WO2008148854A1 - Ghrelin modulators - Google Patents

Abstract

The present invention relates to novel compounds of formula (I) or a pharmaceutically acceptable salt or solvate thereof, processes for their preparation, intermediates useble in these processes, pharmaceutical compositions containing them and their use in therapy, for example as modulators of of the growth hormone secretagogue receptor (also referred to as the ghrelin receptor or GHSR1a receptor) and/or for the treatment and/or prophylaxis of a disorder mediated by the ghrelin receptor.

Description

GHRELIN MODULATORS

The present invention relates to novel acetohydrazide compounds, processes for their preparation, intermediates usable in these processes, and pharmaceutical compositions containing the compounds. The invention also relates to the use of the acetohydrazide compounds in therapy, for example as modulators of the growth hormone secretagogue receptor (also referred to as the ghrelin receptor or GHSRIa receptor) and/or for the treatment and/or prophylaxis of a disorder mediated by the ghrelin receptor.

Ghrelin is the endogenous ligand for the growth hormone (GH) secretagogue receptor. It was originally purified from stomach and is a 28 amino acid peptide hormone in which the serine at position 3 is n-octanoylated. It has potent GH releasing activity and thus is believed to play an important role in maintaining GH release and energy homeostasis. In particular, it appears to exert potent appetite-stimulating activities.

It is therefore desirable to find new compounds which modulate ghrelin receptor activity.

According to the invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof:

It will be appreciated by the person skilled in the art that the compounds of formula (I) may exist either in racemic form or homochiral form. All such forms are included within the scope of the present invention. Diastereoisomer 1 or Diastereoisomer 2 means a compound of the invention in homochiral form as a single diastereoisomer.

In one embodiment there is provided a compound of formula (I) selected from the group consisting of:

and

or a pharmaceutically acceptable salt or solvate thereof.

In a further embodiment there is provided a compound of formula (I) selected from the group consisting of:

(2R)-/V-[3_!5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 _!2-a]pyrazin-2(1 H)-yl]-2- (3-pyridinyl)ethanohydrazide,

(2S)-Λ/^I-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 _!2-a]pyrazin-2(1 H)-yl]-2- (3-pyridinyl)ethanohydrazide, and

N'-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2-(3- pyridinyl)acetohydrazide (diastereomeric mixture) or a pharmaceutically acceptable salt or solvate thereof.

In a further embodiment there is provided a compound of formula (I) which is:

(2R)-N'-[3_!5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2- (3-pyridinyl)acetohydrazide - diastereoisomer 2, or a pharmaceutically acceptable salt or solvate thereof, wherein "diastereoisomer 2" means a single diastereoisomer in homochiral form, prepared according to the procedure described in the Experimental hereinbelow.

In a further embodiment there is provided a compound of formula (I) which is: (2S)-N'-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2- (3-pyridinyl)acetohydrazide - diastereoisomer 1 , or a pharmaceutically acceptable salt or solvate thereof, wherein "diastereoisomer 1" means a single diastereoisomer in homochiral form, prepared according to the procedure described in the Experimental hereinbelow.

It is intended in the context of the present invention that stereochemical isomers enriched in configuration (2R) or (2S) of formula (I) correspond in one embodiment to at least 90% d.e (diastereoisomeric excess). In another embodiment the isomers correspond to at least 95% d.e. In another embodiment the isomers correspond to at least 98% d.e.

Salts of the compounds of the present invention are also encompassed within the scope of the invention. Because of their potential use in medicine, the salts of the compounds of formula (I) are preferably pharmaceutically acceptable. Suitable pharmaceutically acceptable salts can include acid addition salts. A pharmaceutically acceptable acid addition salt can be formed by reaction of a compound of formula (I) with a suitable inorganic or organic acid (such as hydrobromic, hydrochloric, hydroiodic, sulfuric, nitric, phosphoric, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid), optionally in a suitable solvent such as an organic solvent, to give the salt which is usually isolated for example by crystallisation and filtration. Examples of pharmaceutically acceptable acid addition salts of a compound of formula (I) include the HCI, HBr, HI, sulfate or bisulfate, nitrate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, saccharate, fumarate, maleate, lactate, citrate, tartrate, gluconate, camsylate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate and pamoate salts. For reviews on suitable pharmaceutical salts see Berge et al, J. Pharm, ScL, 66, 1-19, 1977; P L Gould, International Journal of Pharmaceutics, 33 (1986), 201-217; and Bighley et al, Encyclopedia of Pharmaceutical Technology, Marcel Dekker Inc, New York 1996, Volume 13, page 453-497. Other salts, which are not pharmaceutically acceptable, for example the trifluoroacetate salt, may be useful in the preparation of compounds of this invention and these form a further aspect of the invention. The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of formula (I).

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compound of the invention are within the scope of the invention.

The invention also includes all suitable isotopic variations of a compound of the invention. An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶CI, respectively. Certain isotopic variations of the invention, for example, those in which a radioactive isotope such as ³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the compounds of the invention can generally be prepared by conventional procedures such as by the illustrative methods or by the preparations described in the Examples hereafter using appropriate isotopic variations of suitable reagents.

The compounds of this invention may be made by a variety of methods, including standard chemistry. Illustrative general synthetic methods are set out below and then specific compounds of the invention are prepared in the working Examples. Scheme 1

(II) (I)

Compounds of formula (I) may be prepared according to the process depicted in Scheme 1 , which comprises:

(A) reacting a compound of formula (II) with [3,5-bis(trifluoromethyl)phenyl]hydrazine of formula (III), together in the presence of a carbamoylating reagent and an organic catalyst in an aprotic solvent; followed by

(B) chiral separation, if required; or in the alternative:

(C) reacting a compound of formula (IV) with with [3,5-bis(trifluoromethyl)phenyl]- hydrazine of formula (III)

(IV) (I) together in the presence of an amide coupling reagent and a suitable base in an aprotic solvent; followed by (D) chiral separation if required.

In step (A) a carbamoylating reagent may be for example tert-Butyl dicarbonate (BOC₂O). In step (C) M means an appropriate metal cation selected from a group consisting of alkaline -earth-metal or alkaline metal, for example potassium.

