WO2007068739A1

WO2007068739A1 - 4-amino-benzamide derivatives as 5-ht4 receptor agonists for the treatment of gastrointestinal, neurological and cardiovascular disorders

Info

Publication number: WO2007068739A1
Application number: PCT/EP2006/069721
Authority: WO
Inventors: Mahmood Ahmed; Neil Derek Miller; Peter Henry Milner; Stephen Frederick Moss; Mervyn Thompson
Original assignee: Glaxo Group Limited
Priority date: 2005-12-16
Filing date: 2006-12-14
Publication date: 2007-06-21
Also published as: GB0525661D0

Abstract

The present invention relates to novel benzamide derivatives of formula (I) having pharmacological activity, to processes for their preparation, to compositions containing them and to their use in the treatment of diseases treatable by 5-HT4 agonism.

Description

4-AMINO-BENZAMIDE DERIVATIVES AS 5-HT4 RECEPTOR AGONISTS FOR THE TREATMENT OF GASTROINTESTINAL, NEUROLOGICAL AND CARDIOVASCULAR DISORDERS

The present invention relates to novel benzamide derivatives having pharmacological activity, to processes for their preparation, to compositions containing them and to their use in the treatment of diseases treatable by 5- HT4 agonism.

EP 0445862, EP 0389037, US 5,374,637, WO 99/02156 and WO 99/02494 (Janssen Pharmaceutica N.V.) all describe benzamide derivatives having gastrointestinal motility stimulating properties. EP 0213775 and GB 2207673 (Fordonal S.A.) describes a series of substituted benzamides having gastrokinetic activity. WO 94/08995 (SmithKline Beecham pic) discloses a series of heterocyclic condensed benzoic acid derivatives as 5-HT4 receptor antagonists. The compounds are claimed to be useful in the treatment of gastrointestinal disorders, cardiovascular disorders and CNS disorders. WO2005/092882 (Pfizer Japan Inc.) describes a series of 4-amino-5- halogeno-benzamide derivatives. The compounds are stated to have 5-HT4 agonistic activity and are indicated to be useful in the treatment of gastrointestinal disorders.

Alzheimer's disease is a chronic neurological disorder characterised by progressive cognitive decline, behavioural impairment and ultimately death. In the US alone, it is estimated that as many as 4.5 million people suffer from the disease, including nearly half of all people over 85 years of age. With an ageing world population, there is a clear need for effective therapies for Alzheimer's disease.

Although Alzheimer's disease is classically associated with loss of cholinergic neurons, deficits in a number of other neurotransmitter systems have also been reported, including that of the serotonergic system. With regard to 5-HT4 receptors, there is a wealth of pre-clinical data to support the use of 5-HT4 agonists as cognitive enhancers, both from in vivo and in vitro functional studies, such as rodent cognition models and electrophysiology (e.g. Moser P.C. et al., JPET 302(2):731-41 , Matsumoto M., JPET 296(3):676-82, Lucas G. et al., Biol. Psychiatry 57(8):918-25). The expression profile of the 5-HT4 receptor in the central nervous system also supports this, with high levels of expression in the hippocampus, striatum, prefrontal cortex and other limbic regions. There is also evidence that 5-HT4 receptor expression may be reduced in the course of Alzheimer's disease. These data suggest that 5-HT4 agonists such as the compound of the present invention could be useful for the treatment of cognitive impairments in neurological diseases such as Alzheimer's disease and related neurological disorders.

In addition, recent reports (e.g. Maillet M. et al., Curr. Alzheimer Res. 1 (2):79- 85) suggest that activation of the 5-HT4 receptor can increase the release of soluble APP-alpha, which has potent neuroprotective and memory-enhancing actions, and in addition enhance the production of neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Thus 5-HT4 agonism could also provide a disease-modifying treatment for Alzheimer's disease.

5-HT4 receptor agonists also have utility in the treatment of gastrointestinal disorders, especially those associated with reduced esopageal, gastric or intestinal motility such as gastro-esophageal reflux disease, dyspepsia conditions, including functional dyspepsia, irritable bowel syndrome and conditions associated with constipation, including those which are age-, disease- or drug-induced and those which may lead to additional symptoms such as incontinence. An exemplar of such a drug is tegaserod maleate, currently marketed by Novartis for the treatment of irritable bowel syndrome. The present invention provides, in a first aspect, a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof:

(I)

wherein R¹ represents a group of formula (a), (b), (c) or (d):

and wherein:

R² represents C_1-4 alkyl, -CH₂-C_3-6cycloalkyl or C_3-6cycloalkyl;

R³ represents hydrogen or halogen;

R⁴ represents Ci_-4 alkyl;

R⁵ represents hydrogen, hydroxy or carboxy;

R⁶ represents hydrogen, hydroxy or methoxy; m and n independently represent 0 or 1 , provided that when m represents 0,

R⁵ cannot be hydroxy; p and q independently represent 1 or 2; and s represents 0, 1 or 2 (hereafter 'the compounds of the invention'). The term 'Ci_-4 alkyl' as used herein as a group or a part of the group refers to a linear or branched saturated hydrocarbon group containing from 1 to 4 carbon atoms. Examples of C_1-4alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

The term 'C_3-6 cycloalkyl' as used herein refers to a saturated monocyclic hydrocarbon ring of 3 to 6 carbon atoms. Examples of C₃.₆ cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term 'halogen' as used herein refers to a fluorine, chlorine, bromine or iodine atom.

In one embodiment, R¹ represents a group of formula (a). In one embodiment, R¹ represents a group of formula (b). In one embodiment, R¹ represents a group of formula (c). In one embodiment, R¹ represents a group of formula (d). In one embodiment, R¹ represents a group of formula (c) or (d).

In another embodiment, R² represents Ci_-4 alkyl, particularly methyl.

In a further embodiment, R³ represents a halogen, particularly chlorine or bromine. More particularly, R³ represents chlorine.

In one embodiment, R⁴ represents methyl.

In another embodiment, R⁵ represents hydrogen.

In yet another embodiment, R⁶ represent hydrogen.

In a further embodiment, m represents 1.

In one embodiment, n represents 0. In another embodiment, n represents 1. In one embodiment, p represents 1. In one embodiment, p represents 2.

In another embodiment, q represents 1. In another embodiment, q represents 2.

In a further embodiment, s represents 0 or 1 , particularly 0.

Compounds according to the invention include the compounds of examples E1 to E10 as shown below, or pharmaceutically acceptable salts or solvates thereof.

In a more particular aspect compounds of the invention include:

8-Amino-7-chloro-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]methyl}-2,3- dihydro-1 ,4-benzodioxin-5-carboxamide;

5-Amino-6-chloro-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]methyl}-3,4- dihydro-2H-chromene-8-carboxamide;

4-Amino-5-chloro-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]methyl}-1- benzofuran-7-carboxamide;

4-Amino-5-chloro-2-methyl-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-1-benzofuran-7-carboxamide;

8-Amino-7-chloro-Λ/-[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]-2,3-dihydro-

1 ,4-benzodioxin-5-carboxamide; or

4-Amino-5-chloro-Λ/-[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]-1- benzofuran-7-carboxamide; and/or a pharmaceutically acceptable salt or solvate thereof.

In a most particular aspect, a compound of the invention includes 4-amino-5- chloro-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]methyl}-1-benzofuran- 7-carboxamide and 5-Amino-6-chloro-Λ/-({1 -[(3S)-tetrahydro-3-furanylmethyl]- 4-piperidinyl}methyl)-3,4-dihydro-2H-chromene-8-carboxamide and/or a pharmaceutically acceptable salt or solvate thereof.

Compounds of the present invention may form acid addition salts with acids. Such salts can be formed by reaction of the free base molecule (I) with a suitable inorganic or organic acid (such as hydrobromic, hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic, mandelic, benzoic, salicylic, glutamic, aspartic, p- toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, naphthalenesulfonic such as 2- naphthalenesulfonic, or hexanoic 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.

When the compounds of the invention are allowed to crystallise or are recrystallised from organic solvents, solvent of crystallisation may be present in the crystalline product. This invention includes within its scope such solvates. Similarly, the compounds of this invention may be crystallised or recrystallised from solvents containing water. In such cases water of hydration may be formed. This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lypohilisation. In addition, different crystallisation conditions may lead to the formation of different polymorphic forms of crystalline products. This invention includes within its scope all polymorphic forms of the compounds of the invention.

Thus, the invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the compounds of the invention including anhydrates, hydrates, solvates and polymorphs thereof.

Since the compounds of the invention are intended for use in pharmaceutical compositions, it will be understood that they are ideally used in substantially pure form, for example at least 75% pure and preferably at least 95% pure (% are on a wt/wt basis). Impure preparations of the compounds of the invention may be used for preparing the more pure forms used in the pharmaceutical compositions. Although the purity of intermediate compounds of the present invention is less critical, it will be readily understood that the substantially pure form is preferred as for the compounds of the invention. Whenever possible, the compounds of the invention are obtained in crystalline form.

The present invention also includes within its scope isotopically-labelled forms of the compounds of the invention. Such compounds are identical to the compounds of the invention except that one or more atoms therein are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the invention and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, bromine and chlorine, such as 2H, 3H, 11 C, 13C, 14C, 15N, 170, 180, 82Br and 36Cl.

Isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as 3H, 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. 11 C isotopes are particularly useful in PET (positron emission tomography), and are useful in brain imaging. Further substitution with heavier isotopes such as deuterium, i.e., 2H, can 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, lsotopically labelled forms of the compounds of the invention may be prepared by carrying out the synthetic procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily available isotopically labelled reagent for a non- isotopically labelled reagent.

The present invention also provides a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof wherein m represents 1 , which process comprises reacting a compound of formula (II),

(H)

wherein R¹, R⁶ and n are as defined in relation to formula (I), with a compound of formula (III),

(III)

wherein R⁵ is defined in relation to formula (I); and optionally thereafter performing one or more of the following steps,

(i) deprotecting a compound of formula (I) which is protected;

(ii) converting from one compound of formula (I) to another; and/or

(iii) forming a salt or solvate of the compound so formed;

(hereafter referred to as 'Process (a)').

In another embodiment of the invention there is provided a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof wherein m represents 1 , which process comprises reacting a compound of formula (II),

(H) wherein R¹, R⁶ and n are as defined in relation to formula (I), with a compound of formula (IV),

(IV)

wherein R⁵ is defined in relation to formula (I) and L¹ represents a suitable leaving group such as a halogen atom (e.g. bromine) or a methanesulfonate group; and optionally thereafter performing one or more of the following steps, (i) deprotecting a compound of formula (I) which is protected; (ii) converting from one compound of formula (I) to another; and/or (iii) forming a salt or solvate of the compound so formed; (hereafter referred to as 'Process (b)').

(H) wherein R¹, R⁶ and n are as defined in relation to formula (I), with a compound of formula (V),

(V)

wherein R⁵ is hydrogen or carboxy and L² represents a suitable leaving group such as a halogen atom (e.g. bromine); and optionally thereafter performing one or more of the following steps, (i) deprotecting a compound of formula (I) which is protected; (ii) converting from one compound of formula (I) to another; and/or (iii) forming a salt or solvate of the compound so formed; (hereafter referred to as 'Process (c)').

Process (a) typically comprises the use of reductive conditions (such as treatment with a borohydride e.g. sodium triacetoxyborohydride), optionally in the presence of an acid, such as acetic acid, in an appropriate solvent such as 1 ,2-dichloroethane at a suitable temperature such as between room temperature and 7O⁰C.

Processes (b) and (c) may be performed in a suitable solvent such as acetonitrile at a suitable temperature such as reflux.

Examples of protecting groups and the means for their removal can be found in T. W. Greene 'Protective Groups in Organic Synthesis' (J. Wiley and Sons, 1991). Suitable amine protecting groups include sulfonyl (e.g. tosyl), acyl (e.g. acetyl, 2¹,2',2'-trichloroethoxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl) and arylalkyl (e.g. benzyl), which may be removed by hydrolysis (e.g. using an acid such as hydrochloric acid in dioxan or trifluoroacetic acid in dichloromethane) or reductively (e.g. hydrogenolysis of a benzyl group or reductive removal of a 2',2',2'-trichloroethoxycarbonyl group using zinc in acetic acid) as appropriate. Other suitable amine protecting groups include trifluoroacetyl (-COCF₃) which may be removed by base catalysed hydrolysis or a solid phase resin bound benzyl group, such as a Merrifield resin bound 2,6-dimethoxybenzyl group (Ellman linker), which may be removed by acid catalysed hydrolysis, for example with trifluoroacetic acid.

Conventional interconversion procedures may be used to convert one compound of formula (I) to another, such as epimerisation, oxidation, reduction, alkylation, nucleophilic or electrophilic aromatic substitution, ester hydrolysis, amide bond formation or transition metal mediated coupling reactions. Examples of transition metal mediated coupling reactions useful as interconversion procedures include the following: Palladium catalysed coupling reactions between organic electrophiles, such as aryl halides, and organometallic reagents, for example boronic acids (Suzuki cross-coupling reactions); Palladium catalysed amination and amidation reactions between organic electrophiles, such as aryl halides, and nucleophiles, such as amines and amides; Copper catalysed amidation reactions between organic electrophiles (such as aryl halides) and nucleophiles such as amides; and Copper mediated coupling reactions between phenols and boronic acids.

Compounds of formula (II) wherein R¹ is a group of formula (a) and wherein n represents 1 may be prepared by the general methods described in WO9902494 and WO2005092882.

Compounds of formula (II) wherein R¹ is a group of formula (b) and wherein n represents 1 may be prepared by the general methods described in WO9316072, WO9902156 and WO2005092882.

Compounds of formula (II) wherein R¹ is a group of formula (c) and wherein n represents 1 may be prepared by the general methods described in WO9305038 and WO9902156.

Compounds of formula (II) wherein R¹ is a group of formula (d) and wherein n represents 1 may be prepared by the general method described in WO9408995.

Compounds of formula (II) wherein R¹ is a group of formula (a) and wherein n represents 0 may be prepared by the general method described in EP0213775 and GB2207673.

Compounds of formula (II) wherein R¹ is a group of formula (b) and wherein n represents 0 may be prepared by the general methods described in US5374637, EP0389037 and EP0445862. Compounds of formula (II) wherein R¹ is a group of formula (c) and wherein n represents 0 may be prepared by the general methods described in US5374637 and EP0389037.

Compounds of formula (II) wherein R¹ is a group of formula (d) and wherein n represents 0 may be prepared by the general method described in WO9408995.

Compounds of formulae (III), (IV) and (V) are either commercially available or may be prepared from commercially available materials by conventional procedures.

The compounds of the present invention are partial agonists of the 5-HT4 receptor. Hence, the compounds of the invention are believed to be of potential use in the treatment of diseases treatable by 5-HT4 agonism. Diseases treatable by 5-HT4 agonism include diseases of the central nervous system such as Alzheimer's disease and related neurological disorders, such as other dementias, cognitive disorder (especially mild cognitive impairment), generalised anxiety disorder, migraine, Parkinson's disease, multiple sclerosis, depression and schizophrenia. Diseases which may benefit from the application of a 5-HT4 receptor agonist also include functional gastrointestinal (Gl) diseases such as gastroesophageal reflux disease, gastric motility disorders such as gastroparesis, non-ulcer dyspepsia, functional dyspepsia, irritable bowel syndrome, constipation, dyspepsia, esophagitis, gastroesophageal disease, nausea, emesis, inflammatory bowel disease, post-operative ileus and visceral hypersensitivity as well as pain, urinary dysfunction, urinary incontinence, overactive bladder, diabetes and apnea syndrome, (especially caused by opioid administration), and cardiovascular disorders such as cardiac failure and heart arrhythmia (hereafter 'the disorders of the invention'). Thus the invention also provides a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for use as a therapeutic substance; in particular in the treatment of the disorders of the invention; and more particularly in the treatment of Alzheimer's disease and related neurological disorders, and also functional Gl diseases.

It is believed that the compounds of the present invention exhibit reduced potential to inhibit the human cytochrome P450 3A4 isoform. This isoform is the predominant isoform involved in P450-mediated clearance of xenobiotics. Inhibition of this isoform may be associated with drug-drug interactions leading to toxic events. Accordingly, it is believed that the compounds of the invention may have a reduced likelihood of P450-mediated toxicity.

