NZ621089B2

NZ621089B2 - Benzothiazolone compound

Info

Publication number: NZ621089B2
Application number: NZ621089A
Authority: NZ
Inventors: Jun Cao; Bernhard Erb; Robin Alec Fairhurst; Arnaud Grandeury; Shinji Hatakeyama; Magdalena Koziczakholbro; Xinzhong Lai; Philipp Lustenberger; Bernd Riebesehl; Nicola Tufilli; Holbro Magdalena Koziczak
Original assignee: Novartis Ag
Priority date: 2011-09-06
Filing date: 2012-09-05
Publication date: 2015-11-03

Abstract

Provided is the benzothiazolone compound (R)-7-(2-(1-(4-butoxyphenyl)-2-methylpropan-2-ylamino)-1-hydroxyethyl)-5-hydroxybenzo[d]thiazol-2(3H)-one. The compound is a beta-2 adrenoreceptor agonist. The compound may be useful in the treatment of muscle wasting diseases such as muscular dystrophy, disuse-related atrophy, cachexia or sarcopenia. se-related atrophy, cachexia or sarcopenia.

Description

PCT/IBZOlZ/054580 Benzothiazolone nd The present invention relates to a benzothiazolone compound, to its preparation, to its medical use as a beta-2 adrenoceptor agonist and to medicaments, pharmaceutical compositions and combinations comprising it.

Benzothiazolone compounds which are beta—2—adrenoceptor agonists are described in /16601 and W02006/056471. WOZOOS/HOQQO also describes benzo—condensed heterocycles as beta-2 agonists.

While beta-2 agonists have long been known for their bronchodilating properties, they are also known for their capability to produce al muscle hypertrophy.

Numerous studies have focused on therapeutic applications of the anabolic properties of beta-2 agonists for ameliorating muscle wasting and improving muscle function. However, this class of nds has also been associated with undesirable side-effects, including sed risk of adverse cardiovascular-related events. Thus, the use of beta-2 agonists in muscle wasting diseases has hitherto been limited by cardiac hypertrophy and potentially deleterious effects on cardiovascular function.

There is a need to provide new beta-2 agonists that are good drug candidates. in particular, a new beta-2 agonist should bind potently to the beta-2 adrenoceptor whilst showing little affinity for other receptors, such as e.g. the beta-1 adrenoceptor, the 1A adrenoceptor, or the 5HTZC receptor, and show functional activity as an agonist. It should be metabolically stable and possess favourable cokinetic properties. It should be non—toxic and demonstrate few side—effects, in particular fewer cardiac ffects than known marketed beta-2 agonists, such as e.g. formoterol. Furthermore, the ideal drug candidate will exist in a physical form that is stable, non-hygroscopic and easily formulated.

The nd of the invention is a selective beta-2 t. in particular, it shows an increased afﬁnity for the beta-2 adrenoceptor which is greater than its afﬁnity for the beta—1 adrenoceptor or the alpha-1A adrenoceptor, compared to known beta—2 agonists such as formoterol. Surprisingly, it also shows a lower afﬁnity for the serotonin receptor (5HT2C) and lower onal potency in 5HT2C expressing cells than its racemate or its ponding enantiomer, indicating that it does not affect tor activity and food intake which may cause body weight reduction, potentially counteracting beta—2 agonist-induced al muscle hypertrophy. The negative effects of 5HT2C receptor agonists on energy intake and body weight are described by J. Halford and J. Harrold in Handb Exp Pharmacol. 2012; (209) 349-56.

The compound of the present invention is therefore potentially useful in the treatment of a wide range of disorders, particularly in the treatment or prevention of muscle-wasting diseases such as muscular dystrophy, disuse-related atrophy, cachexia 0r sarcopenia.

The treatment of ia is also a contemplated use. All forms of cachexia are potentially treatable with the compounds of the present invention, including cancer ia for example.

In a ﬁrst aspect of the invention, there is therefore provided a compound of formula (l) in free form or in pharmaceutically acceptable salt form which is /\—_o 1O (1)- The compound of the ion is (R)(2-(1~(4—butoxyphenyl)—2—methylpropanylamino)—1— hydroxyethyl)—5—hydroxybenzo[d]thiazol—2(3H)-one.

The following are embodiments of the ion: Embodiment 1: A compound according to the first aspect of the invention. ment 2: A compound according to ment 1 which is (R)—7—(2—(1-(4— butoxyphenyl)—2-methylpropan-2—ylamino)—1-hydroxyethyl)-5—hydroxybenzo[d]thiazol-2(3H)- one in free form.

Embodiment 3: A compound ing embodiment 1 which is (R)-7—(2—(1-(4-butoxyphenyl)- 2-methylpropan-2—ylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)-one in acetate salt form.

Embodiment 4: A compound according to embodiment 1 which is (R)(2-(1-(4- butoxyphenyl)methylpropan—2—ylamino)—1-hydroxyethy|)—5—hydroxybenzo[d]thiazol-2(3H)- one in glycolate salt form.

WO 35047 Embodiment 5: A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of embodiments 1 to 4 and one or more pharmaceutically acceptable carriers.

Embodiment 6: A pharmaceutical composition according to embodiment 5 wherein one of the pharmaceutically acceptable carriers is benzyl alcohol.

Embodiment 7: A combination comprising a therapeutically effective amount of a compound ing to any one of embodiments 1 to 4 and one or more eutically active co- agents.

Embodiment 8: A method of treatment or prevention of muscular dystrophy, disuse—reiated 1O y, cachexia or sarcopenia comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 4 to a subject in need thereof.

Embodiment 9: A method according to embodiment 8, n the compound is administered by subcutaneous infusion or injection.

Embodiment 10: A compound according to according to any one of embodiments 1 to 4 for use as a medicament. ment 11: A compound according to any one of embodiments 1 to 4 for use in the treatment or prevention of muscular dystrophy, disuse-related y, cachexia or sarcopenia.

Embodiment 12: Use of a compound according to any one of embodiments 1 to 4 in the manufacture of a medicament for the ent or prevention of muscular dystrophy, disuse~ related atrophy, cachexia or sarcopenia.

Embodiment 13: A process for the manufacture of a compound of a (I) in free form or in pharmaceutically acceptable salt form which includes the steps of: a) the reaction of a compound of formula (ila) in free form or in pharmaceutically acceptable salt form i>_ORb (Ha) in which R61 and Rb are protecting groups with 2-(4-butoxy—phenyI)—1,1~ dimethyl-ethylamine; b) the cleavage of protecting groups optionally present; 0) the ry of the so obtainable compound of formula (I) in free form or in pharmaceutically acceptable salt form.

Embodiment 14: A process for the manufacture of a nd of formula (l) in free form or in pharmaceutically acceptable salt form according to embodiment 13 in which compound (Ila) is obtained by the reaction of a compound of formula (Illa) in free form or in ceutically acceptable salt form R o N (Illa) in which in which Ra and Rb are protecting groups and LG is a leaving group with a base and optionally a phase transfer st.

Embodiment 15: A process according to embodiment 14, wherein the base is potassium ate.

Embodiment 16: A process according to embodiment 14, wherein the base is sodium hydroxide.

Embodiment 17: A process according to any of embodiments 14 to 16, wherein the phase transfer catalyst is tetra-butylammonium iodide.

PCT/lBZOl2/054580 Embodiment 18: A process for the manufacture of a nd of formula (I) in free form or in pharmaceutically acceptable salt form according to embodiments 14 to 17 in which compound (llla) is obtained by the stereoselective reduction of a nd of formula (Na- 2) in free form or in pharmaceutically acceptable salt form />_0Rb RO N (Na-2) in which R3 and Rb are ting groups and LG is a leaving group.

Embodiment 19: A process according to embodiment 18 wherein the stereoselective reduction is carried out with [N—[(1S,ZS)(Amino-KN)-1,2-diphenylethyl]—4- methylbenzenesulfonamidato-KMchloro[(1,2,3,4,5,6—n)methyl(1-methy|ethyl)benzene]- ruthenium (RuCl(p-cymene)[(S,S)-Ts—DPEN]). ment 20: A process according to embodiment 18 or 19 wherein LG is chloro.

Embodiment 21: A process according to embodiment 20 in which compound (lVa’—2) in free form or in pharmaceutically acceptable salt form )_0Rb RD N (lVa‘-2) is ed by the reaction of a compound of formula (Va) in free form or in pharmaceutically acceptable salt form W0 2013i035047 R o N ORb (V3) in which R81 and Rb are protecting groups and Hal is a halogen with 2—chloro—N-methoxy-N- methyl-acetamide in the presence of a strong base.

Embodiment 22: A process according to embodiment 21, wherein the strong base is tert- butyllithium.

Embodiment 23: A process for the cture of a compound of formula (I) in free form or in pharmaceutically acceptable salt form which includes the steps of: a) the reaction of a compound of formula (llla) in free form or in ceutically acceptable salt form />_0Rb RD N (Na) in which Ra and Rb are protecting groups and LG is a leaving group with 2-(4- butoxy—phenyl)-1,1—dimethyl-ethylamine; b) the cleavage of any protecting groups still present; 0) the recovery of the so obtainable compound of formula (I) in free form or in pharmaceutically acceptable salt form.

Embodiment 24: A process ing to embodiment 23, n LG is chloro or p- toluenesulfonyl.

Embodiment 25: A process according to any of embodiments 13 to 24 wherein Ra is tert- butyl.

W0 2013f035047 Embodiment 26: A process according to any of embodiments 13 to 25, wherein Rb is isopropyl.

Embodiment 27: A compound of formula (la) in free form or in pharmaceutically acceptable salt form ,>_0Rb ('8) in which Ra and R, are protecting groups.

Embodiment 28: A compound of formula (Ila) in free form or pharmaceutically acceptable salt form (Ila) in which R8 and R, are protecting groups. ment 29: A compound of formula 2) in free form or pharmaceutically acceptable salt form W0 2013f035047 />_0Rb R o N (Illa-2) in which Ra and RD are protecting groups. ment 30: A compound of formula (IVa—2) in free form or pharmaceutically able salt form />_0Rb R o N (lVa-Z) in which Ra and RD are protecting groups and LG is a leaving group.

Embodiment 31: A compound according to embodiment 30, n LG is chloro.

Embodiment 32: A compound according to any of embodiments 27 to 31, wherein Ra is tert- butyl.

Embodiment 33: A compound according to any of embodiments 27 to 32, wherein Rb is isopropyl.

Brief description of figures Figure 1 shows the skeletal muscle mass and heart mass increase in rats ed with formoterol vs compound A (compound of the invention) - (values are expressed as means 1- SEM (n=5-6); pool of skeletal muscles (gastrocnemius-soleus-tlbialis) ized by initial body weight; heart weight normalized by brain weight.

Figure 2a shows the increase of beating rate in isolated rabbit sino—atrial nodes when using formoterol vs compound A (compound of the invention).

W0 2013f035047 PCT/IBZOlZ/054580 Figure 2b shows the increase of pacemaker activity in isolated rabbit hearts when using formoterol vs compound A (compound of the invention).

Figures 3a and 3b show the heart rate change in rats upon a 3.0. bolus injection of Compound A (compound of the invention) or formoterol respectively.

Figure 3c compares the average heart rate change in rats when administering formoterol vs compound A (compound of the ion).

Figure 4a and 4b show the heart rate change in rhesus monkeys upon a 3.0. bolus injection of Compound A (compound of the invention) or erol respectively.

Figure 5 shows the X—ray powder diffraction pattern of crystalline free base Compound A (compound of the invention).

Figure 6 shows the X—ray powder diffraction pattern of the crystalline acetate salt of Compound A und of the ion).

Figure 7 shows the X—ray powder diffraction pattern of the crystalline glycolate salt of nd A und of the invention).

Unless specified otherwise, the term “compound of the present invention 1! it , compound of the invention” or “compound A” refers to the compound of formula (I), salts of the compound, hydrates or es of the compound or salts, as well as tautomers and isotopically labeled compounds (including deuterium substitutions). The compound of the t invention further comprises polymorphs of the compound of formula (l) and salts thereof.

As used herein, the term “halogen” or “halo” refers to fluoro, chloro, bromo, and iodo.

As used herein, the absolute stereochemistry is specified according to the Cahn— lngold- Prelog R—S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon may be speciﬁed by either R or 8. Resolved compounds whose absolute configuration is unknown can be ated (+) or (-) depending on the direction o- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line.

Any asymmetric atom (e.g., carbon or the like) of a compound can be present in racemic or enantiomerically enriched, for example the (R)—, (S)— or (R,S)- configuration. The racemic 50:50 mixture of stereoisomers is designated as (RS) and omerically ed forms by the enantiomeric excess of (R) to (8) respectively or (S) to (R) forms. The enantiomeric excess is represented usually by the equation ee = ((mt-m2)/(m1+m2))*100% where m1 and m2 represent the mass of the respective enatiomeric forms R and S.

W0 2013!035047 The compound of the present invention contains one asymmetric centre which is defined in terms of absolute stereochemistry as (R). Its corresponding enantiomer is defined as (S) which is the less active form. in certain embodiments of the ion, the asymmetric atom has at least 95, 98 or 99 % enantiomeric excess in the (R)- configuration.

Thus in one embodiment of the invention, there is provided (R)—7—(2—(1-(4-butoxyphenyI)—2— methylpropan-2—ylamino)—1~hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3 H)—on e, or a pharmaceutically acceptable salt thereof (for example an acetate or glycolate salt thereof), in at least 95 % enantiomeric excess. 1O In another embodiment of the invention, there is provided (2—(1-(4-butoxyphenyl)—2— methylpropanylamino)—1 -hydroxyethyl)~5-hyd roxybenzo[d]th iazoI-2(3 , or a pharmaceutically acceptable salt thereof (for example an e or glycolate salt thereof), in at least 98 % enantiomeric . in yet another embodiment of the ion, there is provided (R)—7-(2-(1-(4—butoxyphenyl)—2- methylpropan-Z-ylamino)hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)-one, or a pharmaceutically acceptable salt f (for example an acetate or ate salt thereof), in at least 99 % enantiomeric excess. in one embodiment of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of (R)-7—(2-(1~(4-butoxyphenyI)-2— methylpropan-Z—ylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazoI-2(3 H)—one, or a pharmaceutically acceptable salt thereof (for example an acetate or ate salt thereof), and one or more pharmaceutically able carriers wherein the (R)—7—(2-(1-(4— butoxyphenyl)methylpropan—2—ylamino)—‘l-hydroxyethy|)hydroxybenzo[d]thiazol-2(3H)~ one, or pharmaceutically acceptable salt thereof, is present in at least 95 % enantiomeric excess.

