HK1089177B - Modulators of peripheral 5-ht receptors - Google Patents

Description

FIELD OF THE INVENTION

The invention relates to modulators of peripheral 5-HT receptors, particularly 5-HT4, receptors said modulators essentially selective for peripheral 5-HT receptors over receptors of the central nervous system. The invention allows for the treatment, amongst others, of gastrointestinal disorders, lower urinary tract disorders, and cardiovascular disorders, without side effects related to CNS activity, according to the claims.

BACKGROUND OF THE INVENTION

5-Hydroxytryptamine (5-HT) is an important signalling molecule in the human body, and has important effects both as a neurotransmitter and as a locally acting signalling molecule with e.g. vasoactive effects. During the past 20 years 14 different 5-HT receptors have been identified and classified into 7 different subgroups (5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄, 5-HT₅, 5-HT₆ and 5-HT₇), based on structural and pharmacological criteria as well as signal transduction properties. Additional diversity arises from e.g. alternative splicing of e.g. 5-HT₄ (e.g. 5-HT_4(a), 5-HT_4(b)) and 5-HT₇ receptors, and of RNA editing of e.g. 5-HT_2C receptors. 5-HT₄ is found to play a central role in diseases in organs like the heart, the gastrointestinal system, the urinary bladder and central nervous system (CNS).

5-HT₄ receptor modulators, agonists and antagonists alike, are found to be useful for the treatment of a variety of diseases such as gastroesophageal reflux disease, gastrointestinal disease, gastric motility disorder, non-ulcer dyspepsia, functional dyspepsia, irritable bowel syndrome, constipation, dyspepsia, oesophagitis, gastroesophageal disease, nausea, central nervous system disease, Alzheimer's disease, cognitive disorder, emesis, migraine, neurological disease, pain, and cardiovascular disorders such as cardiac failure and heart arrhythmia. Further gastrointestinal disorders suitable for prophylaxis or treatment of the symptoms of Irritable Bowel Syndrome, including abdominal pain and disrupted colonic motility.

Since 5-HT₄ receptors are located both inside and outside the CNS, 5-HT₄ receptor agonists and antagonists will have effects both inside and outside the CNS, unless their design prevents their access to or causes them to preferentially localise to only one of these compartments. When addressing 5-HT₄ receptors located outside the CNS, effects on receptors inside the CNS may represent undesirable side-effects of the treatment, and vice versa. The present invention seeks to avoid this problem by presenting 5-HT₄ receptor agonists and antagonists which will not penetrate the blood-brain barrier and thus will not have access to 5-HT₄ receptors located inside the CNS.

Moreover, the problem of poor targeting of 5-HT receptor ligands is aggravated by the fact that the receptor activity is diminished upon frequent binding. Unwanted or unselective binding is undesired in this context also.

Several modulators with affinity for 5-HT₄ receptors are known in the state of the art. This includes agonists, antagonists and partial agonists. Modulators of 5-HT₄ receptors are today in active development as potential therapeutic drugs.

US 6,552,046 discloses the modification of the piperidinyl nitrogen of cisapride with a moiety wherein an acidic group may be in close proximity to the basic nitrogen. Moreover, despite recognising that cisapride has CNS side effects, it modifies cisapride with an ester moiety for purposes of avoiding cytochrome P-450 due to degradation of the ester by esterases. Most remarkably, US 6,552,046 observes that cisapride enters the central nervous system and binds to 5-HT₄ receptors, indicating that cisapride may have centrally-mediated effects. It further states compounds of US 6,552,046 can be used in the treatment of: 1) cognitive disorders, including Alzheimer's disease; 2) behavioural disorders, including schizophrenia, mania, obsessive-compulsive disorder, and psychoactive substance use disorders; 3) mood disorders, including depression and anxiety; and 4) disorders of control of autonomic function, including essential hypertension and sleep disorders.

US 6,624,163 (Pfizer) discloses imidazopyridines as 5-HT₄ modulators. Notably, none of the embodiments of the invention comprise an acidic moiety. This recent attempt in the area of 5-HT modulators is silent to means of differentiation between 5-HT related CNS disorders to gastrointestinal or cardiac disorders. The novel compounds are directed to everything from neurological diseases to heartburn. Each of the embodiments of US 6,624,163 are suitable substrates for modification with an acidic moiety according to the present invention.

US 6,632,827 seeks to minimise side effects with the use of an optically pure form of norcisapride in the treatment of gastrointestinal disorders yet concerns itself with the associated serious CNS side effects such as memory loss, sleep disorders, depression, and psychoactive distress. Each of the embodiments of US 6,632,827 are suitable substrates for modification with an acidic moiety according to the present invention

US 2001/0031751 provides novel 5-HT₄ antagonists, but does not seek to differentiate the CNS- from the peripherally-located receptors and thus intends their use in both CNS and gastrointestinal or cardiovascular disorders. Notably, none of the embodiments of the invention comprise an acidic moiety. Each of the embodiments of US 2001/0031751 are suitable substrates for modification with an acidic moiety according to the present invention.

Sanger et al. 1998 discloses a new 5-HT4 receptor antagonist (SB-207266) which in a pilot study reduced the symptoms of irritable bowel syndrome. The ability of SB-207266 to antagonize at the human 5-HT4 receptor (human isolated intestine) and to affect the mechanisms of peristalsis (guinea-pig isolated ileum) and defaecation (conscious, fed mice) was examined.

SUMMARY OF THE INVENTION

The scope of the present invention is limited by the appended claims.

A principal object of the invention is providing 5-HT₄ receptor modulators selective to peripheral receptors, essentially to the exclusion of delivery to CNS located receptors. The invention accomplishes this by modifying existing modulators and allows for the design and preparation of new modulators which comprise an acidic moiety so that the modulator is unable to cross the blood-brain barrier.

An essential feature of 5-HT modulators, particularly 5-HT4 modulators, is the presence of a basic nitrogen. The present inventors have found that the presence of an acidic moiety, particularly one wherein the acidic hydrogen of the acidic moiety is at least 2 atoms from the basic nitrogen, dramatically improves the selectivity of these modulators for peripheral 5-HT receptors compared to those of the central nervous system. The present inventors have modified existing 5-HT modulators and prepared entirely novel 5-HT modulators of Formula IV-P, see below which, due to comprising both an acidic moiety and a basic nitrogen, will provide for improved treatment of conditions affected by modulating of peripheral 5-HT receptors and with reduced side effects, due to the selectivity over receptors of the central nervous system.

A general aspect of the invention relates to use of said compounds for the manufacture of medicaments for the treatment of a disease associated, at least in part, with peripheral 5-HT receptor comprising administering a compound of the invention, preferably with a peripheral 5-HT4 receptor, preferably essentially whilst not modulating a 5-HT receptor of the central nervous system.

The invention relates to a compound of Formula IV-P, see below which fulfils the following: i) a binding affinity to a 5-HT receptor with a pK_l of at least 5; ii) comprises at least one basic nitrogen atom; iii) comprises at least one acidic moiety with a pKa of no more than 6.4, or a salt or ester thereof.

The compounds of the invention are compounds according to the formula IV-P

• wherein L is absent or selected from the group consisting of straight chain or branched optionally substituted C1-10-alkyl, optionally substituted C2-10-alkenyl, optionally substituted C2-10-alkynyl, C1-10-alkylamine, C1-10-alkoxy, C2-10-alkenyloxy, C2-10-alkynyloxy, C1-10-alkoxycarbonyl, C2-10-alkenyloxycarbonyl, C2-10-alkynyloxycarbonyl; and A is selected from the group consisting of -C(O)-OR1, -OP(O)OR2OR2, -P(O)OR2OR2,-SO2OR2, SO3H, OSO3H and PO3H; wherein R1 and R2 are independently selected from the group consisting of H, M, C1-15-alkyl, C3-8-cycloalkyl, aryl, and R1,2 wherein R1,2 is R'-O-C(O)-R", R'-O-C(O)-O-R", R'-C(O)-O-R", wherein R' and R" are independently selected from the group consisting of C1-15-alkyl, C3-8-cycloalkyl and aryl;
• R13 is selected from the group consisting of H, halogen, NH2, and C1-6-alkyl; and
• R16 is selected from the group consisting of H, halogen, OH, O-C1-6-alkyl, and C1-6-alkyl.