In another embodiment the present invention provides a method for increasing the yields of (2R)-N'-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2- (3-pyridinyl)ethanohydrazide (diastereoisomer 2), as illustrated in the alternative preparation, by epimerisation of the reaction mixture enriched in diastereoisomer 1 (i.e. mother liquor) obtained after removal of the diastereoisomer 2 by filtration and convenient purification through a silica pad, in presence of a suitable base, such as 1 ,4-diazabicyclo[2.2.2]octane (DABCO) or 1 ,5,7-triazabiciclo[4.4.0]dec-5-ene (TBD), 2-tert-butylimino-2-diethylamino-1 ,3- dimethyl-perhydro-1 ,3,2-diazaphosphorine (BEMP), 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU); in a solvent where the diastereoisomer 2 is unsoluble particularly selected from a group consisting of C2-C4 dialkyl ethers such as Et₂O or iPr₂O or THF at an appropriate temperature such as at solvent reflux.

In a further embodiment the present invention thus also provides compounds of formula (I) and pharmaceutically acceptable salts or solvates thereof, for use in medical therapy, and particularly in the treatment of disorders mediated by the ghrelin receptor.

In a further embodiment the present invention is directed to methods of modulating ghrelin receptor activity for the prevention and/or treatment of disorders mediated by the ghrelin receptor.

In a further embodiment the present invention provides a method of treatment of a mammal suffering from a disorder mediated by the ghrelin receptor, which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof. Such treatment comprises the step of administering a therapeutically effective amount of the compound of formula (I), including a pharmaceutically acceptable salt or solvate thereof. Such treatment can also comprise the step of administering a therapeutically effective amount of a pharmaceutical composition containing a compound of formula (I), including a pharmaceutically acceptable salt or solvate thereof. As used herein, the term "treatment" refers to alleviating the specified condition, eliminating or reducing the symptoms of the condition, slowing or eliminating the progression of the condition, and preventing or delaying the reoccurrence of the condition in a previously afflicted patient or subject.

A further embodiment of the present invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for the treatment of a disorder mediated by the ghrelin receptor.

A further embodiment of the present invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for the treatment of a disorder mediated by the ghrelin receptor.

The action of the endogenous ligand ghrelin at the ghrelin receptor has been shown to result in potent growth-hormone releasing activity, appetite stimulation, stimulation of gastric motility and acid secretion, positive cardiovascular effects and direct action on bone formation. Thus, a ghrelin receptor modulator may achieve a beneficial effect in the treatment of growth-hormone deficiencies, eating disorders, gastrointestinal disease, cardiovascular dieases, osteoporosis, aging and catabolic states or chronic wasting syndromes (Kojima and Kangawa, Nature Clinical Practice, Feb 2006, VoI 2, No.2, 80-88). A ghrelin receptor modulator may also achieve a beneficial effect in the treatment of sleep disorders (Brain Research, 1088 (2006) 131-140). Particular disorders which are associated with the ghrelin receptor and thus may be mediated by the ghrelin receptor such that a ghrelin receptor modulator may achieve a beneficial effect include obesity and risk factors associated with obesity, including but not limited to diabetes, complications associated with diabetes, metabolic syndrome, cardiovascular disorders (including atherosclerosis and dyslipidemia).

Other diseases and/or conditions mediated by the ghrelin receptor wherein a ghrelin include the following, treating a growth hormone deficient state , increasing muscle mass, increasing bone density, treating sexual disfunction in males and females, facilitating a weight gain, facilitating weight maintenance, facilitating appetite increase (for example facilitating weight gain, maintenance or appetite increase is useful in a patient having a disorder, or under going a treatment, accompanied by weight loss). Examples of diseases or disorders accompanied by weight loss include anorexia, bulimia, cancer cachexia, AIDS, wasting, cachexia and wasting in frail elderly. Examples of treatments accompanied by weight loss include chemiotherapy, radiation therapy, temporary or permanent immobilisation, and dialysis.

Further diseases or conditions include sleep disorders, congestive heart failure, metabolic disorder, improvements in memory function, breast cancer, thyroid cancer, ameliorating ischemic nerve or muscle damage.

The compounds of the invention function by modulating the activity of the ghrelin receptor. They may activate/inactivate the receptor by acting as an agonist, partial agonist, inverse agonist, antagonist or partial antagonist.

Eating disorders include Anorexia Nervosa (307.1 ) including the subtypes Restricting Type and Binge-Eating/Purging Type; Bulimia Nervosa (307.51 ) including the subtypes Purging Type and Nonpurging Type; Obesity; Compulsive Eating Disorder; Binge Eating Disorder; and Eating Disorder Not Otherwise Specified (307.50). [the numbers in brackets after the listed diseases above refer to the classification code in Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, published by the American Psychiatric Association (DSM-IV) and/or the International Classification of Diseases, 10th Edition (ICD-10)].

In a further embodiment the invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for the treatment of an eating disorder

In a further embodiment the present invention provides a method of treatment of a mammal suffering from an eating disorder which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof. Gastrointestinal diseases include gastric ileus, gastric ulcer and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. The compounds of the invention may also be useful for treatments to alleviate symptoms associated with gastro-esophageal reflux (GER) and/or with dyspepsia, with or without appetite-/metabolic-related cachexia, and in the treatment of paralytic ileus or pseudo obstruction, and of conditions associated with with constipation, such as constipation-predominant irritable bowel syndrome.

Cardiovascular diseases include heart failure and dilated cardiomyopathy.

Catabolic states or chronic wasting syndromes may be seen in post-operative patients and also include AIDS-associated and cancer-associated wasting syndromes, such as cancer cachexia.

While it is possible that, for use in therapy a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, may be administered as the raw chemical, it is possible to present the active ingredient as a pharmaceutical composition. Thus, in a further embodiment the invention provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, in admixture with one or more pharmaceutically acceptable carriers, diluents, or excipients. The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In a further embodiment the invention also provides a process for the preparation of a pharmaceutical composition including admixing a compound of (I), or a pharmaceutically acceptable salt or solvate thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients.

Pharmaceutical compositions of the invention may be formulated for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Therefore, the pharmaceutical compositions of the invention may be formulated, for example, as tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions. Such pharmaceutical formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatine, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatine, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.

The topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, eye ointments and eye or ear drops, impregnated dressings and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams. The formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.

Pharmaceutical formulations adapted for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid may include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered, dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question.

A therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof will depend upon a number of factors including, for example, the age and weight of the human or other animal, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. However, an effective amount of a compound of formula (I) for the treatment of disorders mediated by the ghrelin receptor will generally be in the range of 0.1 to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 1 to 10 mg/kg body weight per day. Thus, for a 70kg adult mammal, the actual amount per day would usually be from 70 to 700 mg and this amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a pharmaceutically acceptable salt or solvate thereof, may be determined as a proportion of the effective amount of the compound of formula (I) per se.

A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof for use in the instant invention may be used in combination with one or more other therapeutic agents. The invention thus provides in a further embodiment a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof together with a further therapeutic agent, which may be for example an additional anti-obesity agent. In a yet further embodiment the invention also provides the use of a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof with a further therapeutic agent in the treatment of disorders mediated by the ghrelin receptor.

When a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof is used in combination with one or more other therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above optimally together with a pharmaceutically acceptable carrier or excipient comprise a further embodiment of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation and may be formulated for administration. When formulated separately they may be provided in any convenient formulation, conveniently in such a manner as are known for such compounds in the art.

When a compound is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

Experimental The invention is illustrated by the Compounds described below.

Compounds are named using ACD/Name PRO 6.02 chemical naming software (Advanced Chemistry Development Inc., Toronto, Ontario, M5H2L3, Canada).

Proton Magnetic Resonance (NMR) spectra were recorded either on Varian instruments at 300, 400 or 500 MHz, or on a Bruker instrument at 300 or 400 MHz. Chemical shifts are reported in ppm (δ) using the residual solvent line as internal standard. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The NMR spectra were recorded at a temperature ranging from 25 to 9O⁰C.

Mass spectra (MS) were taken on an Agilent MSD 1100 Mass Spectrometer, operating in ES (+) and ES (-) ionization mode or on an Agilent LC/MSD 1100 Mass Spectrometer, operating in ES (+) and ES (-) ionization mode coupled with HPLC instrument Agilent 1100 Series [LC/MS - ES (+/-): analysis performed using a Supelcosil ABZ +Plus (33x4.6 mm, 3μm) (mobile phase: 100% [water +0.1% HCO₂H] for 1 min, then from 100% [water +0.1% HCO₂H] to 5% [water +0.1 % HCO₂H] and 95% [MeCN ] in 5 min, finally under these conditions for 2 min; T=40°C; flux= 1 mL/min; LC/MS - ES (+/-):analysis performed on a Supelcosil ABZ +Plus (33x4.6 mm, 3μm) (mobile phase: 100% [water +0.05% NH₃] for 1 min, then from 100% [water +0.05% NH₃ to 5% [water +0.05% NH₃] and 95% [MeCN ] in 5 min, finally under these conditions for 2 min; T=40°C; flux= 1 mL/min]. In the mass spectra only one peak in the molecular ion cluster is reported. Total ion current (TIC) and DAD UV chromatographic traces together with MS and UV spectra associated with the peaks were taken also on a UPLC/MS Acquity™ system equipped with 2996 PDA detector and coupled to a Waters Micromass ZQ™ mass spectrometer operating in positive or negative electrospray ionisation mode. [LC/MS - ES (+/-): analyses performed using an Acquity™ UPLC BEH C18 column (50 x 21 mm, 1.7 μm particle size), column temperature 4O⁰C (mobile phase: A-water + 0.1% HCOOH / B - MeCN + 0.075% HCOOH, Flow rate: 1.0 mL/min, Gradient: t=0 min 3% B, t=0.05 min 6% B, t= 0.57 min 70% B, t=1.4 min 99% B, t=1.45 min 3% B)].

For the chiral separation and the chiral quality control two different techniques were used: 1 ) Supercritical Fluid Chromatography (SFC): analytical chromatography was performed on a Berger SFC Analytix, while for the preparative SFC, a Gilson SFC series SF3 was used 2) High Performance Liquid Chromatography (HPLC): chiral Preparative HPLC was performed using a Waters 600 HPLC system and Agilent series 1 100 instrument, while for analytical chromatography an Agilent series 1 100 HPLC was used.

For reactions involving microwave irradiation, a Personal Chemistry EmrysTM Optimizer was used.

Thin Layer Chromatography (TLC) was performed on Merck 200 TLC Plates (5 x 10 cm) Silica Gel 60 F₂₅₄

Flash silica gel chromatography was carried out on silica gel 230-400 mesh (supplied by Merck AG Darmstadt, Germany) or over Varian Mega Be-Si pre-packed cartridges or over pre-packed Biotage silica cartridges.

SPE-SCX cartridges are ion exchange solid phase extraction columns by supplied by Varian. The eluent used with SPE-SCX cartridges is MeOH followed by ammonia solution in MeOH.

Oasis HLB-LP extraction cartridges are ion exchange solid phase extraction columns supplied by Waters. The eluent used with HLB cartridges is water followed by MeOH.

In a number of preparations purification was performed using either Biotage manual flash chromatography (Flash+) or automatic flash chromatography on Horizon or ISCO- Companion systems. All these instruments work with Silica cartridge.

SPE-SI cartridges are silica solid phase extraction columns supplied by Varian or IST Isolute.

Where a percentage yield has been quoted for a single diastereoisomer isolated from a racemic mixture this has been calculated on the basis that the maximum possible yield of material (i.e. 100%) is half of the total mass of the racemate.

Abbreviations: d.e.: diastereoisomeric excess Ms: Mesyl

R₁: retention time

CH: cyclohexane

AcOEt: ethyl acetate DCM: dichloromethane

MeOH: methanol

IPA: isopropanol

Et₂O: diethyl ether

EtOH: ethanol MeCN: acetonitrile

DMF: N,N-dimethylformamide

THF: tetrahydrofuran

NMM: 4-methylmorpholine

BOP: (benzotriazol-1-yloxy)tris(dimethyl-amino)phosphonium-hexafluorophosphate TEA: triethylamine n-BuLi: n-Butyllithium n-Hex: n-hexane

DMAP: 2,6-dimethylaminopyridine

RT: room temperature TMSCN: trimethylsilylcyanide

DMSO: dimethylsulfoxide

BoC₂O: tert-Butyl dicarbonate

In the procedures that follow, after each starting material, reference to a Description or Example by number is typically provided. This is provided merely for assistance to the skilled chemist. The starting material may not necessarily have been prepared from the batch referred to.