In another embodiment of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of (R)—7-(2-(1-(4-butoxypheny|)—2- propanylamino)hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)-one, or a pharmaceutically acceptable salt thereof (for e an acetate or glycolate salt thereof), and one or more pharmaceutically acceptable carriers wherein the (R)(2-(1-(4— butoxyphenyl)—2—methylpropan—2—ylamino)—1-hydroxyethyl)hyd roxybenzo[d]thiazol-2(3H)- one, or pharmaceutically acceptable salt thereof, is present in at least 98 % enantiomeric EXCESS.

In yet another embodiment of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of (R)(2-(1-(4-butoxypheny|)-2— methylpropan-Z-ylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)-one, or a pharmaceutically acceptable salt thereof (for example an acetate or ate salt thereof), and one or more pharmaceutically acceptable carriers wherein the (R)(2-(1-(4- butoxyphenyl)methylpropany|amino)—1-hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)- one, or pharmaceutically acceptable salt thereof, is present in at least 99 % enantiomeric EXCESS.

The compound of the present ion contains one tric centre which is defined in terms of te stereochemistry as (R). its corresponding enantiomer is defined as (8).

Depending on the choice of the starting materials and procedures for the chemical synthesis, compounds can be present in the form of one of the possible isomers or as mixtures thereof, for example as pure optical s, or as isomer mixtures, such as racemates. Optically active (R)— and (S)— isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. All tautomeric forms of the compound of the present invention are intended to be included.

Accordingly, as used herein the compound of the present invention can be in the form of tautomers or es f.

Any resulting tes of final products or synthesis intermediates can be resolved into the optical antipodes by known methods, e.g., by separation of the diastereomeric salts thereof, obtained with an optically active acid or base, and liberating the lly active acidic or basic compound. In particular, a basic moiety may thus be employed to resolve the compound of the present invention into its optical antipodes, e.g., by fractional crystallization of a salt formed with an optically active acid, e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di~0,0'—p—toluoy| tartaric acid, mandelic acid, malic acid or camphor—tO- sulfonic acid. c or enantiomerically enriched products can also be resolved by chiral chromatography, e.g., high pressure liquid chromatography (HPLC) using a chiral adsorbent.

As used herein, the terms “salt” or ” refers to an acid addition or base on salt of the nd of the invention. “Salts” e in particular “pharmaceutically able salts”. The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compound of this invention and, which typically are not biologically or otherwise undesirable. The compound of formula I of the present invention is capable of forming a characteristic salt with a defined acid by virtue of the presence of a basic amino group in the side chain. It is also capable to form characteristic salts with defined bases by virtue of the presence of two acidic groups (phenol; lone ring) in the heterocylic moiety.

Pharmaceutically acceptable acid addition salts may be formed with inorganic acids and organic acids, e.g., acetate, aspartate, te, te, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, glycolic, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, naphthoate, napsylate, nate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate salts. in one embodiment of the ion, there is provided (2—(1-(4—butoxyphenyl) methylpropan-2—ylamino)hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)—one in acetate, benzoate, camphorate, fumarate, glycolate, lactate, malonate, te, succinate, sulfate, tartrate or xinafoate salt form. ln one particular embodiment of the invention, there is provided (R)-7—(2—(1-(4—butoxyphenyl)— 2-methylpropan-2—ylamino)—1—hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)-one in acetate salt form. in r particular embodiment of the invention, there is provided (R)-7—(2-(1—(4- butoxyphenyl)—2-methylpropan—2—ylamino)—1—hydroxyethyl)—5—hyd roxybenzo[d]thiazol-2(3H )- one in glycolate salt form.

Inorganic acids from which salts may be derived include, for e, hydrochloric acid, romic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids from which salts may be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, ic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, alicylic acid, and the like.

Pharmaceutically acceptable base addition salts may be formed with nic and organic bases. inorganic bases from which salts may be derived include, for example, ammonium salts and metals from columns | to XII of the periodic table. in certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, ium and iron; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts. c bases from which salts may be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines e isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, zine and tromethamine.

The pharmaceutically acceptable salts of the present invention can be synthesized from a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be 1O prepared by reacting free acid forms of the nd with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like), or by reacting free base forms of the compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, use of ueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, where cable. Lists of additional suitable salts can be found, e.g., in “Remington's Pharmaceutical Sciences”, 20th ed., Mack hing Company, Easton, Pa., (1985); and in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley—VCH, Weinheim, Germany, 2nd revised edition, 2011).

Furthermore, the compound of the present invention, including its salts, may also be obtained in the form of its hydrates, or include other solvents used for its crystallization. The compound of the present ion may ntly or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, it is intended that the ion embrace both solvated and unsolvated forms. The term "solvate“ refers to a molecular complex of the compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those ly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g., water, ethanol, and the like. The term "hydrate" refers to the complex where the solvent molecule is water.

The compound of the present invention, ing salts, hydrates and solvates f, may inherently or by design form polymorphs.

WO 35047 In one embodiment of the invention, there is provided (R)(2-(1-(4-butoxyphenyl)—2- methylpropan—Z-ylamino)—1~hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)-one in crystalline form.

In another embodiment of the invention, there is provided (R)(2—(1—(4—butoxyphenyl)—2- methylpropan-Z—ylamino)—1~hydroxyethyl)hydroxybenzold]thiazoI-2(3H)-one e salt in crystalline form.

In yet another embodiment of the invention, there is provided (R)—7—(2—(1-(4—butoxyphenyI) methylpropan—Z—ylamino)-1~hydroxyethyl)-5—hydroxybenzo[d]thiazol-2(3H)—one glycolate salt in lline form.

In one embodiment of the invention, there is provided crystalline (R)(2-(1—(4— butoxyphenyl)methy|propan~2—ylamino)hydroxyethyI)hyd roxybenzo[d]thiazoI—2(3H )- one in substantially pure form.

In another embodiment of the invention, there is provided crystalline (R)(2—(1-(4- butoxyphenyl)methy|propan—Z—ylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazoI-2(3H)- one acetate salt in substantially pure form.

In yet another embodiment of the invention, there is ed crystalline (R)—7-(2-(1-(4- butoxyphenyl)—2—methylpropan—2—y|amino)—1—hydroxyethyl)~5—hydroxybenzo[d]thiazoI-2(3H)— one glycolate salt in substantially pure form.

As used herein, “substantially pure,” when used in reference to lline (R)—7-(2~(1-(4- butoxyphenyl)-2—methylpropanylamino)hydroxyethyl)—5-hydroxybenzo[d]thiazoI-2(3H)- one, or its pharmaceutically acceptable salt, means having a purity greater than 90 weight %, including greater than 90 , 91 , 92, 93, 94, 95, 96, 97, 98, and 99 weight %, and also including equal to about 100 weight % of (R)(2—(1~(4-butoxyphenyl)—2—methy|propan—2— ylamino)-1—hydroxyethyI)hydroxybenzo[d]thiazoI-2(3H)—one, based on the weight of the compound, or its pharmaceutically acceptable salt.

The presence of reaction impurities and/or processing impurities may be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear ic resonance spectroscopy, mass spectrometry, or infrared oscopy.

In a more focused aspect, the invention relates to a crystalline form of (R)—7-(2-(1-(4— butoxyphenyl)—2—methy|propanylamino)-1~hydroxyethyl)—5-hydroxybenzo[d]thiazol—2(3H)- one which has an X-ray powder ction n with at least one, two or three peaks having angle of refraction 2 theta (8) values selected from 8.5, 13.3, 13.9, 14.4, 15.2, 17.2, 17.5, 18.1, 21.3 and 225" when measured using CuKa radiation, more particularly wherein said values are plus or minus 02° 26. in one embodiment, the invention s to a crystalline form of (R)—7-(2—(1-(4— butoxyphenyl)methy|propan—2-ylamino)hydroxyethyl)-5—hydroxybenzo[d]thiazol-2(3H)— one which has an X—ray powder diffraction pattern with a peak at an angle of refraction 26 value of 152° when measured using Cqu radiation, more particularly wherein said value is plus or minus 02° 26. in one embodiment, the invention relates to a crystalline form of (R)—7-(2—(1-(4~ phenyl)methylpropanylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazol—2(3H)- one which has an X-ray powder diffraction pattern with a peak at an angle of refraction 26 value of 18.1° when measured using CuKu radiation, more particularly wherein said value is plus or minus 02" 26. in one embodiment, the invention relates to a crystalline form of (R)—7-(2-(1-(4- phenyl)methy|propanylamino)-1~hyd roxyethyl )—5-hyd roxybenzo[d]th iazol—2(3H)- one which has an X—ray powder diffraction pattern with a peak at an angle of refraction 26 value of 225° when measured using CuKa radiation, more particularly wherein said value is plus or minus 02° 26. in one embodiment, the ion s to a crystalline form of (R)(2-(1-(4— butoxyphenyl)methylpropan—2-ylamino)—1—hydroxyethyl)—5—hydroxybenzo[d]thiazol-2(3H)- 2O one which has an X—ray powder diffraction pattern substantially the same as the X—ray powder diffraction pattern shown in Figure 5 when measured using CuKu radiation. For details see Example 5. in another , the invention relates to a crystalline form of the acetate salt of (R)—7-(2-(1— (4-butoxyphenyl)—2—methylpropanylamino)-1~hydroxyethyl)—5-hydroxybenzo[d]thiazol— 2(3H)-one which has an X—ray powder diffraction n with at least one, two or three peaks having angle of refraction 2 theta (6) values ed from 8.8, 11.5, 16.4, 17.6, 18.2, 19.6, .1, 20.8, and 21.1° when measured using CuKCl radiation, more particularly wherein said values are plus or minus 02° 26. in one embodiment, the invention relates to a crystalline form of the e salt of (R)(2- (1-(4—butoxyphenyl)—2-methylpropan—2—ylamino)—1—hydroxyethyl)hydroxybenzo[d]thiazol— 2(3H)—one which has an X-ray powder diffraction pattern with a peak at an angle of refraction 26 value of 88" when measured using (3qu radiation, more particularly wherein said value is plus or minus 02" 26.

WO 2013035047 In one embodiment, the invention relates to a crystalline form of the acetate salt of (R)—7-(2- (1-(4-butoxyphenyl)methy|propan—2—ylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazol- one which has an X-ray powder diffraction pattern with a peak at an angle of refraction 26 value of 164° when measured using CuKa radiation, more particularly wherein said value is plus or minus 02° 26.

In one embodiment, the invention relates to a crystalline form of the acetate salt of (R)-7—(2- (1j(4-butoxyphenyl)—2-methylpropan—2-ylamino)—1-hydroxyethyl)—5-hydroxybenzo[dithiazol- 2(3H)—one which has an X—ray powder diffraction pattern with a peak at an angle of refraction 26 value of 20.8° when measured using Cqu radiation, more particularly wherein said value 1O is plus or minus 02" 26. in one embodiment, the invention s to a crystalline form of the acetate salt of (R)-7—(2~ (1—(4—butoxyphenyl)—2-methylpropanylamino)—1~hydroxyethyl)hydroxybenzo[d]thiazol- 2(3H)-one which has an X-ray powder diffraction pattern substantially the same as the X—ray powder diffraction pattern shown in Figure 6 when measured using CuKcl radiation. For details see Example 6. in yet another aspect, the ion relates to a crystalline form of the glycolate salt of (R)—7- (2-(1-(4—but0xyphenyl)—2-methylpropanylamino)—1-hydroxyethyl)—5—hydroxybenzo[d]thiazol— 2(3H)—one which has an X—ray powder diffraction pattern with at least one, two or three peaks having angle of refraction 2 theta (6) values selected from 8.7, 11.6, 16.1, 18.0, 19.8, 20.7, and 21.1° when measured using CuKOK radiation, more particularly wherein said values are plus or minus 02° 26. in one embodiment, the invention relates to a crystalline form of the glycolate salt of (R)—7—(2- (1-(4-butoxyphenyl)—2-methylpropanylamino)—1-hydroxyethy|)hydroxybenzo[d]thiazol- 2(3H)—one which has an X-ray powder diffraction n with a peak at an angle of refraction 26 value of 186° when measured using CuKa radiation, more ularly wherein said value is plus or minus 0.2" 26.

In one ment, the ion relates to a crystalline form of the glycolate salt of (R)(2— (1-(4-butoxyphenyl)methylpropan-2—ylamino)—1-hydroxyethy|)hydroxybenzo[d]thiazol- 2(3H)-one which has an X-ray powder diffraction n with a peak at an angle of refraction 26 value of 19.8° when measured using CuKCl radiation, more particularly n said value is plus or minus 02" 26. ln one embodiment, the invention relates to a crystalline form of the glycolate salt of (R)—7-(2— (1-(4-butoxyphenyl)-2—methylpropan-2—ylamino)-1—hydroxyethyl)-5—hydroxybenzo[d]thiazol— ZOIZ/054580 one which has an X—ray powder diffraction pattern with a peak at an angle of refraction 26 value of 20.7° when measured using CuKu ion, more particularly wherein said value is plus or minus 02° 26.

In one embodiment, the invention relates to a crystalline form of the glycolate salt of (R)-7—(2— (1 ~(4-butoxyphenyl)—2-methylpropan-2—ylamino)—1 -hyd roxyethyl )hyd roxybenzoid1thiazol- 2(3H)—one which has an X—ray powder diffraction pattern ntially the same as the X—ray powder diffraction pattern shown in Figure 7 when measured using CuKu ion. For details see Example 7.

