl)oxy] -4-glycoloyl- 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one (MH+ 416);
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(tetrahydrofuran-2-ylcarbonyl)-l,4- diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(lH-pyrrol-2-ylcarbonyl)-l,4-diazepan-2- one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4- [(5 -methyl- lH-pyrazol-3 -yl)carbonyl] -1,4- diazepan-2-one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4- [(5 -methylisoxazol-3 -yl)carbonyl] -1,4- diazepan-2-one; 6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(2-methyl-l,3-thiazol-4-yl)carbonyl]-l,4- diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(isoxazol-5-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2- one;
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4- [(5 -methyl- 1 ,3-oxazol-4-yl)carbonyl] -1,4- diazepan-2-one;
propyl 6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxylate;
4-butyryl-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)-4-( 1 H-pyrazol-3 -ylcarbonyl)- 1 ,4-diazepan-2- one;
6-[(2-fluorobenzyl)oxy]-4-(lH-imidazol-4-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan- 2-one;
N-ethyl-6-[(2-fluorobenzyl)oxy]-4-(4-methoxybenzyl)-3-oxo-l,4-diazepane-l-carboxamide;
6-[(2-fluorobenzyl)oxy]-4-(isoxazol-3-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2- one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(lH-imidazol-2-ylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan- 2-one;
6-[(2-fluorobenzyl)oxy]-4-isobutyryl-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-propionyl-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy]-4-(methoxyacetyl)- 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one;
4-acetyl-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-(cyclobutylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-[(l-methyl-lH-pyrazol-3-yl)carbonyl]-l,4- diazepan-2-one;
6- [(2-fluorobenzyl)oxy]-4- [2-furyl(oxo)acetyl]- 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(l,3-thiazol-4-ylcarbonyl)-l,4-diazepan-2- one; or 6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-4-(pyridin-2-ylcarbonyl)-l,4-diazepan-2-one.
32. The compound of claim 1, wherein the compound is
4-({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzonitrile ;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(4-methoxybenzyl)oxy]-l,4-diazepan-2- one;
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-3-ylmethoxy)-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-6-[(4-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-2-ylmethoxy)-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-6-[(3-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(2-methoxybenzyl)oxy]-l,4-diazepan-2- one;
6-[(2-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzoic acid ;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-(pyridin-4-ylmethoxy)-l,4-diazepan-2-one;
2- ({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzonitrile ;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(4-methylbenzyl)oxy]-l,4-diazepan-2-one;
6-(benzyloxy)-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
6-[(4-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
3- ({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzonitrile ;
4- (cyclopropylcarbonyl)-6-[(3,5-dimethylisoxazol-4-yl)methoxy]-l-(4-methoxybenzyl)-l,4- diazepan-2-one;
3- ({ [4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-2-oxo-l,4-diazepan-6- yl] oxy } methyl)benzoic acid ;
4- (cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(2-methylbenzyl)oxy]-l,4-diazepan-2-one; 4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(3-methylbenzyl)oxy]-l,4-diazepan-2-one;
4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-6-[(3-methoxybenzyl)oxy]-l,4-diazepan-2- one; or
6-[(3-chlorobenzyl)oxy]-4-(cyclopropylcarbonyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one.
33. The compound of claim 1, wherein the compound is
4-{ [6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l yl] methyl }benzonitrile; l-benzyl-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
l-(2-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-phenylethyl)-l,4-diazepan-2-one;
l-butyl-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-2-ylmethyl)-l,4-diazepan-2-one;
l-(cyclobutylmethyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(l,3-thiazol-4-ylmethyl)-l,4-diazepan-2-one;
l-(4-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6- [(2-fluorobenzyl)oxy] -4-(2-furoyl)- 1 - [2-( 1 H-pyrazol- 1 -yl)ethyl] - 1 ,4-diazepan-2-one ; l-(cyclopropylmethyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
l-[(3,5-dimethylisoxazol-4-yl)methyl]-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2- one;
l-(2-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(2-methyl-l,3-oxazol-4-yl)methyl]-l,4-diazepan-2- one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-phenylpropyl)-l,4-diazepan-2-one;
l-(4-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
2-{ [6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile; 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbut-2-en-l-yl)-l,4-diazepan-2-one;
3-{ [6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-2-oxo-l,4-diazepan-l-yl]methyl}benzonitrile; 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methoxybenzyl)-l,4-diazepan-2-one; 6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(4-methylbenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(2-methyl-l,3-thiazol-4-yl)methyl]-l,4-diazepan-2- one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-[(3-methylisoxazol-5-yl)methyl]-l,4-diazepan-2-one; l-(3-chlorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-4-ylmethyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(pyridin-3-ylmethyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-methylbenzyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methoxybenzyl)-l,4-diazepan-2-one;
