WO2008019825A1

WO2008019825A1 - Use of tricyclic indole derivatives for the treatment of muscular diseases

Info

Publication number: WO2008019825A1
Application number: PCT/EP2007/007183
Authority: WO
Inventors: Holger Deppe; Alexandre Briguet; Günther Metz; Michael Erb
Original assignee: Santhera Pharmaceuticals (Schweiz) Ag
Priority date: 2006-08-14
Filing date: 2007-08-14
Publication date: 2008-02-21

Abstract

The present invention relates to the use of a compound for the preparation of a medicament for the treatment of muscular atrophy.

Description

Use of tricyclic indole derivatives for the treatment of muscular diseases

The present invention relates to the use of a compound for the preparation of a medicament for the prophylaxis and/or treatment of muscular atrophy.

A general loss of skeletal muscle mass is a characteristic, debilitating response to fasting, as well as many severe diseases, including advanced cancer, renal failure, sepsis, and diabetes. In addition, atrophy of specific muscles results from their disuse or denervation. In most types of muscle atrophy overall rates of protein synthesis are suppressed and rates of protein degradation are consistently elevated; this response accounts for the majority of the rapid loss of muscle protein. In a variety of animal models of human diseases e.g., fasting, diabetes, cancer cachexia, acidosis, sepsis, disuse atrophy, denervation, and glucocorticoid treatment, most of the accelerated proteolysis in muscle appears to be due to an activation of the Ub-proteasome pathway. This activation is associated with the upregulation of muscle specific ligases.

Muscle specific ubiquitin ligases are an essential part of the ubiquitin-proteasome pathway for protein degradation. Indeed, increased expression of the ubiquitin ligases MAFbx and MURF1 is a critical component in muscle atrophy. These so called atrogins are upregulated in a rat atrophy model (Gomes et al., PNAS 98, 2001 , 14440-14445). Moreover, mice deficient of MAFbx, which is sometimes also referred to as atrogini, or MURF1 are resistent to atrophy (Bodine et al., Science 294, 2001 , 1704-1708). FOXO transcription factors induce are essential to upregulate the atrophy-related ubiquitin ligases MAFbx and MURF and thus contribute to developing skeletal muscle atrophy (Goldberg et al., Cell 117, 2004, 399-412). Skeletal muscle FOXO1 transgenic mice have less skeletal muscle mass, down-regulated Type I (slow twitch/red muscle) fiber genes, and impaired glycemic control. (Kamei et al., J. Biol. Chem. 279, 2004, 41114- 411123).

FOXO transcription factors belong to the large Forkhead family of proteins, a family of transcriptional regulators characterized by a conserved DNA-binding domain termed the 'forkhead box'. The Forkhead family is present in all eukaryotes. In humans, the FOXO Forkhead subgroup contains four members (FOXO1, FOXO3, FOXO4, and FOXO6). The transcriptional activity of FOXO transcription factors is regulated via its acetylation level. Whereas the majority of data describe that acetylation of FOXO factors represses target gene transcription, some suggest that FOXO acetylation increases target gene transcription. This discrepancy has been attributed to cell type and the target-gene-specific effects of FOXO. (Greer et al., Oncogene 24, 2005, 7410- 7425).

Protein acetylation regulates a wide variety of cellular functions, including the recognition of DNA by proteins, protein-protein interactions, and protein stability. Post- translational modification of proteins at lysine residues by reversible acetylation is catalyzed by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs), which act on both histone and non-histone substrates despite their names. HDACs are grouped into three classes on the basis of their homology to yeast transcriptional repressors. The class III HDACs are homologous to the yeast transcriptional repressor Sir2p and have no sequence similarity to class I and Il HDACs; these Sir2 proteins are also called sirtuins (Denu, Curr. Opin. Chem. Biol. 9, 2005, 431-440).

Sirtuins have been found in bacteria to eukaryotes. The hallmark of the family is a domain of approximately 260 amino acids that has a high degree of sequence similarity in all sirtuins. The family is divided into five classes (I-IV and U) on the basis of a phylogenetic analysis of 60 sirtuins from a wide array of organisms. Class I and class IV are further divided into three and two subgroups, respectively. The U-class sirtuins are found only in Gram-positive bacteria. The human genome encodes seven sirtuins, with representatives from classes I-IV.

Non-histone substrates of the most studied human Sir2 homolog, SIRT1, were shown to include p53, FOXO proteins, p300, NFkB and PGC-Ia, implicating sirtuins in apoptosis, cell survival, transcription and metabolism. Data indicate that SIRT1 is one of multiple deacetylases involved in the regulation of FOXO dependent transcription in mammalian cells (Brunet et al., Science 303, 2004, 2011-2015).

Atrophy is a common form of muscle tissue loss which can be long-lasting but, nevertheless, is frequently reversible. The classic example would be the muscle deterioration seen in bed ridden patients but genetically determined diseases such as for spinal muscular atrophy can cause life-threatening muscle atrophy as well. Loss of muscle tissue is also observed as a consequence of cancer, cardiac disease, infectious disease, chronic obstructive pulmonary disease (COPD), rheumatic disease, inflammatory bowel disease, end-stage renal disease, liver cirrhosis, severe injury, where it is referred to as cachexia.

Finally, severe and life threatening loss of muscle tissue is observed in genetic diseases causing muscle dystrophies exemplified in Duchenne Muscular Dystrophy, limb girdle muscular dystrophies and congenital muscle dystrophies.

Preventing loss of muscle tissue or accelerating the recovery from muscle weakness and/or loss due to atrophy, cachexia or dystrophy would be of great clinical benefit and highly desirable since the current treatment options are very limited. Therapeutic options for muscle atrophy include physiotherapy and similar interventions such as electro-stimulation of affected muscle groups, while pharmacological interventions available to prevent muscle loss or accelerate recovery from muscle weakness are not at all satisfying. Those agents include appetite stimulants such as megestrol, fish oil and certain unsatured fatty acids and canabinoid-like substances.

It is the object of the present invention to provide a compound for the treatment of muscular atrophy which has superior properties compared to known compounds conventionally used in the therapy of muscular atrophy, such as a better efficacy and improved patient compliance.

Said object is achieved by the use of tricyclic indole derivatives according to formula (I) for the preparation of a medicament for the prophylaxis and/or treatment of muscular atrophy.

Brief description of the drawings

Figure 1 shows a histogram representing the mRNA levels of atrogin-1 measured in cultured C2C12 myotubes treated for 24 h with solvent only (1% DMSO), 10 μM Dex, 10 μM Dex and 10 ng/ml IGF-1 or 10 μM Dex and 10 μM Compound 16. The invention relates to the use of a compound according to formula (I):

wherein each of R¹ and R² is, independently from each other, halo, hydroxy, C1-C10 alkyl, Ci-C₆ haloalkyl, C1-C10 alkoxy, Ci-C₆ haloalkoxy, Z-C₆-Ci₀ aryl, Z-C₅-Ci₀ heteroaryl, Z-C₃-C₈ heterocyclyl, Z-C₃-C₈ cycloalkyl, C₂-Ci₂ alkenyl, C₂-Ci₂ alkynyl, Z-C₅-Ci₀ cycloalkenyl,