The term “substantially the same” with reference to X-ray diffraction peak positions means that typical peak position and intensity variability are taken into account. For example, one skilled in the art will appreciate that the peak positions (29) will show some inter-apparatus variability, typically as much as 0.2°. Further, one skilled in the art will appreciate that relative peak intensities will show inter—apparatus variability as well as variability due to degree of crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art, and should be taken as qualitative measures only.

One of ordinary skill in the art will also appreciate that an X—ray diffraction pattern may be obtained with a ement error that is dependent upon the measurement conditions ed. In particular, it is lly known that intensities in an X—ray diffraction pattern may fluctuate depending upon measurement conditions employed. It should be further understood that relative intensities may also vary depending upon experimental conditions and, accordingly, the exact order of intensity should not be taken into account. Additionally, a measurement error of ction angle for a conventional X—ray diffraction pattern is typically about 5% or less, and such degree of measurement error should be taken into account as pertaining to the aforementioned diffraction angles. uently, it is to be understood that the crystal forms of the instant invention is not limited to the crystal form that provides an X- ray diffraction pattern completely identical to the X-ray diffraction pattern depicted in the accompanying s 5, 6 and 7 disclosed herein. Any crystal forms that provide X- ray diffraction patterns ntially identical to those disclosed in the accompanying Figures 5, 6 and 7 fall within the scope of the t invention. The ability to ascertain substantial identities of X-ray diffraction patterns is within the purview of one of ordinary skill in the art.

Pharmaceutically acceptable solvates in accordance with the invention e those wherein the solvent of crystallization may be isotopically substituted, 9,9. D20, de—acetone, ds-DMSO.

PCT/IBZOIZ/054580 The formula given herein is also intended to represent led forms as well as isotopically labeled forms of the compound. An isotopically d nd of the invention has a structure depicted by the formula given herein except that one or more atoms are ed by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compound of the invention e es of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N, 18F 31P, 32F, 358, 36Cl, 125l respectively. The invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as 3H and 14C, or those into which non-radioactive isotopes, such as 2H and 13C are present. Such isotopically labelled nds are useful in metabolic studies (with 14C), on kinetic studies (with, for example 2H or 3H), detection or imaging techniques, such as positron emission tomography (PET) or -photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. in particular, an 18F or labeled compound may be particularly desirable for PET or SPECT studies. An isotopically-labeled compound of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the anying Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

Further, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. it is understood that deuterium in this context is regarded as a substituent of a compound of the formula (l). The concentration of such a heavier isotope, specifically deuterium, may be defined by the isotopic enrichment factor. The term "isotopic enrichment “ as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. lf a substituent in the compound of this invention is denoted deuterium, such compound has an ic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium oration), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% ium incorporation), at least 6000 (90% deuterium oration), at least 6333.3 (95% ium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

The compound of the invention may be capable of forming co—crystals with suitable co—crystal s. These co-crystals may be prepared from a compound of formula (I) by known co- crystal forming procedures. Such procedures include grinding, heating, co-subliming, co- melting, or contacting in solution a compound of formula (I) with the co-crystal former under crystallization conditions and isolating co-crystals thereby formed. Suitable co-crystal s include those described in . Hence the invention further provides co— crystals sing a compound of formula (i).

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, gs, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal ), isotonic agents, tion delaying agents, salts, preservatives, drug stabilizers, s, excipients, egration agents, lubricants, sweetening agents, 1O flavoring agents, dyes, and the like and combinations f, as would be known to those d in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289— 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The term "a therapeutically effective amount" of the compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or tion of an enzyme or a protein activity, or ameliorate symptoms, ate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the t invention that, when stered to a subject, is effective to (1) at least partially alleviating, inhibiting, preventing and/or ameliorating a condition, or a disorder or a disease ated with beta-2—adrenoceptor activity; or (2) increasing or promoting the activity of beta-Z—adrenoceptor.

In another non—limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a cell, or a , or a non—cellular biological material, or a medium, is effective to at least partially increase or e the activity of -adrenoceptor. The meaning of the term “a therapeutically effective amount” as illustrated in the above embodiment for beta-2— ceptor also applies by the same means to any other relevant proteins/peptides/enzymes, such as IGF—1 mimetics or ActRlIB/myostatin blockers and the like.

As used herein, the term “subject” refers to an animal. Typically the animal is a mammal. A subject also refers to for example, primates (e.g., humans, male or female), cows, sheep, WO 35047 goats, horses, dogs, cats, s, rats, mice, fish, birds and the like. In certain embodiments, the t is a primate. In yet other embodiments, the subject is a human.

As used herein, the term “inhibit”, "inhibition" or “inhibiting" refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a signiﬁcant decrease in the baseline activity of a biological activity or process.

As used herein, the term “treat”, “treating" or "treatment" of any disease or er refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treat”, ing" or "treatment" refers to alleviating or rating at 1O least one al parameter including those which may not be discernible by the patient. in yet another embodiment, “treat”, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., ization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. in yet another embodiment, “treat”, "treating" or "treatment" refers to ting or delaying the onset or development or progression of the e or disorder.

As used herein, a subject is “in need of’ a ent if such subject would benefit ically, medically or in quality of life from such treatment.

As used herein, the term "a,” "an,” "the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise ciearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

The compound of formula (I) can be prepared according to the Scheme provided infra.

The process steps are described in more details below.

Step 1: A compound of formula (Vla) wherein Hal represents halogen and Ra is a protecting group is d with a compound of formula RbOH wherein Rb is a ting group in the presence of a le base, e.g. ylamine, to give a compound of formula (Va) wherein Hal represents halogen and Ra and Rb are protecting groups.

Step 2: A compound of formula (Va) is reacted with a suitable strong base, e.g. tert- ithium, in a suitable solvent, e.g. tetrahydrofuran (THF) in the presence of a suitable carbonylating agent, e.g. a suitable amide, to give a compound of formula (IVa) wherein Ra and Rh are protecting groups and Rc is hydrogen or any moiety derived from the carbonylating agent.

Step 3: A compound of formula (lVa) is optionally functionalised prior to stereoselective conversion to give a compound of formula (llla) wherein Ra and Rb are protecting groups and LG is a leaving group.

PCT/132012/054580 Step 4: A compound of formula (Illa) is treated with a le base, e.g. sodium bicarbonate, to give a compound of a (Ila) wherein R2, and Rb are protecting groups.

Step 5: A compound of formula (Ila) or (Illa) is reacted with 2-(4-butoxy—phenyI)—1,1-dimethyl- ethylamine in a le solvent e.g. toluene, optionally in the presence of a suitable base, e.g. potassium carbonate, followed by deprotection in the presence of a suitable acid, e.g. hydrochloric acid, to give a compound of formula (I).

In a further aspect, the invention relates to a process for the preparation of a compound of formula (I), in free from or in pharmaceutically acceptable salt form, comprising a) the reaction of a compound of formula (Ila) in free form or in pharmaceutically 1O acceptable salt form />_OR, R o N (Ila) in which R, and Rb are protecting groups with utoxy—phenyI)-1,1— dimethyI-ethylamine; b) the cleavage of any protecting groups still present; c) the recovery of the so obtainable compound of formula (I) in free form or in pharmaceutically acceptable salt form.

In a further aspect, the invention relates to a process for the manufacture of a compound of formula (I) in free form or in pharmaceutically acceptable salt form which es the steps a) the reaction of a nd of formula (Illa) in free form or in pharmaceutically acceptable salt form PCT/IBZOIZ/054580 />_0Rb R o N (llla) in which Ra and R, are protecting groups and LG is a leaving group with 2-(4~ butoxy—phenyl)—1,1-dimethyl-ethylamine; b) the cleavage of any protecting groups still present; c) the recovery of the so able compound of formula (I) in free form or in pharmaceutically acceptable salt form.

In another aspect, the invention relates to a process for the preparation of a compound of formula (Illa), in free from or in pharmaceutically acceptable salt form, />_0Rb R o N (Illa) comprising the stereoselective reduction of a nd of formula (lVa-2) in free form or in pharmaceutically acceptable salt form ,>_0Rb R o N (Na-2) in which Ra and RD are protecting groups and LG is a leaving group to give a compound of formula (llla) in free form or in pharmaceutically acceptable salt form.

W0 20131’035047 In the processes of the invention, typical protecting groups include pyl, ted-butyl, tert- butyldimethylsilyl. in the processes of the invention, typical leaving groups include chloride, p-toluenesulfonyl, bromide, methanesulfonyl, benzenesulfonyl, iodide.

The reactions can be effected according to conventional methods, for example as described in the Examples. The work-up of the reaction mixtures and the purification of the compounds thus obtainable may be carried out in accordance with known procedures. Acid addition salts may be produced from the free bases in known manner, and vice-versa. Compound of formula (I) can also be prepared by further conventional processes, for example as described in the Examples, which ses are r aspects of the invention.

The starting als used are known or may be prepared according to tional procedures starting from known nds, for example as described in the Examples.

The invention r includes any t of the present processes, in which an intermediate product obtainable at any stage thereof is used as starting material and the ing steps are carried out, or in which the starting materials are formed in situ under the reaction conditions, or in which the on components are used in the form of their salts or optically pure material.

The compound of the invention and intermediates can also be converted into each other according to methods generally known to those skilled in the art.

In a further aspect, the invention relates to a compound of formula (Ila) in free form or in pharmaceutically acceptable salt form />_OR, R o N (lla) wherein R8 and Rb are protecting groups.

RE] and R, may be independently selected from the group including tert-butyl, isopropyl and tert-butyldimethylsilyl.

In a r , the invention relates to a compound of formula (Illa—2) in free form or in ceutically acceptable salt form R o N (Illa-2) wherein R8 and Rb are protecting groups.

Ra and R, may be ndently selected from the group including tert—butyl, isopropyl and tert-butyldimethylsilyl.

In a further aspect, the invention relates to a compound of formula (la) in free form or in 1O pharmaceutically acceptable salt form RD N wherein R6] and R, are protecting groups.

Ra and Rb may be independently selected from the group including tert-butyl, isopropyl and tert-butyldimethylsilyl.

In another , the present invention provides a pharmaceutical composition comprising the compound of the present invention in free form or in pharmaceutically acceptable salt form and a pharmaceutically acceptable r. in particular, the invention relates to a 012/054580 pharmaceutical composition comprising a therapeutically ive amount of a compound of formula (I) in free form and one or more pharmaceutically acceptable carriers. in one embodiment, the invention relates to a pharmaceutical composition comprising a eutically effective amount of a compound of formula (I) in pharmaceutically acceptable salt form and one or more pharmaceutically acceptable carriers. In another embodiment, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a nd of a (I) in acetate salt form and one or more pharmaceutically acceptable carriers. In yet another embodiment, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of a (I) in 1O glycolate salt form and one or more pharmaceutically acceptable rs.

The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, transdermal application, parenteral administration, rectal administration, subcutaneous stration etc. ln addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or itories), or in a liquid form (including without limitation solutions, suspensions or emulsions). The pharmaceutical itions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain tional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.

Typically, the pharmaceutical compositions are tablets or n capsules comprising the active ingredient together with a) ts, e.g., e, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, , stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also 0) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners.

WO 35047 Tablets may be either film coated or enteric coated according to methods known in the art.

Suitable compositions for oral administration include an effective amount of a compound of the ion in the form of s, lozenges, aqueous or oily suspensions, dispersible s or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use are prepared ing to any method known in the art for the cture of ceutical compositions and such itions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with nontoxic 1O pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.

These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium ate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, , gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for e, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

The compound of the invention may be administered orally to preclinical species as a liquid dosage form with the drug in a on or in a suspension vehicle. Solution vehicles can be composed of surfactant (e.g., hor or solutol), solvent (e.g., propylene glycol) and buffer agent (e.g. citric buffer). Suspension formulations can contain surfactant (e.g. Tween 80), a polymer agent (e.g., methyl cellulose (MC)) and a buffer agent (e.g., phosphate).

Examples of solution formulations suitable for nical studies are set out below: Preparation: free base or acetate salt (R)~7~(2-(1-(4-butoxyphenyl)—2—methylpropan 3O ylamino)—1—hydroxyethy|)hydroxybenzo[d]thiazol-2(3H)-one) is first dissolved in the W0 35047 surfactant and mixed until a solution is obtained. Next the buffer is added and the solution mixed to provide a clear solution. Solution formulations 1 and 2 are able to support up to a 10 mg/mL dose. Both formulations are chemically and physically stable after 1 week at RT.

Examples of suspension formulations suitable for preclinical studies are set out below: ient (%w/w) WW 0.5%MCin50mMpH6.8 ate buffer 99.5 Preparation: (R)—7-(2-(1-(4-butoxyphenyl)—2—methylpropan—2—ylamino)—1-hydroxyethyl)—5- ybenzo[d]thiazol-2(3H)—one) is dispersed in the surfactant and mixed to homogenize the sion. The r solution is then added drop wise and mixed. A homogeneous suspension is obtained with small particles. The suspension is chemically and ally stable after 1 week at RT.

Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.

Said itions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 01-75%, or contain about 1—50%, of the active ingredient.

Suitable itions for subcutaneous application include, for example, the compound of the invention with 2.5% poloxamer 407 in 0.9% sodium chloride. Examples of suitable devices for injectable compositions include infusion pumps such as lnsulet’s OmniPod system.

The compound of the invention may also be administered by multidose subcutaneous ion using an auto injector or jector. Formulation compositions suitable for such subcutaneous injection are set out below.

Component Formulation 1 Formulation 2 nd A 1.00 mg 1.00 mg acetic acid 0.60 mg 0.60 mg mannitol 50 mg 50 mg benzyl alcohol 8.00 mg 10.00 mg sodium ide 1N adjusted to pH 5.0 adjusted to pH 5.0 water for injection add up to 1.016 9 add up to 1.016 g Benzyl alcohol (in comparison to phenol or m-cresol) was found to be a particularly suitable preservative for a subcutaneous injection formulation.