A further aspect of the invention relates to a use of said compounds for the manufacture of medicaments for treating a cardiovascular disorder comprising administering a compound of the invention. Suitably, the cardiovascular disorder is selected from tachycardia, bradycardia, cardioexcitation, cardiodepression, arrhythmia, fibrillation, atrial fibrillation, Paroxysmal Supraventricular Tachycardia (PSVT), thromoembolisms and VTE.

A particularly interesting aspect of the invention relates to a use of said compounds for the manufacture of medicaments for treating gastrointestinal disorders, such as irritable bowel syndrome, comprising administering a compound of the invention. Alternatively stated, an aspect of the invention relates to the use of the compounds of the invention for the preparation of a medicament for the prophylaxis or treatment of gastrointestinal disorders suitable and for symptoms of Irritable Bowel Syndrome, including abdominal pain and disrupted colonic motility; diarrhea; constipation; urinary incontinence and anal incontinence

The use of said compounds for the manufacture of medicaments for the treatment of lower urinary tract disorders, such as e.g. hyperactive bladder, comprising administering a compound of the invention is a further aspect of the invention.

DESCRIPTION OF THE INVENTION

The invention relates to a compound of Formula IV-P which fulfils the following: i) a binding affinity to a 5-HT receptor with a pK_l of at least 5; ii) comprises at least one basic nitrogen atom; iii) comprises at least one acidic moiety with a pKa of no more than 6.4, or a salt or ester thereof. It is anticipated that the acidic moiety is to be spaced from the acidic nitrogen atom such that the acidic moiety does not interfere with the binding capacity of the nitrogen atom believed to constitute part of the pharmacophore in 5-HT modulators.

The acidic moiety (A) is thus suitably spaced from the basic nitrogen (BN) by at least 2 atoms. Correspondingly, L in compounds of the invention preferably comprises at least 2 atoms.

The compounds of the invention i.e the compounds of Formula IV-P further comprise iv) an aromatic or heteroaromatic ring, more typically an aromatic ring. The acidic moiety may be covalently linked to the aromatic or heteroaromatic ring. It is believed that the acidic moiety is to be within a 20 atom space from either the basic nitrogen or the aromatic/heteroaromatic ring. Typically, the basic nitrogen or the aromatic/heteroaromatic ring is less than 16 atoms, such as less than 10, from the acidic moiety.

An acidic moiety is a group that is at least 90 % ionic form at physiological pH, more typically at least 95%, even more typically at least 99% of the group is in ionic form. In a preferred embodiment, the acidic moiety has a pKa of no more than 6, more preferably, no more than 5.5, such as no more than 5.4, 5.3, 5.3, 5.2, 5.1, 5.0. In a most preferred form, the compounds of the invention have a pKa of less than 5.0, such as less than 4.5.

The acidic moiety may be in the form of its ester, in its free ion form, or in a salt form. In the embodiment wherein the acidic moiety is in the form of its ester, after hydrolysis of the ester to the acid or to its free ion form is at least 90 % ionic form at physiological pH, more typically at least 95%, even more typically at least 99% of the group is in ionic form. It is to be understood that esters of the acidic moiety are characterised in that their hydrolysis consequently result in the presence of the acid in its protonated form or in its free ion form.

Suitable salts include the counter-ion M selected from the group comprising sodium, potassium, calcium, magnesium, aluminium, iron, and zinc ions. The inventor contemplates salts with ammonia and organic nitrogenous bases strong enough to form salts with carboxylic acids. Bases useful for the formation of pharmaceutically acceptable nontoxic base addition salts of the compound of the present invention form a class whose limits are readily understood by those skilled in the art.

In the present context, the term "halogen" includes fluorine, chlorine, bromine and iodine.

In the present context the term "aryl" is intended to mean a carbocyclic aromatic ring or ring system. Moreover, the term "aryl" includes fused ring systems wherein at least two aryl rings, or at least one aryl and at least one C_3-8-cycloalkyl share at least one chemical bond. Examples of "aryl" rings include optionally substituted phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl, and indanyl. A preferred aryl group is phenyl. The term "aryl" relates to aromatic, preferably benzenoid groups connected via one of the ring-forming carbon atoms, and optionally carrying one or more substituents selected from halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, C_1-6 alkoxy, C_1-6 alkyl, C_1-6 hydroxyalkyl, C_1-6 aminoalkyl, C_1-6 alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl. As stated, preferred aryl groups are phenyl, and, most suitably, substituted phenyl groups, carrying one or two, same or different, of the substituents listed above.

The term "heterocyclic ring" is intended to mean three-, four-, five-, six-, seven-, and eight-membered rings wherein carbon atoms together with from 1 to 3 heteroatoms constitute said ring. A heterocyclyl may optionally contain one or more unsaturated bonds situated in such a way, however, that an aromatic π-electron system does not arise. The heteroatoms are independently selected from oxygen, sulphur, and nitrogen.

A heterocyclic ring may further contain one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo-systems and thio-systems such as lactams, lactones, cyclic imides, cyclic thloimides and cyclic carbamates..

Heterocyclic rings may optionally also be fused to aryl rings, such that the definition includes bicyclic structures. Preferred such fused heterocyclyl groups share one bond with an optionally substituted benzene ring. Examples of benzo-fused heterocyclyl groups include benzimidazolidinone, tetrahydroquinoline, and methylenedioxybenzene ring structures.

Examples of "heterocyclic rings" are the heterocycles tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin, 1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane, 1,4-oxathiin, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine , maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane, hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline, pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3-dioxolane, 1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline, oxazolidine, thiazoline, thiazolidine, 1,3-oxathiolane. Binding to the heterocycle may be at the position of a heteroatom or via a carbon atom of the heterocycle, or, for benzo-fused derivatives, via a carbon of the benzenoid ring.

Suitable embodiments of the acidic moiety or an ester thereof selected from the group consisting of C(O)-OR¹, OP(O)OR²OR², P(O)OR²OR², SO²OR², SO₃H, OSO₃H, and PO₃H wherein R¹ and R² are independently selected from the group consisting of H, M, C_1-15-alkyl, C_3-8-cycloalkyl, aryl, and R^1,2 wherein R^1,2 is R'-O-C(O)-R", R'-O-C(O)-O-R", R'-C(O)-O-R", wherein R' and R" are independently selected from the group consisting of C_1-15-alkyl, C_3-8-cycloalkyl and aryl. Salts of these acid moieties imply that at least one of R1 and R2 is M. M is a counterion as defined above.

A particularly interesting embodiment of the compounds of the invention are esters of the previously described acidic moiety. Typical esters include alkyl esters, substituted alkyl esters, aryl esters, substituted aryl esters and acyloxyalkyl esters. Exemplary embodiments of esters of acidic moieties include

In another suitable embodiment, the compound of the invention has a pK_l of at least 5.5, such as at least 6.

The compounds of the invention may be agonist, antagonists, reverse agonists, partial agonists, or partial antagonists of a 5-HT receptor. Typically, the compound of the invention will have either agonist or partial agonistic activity towards at least one receptor sub-group and optionally concomitant antagonist or partial antagonistic activity toward at least one other receptor sub-group. In a preferred embodiment of the invention, the compounds of the invention have a binding affinity with a pK_l of at least 5, such as at least 5.5, preferably at least 6 to the 5-HT4 or 5-HT3 receptor subgroup.

A further aspect of the invention is directed to the use of a compound of the invention as defined supra, or a composition comprising said compound or a salt of said compound for the preparation of a medicament for the treatment of a cardiovascular disorder. The cardiovascular disorder is typically selected from the group consisting of tachycardia, bradycardia, cardioexcitation, cardiodepression, arrhythmia, fibrillation, atrial fibrillation, Paroxysmal Supraventricular Tachycardia (PSVT), thromoembolisms and VTE.

A related aspect of the invention is directed to the use of a compound of the invention as defined herein, or a composition comprising said compound or a salt of said compound for the preparation of a medicament for the treatment of a gastrointestinal disorder or lower urinary tract disorder. The gastrointestinal disorder may be selected from the group consisting of irrital bowel syndrome; gastrointestinal hypomotility disorders; gastroesophageal reflux, such as heartburn or mild oesophagitis; functional or nonulcer dyspensia; gastroparesis; nausea and vomiting; early satiety in the elderly; paraneoplastic of HIV-associated gastroparesis; drug-induced delays in gastric emptying and functional bowel obstructions, such as bowel obstructions caused by pancreatic cancer or drugs; and emesis. An related aspect of the invention relates to the use of the compounds of the invention for the preparation of a medicament for the prophylaxis or treatment of gastrointestinal disorders suitable and for symptoms of Irritable Bowel Syndrome, including abdominal pain and disrupted colonic motility; diarrhea; constipation; urinary incontinence and anal incontinence.