Z-C₅-Ci₀ heterocycloalkenyl, carboxy, carboxylate, cyano, nitro, amino, CrC₆ alkyl amino, Ci-C₆ dialkyl amino, mercapto, S(O)NH₂, S(O)₂NH₂, CrC₄ alkylenedioxy, oxo, acyl, aminocarbonyl, Ci-Ci₀ alkoxycarbonyl, hydrazinocarbonyl, Ci-C₆alkyl hydrazinocarbonyl, d-C₆ dialkyl hydrazinocarbonyl, hydroxyamino carbonyl, or alkoxyamino carbonyl, wherein each alkyl, heterocyclyl, cycloalkyl and cycloalkenyl is optionally substituted by one or more R³; and wherein each aryl and heteroaryl is optionally substituted by one or more R⁴;

R³ is independently from each other fluoro, methyl, methoxy, hydroxy or amino; R⁴ is independently from each other fluoro, chloro, methyl, methoxy, cyano, hydroxy, amino, CF₃, CHF₂, CH₂F, OCF₃, OCHF₂ or OCH₂F;

Z is a bond or d-C₆ alkylene, wherein the Ci-C₆ alkylene group is optionally substituted with one or more fluorine atoms; m is 1, 2, 3 or 4; n is 1 , 2, 3 or 4; and p is 0, 1 , 2 or 3 or its tautomeric, enantiomeric, diastereomeric or pharmaceutically acceptable salts thereof for the preparation of a medicament for the prophylaxis and/or treatment of muscular atrophy.

Compounds of formula (I) preferably used in the present invention are those compounds in which one or more of the residues contained therein have the meanings given below. It is understood, that the preferably used compounds cover any compound obtained by combining any of the definitions disclosed within this description for the various substituents. With respect to all compounds of the formulas (I) the present invention also includes all tautomeric and stereoisomers forms and mixtures thereof in all ratios, and their pharmaceutically acceptable salts.

In preferred embodiments of the present invention, the substituents R¹ and R² as well as the indices m, n and p of the formula (I) independently from each other have the following meaning. Hence, one or more of the substituents R¹ and R² as well as the indices m, n and p can have the preferred or more preferred meanings given below.

In a preferred embodiment of the present invention, R¹ is halo, C1-C6 alkyl, Z-Ce-Ci₀ aryl, Z-C₅-Ci₀ heteroaryl or Z-C₃-C₈ cycloalkyl, wherein alkyl, aryl, heteroaryl and cycloalkyl are optionally substituted with one or more substituents R³. More preferably, R¹ is halo, preferably chloro or fluoro, more preferably chloro, or Z-cyclopropyl which is unsubstituted or substituted with one or more substituents R³.

In a preferred embodiment, the index m is 1 or 2. More preferably, the index m is 2.

R² is as defined above. Preferably, R² is aminocarbonyl.

It is further preferred in the present invention that the index n is 1.

It is further preferred that each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocycloalkenyl or cycloalkenyl group in the definition of R¹ and R² is unsubstituted or substituted by 1 to 4 substituents R³, more preferably 1 to 3 substituents R³, most preferably 1 to 2 substituents R³. In one preferred embodiment, two substituents R³ are simultaneously attached to the same carbon atom of the alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or cycloalkenyl group.

It is further preferred that each aryl or heteroaryl group in the definition of R¹ and R² is unsubstituted or substituted by 1 to 4 substituents R⁴, more preferably 1 to 3 substituents R⁴, most preferably 1 to 2 substituents R⁴.

R³ is as defined as above. Preferably, R³ is fluoro, methyl or methoxy. Most preferably, R³ is fluoro. In the case of a Z-C₃-C₈ cycloalkyl group, R³ is preferably attached to the carbon atom connected to the tricyclic ring system if Z is a bond or to the carbon atom connected to the alkylene chain if Z is a d-C₆ alkylene group. R⁴ is as defined above. Preferably, R⁴ is fluoro, methyl, methoxy, hydroxy, amino or CF₃.

Z is as defined above. Preferably, Z is a bond or a Ci-C₂ alkylene optionally substituted with 1 to 4 fluoro.

In a preferred embodiment, Z is -CF₂-, -CH₂-CF₂-, -CH₂-CH₂-CF₂- or -CH₂-CH₂-CH₂- CF₂-, wherein the substituted alkyl chain is connected with the tricyclic moiety via the CF₂ group.

The index m is as defined above. Preferably, m is 1 , 2 or 3, more preferably, m is 1 or 2. If m = 1 then the substituent R¹ is preferably located on position 6 of the tricyclic indole ring system.

The index n is as defined above. Preferably, n is 1 , 2 or 3, more preferably, n is 1 or 2. In a preferred embodiment, n is 1 and R² is attached to position 1 of the ring.

The index p is as defined above. Preferably, p is 1 or 2.

Where tautomerism, like e.g. keto-enol tautomerism, of compounds of general formula (I), may occur, the individual forms, like e.g. the keto and enol form, and together as mixtures in any ratio are also within the scope of the invention. Same applies for stereoisomers, like e.g. enantiomers, cis/trans isomers, conformers and the like.

Preferably, the compounds of general formula (I) are separated into their enantiomers and used in accordance with the present invention as such.

Preferred embodiments of the compounds according to present invention are shown below.

In case the compounds according to formula (I) contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts. Thus, the compounds of the formula (I) which contain acidic groups can be present on these groups and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. Compounds of the formula (I) which contain one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfoπic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art. If the compounds of the formula (I) simultaneously contain acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions).

Within the meaning of the present invention the terms are used as follows:

"C₁-C₁0 Alkyl" means a straight-chain or branched carbon chain having 1 - 10 carbon atoms, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- pentane, n-hexane, n-heptane, n-octane, n-nonane or n-decane. Each hydrogen of a Ci- C₁0 alkyl carbon may be replaced by a substituent.

"C₂-C1₂ Alkenyl" means a straight-chain or branched carbon chain of 2- 12 carbon atoms having one or more double bonds in the chain. Examples of C₂-Ci₂ alkenyl include -CH=CH₂, -CH=CH-CH₃, -CH₂-CH=CH₂, -CH=CH-CH₂-CH₃, -CH=CH-CH=CH₂, -CH₂-CH₂- CH₂-CH=CH₂, -CH₂-CH₂-CH₂-CH₂-CH=CH₂, -CH₂-(CH₂J₄-CH=CH₂, -CH₂-(CH₂)_S-CH=CH₂, -CH₂-(CHz)₆-CH=CH₂, -CH₂-(CH₂JrCH=CH₂, -CH₂-(CH₂)₈-CH=CH₂ and -CH₂-(CHz)₉- CH=CH₂. Each hydrogen of a C₂-Ci₂ alkenyl carbon may be replaced by a substituent.

"C₂-Ci₂ Alkynyl" means a straight-chain or branched carbon chain of 2- 12 carbon atoms having one or more triple bonds in the chain. Examples of C₂-Ci₂ alkynyl include -C≡CH, -C=C-CH₃, -CH₂-C=CH, -C=C-CH₂-CH₃, -C≡C-C≡CH, -CH₂-CH₂-CH₂-C=CH, -CH₂- CH₂-CH₂-CH₂-C=CH, -CH₂-(CHz)₄-C=CH, -CH₂-(CHz)₅-C=CH, -CH₂-(CHz)₆-C=CH, -CH₂- (CH₂)₇-C≡CH, -CH₂-(CH_Z)₈-C=CH and -CH₂-(CH₂)₉-C≡CH. Each hydrogen of a C₂-Ci₂ alkynyl carbon may be replaced by a substituent.

