Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/56—Ring systems containing three or more rings
  • C07D209/80—[b, c]- or [b, d]-condensed
  • C07D209/94—[b, c]- or [b, d]-condensed containing carbocyclic rings other than six-membered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/06—Anabolic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
  • A61P5/26—Androgens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

• Endogenous steroidal androgens e.g. testosterone and 5 ⁇ -dihydrotestosterone (DHT) exert profound influences on a multitude of physiological functions.
• steroidal androgen therapy has limitations.
• unwanted side effects of steroidal androgen therapy include growth stimulation of the prostate and seminal vesicles.
• PSA prostate specific antigen
• preparations of steroidal androgens have been found to suffer from rapid degradation in the liver leading to poor oral bioavailability and short duration of activity following parenteral administration, variations in plasma levels, hepatotoxicity, or cross reactivity with other steroid hormone receptors (e.g. the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the progesterone receptor (PR)).
• GR glucocorticoid receptor
• MR mineralocorticoid receptor
• PR progesterone receptor
• the use of steroidal androgens may lead to hirsutism or virilization.
• tissue selective androgen receptor modulators which display androgen agonist activity in muscle or bone, but only partial agonist, partial antagonist or antagonist activity in androgenic tissues such as the prostate or seminal vesicle.
• the present invention is directed to certain tetrahydrocyclopenta[b] indole compounds, as defined by Formula (I) below, which have particular profiles of activity in in vitro and in vivo testing which suggest they are useful in the treatment of disorders responsive to steroidal androgen therapy. Accordingly, the present invention provides a compound of Formula (I):
• R represents a substitutent selected from the group consisting of
• the present invention provides a method of treating or preventing hypogonadism, reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength, male or female sexual dysfunction, erectile dysfunction, or reduced libido comprising administereing to a patient in need thereof an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
• the present invention provides a method for treating or preventing reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength, or erectile dysfunction.
• the present invention provides a method for treating or preventing reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength induced by immobilization, disuse or trauma, or erectile dysfunction.
• the present invention provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of hypogonadism, reduced bone mass or density, osteoporosis, osteopenia, reduced muscle -A-
• the invention provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength, or erectile dysfunction.
• the present invention provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength induced by immobilization, disuse or trauma, or erectile dysfunction.
• the present invention provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.
• the present invention provides the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of hypogonadism, reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength, male or female sexual dysfunction, erectile dysfunction, or reduced libido. More particularly, the present invention provides the use of a compound of Formula (I) for the manufacture of a medicament for the treatment or prevention of reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength, or erectile dysfunction.
• the present invention provides the use of a compound of Formula (I) for the manufacture of a medicament for the treatment or prevention of reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength induced by immobilization, disuse or trauma, or erectile dysfunction.
• the present invention provides a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with one of more pharmaceutically acceptable carriers, diluents, or excipients. More particularly, the present invention provides a pharmaceutical composition for the treatment of reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength, or erectile dysfunction comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with with one of more pharmaceutically acceptable carriers, diluents or excipients. Even more particularly, the present invention provides a pharmaceutical composition for the treatment of reduced bone mass or density, osteoporosis, osteopenia, reduced muscle mass or strength induced by immobilization, disuse or trauma, or erectile dysfunction.
• the present invention also encompasses novel intermediates and processes useful for the synthesis of a compound of Formula (I).
• the present invention also relates to pharmaceutically acceptable salts of the compound of Formula (I). Examples of pharmaceutically acceptable salts and methods for their preparation are well within the knowledge of those skilled in the art.
• the present invention also relates to solvates of the compound of Formula (I) or the pharmaceutically acceptable salts of compounds of Formula (I). As such, when used herein the term "Formula (I)", or any particular compound of Formula (I), includes within its meaning any solvate of the compound.
• the compounds of Formula (I) may have one or more chiral centers and, therefore, may exist in particular stereoisomeric configurations.
• R and “S” are used herein as commonly used in organic chemistry to denote specific configurations of a chiral center.
• the terms “(+)” or “RS” refer to a configuration of a chiral center comprising a racemate.
• stereoisomers and enantiomers of compounds of Formula I can be prepared by one of ordinary skill in the art utilizing well known techniques and processes, such as those disclosed by Eliel and Wilen, "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., 1994, Chapter 7; Separation of Stereoisomers, Resolution, Racemization; and by Collet and Wilen, "Enantiomers, Racemates, and Resolutions", John Wiley & Sons, Inc., 1981.
• specific stereoisomers and enantiomers can be prepared by stereospecific syntheses using enantiomerically and geometrically pure, or enantiomerically or geometrically enriched starting materials.
• stereoisomers and enantiomers can be resolved and recovered by techniques such as chromatography on chiral stationary phases, enzymatic resolution, fractional recrystallization of addition salts, as well as those techniques provided in the Schemes and Examples herein.
• the designation " - ⁇ * " refers to a bond that protrudes forward out of the plane of the page.
• the designation " "” refers to a bond that protrudes backward out of the plane of the page.
• the designation “ “ ⁇ “ refers to a bond that exists as a mixture of bonds that protrude both forward and backward out of the plane of the page.
• the term "patient” refers to a human or nonhuman mammal such as a dog, cat, cow, monkey, horse, or sheep. More particularly, the term “patient” refers to a human.
• the term “treating” (or “treat” or “treatment”) as used herein includes prohibiting, restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.