The compound of the invention is a derivative of piboserod, namely of the formula IV-P

• wherein L is absent or selected from the group consisting of straight chain or branched optionally substituted C1-10-alkyl, optionally substituted C2-10-alkenyl, optionally substituted C2-10-alkynyl, C1-10-alkylamine, C1-10-alkoxy, C2-10-alkenyloxy, C2-10-alkynyloxy, C1-10-alkoxycarbonyl, C2-10-alkenyloxycarbonyl, C2-10-alkynyloxycarbonyl or combinations thereof; and
• A is selected from the group consisting of C(O)-OR1, OP(O)OR2OR2, P(O)OR2OR2, SO2OR2, SO3H, OSO3H, and PO3H; wherein R1 and R2 are independently selected from the group consisting of H, M, C1-15-alkyl, C3-8-cycloalkyl, aryl, and R1,2 wherein R1,2 is R'-O-C(O)-R", R'-O-C(O)-O-R", R'-C(O)-O-R", wherein R' and R" are independently selected from the group consisting of C1-15-alkyl, C3-8-cycloalkyl and aryl;
• R13 is selected from the group consisting of H, halogen, NH2, and C1-6-alkyl; and
• R16 is selected from the group consisting of H, halogen, OH, O-C1-6-alkyl, and C1-6-alkyl.

L is typically selected from straight chain or branched optionally substituted C_1-10-alkyl, C_1-10-alkoxycarbonyl, C_1-10-alkylamine or C_1-10-alkoxy. In embodiments wherein L is a branched chain optionally substituted C_1-10-alkyl, C_1-10-alkylamine or C_1-10-alkoxy, there may be one or two acidic moieties A, namely L-A may be of the formula where X is selected from the group consisting of C and N and i, j, and k are independently selected from a whole number selected from the group consisting of 0-10, wherein the sum i + j+ k is typically less than 10; and one or both of the A groups is as defined above, the other being absent.

An exemplary embodiment of branched optionally substituted C_1-10-alkyl, C_1-10-alkoxycarbonyl, C_1-10-alkylamine or C_1-10-alkoxy is

In a most preferred embodiment of compounds of the formula IV-P, L-A is selected from the group consisting of a straight chain C_1-10-alkyl-CO₂H, a straight chain C_1-10-alkyl- C(O)-OR¹, a branched C_1-10-alkyl-CO₂H, a branched C_1-10-alkyl- C(O)-OR¹and di(C_1-10-alkoxycarbonyl)s of the formula C_1-10-alkyl-C(O)O-CH(C_1-10-alkyl)-OC(O)O-C_1-10-alkyl, C_1-10-alkyl-C(O)O-CH(C_1-10-alkyl)-C(O)O-C_1-10-alkyl, and C_1-10-alkyl-C(O)O-CH(C_1-10-alkyl)-OC(O)-C_1-10-alkyl.

As stated, the compounds of the invention are 5-HT modulators, typically 5-HT4 modulators. In a suitable embodiment, the compounds of the invention are 5-HT4 agonists. In a further suitable embodiment, the compounds of the invention are 5-HT4 antagonists. In a still further suitable embodiment of the invention, the compounds of the invention are partial agonists.

The subject invention provides novel compounds and compositions for the safe and effective treatment of gastroesophageal reflux and related conditions. These compositions possess potent activity in treating gastroesophageal reflux disease and substantially reduce adverse effects associated with the administration of 5-HT modulators. These adverse effects include diarrhea, abdominal cramping and elevations of blood pressure and heart rate.

The compounds of the invention are anticipated for the treatment of dyspepsia, gastroparesis, constipation, post-operative ileus, and intestinal pseudo-obstruction. Dyspepsia is a condition characterized by an impairment of the power or function of digestion that can arise as a symptom of a primary gastrointestinal dysfunction or as a complication due to other disorders such as appendicitis, gallbladder disturbances, or malnutrition. Gastroparesis is a paralysis of the stomach brought about by a motor abnormality in the stomach or as a complication of diseases such as diabetes, progressive systemic sclerosis, anorexia nervosa or myotonic dystrophy. Constipation is a condition characterized by infrequent or difficult evacuation of feces resulting from conditions such as lack of intestinal muscle tone or intestinal spasticity. Post-operative ileus is an obstruction in the intestine due to a disruption in muscle tone following surgery. Intestinal pseudo-obstruction is a condition characterized by constipation, colicky pain, and vomiting, but without evidence of physical obstruction.

An important aspect of the invention relates to the use of a compound of formula IV-P for the manufacture of a medicament for treating a cardiovascular disorder comprising administering a compound having a binding affinity to a 5-HT receptor with a pK_l of at least 5; ii) comprises at least one basic nitrogen atom; iii) comprises at least one acidic moiety with a pKa of no more than 6.4, or a salt or ester thereof. This method treating a cardiovascular disorder is done essentially free of CNS-related side effects. Typically, the compound is a compound of Formula IV-P having a binding pK_l for a 5-HT receptor of at least 5; and -A is an acidic moiety with a pKa of no more than 6.4 or an ester thereof.

In an exemplary embodiment of the use of a compound of formula IV-P for the manufacture of a medicament for treating a cardiovascular disorder, the disorder is selected from the group consisting of tachycardia, bradycardia, cardioexcitation, cardiodepression, arrhythmia, fibrillation, atrial fibrillation, Paroxysmal Supraventricular Tachycardia (PSVT), thromoembolisms and VTE..

A further aspect of the invention relates to a use of a compound of formula IV-P for the manufacture of a medicament for treating gastrointestinal disorders comprising administering a compound of the invention. In exemplary embodiments of this aspect of the invention, the gastrointestinal disorder is selected from the group consisting of irritial bowel syndrome, gastrointestinal hypomotility disorders such as gastroesophageal reflux (heartburn, mild oesophagitis); functional or nonulcer dyspensia; gastroparesis, nausea and vomiting; early satiety in the elderly; paraneoplastic of HIV-associated gastroparesis; drug-induced delays in gastric emptying and functional bowel obstructions, such as bowel obstructions caused by pancreatic cancer or drugs; and emesis.

A further aspect of the present invention includes a use of a compound of formula IV-P for the manufacture of a medicament for treating a condition caused by gastrointestinal motility dysfunction in a mammal which comprises administering to a mammal in need of treatment for gastrointestinal motility dysfunction, a therapeutically effective amount of a compound of the invention or a pharmaceutically compositions thereof. Conditions caused by gastrointestinal motility dysfunction include dyspepsia, gastroparesis, constipation, post-operative ileus, and intestinal pseudo-obstruction. Preferably, the mammal is a human. A further aspect of the invention relates to a use of a compound of formula IV-P for the manufacture of a medicament for treating lower urinary tract disorders, such as e.g. hyperactive bladder, comprising administering a compound of the invention.

The use of a compound of formula IV-P for the manufacture of a medicament for the treatment of primary or secondary hyperaldosteronism comprising administering a compound of the invention is a further aspect of the invention.

The person skilled in the art will appreciate that the compounds of the invention are applicable for use in the treatment of all diseases associated with peripheral 5-HT receptors. Thus, a further aspect of the invention relates to a use of a compound of formula IV-P for the manufacture of a medicament for treating a disease associated, at least in part, with a peripheral 5-HT receptor subgroup comprising administering a compound of the invention. Pharmacologically the active principle according to the invention has the advantage of facilitating absorption over the gastrointestinal membranes due to the relatively lipophilic moiety and/or nonionic moiety, such as an ester group. Subsequently to being absorbed through the biomembrane of the gastrointestinal tract the facilitating moiety is cleavaged from the active principle resulting in leaving an acid moiety on the active principle. The active principle comprising the acid moiety is then present in the blood circulation for systemic action. As a consequence of the residual acid moiety the access to the brain over the BBB is prevented.

Thus the active principle is in one aspect of the invention acting as a pro-drug facilitating the absorption over the biomembrane of the gastrointestinal tract. A further aspect of the invention relates to the systemically circulating drug that is provided with an moiety, such as an acid moiety, that prevents the absorption over the BBB.