"C₃-C₈ Cycloalkyl" as employed herein includes saturated monocyclic hydrocarbon group having 3 to 8 carbons., e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. Each hydrogen of a C₃-C₈ cycloalkyl carbon may be replaced by a substituent. "C₅-Ci₀ Cycloalkenyl" means a cyclic alkyl chain having 5 - 11 carbon atoms, which is partially unsaturated e.g. cyclopentenyl, cyclopentadienyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl or cyclodecenyl. Each hydrogen of a C₅-Ci₀ cycloalkenyl carbon may be replaced by a substituent.

"Halo" means fluoro, chloro, bromo or iodo. It is generally preferred that halogen is fluoro or chloro.

"C₃-C₈ Heterocyclyl" refers to a nonaromatic 3-8 membered monocyclic ring system having 1-3 heteroatoms selected from O, N, or S. Any ring atom can be substituted. Examples of C₃-C₈ heterocyclyl include tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, imidazolidinyl and pyrrolidinyl.

"C₅-C₁₀ Heterocycloalkenyl" means a partially saturated, nonaromatic 5-10 membered monocyclic ring system having 1-3 heteroatoms selected from O, N, or S. Any ring atom can be substituted. Examples of C₅-Ci₀ heterocycloalkenyl include tetrahydropyridyl and dihydropyranyl.

"C₆-Ci₀ Aryl" refers to an aromatic monocyclic or bicyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted. Examples of C₆-Ci₀ aryl moieties include phenyl and naphthyl.

"C₅-Ci₀ Heteroaryl" refers to an aromatic 5-10 membered monocyclic or bicyclic ring system having 1-3 heteroatoms selected from O, N, N(O) or S. Any ring atom can be substituted. Examples of C₅-Ci₀ Heteroaryl include

"Acyl" means an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted.

The terms "aminocarbonyl," "C1-C10 alkoxycarbonyl", "hydrazinocarbonyl", and "hydroxyaminocarbonvl" refer to the radicals -C(O)NH, -C(O)O-Ci-Ci₀ alkyl, -C(O)NHNH₂ and -C(O)NH₂NH₂, respectively. The terms " Ci-C₆ alkylamino" and " Ci-C₆ dialkylamino" refer to -NH-Ci-C₆ alkyl and - N(CrC₆ alkyl)₂ radicals, respectively.

The term "alkoxy" refers to an -O- C₁-C₁₀ alkyl radical. The term "mercapto" refers to an SH radical.

Pharmacological agents presently available to prevent muscle loss or accelerate recovery from muscle weakness are not at all satisfying. Those agents include appetite stimulants such as megestrol, fish oil and certain unsatured fatty acids and canabinoid- like substances. An alternative approach to reduce atrophy, cachexia or dystrophy or to improve recovery from atrophy is offered by interventions that slow or stop the degradation of muscle proteins which frequently is mediated by the ubiquitin- proteasome system. Activation of this proteolytic enzyme complex as seen in clinical conditions of atrophy, cachexia and dystrophy critically depends on the ligation of peptides and proteins to ubiquitin moieties. Atrogins are a class of muscle-specific ligases that couple target proteins and peptides with ubiquitin moieties thereby mediating proteolysis of muscle proteins.

A method for treatment of muscular atrophies is offered by identifying pharmacological agents that would appropriately modulate any of the molecules involved in the signaling cascade that leads to pathologically increased muscle protein degradation. Specifically, a solution to this problem would be offered by identifying pharmacological modulators of either of the known atrogins or, alternatively, pharmacological modulators of a suitable member of upstream activators of atrogin expression. The identification of such pharmacological modulators offers a broad commercial application since atrophy, cachexia and dystrophy is common to a number of chronic disease states characterized by long periods of immobility. There is also a potential to apply this treatment approach in a subset of the geriatric population.

Surprisingly, it has been found, that inhibitors of sirtuins downregulate atrogin levels in a model for muscle atrophy in cells. Without being bound to a theory it is contemplated that the treatment of differentiated myotubes with an inhibitor of SIRT1 results in the suppression of atrogin-1 mRNA levels. As discussed above, muscular atrophy is associated with the upregulation of muscle specific ligases, e.g. atrogins, therefore, inhibitors of sirtuins can be used for the treatment of muscular atrophies. The compounds of formula (I) have been previously described as inhibitors of sirtuins and as such have been proposed for the treatment of various diseases such as cancer, diabetes, Parkinson's or Alzheimer's disease or obesity (e.g. US-A-2005/0209300). It has now been shown for the first time that tricyclic indol derivatives exhibit an unexpectedly high activity in the modulation of atrogin levels. Said compounds therefore are potential drugs for treating muscular atrophy.

In practical use, the compounds of formula (I) can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. Such compositions and preparations should contain at least 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained. The active compounds can also be administered intranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor.

Compounds of formula (I) may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dose of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. Preferably compounds of formula (I) are administered orally.

The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.

Preferably, the effective dosage is chosen in a range between 0.01 milligram to about 100 milligram per kilogram of mammal body weight, preferably given as a single daily dose or in divided doses two to six times a day, or in sustained release form. More preferably, the dosage is between 0.01 milligram to about 50 milligram per kilogram of animal body weight. This dosage regimen may be adjusted to provide the optimal therapeutic response.

In the schemes and examples below, the abbreviations used therein have the following meanings:

AcOH acetic acid

Boc tert-butoxycarbonyl

CDI 1 , 1 '-carbonyldiimidazole

CHx cyclohexane

DCM dichloromethane

DIPEA ethyl-diisopropylamine

DMA N,N-dimethylacetamide

DMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

EDC 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

Et₂O diethyl ether

EtOAc ethyl acetate

EtOH ethanol

HOBt 1 -hydroxybenzotriazole h hour(s)

LiOH lithiumhydroxide

MeCN acetonitrile

MeOH methanol

Mes-CI mesylchloride

MW molecular weight

NHS N-hydroxysuccinimid

TEA triethylamine

TFA trifluoroacetic acid

TFAA trifluoroacetic acid anhydride

THF tetrahydrofurane t_R (min) HPLC retention time The compounds of the present invention can be prepared by the synthesis routes shown below or by methods described previously (DE 2226703, DE 2431292, V.A. Parshin, H.V. Alekseeva, A.I. Bokanov, LM. Alekseeva, V.G. Granik, "Synthesis and pharmacological activity of 1 ,2,3,4-Tetrahydrocarbazole-i-carboxamides", Voprosy Biologicheskoi, Meditsinskoi i Farmatsevticheskoi Khimii 2001, (4), 40-45).

Reaction Scheme 1:

Synthesis of 3-Bromo-2-oxo-cycloalkane-carboxylic acid esters:

As shown in reaction Scheme 1 , optionally substituted keto-esters are reacted in an organic solvent such as Et₂O with a brominating agent such as bromine at a suitable temperature for a given time to yield the corresponding 3-Bromo-2-oxo-cycloalkane- carboxylic acid esters.

Reaction Scheme 2:

Synthesis of 2,3,4,9-Tetrahydro-1H-carbazole-1-carboxylic acid esters

As shown in Reaction Scheme 2, optionally substituted α-bromo-ketoesters are reacted with optionally substituted anilines in a given solvent or without a solvent at an appropriate temperature for a given time to yield of 2,3,4,9-tetrahydro-1H-carbazole-1- carboxylic acid esters. The reaction can be carried out either in a flask without a solvent by carefully mixing the two starting materials or in a microwave reaction system. The reaction can be carried out at elevated temperatures, for example at an oil bath temperature higher than 130° C. The products can be purified by standard procedures or may precipitate directly from a solution of the reaction mixture dissolved in an appropriate solvent upon cooling and may then be used in subsequent reactions without further purification.