Intermediate 1 : (8a/?)-Octahvdropyrrolo[1 ,2-a1pyrazine dihvdrochloride

To a stirred solution of 10 g of (8af?)-hexahydropyrrolo[1 ,2-a]pyrazin-6(2H)-one (prepared as set out in WO03066635A1 , 71.4 mmole) in 150 ml. of dry THF, were added in 20 min, under Argon atmosphere, 214 ml. of 1 M solution of BH₃THF (214 mmole, Aldrich) at O⁰C. The solution was then stirred at RT for 3 h, heated at 5O⁰C for 3 h, left at RT overnight and then heated again at 5O⁰C for 2 h. The reaction mixture was then cooled to O⁰C and further 71 ml_ of BH₃THF (71 mmole, Aldrich) were added dropwise. The solution was heated at 5O⁰C to allow the reaction to be completed. At O⁰C, 80 ml. of MeOH were added followed by 300 ml_ of HCI 1 M in Et₂O. The solution was heated at reflux for 5 h and then left at RT over the weekend. Solvents were removed under reduced pressure to give the title compound (13g, 93%). ¹H-NMR (400 MHz, DMSO-d₆): δ 1.15-4.05 (13H, m), 9.2-9.9 (2H, br s), 1 1.84-12.04 (1 H, br s).

19.78 ml. of nBuLi 1.6M in hexanes (31.65 mmoles, ALDRICH) were added to 7.91 ml. of iPrMgCI 2M diluted in 66 ml. of THF at O⁰C (7⁰C int. T). After stirring for 15 min at O⁰C, the mixture was cooled to -1O⁰C (-8⁰C int. T) and a solution of 3.05 ml. of 3-bromopyridine (31.65 mmoles, ALDRICH) in 31.65 mL of THF was added. The mixture was then stirred at - 1O⁰C for 30 min. Then 12.92 mL of a solution of ethyl glyoxylate 50 % in toluene (63.29 mmoles, FLUKA) were added and the mixture was stirred at O⁰C for 2 h. The reaction mixture was then partitioned between 100 mL of AcOEt and 100 mL of a saturated solution of K₂CO₃. The precipitate was filtered and the resulting biphasic solution was transferred to a separatory funnel. The aqueous phase was extracted with 50 mL of AcOEt. The combined organic layers were dried over Na₂SO₄, filtered and evaporated to dryness. The resulting crude was then purified by flash chromatography on silica gel with the following gradient: A: CH + 3%TEA/AcOEt + 3%TEA: 0%B for 2 min, 0% to 100% B in 24 min, 100%B for 6 min. Evaporation of the solvents under reduced pressure and further purification gave the title compound as a yellow oil (2.75 g, 48%). ¹H-NMR (400 MHz, CDCI₃): δ 1.23 (3H, t), 4.14-4.33 (2H, m), 5.23 (1 H, s), 7.31 (1 H, dd), 7.79 (1 H, dt), 8.55 (1 H, dd), 8.68 (1 H, d); LC-MS [Supelcosil ABZ+Plus, 33x4.6mm, 3 μm, gradient: A: H₂O +0.1% HCOOH/B: MeCN: 0% to 95%B in 3 min., 95%B for 1 min., 95%B to 0%B in 0.1 min., flow rate: 2mL/min]: R_t = 0.24 min and 0.36 min. (96.8%) m/z (ES): 182.0 [M+H]⁺.

Intermediate 3: Ethyl (8aR)-hexahvdropyrrolo[1 ,2-a1pyrazin-2(1 H)-yl(3-pyridinyl)acetate (mixture of diastereoisomers)

1.5 g of (±)-ethyl hydroxy(3-pyridinyl)acetate (Intermediate 2, 8.28 mmoles) were dissolved in 52.5 mL of DCM. The solution was cooled to O⁰C (+4⁰C int. T) and 4.05 mL of TEA (28.97 mmoles, ALDRICH) were added followed by 704 μL of MsCI (9.1 1 mmoles, ALDRICH). The mixture was then stirred at O⁰C for 1.3 h. Then 1.41 g of (8aR)-octahydropyrrolo[1 ,2- a]pyrazine dihydrochloride (Intermediate 1 , 8.7 mmoles) were added and the solution was warmed to RT and stirred at RT for 13.5 h. 50 mL of a saturated solution of K₂CO₃ were added and the biphasic solution was transferred to a separatory funnel. The aqueous phase was removed and the organic phase was dried over Na₂SO₄, filtered and evaporated to dryness. The resulting crude was then purified by flash chromatography on silica gel using the following gradient: A: CH +3%TEA/ AcOEt +3% TEA: 0%B for 2 min, 0% to 100%B in 25 min, 100%B for 5 min. Evaporation of the solvents under reduced pressure and further purification gave the title compound as an orange oil (896.3 mg, 37%). ¹H-NMR (400 MHz, CDCI₃): δ 1.22-1.41 (5H, m), 1.68-1.90 (3H, m), 2.05-2.20 (2H, m), 2.22-2.50 (2H, m), 2.69- 2.83 (1 H, m), 2.90-3.10 (3H, m), 4.12-4.29 (3H, m), 7.30-7.32 (1 H, m), 7.82-7.84 (1 H, m), 8.57-8.58 (1 H, m), 8.64 (1 H, d); LC-MS [Supelcosil ABZ+Plus, 33x4.6mm, 3 μm, gradient: A: H₂O +0.1% HCOOH/B: MeCN: 0% to 95%B in 3 min., 95%B for 1 min., 95%B to 0% B in 0.1 min., flow rate: 2ml_/min]: R_t = 1.35 min (82.8%) m/z (ES): 290.1 [M+H]⁺.