In embodiments of the invention wherein known compounds are modified according to the invention, that is to say with an acidic moiety, the compounds of this invention are anticipated to have therapeutic properties similar to those of the unmodified parent compounds. Accordingly, dosage rates and routes of administration of the disclosed compounds are similar to those already used in the art and known to the skilled artisan (see, for example, Physicians' Desk Reference, 54th Ed., Medical Economics Company, Montvale, N.J., 2000).

Preferably, a daily dose range should be between about 1 mg to about 100 mg, in single or divided doses, while most preferably, a daily dose range should be between about 2 mg to about 75 mg, in single or divided doses. It is preferred that the doses are administered from 1 to 4 times a day. It may be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art.

A further aspect of the invention relates to a composition comprising a compound of the invention and a pharmaceutically acceptable excipient. The compounds of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E. W. Martin describes formulations which can be used in connection with the subject invention. In general, the compositions of the subject invention are formulated such that an effective amount of the bioactive compound(s) is combined with a suitable carrier in order to facilitate effective administration of the composition.

In one embodiment of the invention the active component is formulated in a pharmaceutical dosage unit. The dosage unit can be formulated for release of the active principle in the stomach. The dosage unit can be formulated for release of the active principle in the duodenum. The dosage unit can be formulated as a sustained release formulation, implying that the active principle is not immediately released. The release can be sustained to take place in the intestinal tract. In one embodiment the sustained release can take place in the small intestine. In a suitable embodiment, the sustained release takes place in the colon.

The dosage unit comprises pharmaceutically acceptable excipients. The dosage unit is typically a tablet or capsule. The dosage unit can be coated with an exipient known in the art for controlling the disintegration of the dosage unit. Such coating excipients comprises one or more of various polymers such as polymethacrylates, tributylesters, cellulose and modified celluloses, carboxymethylcellulose and salts thereof, natural and synthetic waxs, camaubawax, polyvinylpyrrolidone, sugar alcohols, starch and modified starch, gelatine, chitosan and shellac though is not limited hereto.

In still another embodiment the release is controlled in a manner that implies for a targeted release. The dosage unit formulated as a targeted release formulation can control the release to take place at the desired site of action. The release can be controlled in a manner where a burst dose of the active principle is initially released, followed by a secondary release at a later time point and/or at another site in the gastrointestinal tract. The release can be controlled by means of pH as the pH varies in the gastrointestinal tract. Alternatively, the release can be controlled by a dosage unit that is eroded in a time dependent manner. In one embodiment the dosage unit is formulated to have a prolonged release, by which is intended that the active principle is slowly released over a longer time period compared with that of an immediate release dosage unit.

The sustained or controlled release effect can be achieved by a coating or by a matrix type of dosage unit, as is well-known in the art.

In one embodiment the tablet is a matrix type of tablet. In another embodiment the tablet is coated.

In still another embodiment the active principle is formulated to give a fast onset of action. The fast onset of action can be achieved by formulating the dosage unit in formulations that implies absorption of the active principle transmucosally. In one aspect the active principle is formulated into a buccal formulation providing the active principle to buccal absorption. A buccal formulation comprises a tablet, a sublingual tablet, a buccal patch, a buccal spray, a chewing gum.

Administration routes like nasal, transdermal and buccal administration have special advantages when treating conditions implying nausea and/or vomiting.

Disclosed is also formulating the active principle into a formulation for nasal administration. By the nasal administration is intended a systemic action. Though administering the active principle nasally, the intention being to treat conditions peripherally and not for the active principle to cross the blood-brain-barrier. Nasal administration has the advantage of a fast onset of action. However, nasal administration is also known to provide direct access to the CNS, which is to be avoided when targetting peripheral 5-HT, particularly 5-HT4 receptors.

As can be realised by the artisan, the chemical characteristics of the active substance are such that the compound has one lipophilic site and one hydrophilic site of the molecule. The lipophilic part will tend to be readily absorbed into biological membranes. The hydrophilic part will in the circumstances that it is ionised not be readily absorbed through biological membranes. In the embodiment where the hydrophilic end comprises an acid moiety, the acid moiety will be non-ionised in an environment more acidic than the pKa value. In the embodiment where the hydrophilic site comprises a basic moiety the basic moiety will be unionised in an environment more basic than the pKa value.

In the embodiment where the hydrophilic site comprises a acid moiety the artisan will often prefer to formulate the active principle as the salt of the acid. This will also be the case in the event that the hydrophilic site comprises a basic moiety. Formulating the active principle as a salt can improve the solubility and the solubility rate. Pharmaceutical salts of basic active principles are salts of strong or medium strong acids; hydrochloric acid; sulphate; phosphate or weak acids like tartrate; acetate. Salts of weak acids are typically salts of sodium; potassium; or calcium.

Disclosed is also that the active principle is formulated in a formulation comprising lipid excipients. The lipid excipients are providing compositions wherein the active principle of the invention is encapsulated. By formulating the active principle in a lipid formulation the absorption over the biological membrane of the intestinal tract is facilitated. Once absorbed systemically the active principle will be released from the lipid formulation. The active principle will target itself to the desired site of action while being hindered to pass the BBB due to the character, such as an ionic character, of the hydrophilic moiety. In preparing the lipid formulations, lipid components including neutral lipids, positively-charged lipids, such as sphingosine and ceramide, negatively-charged lipids, amphoteric lipids such as phospholipids, and cholesterol are advantageously used. As defined herein, the "lipid component" of the compositions of the invention are intended to encompass a single species of lipid (such as a particular phospholipid) or combinations of such lipids, either of one type such as combinations of phospholipds (for example, phophatidylcholine plus phosphatidyl enthanolamine) or of different types (such as a phospholipid plus a charged lipid or a neutral lipid). Combinations comprising a multiplicity of different lipid types are also advantageously encompassed by the proliposomal compositions of the invention.

Chemical substances crosses the blood brain barrier (BBB) via differents routes such as opening of tight junctions, increased pinocytosis, decreased membrane rigidity, by pore formation or other mechanisms. There are four basic mechanisms by which solute molecules move across membranes. First is by simple diffusion, which proceeds from low to high concentrations. Secondly is by facilitated diffusion, a form of carrier-mediated endocytosis, in which solute molecules bind to specific membrane protein carriers, also from low to high concentration. Thirdly is simple diffusion through an aqueous channel, formed within the membrane. Fourthly is by active transport through a protein carrier with a specific binding site that undergoes a change in affinity. Active transport requires ATP hydrolysis and conducts movement against the concentration gradient. Movement between cells is referred to as paracellular diffusion. The BBB has a number of highly selective mechanisms for transport of nutrients into the brain.

Diffusion of substances into the brain can be divided into paracellular (i.e. between cells) and transcellular_(i.e. across cells) diffusion, both of which are non-saturable and non-competitive. Paracellular diffusion does not occur to any great extent at the BBB, due to the "tight junctions". In the case of transcellular diffusion, the general rule is the higher the lipophilicity of a substance, the greater the diffusion into the brain. Another general rule is the smaller size of the molecule the greater the diffusion into the brain. In the present invention the active principle is chemically modified comprising a polar site that prevents the active principle from being transported into the brain. Further the transportation through the BBB can be prevented by modifying the active principle with a bulky moiety as the BBB is most permeable towards small, lipid-soluble molecules.

The compositions of the subject invention include compositions such as suspensions, solutions and elixirs; aerosols; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrating agents, in the case of oral solid preparations (such as powders, capsules, and tablets) with the oral solid preparations being preferred over the oral liquid preparations. A preferred oral solid preparation is capsules. The most preferred oral solid preparation is tablets.

Further, acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules. A solid carrier can be one or more substances which may act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.

The pharmaceutical compositions may be subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, such as packeted tablets, capsules, and powders in paper or plastic containers or in vials or ampules. Also, the unit dosage can be a liquid based preparation or formulated to be incorporated into solid food products, chewing gum, or lozenge.

Any suitable route of administration may be employed for providing the patient with an effective dosage. For example, oral, rectal, parenteral (subcutaneous, intramuscular, intravenous), transdermal forms of administration may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules and patches.

When formulating the active principle into a transdermal formulation special care should be taken to select a suitable enhancer.

One aspect of the invention relates to formulating the active principle into a dosage unit that displays a stability that implies a shelf-life of the dosage units of 5 years; such as 4 years; such as 3 years; such as 2 years. In one embodiment the stability during the shelf-life is obtained by means of the packaging material. In one embodiment packaging material displays a high resistance towards water vapour.