Thus in one embodiment of the invention, there is provided a pharmaceutical ition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof (for example Compound A in acetate salt form), and benzyl alcohol. in a further embodiment of the invention, there is provided a pharmaceutical composition comprising a eutically effective amount of Compound A, or a pharmaceutically acceptable salt f (for example Compound A in acetate salt form), and between 0.1 and ; 0.1 and 5; 0.5 and 2; 0.5 and 1.5; or 0.9 and 1.1 % (w/v) benzyl alcohol.

Suitable compositions for transdermal application include an ive amount of a compound of the invention with a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable ts to assist passage through the skin of the host. A combination of PG/OA (propylene glycol / oleyl alcohol) is an example of suitable solvent. For example, transdermal devices may be in the form of a bandage sing a g member, a reservoir containing the compound optionally with rs, optionally a rate controlling barrier to deliver the compound of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

Suitable itions for topical ation, e.g., to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like. Such topical delivery systems will in particular be appropriate for dermal application. They are thus particularly suited for use in topical, including cosmetic, formulations well—known in the art. Such may n solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein a topical application may also pertain to an inhalation or to an intranasal application. They may be conveniently delivered in the form of a dry powder (either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids) from a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray, atomizer or nebuliser, with or without the use of a suitable propellant.

The present invention further provides ous pharmaceutical compositions and dosage forms comprising the compound of the t invention as active ient, since water may facilitate the degradation of certain compounds.

Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ients and low moisture or low humidity conditions. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, ous compositions are packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, cs, unit dose containers (e. g., vials), blister packs, and strip packs.

The invention further provides pharmaceutical compositions and dosage forms that se one or more agents that reduce the rate by which the nd of the present ion as an active ient will decompose. Such agents, which are referred to herein as "stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers, etc.

The compound of formula (I) in free form or in pharmaceutically acceptable salt form, exhibits valuable pharmacological properties, eg. betaadrenoceptor modulating properties, eg. as indicated in in vitro and in vivo tests as provided in the next sections and is therefore indicated for y or for use as research chemicals, eg. as a tool compound.

The compound of the ion may be useful in the treatment of an indication ed from: muscular dystrophy, disuse-related atrophy, cachexia or sarcopenia.

PCT/IBZOIZ/054580 Thus, as a further embodiment, the present invention provides the compound of a (I) as deﬁned herein, as a medicament. In an embodiment, the t ion relates to the compound of formula (I) for use as a medicament. In a further embodiment, the present invention relates to the compound of formula (I) for use in the treatment or prevention of muscular dystrophy, disuse-related y, cachexia or sarcopenia.

Thus, as a further embodiment, the present invention provides the use of a compound of formula (I) in therapy. In a further embodiment, the therapy is selected from a e which may be treated by activation of betaadrenoceptor. In another embodiment, the disease is selected from muscular dystrophy, disuse-related y, cachexia or sarcopenia.

In another embodiment, the invention provides a method of treating a disease which is d by activation of beta—2~adrenoceptor comprising administration of a therapeutically acceptable amount of a compound of formula (I). In a further embodiment, the e is selected from muscular dystrophy, disuse—related y, cachexia or sarcopenia.

A further aspect of the invention thus relates to a method of treatment or prevention of muscular dystrophy, disuse-related atrophy, cachexia or sarcopenia comprising administering a therapeutically effective amount of a compound of formula (I) in free form or in pharmaceutically able salt form to a subject in need thereof.

As a further embodiment, the present invention provides the use of a compound of formula (I) for the manufacture of a medicament. In a further ment, the medicament is for the treatment of a disease or disorder which may be treated by activation of beta—2 adrenoceptor. In another embodiment, the disease is selected from the afore—mentioned list, suitably muscle g diseases, more ly muscular phy, disuse—related atrophy, cachexia or sarcopenia.

The compound of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agent. The compound of the present ion may be administered separately, by the same or different route of administration, or er in the same pharmaceutical composition as the other agents.

In one embodiment, the ion provides a product comprising a compound of formula (I) and at least one other eutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a disease or condition modulated by beta-2 adrenoceptor agonism. Products provided as a combined preparation include a composition comprising the compound of formula (I) and the other therapeutic agent(s) together in the same pharmaceutical composition, or the compound of formula (I) and the other therapeutic agent(s) in separate form, e.g. in the form of a kit.

In one embodiment, the invention provides a pharmaceutical composition comprising a compound of formula (I) and another therapeutic agent(s). Optionally, the pharmaceutical ition may comprise a pharmaceutically acceptable excipient, as described above.

A further aspect of the invention thus relates to a combination comprising a therapeutically effective amount of a compound of formula (I) and one or more eutically active co— In one ment, the invention provides a kit comprising two or more separate ’IO pharmaceutical compositions, at least one of which contains a compound of formula (I). In one embodiment, the kit ses means for separately ing said compositions, such as a ner, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as lly used for the packaging of tablets, capsules and the like.

The kit of the invention may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the invention typically comprises directions for administration.

In the combination therapies of the invention, the nd of the invention and the other therapeutic agent may be manufactured and/or formulated by the same or different 2O manufacturers. er, the nd of the invention and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the compound of the invention and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential .25 administration of the compound of the invention and the other therapeutic agent.

Accordingly, the invention provides the use of a compound of a (I) for treating a disease or condition modulated by beta-2 adrenoceptor agonism, wherein the medicament is prepared for stration with r therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition modulated by beta-2 adrenoceptor agonism, wherein the ment is administered with a compound of formula (I).

PCT/IBZOlZ/054580 The invention also provides a compound of a (I) for use in a method of treating a disease or condition modulated by beta—2 adrenoceptor agonism, wherein the compound of formula (I) is prepared for administration with another therapeutic agent. The ion also provides another eutic agent for use in a method of treating a disease or condition modulated by beta-2 adrenoceptor agonism, n the other therapeutic agent is prepared for administration with a compound of formula (I). The invention also provides a compound of formula (I) for use in a method of treating a disease or condition modulated by beta-2 adrenoceptor agonism, wherein the compound of formula (I) is administered with another therapeutic agent. The ion also provides another therapeutic agent for use in a method of treating a disease or condition modulated by beta-2 adrenoceptor m, wherein the other therapeutic agent is administered with a nd of a (I).

The invention also provides the use of a compound of formula (I) for treating a e or ion modulated by beta-2 adrenoceptor, wherein the patient has previously (e.g. within 24 hours) been treated with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition moduIated by beta-2 adrenoceptor, wherein the patient has previously (e.g. within 24 hours) been treated with a compound of a (I).

In one embodiment, the other therapeutic agent is selected from testosterone, androgen agonists, or SARM tive androgen receptor modulators); IGF-l mimetics; myostatin and its receptor ActRlIA/B blockers; TGFbeta and n blockers (as anti—atrophy agents); Muft/MAFbx E3 Iigase inhibitors; HDAC inhibitors or any tic agents (e.g. for cancer cachexia); anti—inflammatory agents like NSAIDs, TNF or IL-1b blockers; metabolic modulators like PPAR agonists or lL-15 mimetics; cardiovascular agents like b(1) blockers (e.g. nebivolol) or ARB (e.g. for cardiac cachexia); antisense oligos for axon-skipping (e.g. for dystrophy); an te enhancer such as ghrelin, progestin or MC-4 antagonists; high protein nutrient supplements and the like.

The pharmaceutical composition or combination of the present invention can be in unit dosage of about 0.05-1000 mg of active ient(s) for a subject of about 50—70 kg, or about 005-500 mg or about 005—250 mg or about 005-150 mg or about 0.05—100 mg, or about 0.05-50 mg or about 0.05-10 mg of active ingredients. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations f, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian W0 35047 of ordinary skill can readily determine the effective amount of each of the active ingredients ary to prevent, treat or inhibit the progress of the disorder or disease.

The above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously s, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations f. The compound of the present invention can be applied in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either enterally, erally, advantageously subcutaneously, e.g., as a suspension or in aqueous on. The dosage in Vitro may range between about 10'3 molar and 10'9 molar concentrations. A therapeutically effective amount in vivo may range depending on the route of administration, between about 001-500 mg/kg, or between about 001-100 mg/kg, or between about 0.01-1 mg/kg, or between about .1 mg/kg.

The activity of the compound of the present invention can be assessed by the following in vitro method. Further in vivo methods are described further in the Examples.

Test 1: in vitro cellular functional assay using CHO cells and skeletal muscle cells M: Human skeletal muscle cells (skMC) were obtained from Cambrex (catalog no CC- 2561) and cultured in Skeletal Basal Medium (SKBM) obtained from Cambrex (catalog no #CC-3161). The CAMP responses were measured using CAMP c 2 bulk HTRF-Assay kit obtained from Cisbio or Cis Competitive Intelligence og no 62AM4PEC). skMC cells were cultured for 1 day in SKBM cell culture medium supplemented with 20% FCS in 384— well plates at 37°C, 5% C02. The next day, the cells were washed twice with 50 uL PBS, and differentiated for 3 days in serum—free SKBM in presence of 1 uM SB431542, a ALK 4/5 Inhibitor obtained from Sigma (catalog no S4317) at 37°C, 7.5% 002. On day 4, serum-free SKBM supplemented with 1 uM 42 was removed, cells were washed twice with 50 uL PBS and further differentiated for 1 day in serum-free SKBM without 88431542 (50 uL per well) at 37°C, 7.5% COZ. Rat skMC and cardiomyocytes cells were isolated from neonatal rats in a standard way and treated as described above. Chinese hamster ovary (CHO) cells stably transfected with human (3 adrenoceptors ((31 or 62) were produced at is Institutes for ical Research and cultured as described before (J Pharmacol Exp Ther. 2006 May;317(2):762—70).

Compounds were made up in stimulation buffer at 2 x required concentration and 1:10 serial dilutions in stimulation buffer were prepared in 96-well plate m). DMSO l was normalized to the DMSO content of the highest dilution, e.g. 0.1 % DMSO (x 2) for 10‘5 M (x 2) concentration of the first compound dilution. The assay was carried out in 384-well plates, 2012/054580 in a 20 uL stimulation volume, and a ﬁnal assay volume of 40 uL per well. On the day of experiment, culture medium was removed from ll cell culture plates by inverting and ﬂicking the plate on stack of paper 2-3 times. 10 uL of fresh culture medium per well was first added in the 384-well plate. After 10 minutes of incubation at room temperature, 10 uL per well of working compounds ons were added to the cells and incubated for 30 minutes at room temperature in the dark. During this time, working solutions of reagents were ed by diluting stock solutions of anti CAMP cryptate and CAMP D2 1:20 in lysis buffer, supplied with the kit. After 30 s of compound incubation, 10 uL of CAMP-D2 and 10 uL of anti CAMP cryptate were sequentially added to the assay plates. After 1 hour of incubation time at room temperature in the dark, the measurement was performed with the PheraStar (Excitation wavelength: 337 nm, Emission wavelengths: 620 and 665 nm).

Ca2+: The human adrenergic AlphatA CHO-K1 cell line was purchased from Perkin Elmer (ValiScreenTM Stable inant GPCR Cell line, catalog no ES-O36-C, Lot no M1W—C1, Boston, Massachusetts, USA). One day before the experiment, A frozen cells (10 ns per ml and per vial) were thawed in a water bath at 37°C. The cell suspension was centrifuged for 5 minutes at 1,000 rpm and the cell pellet was resuspended in cell e medium. Cells were seeded into black 384-well plates with clear bottom at a density of 8,000 cells per well in 50 uL of cell culture medium. Plates were incubated for about 24 hours at 37°C, 5% 002. The day of the experiment, the medium was removed using a cell washer 2O (TECAN PW3). After the final wash there was 10 uL left in the wells. 40 uL of loading buffer were added and cells were loaded for 60 min at 37°C, 5% 002. Plates were washed with TECAN PW3 with 20 uL assay buffer left and were incubated for at least 20 minutes at RT before ming the FLlPR experiment. Compounds were then characterized in the agonist and/or antagonist mode. For assay validation, the same protocol was used with the fresh cells. In this case, cells were detached from a 150 cm2 ﬂask using 3 ml of Trypsin—EDTA, centrifuged and resuspended in cell culture medium.

Cells were stimulated by adding 5 uL of compounds (5X), using the FLIPR head. nds acting as agonists induce a transient increase of intracellular calcium. This was recorded on the FLlPR system. A measurement of the signal baseline was first recorded every second for 2 minutes before the injection of the compounds. Calcium measurements were performed by exciting the cells with the argon ion laser at 488 nm at 0.6 W laser power and recording the fluorescence signal with a CCD camera (opening of 0.4 sec) for 2 minutes.

Low controls mulated cells) were determined with the addition of 5 uL of assay buffer.

High controls were determined with the addition of 5 uL of a known agonist at high ZOIZ/054580 concentration EC100 (A-61603 at 1 uM) and a reference agonist compound was also added in each plate.

The compound of the invention exhibits efficacy in test assay 1 with an E050 of less than 10nM. Specific activity is shown in example 10 Further specific activities of the compound of the invention are described in examples 11 to The following examples are ed to illustrate the ion and are not to be construed as being tions thereon. Temperatures are given in degrees s. If not mentioned otherwise, all evaporations are performed under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20—133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR. Abbreviations used are those conventional in the art.

All starting materials, ng blocks, reagents, acids, bases, dehydrating , solvents, and sts utilized to synthesise the compound of the present invention are either commercially ble or can be produced by organic synthesis methods known to one of ordinary skill in the art (Houben-Weyl 4th Ed. 1952, Methods of Organic sis, Thieme, Volume 21). Further, the compound of the present invention can be produced by organic synthesis methods known to one of ordinary skill in the art as shown in the following examples.