Intermediate 4: N'-[3,5-bis(trifluoromethyl)phenyl1-2-[(8aR)-hexahvdropyrrolo[1 ,2-aipyrazin- 2(1 H)-yl1-2-(3-pyridinyl)acetohydrazide (mixture of diastereoisomers)

896 mg of ethyl (8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl(3-pyridinyl)acetate (Intermediate 3, 3.1 mmoles) were dissolved in 10.1 ml. of MeOH. 191 mg of KOH (3.4 mmoles, FLUKA) were added followed by 2.23 mL of water. The mixture was then stirred at RT for 43.5 h. Solvents were removed under reduced pressure and the resulting compound was dried under high vacuum for a couple of hours to give an orange oil (984 mg). This compound was dissolved in 20.5 mL of DMF at RT. Then 883 mg of 3,5- bis(trifluoromethyl)phenylhydrazine (3.62 mmol, LANCASTER) were added followed by 1.60 g of BOP (3.62 mmole, FLUKA). The mixture was stirred at RT for 3.5 h. Solvents were removed under reduced pressure and the residue was dissolved in MeOH. The solution was then passed through a SCX cartridge (25g). The cartridge was washed with 100 mL of DCM, 100 mL of MeOH and the compound was released with 60 mL of NH3 2M in MeOH. Solvents were removed under reduced pressure and the resulting crude was then purified by flash chromatography on silica gel with the following gradient: A: AcOEt+3%TEA/B: MeOH+3%TEA: 0%B for 2 min, 0% to 20%B in 20 min, 20%B for 4 min. Solvents were removed under reduced pressure and the resulting compound was then triturated twice with 10 mL of Et2θ. Supernatant was removed and the resulting solid was dried under high vacuum for a couple of hours to give the title compound as a yellow solid (673 mg, 44%). ¹H- NMR (400 MHz, CD₃OD): δ 1.40-1.60 (1 H, m), 1.70-2.00 (3H, m), 2.22-2.59 (5H, m), 2.73- 2.80 (1 H, m), 3.06-3.33 (3H, m), 4.23-4.25 (1 H, 2 s), 7.13 (2H, d), 7.34 (1 H, s), 7.58-7.61 (1 H, m), 8.14-8.16 (1 H, m), 8.66 (1 H, d), 8.79 (1 H, s). LC-MS [Supelcosil ABZ+Plus, 33x4.6mm, 3 μm, gradient: A: H₂O +0.1% HCOOH/B: MeCN: 0% to 95%B in 3 min., 95%B for 1 min., 95%B to 0%B in 0.1 min., flow rate: 2mL/min]: R_t = 1.52 min (100%) m/z (ES): 488 [M+H]⁺, 244.5 [M+H/2]⁺. Compound 1 : (2S)-ΛM3,5-bis(trifluoromethyl)phenyl1-2-[(8a/?)-hexahvdropyrrolo[1 ,2- a1pyrazin-2(1 H)-yl1-2-(3-pyridinyl)ethanohydrazide (diastereoisomer 1 )

673 mg of Λ/'-[3_!5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 _!2-a]pyrazin-2(1 H)- yl]-2-(3-pyridinyl)acetohydrazide (Intermediate 4, 1.38 mmoles) were purified by semipreparative chiral SFC [Chiralpak AD-H, 25x2.0 cm, pressure: 182 bar, Flow rate: 22 mL/min, UV detection: 220 nm, Injection, 20 mg each in EtOH, modifier: EtOH +0.1 % isopropylamine]. Solvents were removed under reduced pressure to give the title compound as a yellow solid (258.4 mg, 38%). ¹H-NMR (400 MHz, CDCI₃): δ 1.31-1.36 (2H, m), 1.64- 1.84 (3H, m), 2.12-2.22 (2H, m), 2.41 (1 H, td), 2.58 (1 H, dt), 2.88 (1 H, dt), 3.00-3.14 (3H, m), 4.31 (1 H, s), 6.45 (1 H, br s), 7.14 (2H, s), 7.34-7.38 (2H, m), 7.69 (1 H, dt), 8.60 (1 H, s), 8.63 (1 H, dd), 8.91 (1 H, s); LC-MS [Supelcosil ABZ+Plus, 33x4.6mm, 3 μm, gradient: A: H₂O +0.1% HCOOH/B: MeCN: 0% to 95%B in 3 min., 95%B for 1 min., 95%B to 0% B in 0.1 min., flow rate: 2ml_/min]: R_t = 1.52 min (100%) m/z (ES): 488 [M+H]⁺, 244.5 [M+H/2]⁺; Chiral SFC [Chiralpak AD-H, 25x0.46 cm, pressure: 100 bar, Flow rate: 2.0 mL/min, UV detection: 240 nm, modifier: EtOH+0.1 % isopropylamine]: R₁ = 9.61 min. (100 % d.e.).

Compound 2: (2/?VΛH3.5-bis(trifluoromethvnphenyll-2-r(8a/?Vhexahvdropyrrolori .2- alpyrazin-2(1 H)-yll-2-(3-pyridinyl)ethanohvdrazide (diastereoisomer 2):

673 mg of /V-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)- yl]-2-(3-pyridinyl)acetohydrazide (Intermediate 4, 1.38 mmoles) were purified by semipreparative chiral SFC [Chiralpak AD-H, 25x2.0 cm, pressure: 182 bar, Flow rate: 22 mL/min, UV detection: 220 nm, Injection, 20 mg each in EtOH, modifier: EtOH +0.1 % isopropylamine]. Solvents were removed under reduced pressure to give the title compound as a yellow solid (285 mg, 42%). ¹H-NMR (400 MHz, CDCI₃): δ 1.42-1.46 (1 H, m), 1.75-1.87 (3H, m), 2.09-2.23 (4H, m), 2.33-2.37 (1 H, t), 2.71 (1 H, d), 3.02-3.08 (2H, m), 3.19 (1 H, d), 4.27 (1 H, s), 6.38 (1 H, br s), 7.15 (2H, s), 7.33-7.39 (2H, m), 7.70 (1 H, dt), 8.61-8.63 (2H, m), 8.86 (1 H, s); LC-MS [Supelcosil ABZ+Plus, 33x4.6mm, 3 μm, gradient: A: H₂O +0.1% HCOOH/B: MeCN: 0% to 95%B in 3 min., 95%B for 1 min., 95%B to 0% B in 0.1 min., flow rate: 2ml_/min]: R₁ = 1.53 min (100%) m/z (ES): 488 [IVH-H]⁺, 244.5 [M+H/2]⁺; Chiral SFC [Chiralpak AD-H, 25x0.46 cm, pressure: 100 bar, Flow rate: 2.0 mL/min, UV detection: 240 nm, modifier: EtOH+0.1 % isopropylamine]: R₁ = 13.08 min. (100 % d.e.).