One embodiment of the invention provides a method of treating gastroesophageal reflux disease in a mammal, while substantially reducing the concomitant adverse effects associated with the administration of the compound devoid of the acidic moiety, comprising administering to a human in need of such treatment, a therapeutically effective amount of a compound of the invention.

Yet another embodiment of the present invention provides a method of eliciting an anti-emetic effect in a mammal, while substantially reducing the adverse effects associated with the administration of the compound devoid of the acidic moiety, comprising administering to a mammal in need of such anti-emetic therapy, a therapeutically effective amount a compound of the invention.

The invention will hereinafter be described by way of the following Examples.

Table 1 in the Examples demonstrate the proof of concept that a representative sample of all of the compounds of the invention have a good binding affinity for the serotonin recepetor.

EXAMPLES Synthetic Chemistry Reference example 1 Preparation of intermediate 2,2,2-trichloroethyl 4-bromobutyrate

A stirred solution of 4-bromobutyric acid (3.34 g, 20.0 mmol) in toluene (50 ml) was added 2,2,2-trichloroethanol (14.94 g, 0.10 mol) and p-toluenesulfonic acid monohydrate (7.60 g, 40.0 mmol) and refluxed with a Dean-Stark trap attached for 6 h. Water was removed continuously. The reaction mixture was cooled to room temperature and concentrated in vacuo. The mixture was added CH₂Cl₂ (75 ml) and washed with H₂O (3 x 25 ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to leave an oil. The residue was distilled to leave the title compound as a colourless oil (4.77 g, 79.9 %) (bp 100 °C at 0.5 mmHg). ¹H-NMR (300 MHz, CDCl₃):δ 4.74 (s, 2H), 3.48 (t, 2 H), 2.65 (t, 2 H), 2.21-2.13 (m, 2 H)

Reference example 2 Preparation of intermediate 4-(4-hydroxymethyl-piperidin-1-yl) butyric acid 2,2,2-trichloroethylester

A stirred solution of 4-piperidinemethanol (1.72 g, 15.0 mmol) in acetone (100 ml) was added K₂CO₃ (4,14 g, 30. mmol) and 2,2,2-trichloroethyl 4-bromobutyrate (4.47 g, 15.0 mmol) and heated under reflux for 3 h. The reaction mixture was cooled to room temperature, filtered and the filtrate concentrated in vacuo. The residue was added CH₂Cl₂ (75 ml) and washed with brine (25 ml) and H₂O (2 x 25 ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to leave the title compound as a viscous oil (4.70 g, 94.1 %). ¹H-NMR (300 MHz, CDCl₃):δ 4.74 (s, 2H), 3.50 (d, 2 H), 2.92 (d, 2 H), 2.52-2.35 (m, 4 H), 1.97-1.70 (m, 7 H),1.52-1.45 (m, 1 H), 1.32-1.23 (m, 2 H)

Reference example 3 Synthesis of 1H-indole-3-carboxylic acid 1-[3-(2,2,2-trichloroethyl-ethoxycarbonyl)-propyl]-piperidine-4-ylmethyl ester

A suspension of indole-3-carboxylic acid (2.90 g, 18.0 mmol) in CH₂Cl₂ (75 ml) was treated with oxalyl chloride (1.84 ml, 20.7 mmol) and DMF (1 drop) and the mixture stirred at room temperature for 2 h, then concentrated in vacuo to leave the acid chloride as a yellow solid. This was dissolved in a mixture of CH₂Cl₂ (30 ml) and THF (10 ml) and added dropwise (30 min) to a stirred solution of 4-(4-hydroxymethyl-piperidin-1-yl) butyric acid 2,2,2-trichloroethyl ester (from example 2) (4.98 g, 15.0 mmol) and NEt₃ (1.82 g, 18.0 mmol) in CH₂Cl₂ (30 ml). The reaction mixture was stirred at room temperature overnight, treated with an aqueous satd. NaCl solution (25 ml) and 10 % aqueous NaHCO₃ solution (25 ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to a brown viscous oil. The residue was separated with flash chromatography (SiO₂, EtOAc). The product was obtained as a pale yellow solid (1.83 g, 25.6 %). Conversion to the hydrochloride salt was effected using etheral HCl. ¹H-NMR (300 MHz, CDCl₃):δ 9.02 (br s, 1 H), 8.22-8.18 (m,1 H), 7.92 (d, 1 H), 7.48-7.41 (m, 1 H), 7.35-7.28 (m, 2 H), 4.77 (s, 2 H), 4.24 (d, 2 H) 3.03 (d, 2 H), 2.59-2.44 (q, 5 H), 2.13-1.85 (m, 7 H), 1.60-1.43 (m, 2 H) ¹³C-NMR (75 MHz, CDCl₃): δ 171.7, 165.5, 136.2, 131.5, 125.7, 122.8, 121.7, 121.0, 111.7, 108.0, 94.8, 73.7, 67.9, 57.5, 53.1, 35.4, 31.7, 28.8, 21.8 MS (ES): 477.1 [M + H] ⁺

Reference example 4 Synthesis of 1H-indole-3-carboxylic acid 1-(3-carboxy-propyl)-piperidin-4-ylmethyl ester

1H-indole-3-carboxylic acid 1-[3-(2,2,2-trichloroethyl-ethoxycarbonyl)-propyl] piperidine-4-ylmethyl ester (0.48 g, 1.0 mmol) was dissolved in a mixture of THF (25 ml) and aqueous 1 M KH₂PO₄ (5 ml). Zn-powder (0.66 g, 10.0 mmol) was added and the resulting mixture stirred at room temperature for 24 h. The reaction mixture was filtered through a pad of kiselguhr and the filtrate evaporated in vacuo. The residue was separated with flash chromatography (SiO₂, EtOAc/MeOH (2 : 1)). The expected product was obtained as a white solid (0.29 g, 84.2 %). Conversion to the hydrochloride salt was effected using etheral HCl. ¹H-NMR (300 MHz, DMSO): δ 11.98 (s, 1 H), 8.08-7.97 (m, 2 H), 7.47 (d, 1 H), 7.20-7.17 (m 2 H), 4.11 (d, 2 H), 2.96 (d, 2 H), 2.50-2.37 (m, 4 H), 2.05 (t, 2 H), 1.77-1.66 (m, 6 H), 1.42-1.35 (m, 2 H) ¹³C-NMR (75 MHz, DMSO):δ 171.7, 165.5, 136.2, 131.5, 125.7, 122.8, 121.7, 121.0, 111.7, 108.0, 94.8, 73.7, 67.9, 57.5, 53.1, 35.4, 31.7, 28.8, 21.8 MS (ES): 345.2 [M + H] ⁺

Reference example 5 Preparation of intermediate N-(1-benzylpiperidin-4-yl)napth-1-yl carboxamide

A stirred suspension of 1-napthoic acid (8.61 g, 0.050 mol) in CH₂Cl₂ (150 ml) was added SOCl₂ (23.79 g,0.20 mol) and the mixture heated under reflux for 4 h. The mixture was evaporated in vacuo to leave the acid chloride as a solid material. This was dissolved in CH₂Cl₂ (150 ml ) and added dropwise to a stirred solution of 4-amino-1-benzylpiperidine (9.51 g, 0.050 mol) and NEt₃ (5.06 g, 0.05 mol) in CH₂Cl₂ (100ml) at 0 °C. The mixture was stirred to room temperature for 24 h and washed with H₂0 (3 x 75 ml) The organic layer was dried over Na₂SO₄ and evaporated in vacuo to a solid material. This was recrystallized from ethanol/water (40/60) to leave the product as a white solid (7.8 g, 45.3 %). ¹H-NMR (300 MHz, CDCl₃): δ 8.32-8.27 (m, 1 H), 7.90 (t, 2 H), 7.57-7.30 (m, 9 H), 6.17 (d, 2 H), 4.17-4.06 (m, 1 H), 3.55 (s, 2H), 2.88 (d, 2 H), 2.27-2.05 (m, 4 H), 1.69-1.50 (m, 2 H)

Reference example 6 Preparation of intermediate N-(piperidin-4-yl)napth-1-yl carboxamide hydrochloride