The invention further provides a method of treatment of the disorders of the invention, in mammals including humans, which comprises administering to the sufferer a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for use in the treatment of the disorders of the invention.

It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment.

When used in therapy, the compounds of the invention are usually formulated in a standard pharmaceutical composition. Such a composition can be prepared using standard procedures, such as those described in, for example, Remington's Pharmaceutical Sciences (Mack Publishing Company).

Thus, the present invention further provides a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier. The present invention further provides a pharmaceutical composition for use in the treatment of the disorders of the invention which comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

The compounds of the invention may be used in combination with other therapeutic agents.

When the compounds of the invention are intended for use in the treatment of Alzheimer's disease, they may be used in combination with medicaments claimed to be useful as either disease modifying or symptomatic treatments of Alzheimer's disease. Suitable examples of such other therapeutic agents may be agents known to modify cholinergic transmission such as M1 muscarinic receptor agonists or allosteric modulators, nicotinic receptor agonists or allosteric modulators, symptomatic agents such as 5-HT6 receptor antagonists or H3 receptor antagonists, also NMDA receptor antagonists (such as memantine hydrochloride) or modulators or acetylcholinesterase inhibitors (such as donepezil hydrochloride), and disease modifying agents such as β or γ-secretase inhibitors.

When the compounds of the invention are intended for use in the treatment of gastrointestinal disease, they may be used in combination with medicaments which induce symptoms treated by 5-HT4 receptor agonists and/or medicaments claimed to be useful as treatments of the same or different aspects of such disease. Suitable examples of therapeutic agents which evoke symptoms treated by 5-HT4 receptor agonists include those which evoke constipation, such as morphine or other opiate receptor ligands. Suitable examples of other therapeutic agents used to treat gastrointestinal disease include those known to modify gastric acid secretion, such as ranitidine or lansoprazole, gastrointestinal motility, such as almivopan, or visceral pain, such as codeine, as well as the use of these compounds to aid the therapeutic use of agents designed as laxatives.

When the compounds of the invention are used in combination with other therapeutic agents, the compound and agent may be administered either sequentially or simultaneously by any convenient route.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof together with a further therapeutic agent or agents.

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

When a compound of the present invention or a pharmaceutically acceptable salt or solvate thereof is used in combination with a second therapeutic agent active against the same disease state 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.

A pharmaceutical composition of the invention, which may be prepared by admixture, suitably at ambient temperature and atmospheric pressure, is usually adapted for oral, parenteral or rectal administration and, as such, may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable or infusible solutions or suspensions or suppositories. Orally administrable compositions are generally preferred. Tablets and capsules for oral administration may be in unit dose form, and may contain conventional excipients, such as binding agents, fillers, tabletting lubricants, disintegrants and acceptable wetting agents. 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 suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and, if desired, conventional flavourings or colorants.

For parenteral administration, fluid unit dosage forms are prepared utilising a compound of the invention or pharmaceutically acceptable salt thereof and a sterile vehicle. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, the compound can be dissolved for injection and filter sterilised before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilisation cannot be accomplished by filtration. The compound can be sterilised by exposure to ethylene oxide before suspension in a sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The composition may contain from 0.1 % to 99% by weight, preferably from 10 to 60% by weight, of the active material, depending on the method of administration. The dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses may be 0.05 to 1000 mg, more suitably 0.1 to 200 mg and even more suitably 1.0 to 200 mg. In one aspect, a suitable unit dose would be 0.1-50 mg. Such unit doses may be administered more than once a day, for example two or three a day. Such therapy may extend for a number of weeks or months.

The following Descriptions and Examples illustrate the preparation of compounds of the invention.

Where indicated, chromatography was carried out on silica gel cartridges on a Flashmaster Il automated chromatography system (Argonaut) and eluting with mixtures of methanol/dichloromethane or ethyl acetate/pentane.

¹H NMR spectra were recorded on a Bruker AVANCE 400 NMR spectrometer or a Bruker DPX250 NMR spectrometer. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants (J) are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), dt (double triplet), m (multiplet), br (broad).

LC/Mass spectra were obtained using an Agilent 1100 series HPLC system coupled with a Waters ZQ Mass Spectrometer. LC analysis was performed on a Waters Atlantis™ dCi₈ column (50 x 4.6 mm, 3μm) (mobile phase: 97% [water +0.05% HCO₂H]/ 3% [CH₃CN +0.05% HCO₂H] for 0.1 min, then a gradient to 3% [water +0.05% HCO₂H]/97% [CH₃CN +0.05% HCO₂H] over 3.9 min, and then held under these conditions for 0.8 min); temperature = 30 ⁰C; flow rate = 3 mL/min; Mass spectra were collected using electrospray and/or APCI. In the mass spectra only one peak in the molecular ion cluster is reported. The UV detection range is from 220 to 330nm.

All reactions were monitored by thin-layer chromatography on 0.25 mm E. Merck silica gel plates (60F-254), visualised with UV light, 5% ethanolic phosphomolybdic acid, p-anisaldehyde solution, aqueous potassium permanganate or potassium iodide / platinum chloride solution in water.

Mass Directed Auto Preparation (MDAP) Column

Waters Atlantis: 19mm x 100mm (small scale); and 30mm x 100mm (large scale).

Stationary phase particle size, 5μm.

Solvents

A: Aqueous solvent = Water + 0.1 % Formic Acid B: Organic solvent = Acetonitrile + 0.1 % Formic Acid Make up solvent = Acetonitrile: DMSO 50:50 Needle rinse solvent = Methanol

Methods

Five methods were used depending on the analytical retention time of the compound of interest:

(1 ) Large/Small Scale 1.0-1.5 = 5-30% B

(2) Large/Small Scale 1.5-2.2 = 15-55% B

(3) Large/Small Scale 2.2-2.9 = 30-85% B

(4) Large/Small Scale 2.9-3.6 = 50-99% B

Runtime, 13.5 minutes, comprising 10-minute gradient followed by a 3.5 minute column flush and re-equilibration step.

(5) Large/Small Scale 3.6-5.0 = 80-99% B

Runtime, 13.5 minutes, comprising 6-minute gradient followed by a 7.5 minute column flush and re-equilibration step. Flow rate

20mls/min (Small Scale) or 40mls/min (Large Scale).

Abbreviations

DCM Dichloromethane

DCE 1 ,2-Dichloroethane

DMAP N,N-dimethylamino-4-aminopyridine

EDC/EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ee Enantiomeric excess

EtOAc Ethyl acetate

Et₂O Diethyl ether

LCMS Liquid Chromatography Mass Spectrometry

MDAP Mass Directed Autopreparation

MeOH Methanol

NBS N-Bromosuccinimide

SCX Strong Cation Exchange

THF Tetrahydrofuran

Preparation of Intermediates

Description 1

1 ,1 -Dimethylethyl 4-({[(5-amino-6-chloro-3,4-dihydro-2H-chromen-8- yl)carbonyl]amino}methyl)-1 -piperidinecarboxylate (D1 )

A solution of 1 ,1 -dimethylethyl 4-(aminomethyl)-1 -piperidinecarboxylate (1.65g, 7.7mmol; available commercially from e.g. Fluka) in dichloromethane (100ml) was added to a solution of 5-amino-6-chloro-3,4-dihydro-2H- chromene-8-carboxylic acid (may be prepared according to the procedure described in Chem. Pharm. Bull., 1998, 46(12): 1881 ) (1.75g, 0.77mmol) in dichloromethane (100ml). Successive additions of 1-ethyl-3(3- dimethylaminopropyl)carbodiimide hydrochloride (2.45g, 12.8mmol) and A- (N,N-dimethylamino)pyridine (151 mg, 1.23mmol) were then made to the stirred mixture and the whole reaction mixture was left to stir at ambient temperature for 24h. After this time the solvent was evaporated under reduced pressure and the residue dissolved in dichloromethane. This solution was washed with water and the aqueous layer re-extracted with dichloromethane (3 x). The combined organic extracts were washed with saturated sodium hydrogen carbonate solution and then dried (MgSO₄) and evaporated under reduced pressure to a solid. This solid was purified by chromatography over silica gel eluting with a solvent gradient of ethyl acetate/hexane to afford the title compound (D1 ) as a solid (2.62g, 6.2mmol, 80%). δH (CD₃OD, 400MHz) 1.10-1.20 (2H, m), 1.45 (9H, s), 1.73-1.80 (3H, m), 2.05-2.11 (2H, m), 2.55 (2H, t, J = 6.4Hz), 2.70-2.85 (2H, br, s), 3.27-3.30 (2H, m), 4.06-4.11 (2H, m), 4.27 (2H, t, J = 4.8Hz), 7.71 (1 H, s). Mass Spectrum: C_2IH₃₀CIN₃O₄ requires 423/425; found 424/426 (MH⁺)

Description 2

S-Amino-β-chloro-N^-piperidinylmethylJ-S^-dihydro^H-chromene-δ- carboxamide (D2)

Water (5ml) was added to a mixture of 1 ,1-dimethylethyl 4-({[(5-amino-6- chloro-3,4-dihydro-2/-/-chromen-8-yl)carbonyl]amino}methyl)-1- piperidinecarboxylate (may be prepared as described in Description 1 ) (2.62g, 6.9mmol) in 4M hydrogen chloride in 1 ,4-dioxan (20ml). After 2h stirring at ambient temperature, dichloromethane (150ml) and water were added to the solution with stirring. The aqueous layer of the stirred mixture was basified to pH14 by the addition of sodium hydroxide solution and then saturated with sodium chloride. The mixture was shaken but the emulsion did not separate. The organic solvent was removed from this mixture by evaporation and the remaining aqueous layer was diluted with water and the solution adjusted to pH7. Attempts to extract this solution with dichloromethane again gave an emulsion. The organic solvent was again evaporated off and the aqueous residue re-evaporated with toluene (2x) to give a solid residue. This solid was stirred at reflux with methanol (50ml) for 10 minutes and the solution decanted off and evaporated to afford the title compound (D2) as a yellow solid (0.777g, 2.4mmol, 35%). δH (CD₃OD, 400MHz) 1.35-1.48 (2H, m), 1.80-1.95 (3H, m), 2.05-2.12 (2H, m), 2.55 (2H, t, J = 6.8Hz), 2.85-2.906 (2H, m), 3.30-3.48 (integral masked by solvent, m), 4.28 (2H, t, J = 5.6Hz), 7.72 (1 H, s). Mass Spectrum: Ci₆H₂₂CIN₃O₂ requires 323/325; found 324/326 (MH⁺)

Description 3 4-Amino-5-chloro-1 -benzofuran-7-carboxylic acid (D3)

To a suspension of methyl 4-(acetylamino)-5-chloro-1-benzofuran-7- carboxylate (may be prepared according to the procedure described in Synlett., 1993, 4, 269) (1.25g, 4.67mmol) in ethanol (20ml) was added 5N sodium hydroxide (20ml) and the mixture stirred for 6Oh. The reaction was found to be incomplete. The mixture was then heated at reflux for 3h followed by removal of ethanol in vacuo and addition of water (10ml). The aqueous layer was washed with ethyl acetate (50ml), acidified using 5N HCI and the resulting precipitate filtered. This residue was combined with the solid isolated from filtration of the organic washings to afford the title compound (D3) (1.12g, 100%). δH (DMSO-d6, 400MHz) 6.66 (2H, br s), 7.23 (1 H, s), 7.65 (1 H, s), 7.90 (1 H, s), 12.50 (1 H, br s). Mass Spectrum: C₉H₆CINO₃ requires 211/213; found 212/214 (MH⁺).

Description 4

1 ,1 -Dimethylethyl 4-({[(4-amino-5-chloro-1 -benzofuran-7- yl)carbonyl]amino}methyl)-1 -piperidinecarboxylate (D4)

To a stirred solution of 4-amino-5-chloro-1-benzofuran-7-carboxylic acid (may be prepared as described in Description 3) (0.2g, 0.95mmol) in dichloromethane (20ml) was added 1 ,1 -dimethylethyl 4-(aminomethyl)-1- piperidinecarboxylate (0.3g, 1.42mmol; available commercially from e.g. Fluka), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (0.36g, 1.89mmol) and 4-(N,N-dimethylamino)pyridine (23mg, 0.19mmol). The reaction mixture was stirred for 19h followed by addition of dichloromethane (20ml), washing with saturated sodium hydrogen carbonate solution (20ml), filtration through a phase-separation cartridge and evaporation of the organic phase. The resulting residue was purified by chromatography over silica gel (eluting with a solvent gradient of 0-95% ethyl acetate/pentane) to afford the title compound (D4) (0.27g, 69%). δH (CDCI₃, 400MHz) 1.15-1.35 (2H, m), 1.45 (9H, s), 1.70-1.90 (3H₁ m), 2.65-2.77 (2H, m), 3.40-3.47 (2H, m), 4.05- 4.20 (2H, m), 4.67 (2H, br s), 6.80 (1 H, d, J = 2.4Hz), 7.33 (1 H, br s), 7.64 (1 H, d, J = 2.4Hz), 8.05 (1 H, s). Mass Spectrum: C₂₀H_2GCIN₃O₄ requires 407/409; found 406/408 (M-H)^".

Description 5

4-Amino-5-chloro-Λ/-(4-piperidinylmethyl)-1-benzofuran-7-carboxamide hydrochloride (D5)

4M HCI/1 ,4-dioxan (5ml) was added to 1 ,1-dimethylethyl 4-({[(4-amino-5- chloro-1-benzofuran-7-yl)carbonyl]amino}methyl)-1-piperidinecarboxylate (may be prepared as described in Description 4) (0.27g, 0.66mmol) and the resulting mixture was left to stand at room temperature for 0.5h. The solvent was then removed in vacuo to afford the title compound (D5) (0.25g, 100%). δH (MeOD, 400MHz) 1.46-1.59 (2H, m), 1.95-2.10 (3H, m), 2.90-3.05 (2H, m), 3.35-3.50 (4H, m), 7.11 (1 H, d, J = 2.4Hz), 7.76 (1 H, s), 7.79 (1 H, d, J = 2Hz). Mass Spectrum: Ci₅Hi₈CIN₃O₂ requires 307/309; found 308/310 (MH⁺).

Description 6 (2S)-Tetrahydro-2-furanylmethanol (D6)

Lithium aluminium hydride (3.76ml_, 13.76mmol, 1 M in THF) was measured into a 25OmL round bottom flask containing 1OmL THF, fitted with a thermometer, condenser and pressure equalising dropping funnel, under argon. (2S)-tetrahydro-2-furancarboxylic acid (1g, 8.6mmol) in 1OmL THF was added drop-wise from the dropping funnel with stirring, keeping the mixture under 4O⁰C. The mixture was stirred at room temperature overnight. The reaction mixture was quenched by slowly adding portion-wise saturated ammonium chloride solution (75mL) followed by ethyl acetate (5OmL). The mixture was filtered through a sinter funnel. The ethyl acetate layer was isolated and the aqueous layer was washed with ethyl acetate (25mL). The ethyl acetate washings were combined and dried over sodium sulfate. The solvent was removed in vacuo to yield 368mg of a pale yellow oil (D6). (368mg, 42%). δH (CDCI₃, 400MHz) 1.60-1.71 (1 H, m), 1.85-2.00 (4H, m), 3.45-3.55 (1 H, m), 3.64-3.72 (1 H, m), 3.75-3.83 (1 H, m), 3.84-3.90 (1 H, m), 3.98-4.06 (1 H, m).

Description 7

(2S)-Tetrahydro-2-furanylmethyl methanesulfonate (D7)

A solution of (2S)-tetrahydro-2-furanylmethanol (may be prepared as described in Description 6) (368mg, 3.6mmol) in DCM (1OmL) was made. Triethylamine (478mg, 4.7mmol) was added and the mixture was cooled to O⁰C. Methanesulfonyl chloride (456mg, 3.96mmol) was added drop-wise. The mixture was stirred and allowed to warm to room temperature overnight. The reaction mixture washed with saturated aqueous sodium bicarbonate and brine. Solvent was then removed in vacuo from organic layer to yield a yellow oil (D7) (766mg, 81 %). δH (CDCI₃, 400MHz) 1.65-1.75 (1 H, m), 1.86-1.98 (2H, m), 1.99-2.10 (1 H, m), 3.07 (3H, s), 3.76-3.85 (1 H, m), 3.86-3.93 (1 H, m), 4.14-4.23 (2H, m), 4.24-4.30 (1 H, m).