1- (3-fluorobenzyl)-6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l,4-diazepan-2-one;
6-[(2-fluorobenzyl)oxy]-4-(2-furoyl)-l-(2-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]- l-(2-methoxyethyl)- 1 ,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(4-methylbenzyl)-l,4-diazepan-2-one;
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [( 1 -methyl- lH-imidazol-5 -yl)methyl] - 1 ,4-diazepan-2- one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(l,3-thiazol-4-ylmethyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methylbenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(3-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(3-methylbenzyl)-l,4-diazepan-2-one;
4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(5 -methyl- 1 ,2,4-oxadiazol-3 -yl)methyl] - 1 ,4-diazepan-2- one;
4-acetyl-l-(cyclopropylmethyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
2- {4-acetyl-6- [(3-fluorobenzyl)oxy]-2-oxo- 1 ,4-diazepan- 1 -yl } -N,N-dimethylacetamide; 4-acetyl-6- [(3 -fluorobenzyl)oxy] - 1 - [(3 -methylisoxazol-5 -yl)methyl] - 1 ,4-diazepan-2-one; 4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(pyridin-2-ylmethyl)-l,4-diazepan-2-one;
4-acetyl-l-(4-fluorobenzyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-[(4-methoxypyridin-2-yl)methyl]-l,4-diazepan-2-one; 4-acetyl-6-[(3-fluorobenzyl)oxy]-l-[(2-methyl-l,3-oxazol-4-yl)methyl]-l,4-diazepan-2-one; 4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-phenylethyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(pyridin-4-ylmethyl)-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(l,3-thiazol-2-ylmethyl)-l,4-diazepan-2-one; 4-acetyl- 1 -benzyl-6- [(3 -fluorobenzyl)oxy] - 1 ,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(2-methoxybenzyl)-l,4-diazepan-2-one;
4-acetyl-l-(cyclohexylmethyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl-l-(3-fluorobenzyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl-l-(2-fluorobenzyl)-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one;
4-acetyl-6-[(3-fluorobenzyl)oxy]-l-(pyridin-3-ylmethyl)-l,4-diazepan-2-one;
4-acetyl- 1 -(cyclobutylmethyl)-6- [(3 -fluorobenzyl)oxy] - 1 ,4-diazepan-2-one; or
4-acetyl- 1 - [(3 ,5 -dimethylisoxazol-4-yl)methyl] -6- [(3-fluorobenzyl)oxy] - 1 ,4-diazepan-2-one.
34. The compound of claim 1, wherein the compound is 6-[(3-fluorobenzyl)oxy]-4-(2- furoyl)- 1 -(3-methylbenzyl)- 1 ,4-diazepan-2-one
Figure imgf000096_0001
35. The compound of claim 1, wherein the compound is 4-(cyclopropylcarbonyl)-6-[(2- fluorobenzyl)oxy] - 1 -(4-methoxybenzyl)- 1 ,4-diazepan-2-one
Figure imgf000096_0002
36. The compound of claim 1, wherein the compound is 4-acetyl- 1- [2- (dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
Figure imgf000097_0001
37. The compound of any one of claims 1-36, wherein the compound is its isomer, pharmaceutically acceptable salt, or tautomer, or any combinations thereof.
38. The compound of claim 37, wherein said compound is
(6R)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
39. The compound of claim 37, wherein said compound is
(65)-(6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one).
40. A pharmaceutical composition comprising a compound of any one of claims 1-39 and at least one pharmaceutically acceptable carrier.
41. The pharmaceutical composition of claim 40, wherein said compound is (6R)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
42. The pharmaceutical composition of claim 40, wherein said compound is (65)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
43. A method of treating, preventing, suppressing, reducing the severity of, or reducing the risk of a metabolic disease or disorder in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of any one of claims 1-39..
44. The method of claim 43, wherein said metabolic disease comprises diabetes mellitus, obesity, dyslipidemia, insulin resistance, or insulin sensitivity, or any combination thereof.
45. The method of claim 43, wherein said compound has diminished adverse effects on cardiac and gallbladder function.
46. The method of claim 43, wherein said compound selectively increases GLP-1 secretion without producing an anti-inflammatory effect.
47. The method of claim 43, wherein said compound comprises
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one
Figure imgf000098_0001
4-(cyclopropylcarbonyl)-6-[(2-fluorobenz l)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one
Figure imgf000098_0002
4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
Figure imgf000098_0003
48. The method of claim 43, wherein said compound comprises an R- enantiomer of the compound.
49. The method of claim 43, wherein said compound comprises an S- enantiomer of the compound.
50. The method of claim 48, wherein said R- enantiomer comprises (6R)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
51. A method of treating, preventing, suppressing, reducing the severity of, or reducing the risk of an inflammatory disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of any one of claims 1-39.
52. The method of claim 51, wherein said inflammatory disease comprises rheumatoid arthritis, systemic lupus erythematosus, scleroderma, atherosclerosis, coronary artery disease, stroke, allergy, osteoarthritis, appendicitis, bronchial asthma, pancreatitis, or psoriasis, or any combination thereof.