Reaction Scheme 3:

Synthesis of 2,3,4,9-Tetrahydro-IH-carbazole-i-carboxylic acid amides:

As shown in Reaction Scheme 3, 2,3,4,9-tetrahydro-IH-carbazole-i-carboxylic acid- amides can be obtained from the corresponding esters by treatment with a inorganic base such as LiOH in a mixture of solvents such as water, THF and EtOH followed by the conversion to the carboxylic acid amide with a coupling reagent and NH₃ in water, dioxane or EtOH. Such compounds can then be purified by standard purification procedures such as flash chromatography or preparative HPLC.

Generally, after the reaction is complete, the solvent is evaporated and the reaction mixture can be diluted with an appropriate organic solvent, such as EtOAc or DCM, which is then washed with aqueous solutions, such as water, HCI, NaHSO₄, bicarbonate, NaH₂PO₄, phosphate buffer (pH 7), brine, Na₂CO₃ or any combination thereof. The combined organic solvents can then be concentrated and the reaction mixture purified by chromatography. The following examples are provided to illustrate the invention and are not limiting the scope of the invention in any manner.

Example 1

Synthesis of the intermediate 1,3-bromo-2-oxo-cyclohexane-carboxylic acid ethylester (i)

(1) Ethyl^-oxocyclohexanecarboxylate (10 g, 58.7 mmol) was dissolved in Et₂O and cooled with stirring to 0⁰C. Bromine was added dropwise over 15 min and the reaction was allowed to warm to room temperature over 90 min. The reaction mixture was poured into ice-cold saturated aqueous Na₂CO₃ and extracted with EtOAc (3x). The combined organic layers were dried (Na₂SO₄), the solvent was evaporated to yield the final product, 3-Bromo-2-oxo-cyclohexane-carboxylic acid ethylester (1) (14.33 g), which was used without further purification.

General procedure (I):

Synthesis of intermediate 2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid ethyl esters

In a flask or a sealable reaction vessel 3-bromo-2-oxo-cyclohexane-carboxylic acid ethylester (1 eq) and an aniline (2.5 eq) were mixed without solvent and the mixture was heated to 150° C in an oil bath for 30 min to 3h under argon. The reaction was monitored either by LCMS and/or TLC. After the heating was stopped, the reaction mixture was purified by flash-chromatography (AcOEt/cyclohexane) to yield the intermediate 2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid ethylesters. In the case of polysubstituted anilines the resulting mixtures of isomeric 2,3,4,9-tetrahydro-1 H- carbazoles were separated as described, if possible. In other cases the isomers were used without separation in the following steps and were separated after the last step via preparative HPLC.

Example 2: Synthesis of 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid ethylester (2)

(2) According to the general procedure (I)₁ 3-bromo-2-oxo-cyclohexane-carboxylic acid ethylester (800 mg) and 4-chloroaniline (1024 mg) were reacted for 3h to yield 6- chloro-2,3,4,9-tetrahydro-1 H-carbazole-1-carboxylic acid ethylester (2) (422 mg, 45% yield) after purification.

Example 3: Synthesis of β-chloro-S-fluoro^AS-tetrahydro-IH-carbazole-i- carboxylic acid ethylester (3) and 6-chloro-7-fluoro-2,3,4,9-tetrahydro-1H- carbazole-1-carboxylic acid ethylester (4)

(3) (4) According to the general procedure (I), 3-bromo-2-oxo-cyclohexane-carboxylic acid ethylester (1000 mg) and 4-chloro-3-fluoro-aniline (1455 mg) were reacted for 3h to yield a mixture of δ-chloro-S-fluoro^.S^.Θ-tetrahydro-IH-carbazole-i-carboxylic acid ethylester (3) and e-chloro-T-fluoro^.SAΘ-tetrahydro-IH-carbazole-i-carboxylic acid ethylester (4) (1250 mg) after purification. The two isomers could not be separated by column chromatography and were used as a mixture in the next step. Example 4: Synthesis of S-chloro-e-fluoro^AΘ-tetrahydro-IH-carbazole-i- carboxylic acid ethylester (5) and 7-chloro-6-fluoro-2,3,4,9-tetrahydro-1H- carbazole-1-carboxylic acid ethylester (6)

(5) (6)

According to the general procedure (I), 3-bromo-2-oxo-cyclohexanecarboxylic acid ethylester (544 mg) and 3-chloro-4-fluoro-aniline (795 mg) were reacted for 90 min to yield the two isomers 5-chloro-6-fluoro-2,3A9-tetrahydro-1H-carbazole-1 -carboxylic acid ethylester (5) (118mg) and 7-chloro-6-fluoro-2,3A9-tetrahydro-1H-carbazole-1- carboxylic acid ethylester (6) (213 mg) after column chromatography. The two isomers were used separately in the next steps.

Example 5: Synthesis of 5,6-dichloro-2,3,4,9-tetrahydro-1H-carbazole-1- carboxylic acid ethylester (7) and 6,7-dichloro -2,3,4,9-tetrahydro-1H-carbazole-1- carboxylic acid ethylester (8)

(7) (8)

According to the general procedure (I), 3-bromo-2-oxo-cyclohexane-carboxylic acid ethylester (500 mg) and 3,4-dichloroaniline (815 mg) were reacted for 2h to yield the two isomeric compounds 5,6-dichloro-2,3,4,9-tetrahydro-1H-carbazole-1 -carboxylic acid ethylester (7) (158 mg) and 6,7-dichloro-2,3A9-tetrahydro-1H-carbazole-1- carboxylic acid ethylester (8) (105 mg) after column chromatography. The two isomers were used separately in the next steps. General procedure (II):

Synthesis of 2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acids

In a flask the intermediate 2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid ethyl- esters were dissolved in a mixture of THF/EtOH/H₂O and a 0.5 M solution of LiOH was added. The reaction was stirred at room temperature for a given time and monitored by TLC or HPLC. After the reaction was finished, the solvent was removed by evaporation and the residue was extracted with DCM (2x). The combined organic layers were dried (Na₂SO₄) and the solvent was evaporated to yield the final product, which was used without further purification.

Example 6: Synthesis of 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid (9)

(9)

According to the general procedure (II) e-chloro^.SAΘ-tetrahydro-IH-carbazole-i- carboxylic acid ethylester (2) (422 mg) was converted to 6-chloro-2,3,4,9-tetrahydro- 1 H-carbazole-1-carboxylic acid (9), which was used directly in the next step. Example 7: Synthesis of 6-chloro-5-fluoro-2,3,4,9-tetrahydro-1 H-carbazole-1- carboxylic acid (10) and 6-chloro-7-fluoro-2,3,4,9-tetrahydro-1H-carbazole-1- carboxylic acid (11)

(10) (11 )

According to the general procedure (II) a mixture of the two isomers 6-chloro-5-fluoro- 2,3,4,9-tetrahydro-1H-carbazole-1 -carboxylic acid ethylester (3) and 6-chloro-7-fluoro- 2,3,4,9-tetrahydro-1H-carbazole-1 -carboxylic acid ethylester (4) (1250 mg) was converted to their corresponding acids β-chloro-δ-fluoro^.SΛΘ-tetrahydro-I H- carbazole-1 -carboxylic acid (10) and e-chloro-T-fluoro^.SAΘ-tetrahydro-IH-carbazole- 1 -carboxylic acid (11) (1000 mg total), which were used directly as a mixture in the next step.