• the term “preventing” (or “prevent” or “prevention”) as used herein refers to prohibiting, restraining, or inhibiting the incidence or occurrence of a symptom or disorder.
• physiological disorders may present as a "chronic" condition, or as an "acute” episode.
• the treatment of disorders contemplates both acute events and chronic conditions. In an acute event, compound is administered at the onset of symptoms and discontinued when the symptoms disappear, whereas a chronic condition is treated throughout the course of the disease.
• Compounds of the present invention may be formulated as part of a pharmaceutical composition.
• a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, diluent or excipient is an important embodiment of the invention.
• examples of pharmaceutical compositions and methods for their preparation are well known in the art.
• a compound of Formula (I), or a composition comprising a compound of Formula (I) may be administered by any route which makes the compound bioavailable, including oral and parenteral routes.
• the term "effective amount” refers to the amount or dose of a compound of Formula (I) which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment.
• An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by considering a number of factors such as the species of mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; and the use of any concomitant medications.
• the compounds and compositions of the present invention may be administered, or used in a medicament, either alone or in combination with conventional therapeutic agents used to a particular disorder. Where the compounds or compositions of the present invention are used as part of a combination, the compound or composition comprising Formula (I) may be administered either separately, or as part of a formulation comprising the therapeutic agent with which it is to be combined.
• conventional therapeutic agents for the treatment of erectile dysfunction may be advantageously combined with the compounds of Formula (I), or compositions comprising a compound of Formula (I).
• Conventional agents for the treatment of erectile dysfunction include the phosphodiesterase type 5 (PDE5) inhibitors tadalafil (CialisTM), sildenafil citrate (ViagraTM) and vardenafil hydrochloride (LevitraTM).
• the present invention also provides a method of treating or preventing erectile dysfunction comprising administering to a patient in need thereof an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with an agent selected from the group consisting of tadalafil, sildenafil citrate, and vardenafil hydrochloride. More particularly, the present invention provides a method of treating or preventing erectile dysfunction comprising administering to a patient in need thereof an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with an agent selected from the group consisting of CialisTM, ViagraTM and LevitraTM.
• the present invention further provides a combination therapy formulation which comprises: (a) a compound of Formula (I); (b) one or more co-agents that are conventional for the treatment of erectile dysfunction selected from the group consisting of tadalafil, sildenafil citrate, and vardenafil hydrochloride; and optionally (c) a pharmaceutically acceptable carrier, diluent or excipient. More particularly, the present invention provides a combination therapy formulation which comprises: (a) a compound of Formula (I); (b) one or more co-agents that are conventional for the treatment of erectile dysfunction selected from the group consisting of CialisTM, ViagraTM and
• a particular aspect of the present invention is a compound of Formula (I) wherein:
• the compounds of formula (I) may be prepared by a variety of procedures known in the art as well as those described in the Schemes, Preparations, and Examples below. However, the following discussion is not intended to be limiting to the scope of the present invention in any way. For example, the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare additional compounds of Formula (I).
• MeOH refers to methanol
• EtOH refers to ethanol
• i-PrOH refers to isopropanol
• EtOAc refers to ethyl acetate
• DMF dimethylformamide
• DMSO refers to methyl sulfoxide
• NMP refers to l-methyl-2-pyrrolidinone
• TFA trifluoroacetic acid
• THF tetrahydrofuran
• DEAD refers to diethyl azodicarboxylate
• DIAD refers to diisopropyl diazodicarboxylate
• NBS refers to N- bromosuccinimide
• TDMS refers to t- butyldimethylsilyl
• Ptg refers to t- butyldimethylsilyl
• Ptg refers to t- butyldimethylsilyl
• Step A cyclopentenone is reacted with phthalimide in a Michael addition to give (R,5)-2-(3-oxo-cyclopentyl)-isoindole-l,3-dione (1).
• the reaction is performed in methanol/2 N Na 2 C ⁇ 3 in a ratio of 10/1 by volume preferably at ambient temperature using conditions similar to those described by O.
• Step B (R,5')-2-(3-oxo-cyclopentyl)-isoindole-l,3-dione (1) is reacted with 4- cyano-phenylhydrazine in a typical Fischer indole synthesis to give a tetrahydrocyclopenta[b] indole of formula (2).
• the skilled artisan will recognize that there are a variety of acidic conditions to effect a Fischer indole synthesis, including both proton and Lewis acids.
• the preferred conditions use a mixture of glacial acetic acid with 4 N HCl in dioxane, at a temperature of about 50 0 C to the reflux temperature of the solvent, for about 4 to 24 hours.
• the product is isolated by addition of water followed by filtration of the resulting solid.
• the solid is sonicated in methanol to give material of sufficient purity.
• the reaction is effected using a Lewis acid, such as zinc chloride, in an amount of about 2 to 4 equivalents.
• Other preferred conditions for Step B use ethanol at reflux temperature for about 4 to 24 hours.
• the product is isolated and may be purified by filtration of the reaction mixture, followed by silica gel chromatography of the filtrate.
• Step C the phthalimide group of the tetrahydrocyclopenta[b] indole of formula (2) is cleaved with hydrazine or hydrazine hydrate to provide an aminotetrahydrocyclopenta[b]indole of formula (3) using conditions as described by M. Alajarin, et al (Eur. J. Org. Chem. 2002, 4222-4227).