Examples List of Abbreviations: 1M one molar APCI atmospheric—pressure chemical ionization aq aqueous AR adrenoceptor atm atmosphere br broad cm centimeters d doublet dd double doublet ddd double double doublet (DHDQ)2PHAL Hydroquinidine 1,4-phthalazinediyl diether PCT/[82012/054580 DMAC dimethylacetamide DMSO dimethylsulfoxide DSC differential scanning calorimetry ee enantiomeric excess equiv equivalent ES electron—spray 9 grams h hours HPLC high performance liquid tography HRMS high resolution mass spectroscopy m multiplet MC methyl cellulose mbar millibar MeOH methanol min minutes ml milliliters MS mass spectroscopy MTBE methyl tert-butyl ether nm nanometers NMR nuclear magnetic resonance RT retention time r.t. room temperature 8 singlet sat. saturated sept septet t triplet TFA roacetic acid um eters w/v weigh/volume XRPD x-ray powder diffraction Unless otherwise indicated, HPLC/MS spectra were recorded on an Agilent 1100 series LC / Agilent MS 6210 pole. A Waters Symmetry C8 column (3.5 um; 2.1 x 50 mm) (WAT200624) was used. The following gradient method was applied (% = percent by ): A = water + 0.1% TFA/ B = acetonitrile + 0.1% TFA; 0.0 -— 2.0 min 90A:1OB — PCT/IBZOIZ/054580 5A2958; 2.0 — 3.0 min 5Az9SB; 3.0 ——3.3 min 5A:9SB — B; flow 1.0 ml/min; column temperature 50 °C. All nds were ionized in APCI mode. 1H-NMR spectra were recorded on a Varian Mercury (400 MHz) or Bruker e (600 MHz) machine.

Optical rotation was measured on a Perkin Elmer Polarimeter 341.

LCMS condition for example 2b, 2c, 2d, 2e, 2g: Mass spectra station: Agilent 6130 quadrupole LC/MS with Agilent 1200 HPLC; Column: Agilent Zorbax SB-C18 (Rapid resolution), 2.1*30mm, 3.5 pm; Mobile phases: B: 0.1% formic acid in water; C: 0.1% formic acid in MeCN; 1.0 min to 6.0 min, 95% B to 5% B, and 5% C to 1O 95% C; 6.0 min to 9.0 min, 5% B and 95% C; post time: 2.0 min; flow rate: 0.8 ml/min; column temperature: 30 °C; UV detection: 210 nm and 254 nm; MS scan positive and ve: 80-1000; Ionization method: APl-ES.

HRMS conditions for example 2f: Instrument: Waters Acquity UPLC coupled with Synapt Q-TOF MS; Column: Waters Acquity UPLC BEH C18, 2.1*5O mm, 1.7 pm Mobile Phase: A: 0.1% formic acid in water, B: 0.1% formic acid in Acetonitrile; Column temperature: at room temperature; UV detection: scan from 190nm to 400nm; Flow rate: 0.5 ; Gradient condition: Time [min.] Phase B [%] 0 5 1 5 Start of acquisition 9 95 11 95 End of acquisition 11.10 5 14 5 Next injection Ionization method: ESI+; MS scan range: 00 m/z.

Intermediate A: 2-(4-butoxyphenyl)-1,1-dimethyl-ethylamine a) 4—(2—methyl-2—nitropropyl)phenol W0 35047 PCT/IBZOIZ/054580 A e of 4-(hydroxymethyl)phenol (20 g), KOtBu (27.1 g) and DMAC (200 mL) was stirred with magnetic stirrer. 2-nitropropane (21.5 g) was added slowly within 20 min. The mixture was heated to 140 °C for 5 hr before cooled to r.t. The mixture was added slowly to cool HCl aqueous solution (3.0 %, 600 mL), then extracted with MTBE (300 ml* 1, 200ml* 1).

The organic layers were combined, washed with water (300 ml* 2) and sat. NaCl aqueous solution (50 ml* 1), then dried with ous Na2804. The mixture was filtered and concentrated under vacuum to give light-yellow solid (28.5g), which was used for next step without further purification.

[M-1]+ =194.2; RT= 5.3 minutes 1O 1H—NMR (400 MHz, CDCIs) ppm 6.96 (d, J: 8.5 Hz, 2H), 6.75 (d, J: 8.5 Hz, 2H), 3.11 (s, 2H), 1.56 (s, 6H). b) 1-butoxy(2—methyl-2—nitropropyl)benzene The mixture of 4-(2-methylnitropropyl)phenol (20.4 g), 1-bromobutane (28.7 g), DMAC(200 ml), K2003 (21.6 g), tetrabutylammonium iodide (38.7 g) was stirred with magnetic stirrer and heated to 85 °C for 17 h. The mixture was cooled to 0-10 °C and water (700 ml) was added. The mixture was extracted with MTBE (300 ml*1, 200 ml* 1). The combined organic phases were washed with water (250 ml* 2), then concentrated under vacuum to give a red-brown oil (27.8g), which was used in the next step without further purification. 1H-NMR (400 MHz, CD013) ppm 7.0 (d, J: 8.8 Hz, 2H), 6.81 (d, J: 8.8 Hz, 2H), 3.93 (t, J: 6.6 Hz, 2H), 3.12 (s, 2H), 1.74 (m, 2H), 1.56 (s, 6H), 1.48 (m, 2H), 0.97 (t, 3H). (3) 2—(4-butoxyphenyl)-1,1-dimethyl-ethylamine In a hydrogenating reactor (1 L), a on of 1-butoxy(2-methyl-2—nitropropyl) benzene (27.8 g) in AcOH (270 ml) was added followed by wet Raney Ni (7.0 g). The mixture was purged with Hg for 3 times, then heated to 60 °C and kept stirring under 5.0 atm for 16 h. The mixture was ﬁltered, the total te was concentrated under vacuum. The resulting residue was d with water (150 ml)/n-heptane (80 ml), the aqueous layer was washed with n— heptane (80 ml) again. The aqueous layer was adjusted with NaOH (~20 %) to pH ~ 11, then ted with MTBE (100 ml* 1) and EtOAc (150 ml* 2). The medium layer was discarded.

All top layers were combined and washed with saturated NaHCOa (100 ml) and saturated NaCl (100 ml) before being dried with anhydrous Na2804. After filtration, the mixture was concentrated. The ing residual was stirred and HCl solution in isopropyl alcohol (2M, 40 ml) was added. The slurry was heated to 60 °C and n-heptane (120 ml) was added. The W0 201311135047 PCT/IBZOlZ/054580 mixture was cooled to 20 °C, then filtered, the cake was washed with some n-heptane. The white solid was dried in air for 2days to give 10 g of pure HCI salt of product. Yield: 35.2 %.

[MH]+ =222.2; RT= 5.0 minutes 1H—NMR (400 MHz, d-DMSO) ppm 8.13 (s, 3H), 7.12 (d, J: 8.6 Hz, 2H), 6.88 (d, J: 8.5 Hz, 2H), 3.93 (t, J: 6.4 Hz, 2H), 2.80 (s, 2H), 1.67 (m, 2H), 1.42 (m, 2H), 1.18 (s, 6H), 0.92 (t, 3H). h drox benzo d thiazol-2 3H -one osgene K2003 water/GHQ]3 . S tBULI DMF , />-O PhSPMeBr nBuLi o N >— (IV-1) K2003, K3Fe(CN)6 toluenesulfonyl (DHQD)2PHAL, OsO4 chloride t—BuOH/HZO pyridine utoxy-pheny')‘1 1‘ di methyl-ethylamine —__p toluene PCT/lBZOlZ/054580 a )1 -tert—Butoxy-3—ﬂuoroisothiocyanatobenzene Thiophosgene (33.6 g) in CHC|3 (250 ml) and K2003 (64.7 g) in H20 (450 ml) are added, separately and aneously, drop wise to a solution of 3-tert—Butoxy—5—tluoro—phenyl- amine (42.9 g) in CHCI3 (350 ml) at 0 °C. The reaction mixture is warmed to room temperature over night. The organics are separated and washed with water (3x), brine (1x), dried over M9804, ﬁltered and the solvent removed in vacuo. The title compound is obtained by flash column chromatography (silica, eluent romethane/ xane 1:3). 1H NMR(CDC13, 400 MHz); 6.70 (m, 3H), 1.40 (s, 9H). b 3-tert-Buto -5—fluoro— hen l-thiocarbamic acid O—iso ro lester 1~tert—Butoxy-3—fluoroisothiocyanatobenzene (24.0 g) and triethylamine (10.9 g) are dissolved in iso-propanol (150 ml). The reaction e is ed for 18 hours and the solvent is removed by vacuo. The crude product is dissolved in hexane: diethyl ether (19:1).

The diethyl ether is removed in vacuo and the solution is cooled to 0 °C for 3 hours. The solution is filtered to give the title compound. 1H NMR (CDCI3, 400 MHz); 8.10 (br s, 1H), 6.65 (br s, 2H), 6.45 (ddd, 1H) 5.60 (sept, 1H), 1.35 (d, 6H), 1.30 (s, 9H). c) 5-tert-Butoxy-2—isopropoxy—benzothiazolecarbaldehyde (3—tert-Butoxyfluoro—phenyl)—thiocarbamic acid O-isopropyl ester (2.2 g) is dissolved in dry tetrahydrofuran (20 ml) The reaction mixture is cooled to —78 oC and tert-butyl lithium (15.2 ml, of 1.5 M solution) is added over 20 minutes. The reaction mixture is then warmed to -10 0C for 75 minutes. The reaction mixture is then re-cooled to —78 °C, N,N-dimethyl-formamide (1.5 g) is added and the reaction mixture is slowly warmed to room temperature then stirred at ~10 °C for 1 hour. The reaction e is ed with HCIM) (5 ml, of a 2 M solution), the organics are separated between ethyl e/water and removed in vacuo. The title compound is obtained by ﬂash column chromatography a, eluent ethyl acetate/iso- hexane 1:9).

MS (ES+) mIe 294 (MH*). d) 5-tert—Butoxy-Z—isopropoxy—7-vinylbenzothiazole PthMeBr (5.0 g) is dissolved in dry tetrahydrofuran (100 ml) under argon. N—butyl lithium (8.8 ml, of 1.6 M solution) is added at room temperature over 10 minutes and reaction mixture stirred for a further 30 minutes. A solution of 5-tert—Butoxy—Z-isopropoxy— WO 35047 benzothiazolecarbaIdehyde (1.25 g) in dichloromethane (40 ml) is added drop wise to the reaction mixture and the reaction mixture is d for 4.5 hours at room temperature. The solvent is removed in vacuo. redissolved in ethyl acetate, washed with water (3x), brine (1x), dried over M9804, ed and the solvent removed in vacuo. The title compound is obtained by flash column chromatography (silica, eluent ethyl acetate/iso-hexane 1:9).

MS (ES+) mfe 292 (MH*). e R —1- 5-tert—Butox —benzothiazol l -ethane-1.2—diol K3Fe(CN)6 (1.2 g), K2C03 (0.5 g), (DHQD)2PHAl (19 mg) are dissolved in tert—butanol/water (15 ml, 1:1 mix) under argon and stirred for 15 minutes. The reaction mixture is cooled to 0 1O °C and 0804 (3.1 mg) is added followed by 5-tert-Butoxy isopropoxy—7—vinylbenzothiazole (0.35 g). The reaction mixture is stirred over night at room temperature. The on mixture is ed with sodium-meta-bisulphate (1 g) and stirred for 1.5 hours. Ethyl acetate is added, the organics are separated, washed with (2X) water, (1x) brine, dried over M9804, ﬁltered and the solvent removed in vacuo. The title nd is obtained by flash column chromatography (silica, eluent ethyl acetate/iso- hexane 2:5).

MS (ES+) m/e 326 (MW). 5-ten‘—b methylbenzenesulfonate lnto a 500—ml 3—necked round-bottom flask, purged and maintained with an inert here of nitrogen, was placed a solution of (R)(5-tert-butoxy-2—isopropoxy-benzo[d]thiazol—7- yl)ethane-1,2—diol (20 g, 59.05 mmol) in pyridine (240 ml) and 4A molecular sieves (5 g). This was followed by the addition of a on of toluenesulfonic acid chloride (tosyl chloride) (15.3 g, 79.73 mmol) in pyridine (60 ml) dropwise with stirring at 0°C. The resulting solution was stirred for 4 h at room temperature. The reaction was then quenched by the on of 1000 ml of 1M hydrogen chloride. The resulting solution was extracted with 2x300 ml of ethyl acetate and the organic layers are combined. The organic phase was washed with 1x500 ml of 1M hydrogen chloride, 1x500 ml of 10% sodium bicarbonate and 300 mi of brine. The mixture was dried over anhydrous sodium sulfate and trated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:10). This resulted in 26 g (87%) of (R)(5-tert—butoxy—2—isopropoxybenzo[d]thiazoIyl) hydroxyethyl 4—methylbenzenesulfonate as yellow oil.

LC/MS RT = 2.47 min; (m/z): 480 [M+H}+ 1H-NMR: (400 MHz, s): 5 (ppm) 7.57 (d, 2H); 7.36 (d, 2H); 7.17 (d, 1H); 6.79 (d, 1H); 6.32 (d, 1H); 5.37-5.26 (m, 1H); 4.97-4.90 (m, 1H); 4.12-4.00 (m, 2H); 2.40 (s, 3H); .38 (m, 6H); 1.32 (s, 9H). methylgrogan—Z—ylamino)ethanol into a Lml 4-necked bottom flask was placed a solution of (R)(5-tert—butoxy- 2—isopropoxybenzo[d]thiazol—7-yl)hydroxyethyl-4—methylbenzenesulfonate (26 g, 51.55 mmol, 1.00 equiv) in toluene (320 mLml) and 2~(4—butoxyphenyl)-1,1-dimethyl-ethylamine (intermediate A) (22 g, 99.47 mmol, 1.93 equiv). The solution was stirred for 24 h at 90 °C in 1O an oil bath. The resulting mixture was concentrated under vacuum. The residue is applied onto a silica gel column with ethyl acetate/petroleum ether (1:8). This resulted in 16 g (58%) of (R)(5-tert—butoxy—Z—isopropoxybenzo[d]thiazol—7—yl)-2—(1-(4—butoxyphenyl) methylpropanylamino)ethanol as light yellow oil.