Further compounds which were synthesized according to general procedure (II) are:

5-Chloro-6-fluoro-2,3A9-tetrahydro-1 H-carbazole-1 -carboxylic acid (12) 7-Chloro-6-fluoro-2,3 A9-tetrahydro-1 H-carbazole-1 -carboxylic acid (13) 5,6-Dichloro-2,3A9-tetrahydro-1 H-carbazole-1 -carboxylic acid (14) 6,7-Dichloro-2,3,4,9-tetrahydro-1 H-carbazole-1 -carboxylic acid (15)

General procedure (III):

Synthesis of 2,3,4,9-tetrahydro-1 H-carbazole-1 -carboxylic acid amides

In a flask the intermediate 2,3,4,9-tetrahydro-IH-carbazole-i-carboxylic acid was dissolved in dry DCM. If necessary, some dry DMF was added to dissolve the starting material. To this mixture, NHS (1.4 eq) and EDC (2 eq) were added and the reaction was stirred at room temperature for 1h. The formation of the intermediate NHS-ester was monitored by TLC and/or LCMS. The solution was then washed with H₂O₁ the aqueous layer was extracted with DCM. From the combined organic layers the solvent was removed in vacuo. The resulting residue was redissolved in THF and a solution of NH₃ in H₂O was added. The reaction mixture was stirred at room temperature for a given time. After the reaction was finished, the solvent was evaporated and the final product was purified by prep. HPLC.

Example 8: Synthesis of 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid amide (16)

(16) According to the general procedure (III), δ-chloro^.SAΘ-tetrahydro-IH-carbazole-i- carboxylic acid (9) (400 mg) was converted to 6-chloro-2,3,4,9-tetrahydro-1 H- carbazole-1-carboxylic acid-amide (16) (183 mg).

Example 9: Synthesis of 5-chloro-6-fluoro-2,3,4,9-tetrahydro-1H-carbazole-1- carboxylic acid amide (17)

(17)

According to the general procedure (III) 5-chloro-6-fluoro-2,3,4,9-tetrahydro-1 H- carbazole-1 -carboxylic acid (12) (97 mg) was converted to the corresponding 5-chloro- 6-fluoro-2,3,4,9-tetrahydro-1 H-carbazole-1 -carboxylic acid amide (17) (22 mg). Example 10: Synthesis of 7-chloro-6-fluoro-2,3,4,9-tetrahydro-1 H-carbazole-1- carboxylic acid amide (18)

(18)

According to the general procedure (III) 7-chloro-6-fluoro-2,3,4,9-tetrahydro-1 H- carbazole-1 -carboxylic acid (13) (187mg) was converted to the corresponding 7-chloro- 6-fluoro-2,3,4,9-tetrahydro-1 H-carbazole-1 -carboxylic acid-amide (18) (48 mg).

Example 11: Synthesis of 5,6-dichloro-2,3,4,9-tetrahydro-1 H-carbazole-1 - carboxylic acid amide (19)

(¹⁹)

According to the general procedure (III) 5,6-dichloro-2,3,4,9-tetrahydro-1 H-carbazole-1 - carboxylic acid (14) (110mg) was converted to the corresponding 5,6-dichloro-2, 3,4,9- tetrahydro-1 H-carbazole-1 -carboxylic acid amide (19) (86 mg).

Example 12: Synthesis of 6,7-dichloro-2,3,4,9-tetrahydro-1 H-carbazole-1 - carboxylic acid amide (20)

(20)

According to the general procedure (III) 6,7-dichloro-2,3,4,9-tetrahydro-1 H-carbazole-1 - carboxylic acid (15) (110 mg) was converted to the corresponding 6,7-dichloro -2,3,4,9- tetrahydro-1 H-carbazole-1 -carboxylic acid amide (20) (35 mg). Example 13: Synthesis of β-chloro-S-fluoro^.SAS-tetrahydro-IH-carbazole-i- carboxylic acid-amide (21) and 6-chloro-7-fluoro-2,3,4,9-tetrahydro-1H-carbazole- 1-carboxylic acid-amide (22)

(21 ) (22)

According to the general procedure (III) a mixture of 6-chloro-5-fluoro-2, 3,4,9- tetrahydro-1 H-carbazole-1-carboxylic acid (10) and 6-chloro-7-fluoro-2,3,4,9- tetrahydro-1 H-carbazole-1-carboxylic acid (11) was converted to the corresponding mixture of 6-chloro-5-fluoro-2,3,4,9-tetrahydro-1 H-carbazole-1-carboxylic acid amide (21) and β-chloro-T-fluoro^.S^.Θ-tetrahydro-I H-carbazole-i-carboxylic acid amide (22), which could not be separated by either column chromatography or preparative HPLC. The mixture was further resolved by chiral separation, as described on pages 26 to 27.

General procedure (IV): Chiral separation of 2,3,4,9-tetrahydro-1H-carbazole-1- carboxylic acid-amides

In order to obtain the pure enantiomers of the corresponding racemic 2,3,4,9- tetrahydro-1H-carbazole-1-carboxylic acid-amides the racemate was submitted to chiral separation using a given method (Method H or K) described on page 29.

The absolute stereochemistry of the compounds was not determined.

Example 14: 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid amides (16a) and (16b)

(16a) (16b) According to the general procedure (IV) chiral separation (Method H) of the racemic 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid amide gave the two enantiomers 16a and 16b. Analytical data: The two compounds were analysed (Method G) and had a retention time of 10.8 (purity > 90%) and 16.8 (purity > 90% ), respectively.

Example 15: 6-chloro-5 fluoro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid- amides (21a) and (21b)

(21a) (21 b)

According to the general procedure (IV) a chiral separation (Method K) of the racemic 6-chloro-5-fluoro-2,3,4,9-tetrahydro-1 H-carbazole-1-carboxylic acid amide mixture gave the two enantiomers 21a and 21b. Analytical data: The two compounds were analysed (Method J) and had a retention time of 9.3 (purity > 90%) and 11.9 ( purity > 90%), respectively.

Example 16: 6-chloro-7-fluoro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid amides (22a) and (22b)

(22a) (22b)

According to the general procedure (IV) a chiral separation (Method K) of the racemic 6-chloro-7-fluoro-2,3A9-tetrahydro-1H-carbazole-1-carboxylic acid amide gave the two enantiomers 22a and 22b. Analytical data: The two compounds were analysed (Method J), and had a retention time of 7.7 (purity > 90%) and 10.6 (purity > 90%), respectively.

Analytical LC-MS

The compounds of the present invention according to formula (I) were analyzed by analytical LC-MS. The conditions are summarized below.