A solution of N-(1-benzylpiperldin-4-yl)napth-1-yl carboxamide (1.38 g, 4.0 mmol) in dry CH₂Cl₂ (15 ml) was cooled to 0 °C and added α-chloroethyl chloroformate (1.14 g, 8.0 mmol) and stirred for 30 minutes. The mixture was evaporated in vacuo, added MeOH (15 ml) and heated under reflux for 1 h. The reaction mixture was evaporated in vacuo and the residue recrystallized from acetonitrile to give the product as a white powder (1.01 g, 86.8 %). ¹H-NMR (300 MHz, CDCl₃):δ 8.32-8.27 (m, 1 H), 7.90 (t, 2 H), 7.57-7.30 (m, 9 H), 6.17 (d, 2 H), 4.17-4.06 (m, 1 H), 3.55 (s, 2H), 2.88 (d, 2 H), 2.27-2.05 (m, 4 H), 1.69-1.50 (m, 2 H)

Reference Example 7 Alkylation of N-(piperidin-4-yl)napth-1-yl carboxamide hydrochloride with 2,2,2-trichloroethyl 4-bromobutyrate

A stirred suspension of N-(piperidin-4-yl)napth-1-yl carboxamide hydrochloride (0.58 g, 2.0 mmol) in acetone (20 ml) was added K₂CO₃ (1.10 g, 8.0 mmol) and 2,2,2-trichloroethyl 4-bromobutyrate (0.89 g, 3.0 mmol) and heated under reflux for 24 h. The mixture was cooled to room temperature and filtered. The filtrate was evaporated in vacuo and the residue added CH₂Cl₂ (50 ml) and washed with H₂O (3 x 25 ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to an oil. The oil was separated with flash chromatography (SiO₂, EtOAc/MeOH (1 : 1)) to give the product as a white solid (0.87 g, 92.2 %). ¹H-NMR (200 MHz, CDCl₃):δ 8.26-8.21 (m, 1 H), 7.83 (t, 2 H), 7.56-7.37 (m, 4 H), 5.89 (d, 2 H), 4.71 (s, 2 H), 4.17-4.06 (m, 1 H), 2.84 (d, 2 H), 2.49 (t, 2 H), 2.39 (t, 2 H), 2.21-2.11 (m, 4 H), 1.93-1.82 (p, 2 H), 1.62-1.49 (m, 2 H) ¹³C-NMR (75 MHz, CDCl₃):δ 173.0, 168.4, 134.1, 133.0, 129.8, 129.4, 127.7, 126.4, 125.6, 124.8, 124.2, 124.1, 73. 8, 60.0, 57.0, 52.0, 46.5, 31.6, 22.0, 14.1 MS (ES): 494.2 [M + Na]⁺

Reference example 8 Hydrolysis of the trichloroethyl ester from example 7

Following the procedure outlined in example 4, the trichloroethyl ester from example 7 (0.67 g, 1.4 mmol) was converted to the title compound as a white solid (0.38 g, 79.6 %). ¹H-NMR (200 MHz, DMSO- _d6 ): δ 8.50 (d, 2 H), 8.39-8.18 (m, 1 H), 8.04-7.96 (m, 2 H), 7.62-7.50 (m, 4 H),3.91 (br s, 1 H), 2.93 (d, 2 H), 2.37 (t, 2 H), 2.23-2.06 (m, 4 H), 1.92 (d, 2 H), 1.72-1.57 (m, 4 H) ¹³C-NMR (50 MHz, DMSO- _d6 ): δ 167.9, 135.1, 133.0, 129.7, 129.5, 128.1, 126.6, 126.1, 125.3, 125.0, 124.9, 57.4, 52.0, 46.6, 38.6, 33.5, 31.2, 22.2 MS (ES): 363.1 [M + Na]⁺

Reference example 9 Alkylation of N-(piperidin-4-yl)napth-1-yl carboxamide hydrochloride with ethyl 4-bromobutyrate

Following the procedure outlined in example 7, N-(piperidin-4-yl)napth-1-yl carboxamide hydrochloride 6 (0.58 g, 2.0 mmol) was converted to the title compound as a white solid (0.67 g, 91.4 %). ¹H-NMR (300 MHz, CDCl₃):δ 8.24 (d, 1 H), 7.89-7.82 (m, 2 H), 7.54-7.49 (m, 3 H), 7.40 (t, 1 H), 6.07 (d, 2 H), 4.14-4.07 (m, 3 H), 2.84 (d, 2 H), 2.36-2.28 (m, 4 H), 2.13-2.04 (m, 4 H), 1.81-1.76 (p, 2 H), 1.55-1.51 (m, 2 H), 1.26 (t, 3 H) ¹³C-NMR (75 MHz, CDCl₃):δ 173.3, 168.7, 134.5, 133.4, 130.2, 129.8, 128.1, 126.8, 126.1, 125.1, 124.6, 124.5, 60.1, 57.4, 52.1, 46.9, 32.0, 22.2, 14.0 MS (ES): 391.2 [M + Na]⁺

Reference example 10 Preparation of intermediate (piperidin-4-yl)ethylcarboxylate hydrochloride

To a stirred solution of isonipectoic acid (12.9 g, 0.10 mol) in absolute ethanol (200 ml) was cooled to 0 °C and SOCl₂ (47.5 g, 0.40 mol) dropwise added. The mixture was stirred to room temperature and heated to reflux for 3 h. The reaction mixture was evaporated in vacuo and the residue dissolved in a 10 % aqueous solution of NaOH (250 ml). The aqueous solution was extracted with CH₂Cl₂ (3 x 100 ml). The organic extracts was dried over NaSO₄, filtered and evaporated in vacuo. The residue was dissolved in dry ethanol and HCl bubbled into the solution to give the hydrochloride precipitate. The residue was recrystallized from absolute ethanol to give the product as a white solid (17.46 g, 90.2 %). ¹H-NMR (300 MHz, CDCl₃): δ 9.40 (br s, 2 H), 4.09-4.02 (q, 2 H), 3.30 (d, 2 H), 3.01-2.95 (m, 2 H), 2.56- 2.47 (m, 1 H), 2.14-1.95 (m, 4 H), 1.30 (t, 3 H)

Reference example 11 Preparation of intermediate (1-benzylpiperidin-4-yl)ethylcarboxylate hydrochloride

A suspension of (piperidin-4-yl)ethylcarboxylate hydrochloride (8.6 g, 44.4 mmol) and K₂CO₃ (24.5 g, 0.17 mol) in acetone (200 ml) was added benzylbromide (9.11 g, 53.3 mmol) and heated to reflux for 12 h. The solvent was evaporated in vacuo and the residue added H₂O (200 ml). The aqueous layer was extracted with Et₂O (3 x 100 ml) and the organic extracts dried over Na₂SO₄, filtered and evaporated in vacuo. The residue was dissolved in acetone and HCl bubbled into the solution to give the hydrochloride precipitate. The precipitate was filtered, dried and recrystallized from acetone to give the expected product as a white solid (11.03 g, 87.6 %). ¹H-NMR (300 MHz, DMSO- _d6 ):δ 11.48 (br s, 1 H), 7.67 (s, 2 H), 7.42 (s, 3 H), 4.30-4.25 (m, 2 H), 4.11-4.01 (m, 2 H), 3.28 (d, 2 H), 2.97-2.84 (m, 2 H), 2.15-1.99 (m, 4 H) 1.15 (t, 3 H)

Reference example 12 Preparation of intermediate 1-[(1-benzylpiperidin-4-yl)methanol

A suspension of LiAlH₄ (1.52 g, 40.0 mmol) in dry THF (30 ml) was stirred at 0 °C and dropwise added a solution of 1-benzylpiperidin-4-yl)ethylcarboxylate hydrochloride (2.47 g, 10.0 mmol) in dry THF (50 ml). The obtained mixture was heated under reflux for 4 h and then cooled to room temperature. EtOAc (200 ml), water (40 ml), and a 2 N aqueous solution of NaOH (10 ml) were added. The obtained mineral precipitate was filtered through a pad of kiselguhr, the filtrate evaporated in vacuo and water (50 ml) added to the residue. The aqueous layer was extracted with CH₂Cl₂ (3 x 50 ml) and the organic extracts combined and dried over Na₂SO₄, filtered and evaporated in vacuo to give the product as a colourless oil (1.75 g, 85.6 %). ¹H-NMR (200 MHz, CDCl₃):δ 7.40-7.26 (m, 5 H), 3.53-3.46 (m, 4 H), 2.94 (d, 2 H), 2.61 (br s, 1 H), 2.00 (t, 2 H), 1.75 (d, 2 H), 1.48-1.26 (m, 3 H)

Reference example 13 Synthesis of 1-[(1-benzylpiperidin-4-yl)carboxymethyl]napthalene