53. The method of claim 51, wherein said compound has diminished adverse effects on cardiac and gallbladder function.
54. The method of claim 51, wherein said compound comprises
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one
Figure imgf000099_0001
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one
Figure imgf000099_0002
4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
Figure imgf000100_0001
55. The method of claim 51, wherein said compound comprises an R- enantiomer of said compound.
56. The method of claim 51, wherein said compound comprises an S- enantiomer of said compound.
57. The method of claim 56, wherein said S- enantiomer comprises (65)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)- l-(3-methylbenzyl)-l ,4-diazepan-2-one.
58. The method of claim 57, wherein said compound is selective and wherein GLP- 1 is not increased.
59. The method of claim 51, wherein said compound decreases expression or elaboration of pro-inflammatory cytokines.
60. A method of treating, preventing, suppressing, reducing the severity of, or reducing the risk of atherosclerosis in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of any one of claims 1-39.
61. The method of claim 60, wherein said compound has diminished adverse effects on cardiac and gallbladder function.
62. The method of claim 60, wherein said compound comprises
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)- l ,4-diazepan-2-one
Figure imgf000100_0002
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one
Figure imgf000101_0001
4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
Figure imgf000101_0002
63. The method of claim 60, wherein said compound comprises an R- enantiomer of the compound.
64. The method of claim 60, wherein said compound comprises S- enantiomer of the compound.
65. The method of claim 64, wherein said S- enantiomer comprises (65)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
66. The method of claim 60, wherein said compound is selective and wherein GLP-1 is not increased.
67. The method of claim 60, wherein said compound decreases expression or elaboration of pro-inlfammatory cytokines.
68. A method of reducing plasma triglyceride and LDL-cholesterol levels in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a compound of any one of claims 1-39.
69. The method of claim 68, wherein said compound has diminished adverse effects on cardiac and gallbladder function.
70. The method of claim 68, wherein said compound comprises
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one
Figure imgf000102_0001
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one
Figure imgf000102_0002
4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l ,4-diazepan-2-one
Figure imgf000102_0003
71. The method of claim 68, wherein said compound comprises an R- enantiomer of said compound.
72. The method of claim 68, wherein said compound comprises an S- enantiomer of said compound.
73. The method of claim 72, wherein said S- enantiomer comprises (65)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
74. A method for inducing increased GLP-1 secretion in a cell, the method comprising contacting the cell with effective amount of a compound of any one of claims 1-39.
75. The method of claim 74, wherein said inducing is in vivo.
76. The method of claim 75, wherein said compound has diminished adverse effects on cardiac and gallbladder function.
77. The method of claim 74, wherein said compound comprises
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one
Figure imgf000103_0001
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one
Figure imgf000103_0002
4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
Figure imgf000104_0001
78. The method of claim 74, wherein said compound comprises an R- enantiomer of the compound.
79. The method of claim 78, wherein said R-enantiomer comprises (6R)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
80. A method for decreasing expression or elaboration of pro-inflammatory cytokines in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of claims 1-39.
81. The method of claim 80, wherein said decreasing expression or elaboration is in vivo.
82. The method of claim 81, wherein said compound has diminished adverse effects on cardiac and gallbladder function.
83. The method of claim 80, wherein said compound comprises
6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one
Figure imgf000104_0002
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]-l-(4-methoxybenzyl)-l,4-diazepan-2-one
Figure imgf000105_0001
4-acetyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l,4-diazepan-2-one
Figure imgf000105_0002
84. The method of claim 80, wherein said compound does not increase secretion of GLP- 1.
85. The method of claim 80, wherein said compound comprises an R- enantiomer of the compound.
86. The method of claim 80, wherein said compound comprises an S- enantiomer of the compound.
87. The method of claim 86, wherein said S- enantiomer comprises
(65)-6-[(3-fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
88. A method of binding a compound of any one of claims 1-39 to a G-protein coupled bile acid receptor 1 (TGR5 receptor), the method comprising contacting the TGR5 receptor with the compound under conditions effective to bind the compound to the TGR5 receptor.
89. The method of claim 88, wherein said compound comprisies 6-[(3-fluorobenzyl)oxy]- 4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one
Figure imgf000106_0001
4-(cyclopropylcarbonyl)-6-[(2-fluorobenzyl)oxy]- l-(4-methoxybenzyl)-l ,4-diazepan-2-
Figure imgf000106_0002
:etyl-l-[2-(dimethylamino)ethyl]-6-[(3-fluorobenzyl)oxy]-l ,4-diazepan-2-
Figure imgf000106_0003
90. The method of claim 88, wherein said compound comprises an R- enantiomer of the compound.
91. The method of claim 90, wherein said R- enantiomer comprises (6R)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)- l-(3-methylbenzyl)-l ,4-diazepan-2-one.
92. The method of claim 88, wherein said compound comprises an S- enantiomer of the compound.
93. The method of claim 92, wherein said S- enantiomer comprises (65)-6-[(3- fluorobenzyl)oxy]-4-(2-furoyl)-l-(3-methylbenzyl)-l,4-diazepan-2-one.
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