LC/MS: RT = 2.24 min (m/z): 529 [M+H]+ 1H-NMR: (600MHz, DMSO-dg): 5 (ppm) 7.12 (s, 1H); 6.83 (d, 2H); 6.77 (s, 1H); 6.63 (d, 2H); .80 (br. s, 1H); 5.38-5.30 (m, 1H); 4.70-4.66 (m, 1H); 3.90 (t, 2H); 2.81-2.61 (m, 2H); 2.50- 2.39 (m, 2H); 1.71-1.62 (m, 2H); 1.47-1.41 (m, 2H); 1.41 (d, 6H); 1.22 (s, 9H); 0.91 (q, 3H); 0.88 (s, 3H); 0.83 (s, 3H). hydroxybenzold |thiazol-2(3H )-one A solution of (R)—1~(5-tert—butoxy-2—isopropoxybenzo[d]thiazol~7~yl)—2—(1-(4-butoxyphenyl)—2— methylpropan-2—ylamino)ethanol (3.5 g) in formic acid (40 ml) was d for 68 h at ambient temperature. 50 ml of water was added, and the resulting mixture was evaporated to s (rotary evaporator, 15 mbar, 40°C) to give 3.8 g of crude product. This material was partitioned between ted aqueous sodium bicarbonate (50 ml) and ethyl acetate (50 ml) in order to remove formic acid. The aqueous layer was ted 3x with ethyl acetate (30 ml each). The combined organic extracts were dried over magnesium sulfate, filtered and concentrated to give 3 g of crude free—base. This material was flash-chromatographed (silica gel; gradient 0-60% methanol in romethane). Pure fractions were collected and evaporated to dryness to give 1.74 g of an amorphous semi-solid.

This material was subjected to chiral preparative chromatography [column: Chiralpak IC (20 um) 7.65 x 37.5 cm; eluent: n—heptane/dichloromethane/ethanol/diethylamine 50:30:20 WO 2013203504? PCT/lBZOlZ/054580 (+0.05 diethylamine); flow rate = 70 ml/min; concentration: 2.5 g / 50 ml eluent; detection: UV, 220 nm] to give pure enantiomer (100% ee).

This material was dissolved in 45 ml of acetonitrile at 60°C. The solution was allowed to cool to ambient temperature over 18 h, upon which precipitation occured. The mixture was diluted with 5 ml of cold (4°C) acetonitrile and ﬁltered through a Buchner . The filter cake was washed twice with cold itrile. Then the wet solid was collected and dried in vacuo (0.2 mbar) at ambient temperature overnight to give 1.42 g of (R)—7—(2-(1-(4—butoxyphenyl)—2- methylpropan-Z—ylamino)—1—hydroxyethyl)~5-hydroxybenzo{d]thiazol-2(3H)—one as a ess 1O LC/MS: RT = 1.81 min (m/Z): 431 [M+H] + 1H-NMR: (600MHz, DMSO-dg): 5 (ppm) 11.5 (br. s, 1H); 9.57 (br. s, 1H); 6.99 (d, 2H); 6.76 (d, 2H); 6.52 (s, 1H); 6.47 (s, 1H); 5.63 (br. s, 1H); 4.53-4.48 (m, 1H); 3.90 (t, 2H); 2.74—2.63 (m, 2H); 2.54-2.45 (m, 2H); 1.71—1.62 (m, 2H); 1.49-1.40 (m, 2H); 0.93 (q, 3H); 0.89 (s, 6H).

Optical rotation: [011922 = -43° (c = 1.0 g/100 ml MeOH).

Exam le 2: alternative route to R 24-butox hen lmeth I re an lamino h drox eth | h drox benzo d thiazol-Z 3H -one F F thiocarbonyl— diimidazole S iPrOH JSL J\ NH2 0 N/// N 0 #\(VII) (Vl) (V) CI CI 0 “.IOH 2—chloro—N-methoxy- RuCI (p-cymene) ‘ N-methyl-acetamide S [(S,S)-Ts—DPEN] ———-—-——————-—-———’ />_O —————-——’ />-O t-BuLi HCOOH o N )_ o N )— +(IV-2) “)\ (III-2) NaOH 2—-(4 Butoxy-phenyl))-1 1- TBAI :/>-O yl-ethylamine a 1-tert-Buto -3—fluoroisothioc anato-benzene 1,1'-Thiocarbonyldiimidazole (423 g, 2.37 mol) was dissolved in dichloromethane (3200 ml).

The e was stirred under N2 here while a solution of 3-tert—butoxy—5-fluoroaniline (435 g, 2.37 mol) in dichloromethane (800 ml) was added slowly within 2 h. Then the mixture was kept stirring at 20 °C for 16 h. The mixture was diluted with water (3000 mi). The separated diohloromethane phase was washed again with water (3000 ml) before dried with WO 35047 anhydrous Nazso4 for 2 h. The mixture was filtered and the ﬁltrate was concentrated under vacuum to remove solvent to give 1-tert-butoxy—3-fluoro—5-isothiocyanato-benzene (499 g). 1H-NMR (400 MHz, CDCls): 6.63-6.68 (m, 3 H), 1.37 (s, 9H). b 3-tert—Butox -5—fluoro- hen carbamic acid O-iso ro lester To a solution of 1—tert-butoxy-3—fluoro—5-isothiocyanatobenzene (460 g, 2.04 mol) in anhydrous isopropyl alcohol (3250 ml) was added triethylamine (315 g. 3.06 mol). The mixture was heated to reflux under N2 here for 16 h and the temperature was cooled to 40—50 °C. After concentration, the resulting dark residue was d with n-heptane (1000 ml) and heated to 60°C. The mixture was slowly cooled to 25 °C, at the same time seeding 1O was added. A slurry was observed and stirred at 25 °C for 16 h before being cooled slowly to 0-10 °C within 2 h. After filtration and washing with n-heptane (200 ml), the collected solid was dried in oven under vacuum at 40-45 °C for 18 h to give (3-ted-Butoxyfluoro-phenyl)- thiocarbamic acid O-isopropyl ester (453.1 g).

LCMS: [M+H]+ = 286.1 ; RT= 7.2 minutes ‘H-NMR (400 MHz, 00013): 8.18 (s, 1H), 6.81 (m, 2H), 6.51 (dt, J: 10.2 Hz, 1H), 5.66 (heptet, J: 6.3 Hz, 1H), 1.42 (d, J: 6.2 Hz, 6H), 1.37 (s, 9H).

C 1— 5—tert—Butox Under a nitrogen atmosphere, a solution of tert—butyllithium (481 ml, 737.6 mmol, 1.6 M) was added dropwise to a solution of (3—tert-Butoxy—5-fluoro-phenyl)—thiocarbamic acid O-isopropyl ester (200 g, 700.83 mmol) in 2-Me-THF (1600 ml) at temperature below -65 °C. The reaction ature was warmed to -35°C, and a second portion of tert—butyllithium (388 ml, 737.6 mmol, 1.9 M) was added slowly while keeping the temperature below -35 °C. The reaction mixture was then stirred at this temperature for 3 h and cooled down to —70 °C. A solution of N-methyl-N-methoxy chloroacetamide (96.4 9, 700.83 mmol) in F (300 ml) was added to the reaction mixture while keeping the temperature below -70 °C. The mixture was then warmed to -30 °C and stirred for 45 minutes. The cold reaction e was quenched by dropwise on of 30% HCI in isopropanol (240 9) followed by the on of 1500 ml water. The organic layer was washed sequentially with 1000 ml water, 1500 ml saturated aqueous NaHCOs and 1500 ml brine. After concentration, the resulting light brown residue was added to isopropanol (135 ml). The mixture was warmed to 50 °C and cooled down slowly to 25 °C. n-heptane (135 ml) was added dropwise to the solution and the mixture was stirred overnight. The slurry was ﬁltered and the filter cake was washed with n- heptane (40 ml) followed by r portion of n—heptane (20 ml). The cake was dried under vacuum to yield 1-(5—tert—butoxy—2-isopropoxy-ben20thiazoI—7-yl)chloro—ethanone as off- white powder (42.8 g, 17.9% yield). 1H NMR (400 MHz, CDClg): 7.60 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 5.40 (heptet, J = 6.3 Hz, 1H), 4.77 (s, 2H), 1.47 (d, J = 6.3 Hz, 6H), 1.40 (s, 9H).

LCMS: [M+H]+ = 342.1, RT = 7.29 min. d R -1— 5-tert—Butox A suspension of ert-butoxyisopropoxybenzoidithiazol-7—yl)—2—chloro—ethanone (70 9, 204.8 mmol) and RuCI(p-cymene)[(S,S)-Ts~DPEN] (1.954 g, 3.07 mmol) in methanol/DMF (1330 ml/70 ml) was degassed and refilled with N2 three times. A degassed med 1O mixture of formic acid (28.3 g) in Eth (124.3 g) was added slowly while keeping the internal ature between 15 to 20 °C. The resulting yellow suspension was warmed up to 30 °C.

After 2h the reaction mixture is cooled to 25°C, water (750 ml) was then added into the reaction mixture followed by the addition of acetic acid (56 ml) in one portion. The mixture was concentrated and then d with TBME (1000 ml). Aqueous phase was separated and extracted with TBME (1000 ml). The combined organic phase was washed sequentially with water and brine and then dried with NaZSO4 and concentrated under vacuum to give (R) (5-tert—Butoxy—2—isopropoxy—benzothiazol—7-yl)chloro-ethano| (72 g).

LCMS d A): [M+H]+ = 343.1, RT = 5.67 min. 1H NMR (400 MHZ, CDCIg): 7.29 (d, J = 2.0 Hz, 1H), 6.83 (d, J = 2.0 HZ, 1H), 5.37 (heptet, J = 6.3 HZ, 1H), 4.96 (m, 1H), 3.74 (m, 2H), 3.01 (s, 1H), 1.46 (d, J = 6.2 HZ, 6H), 1.36 (s, 9H). e R —5—ten‘-Butox iso ro ox -7—oxiran l-benzothiazole To a solution of (R)—1-(5-tert—butoxyisopropoxybenzo[d]thiazol—7-yl)chloro—ethanol (140 9, 407.1 mmol) in TBME (420 ml) was added dropwise NaOH aqueous on (2M, 420 ml) followed by tetrabutylammonium iodide (7.52 g, 20.36 mmol) added in one portion. After 4 h at 26 °C, 400 ml TBME was added and the organic layer was separated. The aqueous layer was extracted with TBME (400 ml). The combined organic layers were washed with water (400 ml) and brine (400 ml) to give (R)tert—butoxy—2-isopropoxyoxiranyl-benzothiazole (122 g).

LCMS: [M+H]+ = 308.0, RT = 6.80 min. 1H NMR (400 MHz, CDClg) ppm 7.28 (d, J = 2.0 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 5.38 (m, 1H), 3.96 (m, 1H), 3.15 (dd, J =43, 5.5 Hz, 1H), 2.94 (dd, J :43, 5.5 Hz, 1H), 1.45 (d, J = Hz, 6H), 1.37 (s, 9H). 1 R —1— 5-tert—butox methylgrogan-Z—ylamino)ethanol (R)—5-z‘en‘-butoxyisopropoxyoxiranyl-benzothiazole (145 9, 471.7 mmol) and 2—(4~ butoxy-phenyl)—1,1-dimethyl—ethylamine (114.8 9, 518.9 mmol) were dissolved in DMSO (850 ml). The reaction mixture was heated to 80 °C and stirred for 27 h. The mixture was then cooled to 25 °C and added to a stirred e of water (1500 ml) and TBME (1500 ml). The aqueous layer was ted and extracted with TBME (1000 ml). The combined organic layers were sequentially washed with water (1500 ml) and brine (1000 ml), dried with anhydrous Na2804. After concentration, the residue was puriﬁed by column chromatography (eluting with 10% of EtOAc in n-heptane to 33% of EtOAc in n-heptane). Solid product (R)-1— (5-tert—butoxyisopropoxybenzolthhiazolyl)-2—(1-(4-butoxyphenyl)methylpropan ylamino)ethanol was obtained (140 g) as off-white solid.

HRMS: [M+1] 529.2996 1H NMR (400 MHz, 00013): 7.26 (m, 1H), 7.01 (m, 1H), 6.99 (m, 1H), 6.78-8.80 (m, 3H), 5.39 (m, 1H), 4.65 (dd, J = 3.8, 8.8Hz, 1H), 3.83 (t, J = 6.4 Hz, 2H), 2.96 (dd, J = 3.8, 12 Hz, 1H), 2.74 (dd, J = 8.8, 12 Hz, 1H), 2.60 (dd, J = 13.6, 17.6 Hz, 2H), 1.72—1.79 (m, 2H), 1.50 (m, 2H), 1.46 (d, J = 2.0 Hz, 3H), 1.45 (d, J = 2.0 Hz, 3H), 1.35 (s, 9H), 1.06 (s, 3H), 1.04 (s, 3H), 0.98 (t, J = 7.2 Hz, 3H). hydroxybenzol d lthiazol-2(3H )—one To (5-tert—butoxy—2-isopropoxybenzoldhhiazolyI)(1-(4-butoxyphenyl)-2— methylpropan-2—ylamino)ethanol (7.5 g) in isopropanol (30 ml) and water (25 ml) was added 1M HCl aqueous solution (43 ml). The reaction mixture was then heated to 60 °C and stirred for 2.5 h. The mixture was cooled to 50 °C, and then 2M NaOH aqueous solution (18 ml) was added slowly to adjust pH between 4. The reaction mixture was then cooled to 30 °C, followed by extraction with TBME (first time with 40 ml, the second time with 25 ml). Two organic layers were ed and washed with water (38 ml for two times) before drying with anhydrous Na2804. After ion, the filtrate was concentrated, and then dissolved in MeCN (145 ml). The solution was treated with active carbon (0.6 g) and heated to 60 °C.