Compound 3: (2/?)-ΛM3,5-bis(trifluoromethyl)phenyl1-2-[(8a/?)-hexahvdropyrrolo[1 ,2- 2):

285 mg of (2R)-Λ/'-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin- 2(1 H)-yl]-2-(3-pyridinyl)ethanohydrazide (diastereoisomer 2) (Compound 2) were dissolved in 8 ml. of MeOH and 585 μl_ of HCI 1 N/Et₂O were added. The mixture was stirred at RT for 5 min and solvents were removed. 5 ml. of Et₂O were added and solvents were removed. The resulting solid was then dried under high vacuum for a couple of hours to give the title compound as a white solid (313.7 mg, quantitative). ¹H-NMR (400 MHz, CD₃OD): δ 1.70-1.80 (0.5H, m), 2.1 1-2.27 (3.5H, m), 2.56-2.94 (4H, m), 3.05-3.25 (2H, m), 3.37-3.52 (3H, m), 3.60-3.70 (1 H, m), 4.43 (0.5H, s), 4.58 (0.5H, s), 7.05-7.1 1 (2H, m), 7.29 (1 H, m), 7.75 (1 H, m), 8.28 (1 H, m), 8.72 (1 H, m), 8.83 (1 H, s); LC-MS [Supelcosil ABZ+Plus, 33x4.6mm, 3 uM, gradient: A: H2O+0.1 % HCOOH/B: MeCN: 0% to 95%B in 3 min, 95 %B for 1 min, 95%B to 0%B in 0.1 min, flow rate: 2 mL/Min]: R₁ = 1.53 min (100%) m/z (ES): 488 [M+H]⁺, 244.5 [M+H/2]⁺; Chiral SFC [Chiralpak AD-H, 25x0.46 cm, pressure: 100 bar, Flow rate: 2.0 mL/min, UV detection: 240 nm, modifier: EtOH+0.1 % isopropylamine]: R_t = 13.08 min. (100 % d.e.).

Alternative Preparation of Compound 2

Intermediate 1 : (8a/?)-Octahvdropyrrolo[1 ,2-a1pyrazine dihvdrochloride

62 g of (8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-6(2H)-one (prepared as set out in WO03066635A1 , 442 mmole) were suspended in 1 L of anhydrous THF (15 vol.) and the mixture was cooled to 7°C (internal). 1.33 L of Borane 1 M sol. in THF (1327 mmol, Aldrich) were added dropwise in 20 min. The mixture was heated at 50⁰C (internal) for 5 h. Then the solution was cooled to 7°C (internal) and 500 mL of MeOH were added. The solution was left at 25°C overnight. 1 L of HCI 1 M in ether (16 vol.) were added and the mixture was heated at 50⁰C (internal) for 4 h. Further 200 mL of HCI 1 M in ether were added and the mixture was heated at 50⁰C for another hour. The mixture was cooled to 25°C and the solvent was evaporated. MeOH (2x1250 ml) was added and evaporated to give a white solid (89.7 g, 100%). ¹H-NMR (400 MHz, DMSO-d₆): δ 1.15-4.05 (13H, m), 9.2-9.9 (2H, br s), 11.84-12.04 (1 H, br s).

Intermediate 5: (8a/?)-hexahydropyrrolori ,2-alpyrazin-2(1 /-/)-yl(3-pyridinyl)acetonitrile (mixture of diastereoisomers)

To a solution of 30.8 ml. of 3-pyridinecarbaldehyde (326 mmole, Aldrich) in 52.5 ml. of diethyl ether (1.5 vol.) were added 52.5 ml. of TMSCN (392 mmole, Aldrich) and 5.21 g of zinc iodide (16.32 mmole, Aldrich) at RT under nitrogen. The orange reaction mixture was cooled to 10⁰C (internal) and it was stirred for 5 min. Then a solution of 70 g of (8a/?)- octahydropyrrolo[1 ,2-a]pyrazine dihydrochloride (Intermediate 1 , 352 mmole) and 95 ml. of TEA (685 mmol) in 286 ml. of methanol (8.1 vol.) was added dropwise to the reaction mixture and it was refluxed for 3 h. The solution was cooled to RT and 700 ml. of a saturated solution of K₂CO₃ in water and 700 ml. of AcOEt were added. The phases were separated and the water phase was back extracted twice with AcOEt (700 ml. and 350 ml_). The combined organic layers were dried over Na₂SO₄, filtered and evaporated to dryness. The crude was purified by flash chromatography on silica gel eluting with AcOEt + TEA 2% v/v to give the title compound as an orange liquid that solidified at +5°C (71.3 g, 90%) ¹H-NMR (400 MHz, CDCI₃): δ 1.30-1.58 (1 H, m), 1.64-2.05 (4H, m), 2.05-2.26 (2H, m), 2.35-2.54 (2H, m), 2.56- 3.02 (2H, m), 3.05-3.18 (2H, m), 4.88-4.99 (1 H, m), 7.34-7.41 (1 H, m), 7.89 (1 H, d), 8.65 (1 H, ddd), 8.77-8.83 (1 H, m). TLC plate (DCM/MeOH 9:1 ), R_f=0.58.

Intermediate 6: 2-[(8a/?)-hexahydropyrrolo[1 ,2-a1pyrazin-2(1 /-/)-yl1-2-(3-pyridinyl)acetamide

71 g of (8a/?)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl(3-pyridinyl)acetonitrile (Intermediate 5, 293 mmole) were suspended in 710 ml. of n-Hexane and cooled down to O⁰C. Then 437 ml_ of concentrated sulfuric acid (8204 mmole) were added dropwise and the obtained mixture was allowed to reach RT and was stirred mechanically for 2 days. The mixture was cooled down to O⁰C and 1 volume of ice was added. The mixture was slowly alkalinized with 1.4 L of a 28 % solution of ammonia in water. The aqueous phase was extracted twice with 1.5 L of DCM, The organic phase was dried over Na₂SO₄, filtered and evaporated to dryness to give a first batch of the title compound as a yellow solid (39 g). The aqueous phase was extracted twice with 2 L of DCM. The combined organic layers were then dried over Na₂SO₄, filtered and evaporated to dryness to give a second batch of the title compound as a yellow solid (15 g). Finally, the aqueous layer was concentrated under reduced pressure to approximately 1 L, solids were removed by filtration and mother liquors were extracted twice with 1.5 L of DCM. The combined organic layers were then dried over Na₂SO₄, filtered and evaporated to dryness to give the third batch of the title compound as a yellow solid (11.7 g) (global yield: 86 %). ¹H-NMR (400 MHz, CD₃OD): δ 1.24-1.50 (1 H, m), 1.70-2.28 (6H, m), 2.53 (2H, m), 2.76 (1 H, m), 2.89-3.24 (3H, m), 4.01-4.09 (1 H, m), 7.47 (1 H, dd), 7.92-8.01 (1 H, m), 8.52 (1 H, m), 8.58-8.64 (1 H, m); TLC Plate R_f = 0.15 (AcOEt/MeOH 9/1 + Et₃N 3%v/v)