Analytical conditions summary:

LC10Advp-Pump (Shimadzu) with SPD-M10Avp (Shimadzu) UV/Vis diode array detector and QP2010 MS-detector (Shimadzu) in ESI+ modus with UV-detection at 214, 254 and 275 nm,

Column: Waters XTerra MS C18, 3.5 μm, 2.1 * 100 mm, linear gradient with acetonitrile in water (0.15% HCOOH) Flow rate of 0,4 ml/min; Mobile Phase A: water (0.15% HCOOH) Mobile Phase B: acetonitrile (0.15% HCOOH)

Methods are:

A : linear gradient from 5% to 95% acetonitrile in water (0.1% HCOOH)

0.00 mm 5% B

5.00 min 95 % B

5.10 min 99 % B

6.40 min 99 % B

6.50 min 5 % B

8.00 min Pump STOP

B : linear gradient from 5% to 95% acetonitrile in water (0.1% HCOOH) 0.00 min 10% B

5.00 min 90 % B 5.10 min 99 % B 6.40 min 99 % B

6.50 min 5 % B

8.00 min Pump STOP

C : linear gradient from 5% to 95% acetonitrile in water (0.1% HCOOH) 0.00 min 5% B

10.00 min 95% B

10.10 min 99% B

11 .40 min 99% B I

11 .50 min 5% B

13 .00 min Pump STOP

D : start concentration 1 % acetonitrile

9.00 B.Conc 30

10 .00 B.Curve 3

12 .00 B.Conc 99

15. 00 B.Conc 99

15. 20 B.Conc 1

18.00 Pump STOP

E : start concentration 10% acetonitrile

10.00 B.Conc 60

11.00 B.Curve 2

12.00 B.Conc 99

15.00 B.Conc 99

15.20 B.Conc 10

18.00 Pump STOP

F : start concentration 15% acetonitrile 12.00 B.Conc 99 15.00 B.Conc 99 15.20 B.Conc 15

18.00 STOP 0

G;

Analytical conditions:

LC10Advp-Pump (Shimadzu) with SPD-M10Avp (Shimadzu) UV/Vis diode array detector and QP2010 MS-detector (Shimadzu) in ESI+ modus with UV-detection at 214, 254 and

220 nm, Column: Daicel CHIRALPAK AD-H 0.46cm *25cm

Gradient: lsocratic with 20% Ethanol (0.1% DEA) + 80% Heptan (0.1%DEA)

Flow rate: 0,6 ml/min

Runtime: 30 min

Mobile Phase A: Ethanol + 0.1% DEA Mobile Phase B: Heptan + 0.1% DEA

H;

Prep, conditions :

LC8A-Pump (Shimadzu) with SPD-M 10A vp (Shimadzu) UV/Vis diode array detector and QP2010 MS-detector in ESI+ modus with UV-detection at 214 and 254, 275 nm

Column: Daicel CHIRALPAK AD-H 2.0cm *25cm

Gradient: lsocratic with 20% Ethanol (0.1% DEA) + 80% Heptan (0.1%DEA)

Flow rate: 6 ml/min

Runtime: 40 min

J;

Analytical conditions:

LC10Advp-Pump (Shimadzu) with SPD-M 10A vp (Shimadzu) UV/Vis diode array detector and QP2010 MS-detector (Shimadzu) in ESI+ modus with UV-detection at 214, 254 and 220 nm,

Column: Daicel CHIRALPAK AD-H 0.46cm *25cm

Gradient: lsocratic with 80% Ethanol (0.1% DEA) + 20% Methanol (0.1%DEA)

Flow rate: 0,6 ml/min

Runtime: 30min Mobile Phase A: Ethanol + 0.1% DEA

Mobile Phase B: Methanol + 0.1 % DEA K;

Prep, conditions :

LC8A-Pump (Shimadzu) with SPD-M10Avp (Shimadzu) UVΛ/is diode array detector and

QP2010 MS-detector in ESI+ modus with UV-detection at 214 and 254, 275 nm

Column: Daicel CHIRALPAK AD-H 2.0cm *25cm

Gradient: lsocratic with 80% Ethanol (0.1% DEA) + 20% Methanol (0.1%DEA)

Flow rate: 6 ml/min

Runtime: 60 min

In table 1 below detailed examples of the invention are described which can be prepared via the reaction schemes 1 to 3.

The positions of the substituents (Pos 5-7) refer to the scheme shown below.

Table 1:

BIOLOGICAL ASSAYS

A. Enzymatic Assay - SIRT1 Deacetylase Activity Assay

The SIRT1 deacetylase activity assay is an assay system designed to measure the lysine deacetylase activity of recombinant human SIRT1. The assay procedure has two main steps. First, the Fluor-de Lys-SIRT1 substrate, which comprises the p53 sequence Arg-His-Lys-Lys(ε-acetyl), is incubated with human recombinant SIRT1 together with the cosubstrate NAD+. Deacetylation of Fluor de Lys-SIRT1 sensitizes it so that, in the second step, treatment with the Fluor de Lys™ developer (trypsin) produces a fluorophore. The fluorophore is excited with 360 nm light and the emitted light (460 nm) is detected on a fluorometric plate reader. NAD+ is consumed in the reaction to produce nicotinamide (NAM) and O-acetyl-ADP-ribose.

In detail: recombinant human SIRT1 is incubated for 60 min at 37°C with 25 μM of the indicated fluorogenic acetylated peptide substrate and 1.25 mM NAD+. Reactions were stopped by the addition of trypsin/2 mM nicotinamide and the deacetylation-dependent fluorescent signal was allowed to develop for 45 min. Fluorescence was then measured in the wells of a white microplate with a FARCyte fluorescence plate reader (Ex. 360 nm, Em. 450 nm, gain=24).

Table 2: Biological data for selected examples of the invention

In the table are listed the IC₅₀ values of the SIRT 1 enzymatic assay. The IC₅₀ values were grouped in 2 classes: a < 0.1 μM and 0.1 < b < 1 μM.

B. Cell based assay

Atrogin-1 levels in C2C12 myotubes

For the determination of atrogin-1 mRNA levels, C2C12 myoblasts were seeded at a density of 40O00 cells per well in 6-well plates previously coated with 0.1% gelatin. The cells were then grown for 2 days in growth medium containing 20% fetal calf serum. To initiate myotube differentiation, the growth medium was replaced by fusion medium containing 5% horse serum and the cells were incubated for another 4 days until myotubes formed.

Differentiated myotubes were then pre-treated with 10 ng/ml IGF-1 , 10 μM Compound 16 (6-Chloro-2,3,4,9-tetrahydro-1 H-carbazole-1-carboxylic acid amide) or solvent only (1% DMSO) for 1 hour. After this pre-incubation, myotube atrophy was induced by adding 10 μM of the glucocorticoid dexamethasone (Dex) and the cultures were incubated for another 24 hours.

At the end of this treatment the cultures were rinsed once with PBS and total RNA was extracted. For each culture, 0.5 μg RNA were then reversed transcribed and atrogin-1 mRNA levels were determined relative to the 18 S mRNA levels using real-time PCR. For each treatment condition atrogin-1 mRNA levels were quantified in three separate cultures. The results are shown in Figure 1.

As was also shown by others (Sandri et al., Cell 117, 2004, 399-412) treatment of differentiated myotubes with the glucocorticoid dexamethasone (Dex) induces atrogin-1 mRNA levels 2-fold. This increase in atrogin-1 ^mRNA levels can be suppressed by co- treatment with 10 ng/ml of IGF-1 to the level measured in solvent treated myotubes. IGF-1 is a protein which acts through the IGF-Akt pathway.

As can be taken from Figure 1 , co-treatment of differentiated myotubes with compound 16 (6-Chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid amide, 10μM) for 1h also suppresses the increase in atrogin-1 mRNA in Dex treated myotubes to the level measured in solvent treated myotubes or IGF-1 treated myotubes.