Description 8

1 ,1 -Dimethylethyl 4-({[(4-amino-5-chloro-2-methyl-1 -benzofuran-7- yl)carbonyl]amino}methyl)-1 -piperidinecarboxylate (D8)

To a solution of 4-amino-5-chloro-2-methyl-1-benzofuran-7-carboxylic acid (may be prepared according to the procedure described in Chem. Pharm. Bull., 1998, 46, 42) (221 mg, 0.98mmol) in DCM (1OmL) was added 1 ,1- dimethylethyl 4-(aminomethyl)-1 -piperidinecarboxylate (315mg, 1.47mmol; available commercially from e.g. Fluka), EDC (376mg, 1.96mmol) and DMAP (24mg, 0.19mmol). The mixture was stirred under argon at room temperature. The reaction was shown to be complete by LCMS after stirring at room temperature for 3h. The reaction mixture was transferred to a separating funnel. 2OmL DCM and 2OmL sodium bicarbonate were added, and the DCM layer was separated using a phase separation cartridge. The DCM was removed in vacuo and the residue was purified on silica, eluting with 0-95% EtOAc/pentane. The product containing fractions identified were combined, and the solvent was removed in vacuo to yield the desired product as a yellow paste (D8) (202mg, 49%). δH (CDCI₃, 400MHz) 1.17-1.32 (2H, m, masked by solvent peak), 1.45 (9H, s), 1.72-1.90 (3H, m), 2.50 (3H, s), 2.65-2.80 (2H, m), 3.43 (2H, br s), 4.06-4.20 (2H, m, masked by solvent peak), 4.54 (2H, br s), 6.37 (1 H, s), 7.30 (1 H, br s), 7.95 (1 H, s). Mass Spectrum: C_2IH₂₈CIN₃O₄ requires 421/423; found 422/424 (MH⁺).

Description 9

4-Amino-5-chloro-2-methyl-Λ/-(4-piperidinylmethyl)-1-benzofuran-7- carboxamide hydrochloride (D9)

4M HCI/dioxan (4ml_) was added to the 1 ,1-dimethylethyl 4-({[(4-amino-5- chloro-2-methyl-1 -benzofuran-7-yl)carbonyl]amino}methyl)-1 - piperidinecarboxylate (may be prepared as described in Description 8) (202mg, 0.48mmol). The reaction mixture was stirred at room temperature for 20 minutes. The reaction was shown to be complete by LCMS. The solvent was removed in vacuo to yield the title compound as an off-white solid (D9) (174mg, 100%). δH (MeOD, 400MHz) 1.45-1.58 (2H, m), 1.95-2.08 (3H, m), 2.51 (3H, s), 2.95-3.05 (2H, m), 3.38-3.45 (4H, m), 6.69 (1 H, s), 7.67 (1 H₁ s). Mass Spectrum: Ci₆H₂₀CIN₃O₂ requires 321/323; found 322/324 (MH⁺).

Description 10

Ethyl 8-amino-7-bromo-2,3-dihydro-1 ,4-benzodioxin-5-carboxylate (D10)

To a 3-neck flask fitted with a thermometer was added ethyl 8-amino-2,3- dihydro-1 ,4-benzodioxin-5-carboxylate (5g, 22.4mmol) (may be prepared according to the procedure described in WO9717345) followed by 1 ,4-dioxan (80ml). The resulting mixture was stirred for 15 minutes to provide a homogeneous mixture. To this was added NBS (4.19g, 23.5mmol) in portions over 20 minutes (keeping internal temperature below 25⁰C, slight exotherm observed). The resulting orange coloured solution was stirred at room temperature for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was then diluted with DCM (150ml), washed with saturated aqueous NaHCO₃ (I OOmI), water (100ml) and dried (MgSO₄). Evaporation of the solvent gave crude material (brown solid, 7.5g). This was purified on silica (Biotage 75), eluting with hexane (2.5L), 20% EtOAc/hexane (2.5L) and 40%

EtOAc/hexane (2.5L). The product came off (eluted) at 40% EtOAc/hexane.

Evaporation of solvent gave the desired product as a white solid (D10) (6.5g,

96%). δH (CDCI₃, 400MHz) 1.35 (3H, t, J = 7.2Hz), 4.29 (2H, q, J = 7.2Hz), 4.33-

4.38 (4H, m), 4.50 (2H, br s), 7.66 (1 H, s)

Mass Spectrum: CnH₁₂BrNO₄ requires 301/303; found 302/304 (MH⁺).

Description 11

8-Amino-7-bromo-2,3-dihydro-1 ,4-benzodioxin-5-carboxylic acid (D11 )

To a suspension of ethyl 8-amino-7-bromo-2,3-dihydro-1 ,4-benzodioxin-5- carboxylate (may be prepared as described in Description 10) (6g, 19.9mmol) in MeOH (60ml) in a 25OmL flask was added NaOH (3.97g, 99.3mmol, solution in 6OmL water) and the resulting suspension was heated at 66⁰C (bath temp.). The mixture became homogeneous after 30 minutes. After stirring at reflux for 1 h, LCMS indicated that the reaction was complete (pale yellow colour at this stage). After allowing the reaction mixture to cool to room temperature, MeOH was removed in vacuo and a further 10ml of water was added to dissolve the precipitate. The resulting solution was washed with EtOAc (2x50ml) and the aqueous phase was acidified to pH<4 (1OmL 5N HCI). The suspension containing the resulting white precipitate was cooled in ice (5 minutes) and filtered, washing with 2OmL ice-cold water. The solid was collected and dried in vac-oven (4O⁰C, 1 h). This gave desired product as a white solid (D11 ) (5.2g, 96%). δH (DMSO-d6, 400MHz) 4.27-4.29 (4H, m), 7.43 (1 H, s), 12.15 (1 H, br s) Mass Spectrum: C₉H₈BrNO₄ requires 272/274; found 273/275 (MH⁺).

Description 12

1 ,1 -Dimethylethyl 4-({[(8-amino-7-bromo-2,3-dihydro-1 ,4-benzodioxin-5- yl)carbonyl]amino}methyl)-1-piperidinecarboxylate (D12)

8-Amino-7-bromo-2,3-dihydro-1 ,4-benzodioxin-5-carboxylic acid (may be prepared as described in Description 11 ) (500mg, 1.82mmol) was weighed into a round bottom flask and DCM (20ml) was added with stirring. 1 ,1- dimethylethyl 4-(aminomethyl)-1-piperidinecarboxylate (470mg, 2.19mmol; available commercially from e.g. Fluka) was added followed by EDC (700mg, 3.64mmol) and DMAP (44mg, 0.36mmol). The mixture was stirred under argon for 2.5h. Product formation was confirmed by LCMS. The reaction mixture was then transferred to a separating funnel and extracted with DCM and sodium bicarbonate. The DCM layer was isolated using a phase separation cartridge. The product was purified using a silica column, eluting with 0-100% EtOAc/pentane. The product containing fractions were combined and the solvent was removed in vacuo to give the title compound (D12) (766mg, 89%). δH (MeOD, 400MHz) 1.09-1.17 (2H, m), 1.71-1.81 (3H, m), 2.70-2.82 (2H, m), 3.26-3.29 (2H, m), 4.06-4.10 (2H, m), 4.35-4.42 (4H, m), 7.60 (1 H, s) Mass Spectrum: C₂₀H_2SBrN₃O₅ requires 469/471 ; found 470/472 (MH⁺).

Description 13

8-Amino-7-bromo-Λ/-(4-piperidinylmethyl)-2,3-dihydro-1 ,4-benzodioxin-5- carboxamide hydrochloride (D13)

4M HCI/1 ,4-dioxan (5ml) was added to 1 ,1 -dimethylethyl 4-({[(8-amino-7- bromo-2,3-dihydro-1 ,4-benzodioxin-5-yl)carbonyl]amino}methyl)-1- piperidinecarboxylate (may be prepared as described in Description 12) (766mg, 1.63mmol) in a round bottom flask. A small quantity of methanol was added to aid dissolution. The mixture was left to stand at room temperature, open to air, for 1.5h. The solvent was removed in vacuo to yield the product (D13) (671 mg, 100%). δH (MeOD, 400MHz) 1.40-1.51 (2H, m), 1.90-1.99 (3H, m), 2.92-3.00 (2H, m), 3.33-3.35 (2H, m), 3.39-3.42 (2H, m), 4.38-4.44 (4H, m), 7.61 (1 H, s) Mass Spectrum: Ci₅H₂₀BrN₃O₃ requires 369/371 ; found 370/372 (MH⁺).

Description 14

Methyl 5-(acetylamino)-6-bromo-3,4-dihydro-2H-chromene-8-carboxylate

(D14)

NBS (5.9g, 33mmol) was added portionwise to a solution of methyl 5- (acetylaminoJ-S^-dihydro^H-chromene-δ-carboxylate (may be prepared according to the procedure described in US2004181064) (7.9g, 32mmol) in

1 ,4-dioxan (100ml) and acetic acid (100ml) at ambient temperature. After stirring for 22h, the mixture was evaporated to a solid. The solid was redissolved in dichloromethane (600ml) and the solution extracted with saturated sodium hydrogen carbonate solution (400ml), dried (MgSO₄) and evaporated to a solid. The solid was stirred with diethyl ether to afford the title compound (D14) as a white solid (9.2g, 28mmol, 88%) δH (CDCI₃, 400MHz) 1.94-2.01 (2H, m), 2.25 (3H, s), 2.71-2.75 (2H, m), 3.87

(3H, s), 4.29 (2H, t, J = 4.8Hz), 6.99 (1 H, br, s), 7.88 (1 H, s).

Mass Spectrum: C₁₃Hi₄BrNO₄ requires 327/329; found 328/330 (MH⁺)

Description 15 S-Amino-θ-bromo-S^-dihydro^H-chromene-δ-carboxylic acid (D15)

A stirred suspension of methyl 5-(acetylamino)-6-bromo-3,4-dihydro-2H- chromene-8-carboxylate (may be prepared as described in Description 14)

(9.2g, 28mmol) in 10% sodium hydroxide solution (200ml) was heated under reflux for 16h. The solution was then cooled to ambient temperature and brought to pH4 by the addition of concentrated hydrochloric acid, whereupon precipitation of a white solid occurred. The solid was filtered, washed with water, dried at 5O⁰C and identified as the title compound (D15) (7.7g, 28mmol,

100%) δH (DMSO-d6, 400MHz) 1.91-1.97 (2H, m), 2.46 (2H, t, J = 5.2Hz), 4.11 (2H, t, J = 5.2Hz), 5.69 (2H, br, s), 7.66 (1 H, s), 11.8 (1 H, br, s).

Mass Spectrum: Ci₀H₁₀BrNO₃ requires 271/273; found 272/274 (MH⁺).

Description 16

1 ,1 -Dimethylethyl 4-({[(5-amino-6-bromo-3,4-dihydro-2H-chromen-8- yl)carbonyl]amino}methyl)-1 -piperidinecarboxylate (D16)

A solution of 1 ,1 -dimethylethyl 4-(aminomethyl)-1 -piperidinecarboxylate (0.78g, 3.7mmol; available commercially from e.g. Fluka) in DCM (40ml) was added to a solution of 5-amino-6-bromo-3,4-dihydro-2H-chromene-8- carboxylic acid (may be prepared as described in Description 15) (1.0g, 3.7mmol) in DCM (80ml). Successive additions of EDC (1.17g, 6.1 mmol) and DMAP (72mg, 0.59mmol) were then made to the stirred mixture and this was left to stir at ambient temperature for 3.5h. After this time the solvent was evaporated under reduced pressure and the residue dissolved in DCM. This solution was washed with water and the aqueous layer re-extracted with DCM (3 x). The combined organic extracts were successively washed with saturated sodium hydrogen carbonate solution and brine then dried (MgSO₄) and evaporated under reduced pressure to give an oily solid. This solid was purified by chromatography over silica gel eluting with a solvent gradient of EtOAc/hexane to afford the title compound (D16) as a solid (1.39g, 2.96mmol, 80%). δH (MeOD, 400MHz) 1.15 (2H, m), 1.54 (9H, s), 1.75 (2H, d, J = 13.2Hz), 1.80 (1 H, m), 2.12 (2H, m), 2.80 (2H, m), 3.30 (2H, t, J = 6Hz), 4.17 (2H, m), 4.35 (2H, t, J = 4.8Hz), 7.88 (1 H, s), 8.11 , (1 H, t). Mass Spectrum: C_2IH₃₀BrN₃O₄ requires 467/469; found 468/470 (MH⁺).

Description 17

5-Amino-6-bromo-N-(4-piperidinylmethyl)-3,4-dihydro-2H-chromene-8- carboxamide (D17)

Water (5ml) was added to a mixture of 1 ,1-dimethylethyl 4-({[(5-amino-6- bromo-3,4-dihydro-2H-chromen-8-yl)carbonyl]amino}methyl)-1- pipehdinecarboxylate (may be prepared as described in Description 16) (1.39g, 2.97mmol) in 4M hydrogen chloride in 1 ,4-dioxan (12ml). After 2h stirring at ambient temperature, dichloromethane (75ml) and water were added to the solution with stirring. The aqueous layer of the stirred mixture was basified to pH14 by the addition of sodium hydroxide solution and then saturated with sodium chloride. The mixture was shaken and the layers separated. The aqueous layer was further extracted with dichloromethane (5x) and the combined organic extracts were dried (MgSO₄) and evaporated to afford the title compound (D17) as a pale yellow solid (0.821 g, 2.2mmol, 75%). δH (MeOD, 400MHz) 1.20 (2H, m), 1.73 (3H, m), 2.08 (2H, m), 2.55 (2H, t, J = 6.8Hz), 2.65 (2H, m), 3.12 (2H, d, J = 12.4Hz), 3.35 (2H, m), 4.28 (2H, t, J = 4.8Hz), 7.88 (1 H, s). Mass Spectrum: C₁₆H₂₂BrN₃O₂ requires 367/369; found 368/370 (MH⁺)

Description 18

1 ,1 -Dimethylethyl 4-{[(8-amino-7-chloro-2,3-dihydro-1 ,4-benzodioxin-5- yl)carbonyl]amino}-1-piperidinecarboxylate (D18)

To a solution of 8-amino-7-chloro-2,3-dihydro-1 ,4-benzodioxin-5-carboxylic acid (may be prepared according to the procedure described in WO9305038) (0.5g, 2.17mmol) in DCM (5OmL) was added 1 ,1 -dimethyl ethyl 4-amino-1- piperidinecarboxylate (0.52g, 2.6mmol; available commercially from e.g. Fluka), EDC (0.8g, 4.2mmol) and DMAP (53mg, 0.43mmol), and the reaction mixture was stirred at room temperature for 2h. After 2h stirring, the reaction was shown to be complete by LCMS and the organic solution was washed with NaHCO₃ solution. The organic phase was then washed with brine, dried (phase separation cartridge) and the solvents were removed to afford crude material. The crude material was then purified on silica (0-100% EtOAc/pentane) to afford the title compound as a colourless paste (D18) (848mg, 95%). δH (CDCI₃, 400MHz) 1.35-1.45 (2H, m), 1.47 (9H, s), 1.95- 2.03 (2H, m), 2.98 (2H, t, J = 11.6Hz), 3.99 (2H, br s), 4.10-4.20 (1 H, m, integration masked by EtOAc), 4.33-4.42 (6H, m), 7.40 (1 H, d, J = 7.6Hz), 7.75 (1 H, s) Mass Spectrum: C₁₉H₂₆CIN₃O₅ requires 412/414; found 412/414 (MH⁺)

Description 19

8-Amino-7-chloro-Λ/-4-piperidinyl-2,3-dihydro-1,4-benzodioxin-5- carboxamide hydrochloride (D19)