A stirred solution of (1-benzylpiperidin-4-yl)methanol (2.79 g, 13.7 mmol) and NEt₃ (1.65 g, 16.3 mmol) in CH₂Cl₂ (50 ml) was cooled to 0 °C and dropwise added a solution of napthoyl chloride (prepared as in example 7) (3.11 g, 16.3 mmol) dissolved in CH₂Cl₂/THF (1 :1 , 50 ml). The resulting mixture was stirred to room temperature overnight, evaporated in vacuo and the residue added EtOAc (100 ml). The organic layer was washed with water (50 ml), brine (50 ml) and water (50 ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to give an oil. The oil was separated with flash chromatography (SiO₂, EtOAc) to give the expected product as a yellow oil (3.12 g, 63.3 %). ¹H-NMR (300 MHz, CDCl₃):δ 8.94 (d, 1 H), 8.22-8.19 (m, 1 H), 8.04 (d, 1 H), 7.90 (d, 1 H), 7.64-7.49 (m, 3 H), 7.49-7.25 (m, 5 H), 4.30 (d, 2 H), 3.52 (s, 2 H), 2.98 (d, 2 H), 2.09-2.00 (m, 2 H), 1.93-1.83 (m, 3 H), 1.57-1.45 (m, 2 H) MS (ES): 360.1 [M + H] ⁺

Reference example 14 Preparation of intermediate 1-[(piperidin-4-yl)methyloxycarbonyl]napthalene hydrochloride

Following the procedure outlined in Example 6, 1-[(1-benzylpiperidin-4-yl)carboxymethyl]napthalene (1.69 g, 4.70 mmol) was converted to the title compound as a yellow solid (1.07 g, 74.5 %). ¹H-NMR (300 MHz, CDCl₃):δ 9.60 (br s, 2 H), 8.86 (d, 1 H), 8.17 (d, 1 H), 8.00 (d, 1 H), 7.85 (d, 1 H), 7.61-7.34 (m, 3 H), 4.28 (d, 2 H), 3.55 (d, 2 H), 2.89 (d, 2 H), 2.04-1.68 (m, 5 H)

Reference example 15 Alkylation of 1-[(piperidin-4-yl)methyloxycarbonyl]napthalene hydrochloride with 2,2,2-trichloroethyl 4-bromobutyrate

Following the procedure outlined in Example 7, 1-[(piperldin-4-yl) methyloxycarbonyl]napthalene hydrochloride (0,30 g, 1.0 mmol) was converted to the title compound as a white solid (0.43 g, 89.5 %). ¹H-NMR (300 MHz, CDCl₃):δ 8.89 (d, 1 H), 8.18-8.14 (m, 1 H), 8.00 (d, 1 H), 7.88-7.84 (m, 1 H), 7.63-7.43 (m, 3 H), 4.72 (s, 2 H), 4.24 (d, 2 H), 2.93 (d, 2 H), 2.49 (t, 2 H), 2.38 (t, 2 H), 1.96-1.79 (m, 8 H), 1.46-1.27 (m, 2 H) ¹³C-NMR (75 MHz, CDCl₃):δ 171.9, 167.5, 133.8, 133.2, 131.3, 130.0, 128.5, 127.7, 127.2, 126.1, 125.7, 124.4, 95.0, 73.9, 69.3, 60.3, 57.7, 53.3, 35.5, 31.9, 29.1, 22.1, 21.0, 14.1 MS (ES): 487.1 [M + H]⁺

Reference example 16 Alkylation of 1-[(piperidin-4-yl)methyloxycarbonyl]napthalene hydrochloride with ethyl 4-bromobutyrate

Following the procedure outlined in example 7, 1-[(piperidin-4-yl)methyloxycarbonyl]napthalene hydrochloride (0,39 g, 1.27 mmol) was converted to the title compound as a yellow oil. The oil was dissolved in Et₂O and HCl bubbled into the solution to give the hydrochloride precipitate. The precipitate was filtered off, dried and recrystallized from acetonitrile to leave the hydrochloride salt (0.31 g, 73.8 %). ¹H-NMR (300 MHz, DMSO- _d6 ):δ 10.7 (br s, 1 H), 8.76 (d, 1 H), 8.23 (t, 2 H), 8.04 (d, 1 H), 7.71-7.59 (m, 3 H), 4.28 (d, 2 H), 4.11-4.03 (q, 2 H), 3.49 (d, 2 H), 3.51-2.96 (m, 5 H), 2.41 (t, 2 H), 2.00-1.83 (m, 7 H), 1.19 (t, 3 H) ¹³C-NMR (75 MHz, DMSO- _d6 ):δ 172.8, 167.4, 134.4, 134.2, 131.3, 131.0, 129.6, 128.8, 127.3, 127.2, 125.8, 125.7, 68.6, 60.9, 56.0, 52.0, 33.7, 31.4, 26.4, 19.6, 14.9 MS (ES): 406.2 [M + Na]⁺

Reference example 17 Hydrolysis of the trichloroethyl ester from example 16

Following the procedure outlined in example 4, the trichloroethyl ester from example 16 (0.43 g, 0.88 mmol) was converted to the title compound as a white solid (0.25 g, 79.9 %). ¹H-NMR (300 MHz, DMSO- _d6 ):δ 10.11, 8.75 (d, 1 H), 8.15 (t, 2 H), 8.01 (d, 1 H), 7.68-7.57 (m, 3 H), 4.20 (d, 2 H), 2.90 (d, 2 H), 2.33 (t, 2 H), 2.20 (t, 2 H), 2.03-1.92 (t, 2 H), 1.75-1.60 (m, 5 H), 1.45-1.30 (m, 2 H) ¹³C-NMR (75 MHz, DMSO- _d6 ):δ 175.0, 166.6, 133.4, 133.3, 130.4, 129.8, 128.6, 127.8, 126.7, 126.3, 125.0, 124.8, 68.7, 57.4, 52.3, 34.7, 33.2, 28.0, 21.6 MS (ES): 378.1 [M + Na] ⁺

Reference example 18 Alkylation of 1-[(piperidin-4-yl)methyloxycarbonyl]napthalene hydrochloride with diethyl 2-bromoethylphosphonate

Following the procedure outlined in example 7, 1-[(piperidin-4 yl)methyloxycarbonyl]-napthalene hydrochloride (0,63 g, 2.06 mmol) was converted to the title compound as a yellow oil. The oil was dissolved in Et₂O and HCl bubbled into the solution to give a white precipitate. The precipitate was filtered off, dried and recrystallized from acetonitrile to leave the hydrochloride salt (0.37 g, 37.9 %) ¹H-NMR (300 MHz, DMSO- _d6 ):δ 11.0 (br s, 1 H), 8.78 (d, 1 H), 8.28-8.21 (m, 2 H), 8.09-8.05 (m, 1H), 4.30 (d, 2 H), 4.14-4.03 (q, 4 H), 3.63-3.52 (m, 2 H), 3.20-2.95 (m, 5 H), 2.48-2.37 (m, 1 H), 2.02-1.81 (m, 5 H), 1.28 (t, 6 H) ¹³C-NMR (75 MHz, DMSO- _d6 )δ 166.5, 133.4, 133.3, 130.4, 130.1, 128.7, 127.9, 126.4, 126.3, 124.9, 124.8, 67.7, 61.6, 61.5, 50.7, 50.0, 32.9, 26.5, 21.1, 19.3, 16.2, 16.1 MS (ES): 456.2 [M + N] ⁺

Reference example 19 Preparation of intermediate N-(1-benzylpiperidin-4-yl)-indazole-3-carboxamide

A stirred solution of 1-H-indazole-3-carboxylic acid (8.11 g, 50.0 mmol) in dry DMF (140 ml) under argon atmosphere was added CDI (8.92 g, 55 mmol) and heated at 60 °C for 2 h. The mixture was cooled to room temperature, dropwise added 4-amino-1-benzylpiperidine (9.51 g, 50.0 mmol) previously dissolved in DMF (20 ml). The mixture was heated at 60 °C for 2 h, cooled to room temperature and the solvent evaporated In vacuo. The residue was added CH₂Cl₂ (250 ml) and the organic layer washed with H₂O (100 ml), 1 N aqueous NaOH (100 ml), H₂O (100 ml) and brine (100 ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo. The residue was recrystalllized from EtOH to leave the expected product as a white solid (14.23 g, 85.1 %). ¹H-NMR (200 MHz, DMSO- _d6 ): δ 13.59, 8.20 (t, 2 H), 7.61 (t, 1 H), 7.38-7.21 (m, 7 H), 3.95-3.87 (m, 1 H), 3.49 (s, 2 H), 2.80 (d, 2 H), 2.04 (t, 2 H), 1.78-1.67 (4 H)