After a second ﬁltration, the cake was washed with MeCN (10 ml for two times), the filtrate 2012/054580 was crystallized at 60 °C to gain (R)(2-(1—(4—butoxyphenyl)—2-methylpr0panylamino)-1~ hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)-one (3.8 g). e.e. = 97.6 %.

LCMS (method A): [M+H]+ =431.2 1H NMR (400 MHz, DMSO- d5): 9.5 (br. s, 1H), 6.81 (d, J = 8.5 Hz, 2H), 6.57 (d, J = 8.6 Hz, 2H), 6.33 (d, J = 2.2 Hz, 1H), 6.30 (d, J = 2.2 Hz, 1H), 4.43 (br. s, 1H), 3.69 (t, J = 6.4Hz, 2H), 2.58—2.59 (m, 2H), 2.24—2.31 (m, 2H), 1.41—1.48 (m, 2H), 1.15—1.25 (m, 2H), 0.78 (s, 6H), 0.70 (t, J = 7.4Hz, 3H).

Exam le 3: R24-butox hen Imeth I ro an lamino h drox eth l hydroxybenzold|thiazol~2§3H)-one acetate salt 500 mg (1.161 mmol) of free base (2-(1-(4—butoxyphenyl)methy|propanylamino)— 1-hydroxyethyl)-5—hydroxybenzo{d]thiazol-2(3H)—one was suspended in 10.0 ml acetonitrile and 0.25 ml water in a 50 ml four~necked flask and paddle stirred at r.t. The suspension was heated at an internal ature of 50°C (jacket temperature 75°C) and 72 mg acetic acid (1.161 mmol) was added (a clear yellow solution was formed). The solution was cooled down over 30 min. at r.t. and 0.15 ml water added.

The solution was then seeded with (R)(2—(1—(4-butoxyphenyl)-2—methylpropan—2-ylamino)— 1-hydroxyethyI)-5—hydr0xybenzo[d]thiazoI-2(3H)—one acetate and stirred ght (16 h) at r.t. The suspension was then filtered at r.t. h a glass filer and washed three times with 1 ml acetonitrile. 510 mg of wet filter cake was dried in a drying oven overnight (16h) at r.t. to dryness. Yield: 508 mg white powder (89.1%) Pre aration of hydroxybenzoldlthiazol-213H)—one acetate seeds 57.0 mg (0.132 mmol) of free base (R)—7-(2—(1—(4-butoxyphenyl)—2—methylpropan—2-ylamino)- 1-hydroxyethyl)—5—hydroxybenzo[d]thiazol—2(3H)—one and 8.03 mg (0.132 mmol) acetic acid were dissolved in 1.0 ml itrile and 0.05 ml water. The solution was stirred at r. t. with a magnetic stirrer stirred. Precipitation took place over night. The solution was then filtered at r.t. through a glass ﬁlter and washed three times with 0.5 ml acetonitrile. The wet filter cake was dried in a drying oven overnight (16h) at r. t. to dryness. Yield: 57 mg white powder Example 3a: Alternative procedure for the formation of {RH-(241-(4-butoxyphenyl)—2- methylgropan-Z-ylamino)hydroxyethyl)hydroxybenzoldIthiazol-213H1-one acetate 2012/054580 (R)(5-tenf—butoxy—2-isopropoxybenzo[d]thiazolyl)(1~(4-butoxyphenyl)—2-methylpropan- 2-ylamino)ethanol, (1 equiv.) was suspended in isopropanol. At 50 to 60°. a 1M aqueous hydrochloric acid solution (3 ) was added within about 30 - 60 min. After te reaction (approx. 2.5 hours at 60°C) the solution was cooled to 20°C and sodium ide 2M (3 equiv.) added gradually at this temperature. After complete addition the emulsified free base (R)—7—(2—(1-(4—butoxyphenyl)-2—methy|propan-Z-ylamino)—1—hydroxyethyl) hydroxybenzoid]thiazoI—2(3H)-one was extracted into ethylacetate and the organic layer washed with water. The organic layer was treated with activated carbon and filtered using microcrystalline cellulose as a filter aid. The filter cake was washed with ethyl acetate. The filtrate, containing the free base (R)—7—(2-(1-(4—butoxyphenyl)-2~methylpropanylamino)—1- hydroxyethyl)hydroxybenzoid]thiazol-2(3H)-one, was carefully concentrated to a deﬁned residual volume by distillation at a jacket temperature of 55°C under reduced pressure. pylacetate was then added and partly removed by distillation to a defined residual volume at a jacket temperature of 55°C under reduced pressure. Further isopropylacetate and a solution of acetic acid in isopropylacetate were added to the warm distillation residue at 50-55°C. During the acetic acid addition the batch was seeded with (R)—7-(2—(1-(4- pheny|)methylpropan-Z—ylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)- one acetate salt to initiate the controlled crystallization of the acetate salt early at 50-55°C.

After gradually cooling to 0°C the product suspension was filtered and washed twice with cold isopropylactetate. The ﬁlter cake was dried at 50 to 90°C under reduced pressure until constant weight to give crystalline (R)(2—(1-(4—butoxyphenyl)methylpropan-2—ylamino) hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)—one acetate salt at a typical yield of . 80%.

Exam le 4: R 2 4-butox hen l meth i re an lamino h drox benzo d thiazol-Z 3H -one | colate salt 500 mg (1.161 mmol) of free base (R)—7—(2-(‘l-(4—butoxyphenyl)methylpropan-2—ylamino)- oxyethy|)—5—hydroxybenzoid]thiazol~2(3H)-one was suspended in 10.0 ml acetonitrile and 0.25 ml water in a 50 mL four—necked flask and paddle stirred at r.t.. The suspension was heated at an al temperature of 60°C (jacket temperature 85°C) and 90 mg 2- hydroxyacetic acid (1.161 mmol) added to the solution. 0.25 ml water was then added at an internal temperature of 60°C. The solution was seeded with (R)(2-(1-(4-butoxypheny|)—2- methylpropan-Z-ylamino)hydroxyethyl)—5—hydroxybenzo[d]thiazol—2(3H)—one glycolate at an internal temperature of 30°C and stirred overnight (16 h) at r.t.. r 10 ml acetonitrile was added and stirred over the weekend at r.t.. The suspension was filtered at r.t. through a glass filter and washed once with 1.0 ml acetonitrile/water 9:1 v/v and twice with 1.0 ml PCT/IBZOIZ/054580 acetonitrile. 320 mg wet filter cake was dried in a drying oven overnight (16h) at r.t. to dryness.

Pre aration of hydroxybenzold |thiazol-2(3H )—one glycolate seeds 63.0 mg (0.146 mmol) of free base (R)—7-(2-(1-(4-butoxyphenyl)-2—methylpropan-2—ylamino)— 1—hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)—one and 11.24 mg (0.146 mmol) glycolic acid were dissolved in 1.0 ml itrile and 0.05 ml water. The solution was stirred at r.t. with a magnetic stirrer. Precipitation took place overnight. The suspencion was filtered at r.t. through a glass filter and washed three times with 0.5 ml acetonitrile. The wet filter cake was dried in a drying oven overnight (16h) at r.t. to dryness. Yield: 52 mg white powder Examples 5: 6 and 7: XRPD and DSC analysis of cgstalline (RH-(24114- butoxyphenyl1methylproganylaminothydroxyethyl)hydroxybenzo|d|thiazo|- 2 3H -one and its acetate and l colate salt forms XRPD analysis of free base crystalline (R)—7—(2-(1-(4-butoxyphenyl)-2—methylpropan ylamino)hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)-one and its acetate and glycolate salt forms was carried out under the following experimental conditions: XRPD method instrument Bruker D8 Advance (reflection) irradiation CuKa (40 kV, 30 mA) Step rd Scan type uous scan Scan time 107.1 3 Scan range 2°-40° (2 theta value) DSC analysis was carried out under the ing experimental conditions: DSC method Instrument Perkin Elmer Diamond ature range 30°— 300C Sample mass 2-3mg Sample pan Aluminium closed Nitrogen flow 20-50 K/min Example 5: XRPD analysis of line (R1(2-(1-(4-butoxyphenyl1methylpropan- ino)hydroxyethy|)hydroxybenzo|d]thiazo|-2(3H)-one Free base (R)—7-(2-(1-(4-butoxyphenyl)—2—methylpropan-2—ylamino)—1-hydroxyethy|)-5— hydroxybenzo[d]thiazol-2(3H)—one was recrystallised as described below prior to XRPD analysis. 4.09 (2.232 mmol) of (R)(2—(1~(4-butoxyphenyl)methylpropan—2—ylamino)—1— hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)-one was suspended in 24.0 ml ethyl acetate in a 100 ml four-necked flask and paddle stirred at r.t.. The sion was dissolved at an internal temperature of 70°C (jacket temperature 90°C) to provide a clear yellow solution. 1O The solution was cooled down over 30 min. at r.t. and seeded with free base (R)—7-(2—(1-(4— butoxyphenyl)methylpropan—2—ylamino)—1-hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)- one at an internal temperature of 35°C (crystallisation taking place very slowly) and stirred overnight (16 h) at r.t.. The on was then ﬁltered at r.t. through a glass ﬁlter (fast ﬁltration, duration: <1 min.) and washed 3 x with 2.0 ml ethyl acetate (clear yellow mother liquor). 5.82 g wet ﬁlter cake was dried in a drying oven overnight 16h at r.t. and 16h at 40°C.

Yield: 3.63 9 white powder %) The crystalline (R)(2-(1-(4-butoxyphenyl)—2-methylpropan—2—ylamino)—1-hydroxyethyl) hydroxybenzo[d]thiazol-2(3H)-one was analysed by XRPD and the characteristic peaks are shown in the table below (see also Figure 5). Of these, the peaks at 8.5, 13.3, 13.9, 14.4, 15.2, 17.2, 17.5, 18.1, 21.3 and 225° 2-theta are the most characteristic.

Angle (2-Theta °) lntensity‘Yo Intensity % medium 13.9 medium medium 14.4 medium 21.3 medium medium Crystalline free base (2—(1-(4—butoxyphenyl)methylpropanyla mino)—1 - hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)—one was analysed by DSC and found to have an onset of melting at about 115 °C.

Example 6: XRPD analysis of crystalline (RH-(241-t4-butoxyphenyl)methylgrogan- 2- lamino h drox eth l h drox benzo d thiazol-Z 3H -one acetate salt lline (R)—7—(2-(1—(4-butoxyphenyl)-2—methylpropan-2—ylamino)—1-hydroxyethyl)—5- hydroxybenzo[d]thiazol-2(3H)-one acetate salt was analysed by XRPD and the characteristic peaks are shown in the table below (see also Figure 6). Of these, the peaks at 8.8, 11.5, 16.4, 17.6, 18.2, 19.6, 20.1, 20.8, and 21.1° 2-theta are the most characteristic.

Angle (2-Theta °) ity % Angle (2-Theta °) Intensity % .0 low 11.5 high 14.6 low .7 low medium 16.4 high 17.6 medium 18.2 high Crystalline (R)—7-(2—(1-(4—butoxyphenyl)—2-methylpropan-2—ylamino)—1—hydroxyethy|) hydroxybenzo[d]thiazol-2(3H)-one acetate salt was analysed by DSC and found to have a broad endotherm at around 170 °C.

W0 2013f035047 Example 7: XRPD is of crystalline (RH-(241-(4-butoxyphenyl)methylpropan- 2- lamino ~1-h drox eth lh drox benzo d thiazol-2 3H -one l colate salt Crystalline (R)—7-(2—(1—(4-butoxyphenyl)—2—methy|propan-2—ylamino)—1-hydroxyethyl)—5- hydroxybenzo[d]thiazol-2(3H)—one glycolate salt was analysed by XRPD and the Characteristic peaks are shown in the table below (see also Figure 7). Of these, the peaks at 8.7, 11.6, 16.1, 18.0, 19.8, 20.7, and 21.1° a are the most Characteristic.

Angle (2-Theta °) lntensity % Intensity % 21.1 high Crystalline (R)—7-(2—(1-(4—butoxyphenyl)methylpropan—Z—ylamino)hydroxyethyl)—5- hydroxybenzo[d]thiazol—2(3H)-one glycolate salt was analysed by DSC and found to have an 1O onset of melting at about 188 °C.

Exam le 8: Method for the re aration of a harmaceutical formulation suitable for subcutaneous administration of Compound A in acetate salt form For 1.00 liter drug t solution approximately 900 g of water for injection is placed into a clean vessel suitable for pharmaceutical compounding. 50 g mannitol, 0.60 g acetic acid and 10 g benzyl alcohol is added and dissolved in the water for injection. 1.00 g of Compound A is then added and ved. pH is adjusted to the target value, for e 5.0, with 1N sodium hydroxide solution. Water for injection is then added to the target product solution weight of 1.016 kg. The drug product solution is sterile filtered through a 0.22 pm PVDF membrane into washed, depyrogenized vials, closed with sterile rubber rs and crimped. The vials are terminally sterilized by autoclaving.

Example 8a: Alternative method for the ation of a pharmaceutical formulation suitable for the subcutaneous administration of Compound A in acetate salt form WO 35047 PCT/IBZOlZ/054580 For 1.00 liter drug t solution approximately 900 g of water for injection is placed into a clean vessel suitable for pharmaceutical compounding. 50 g mannitol and 10 g benzyl l is added and dissolved in the water for injection. 1.14 g of the acetate salt of Compound A is then added and dissolved. pH is adjusted to the target value, for example .0, with acetic acid on. Water for injection is then added to the target product solution weight of 1.016 kg. The drug product solution is sterile filtered through a 0.22 pm PVDF membrane into washed, depyrogenized vials, closed with sterile rubber stoppers and crimped. The vials are terminally sterilized by autoclaving.