To a 10OmL flask was added 1 ,1 -dimethylethyl 4-{[(8-amino-7-chloro-2,3- dihydro-1 ,4-benzodioxin-5-yl)carbonyl]amino}-1 -piperidinecarboxylate (may be prepared as described in Description 18) (0.62g, 1.51 mmol) followed by 4M HCI/dioxan (1OmL) and methanol (1OmL). After stirring the reaction at room temperature for 45 minutes, LCMS analysis indicated that the reaction was complete. The solvents were then removed by evaporation. The resulting solid was dried in the vacuum oven (4O⁰C) overnight to yield a white solid (D19) (545mg). δH (MeOD, 400MHz) 1.77-1.90 (2H, m), 2.15-2.25 (2H, m), 3.10-3.23 (2H, m), 3.40-3.50 (2H, m), 4.08-4.18 (1 H, m), 4.35-4.48 (4H, m),

7.44 (1 H, s)

Mass Spectrum: C₁₄H₁₈CIN₃O₃ requires 311/313; found 312/314 (MH⁺)

Description 20

1 ,1 -Dimethylethyl 4-{[(4-amino-5-chloro-1 -benzofuran-7- yl)carbonyl]amino}-1 -piperidinecarboxylate (D20)

To a solution of 4-amino-5-chloro-1-benzofuran-7-carboxylic acid (may be prepared as described in Description 3) (0.5g, 2.37mmol) in DCM (5OmL) was added 1 ,1 -dimethylethyl 4-amino-1 -piperidinecarboxylate (712mg, 3.56mmol), EDC (905mg, 4.74mmol) and DMAP (57mg, 0.47mmol). The mixture was stirred under argon at room temperature for 2 days. After this time, LCMS showed that the reaction was complete. The reaction mixture was transferred to a separating funnel and 4OmL DCM and 4OmL sodium bicarbonate was added. The DCM layer was separated using a phase separation cartridge. The DCM was removed in vacuo and the resulting yellow oil was purified on a 5Og silica column eluting with 0-95% EtOAc/pentane. Appropriate fractions were combined and evaporated to give the desired product as a yellow oil (D20) (730mg, 78%). δH (CDCI₃, 400MHz) 1.48 (9H, s), 1.50-1.60 (2H, m), 2.90-3.10 (2H, m), 3.95-4.08 (2H, m), 4.18-4.30 (1 H, m), 4.77-4.87 (2H, m), 6.84 (1 H, d, J = 3.6Hz), 7.17 (1 H, d, J = 12.4Hz), 7.63 (1 H, d, J = 4Hz), 8.03 (1 H, s) Mass Spectrum: C₁₉H₂₄CIN₃O₄ requires 393/395; found 392/394 [M-H]^"

Description 21

4-Amino-5-chloro-Λ/-4-piperidinyl-1-benzofuran-7-carboxamide hydrochloride (D21)

To a 5OmL flask was added 1 ,1 -dimethylethyl 4-{[(4-amino-5-chloro-1- benzofuran-7-yl)carbonyl]amino}-1 -piperidinecarboxylate (may be prepared as described in Description 20) (730mg, 1.86mmol) followed by methanol (1OmL) and 4M HCI/dioxan (1OmL). The reaction mixture was stirred at room temperature under argon for 1 h. After this time, LCMS showed that the reaction was complete. The solvents were removed by evaporation. The resulting solid was dried in the vacuum oven (4O⁰C) overnight, to yield a white solid (D21 ) (400mg). δH (MeOD, 400MHz) 1.80-2.00 (2H, m), 2.20-2.30 (2H, m), 3.10-3.25 (2H, m), 3.40-3.55 )2H, m), 4.15-4.30 (1 H, m), 7.11 (1 H, d, J = 3.6Hz), 7.75 (1 H, s), 7.80 (1 H, d, J = 3.6Hz) Mass Spectrum: CuH₁₆CIN₃O₂ requires 293/295; found 294/296 (MH⁺)

Description 22

1 ,1 -Dimethylethyl 4-({[4-amino-5-chloro-2-

(methyloxy)phenyl]carbonyl}amino)-1-piperidinecarboxylate (D22)

To a solution of 4-amino-5-chloro-2-(methyloxy)benzoic acid (1.16g, 5.79mmol; available commercially from e.g. Aldrich) in DCM (12OmL) was added 1 ,1 -dimethylethyl 4-amino-1-piperidinecarboxylate (1.74g, 8.69mmol; available commercially from e.g. Fluka), EDC (1.21g, 11.58mmol) and DMAP (142mg, 1.16mmol). The mixture was stirred under argon at room temperature for 2h. After this time, LCMS showed that the reaction mixture contained 81 % desired product. The reaction mixture was transferred to a separating funnel and 10OmL sodium bicarbonate was added. The DCM layer was separated using a phase separation cartridge. The resulting oil was purified on a 100g silica column eluting with 0-95% EtOAc/pentane. Appropriate fractions were combined and evaporated to give the desired product as a white solid (D22) (1.63g, 74%). δH (CDCI₃, 400MHz) 1.35-1.45 (2H, m), 1.47 (9H, s), 1.95-2.03 (2H, m), 2.95-3.05 (2H, m), 3.89 (3H, s), 3.93- 4.07 (2H, m), 4.08-4.20 (1 H, m, integration masked by EtOAc), 4.38 (2H, s), 6.29 (1 H, s), 7.33 (1 H, d, J = 7.6Hz), 8.10 (1 H, s) Mass Spectrum: Ci₈H₂SCIN₃O₄ requires 383/385; found 384/386 (MH⁺)

Description 23

4-Amino-5-chloro-2-(methyloxy)-Λ/-4-piperidinylbenzamide hydrochloride (D23)

To a 50OmL flask was added 1 ,1 -dimethylethyl 4-({[4-amino-5-chloro-2- (methyloxy)phenyl]carbonyl}amino)-1-piperidinecarboxylate (may be prepared as described in Description 22) (1.63g, 4.15mmol) followed by methanol (2OmL) and 4M HCI/dioxan (1 OmL). After stirring the reaction at room temperature for 30 minutes, LCMS showed that the reaction mixture contained 85% desired product and 15% starting material. The mixture was stirred at room temperature under argon overnight. After this time, LCMS indicated that the reaction was complete. The solvents were removed by evaporation. The resulting solid was dried in the vacuum oven (4O⁰C) for 2h, to yield a white solid (D23) (1.6g). δH (MeOD, 400MHz) 1.72-1.92 (2H, m), 2.13-2.27 (2H₁ m), 3.06-3.24 (2H, m), 3.37-3.50 (2H, m), 3.94 (3H, s), 4.05- 4.20 (1 H, m), 6.66 (1 H, s), 7.80 (1 H, s) Mass Spectrum: C₁₃H₁₈CIN₃O₂ requires 283/285; found 284/286 (MH⁺)

Description 24 Tθtrahydro-3-furancarbonyl chloride (D24)

Tetrahydro-3-furancarboxylic acid (10.0g, 86.0 mmol) and DMF (3 drops) in dry DCM (10OmL) at room temperature were treated dropwise with a solution of oxalyl chloride (16.41g, 129.0mmol) in DCM (25mL) over 15 mins. The reaction solution was then stirred overnight at room temperature before being evaporated to dryness to afford the crude product as a yellow oil. This was purified by vacuum distillation to afford the title compound (D24) as a colourless liquid (8.96g, 77%) (b.pt 43°C/ 4.7 mbar). δH (CDCI₃, 400MHz) 2.22 (1 H, m), 2.35 (1 H, m), 3.57 (1 H, m), 3.84 (1 H, m), 3.93 (1 H, m), 4.01 (1 H, m) and 4.01 (1 H, m).

Description 25

(4S)-4-(Phenylmethyl)-3-[(3/?)-tetrahydro-3-furanylcarbonyl]-1 ,3- oxazolidin-2-one (D25a) and (4S)-4-(phenylmethyl)-3-[(3S)-tetrahydro-3- furanylcarbonyl]-1 ,3-oxazolidin-2-one (D25b)

1.6M n-Butyl lithium in hexanes (35.3mL, 56.4mmol) was added dropwise with stirring over 25 mins to a solution of (4S)-4-(phenylmethyl)-1 ,3-oxazolidin-2- one (10.0g, 56.4mmol) in dry THF (15OmL) under argon at -70⁰C. The addition was carried out at such a rate to keep the temperature below -65°C. A solution of tetrahydro-3-furancarbonyl chloride (may be prepared as in Description 24) (7.59g, 56.4mmol) in dry THF (1OmL) was then added dropwise over 20 mins, again maintaining the temperature below -65⁰C. The reaction solution was stirred at -7O⁰C for 0.5h and then allowed to warm to room temperature over 2.5h. Saturated aqueous ammonium chloride solution (2OmL) was then added and the reaction mixture was evaporated to near dryness. EtOAc (20OmL) and water (5OmL) were added; the organic layer was separated and washed with dilute aqueous NaHCO₃ (2 x 5OmL) and brine (1 x 5OmL). It was dried (MgSO₄) and evaporated to afford the mixture of diastereomers as a pale yellow oil (15.72g). This was purified on silica eluting with a gradient of 0-2.5% diethyl ether in toluene to afford firstly (4S)-4- (phenylmethyl)-3-[(3f?)-tetrahydro-3-furanylcarbonyl]-1 ,3-oxazolidin-2-one (D25a) (6.23g). δH (CDCI₃, 400MHz) 2.14 (1 H₁ m), 2.33 (1 H, m), 2.80 (1 H, dd, J = 13.2 and 9.2Hz), 3.27 (1 H, dd, J = 13.2 and 3.2Hz), 3.85 (1 H, m), 3.98 (2H, m), 4.10 (2H, m), 4.24 (2H, m), 4.69 (1 H, m), 7.20 (2H, m) and 7.30 (3H, m). HPLC (Waters C18; eluting with a gradient of acetonitrile in 0.1 % TFA/water) showed >98% d.e.

Further elution afforded (4S)-4-(phenylmethyl)-3-[(3S)-tetrahydro-3- furanylcarbonyl]-1 ,3-oxazolidin-2-one (D25b) (5.72g) δH (CDCI₃, 400MHz) 2.26 (2H, m), 2.79 (1 H, dd, J = 13.2 and 9.2Hz), 3.31 (1 H, dd, J = 13.2 and 3.2Hz), 3.85 (1 H, m), 3.97 (1 H, m), 4.02 (2H, m), 4.21 (3H, m), 4.69 (1 H, m), 7.21 (2H, m) and 7.30 (3H, m). HPLC (Waters C18; eluting with a gradient of acetonitrile in 0.1 % TFA/water) showed >99% d.e.

See JP2001-031667 (A) and H. C. Brown, Heterocycle, 1989, 28, 283 for enantiomer resolution and assignments.

Description 26 (3S)-Tetrahydro-3-furanylmethanol

2M Lithium borohydride in THF (10.7mL, 21.45mmol) was added dropwise with stirring to a solution of (4S)-4-(phenylmethyl)-3-[(3/?)-tetrahydro-3- furanylcarbonyl]-1 ,3-oxazolidin-2-one (may be prepared as in Description D25a) (5.37g, 19.5mmol) and water (0.386g, 21.45mmol) in diethyl ether (40OmL) at 0-5⁰C. The resulting mixture was stirred at 0-5°C for 0.5h followed by 3h at room temperature. 2M Aqueous NaOH solution (9.75mL, 19.5mmol) was then added dropwise and the mixture was stirred vigorously at room temperature for 20 mins. The organic layer was separated, dried over MgSO₄, filtered through celite, and evaporated to dryness to leave the crude product mixture as an oily solid (5.4Og). Repeated chromatography on silica eluting with 25-70% EtOAc in 40-60 petroleum ether afforded (3S)-tetrahydro-3- furanylmethanol (D26) (1.44g, 72%). δH (CDCI₃, 400MHz) 1.54 (1 H, m), 1.64 (1 H, m), 2.04 (1 H, m), 2.47 (1 H, m), 3.60 (3H, m), 3.77 (1 H, m) and 3.87 (2H, m).

Description 27

(3/?)-Tetrahydro-3-furanylmethyl methanesulfonate

(3S)-Tetrahydro-3-furanylmethanol (may be prepared as in Description 26) (1.44g, 14.1 mmol) in DCM (12ml_) was treated with pyridine (1.67g, 21.2mmol) and DMAP (0.172g, 1.4mmol) followed by dropwise addition of methanesulfonyl chloride (1.54g, 13.4mmol) in DCM (12ml_). The mixture was stirred at room temperature for 2Oh. It was then diluted with DCM (3OmL) and washed sequentially with dil. HCI (3 x 15mL), dil. NaHCO₃ (2 x 15ml_) and brine (1 x 1OmL). The solution was dried (MgSO₄) and evaporated to leave (3/?)-tetrahydro-3-furanylmethyl methanesulfonate (D27) as a pale yellow oil (1.91g, 79%). δH (CDCI₃, 400MHz) 1.56 (1 H, m), 2.11 (1 H, m), 2.69 (1 H, m), 3.03 (3H, s), 3.65 (1 H, m), 3.77 (1 H, m), 3.87 (2H, m), 4.11 (1 H, m) and 4.20 (1 H, m).

Description 28

1 ,1 -Dimethylethyl 4-[({[(phenylmethyl)oxy]carbonyl}amino)methyl]-1 - piperidinecarboxylate

Benzyl chloroformate (10.53g, 61.7mmol) in DCM (3OmL) was added dropwise with stirring to an ice-cooled solution of 1 ,1 -dimethylethyl 4- (aminomethyl)-i-piperidinecarboxylate (12.Og, 56.1mmol) and pyridine (6.64g, 84.1 mmol) in DCM (8OmL). After the addition, the solution was stirred at 0- 5°C for 0.5h followed by 3.5h at room temperature. The reaction solution was then washed with 1 M HCI (3 x 3OmL) and brine (3OmL). It was then treated with decolourising charcoal and MgSO4, filtered and evaporated to dryness to leave a pale yellow oil. This was stirred and treated with EtOAc (2OmL), causing crystallisation of the product. Hexane (20OmL) was added and stirring was continued for 0.5h. The resulting solid was filtered and dried to afford 1 ,1- dimethylethyl 4-[({[(phenylmethyl)oxy]carbonyl}amino)methyl]-1- piperidinecarboxylate (D28) as a white solid (11.15g, 57%). δH (CDCI₃, 400MHz) 1.10 (2H, br.m), 1.45 (9H, s), 1.67 (3H, br.m), 2.68 (2H, br.m), 3.09 (2H, br.m), 4.10 (2H, br.m), 4.82 (1 H, br.m), 5.10 (2H, s) and 7.34 (5H, m).

Description 29

Phenylmethyl (4-piperidinylmethyl)carbamate

1 , 1 -Dimethylethyl 4-[({[(phenylmethyl)oxy]carbonyl}amino)methyl]-1 - piperidinecarboxylate (may be prepared as in Description 28) (11.1g, 31.9mmol) in dioxane (6OmL) was treated with 4M HCI in dioxane (12OmL) and the reaction mixture was stirred at room temperature for 2.5h. It was then evaporated to dryness and the resulting solid was further dried under vacuum overnight to afford phenylmethyl (4-piperidinylmethyl)carbamate as its HCI salt (9.21 g) slightly contaminated with residual dioxane. δH (DMSO d-6, 400MHz) 1.30 (2H₁ m), 1.71 (3H, m), 2.81 (2H, m), 2.92 (2H, m), 5.01 (2H, s), 7.36 (5H, m), 8.65 (1 H, br. s) and 8.95 (1 H, br.s). A portion of this material (5.5Og, 19.3mmol) was suspended in EtOAc (15OmL) and MeOH (1OmL) and stirred vigorously with satd. K₂CO₃ solution (5OmL). After 1.5h water (3mL) was added and stirring continued for 0.5h. The two phases were separated and the aqueous phase was extracted with EtOAc (3OmL). The combined organic extracts were dried (MgSO₄) and evaporated to leave a yellow oil which solidified on standing to afford the title compound as the free base (D29) (4.76g).