C. In vivo assay

1) In vivo starvation experiment Food deprivation/starvation is known to induce the transcription of the atrophy relevant atrogin-1 messenger RNA (Sandri et al., Cell 117, 2004, 399-412). In a typical setup 2- month-old female C57/BI6 mice arestarved for 1 day. The food is removed at 8.00 am and the mice are sacrificed on the next day at 2.00 pm. Total RNA is isolated from the medial gastrocnemius muscles (using the RNeasy Fibrous Tissue Mini Kit from Qiagen). From each muscle 0.5 μg RNA is reversed transcribed and atrogin-1 mRNA levels can be determined relative to the 18S mRNA levels using real-time PCR. Four groups of mice can be included in this analysis: untreated non-starved mice, compound treated non-starved mice, untreated starved mice and compound treated starved mice. The bi-daily treatment typically starts on the day before the food deprivation and the last application takes place one hour before the mice are sacrificed. As vehicle 10% Cyclodextrin 4% DMSO in PBS can be used. A volume of 10 micro liters per gram bodyweight can be applied.

D. In vitro ADME Assays

1. Microsomal Stability

Experimental Procedure

Pooled human liver microsomes (pooled male and female) and pooled rat liver microsomes (male Sprague Dawley rats) are prepared. Microsomes are stored at - 80⁰C prior to use.

Microsomes (final concentration 0.5 mg/ml), 0.1 M phosphate buffer pH7.4 and test compound (final substrate concentration = 3 μM; final DMSO concentration = 0.25%) are pre-incubated at 37°C prior to the addition of NADPH (final concentration = 1 mM) to initiate the reaction. The final incubation volume is 25 μl. A control incubation is included for each compound tested where 0.1 M phosphate buffer pH7.4 is added instead of NADPH (minus NADPH). Two control compounds are included with each species. All incubations are performed singularly for each test compound.

Each compound is incubated for O₁ 5, 15, 30 and 45 min. The control (minus NADPH) is incubated for 45 min only. The reactions are stopped by the addition of 50 μl methanol containing internal standard at the appropriate time points. The incubation plates are centrifuged at 2,500 rpm for 20 min at 4°C to precipitate the protein.

Quantitative Analysis

Following protein precipitation, the sample supernatants are combined in cassettes of up to 4 compounds and analysed using generic LC-MS/MS conditions. Data Analysis

From a plot of the peak area ratio (compound peak area/internal standard peak area) against time, the gradient of the line is determined. Subsequently, half-life and intrinsic clearance are calculated using the equations below:

Elimination rate constant (k) = (- gradient)

0.693 Half life (t_1/2) (min) = k

VxO.693

Intrinsic Clearance (CL_int) (μl/min/mg protein) = ^tl/2 where V = Incubation volume μl/mg microsomal protein.

Two control compounds are included in the assay and if the values for these compounds are not within the specified limits the results are rejected and the experiment repeated.

2. Hepatocyte Stability

Experimental Procedure

Suspensions of cryopreserved hepatocytes are used for human hepatocyte stability assay (pooled from 3 individuals). All cryopreserved hepatocytes are purchased from In Vitro Technologies, Xenotech or TCS.

Incubations are performed at a test or control compound concentration of 3 μM at a cell density of 0.5x10⁶ viable cells/mlL The final DMSO concentration in the incubation is 0.25%. Control incubations are also performed in the absence of cells to reveal any non-enzymatic degradation.

Duplicate samples (50 μl) are removed from the incubation mixture at 0, 5, 10, 20, 40 and 60 min (control sample at 60 min only) and added to methanol, containing internal standard (100 μl), to stop the reaction. Tolbutamide, 7-hydroxycoumarin, and testosterone are used as control compounds. The samples are centrifuged (2500 rpm at 4⁰C for 20 min) and the supernatants at each time point are pooled for cassette analysis by LC-MS/MS using generic methods. Data Analysis

From a plot of In peak area ratio (compound peak area/internal standard peak area) against time, the gradient of the line is determined. Subsequently, half-life and intrinsic clearance are calculated using the equations below:

Elimination rate constant (k) = (- gradient)

0.693 Half life (t_1/2)(min) = k

Intrinsic Clearance (CL_int)(μl/min/million cells) = ^tl/2 where V = Incubation volume (μl)/number of cells

3. Caco-2 Permeability (Bi-directional)

Experimental Procedure

Caco-2 cells obtained from the ATCC at passage number 27 are used. Cells (passage number 40-60) are seeded on to Millipore Multiscreen Caco-2 plates at 1 x 105 cells/cm². They are cultured for 20 days in DMEM and media is changed every two or three days. On day 20 the permeability study is performed.

Hanks Balanced Salt Solution (HBSS) pH7.4 buffer with 25 mM HEPES and 10 mM glucose at 37°C is used as the medium in permeability studies. Incubations are carried out in an atmosphere of 5% CO₂ with a relative humidity of 95%. On day 20, the monolayers are prepared by rinsing both basolateral and apical surfaces twice with HBSS at 37⁰C. Cells are then incubated with HBSS in both apical and basolateral compartments for 40 min to stabilize physiological parameters. HBSS is then removed from the apical compartment and replaced with test compound dosing solutions. The solutions are made by diluting 10 mM test compound in DMSO with HBSS to give a final test compound concentration of 10 μM (final DMSO concentration 1 %). The fluorescent integrity marker lucifer yellow is also included in the dosing solution. Analytical standards are made from dosing solutions. Test compound permeability is assessed in duplicate. On each plate compounds of known permeability characteristics are run as controls. The apical compartment inserts are then placed into 'companion' plates containing fresh HBSS. For basolateral to apical (B-A) permeability determination the experiment is initiated by replacing buffer in the inserts then placing them in companion plates containing dosing solutions. At 120 min the companion plate is removed and apical and basolateral samples diluted for analysis by LC-MS/MS. The starting concentration (C₀) and experimental recovery is calculated from both apical and basolateral compartment concentrations.

The integrity of the monolayers throughout the experiment is checked by monitoring lucifer yellow permeation using fluorimetric analysis. Lucifer yellow permeation is low if monolayers have not been damaged. Test and control compounds are quantified by LC-MS/MS cassette analysis using a 5-point calibration with appropriate dilution of the samples. Generic analytical conditions are used.

If a lucifer yellow P_app value is above QC limits in one individual test compound well, then an n=1 result is reported. If lucifer yellow P_app values are above QC limits in both replicate wells for a test compound, the compound is re-tested. Consistently high lucifer yellow permeation for a particular compound in both wells indicates toxicity. No further experiments are performed in this case.

Data Analysis

The permeability coefficient for each compound (P_app) is calculated from the following equation:

C₀ X A

Where dQ/dt is the rate of permeation of the drug across the cells, C₀ is the donor compartment concentration at time zero and A is the area of the cell monolayer. C₀ is obtained from analysis of donor and receiver compartments at the end of the incubation period. It is assumed that all of the test compound measured after 120 min incubation was initially present in the donor compartment at 0 min. An asymmetry index (Al) is derived as follows:

Al = P_apP (B-A) P_app (A-B) An asymmetry index above unity shows efflux from the Caco-2 cells, which indicates that the compound may have potential absorption problems in vivo.