• the reaction proceeds in a solvent mixture of tetrahydrofuran/ethanol in a ratio of about 5.5/1 by volume at a temperature of 0 to 50 0 C, preferably at about room temperature, for 4 to 72 hours.
• the resulting phthalhydrazide is removed by filtration and the product isolated by concentration of the filtrate. It may be purified by chromatography using techniques known in the art.
• Step D the racemic aminotetrahydrocyclopenta[b]indole of formula (3) is resolved to the chiral (5)-aminotetrahydrocyclopenta[b]indole of formula (4).
• the amine is dissolved in an appropriate solvent, such as ethanol and recrystillized as the salt of D-pyroglutamic acid.
• an appropriate solvent such as ethanol
• the free base of formula (4) is obtained by dissolution in water and adjustment of the pH to 9 using concentrated aqueous ammonia.
• the resulting solid is filtered to provide (5)-2-amino-l,2,3,4-tetrahydro-
• Step E the amine of formula (4) is acylated with isopropyl chloroformate to give a carbamate of formula (5), using conditions well known to those skilled in the art.
• the amine is combined with an excess of an organic amine base such as triethylamine or diisopropylethylamine in an inert solvent such as tetrahydrofuran, toluene, dichloroethane or dichloromethane, N-methylpyrrolidinone, or N,N-dimethylformamide, or a mixture thereof.
• an organic amine base such as triethylamine or diisopropylethylamine
• an inert solvent such as tetrahydrofuran, toluene, dichloroethane or dichloromethane, N-methylpyrrolidinone, or N,N-dimethylformamide, or a mixture thereof.
• Preferred conditions use diisopropylethylamine in dichloromethane in the presence of isopropylchloro formate at a temperature of about 0 to 40 0 C for 1 to 72 hours.
• the product is isolated by addition of water and diethyl ether, followed by stirring and collection of the resulting solid. If the product is sufficiently soluable in an appropriate organic solvent it may be isolated by extractive techniques.
• Step F the racemic amine of formula (3) is protected as the ⁇ -butoxycarbonyl (boc) protected amine of formula (6) using conditions common in the art.
• Step G the racemic mixture is then resolved using chiral chromatography to obtain the (S) enantiomer of formula (7).
• the boc group is then removed using typical acidic conditions with HCl or TFA to obtain the chiral amine of formula (4).
• Step A (R,5')-2-(3-oxo-cyclopentyl)-isoindole-l,3-dione (1) is reacted with 4-bromophenylhydrazine hydrochloride in a typical Fischer indole synthesis. Preferred conditions use glacial acetic acid at about 50 to 80 0 C for 2 to 24 h.
• Step B the phthalimide group of the tetrahydrocyclopenta[b]indole of formula (8) is cleaved with hydrazine or hydrazine hydrate to provide an aminotetrahydrocyclopenta[b]indole of formula (9).
• Preferred conditions use tetrahydrofuran/ethanol in a mixture of about 20/1 by volume at a temperature of about 40 to 70 0 C, for 1 to 12 hours.
• the resulting phthalhydrazide is removed by filtration and the product isolated by concentration of the filtrate.
• Step C the amine of formula (9) is protected with a t-boc group to give the protected amine of formula (10).
• Preferred conditions use di-tert- butyldicarbonate in an inert solvent such as THF or dioxane, in the presence of an inorganic base such NaHC ⁇ 3.
• Step D the racemic t-boc material of formula (10) is resolved using chiral HPLC to obtain the (S) enatiomer of formula (11).
• Step E the (S) bromo carbamate of formula (11) is converted to the nitrile of formula (5).
• the reaction is run in an inert solvent, such as N,N'-dimethylacetamide in the presence of a mixture of zinc acetate or zinc formate, zinc cyanide, and zinc dust.
• a palladium catalyst is used, such as l,l'-bis(diphenylphosphino)ferrocene) palladium (II) chloride.
• the reaction is heated at about 80 to 120 0 C for about 2 to 24 h.
• Step F the boc group is removed using typical acidic conditions such as HCl/dioxane or TFA to obtain the chiral amine of formula (4) (see Scheme 1) which is then acylated, as described in Scheme 1, Step E, with isopropyl chloro formate to give a carbamate of formula (5).
• Step G the phthalimide group of the tetrahydrocyclopenta[b]indole of formula (8) is cleaved with hydrazine or hydrazine hydrate to provide an aminotetrahydrocyclopenta[b]indole of formula (9) as previously described in Step B. Subsequently, the resulting free amine is converted to thep- toluenesulfonic acid salt in ethanol.
• Step H the racemic aminotetrahydrocyclopenta[b]indole tosylate salt of formula (13) is freed and the resulting free amine is resolved using dibenzyl-D-tartaric acid to obtain the tartaric acid salt of the (5)-aminotetrahydrocyclopenta[b] indole of formula (14).
• the salt formation is preferably done in a solvent mixture of ethanol and water with reflux for about 1 to 6 hours, followed by cooling to obtain the desired enantiomer.
• the salt of formula (14) is neutralized to the free base and then acylated to obtain a carbamate of formula (15).
• the salt is neutralized with an inorganic base, such as sodium hydroxide solution.
• the free base is obtained by extraction followed by acylation with isopropyl chloro formate in the presence of an organic amine such as diisopropylethyl amine in an inert solvent or solvent mixture of tetrahydrofuran and methyl t-butyl ether.