Description 30

Phenylmethyl ({1 -[(3S)-tetrahydro-3-furanylmethyl]-4- piperidinyl}methyl)carbamate

(3f?)-Tetrahydro-3-furanylmethyl methanesulfonate (may be prepared as in Description 27) (1.9Og, 10.6mmol), phenylmethyl (4- piperidinylmethyl)carbamate (may be prepared as in Description 29) (2.62g, 10.6mmol) and K₂CO₃ (2.18g, 15.8mmol) were heated together with stirring in EtOH (4OmL) under argon in an oil bath at 80⁰C for 48h. The mixture was cooled to room temperature and then evaporated to near dryness. The residue was partitioned between EtOAc (15OmL) and water (15mL); the organic phase was separated, washed with brine (1 x 15ml), dried (MgSO₄) and evaporated to leave a yellow oil which crystallised on standing. This was redissolved in DCM (1OmL), filtered to remove a small amount of insoluble material and then purified by chromatography on silica eluting with a gradient of 0-5% (2M NH₃ in MeOH) in DCM to afford the title compound, phenylmethyl ({1-[(3S)-tetrahydro-3-furanylmethyl]-4-piperidinyl}methyl)carbamate (D30), as a waxy, white solid (1.39g, 40%). δH (CDCI₃, 400MHz) 1.25 (2H, m), 1.45 (1 H, m), 1.64 (3H, m), 1.90 (2H, m), 2.00 (1 H, m), 2.29 (2H, m), 2.41 (1 H, m), 2.87 (2H, m), 3.08 (2H, m), 3.47 (1 H, m), 3.72 (1 H, m), 3.83 (2H, m), 4.55 and 4.80 (1 H, 2 x br.s, rotamers), 5.09 and 5.15 (2H, 2 x s, rotamers) and 7.35 (5H, m). Mass spectrum: Ci₉H_2SN₂O₃ requires 332; found 333 (MH⁺). Chiral analytical HPLC (Chiralpak AS eluting with heptane/ EtOH 90:10) showed 99.8% e.e.

Description 31 ({1-[(3S)-Tetrahydro-3-furanylmethyl]-4-piperidinyl}methyl)amine

Phenylmethyl ({1 -[(3S)-tetrahydro-3-furanylmethyl]-4- piperidinyl}methyl)carbamate (may be prepared as in Description 30) (1.36g, 4.1 mmol) in MeOH (3OmL) was hydrogenated over 5% Pd/C (60% H₂O; 0.7Og) at room temperature and pressure for 4h. The catalyst was filtered off and the filtrate was evaporated to dryness to leave an oily white solid. This was dissolved in DCM (3OmL), dried over MgSO₄, filtered and evaporated to afford the title compound (D31 ) (0.79g, 97%). δH (CDCI₃, 400MHz) 1.30 (2H, m), 1.58 (2H, m), 1.79 (2H, m), 2.00 (3H, m), 2.35 (2H, m),2.45 (1 H, m), 2.73 (2H, d J = 6.8Hz), 2.91 (2H, m), 3.48 (1 H, m), 3.73 (1 H, m), 3.84 (2H, m), and 4.92 (v. br. s). Description 32 (3/?)-Tetrahydro-3-furanylmethanol

2M Lithium borohydride in THF (9.7ml_, 19.42mmol) was added dropwise with stirring to a solution of (4S)-4-(phenylmethyl)-3-[(3S)-tetrahydro-3- furanylcarbonyl]-1 ,3-oxazolidin-2-one (may be prepared as in Description D25b) (4.86g, 17.65mmol) and water (0.349g, 19.42mmol) in diethyl ether (40OmL) at 0-5⁰C. The resulting mixture was stirred at 0-5⁰C for 0.5h followed by 3h at room temperature. 2M Aqueous NaOH solution (8.83mL, 17.65mmol) was then added dropwise and the mixture was stirred vigorously at room temperature for 30 mins. The organic layer was separated, dried over MgSO₄, filtered through celite, and evaporated to dryness to leave the crude product mixture as an oily solid (4.87g). Repeated chromatography on silica eluting with 20-70% EtOAc in hexane afforded (3f?)-tetrahydro-3-furanylmethanol (D32) (1.06g, 59%). δH (CDCI₃, 400MHz) 1.51 (1 H, m), 1.64 (1 H, m), 2.04 (1 H, m), 2.47 (1 H, m), 3.60 (3H, m), 3.77 (1 H, m) and 3.87 (2H, m).

Description 33

(3S)-Tetrahydro-3-furanylmethyl methanesulfonate

(3R)-Tetrahydro-3-furanylmethanol (may be prepared as in Description 32) (1.48g, 14.5mmol) in DCM (12mL) was treated with pyridine (1.72g, 21.75mmol) and DMAP (0.177g, 1.45mmol) followed by dropwise addition of methanesulfonyl chloride (1.58g, 13.8mmol) in DCM (12mL). The mixture was stirred at room temperature for 20.5h. It was then diluted with DCM (15mL) and washed sequentially with 1 M HCI (3 x 1OmL), dil. NaHCO₃ (2 x 1OmL) and brine (1 x 1OmL). The solution was dried (MgSO₄) and evaporated to leave (3S)-tetrahydro-3-furanylmethyl methanesulfonate (D33) as a pale yellow oil (2.12g, 85%). δH (CDCI₃, 400MHz) 1.56 (1 H, m), 2.11 (1 H, m), 2.69 (1 H, m), 3.03 (3H, s), 3.65 (1 H, m), 3.77 (1 H, m), 3.87 (2H, m), 4.12 (1 H, m) and 4.20 (1 H, m).

Description 34

Phenylmethyl ({1 -[(3/?)-tetrahydro-3-furanylmethyl]-4- piperidinyl}methyl)carbamate (3S)-Tetrahydro-3-furanylmethyl methanesulfonate (may be prepared as in Description 33) (2.09g, 11.6mmol), phenylmethyl (4- piperidinylmethyl)carbamate (may be prepared as in Description 29) (2.88g, 11.6mmol) and K₂CO₃ (2.4Og, 17.4mmol) were heated together with stirring in EtOH (4OmL) under argon in an oil bath at 80⁰C for 4Oh. The mixture was cooled to room temperature and then evaporated to near dryness. The residue was partitioned between EtOAc (15OmL) and water (15mL); the organic phase was separated, washed with half saturated K₂CO₃ solution (1 x 15mL) and brine (1 x 15mL), dried (MgSO₄) and evaporated to leave an oil (3.51 g). This was redissolved in DCM (1OmL) and then purified by chromatography on silica eluting with a gradient of 0-5% (2M NH₃ in MeOH) in DCM to afford the title compound, phenylmethyl ({1-[(3R)-tetrahydro-3- furanylmethyl]-4-piperidinyl}methyl)carbamate (D34), as a waxy, white solid (1.57g, 41 %). δH (CDCI₃, 400MHz) 1.25 (2H, m), 1.46 (1 H, m), 1.64 (3H, m), 1.90 (2H, m), 2.00 (1 H, m), 2.29 (2H, m), 2.42 (1 H, m), 2.87 (2H, m), 3.08 (2H, m), 3.47 (1 H, m), 3.72 (1 H, m), 3.83 (2H, m), 4.55 and 4.80 (1 H₁ 2 x br.s, rotamers), 5.09 and 5.15 (2H, 2 x s, rotamers) and 7.35 (5H, m). Mass spectrum: C₁₉H_2SN₂O₃ requires 332; found 333 (MH⁺). Chiral analytical HPLC (Chiralpak AS eluting with heptane/ EtOH 90:10) showed 98.7% e.e.

Description 35 ({1-[(3R)-Tetrahydro-3-furanylmethyl]-4-piperidinyl}methyl)amine

Phenylmethyl ({1 -[(3f?)-tetrahydro-3-furanylmethyl]-4- piperidinyl}methyl)carbamate (may be prepared as in Description 34) (1.49g, 4.48mmol) in MeOH (35mL) was hydrogenated over 5% Pd/C (60% H₂O; 0.8Og) at room temperature and pressure for 3.5h. The catalyst was filtered off and the filtrate was evaporated to dryness to leave the title compound, ({1- [(3R)-tetrahydro-3-furanylmethyl]-4-piperidinyl}methyl)amine (D35), as an oily white solid (0.89g, 100%). δH (CDCI₃, 400MHz) 1.28 (2H, m), 1.42 (1 H, m), 1.58 (1 H, m), 1.73 (2H, m), 1.98 (3H, m), 2.32 (2H, m),2.45 (1 H, m), 2.65 (2H, d J = 6.4Hz), 2.89 (2H, m), 3.33 (br.s),3.48 (1 H, m), 3.73 (1 H, m) and 3.85 (2H, m). Mass spectrum: C₁₁H₂₂N₂O requires 198; found 199 (MH⁺).

Preparation of Examples

Example 1

(R/S) 8-Amino-7-chloro-/V-{[1 -(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-2,3-dihydro-1 ,4-benzodioxin-5-carboxamide (E1 )

8-Amino-7-chloro-Λ/-(4-piperidinylmethyl)-2,3-dihydro-1 ,4-benzodioxin-5- carboxamide hydrochloride (may be prepared according to the procedure described in WO9305038) was purified on an SCX cartridge eluting with methanol then 10% ammonia/methanol to yield the free base (389mg). Tetrahydro-3-furancarbaldehyde (324μl_, 1.79mmol, 50% solution in water) was added to a stirring solution of the free base (389mg, 1.18mmol) in DCE (1OmL), followed by sodium triacetoxyborohydride (632mg, 2.98mmol) and acetic acid (3 drops). The mixture was stirred at room temperature for 18h. The reaction was shown to be complete by LCMS. The reaction mixture was then quenched with methanol (5mL) and the solvent removed in vacuo. The crude mixture was purified on silica, eluting with 0-20% MeOH/DCM. The product containing fractions were identified and combined and the solvent was removed in vacuo to give the title compound as a colourless oil (E 1 ) (375mg, 92%). δH (CDCI₃, 400MHz) 1.48-1.70 (3H, m), 1.75-1.87 (3H, m), 2.07-2.17 (1 H, m), 2.23-2.33 (2H, m), 2.53-2.75 (3H, m), 3.20-3.37 (4H, m), 3.40-3.45 (1 H, m), 3.70-3.77 (1 H, m), 3.80-3.88 (1 H, m), 3.90-3.97 (1 H, m), 4.34.4.48 (6H, m), 7.61 (1 H, br s), 7.74 (1 H, m).

Mass Spectrum: C₂₀H₂₈CIN₃O₄ requires 409/411 ; found 410/412 (MH⁺). Preparative chiral HPLC separation carried out on Agilent P. S. System (Chiralpak AD (250mmx20mm, 10um), 50:50 Heptane:Ethanol, flow rate at 17.0ml/min, UV Absorbance at 254nm) Analytical HPLC separation carried out on Agilent 1100 System (Chiralpak AD (250mmx4.6mm, 10um), 50:50 Heptane:Ethanol, flow rate at 1.0ml/min, UV Absorbance at 254nm)

Faster running enantiomer (HCI salt; E1A): (109mg) 99.8% ee (retention time = 9.39 minutes); δH (MeOD, 400MHz) 1.50-1.65 (2H, m), 1.66-1.77 (1 H₁ m), 1.90-2.06 (3H, m), 2.18-2.28 (1 H, m), 2.65-2.78 (1 H, m), 2.90-3.03 (2H, m), 3.15-3.25 (2H, m), 3.30-3.42 (2H, m, masked by solvent peak), 3.45-3.55 (1 H, m), 3.60-3.70 (2H, m), 3.72-3.82 (1 H, m), 3.85-4.00 (2H, m), 4.35-4.48 (4H, m), 7.46 (1 H, m).

Mass Spectrum: C₂₀H₂₈CIN₃O₄ requires 409/411 ; found 410/412 (MH⁺). Slower running enantiomer (HCI salt; E1 B): (117mg) 96.4% ee (retention time = 11.86 minutes); δH (MeOD, 400MHz) 1.50-1.65 (2H, m), 1.66-1.75 (1 H, m), 1.90-2.10 (3H, m), 2.18-2.28 (1 H, m), 2.65-2.78 (1 H, m), 2.90-3.03 (2H, m), 3.15-3.25 (2H, m), 3.30-3.40 (2H, m, masked by solvent peak), 3.45-3.55 (1 H, m), 3.58-3.70 (2H, m), 3.72-3.82 (1 H, m), 3.85-4.00 (2H, m), 4.35-4.48 (4H, m), 7.46 (1 H, m). Mass Spectrum: C₂₀H₂₈CIN₃O₄ requires 409/411 ; found 410/412 (MH⁺).

Example E2

(R/S) 5-Amino-6-chloro-Λ/-{[1 -(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-3,4-dihydro-2H-chromene-8-carboxamide hydrochloride (E2)

5-Amino-6-chloro-N-(4-piperidinylmethyl)-3,4-dihydro-2H-chromene-8- carboxamide (may be prepared as described in Description 2) (100mg, 0.31 mmol) was dissolved in DCE (4mL). A solution of tetrahydro-3- furancarbaldehyde (67μL, 0.37mmol, 50% solution in water) in DCE (1 mL) was added, followed by sodium triacetoxyborohydride (164mg, 0.772mmol). The reaction mixture was stirred overnight. MeOH was then added to the reaction mixture, and this was then loaded onto 10g SCX cartridge, which had been preconditioned with DCM. The product was eluted with 2 column volumes of DCM, followed by 2 column volumes of MeOH and finally 3 column volumes of 2M NH₃ in MeOH. The product was present in the 2M NH₃ in MeOH fraction. Volatiles were removed in vacuo to yield the crude product. This was purified using MDAP. The residue was dissolved in 2 ml_ of 1 :1 MeOH:DCM and HCI (1 M in ether, 2eq.) was added. Volatiles were removed in vacuo and the product transferred to vial in MeOH. The MeOH was then removed (evaporated) in a blow-down unit. The vial was then put in a vacuum oven for 1h to yield the title compound (E2) as an off-white solid (81 mg). δH (DMSO, 400MHz) 1.40-1.55 (2H, m), 1.55-1.70 (1 H, m), 1.70-1.85 (3H, m), 1.90-2.00 (2H, m), 2.03-2.158 (1 H, m), 2.44-2.54 (m, integration masked by DMSO), 2.55-2.65 (1 H, m), 2.80-2.95 (2H, m), 3.05-3.13 (2H, m), 3.15-3.25 (2H, m), 3.30 - 3.50 (m, integration masked by DMSO), 3.60-3.65 (1 H, m), 3.70-3.78 (1 H, m), 3.83 (1 H, t, J = 8.8Hz), 4.16-4.25 (2H, m), 5.45-5.85 (2H, br s), 7.58 (1 H, s), 8.06 (1 H, t, J = 5.6Hz), 9.30 (1 H, br,s). Mass Spectrum: C₂i H₃₀CIN₃O₃ requires 408/410; found 408/410 (MH⁺)

Example E2A

5-Amino-6-chloro-Λ/-({1-[(3S)-tetrahydro-3-furanylmethyl]-4- piperidinyl}methyl)-3,4-dihydro-2H-chromene-8-carboxamide hydrochloride salt

({1 -[(3S)-Tetrahydro-3-furanylmethyl]-4-piperidinyl}methyl)amine (may be prepared as in Description 31 ) (0.773g, 3.90mmol) in DCM (3OmL) was treated with 5-amino-6-chloro-3,4-dihydro-2/-/-chromene-8-carboxylic acid (may be prepared according to the procedure described in Chem. Pharm. Bull., 1998, 46 (12), 1881 ) (0.889g, 3.90mmol) and the mixture was stirred at room temperature for 3 mins. DMAP (0.048g, 0.39mmol) was added and stirring continued for a further 3 mins. EDC (1.12g, 5.86mmol) was then added portionwise and the reaction mixture was stirred at room temperature for 16h. It was diluted with DCM (3OmL) and washed with water (3OmL). This mixture was filtered to remove insoluble material and the filtrate was separated. The organic phase was washed with water (3OmL); the combined aqueous washings were extracted with DCM (2OmL) and then the combined organic extracts were washed with dil. NaHCO₃ (2 x 3OmL). They were dried (MgSO₄) and evaporated to leave the crude product as a pale yellow foam (0.933g). This was purified on silica eluting with a gradient of 0-5% (2M NH₃ in MeOH) in DCM to afford pure free amine (0.812g). This was dissolved in DCM (6mL) and treated with 1 M HCI in diethyl ether (2.4mL). The resulting suspension was treated with diethyl ether (2OmL) and the white solid was filtered, washed with diethyl ether (3 x 1OmL) and dried in vacuo to afford the title compound, 5-amino-6-chloro-/V-({1 -[(3S)-tetrahydro-3-furanylmethyl]-4- piperidinyl}methyl)-3,4-dihydro-2/-/-chromene-8-carboxamide hydrochloride salt (E2A), (0.823g, 48%). δH (DMSO d-6, 400MHz) 1.61 (3H, m), 1.80 (3H, m), 1.98 (2H, m), 2.09 (1 H, m), 2.50 (m, obscured by DMSO), 2.66 (1 H, m), 2.85 (2H, m), 3.08 (2H, m), 3.19 (3H, m), 3.31-3.50 (m, obscured by water), 3.63 (1 H, m), 3.72 (1 H, m), 3.84 (1 H, m ), 4.21 (2H, m), 5.61 (2H, br. s), 7.57 (1 H, s), 8.05 (1 H, t, J = 6.0Hz) and 9.89 and 10.17 (1 H, 2 x br. s, rotamers). Mass spectrum: C₂iH₃₀CIN₃O₃ requires 407/409; found 408/410 (MH⁺).