Compound 2: (2RVN'-r3.5-bis(trifluoromethvnphenyll-2-r(8aRVhexahvdropyrrolon .2- alpyrazin-2(1 H)-yll-2-(3-pyridinyl)ethanohvdrazide (diastereoisomer 2)

60 g of 2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2-(3-pyridinyl)acetamide (Intermediate 6, 230 mmol) were dissolved in 2.1 L of DCM. Then 107 ml. of tert-Butyl dicarbonate (BoC₂O) (461 mmole) and 2.81 g of DMAP (23.03 mmole) were added. The mixture was stirred at RT for 5.5 h. The solution was concentrated to the third and 67.5 g of [3,5-bis(trifluoromethyl)phenyl]hydrazine (277 mmole) were added followed by 5.63 g of DMAP (46.06 mmole). The solution was stirred at RT for 64 h. The solid was filtered, washed with diethyl ether and dried in vacuo to give 38.7 g of a white solid (batch 1 ). Mother liquors were concentrated to a minimum volume and were charged on a silica pad. The compound was eluted with AcOEt/MeOH 9:1 + 3% of TEA. Removal of the solvent gave 50.9 g of a solid. This solid was suspended in 500 ml. of diisopropyl ether and 57.5 g of 1 ,4- diazabicyclo[2.2.2]octane (513 mmole, Aldrich) were added. The suspension was heated at 8O⁰C overnight. The mixture was allowed to reach RT and filtered through a sintered glass funnel to obtain 20 g of a beige solid (batch 2). Batch 1 and batch 2 were mixed together (53.7 g, 1 10 mmole) and were suspended in 540 ml. of acetone and heated to 7O⁰C for 45 min. Then the mixture was allowed to reach RT and stirred for 1 h. The reaction mixture was filtered through a sintered glass funnel, the solid was washed with 150 ml. of acetone, and dried in vacuo to give the title compound as a white solid (49 g, 43%). ¹H-NMR (400 MHz, DMSO-d₆): δ 1.13-1.37 (1 H, m), 1.58-1.76 (3H, m), 1.90-2.20 (5H, m), 2.51-2.56 (1 H, m), 2.75-3.08 (3H, m), 4.07 (1 H, s), 7.03 (2H, s), 7.29 (1 H, s), 7.40 (1 H, dd), 7.88 (1 H, dt), 8.54 (1 H, dd), 8.64 (1 H, d), 8.77 (1 H, s), 10.46 (1 H, s). Chiral HPLC [Chiralpak AD-H, mobile phase: n-Hexane/lsopropanol 92/8 v/v, flow: 1 mL/min]: R₁ = 20.07 min (d.e. >99%)

X-Rav Structure of compound 2 A spatula tip of compound 2, prepared in ana analogous way as above, (ca. 2 mg) was suspended in water (ca. 2 mL) in a soda glass sample vial (50 x 12/13 mm). The solution was heated to 4O⁰C while being stirred. At 4O⁰C acetonitrile was added dropwise until the solid just dissolved (ca 0.35 mL). The solution was then cooled to ambient temperature and filtered through a 0.45 μm syringe tip filter into a soda glass sample vial, which had been blown through with compressed air to remove any particulates. The vial was sealed with a plastic lid, into which 2 holes had previously been pierced with a needle. The solution was allowed to evaporate slowly before isolation of the crystals. X-ray experiment was performed on a Nonius KappaCCD diffractometer.

The ORTEP diagram of compound 2 is shown in Figure 1 : a view of a crystal structure of a molecule of compound 2, showing the numbering scheme employed. Anisotropic atomic displacement ellipsoids for the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. The minor component of the trifluoromethyl disorder is omitted for clarity.

The diffraction experiment unambiguously gave the relative stereochemistry (C8, R; C10, R) and the modelled enantiomer was chosen based on the 'known' stereocentre (C8, see WO03066635A1 ).

Biological assays

The ability of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof to modulate the growth hormone secretagogue receptor may be determined using the following assay:

Cloning of the ghrelin receptor GHS-R

Human GHS-R was cloned from human hypothalamus cDNA and TOPO Ta cloned into pCR2.1. The sequence was confirmed. The full-length gene was transferred into pCDN for expression analysis. The sequence was confirmed again and the plasmid was electroporated into CHO cells. The clones were screened by FLIPR.

Generation of the GHS-R bacmam virus and viral titre determination Virus generation

The open reading frame of GHS-R was transferred from pCDN into pFastBacmam vector. This vector was used to generate recombinant baculoviruses in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active promoter, in this case CMV. This was then used with the Bac to Bac expression system (Invitrogen). Briefly the vector was transformed into DH 10 bac E.coli and the bacmid isolated from the transformed cells. The bacmid was then transfected into Sf9 insect cells grown in ExCeII 420 (JRH) medium in 6-well dishes for the production of recombinant baculovirus particules. The supernatant from these cells was harvested containing the recombinant GHS-R bacmam virus. This PO viral stock was then used to infect 200 ml. of 1x10^"6cells/ml_ Sf9 cells in ExCeII 420 medium to further amplify the virus and provide a P1 stock.

This P1 viral stock was then used to amplify a P2 viral stock of 10x1 L erlemeyer shake flasks again harvesting the supernatant from the cells. This was then used to transduce mammalian cells for assay

Viral titre determination

Viral titres were determined at all stages of the virus scale up with a plaque elisa method using a gp64 envelope protein monoclonal antibody .