The apparent permeability (P_app (A-B)) values of test compounds are compared to those of control compounds, atenolol and propranolol, that have human absorption of approximately 50 and 90% respectively (Zhao, Y.H., et a/., (2001). Evaluation of Human Intestinal Absorption Data and Subsequent Derivation of a Quantitative Structure-Activity Relationship (QSAR) with the Abraham Descriptors. Journal of Pharmaceutical Sciences. 90 (6), 749-784). Talinolol (a known P-gp substrate (Deferme, S., MoIs, R., Van Driessche, W., Augustijns, P. (2002). Apricot Extract Inhibits the P-gp-Mediated Efflux of Talinolol. Journal of Pharmaceutical Sciences. 91(12), 2539-48)) is also included as a control compound to assess whether functional P-gp is present in the Caco-2 cell monolayer.

4. Cytochrome P450 Inhibition (5 lsoform IC₅₀ Determination))

Experimental Procedure

CYP1A Inhibition Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.35%) are incubated with human liver microsomes (0.25 mg/ml) and NADPH (1 mM) in the presence of the probe substrate ethoxyresorufin (0.5 μM) for 5 min at 37°C. The selective CYP1A inhibitor, alpha-naphthoflavone, is screened alongside the test compounds as a positive control. CYP2C9 Inhibition

Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (1 mg/ml) and NADPH (1 mM) in the presence of the probe substrate tolbutamide (120 μM) for 60 min at 37°C. The selective CYP2C9 inhibitor, sulphaphenazole, is screened alongside the test compounds as a positive control. CYP2C19 Inhibition

Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (0.5 mg/ml) and NADPH' (1 mM) in the presence of the probe substrate mephenytoin (25 μM) for 60 min at 37⁰C. The selective CYP2C19 inhibitor, tranylcypromine, is screened alongside the test compounds as a positive control.

CYP2D6 Inhibition

Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (0.5 mg/ml) and

NADPH (1 mM) in the presence of the probe substrate dextromethorphane (5 μM) for

30 min at 37°C. The selective CYP2D6 inhibitor, quinidine, is screened alongside the test compounds as a positive control.

CYP3A4 Inhibition Six test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration 0.26%) are incubated with human liver microsomes (0.25 mg/ml) and

NADPH (1 mM) in the presence of the probe substrate midazolam (2.5 μM) for 5 min at

37°C. The selective CYP3A4 inhibitor, ketoconazole, is screened alongside the test compounds as a positive control.

For the CYP1A incubations, the reactions are terminated by the addition of methanol, and the formation of the metabolite, resorufin, is monitored by fluorescence (excitation wavelength = 535 nm, emission wavelength = 595 nm). For the CYP2C9, CYP2C19, CYP2D6, and CYP3A4 incubations, the reactions are terminated by the addition of methanol containing internal standard. The samples are then centrifuged, and the supematants are combined, for the simultaneous analysis of 4-hydroxytolbutamide, 4- hydroxymephenytoin, dextrorphan, and 1 -hydroxymidazolam plus internal standard by LC-MS/MS. Generic LC-MS/MS conditions are used. Formic acid in deionised water (final concentration = 0.1%) is added to the final sample prior to analysis. A decrease in the formation of the metabolites compared to vehicle control is used to calculate an IC₅O value (test compound concentration which produces 50% inhibition).

5. Plasma Protein Binding (10%)

Experimental Procedure

Solutions of test compound (5 μM, 0.5% final DMSO concentration) are prepared in buffer (pH 7.4) and 10% plasma (v/v in buffer). The experiment is performed using equilibrium dialysis with the two compartments separated by a semi-permeable membrane. The buffer solution is added to one side of the membrane and the plasma solution to the other side. Standards are prepared in plasma and buffer and are incubated at 37°C. Corresponding solutions for each compound are analyzed in cassettes by LC-MS/MS.

Quantitative Analysis

After equilibration, samples are taken from both sides of the membrane. The solutions for each batch of compounds are combined into two groups (plasma-free and plasma- containing) then cassette analyzed by LC-MS/MS using two sets of calibration, standards for plasma-free (7 points) and plasma-containing solutions (6 points). Generic LC-MS/MS conditions are used. Samples are quantified using standard curves prepared in the equivalent matrix. The compounds are tested in duplicate. A control compound is included in each experiment. Data Analysis

fu = 1 - (( PC - PF))

(PC) fu = fraction unbound

PC = sample concentration in protein containing side PF = sample concentration in protein free side fu at 10% plasma is converted to fu 100% plasma using the following equation:

fUioo% ⁼ f U 10%

Claims

1. Use of a compound according to formula (I):

wherein each of R¹ and R² is, independently from each other, halo, hydroxy, C₁-Ci₀ alkyl,

CrC₆ haloalkyl, C1-C10 alkoxy, Ci-C₆ haloalkoxy, Z-C₆-Ci₀ aryl, Z-C₅-Ci₀ heteroaryl,

Z-C₃-C₈ heterocyclyl, Z-C₃-C₈ cycloalkyl, C₂-Ci₂ alkenyl, C₂-Ci₂ alkynyl, Z-C₅-Ci₀ cycloalkenyl, Z-C₅-Ci₀ heterocycloalkenyl, carboxy, carboxylate, cyano, nitro, amino, Ci-C₆ alkyl amino, d-C₆ dialkyl amino, mercapto, S(O)NH₂, S(O)₂NH₂,

Ci-C₄ alkylenedioxy, oxo, acyl, aminocarbonyl, C₁-C₁Q alkoxycarbonyl, hydrazine- carbonyl, CrC₆ alkyl hydrazinocarbonyl, Ci-C₆ dialkyl hydrazinocarbonyl, hydroxyamino carbonyl, or alkoxyamino carbonyl, wherein each alkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkyl and cycloalkenyl is optionally substituted by one or more R³; and wherein each aryl and heteroaryl is optionally substituted with one or more R⁴;

R³ is independently from each other fluoro, methyl, methoxy, hydroxy or amino;

R⁴ is independently from each other fluoro, chloro, methyl, methoxy, cyano, hydroxy, amino, CF₃, CHF₂, CH₂F, OCF₃, OCHF₂ or OCH₂F;

Z is a bond or Ci-C₆ alkylene, wherein the CrC₆ alkylene group is optionally substituted with one or more fluorine atoms; m is 1 , 2, 3 or 4; n is 1 , 2, 3 or 4; and p is O, 1 , 2 or 3 or its tautomeric, enantiomeric, diastereomeric or pharmaceutically acceptable salts thereof for the preparation of a medicament for the prophylaxis and/or treatment of muscular atrophy.

2. The use according to claim 1 , wherein R¹ is halo, Ci-C₆ alkyl, Z-C₆-Ci₀ aryl, Z-C₅- Cio heteroaryl or Z-C₃-C₈ cycloalkyl, wherein alkyl, and cycloalkyl are optionally substituted with one or more R³ and wherein aryl and heteroaryl are optionally substituted with one or more R⁴.

3. The use according to claim 1 or 2, wherein R¹ is halo or Z-cyclopropyl optionally substituted with one or more R³.

4. The use according to any one of claims 1 to 3, wherein R² is SO₂NH₂ or aminocarbonyl.

5. The use according to any one of claims 1 to 4, wherein p is 1 or 2.

6. Use of sirtuin modulators for regulating atrogin levels in a mammal.

7. Use of sirtuin modulators for the preparation of a medicament for the prophylaxis and/or treatment of disorders associated with elevated atrogin levels.

8. The use according to claim 7 for the prophylaxis and/or treatment of muscular atrophy.