Example E2B

5-Amino-6-chloro-Λ/-({1-[(3R)-tetrahydro-3-furanylmethyl]-4- piperidinyl}methyl)-3,4-dihydro-2H-chromene-8-carboxamide hydrochloride salt

({1 -[(3R)-Tetrahydro-3-furanylmethyl]-4-piperidinyl}methyl)amine (may be prepared as in Description 35) (0.889g, 4.48mmol) in DCM (35mL) was treated with 5-amino-6-chloro-3,4-dihydro-2H-chromene-8-carboxylic acid (may be prepared according to the procedure described in Chem. Pharm. Bull., 1998, 46 (12), 1881 ) (1.02g, 4.48mmol) and the mixture was stirred at room temperature for 3 mins. DMAP (0.055g, 0.45mmol) was added and stirring continued for a further 3 mins. EDC (1.29g, 6.72mmol) was then added portionwise and the reaction mixture was stirred at room temperature for 16h. It was diluted with DCM (3OmL) and washed with water (3OmL). This mixture was filtered to remove insoluble material and the filtrate was separated. The organic phase was washed with water (3OmL); the combined aqueous washings were extracted with DCM (2OmL) and then the combined organic extracts were washed with dil. NaHCO₃ (2 x 3OmL). They were dried (MgSO₄) and evaporated to leave the crude product as a pale yellow foam (1.17g). This was purified on silica eluting with a gradient of 0-5% (2M NH₃ in MeOH) in DCM to afford pure free amine (0.972g). This was dissolved in DCM (6mL) and treated with 1 M HCI in diethyl ether (2.87mL). The resulting white solid was filtered, washed with diethyl ether (3 x 5mL) and dried in vacuo to afford the title compound, 5-amino-6-chloro-N-({1-[(3R)-tetrahydro-3- furanylmethyl]-4-piperidinyl}methyl)-3,4-dihydro-2H-chromene-8-carboxamide hydrochloride salt (E2B), (1.03g, 52%). δH (DMSO d-6, 400MHz) 1.60 (3H, m), 1.80 (3H, m), 1.98 (2H, m), 2.09 (1 H, m), 2.50 (m, obscured by DMSO), 2.66 (1 H, m), 2.85 (2H, m), 3.08 (2H, m), 3.19 (3H, m), 3.31-3.55 (m, obscured by water), 3.63 (1 H, m), 3.72 (1 H, m), 3.84 (1 H, m ), 4.21 (2H, m), 5.61 (2H, br. s), 7.57 (1 H, s), 8.06 (1 H, t, J = 6.0Hz) and 9.85 and 10.11 (1 H, 2 x br. s, rotamers). Mass spectrum: C₂i H₃₀CIN₃O₃ requires 407/409; found 408/410 (MH⁺).

Example E3

(R/S) 4-Amino-5-chloro-Λ/-{[1 -(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-1 -benzofuran-7-carboxamide (E3)

To a stirring solution of 4-amino-5-chloro-A/-(4-pipehdinylmethyl)-1- benzofuran-7-carboxamide hydrochloride (may be prepared as described in Description 5) (230mg, 0.67mmol) in DCE (2OmL) was added tetrahydro-3- furancarbaldehyde (181 μL, 1 mmol, 50% solution in water) followed by sodium triacetoxyborohydride (356mg, 1.68mmol) and acetic acid. The mixture was stirred for 65h. LCMS showed that the reaction mixture contained 60% desired product, 40% starting material. Another 1 equivalent of aldehyde and 1 equivalent of sodium triacetoxyborohydride were added and the reaction was stirred for 1 h to complete the reaction. The reaction mixture was washed with sodium bicarbonate solution and extracted into DCM. The residue was purified on silica, eluting with 0-20% MeOH/DCM. The product containing fractions were identified and combined, and solvent was removed in vacuo to give the title compound as a colourless paste (E3) (168mg, 64%). δH (CDCI₃, 400MHz) 1.30-1.42 (2H, m), 1.52-1.70 (2H, m), 1.72-1.80 (2H, m), 1.83-2.05 (3H, m), 2.28-2.38 (2H, m), 2.40-2.50 (1 H, m), 2.83-2.98 (2H, m), 3.38-3.45 (2H, m), 3.46-3.50 (1 H, m), 3.70-3.78 (1 H, m), 3.80-3.90 (2H₁ m), 4.71 (2H, br s), 6.81 (1 H, d, J = 2Hz), 7.33 (1 H, br s), 7.63 (1 H, d, J = 2.4Hz), 8.05 (1 H, s). Mass Spectrum: C2₀H₂₆CIN₃O3 requires 391/393; found 392/394 (MH⁺). Preparative chiral HPLC separation carried out on Agilent P. S. System (Chiralcel OJ (250mmx20mm, 10um), 90:10 Heptane:Ethanol, flow rate at 17.0ml/min, UV Absorbance at 215nm)

Analytical HPLC separation carried out on Agilent 1100 System (Chiralcel OJ (250mmx4.6mm, 10um), 90:10 Heptane:Ethanol, flow rate at LOml/min, UV Absorbance at 215nm)

Faster running enantiomer (HCI salt; E3A): (45mg) 95.6% ee (retention time = 36.94 minutes); δH (MeOD, 400MHz) 1.55-1.75 (3H, m), 1.95-2.12 (3H, m), 2.18-2.30 (1 H, m), 2.65-2.75 (1 H, m), 2.90-3.05 (2H, m), 3.15-3.22 (2H, m), 3.40-3.55 (3H, m), 3.60-3.72 (2H, m), 3.73-3.82 (1 H, m), 3.85-4.00 (2H, m), 7.11 (1 H, d, J = 2Hz), 7.76 (1 H, s), 7.78 (1 H, d, J = 2Hz). Mass Spectrum: C20H26CIN₃O3 requires 391/393; found 392/394 (MH⁺). Slower running enantiomer (HCI salt; E3B): (20mg) 95.1 % ee (retention time = 42.97 minutes); δH (MeOD, 400MHz) 1.52-1.75 (3H, m), 1.95-2.12 (3H, m), 2.15-2.27 (1 H, m), 2.65-2.75 (1 H, m), 2.90-3.05 (2H, m), 3.15-3.25 (2H, m), 3.40-3.55 (3H, m), 3.60-3.72 (2H, m), 3.73-3.80 (1 H, m), 3.85-4.00 (2H, m), 7.11 (1 H, d, J = 2.4Hz), 7.76 (1 H, s), 7.78 (1 H, d, J = 2.4Hz). Mass Spectrum: C₂₀H₂₆CIN₃O₃ requires 391/393; found 392/394 (MH⁺).

Examples E4

(WS) 4-Amino-5-chloro-2-(methyloxy)-Λ/-{[1-(tetrahydro-3-furanylmethyl)-

4-piperidinyl]methyl}benzamide (E4)

To a 10OmL flask was added 4-amino-5-chloro-2-(methyloxy)-Λ/-(4- piperidinylmethyl)benzamide hydrochloride (may be prepared according to the procedure described in Bioorg. & Med. Chem., 2003, 11(19), 4225) (613mg, 1.84mmol) followed by DCE (3OmL), tetrahydro-3-furancarbaldehyde (500μL, 2.76mmol, 50% solution in water), sodium triacetoxyborohydride (975mg, 4.6mmol) and 3 drops of acetic acid. A reflux condenser was put on the flask and the reaction mixture was stirred at 7O⁰C under argon for 12h. After this time, LCMS showed that the reaction mixture contained 47% desired product and 34% starting material. Another 0.5 equivalents of tetrahydro-3- furancarbaldehyde (167uL) and 0.5 equivalents of sodium triacetoxyborohydride (195mg) were added and the reaction mixture was stirred at 7O⁰C for 1 h. Methanol was then added to quench the reaction. The reaction solvent was removed in vacuo and purification was carried out using a 100g silica column (eluting with 0 to 20% MeOH in DCM). Appropriate fractions were combined and evaporated to give the desired product as a yellow oil (E4) (660mg, 94%). δH (CDCI₃, 400MHz) 1.35-1.85 (6H, m), 2.08- 2.23 (m, integration masked by solvent), 2.46-2.62 (m, integration masked by solvent), 3.02-3.22 (m, integration masked by solvent), 3.27-3.38 (2H, m), 3.40-3.52 (m, integration masked by solvent), 3.68-4.00 (7H, m), 4.39 (2H, br s), 6.30 (1 H, s), 7.79 (1 H, t, J = 8.8Hz), 8.10 (1 H, s). Mass Spectrum: C₁₉H₂₈CIN₃O₃ requires 381/383; found 382/384 (MH⁺). Preparative chiral HPLC separation carried out on Agilent P. S. System (Chiralpak AD (250mmx20mm, 10um), 80:20 Heptane:Ethanol, flow rate at 17.0ml/min, UV Absorbance at 215nm) Analytical HPLC separation carried out on Agilent 1100 System (Chiralpak AD (250mmx4.6mm, 10um), 80:20 Heptane:Ethanol, flow rate at 1.0ml/min, UV Absorbance at 215nm)

Faster running enantiomer (HCI salt; E4A): (140 mg) 95.1 % ee (retention time = 15.61 minutes); δH (MeOD, 400MHz) 1.48-1.85 (3H, m), 1.85-2.13 (3H, m), 2.15-2.30 (1 H, m), 2.65-2.82 (1 H, m), 2.87-3.10 (2H, m), 3.15-3.23 (2H, m), 3.25-3.42 (m, integration masked by MeOH), 3.45-3.54 (1 H, m), 3.55-3.72 (2H, m), 3.72-3.83 (1 H, m), 3.83-4.02 (5H, m), 6.70 (1 H, s), 7.83 (1 H, s) Mass Spectrum: C₁₉H_2SCIN₃O₃ requires 381/383; found 382/384 (MH⁺). Slower running enantiomer (HCI salt; E4B): (105 mg) 95.5% ee (retention time = 19.30 minutes); δH (MeOD, 400MHz) 1.48-1.85 (3H, m), 1.85-2.13 (3H, m), 2.15-2.30 (1 H, m), 2.65-2.82 (1 H, m), 2.87-3.10 (2H, m), 3.15-3.23 (2H, m), 3.25-3.42 (m, integration masked by MeOH), 3.45-3.54 (1 H, m), 3.55-3.72 (2H, m), 3.72-3.83 (1 H, m), 3.83-4.02 (5H, m), 6.70 (1 H, s), 7.83 (1 H, s). Mass Spectrum: Ci₉H_2SCIN₃O₃ requires 381/383; found 382/384 (MH⁺)

Example 5

(R/S) 4-Amino-5-chloro-2-methyl-Λ/-{[1 -(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-1 -benzofuran-7-carboxamide hydrochloride (E5)

To a stirring solution of 4-amino-5-chloro-2-methyl-Λ/-(4-piperidinylmethyl)-1- benzofuran-7-carboxamide hydrochloride (may be prepared as described in Description 9) (85mg, 0.24mmol) in DCE (1OmL) was added tetrahydro-3- furancarbaldehyde (65μL, 0.36mmol, 50% solution in water) followed by sodium triacetoxyborohydride (126mg, 0.6mmol) and acetic acid (3 drops). The mixture was stirred under argon. The reaction was complete by LCMS after 4h. The reaction mixture was then quenched with 5mL methanol and the solvent was removed in vacuo. The crude mixture was purified on silica, eluting with 0-20% MeOH/DCM. The product containing fractions were identified and combined and the solvent removed in vacuo. The HCI salt was made by dissolving the product in DCM and adding 0.5mL of 1 M HCI/diethylether. This gave an off-white solid (E5) (66mg, 63%). δH (MeOD, 400MHz) 1.58-1.75 (3H₁ m), 1.95-2.10 (3H, m), 2.15-2.28 (1 H, m), 2.51 (3H, s), 2.65-2.75 (1 H, m), 2.92-3.05 (2H, m), 3.15-3.23 (2H, m), 3.40-3.52 (3H, m), 3.60-3.70 (2H, m), 3.72-3.80 (1 H, m), 3.85-3.90 (1 H, m), 3.90-4.00 (1 H, m), 6.69 (1 H, s), 7.67 (1 H, s). Mass Spectrum: C_2IH₂₈CIN₃O₃ requires 405/407; found 406/408 (MH⁺).

Example 6

(R/S) 8-Amino-7-bromo-Λ/-{[1 -(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-2,3-dihydro-1 ,4-benzodioxin-5-carboxamide hydrochloride

(E6)

A mixture of 8-amino-7-bromo-Λ/-(4-piperidinylmethyl)-2,3-dihydro-1 ,4- benzodioxin-5-carboxamide hydrochloride (may be prepared as described in Description 13) (50mg, 0.12mmol), tetrahydro-3-furancarbaldehyde (45μl_, 0.25mmol, 50% solution in water), sodium triacetoxyborohydride (65mg, 0.31 mmol), acetic acid (3 drops) and DCE (5mL) was stirred overnight. The reaction was then quenched with MeOH and solvent was removed in vacuo. The residue was purified on a silica column, eluting with 0-20% MeOH/DCM. Product containing fractions were identified and combined. Solvent was removed in vacuo. The HCI salt was made using 0.5ml_ of 1 M HCI/diethyl ether to give off-white solid (E6) (45mg, 75%). δH (MeOD, 400MHz) 1.52-1.78 (3H, m), 1.90-2.08 (3H, m), 2.18-2.30 (1 H, m), 2.65-2.78 (1 H, m), 2.92-3.05 (2H, m), 3.15-3.25 (2H, m), 3.30-3.40 (2H, m, masked by solvent peak), 3.45- 3.55 (1 H, m), 3.60-3.70 (2H, m), 3.75-3.81 (1 H, m), 3.85-3.92 (1 H, m), 3.93- 4.00 (1 H, m), 4.40-4.50 (4H, m), 7.62 (1 H, s). Mass Spectrum: C₂QH₂₈BrN₃O₄ requires 453/455; found 454/456 (MH⁺). Example 7

(WS) 5-Amino-6-bromo-/V-{[1 -(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-3,4-dihydro-2H-chromene-8-carboxamide hydrochloride (E7)