• Step J the bromo carbamate of formula (15) is converted to the nitrile of formula (5) as previously described for Step E.
• Step A the cyano tetrahydrocyclopenta[b]indole of formula (5) is alkylated with the alkyl mesylate of formula (16) to provide the alkylated tetrahydrocyclopenta[b] indole of formula (17).
• the reaction proceeds in an inert solvent, such as DMF, in the presence of an inorganic base, such as cesium carbonate with addition of potassium iodide.
• the reaction proceeds at a temperature of about 50 to 100 0 C for about 8 to 72 h.
• Step B the chirality of the hydroxyl group of the pyrrolidine of formula (17) is converted from (35) to (3R).
• the chiral conversion is effected using a Mitsunobu reaction to give the chloroacetoxy pyrrolidine of formula (18).
• a Mitsunobu reaction to give the chloroacetoxy pyrrolidine of formula (18).
• the alcohol of formula (17) is dissolved in a suitable anhydrous solvent like THF, CH 2 CI 2 , or toluene and treated with a trialkyl- or triarylphosphine such as Me 3 P, BU3P, or Ph 3 P and a dialkylazo-dicarboxylate, such as DEAD or DIAD.
• Step C the 3R-chloroacetoxy pyrrolidine of formula (18) is hydrolyzed to the 3R-hydroxypyrrolidine of formula (19a).
• the hydrolysis is accomplished using an inorganic base, such as lithium hydroxide, in a solvent such as methanol, at 0 to 50 0 C for 4 to 24 h.
• Step D the boc protecting group is removed using acidic conditions such as TFA or preferably 4 N HCl in dioxane to provide the 35-hydroxypyrrolidine of formula (19b).
• Step E the hydroxpyrrolidine of formula (19a or b) is alkylated using reductive amination conditions to give a N-alkyl pyrrolidine of formula (20a, b, or c).
• the reaction takes place in an inert solvent, such as THF, dichloromethane, or preferably, chloroform using a reducing reagent, such as sodium triacetoxyborohydride from about 0 to 50 0 C for 8 to 24 h.
• the aldehyde used can be either formaldehyde or acetaldehyde to provide compounds of formula (20a, b, or c).
• (2R,35)-3-Hydroxy-2-methanesulfonyloxymethyl-pyrrolidine- 1 -carboxylic acid tert-butyl ester (16) can be readily prepared by methods similar to those described herein or by using procedures that are established in the art. For example, 3-hydroxy-L-proline can be protected with boc to give (25 r ,35)-l-(tert-butoxycarbonyl)-3-hydroxypyrrolidine- 2-carboxylic acid. The carboxylic acid is reduced to the alcohol and subsequently converted to the mesylate of formula (16).
• Step A the cyano tetrahydrocyclopenta[b]indole of formula (5) is alkylated with the alkyl mesylate of formula (21a & b) to provide the alkylated tetrahydrocyclopenta[b]indole of formula (22a & b).
• the alkyl mesylate of formula (21a & b) is used as a mixture of the two trans enantiomers.
• the alkylation is preformed similarly to conditions described in Scheme 3, Step A, above.
• Step B the boc protecting group is removed using acidic conditions such as TFA or preferably 4 N HCl in dioxane.
• Step C the hydroxyproline of formula (23a & b) is alkylated using reductive amination conditions as previously described for Scheme 3, Step E to provide a mixture of the trans enantiomers as formula (24a & b).
• the enantiomers can be separated using chiral chromatography.
• trans-3 -Hydroxy-2-methanesulfonyloxymethyl-3 -methyl-pyrrolidine- 1 -carboxylic acid tert-butyl ester (21a & b) can be readily prepared by methods similar to those described herein or by using procedures that are established in the art.
• ?r ⁇ ws-2-benzyloxymethyl-3 -methyl -pyrrolidin-3-ol can be obtained by one-pot conversion of the appropriate 2,3-aziridin-l-ol (J. Schomaker and S. Bhattacharjee, J. Am. Chem. Soc, 2007, 129, 1996-2003).
• the nitrogen of the pyrrolidinol is then protected with t-boc and the benzyl group removed by hydrogenation.
• the resulting alcohol is mesylated to give the trans enantiomers of formula (21a & b).
• Step A the cyano tetrahydrocyclopenta[b]indole of formula (5) is alkylated with the benzyl or pyridylmethyl halide of formula (25) to provide the alkylated tetrahydrocyclopenta[b] indole of formula (26).
• the phenyl or pyridyl ring is substituted with hydroxyl functionality, which is protected with protecting groups (Ptg) known in the art, for example as the methyl ether or silyl ether.
• Ptg protecting groups
• the alkylation is performed similarly to conditions described in Scheme 3, Step A, above.
• Step B the protecting group is removed to give the phenol or hydroxy pyridine of formula (27).
• methyl ether is converted to the free hydroxyl by reaction with boron tribromide in an inert solvent such as dichloromethane at a temperature of about -20 to 25 0 C.
• a silyl protecting group can be removed with fluoride anion using reagents such as cesium fluoride or preferably tetrabutylammonium fluoride.
• the benzyl halides of formula (25) can be readily prepared by methods similar to those described herein or by using procedures that are established in the art.
• fluoro or difluoro 2-hydroxy benzaldehydes can be alkylated with iodomethane to provide the 2-methoxy benzaldehyde.