Example 9: Comparative solubilities of free base, acetate salt and glycolate salt forms 1O of Compound A The relative solubilities of the free base form and the acetate and glycolate salt forms of Compound A were analysed and the results are show in the table below. Solutions were ed with addition of HCI or NaOH for pH adjustment. The improved aqueous solubilities of the acetate and glycolate salt forms relative to the free base form of Compound A make the acetate and glycolate salts of nd A more suitable for subcutaneous ion or infusion.

Compound A free Compound A acetate Compound A glycolate base solubility in H20 salt solubility in H20 ﬂ-“ﬂGone in Gone in -—--_ Exam le 10: In vitro ar rofiles of com ound of the invention Com ound A its enantiomer com oundB its racemate com ound A18 and formoterol The compound of the invention (compound A) shows the following EC50 values in Test 1 as described hereinbefore.

W0 2013I035047 CHO cells Primary cells; cAMP response ECso (Emax °/o) ECso (Emax %) Compounds Human Rat 0.7 nM 85 nM Formoterol 2.9 nM " .. (99% ) (88% ) .6 nM 560 nM 0.7 nM 3.4 nM 5.7 nM Compound A (R) (88%“) (82%“) (98%“) (98%) (71%“) 950 nM 280 nM compound B (S) (83%“) (100%) 11 nM 684 nM 0.63 nM Compound “B (87%“) (38%") (100%‘) Compound 2.5 nM 1.7 nM A (R) . ' . I . I acetate salt (91%“) (93%**) skMC: differentiated skeletal myotubes; ': compared to formoterol; ”: compared to naline; #: cAMP for [31 and 82, Ca2+ for 01A; n.d. not determined The compound of the invention (compound A) is a potent and selective [32 AR agonist with very low intrinsic efﬁcacy on [31 AR and no activity on 01A AR. lts enantiomer Compound B is very weak on [32 AR with an E050 of 950 nM.

Exam le 11: Effects of Formoterol and Com ound A on skeletal muscle and heart weight in vivo Male Wistar Han IGS (lnternational Genetic Standard) rats (Crl:Wl(Han)) at the weight of 1O 350-400 g were sed from Charles River Laboratories. Rats were acclimated to the facility for 7 days. Animals were housed in groups of 3 animals at 25 °C with a 12:12 h light- dark cycle. They were fed a standard laboratory diet containing 18.2% protein and 3.0% fat with an energy content of 15.8 MJ/kg (NAFAG 3890, Kliba, Basel, Switzerland). Food and water were provided ad libitum. Formoterol or nd A was dissolved in the vehicle indicated below to achieve a dose range of 0.003 to 0.03 mg/kg/day for erol and 0.01 to 0.1 mg/kg/day for Compound A with the Alzet model 2ML4 for 28 days. Pumps were ﬁlled with the solution and kept for l hours at 37°C in PBS until surgical implantation. Rats were treated aneously with Temgesic at a dose of 0.02 mg/kg with a volume of 1 ml/kg at least 30 minutes before surgery, and then the pumps filled with the solution indicated above were implanted subcutaneously into the back of the rats under anesthesia with isoflurane at a concentration of 3%. ic was administered aneously to the rats 24 h and 48 h after the surgery. Body weights were measured twice per week. Clips were removed 10 days after the surgery under anesthesia. Four weeks after the treatment, the rats were euthanized with COZ, and the tibialis anterior, gastrocnemius and soleus muscles, heart and brain were dissected and weighed. Brain weight was used for normalization of organ weights. Results are sed as mean +/-SEM. tical analysis was carried out using Dunnett's multiple comparison test following one-way analysis of variance to compare 1O the ent groups to the vehicle control group. Differences were considered to be significant when the probability value was < 0.05: *: Statistical analyses were med by GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA). Muscle weight was normalized to the body weight at day 0 (initial body weight) and heart weight was ized by brain weight.

Study 1: Formoterol —_- Alzet minipump 2ML4 for 4 weeks Vehicle: 20% 1:2 Cremophor:Ethanol in saline (0.9% NaCl) Study 2: Compound A -—-— Alzet minipump 2ML4 for 4 weeks Vehicle: 20% 1:2 CremophorzEthanol in saline (0.9% NaCl) Figure 1 shows that formoterol induces both skeletal muscle hypertrophy and heart mass se to the same extent, while Compound A induces skeletal muscle hypertrophy with minimum impact on heart mass, indicating that Compound A exhibits a selective effect on skeletal muscle over cardiac muscle. Compound A signiﬁcantly induces skeletal muscle hypertrophy by 11% at 0.01 mg/kg/day with steady state plasma concentration of ~ 0.2 nM, PCT/IBZOlZ/054580 while there were no ﬁndings on the heart histopathology even at 0.1 mg/kg/day with steady state tration of ~ 2 nM.

Example 12: Effects of Formoterol and Compound A on the function of isolated organs {left atrium contraction, Sino-Atrial node beating rate and automaticity of whole heart) Method Left atrium contraction: The left atrium contraction assay was performed at Ricerca Biosciences, LLC (catalog no 407500 rgic betat), using left atria from Dunkin Hartley Guinea pig with body weight of 600 +/—8O 9 (Arch. lnt. codyn. 1971 :191 :133-141 .).

Sino-Atrial node beating rate: New Zealand white female rabbits were killed by 1O uination after a deep anesthesia using a mixture of ketamine/xylazine, iv The heart was quickly removed and placed in Tyrode's solution. This solution was continuously gassed with 95% Oz, 5% C02, and previously warmed to approximately 36 i 05°C. The right atrium was separated from the rest of the heart. The preparations were mounted in a tissue bath and kept at 37 i 0.5 °C for at least one hour stabilization. Action ials (AP) were intracellularly recorded with a standard glass lectrode filled with 3 M KCI, connected to a high input impedance-neutralizing amplifier (VF-180 microelectrode amplifier, Bio-Logic).

The AP were displayed on a digital oscilloscope (HM-407 oscilloscope, HAMEG), analyzed by means of high resolution data acquisition system (Notooord software hem 4.2, Notocord SA, Croissy, France). After one hour of stabilization, compounds were added to the Tyrode’s solution at the increasing concentrations, each concentration being maintained for 30 minutes. There was no wash—out between two concentrations. Electrophysiological measurements were made by analyzing action potentials during the experimental protocol at the end of the 30 minute perfusion period. The SA neous frequency was evaluated by counting the number of beats every 10 seconds to express the results in number of beats per minute (bpm). Data were sed as mean i SEM.

Automaticity: Automaticity was investigated in the isolated Langendorff perfused rabbit hearts, conducted by hem Pharmaceuticals Consulting N.V., 8-8400 Oostende, m. The tests were run in on hearts from albino female rabbits weighing about 2.5 kg and having an age of imately 3 months. The compound effects were measured in a fully automated model using isolated rabbit heart ed according to the Langendorff technique. The spontaneously beating heart is retrogradely perfused with increasing concentrations of the test item. One electrode is carefully placed on the left atrium in order to record the cycle length of the sinus node automaticity.

PCT/IBZOIZ/054580 Figures 2a and 2b show the results ed when comparing formoterol with compound of the invention (compound A).

Compound A shows no effects on left atrium contraction up to 10 uM and less direct effects on the pacemaker ty, compared to Formoterol.

Left atrium contraction E050 (n=2) > 10 uM Sino-Atrial node beating rate, maximum +45% +62% increase (n=6) Values in figures 2a and 2b are expressed as means 1 SEM; Sino-atrial node (n=6), isolated heart (n=3) Exam le 13: Effects of erol and Com ound A on the heart rate in vivo Wistar Han (W—H) lGS (International Genetic Standard) rats (Crl:Wl(Han)) were purchased from Charles River Laboratories. Femoral arterial and venous catheters were chronically 1O ted and exteriorized through a spring tether—swivel system and housed in specialized cages. Arterial catheter was ted to a pressure transducer to continuously measure pulse pressure, mean arterial pressure and heart rate, which was derived from the blood pressure signal, via a digital data acquisition system. Compounds were stered via s.c catheter implanted through the skin buttun. Values are expressed as means i SEM (n=3).

Compound A shows less heart rate increases compared to formoterol when administered with so. bolus, up to 0.3 mg/kg as shown in Figures 3a, 3b and 30.

Example 14: Effects of Formoterol and Compound A on the heart rate in vivo Rhesus monkeys, 24 females with body weight around 4 to 8 kg, were randomized into 4 groups of n=6. The animals were restrained on a chair up to 4 hours after single subcutaneous administration of compounds, and then returned to their pens. Heart rates were measured using a et V3304 device. Values are expressed as means i SEM (n=6).

Compound A shows less heart rate se compared to formoterol when stered as a so. bolus, up to 0.03 mg/kg as shown in Figures 4a and 4b.

Example 15: Effect of compound A, its enantiomer (compound B1 and its te (Compound AI B) on Serotonin 5'HTgc receptor Human recombinant hr5—HT2C CHO cell membranes (Biosignal Packard, USA) and 3H- Mesulergine (NEN Life Science Products, USA, 1 nM) are used for measuring the binding y of the compounds to human 5-HT20 or. Non-speciﬁc binding is evaluated in the ce of 1 uM Mesulergine. Fifty uL each of membrane, ligand and compound in a total volume of 250 uL are incubated in 96-well plates for 60 min at 22° C in a buffer containing 50 mM Tris, 0.1% ic acid, 10 uM Pargyline, pH 7.7. The plates are filtrated, washed 3 times in ice-cold 50 mM Tris, dred and measured in Topcount.

CHO-K1 cells coexpressing mitochondrial uorin, recombinant Serotonin 5-HT2Cne and the cuous G protein Gone, grown to mid-log phase in culture media without antibiotics were detached with PBS—EDTA, centrifuged and ended in assay buffer (DMEM/HAM’s F12 with HEPES, without phenol red + 0.1 % BSA protease free) at a concentration of 1 x 106 cells/ml. Cells were incubated at room temperature for at least 4 h with coelenterazine h.

Reference agonist was a—methyl~5-HT. For t testing, 50pL of cell suspension were mixed with 50uL of test or reference agonist in a 96-well plate. The resulting emission of light is recorded using Hamamatsu Functional Drug Screening System 6000 (FDSS 6000) luminometer. Agonist activity of test compound was expressed as a percentage of the activity of the reference agonist at its EC100 concentration.

CH0 EC50 (Emax %) Compound A is 50-fold less active on 5-HT2C when compared to [32 AR t activity (5.6 nM), while its enantiomer Compound B is very weak on [32 AR with EC50 of 950 nM but much more potent on 5-HT2C with EC50 of 19.7 nM, showing inversed ivity on the target.

Compound A is also over 10-fold less active on 5—HT2C when compared to the racemate or the (S) enantiomer, suggesting that the side—effect profile of this compound is advantageous.

Claims

What is claimed is

1. A compound of formula (I) in free form or in pharmaceutically acceptable salt form which is HNwow HO N 5 (I).

2. A compound according to claim 1 which is (R)—7-(2~(1-(4—butoxyphenyl)—2— methylpropanylamino)—1-hydroxyethyl)—5-hydroxybenzo[d]thiazol-2(3H)—one in free form.

3. A compound according to claim 1 which is (R)—7-(2-(1-(4—butoxypheny|) 10 methylpropanylamino)—1-hydroxyethyl)hydroxybenzo[d]thiazol-2(3H)-one in glycolate salt form.

4. A pharmaceutical composition comprising a therapeutically effective amount of a nd according to any one of claims 1 to 3 and one or more pharmaceutically acceptable carriers. 15

5. A pharmaceutical composition according to claim 4 wherein one of the pharmaceutically acceptable rs is benzyl alcohol.

6. A combination comprising a therapeutically effective amount of a compound ing to any one of claims 1 to 3 and one or more therapeutically active co- agents. 20

7. A compound according to any one of claims 1 to 3 for use as a medicament.

8. A compound according to any one of claims 1 to 3 for use in the ent or prevention of muscular dystrophy, disuse-related atrophy, cachexia or sarcopenia.

9. Use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prevention of muscular dystrophy, disuse-related atrophy, cachexia or sarcopenia.

10. Use according to claim 9, wherein the medicament is formulated for administration by subcutaneous infusion.

11. A process for the manufacture of a compound of formula (l) as defined in claim 1 in free form or in pharmaceutically acceptable salt form which includes the steps of: a. the on of a nd of formula (lie) in free form or in pharmaceutically acceptable salt form />—0Rb R o N 1O (lla) in which Ra and R, are protecting groups with 2-(4-butoxy-pheny|)-1,1- dimethyl-ethylamine; b. the cleavage of any protecting groups still present; 0. the recovery of the compound of formula (l) in free form or in pharmaceutically 15 acceptable salt form.

12.A process for the manufacture of a compound of formula (l) in free form or in pharmaceutically acceptable salt form according to claim 11 in which compound (lie) is ed by the on of a compound of formula (Illa) in free form or in pharmaceutically acceptable salt form />—0Rb 20 (llla) in which in which Ra and Rb are protecting groups and LG is a leaving group with a base and optionally a phase transfer catalyst.

13.A process for the manufacture of a compound of formula (I) in free form or in pharmaceutically acceptable salt form according to claim 12 in which compound (Illa) is ed by the stereoselective reduction of a compound of formula (lVa-2) in free form or in pharmaceutically acceptable salt form />——0Rb RD N (lVa-2) in which Ra and Rb are protecting groups and LG is a leaving group.

14. A process according to claim 13 wherein the LG is chloro. 1O 15.A process ing to claim 13 in which compound (lVa’-2) in free form or in pharmaceutically able salt form />—ORb RD N (lVa'-2) is obtained by the reaction of a compound of formula (Va) in free form or in ceutically acceptable salt form R o 0Rb

15 (Va) in which Ra and R, are protecting groups and Hal is a n with 2-chloro-N- methoxy—N-methyl—acetamide in the presence of a strong base.

16. A compound of formula (l) obtained by the process of any one of claims 11 to 15.

17. A compound according to claim 1, substantially as herein described with reference to any one of the Examples and/or