5-Amino-6-bromo-N-(4-piperidinylmethyl)-3,4-dihydro-2H-chromene-8- carboxamide (may be prepared as described in description 17) (100mg, 0.27mmol) was dissolved in DCE (4mL). A solution of tetrahydro-3- furancarbaldehyde (59μL, 0.33mmol, 50% solution in water) in DCE (1 mL) was added followed by sodium triacetoxyborohydride (144mg, 0.68mmol). The reaction mixture was stirred at room temperature overnight. A further 0.3 equivalents of tetrahydro-2-furancarbaldehyde was then added and the reaction mixture was stirred for a further 4h. DCM was added to reaction mixture, and the mixture was transferred to a phase separation cartridge. Water was added and the mixture agitated. The DCM layer was run through. NaCI was added to the aqueous layer and the saturated aqueous layer was decanted back into the phase separation cartridge. This was extracted again with DCM. Volatiles were removed in vacuo. The crude sample was purified using MDAP. The product containing fractions were combined and volatiles removed in vacuo. The sample was dissolved in 2mL of 1 :1 DCM:MeOH and HCI (1 M in ether, 2eq) was added to the sample. Volatiles were removed in vacuo to yield the HCI salt. The product was transferred to vial in MeOH, the solvent blown off (evaporated) in a blow-down unit and the product was dried in a vacuum oven to yield a white solid (E7) (28mg, 23%). δH (DMSO, 400MHz) 1.40-1.55 (2H, m), 1.55-1.70 (2H, m), 1.70-1.85 (3H, m), 1.90-2.03 (2H, m), 2.03-2.15 (1 H, m), 2.45-2.65 (m, integration masked by DMSO), 2.80 - 2.90 (2H, m), 3.03-3.12 (2H, m), 3.13-3.26 (4H, m), 3.30-3.56 (m, integration masked by water), 3.58-3.70 (1 H, m), 3.70-3.80 (1 H, m), 3.83 (1 H, t, J = 8.0Hz), 4.15-4.25 (2H, m), 5.55 (2H, br s), 7.73 (1 H, s), 8.05 (1 H, br s), 9.16-9.28 (1 H, br s) Mass Spectrum: C₂IH₃₀BrN₃O₃ requires 452/454; found 452/454 (MH⁺) Example 8

(R/S) 8-Amino-7-chloro-Λ/-[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]-

2,3-dihydro-1 ,4-benzodioxin-5-carboxamide hydrochloride (E8)

To a carousel tube was added 8-amino-7-chloro-Λ/-4-piperidinyl-2,3-dihydro- 1 ,4-benzodioxin-5-carboxamide hydrochloride (may be prepared as described in Description 19) (100mg, 0.29mmol) followed by DCE (5ml_), tetrahydro-3- furancarbaldehyde (69μL, 0.38mmol, 50% solution in water) and sodium triacetoxyborohydride (154mg, 0.73mmol). After stirring for 17h at 7O⁰C, LCMS showed that the reaction mixture contained 54% desired product and 42% starting material. Another 0.5 equivalents of tetrahydro-3- furancarbaldehyde and acetic acid (3 drops) were added and the reaction mixture was stirred at 7O⁰C for 4h. After this time, LCMS showed that the reaction mixture contained 97% desired product. The reaction solvent was removed in vacuo. The residue was purified using a 2Og silica column (0-20% MeOH/DCM gradient). Appropriate fractions were combined and evaporated to give the desired compound (71.6mg). 1 M HCI in Et₂O (1 mL) and MeOH were added to the product to form the HCI salt and then the solvents were removed by evaporation. The product was dissolved in methanol and put in a vial. The blow-down apparatus was used to remove methanol. The resulting oil was dried in the vacuum oven (4O⁰C) overnight to yield a white solid (E8) (65.3mg, 52%). δH (MeOD, 400MHz) 1.67-1.79 (1 H, m), 1.86-2.20 (2H₁ m), 2.09-2.31 (3H, m), 2.67-2.79 (1 H, m), 3.07-3.19 (2H, m), 3.19-3.24 (2H, m), 3.47-3.59 (1 H, m), 3.62-3.72 (2H, m), 3.72-3.81 (1 H, m), 3.84-3.99 (2H, m), 4.04-4.17 (1 H, m), 4.34-4.49 (4H, m), 7.43 (1 H, s). Mass Spectrum: Ci₉H₂BCIN₃O₄ requires 395/397; found 396/398 (MH⁺) Example 9

(R/S) 4-Amino-5-chloro-Λ/-[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]-

1-benzofuran-7-carboxamide hydrochloride (E9)

To a carousel tube was added 4-amino-5-chloro-Λ/-4-piperidinyl-1-benzofuran- 7-carboxamide hydrochloride (may be prepared as described in description 21 ) (80mg, 0.24mmol) followed by DCE (5mL), tetrahydro-3- furancarbaldehyde (87μL, 0.48mmol, 50% solution in water), sodium triacetoxyborohydride (152mg, 0.72mmol) and acetic acid (2 drops). The reaction mixture was stirred at 70⁰C for 2h. After this time, LCMS showed that the reaction mixture contained 100% of the desired product. Methanol was added and the solvents were removed in vacuo. The residue was purified using a 2Og silica column, eluting with 0 to 20% MeOH in DCM. Appropriate fractions were combined to give a colourless oil. 1 M HCI in Et₂O (1 mL) and methanol were added to the product to form the HCI salt. The solvents were removed by evaporation. The product was dissolved in MeOH and put in a vial. The blow-down apparatus was used to remove methanol to yield a white solid (E9) (50mg, 50%). δH (MeOD, 400MHz) 1.65-1.82 (1 H, m), 1.90-2.10 (1 H, m), 2.15-2.40 (3H, m), 2.68-2.82 (1 H, m), 3.08-3.25 (3H, m), 3.28-3.45 (3H, m), 3.48-3.62 (1 H, m), 3.65-3.82 (2H, m), 3.82-4.03 (2H, m), 4.13-4.30 (1 H, m), 7.11 (1 H, d, J = 3.6Hz), 7.75 (1 H, s), 7.78 (1 H, d, J = 3.6Hz). Mass Spectrum: C₁₉H₂₄CIN₃O₃ requires 377/379; found 378/380 (MH⁺)

Example 10

(R/S) 4-Amino-5-chloro-2-(methyloxy)-Λ/-[1-(tetrahydro-3-furanylmethyl)- 4-piperidinyl]benzamide hydrochloride (E10)

To a carousel tube was added 4-amino-5-chloro-2-(methyloxy)-Λ/-4- piperidinylbenzamide hydrochloride (may be prepared as described in description 23) (280mg, 0.88mmol) followed by ethanol (5mL),tetrahydro-3- furancarbaldehyde (239μL, 1.32mmol, 50% solution in water), sodium triacetoxyborohydride (466mg, 2.2mmol) and acetic acid (3 drops). The reaction mixture was stirred at 75⁰C under argon overnight. After this time, LCMS showed 41 % starting material and 57% desired product. Another 0.5eq. of tetrahydro-3-furancarbaldehyde (8OuL) and 0.5eq. of sodium triacetoxyborohydride (93mg) were added and stirring at 75⁰C under argon overnight. After this time, LCMS showed 35% starting material and 64% desired product. The reaction solvent was removed in vacuo. Material was purified using a 5Og silica column eluting with 0 to 20% methanol in DCM. Appropriate fractions were combined and evaporated to give a yellow oil (200mg). 1 M HCI in Et₂O (1 mL) and methanol were added to the product to form the HCI salt. The solvents were removed by evaporation. The product was dissolved in MeOH and put in a vial. The blow-down apparatus was used to remove MeOH. The sample was dried in the vacuum oven to yield a yellow solid (170mg, 49%). δH (MeOD, 400MHz) 1.65-1.82 (1 H, m), 1.82-2.03 (2H, m), 2.10-2.18 (1 H, m), 2.20-2.33 (3H, m), 2.67-2.80 (1 H, m), 3.05-3.17 (1 H, m), 3.18-3.28 (2H, m), 3.45-3.60 (2H, m), 3.60-3.85 (3H, m), 3.85-4.02 (4H, m), 4.03-4.18 (1 H, m), 6.55 (1 H, s), 7.78 (1 H, s). Mass Spectrum: Ci₈H₂₆CIN₃O₃ requires 368/370; found 368/370 (MH⁺)

Preparation of Comparative Examples

Comparative Example 1

(R/S) 8-Amino-7-chloro-Λ/-{[1 -(tetrahydro-2-furanylmethyl)-4- piperidinyl]methyl}-2,3-dihydro-1,4-benzodioxin-5-carboxamide hydrochloride (CE1)

8-Amino-7-chloro-Λ/-(4-piperidinylmethyl)-2,3-dihydro-1 ,4-benzodioxin-5- carboxamide (may be prepared according to the procedure described in WO9305038) (57mg, 0.17mmol) was dissolved in acetonitrile (5ml_). N₁N- diisopropylethylamine (45mg, 0.35mmol) and 2-(bromomethyl)tetrahydrofuran (29mg, 0.17mmol) were added and the mixture was stirred at reflux. After heating for 30 minutes, the mixture became white and cloudy. After 3h it had become clear and slightly yellow coloured. After 6h, LCMS showed that the reaction mixture contained 14% desired product, 73% starting material. Stirring at reflux was continued overnight. LCMS then showed that the reaction mixture contained 45% desired product. Volatiles were removed in vacuo and the residue was purified using MDAP yielding a colourless paste (CE1 ) (10mg). The hydrochloride salt was prepared as a white solid (10mg) (MeOD, 400MHz) 1.48-1.65 (3H, m), 1.88-2.08 (5H, m), 2.10-2.20 (1H, m), 2.92-3.07 (2H₁ m), 3.08-3.17 (1 H, m), 3.20-3.30 (1 H, m), 3.30-3.50 (2H, m, masked by solvent peak), 3.65-3.77 (2H, m), 3.79-3.86 (1 H, m), 3.90-4.00 (1 H, m), 4.22-4.32 (1 H, m), 4.35-4.47 (4H, m), 7.46 (1 H, m). Mass Spectrum: C₂₀H₂₈CIN₃O₄ requires 409/411 ; found 410/412 (MH+).

Comparative Example 2

8-Amino-7-chloro-Λ/-({1 -[(2S)-tetrahydro-2-furanylmethyl]-4- piperidinyl}methyl)-2,3-dihydro-1,4-benzodioxin-5-carboxamide hydrochloride (CE2)

8-Amino-7-chloro-Λ/-(4-piperidinylmethyl)-2,3-dihydro-1 ,4-benzodioxin-5- carboxamide (may be prepared according to the procedure described in WO9305038) (362mg, 1.1 mmol) was dissolved in acetonitrile (5mL). N₁N- diisopropylethylamine (284mg, 2.2mmol) and (2S)-tetrahydro-2-furanylmethyl methanesulfonate (may be prepared as described in Description 7) (200mg, 1.1 mmol) were added and the mixture was stirred at reflux. After refluxing overnight, LCMS showed that the reaction mixture contained 35% desired product. Volatiles were removed and the residue was purified on silica eluting with 0-20% MeOH/DCM. The product containing fractions were combined and solvent removed in vacuo to yield a beige solid (132mg). This material (100mg) was treated with HCI/diethyl ether to yield the HCI salt as a beige solid (CE2) (117mg). 99.8% ee (retention time = 59.92 minutes); (MeOD, 400MHz) 1.48-1.65 (3H, m), 1.88-2.10 (5H, m), 2.10-2.20 (1 H, m), 2.92-3.07 (2H, m), 3.08-3.17 (1 H, m), 3.20-3.28 (1 H, m), 3.30-3.50 (2H, m, masked by solvent peak), 3.68-3.77 (2H, m), 3.79-3.86 (1 H, m), 3.90-4.00 (1 H, m), 4.22-4.32 (1 H, m), 4.35-4.47 (4H, m), 7.46 (1 H, m). Mass Spectrum: C₂₀H₂₈CIN₃O₄ requires 409/411 ; found 410/412 (MH⁺). Analytical HPLC separation carried out on Agilent 1100 System (Chiralcel OJ (250mmx4.6mm, 10um), 95:5 Heptane:Ethanol, flow rate at 1.0ml/min, UV Absorbance at 215nm)

Comparative Example 3

(R/S) 5-Amino-6-chloro-W-{[1 -(tetrahydro-2-furanylmethyl)-4- piperidinyl]methyl}-3,4-dihydro-2H-chromene-8-carboxamide hydrochloride (CE3)

5-Amino-6-chloro-N-(4-piperidinylmethyl)-3,4-dihydro-2H-chromene-8- carboxamide (may be prepared as described in Description 2) (100mg, 0.31 mmol) was dissolved in DCE (4mL). Tetrahydro-2-furancarbaldehyde (37mg, 0.37mmol) was added followed by sodium triacetoxyborohydride (164mg, 0.77mmol). The reaction mixture was then stirred at room temperature for 5h. The reaction mixture was diluted with MeOH and loaded onto a 1Og SCX cartridge that had been preconditioned with DCM. The column was flushed with DCM (2 column volumes), MeOH (2 column volumes) and finally NH₃ (2M in MeOH, 3 column volumes). The fractions eluted with 2M NH₃ in MeOH were concentrated in vacuo to yield a yellow oil. Further purification was achieved using MDAP. The product containing fractions combined and volatiles removed in vacuo. The resultant oil was dissolved in 1 :1 DCM:MeOH (1 mL). HCI (1 M in ether, 1eq) was then added and the solution was left to stand for 10 minutes. Volatiles then removed in vacuo to yield product (CE3) (59mg). δH (DMSO, 400MHz) 1.40-1.60 (3H, m), 1.75-1.87 (5H, m), 1.96 (2H, t, J = 5.2Hz), 2.00-2.10 (1 H, m), 2.45-2.54 (m, integration masked by DMSO), 2.85-2.97 (2H, m), 3.00-3.10 (1 H, m), 3.13- 3.23 (3H, m), 3.24-3.35 (1 H, m), 3.47-3.85 (m, integration masked by H₂O), 4.20 (2H, t, J = 4.8Hz), 4.22-4.30 (1 H, m), 5.30-5.80 (2H, br s), 7.57 (1 H, s), 8.05 (1 H, t, J = 6Hz), 9.70 (1 H, br s). Mass Spectrum: C₂i H₃₀CIN₃O₃ requires 408/410; found 408/410 (MH⁺)

Comparative Example 4

5-Amino-6-chloro-Λ/-({1-[(2S)-tetrahydro-2-furanylmethyl]-4- piperidinyl}methyl)-3,4-dihydro-2H-chromene-8-carboxamide hydrochloride (CE4)

The 5-amino-6-chloro-N-(4-piperidinylmethyl)-3,4-dihydro-2H-chromene-8- carboxamide (may be prepared as described in Description 2) (98mg, 0.3mmol) was dissolved in acetonitrile (5ml_). The N,N-diisopropylethylamine (78mg, 0.61 mmol) and (2S)-tetrahydro-2-furanylmethyl methanesulfonate (may be prepared as described in Description 7) (55mg, 0.3mmol) were added, the mixture was stirred at reflux for 18h. LCMS showed 27% desired product, 68% starting material. Refluxing continued for a further 6h, desired product increased to 32%. Heating at reflux was continued for 17h, followed by removal of volatiles. Purification was carried out using silica gel chromatography, eluting with 0-6% (10% NH₃/MeOH)/DCM to give a pale yellow oil (CE4) (38mg). The HCI salt was prepared as a beige solid (39mg). 99.8% ee (retention time = 15.83 minutes); (MeOD, 400MHz) 1.50-1.70 (3H, m), 1.85-2.02 (5H, m), 2.02-2.20 (3H, m), 2.52-2.60 (2H, m), 2.92-3.05 (2H, m), 3.10-3.17 (1 H, m), 3.20-3.25 (1 H, m), 3.30-3.45 (2H, m, masked by solvent peak), 3.65-3.75 (2H, m), 3.80-3.88 (1 H, m), 3.90-3.98 (1 H, m), 4.23- 4.33 (3H₁ m), 7.72 (1 H, s).