• 2-(Chloromethyl)-3-methoxypyridine can be obtained from 3-methoxy-2- picoline by chlorination with POCI3.
• exemplified compounds of Formula (I) possess profiles of activity which suggest they have utility in the treatment of disorders responsive to steroidal androgen therapy.
• exemplified compounds of Examples 1-10 and 12 of Formula (I) are potent AR ligands which agonize the androgen receptor.
• the exemplified compounds of Examples 1-10 and 12 of Formula (I) selectively bind to AR relative to each of MR, GR, and PR.
• K ( J" refers to the equilibrium dissociation constant for a ligand- receptor complex
• Kj refers to the equilibrium dissociation constant for drug-receptor complex, and is an indication of concentration of drug that will bind to half the binding sites at equilibrium
• K j5 refers to the equilibrium dissociation constant for an antagonist-receptor complex
• IC50 refers to the concentration of an agent which produces 50% of the maximal inhibitory response possible for that agent or, alternatively, to the concentration of an agent which produces 50% displacement of ligand binding to the receptor
• EC50 refers to the concentration of an agent which produces 50% of the maximal response possible for that agent
• ED50 refers to the dose of an administered therapeutic agent which produces 50% of the maximal response for that agent.
• steroid receptor binding assays include radio-labeled ligands, such as 0.25 nM [ ⁇ HJ-aldosterone for MR binding, 0.3 nM PH]- dexamethasone for GR binding, 0.36 nM [ ⁇ HJ-methyltrienolone for AR binding, and 0.29 nM [ 3 H]-methyltrienolone for PR binding, and either 20 ⁇ g 293-MR lysate, 20 ⁇ g 293- GR lysate, 22 ⁇ g 293 -AR lysate, or 40 ⁇ g 293 -PR lysate per well. Assays are typically run in 96-well format.
• Competing test compounds are added at various concentrations ranging from about 0.01 nM to 10 ⁇ M.
• Non-specific binding is determined in the presence of 500 nM aldosterone for MR binding, 500 nM dexamethasone for GR binding, or 500 nM methyltrienolone for AR and PR binding.
• the binding reactions (140 ⁇ L) are incubated overnight at 4 0 C, then 70 ⁇ L of cold charcoal-dextran buffer (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) is added to each reaction. Plates are mixed for 8 minutes on an orbital shaker at 4 0 C. The plates are then centrifuged at 3,000 rpm at 4 0 C for 10 minutes.
• the compounds of Examples 1 -10 and 12 display a Kj in the AR binding assay of ⁇ 500 nM.
• the compounds of Examples 1, 2, 6, and 9 displayed a Ki in the AR binding assay of about 51 nM, 1 InM, 13 nM, and 0.8 nM respectively, thus demonstrating that compounds within the scope of the present invention are potent ligands of human AR.
• Androgens exerts their physiological effects through interaction with the androgen receptor. Following cytoplasmic binding of an androgen to AR, the ligand receptor complex translocates to the cell nucleus where it binds to hormone response elements on DNA to initiate expression of target genes.
• the effects of androgens may be characterized as anabolic or androgenic in nature.
• Anabolic (i.e. tissue building) effects of androgens include increasing muscle mass and strength and bone mass, whereas androgenic (i.e. masculinizing) effects include the development of male secondary sexual characteristics such as the internal reproductive tissues (i.e. prostate and seminal vesicle), the external genetalia (penis and scrotum), libido, and hair growth patterns.
• bioassays are performed which detect functional modulation of target gene expression in cells transiently transfected with a nuclear receptor protein and a hormone response element-reporter gene construct.
• the solvents, reagents, and ligands employed in the functional assay are readily available from commercial sources, or can be prepared by one of ordinary skill in the art. The following provides typical procedures for nuclear hormone receptor functional assays.
• Human embryonic kidney HEK293 cells are transfected with steroid hormone receptor and reporter gene plasmids using a suitable transfection reagent such as FugeneTM. Briefly, the reporter plasmid containing two copies of probasin ARE and
• TK(thymidine kinase) promoter upstream of the luciferase reporter cDNA is transfected into HEK293 cells with a plasmid constitutively expressing human androgen receptor (AR) using viral CMV (cytomegalovirus) promoter.
• the reporter plasmid containing two copies of GRE and TK promoter upstream of the luciferase reporter cDNA is transfected with a plasmid constitutively expressing either human glucocorticoid receptor (GR), human mineralocorticoid receptor (MR), or human progesterone receptor (PR) using viral CMV promoter.
• GR human glucocorticoid receptor
• MR human mineralocorticoid receptor
• PR human progesterone receptor
• Transfected cells are transfected in T 150 cm flasks in DMEM media with 5% charcoal-stripped Fetal Bovine Serum (FBS). After an overnight incubation, transfected cells are trypsinized, plated in 96 well dishes in DMEM media containing 5% charcoal- stripped FBS, incubated for 4 hours and then exposed to various concentrations of test compounds ranging from about 0.01 nM to 10 ⁇ M.
• FBS Fetal Bovine Serum
• the percentage efficacy (compounds with saturated maximum responses) or the percent maximum stimulation (compounds with maximum responses that do not saturate) are determined relative to maximum stimulation obtained with the following reference agonists: 30 nM aldosterone for MR assay, 100 nM methyltrienolone for AR assay, 30 nM promegestone for PR assay, and with 100 nM dexamethasone for GR assay. IC50 values are determined similarly using antagonist mode assay data.