SF9 cells were plated out into a 96 well plate and a dilution range of virus was added to the cells for 1 h. The virus was removed and a 1% methylcellulose and media mix was added to the cells and incubated for 48 h. The cells were then fixed in a formaldehyde and acetone mix for 8 min. The cells were then washed with a phosphate buffered saline solution (PBS) and normal goat serum added for 25 min. This was then removed and a gp64 monoclonal antibody added for 25 min. The wells were then washed with PBS and a goat anti- mouse/HRP conjugated antibody added for 25 min. The wells were again washed with PBS and True Blue peroxidase substrate solution (Kirkegaard & Perry Laboratories) added and incubated for 60 min. Individual wells were counted for blue foci and taking into account the dilution factor, the plaque forming units/mL of the virus was determined.

Generation of U2OS cells transiently expressing the ghrelin receptor GHS-R

24 h prior to assay U2OS cells at confluence 100% were harvested and spun down. The supernatant was removed and the cells resuspended in media (DMEM + 10% FBS + 1% L-

Glutamine). A cell count was performed using the Cedex instrumentation, and the concentration of cells was adjusted using media to give 200K cells per mL (10K cells/ 50 μL).

Human GHSR BACMAM virus was added to the cell suspension at an appropriate % volume (calculated for individual batches of BACMAM virus as viral titres vary). The transduced cell suspension was dispensed into FLIPR 384-well clear bottom plates, 50 μL per well. Cell plates were incubated at 37°C overnight.

Compound preparation Master compound plates were prepared in 100% DMSO. 0.6 mM was the top concentration (giving 3 μM final concentration) and they were serially diluted 1 in 4. 1 μL from the master plate was transferred to a daughter plate, to which was added 50 μL of compound dilution (NaCI (145 mM) KCI (5 mM) CaCI₂ (2 mM) MgCI₂ (1 mM) HEPES (20 mM) D-(+)-Glucose (1 g/L), pluronic acid (0.05%), pH 7.3). This plate was used for the assay. Ghrelin was always prepared in compound dilution buffer containing 0.1 % BSA.

GHSR Agonist BACMAM FLIPR Assay protocol Media was washed from cell plates, using a cell washer (leaving 20 uL of washing buffer (NaCI (145 mM) + KCI (5 mM) + CaCI₂ (2 mM) + MgCI₂ (1 mM) + HEPES (20 mM) + D-(+)- Glucose (1 g/L) + Probenecid (2.5 mM), pH 7.3). Cells were immediately loaded with loading buffer (washing buffer + pluronic acid (0.02%) + Fluo 4 dye (2 μM), and incubated at 37°C, for 60 mins. After a second washing step (same as for the pre-loading, but leaving 30 μl_ of washing buffer), the plates were placed in a Fluorimetric Imaging Plate Reader (FLIPR, Molecular Devices) where 10 μL of compound, prepared according to method C above, was added to the cells and fluorescence measurements were taken. Maximum changes in fluorescence were plotted as a percentage of the maximum response elicited by 100 nM hGhrelin and curves fitted using a 4- parameter logistic equation to generate pEC50 values. Percentage activation of the compounds was calculated by using the maximum asymptote of it's concentration response curve relative to the maximum response of the hGhrelin at 10OnM concentration.

GHSR Antagonist BACMAM FLIPR Assay protocol

Media was washed from cell plates, using a cell washer (leaving 20 μL of washing buffer (NaCI (145 mM) + KCI (5 mM) + CaCI₂ (2 mM) + MgCI₂ (1 mM) + HEPES (2OmM) + D-(+)- Glucose (1 g/L) + Probenecid (2.5 mM), pH 7.3). Cells were immediately loaded with loading buffer (washing buffer + pluronic acid (0.02%) + Fluo 4 dye (2 μM), and incubated at 37°C, for 60 min. After a second washing step (same as for the pre-loading, but leaving 30 μL of washing buffer), 10 μL of compound, prepared according to method C above, was added to cell plates using an FX robot (or similar) and the plate was then incubated at room temperature for a further 30 min before being assayed on a FLIPR, where 10 μL of an EC80 concentration of hGhrelin was added to the cells and fluorescence measurements were taken. Maximum changes in fluorescence were plotted as a percentage of the maximum inhibition, where 100% inhibition was derived from non-stimulated cells in the same plate. Curves were fitted using a 4- parameter logistic equation to generate plC50 values.

Compounds 1 to 3 were tested in the GHSR Antagonist BACMAM FLIPR Assay protocol and found to give plC50 values of greater than 6.0.

Compound 1 was tested in the GHSR Agonist BACMAM FLIPR Assay and found to give a percentage activation value of ≥ 40%.

Compounds 2 and 3 were tested in the GHSR Agonist BACMAM FLIPR Assay and found to give percentage activation values of less than 40%.

Claims

A compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof:

2. A compound according to claim 1 selected from the group consisting of:

and

or a pharmaceutically acceptable salt or solvate thereof.

3. A compound according to claim 1 selected from the group consisting of:

(2R)-N'-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2-

(3-pyridinyl)acetohydrazide,

(2S)-N'-[3,5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2-

(3-pyridinyl)acetohydrazide, and

N'-[3_!5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2-(3- pyridinyl)acetohydrazide (diastereomeric mixture) or a pharmaceutically acceptable salt or solvate thereof.

4. A compound of formula (I) which is:

(2R)-N'-[3_!5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2- (3-pyridinyl)acetohydrazide, or a pharmaceutically acceptable salt or solvate thereof.

5. A compound of formula (I) which is:

(2R)-N'-[3_!5-bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1 ,2-a]pyrazin-2(1 H)-yl]-2- (3-pyridinyl)acetohydrazide.

6. A method of treatment of a mammal suffering from a disorder mediated by the ghrelin receptor, which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to any of claims 1 to 6.

7. A compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, according to any of claims 1 to 5 for use in therapy.

8. A compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, according to any of claims 1 to 5 for use in the treatment of a disorder mediated by the ghrelin receptor.

9. A pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to any of claims 1 to 5 in admixture with one or more pharmaceutically acceptable carriers, diluents or excipients.

10. Use of a compound as claimed in any of claims 1 to 5 in the manufacture of a medicament for the treatment of a condition in a mammal for which modulation of the ghrelin receptor is beneficial.