Mass Spectrum: C_2IH₃₀CIN₃O₃ requires 407/409; found 408/410 (MH⁺). Analytical HPLC separation carried out on Agilent 1100 System (Chiralpak AD (250mmx4.6mm, 10um), 95:5 Heptane:Ethanol, flow rate at 1.0ml/min, UV Absorbance at 215nm)

Comparative Example E5

(R/S) 4-Amino-5-chloro-Λ/-{[1 -(tetrahydro-2-furanylmethyl)-4- piperidinyl]methyl}-1 -benzofuran-7-carboxamide hydrochloride (CE5)

A mixture of 4-amino-5-chloro-Λ/-(4-piperidinylmethyl)-1-benzofuran-7- carboxamide hydrochloride (may be prepared as described in Description 5) (50mg, 0.15mmol), tetrahydro-2-furancarbaldehyde (22mg, 0.22mmol), sodium triacetoxyborohydride (77mg, 0.36mmol), acetic acid (3 drops) and DCE (5mL) was stirred for 65h. LCMS showed that the reaction mixture contained a majority of the desired product. The reaction was quenched with MeOH and solvent was removed in vacuo. The residue was then purified on silica column, eluting with 0-20% MeOH/DCM. The product containing fractions were identified and combined. Solvent was removed in vacuo and the residue was further purified by MDAP. The HCI salt was made using 0.5mL of 1 M HCI/diethyl ether to give an off-white solid (CE5) (21 mg, 33%). (MeOD, 400MHz) 1.50-1.70 (3H₁ m), 1.90-2.20 (6H, m), 2.95-3.09 (2H, m), 3.10-3.15 (1 H, m), 3.20-3.25 (1 H, m), 3.40-3.50 (2H, m, masked by solvent peak), 3.68-3.75 (2H, m), 3.79-3.88 (1 H, m), 3.90-3.98 (1 H, m), 4.22-4.32 (1 H, m), 7.11 (1 H, d, J = 2.4Hz), 7.76 (1 H, s), 7.78 (1 H, d, J = 2Hz). Mass Spectrum: C₂₀H₂₆CIN₃O₃ requires 391/393; found 392/394 (MH⁺).

Comparative Example E6

4-Amino-5-chloro-Λ/-({1-[(2S)-tetrahydro-2-furanylmethyl]-4- piperidinyl}methyl)-1 -benzofuran-7-carboxamide hydrochloride (CE6)

4-Amino-5-chloro-Λ/-(4-piperidinylmethyl)-1-benzofuran-7-carboxamide hydrochloride (may be prepared as described in Description 5) (124mg, 0.4mmol) was dissolved in acetonitrile (5ml_). N,N-diisopropylethylamine (104mg, 0.81 mmol) and (2S)-tetrahydro-2-furanylmethyl methanesulfonate (may be prepared as described in Description 7) (73mg, 0.4mmol) were added and the mixture was stirred at reflux for 18h. LCMS showed that the reaction mixture contained 25% desired product, 70% starting material. The reaction mixture was refluxed for another 6h, and the desired product increased to 30%. Refluxing continued for 17h, and the reaction mixture contained 42% desired product. Volatiles were removed and the residue was purified on silica, eluting with 0-6% (10% NH₃/MeOH)/DCM to give a colourless paste (CE6) (53mg). The HCI salt was prepared as a beige solid (52mg). 99.8% ee (retention time = 6.05 minutes); (MeOD, 400MHz) 1.50- 1.70 (3H, m), 1.90-2.20 (6H, m), 2.95-3.05 (2H, m), 3.09-3.15 (1 H, m), 3.20- 3.25 (1 H, m), 3.40-3.50 (2H, m), 3.68-3.75 (2H, m), 3.79-3.86 (1 H, m), 3.90- 3.98 (1 H, m), 4.22-4.32 (1 H, m), 7.11 (1 H, d, J = 2Hz), 7.76 (1 H, s), 7.79 (1 H, d, J = 2.4Hz). Mass Spectrum: C₂₀H₂₆CIN₃O₃ requires 391/393; found 392/394 (MH⁺). Analytical HPLC separation carried out on Agilent 1100 System (Chiralcel OJ (250mmx4.6mm, 10um), 50:50 Heptane:Ethanol, flow rate at 1.0ml/min, UV Absorbance at 215nm)

Biological Data

Compounds of the invention may be tested for in vitro biological activity in accordance with the following assays:

Yeast functional 5-HT4a agonist assay

Yeast (Saccharomyces cerevisiae) cells expressing the human 5-HT4a receptor were generated by integration of an expression cassette into the ura3 chromosomal locus of yeast strain MMY23. This cassette consisted of DNA sequence encoding the human 5-HT4a receptor flanked by the yeast GPD promoter to the 5' end of 5-HT4a and a yeast transcriptional terminator sequence to the 3' end of 5-HT4a. MMY23 expresses a yeast/mammalian chimeric G-protein alpha subunit in which the C-terminal 5 amino acids of Gpa1 are replaced with the C-terminal 5 amino acids of human Gi1 (as described in Brown et al. (2000), Yeast 16:11-22). Cells were grown at 3O⁰C in liquid Synthetic Complete (SC) yeast media (Guthrie and Fink (1991 ), Methods in Enzymology, Vol. 194) lacking uracil, tryptophan, adenine and leucine to late logarithmic phase (approximately 6 OD600/ml).

Agonists were prepared as 10 mM stocks in DMSO. EC50 values (the concentration required to produce 50% maximal response) were estimated using serial dilutions of between 3- and 5-fold (BiomekFX, Beckman) into DMSO. pEC50 corresponds to negative Iog10 of molar EC50. Agonist solutions in DMSO were transferred into black microtitre plates (96- or 384- well). Cells were suspended at a density of 0.2 OD600/ml in SC media lacking histidine, uracil, tryptophan, adenine and leucine and supplemented with 1 mM 3-aminotriazole, 0.1 M sodium phosphate pH 7.0, and 10^"5 M fluorescein di-β- D-glucopyranoside (FDGIu, Molecular Probes). This mixture (5OuI per well for 384-well plates, 20OuI per well for 96-well plates) was added to agonist in the assay plates, to give final assay concentration 1 % DMSO. After incubation at 3O⁰C for 24 hours, fluorescence resulting from degradation of FDGIu to fluorescein due to exoglucanase, an endogenous yeast enzyme produced during agonist-stimulated cell growth, was determined (excitation wavelength: 485nm; emission wavelength: 535nm) using a Spectrofluor (Tecan) or similar microtitre plate reader. Fluorescence was plotted against compound concentration and iteratively curve fitted using a four parameter fit to generate a concentration effect value. Efficacy (Emax) was calculated from the equation:

Emax = Max[compound X] - Min[compound X] / Max[5-HT] - Min[5-HT] x 100%

Where Max[compound X] and Min[compound X] are the fitted maximum and minimum respectively from the concentration effect curve for compound X, and Max[5-HT] and Min[5-HT] are the fitted maximum and minimum respectively from the concentration effect curve for 5-hydroxytryptamine [5- HT]. Equieffective molar ratio (EMR) values were calculated from the equation:

EMR = EC50 [compound X] / EC50 [5-HT]

Where EC50 [compound X] is the EC50 of compound X and EC50 [5-HT] is the EC50 of 5-HT.

Mammalian functional 5-HT4a agonist assay

Human embryonic kidney cells stably expressing the human macrophage scavenger receptor type Il (HEK-293-MSR-II cells) were established in-house (Lysko PG et al., J Pharmacol Exp Ther. 289(3): 1277-85). These cells were grown in humidified conditions in Minimum Essential Medium containing 10% FCS, 1x non-essential amino acids, 2mM L-glutamine and 0.4 mg/ml geneticin at 37°C/5% CO₂.

BacMam plasmid constructs for the production of viruses for expression in mammalian cells were generated as described (Condreay JP et al., Proc. Natl. Acad. Sci. 96:127-132, Ames R et al., Receptors Channels 10(3-4):117- 24). A Kpnl-EcoRV fragment encoding the human 5-HT4a receptor cDNA (Genbank accession number Y08756) or a Kpnl-EcoRV fragment encoding the human G protein Gα16 (Genbank accession number M63904), both including an upstream Kozak sequence and start codon (GCCACCATG), were inserted separately into the BacMam shuttle vector multiple cloning site (Condreay JP et al., Proc. Natl. Acad. Sci. 96:127-132). Viruses containing these inserts were then generated using the Bac-to-Bac system (Invitrogen) according to manufacturer's instructions, and further propagated in Sf9 cells to generate high-titre virus stocks.

At 90% confluence, HEK-293-MSR-II cells were harvested in PBS by centrifugation and resuspended in the same media except that dialysed FCS was substituted for normal FCS. BacMam viruses encoding the 5HT4a cDNA and the Gα16 cDNA (see above) were both added to the cells at a multiplicity of infection of 5 and 10 respectively. The cells were then plated out in 96-well clear-bottomed, black-walled plates at 35,000 cells/well (in 100μl) and incubated for 24 hours.

The next day 50μl Fluo4am dye (at 6μM in Tyrodes buffer) was added to each well and incubated at 37°C/5% CO₂ for one hour. Cells were then washed five times in Tyrodes buffer, with the final wash leaving 150μl buffer in each well. Compounds to be tested for 5-HT4 agonism were prepared in 96-well plates as half-log dilution series from 4μM to 4OpM in Tyrodes buffer. A Fluorimetric Imaging Plate Reader (FLIPR) was used to add the compounds (50μl per well) and to determine peak fluorescence emitted per well over the whole assay period. These data were analysed in Microsoft Excel using an in-house macro and iteratively curve fitted using a four parameter logistic fit (as described by Bowen and Jerman TiPS, 16, 413-417) to generate a concentration effect value and thus a pEC50 (negative Iog10 of molar EC50) and efficacy (maximal effect of compound expressed as a percentage of the maximal effect of 5-HT). Compounds of the invention were also tested for potential to inhibit the CYP3A4 isoform of human cytochrome P450, as set out below:

Human Cytochrome P450 Inhibition

Inhibition of the CYP3A4 isoform of human cytochrome P450 was determined by quantifying the production of the fluorescent metabolite following incubation of appropriate pro-fluorescent substrates with enzyme in the presence of a test compound.

For each assay, CYP3A4 (available commercially from Cypex Ltd) was mixed with the appropriate substrate in 50 mM phosphate buffer, pH7.4, as indicated in the attached table:

5 μl test compound in methanol was then added. A seven point concentration curve was used for each test compound (0.1 , 0.3, 1 , 3, 10, 33 and 100 μM) in addition to "no compound" controls and positive controls using an established inhibitor. The reactions were incubated for approximately 10 minutes at 37°C prior to the addition of 25 μl of an NADPH regenerating system (1.7mg/ml NADP₁ 7.8 mg/ml Glucose-6-phosphate, 6 units/ml Glucose-6-phosphate dehydrogenase). The production of fluorescence was then measured over 10 minutes using a Perseptive Biosystems Cytofluor, Series 4000 multi-well plate reader. The excitation and emission wavelengths for each substrate are given below:

The control rate of fluorescent metabolite production was established from the no compound controls (uninhibited). The extent of inhibition at each compound concentration was calculated as a percentage of the uninhibited control rate (assigned as 100%) and the IC50 value (the concentration of test compound required to produce 50% inhibition) was determined from these results.

Time Dependent Inhibition of Human Cytochrome P450

The method used is identical to that described above for assessing human cytochrome P450 inhibition except that two pre-incubation times were used: 10 minutes and 40 minutes (to confirm whether any time dependent inhibition seen is NADPH dependent) and the fluorescence was measured over 30 minutes.

The IC50 values were determined for each 5 minute interval (0-5, 6-10, 11-15, 16-20, 21-25, 26-30) of the 10 minute pre-incubation experiments. Where time dependent inhibition was apparent (i.e. when the IC50 measured during the last interval was <50% of that measured initially) the 40 minute preincubation data were also analysed to confirm whether the apparent time dependent inhibition was NADPH dependent.

Results

The compounds of examples 1 , 1A, 1 B, 2, 3, 3A, 3B, 4, 4A, 4B and 5-10 were tested in the yeast functional 5-HT4a agonist assay. These compounds exhibited a pEC50 of >7.5, an efficacy of >80% and an EMR in the range of 0.1-11.5.

The compounds of examples 1 , 1A, 1 B, 2, 2A, 2B, 3, 3A, 3B, 8 and 10 were tested in the mammalian functional 5-HT4a agonist assay. These compounds exhibited a pEC50 of >8 and efficacy in the range of 15-95%.

The compounds of examples 1 , 1A, 1 B, 2, 2A, 2B, 3, 3A, 3B, 4, and 5-10, and the compounds of comparative examples CE3-6 were tested in the human cytochrome P450 inhibition assay. The results are given below: 1

The compounds of examples 1A, 1B, 2, 2A, 2B, 3, 4 and 5 were also tested in the time dependent inhibition of human cytochrome P450 assay. The results are given below:

The time dependent inhibition of human cytochrome P450 assay may highlight potential for idiosynchratic toxicity. However, the predictive value of this assay is currently unclear.

Claims

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

(I) wherein R¹ represents a group of formula (a), (b), (c) or (d):

and wherein:

R² represents Ci_-4 alkyl, -CH₂-C_3-6CyClOaIkVl or C_3-6cycloalkyl;

R³ represents hydrogen or halogen;

R⁴ represents Ci_-4 alkyl;

R⁵ represents hydrogen, hydroxy or carboxy;

R⁵ cannot be hydroxy; p and q independently represent 1 or 2; and s represents 0, 1 or 2

2. A compound according to claim 1 , wherein R¹ represents a group of formula (c) or (d).

3. A compound according to claim 2, wherein R¹ represents a group of formula (d).

4. A compound according to any preceding claim wherein R³ represents a halogen.

5. A compound according to any preceding claim wherein R⁵ represents hydrogen.

6. A compound according to any preceding claim wherein R⁶ represent hydrogen.

7. A compound according to any preceding claim wherein m represents 1.

8. A compound according to any preceding claim wherein n represents 1.

9. A compound according to any preceding claim wherein p represents 2.

10. A compound according to any preceding claim wherein q represents 2.

11. A compound according to any preceding claim wherein s represents 0.

12. A compound according to claim 1 which is a compound of formula E1- E10 or a pharmaceutically acceptable salt or solvate thereof.

13. A compound according to claim 1 which is selected from the group consisting of:

8-Amino-7-chloro-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]methyl}-2,3- dihydro-1 ,4-benzodioxin-5-carboxamide; 5-Amino-6-chloro-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]methyl}-3,4- dihydro-2/-/-chromene-8-carboxamide;

4-Amino-5-chloro-2-methyl-Λ/-{[1-(tetrahydro-3-furanylmethyl)-4- piperidinyl]methyl}-1 -benzofuran-7-carboxamide;

1 ,4-benzodioxin-5-carboxamide;

4-Amino-5-chloro-Λ/-[1-(tetrahydro-3-furanylmethyl)-4-piperidinyl]-1- benzofuran-7-carboxamide; or a pharmaceutically acceptable salt or solvate thereof.

14. A pharmaceutical composition which comprises a compound of formula (I) as defined in any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier or excipient.

15. A compound as defined in any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof for use in therapy.

16. A compound as defined in any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of diseases treatable by 5-HT4 agonism.

17. Use of a compound as defined in any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the treatment of diseases treatable by 5-HT4 agonism.

18. A method of treatment of diseases treatable by 5-HT4 agonism which comprises administering to a host in need thereof an effective amount of a compound as defined in any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof.

19. A pharmaceutical composition for use in the treatment of diseases treatable by 5-HT4 agonism which comprises a compound as defined in any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

20. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof wherein m represents 1 , as defined in claim 1 , which process comprises reacting a compound of formula (II),

(H)

(III)

(i) deprotecting a compound of formula (I) which is protected;

(ii) converting from one compound of formula (I) to another; and/or

(iii) forming a salt or solvate of the compound so formed.

21. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof wherein m represents 1 , as defined in claim 1 , which process comprises reacting a compound of formula (II),

(IV)

wherein R⁵ is defined in relation to formula (I) and L¹ represents a suitable leaving group; and optionally thereafter performing one or more of the following steps,

(i) deprotecting a compound of formula (I) which is protected;

(ii) converting from one compound of formula (I) to another; and/or

(iii) forming a salt or solvate of the compound so formed.

22. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof wherein m represents 1 , as defined in claim 1 , which process comprises reacting a compound of formula (II),

(V)

wherein R⁵ is hydrogen or carboxy and L² represents a suitable leaving group; and optionally thereafter performing one or more of the following steps, (i) deprotecting a compound of formula (I) which is protected; (ii) converting from one compound of formula (I) to another; and/or (iii) forming a salt or solvate of the compound so formed.