• percent inhibitions are determined by comparing test compound activity in the presence of low concentration of agonist (0.08 nM aldosterone for MR, 0.25 nM dexamethasone for GR, 0.66 nM of methyltrienolone for AR, and 0.08 nM of promegestone for PR) to the response produced by the same low concentration of agonist in the absence of test compound.
• the C2C12 AR/ARE reporter assay is performed. Briefly, mouse myoblast C2C12 cells are co-transfected using FugeneTM reagent. A reporter plasmid containing a GRE/ARE (glucocorticoid response element/androgen response element) and TK promoter upstream of the luciferase reporter cDNA, is transfected with a plasmid constitutively expressing human androgen receptor (AR) using viral CMV promoter. Cells are transfected in T 150 cm flasks in DMEM media with 4% charcoal stripped Fetal Bovine Serum (FBS).
• GRE/ARE glucocorticoid response element/androgen response element
• AR human androgen receptor
• transfected cells are trypsinized, plated in 96 well dishes in DMEM media containing 4% charcoal-stripped FBS, incubated for 2 h and then exposed to various concentrations of test compounds ranging from about 0.01 nM to 10 ⁇ M .
• concentrations of test compounds ranging from about 0.01 nM to 10 ⁇ M .
• cells are lysed and luciferase activity is determined by standard techniques. Data is fit to a 4 parameter-fit logistics to determine EC50 values. The % efficacy is determined versus maximum stimulation obtained with 10 nM methyltrienolone.
• mice Male Sprague Dawley rats (24 weeks old) are castrated (gonadectomized or "GDX") according to approved procedures and allowed to waste for eight weeks. Age- matched sham-operated mice are also prepared. Animals are housed in a temperature- controlled room (24 0 C) with a reversed 12 hour light/dark cycle and water and food are available ad libitum.
• GDX gonadectomized mice
• mice are randomized based on body weight prior to ascribing a test slot.
• Compounds of the present invention are administered daily by oral gavage to the castrated thirty two week old rats (body weight about 450-500 g) using a conventional vehicle such as 1% sodium carboxymethylcellulose (CMC) + 0.25% Tween 80 + 0.05% AntiFoam® in sterile H 2 O. Sham operated rats treated with vehicle alone are used as a treatment positive controls whereas castrated rats treated only with vehicle are used as treatment negative control.
• Test animals are dosed daily over a two or eight week timeframe with, for example, 0.3, 1, 2, 3, or 6 mg/kg/day of a compound of the present invention.
• the wet weight of the Levator Ani (LA) muscle and the bulbocavernous muscle in each test group may determined and compared to the wet weight of the Levator Ani and the bulbocavernous muscle from the castrated, vehicle-only control group (after weighing, the bulbocavernous muscle may be flash frozen in liquid nitrogen for later use in measuring nitric oxide synthase mRNA, as described below)
• the wet weight of the prostate from test animals may be similarly compared to the wet weight of the prostate from the castrated, vehicle-only group.
• Sham (vehicle only) showed a mean LA wet weight of about 0.255 g, a mean prostate wet weight of about 824.5 mg, and a mean bulbocavernous wet weight of about 0.93 g;
• the castrated/vehicle only animals showed a mean LA wet weight of about 0.094 g, a mean prostate wet weight of about 103.8 mg, and a mean bulbocavernous wet weight of about 0.362 g;
• the 0.3 mg/kg study groups showed a mean LA wet weight of about 0.137 g, a mean prostate wet weight of about 72.4 mg, and a mean bulbocavernous wet weight of about 0.476 g;
• the 1.0 mg/kg study groups showed a mean LA wet weight of about 0.182 g, a mean prostate wet weight of about 102.8 mg, and a mean bulbocavernous wet weight of about 0.582 g
• treatment with the compound of Example 2 produced a dose dependent increase in Levator ani and bulbocavernous weights in comparison to the castrated control group.
• NOS/cGMP nitric oxide synthase/cyclic guanosine monophosphate pathway
• NOS expression leads to nitric oxide (NO) generation which, in turn, promotes cGMP generation through activation of guanylyl cyclase.
• cGMP promotes protein kinase G (PKG) activity which mediates relaxation of corporal smooth muscle to facilitate penile erection.
• Evidence supports a role for androgens in regulating the expression and activity of NOS isoforms in the corpus cavernorsum in experimental animal models. Traish et ah, European Urology, 52; 54-70 (2007).
• androgen receptor modulators which are capable of increasing the expression of NOS isoforms, are believed to have a role in regulating penile erectile activity.
• RNA is isolated from frozen bulbous and corpus cavernosum tissues that are obtained at necropsy from castrated Sprague Dawley rats that are prepared and dosed essentially as described above for the Model of Efficacy and Selectivity.
• cDNA is synthesized from 2 ⁇ g of RNA using a high capacity cDNA kit according to the manufacturer's instructions.
• PCR Real-time quantitative PCR is then performed according to the fluorescent TaqMan® methodology (Applied Biosystems). Assays-on-DemandTM (Applied Biosystems) probes are used for the rat epithelial nitric oxide synthase transcript (eNOS) while probes are designed for the rat penile specific isoform of neuronal nitric oxide synthase (pnNOS) using probe designer software (Applied Biosystems). The probes are designed to span a 102 bp region of the rat neuronal nitric oxide synthase gene (pnNOS) that is specific to pnNOS (positions 2865-2967).
• pnNOS rat neuronal nitric oxide synthase gene
• MGBTM Primer sequences are 5 'CCGGAACCCTTGCGTTT 3' (SEQ ID NO: 1) (forward) and 5 'CAGACTGTGGGCTTCAGAGTCA 3' (SEQ ID NO:2) (reverse) and the probe sequence is 5 'CCCGT AAAGGGCCT 3' (SEQ ID NO:3) (FAM NFQ).
• Assays-on- DemandTM probe sets for the PPIB transcript are used as an internal control. PCR is performed on an ABI Prism 7700 Sequence Detection System at the following thermocycler conditions: 2 min. at 50 0 C, 10 min. at 95 0 C, and 40 cycles at 95 0 C for 30 s, and 60 0 C for 1 min. All reactions are carried out in triplicate.
• the compound of Example 2 shows a dose dependent increase in penile nitric oxide synthase (pnNOS) mRNA in the bulbocavernosum tissues obtained from castrated Sprague Dawley rats treated over an eight week timeframe.
• pnNOS penile nitric oxide synthase
• the 0.3 mg/kg study groups showed pnNOS expression of about 97% of control; the 1.0 mg/kg study group showed pnNOS expression of about 93% of control; the 3.0 mg/kg study group showed pnNOS expression of about 153% of control; and the 6.0 mg/kg study group showed pnNOS expression of about 248% of control.
• Example 2 shows an increase in epithelial nitric oxide synthase (eNOS) mRNA in the corpus cavernosum tissue obtained from castrated Sprague Dawley rats treated over a two week timeframe at 2mg/kg/day. Specifically, eNOS expression in the study group was about 159% of control. Reductions in muscle mass or strength may occur as a result of immobilization or disuse following, for example bone fractures or hip or knee replacements. In order to determine the ability of compounds of the present invention to treat or prevent loss of muscle mass or strength induced by immobilization, disuse or trauma, the following animal models may be employed. Model of Muscle Loss induced by Immobilization
• a hind limb of Male 12 week ICR mice is immobilized in the plantar flexion mode by placing a cast on the limb. Following seven days of immobilization, the mice are treated with a daily administration of a compound of the present invention for various time periods. Control animals, with and without casts, are similarly treated with vehicle for various time periods. At the end of the treatment protocol, the mice are sacrificed, the wet weights of the casted gastrocnemius are determined and the individual treatment groups are compared to the vehicle controls. See generally Am. J. Endocrinol. Metab. 289: 969-980 (2005). Model of Muscle injury and Trauma Male ICR mice are castrated at 8 weeks of age and allowed to waste for an additional 8 weeks.
• mice are individually caged and maintained on a 12 hr light/dark cycle at 22°C with ad lib access to food and water.
• Mice are anesthetized with isofluorane (1-5 %) and the right gastrocnemius muscle is bilaterally injected with 100 ⁇ L of a 10 ⁇ M cardiotoxin (naja naja atra; Sigma Aldrich) to induce muscle injury. Animals recover from anesthesia and resume normal activity within 5 minutes.
• animals On day 5 post injection animals are treated with various doses of a compound of the present invention. After day 14 post injection, the treated mice are euthanized, weighed, and the gastrocnemius muscle tissue is harvested from both uninjected (contra-lateral control) and cardiotoxin injected legs.
• the muscle weights were matched to the uninjected and untreated control to establish percentage recovery from trauma.
• Other animal models well known to those in the art may be employed. Examples of such models are provided in Y. L. Ma et ah, Japanese Journal of Bone and Mineral Metabolism 23 (Suppl): 62-68 (2005); Y.L. Ma et ah, Endocrinology 144: 2008-2015 (2003); and K. Hanada et ah, Biol. Pharm. Bull. 26(11): 1563-1569 (2003).
• Model of Estrogen Deficiency Osteopenia induced by Ovariectomy Six-month-old, virgin Sprague Dawley female rats weighing about 220 g are housed with ad libitum access to food and water. Bilateral ovariectomies (Ovx) are performed on the animals (except for sham-operated controls) and then randomized into treatment groups of 7-8 rats per group.
• Each assay typically contains at least 2 sets of controls, including sham-ovariectomy (Sham) and ovariectomized controls (Ovx) treated with vehicle.
• Ovx rats are permitted to lose bone for 1 month to establish osteopenia before treatment with test compound.
• Test compounds are administered orally via gavage to Ovx animals for 8 weeks.
• recombinant human PTH (1-38) (about 10 ⁇ g/kg/d, subcutaneously) may be given to a subset of Ovx animals.
• QCT Quantitative computed tomographic
• Biomechanical analyses of three point bending on the femoral midshaft and load to failure on the proximal femur are performed using a material mechanical testing machine and analyzed using TestWorks 4® software.
• Model of Androgen Deficiency Osteopenia induced by Orchidectomy Six-month-old, Sprague Dawley male rats weighing about 485 g are housed with ad libitum access to food and water. Bilateral orchidectomy (Orx) are performed on the animals (except for sham-operated controls) and then randomized into the treatment groups of 7-8 rats per group.
• Each assay typically contains at least 2 sets of controls, including sham-orommectomized (Sham) and orchidectomized controls (Orx) treated with vehicle. Orx rats are permitted to lose bone for 2 months to establish osteopenia before treatment with test compound is initiated. Test compounds are administered orally via gavage to Ovx animals for 8 weeks. As a positive control, recombinant human PTH (1- 38) (about lOug/kg/d, subcutaneous Iy) may be given to a subset of Orx animals.
• the BMD of the vertebra and femur, as well as the biomechanical analyses of the femur may be performed as described above for the ovariectomized female rat model.
• the BMD of the vertebra and femur, as well as the biomechanical analyses of the femur may be performed as described above for the ovariectomized female rat model.
• animal model protocols described above may be readily adapted for use in conjunction with the compounds and methods of the present invention.
• the R or S configuration of compounds of the invention may be determined by standard techniques such as X-ray analysis and correlation with chiral-HPLC retention time.
• the names of the compounds of the present invention are generally provided by Autonom
• the solid is mixed with CH 2 CI 2 (400 mL), cooled in an ice bath, and filtered.
• the solid is rinsed with CH 2 CI 2 and hexanes to recover 28.1 g (34%) of the title compound as a solid.
• An additional 78.8 g (60 %) of the title compound is obtained by concentrating the filtrate and purifying by chromatography (I L silica gel, loaded as a concentrated CH 2 CI 2 solution and eluted with 30% hexanes/CH 2 Cl 2 , 100% CH 2 Cl 2 , then 3% EtOAc/CH 2 Cl 2 ).
• the solid is slurried in dichloromethane (168 mL) and treated with diisopropylethylamine (9.13 g, 12.3 mL, 70.1 mmol) and isopropyl chloroformate (1.0 M in toluene, 34.0 mL, 34.0 mmol) at room temperature. After 4 h the reaction is treated with water (50 mL) and concentrated to give an aqueous slurry of the product. The reaction is further diluted with water (500 mL) and sonicated for 15 min in an ultrasonic bath. A tan solid is filtered off and dried in vacuo at 40 0 C.
• a suspension of £r ⁇ r ⁇ -3-hydroxy-L-proline (5 g, 37.56 mmol) in methanol (100 mL) is treated with diisopropylethylamine (6.55 mL, 37.56 mmol) and subsequently di-t- butyldicarbonate (8.87 g, 39.44 mmol) at room temperature.
• the resulting suspension is stirred for 2 h at room temperature while becoming a homogeneous solution.
• the solvent is removed in vacuo and the residue is dissolved in ethyl acetate (120 mL).
• the organic solution is washed with 1 N aqueous hydrogen chloride.
• Tris(dibenzylideneacetone)dipalladium (110 mg, 120 ⁇ mol) and 2-di-tert-butylphosphino-3,4,5,6-tetramethyl-2',4',6'- triisopropyl-l,l '-biphenyl (230 mg, 479 ⁇ mol) are added and the mixture is heated for 3 h.
• the reaction is diluted with water and dichloromethane.
• the layers are then separated and the aqueous layer is washed with dichloromethane twice.
• the aqueous portion is acidified to pH 1 -2 with a few drops of 5 M HCl and then neutralized to pH 7 with potassium carbonate.
• Potassium carbonate (40 g) is added slowly in portions and after stirring for 5 min a 1 M solution of isopropyl chloroformate in toluene (34.0 mL, 34.0 mmol) is added at room temperature. A portion of the solvent is removed under reduced pressure and the residue is diluted with dichloromethane and ethyl acetate. The brown insoluble residue is filtered off and the phases are separated. The organic phase is washed with 10% potassium carbonate once, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a brown foam. The foam is dissolved in methanol (20 rnL), 2 M sodium hydroxide (10 mL) is added and the reaction is stirred at room temperature for 1 hour.
• the solvent is removed under reduced pressure.
• the residue is dissolved in 0.5 M HCl/EtOAc/CHCl3 and the bilayer is filtered. It is then acidified to pH 7-8 with 5% potassium carbonate.
• the phases are separated and the organic phase is washed with 5% potassium carbonate once, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain an orange foam.
• the foam is purified by silica flash chromatography eluting with 10 to 40% ethyl acetate/chloroform to obtain a red solid (3.33 g).
• the solid is dissolved in 0.5 M sodium hydroxide (30 mL).
• the aqueous layer is washed with dichloromethane twice and the organic layers are discarded.
• the aqueous layer is adjusted to pH 4-5 with 5 M hydrochloric acid. An off-white/yellow solid crashes out of solution.
• the pH is adjusted to 8-9 with potassium carbonate, the yellow solid is collected by filtration and air dried for one hour. It is then suspended in a minimum amount of acetonitrile, filtered, and rinsed with a minimum amount of acetonitrile. The mother liquor is concentrated and the procedure is repeated two times to afford a white powder, which is dried under high vacuum overnight to obtain the title compound as a white solid (1.45 g, 33%).
• Tetrakis(triphenylphosphine)palladium (1.02 g, 0.87 mmol) is added while maintaining the nitrogen atmosphere.
• the reaction is heated at 80 0 C for 3 h and then cooled to room temperature.
• the reaction is filtered through a pad of diatomaceous earth.
• the filtrate is evaporated and the crude material dried under vacuum to afford a brown solid.
• the solid is dissolved in NMP (100 mL) and then added to water (1.2 L).
• a pale cream solid is collected by filtration and dried overnight under vacuum to afford 41 g of crude material.
• the material is purified by recrystallization from acetonitrile and then dried under vacuum to constant weight to afford the title compound (29.5 g, 86%).

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