Reference example 20 Preparation of intermediate N-(1-benzylpiperidin-4-yl)-1-isopropylindazole-3-carboxamide

A solution of N-(1-benzylpiperidin-4-yl)-indazole-3-carboxamide (3.34 g, 10.0 mmol) in dry DMF (70 ml) under argon atmosphere was added sodium hydride (0.25 g, 10.0 mmol) and stirred at room temperature for 3 h. The mixture was added isopropylbromide (1.37 g, 11.0 mmol) and stirred for additional 24h. The reaction mixture was evaporated in vacuo and the residue added EtOAc (100 ml). The organic layer was washed with brine (50 ml) and H₂O (2 x 50ml). The organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to leave an oil that solidified upon standing. The oil was separated with flash chromatography (SiO₂, Et2O/Hexane (2 : 1) to leave the product as a solid (1.22 g, 32.7 %). ¹H-NMR (200 MHz, CDCl₃): δ 8.40 (d, 1 H), 7.44-7.27 (m, 8 H), 6.95 (d, 1 H), 4.92-4.83 (p, 1 H); 4.05-3.95 (m, 1 H), 3.55 (s, 1 H), 2.91 (d, 2 H), 2.21 (t, 2 H), 2.08 (d, 2 H), 1.71-1.60 (m, 8 H)

Reference example 21 Preparation of intermediate N-(1-piperidin-4-yl)-1-isopropylindazole-3-carboxamide hydrochloride

Following the procedure outlined in Example 6, N-(1-benzylpiperidin-4-yl)-1-isopropylindazole-3-carboxamide (1.77 g, 4.28 mmol) was converted to the title compound as a white solid (1.26 g, 91.1 %). ¹H-NMR (200 MHz, CDCl₃): δ 9.80 (br s, 1 H), 9.68 (br s, 1 H), 8.34 (d, 1 H), 7.49-7.31 (m, 2 H), 7.28-7.26. (m, 1 H), 7.05 (d, 1 H), 4.94-4.85 (m, 1 H), 4.35-4.32 (m, 1 H), 3.62 (d, 2 H), 3.15-3.04 (m, 2 H), 2.36-2.32 (m, 2 H), 2.18-2.08 (m, 3 H), 1.65 (d, 6 H)

Reference example 22 Alkylation of N-(1-piperidin-4-yl)-1-isopropylindazole-3-carboxamide hydrochloride with ethyl 4-bromobutyrate.

Following the procedure outlined in Example 7, N-(1-piperidin-4-yl)-1-isopropylindazole-3-carboxamide hydrochloride (0.32 g, 1.0 mmol) was converted to the title compound as a colourless oil (0.37 g, 93.7 %). ¹H-NMR (300 MHz, CDCl₃): δ 8.38 (d, 1 H), 7.43-7.37 (m, 3 H), 7.26 (d, 1 H), 4.90-4.82 (m, 1 H), 4.19-4.08 (m, 1 H), 2.91 (d, 2 H), 2.43-2.31 (m, 4 H), 2.19-2.03 (m, 5 H), 1.87-1.58 (m, 11 H), 1.26 (t, 3 H) ¹³C-NMR (75 MHz, CDCl₃): δ173.3, 162.0, 139.8, 136.7, 126.2, 122.8, 122.7, 122.2, 109.1, 60.1, 57.4, 52.3, 50.7, 46.0, 32.2, 32.1, 22.2, 21.9, 14.1 MS (ES): 423.1 [M + Na⁺] Conversion to the hydrochloride salt was effected using ethereal HCl.

Reference example 23 Alkylation of N-(1-piperldln-4-yl)-1-isopropylindazole-3-carboxamide hydrochloride with 2,2,2-trichloroethyl 4-bromobutyrate

A stirred suspension of N-(1-piperidin-4-yl)-1-isopropylindazole-3-carboxamide hydrochloride (0,32 g, 1.0 mmol) and K₂CO₃ (0,55 g, 4.0 mmol) in acetone (15 ml) was added 2,2,2-trichloroethyl 4-bromobutyrate (0.45 g, 1.5 mmol) and heated under reflux for 12 h. The mixture was cooled to room temperature, filtered and the filtrate evaporated in vacuo. The residue was added EtOAc (30 ml) and the organic layer washed with H₂O (15 ml), brine (15 ml) and H₂O (15 ml). The organic layer was dried over Na₂SO₄, filtered and the solvent evaporated in vacuo to leave an oil. The oil was dissolved in acetone and dropwise added 1.0 M HCl in Et₂O to give a white precipitate. The precipitate was filtered off, dried and recrystallized from acetone to leave the hydrochloride salt as a white powder (0.47 g, 87.0 %). ¹H-NMR (300 MHz, CDCl): δ 12.44 (br s, 1 H), 8.29 (d, 1 H), 7.47-7.37 (m, 2 H), 7.25 (t, 1 H), 7,16 (d, 1 H), 4.89-4.85 (m, 1 H), 4.76 (s, 2 H), 4.19-4.08 (m, 1 H), 3.71 (d, 2H), 3.14-2.69 (m, 4 H), 2.34-2.03 (m, 8 H), 1.61 (d, 6 H) ¹³C-NMR (75 MHz, CDCl₃):δ 170.3, 162.4, 139.9, 135.9, 126.4, 122.8, 122.6, 122.3, 109.3, 94.6, 74.0, 56.2, 52.2, 50.9, 43.8, 30.7, 29.1, 22.0, 18.9, 15.2 MS (ES): 526.2 [M + Na]⁺

Reference example 24 Hydrolysis of the trichloroethyl ester from Example 23

Following the example outlined in example 4, the trichloroethyl ester from example 23 (0.37 g, 0.69 mmol) was converted to the title compound as a white solid (0.20 g, 77.9 %). ¹H-NMR (300 MHz, CD₃OD):δ 8.21 (d, 1 H), 7.61 (d, 1 H), 7.42-7-37 (m, 1 H), 7.24 (t, 1 H), 5.01-4.93 (m, 1 H), 4.24-4.19 (m, 1 H), 3.51 (d, 2 H), 3.05-2.91 (m, 4 H), 2.45 (t, 2 H), 2.18-2.14 (m, 2 H), 1.99-1.88 (m, 4 H), 1.57 (d, 6 H) ¹³C-NMR (75 MHz, CD₃OD):δ 181.1, 164.5, 141.4, 137.6, 127.6, 124.0, 123.7, 123.0, 110.9, 59.1, 52.6, 52.1, 45.6, 37.4, 30.5, 22.3, 21.7 MS (ES): 395.1 [M + Na] ⁺

Reference example 25 Preparation of intermediate 4-bromomethyl benzoic acid 2,2,2-trichloroethyl ester

A solution of 2,2,2-trichloroethanol (2.46 g, 16.5 mmol) and NEt₃ (1.67 g, 16.5 mmol) in CH₂Cl₂ (40 ml) at 0 °C was dropwise added 4-bromomethyl benzoylbromide (4.17 g, 15.0 mmol) in CH₂Cl₂ (20 ml) and stirred to room temperature overnight. The reaction mixture was added H₂O (20 ml) and the organic layer separated. The organic layer was washed with aqueous 1 M HCl (20 ml) and H₂O (20 ml). The organic layer was dried over Na₂SO₄, filtered and the solvent evaporated in vacuo to leave the expected product as a white solid. The product was used directly in the next step without any further purification (to be filled in). ¹H-NMR (300 MHz, CDCl₃): δ 7.86 (d d, 4 H), 5.00 (s, 2 H), 4.54 (s, 2 H)

Reference example 26 Alkylation of N-(1-piperidin-4-yl)-1-isopropylindazole-3-carboxamide hydrochloride with 4-bromomethyl benzoic acid 2,2,2-trichloroethyl ester

Following the procedure outlined in Example 7, N-(1-piperidin-4-yl)-1-isopropylindazole-3-carboxamide hydrochloride (0,41 g, 1.3 mmol) was converted to the title compound as a colourless oil (0.61 g, 85.2 %). ¹H-NMR (300 MHz, CDCl₃):δ 8.38 (d, 1 H), 8.09 (d, 2 H), 7.50-7.36 (m, 4 H), 7.29-7.23 (m, 1 H), 6.95 (d, 1 H) 4.98 (s, 2 H), 4.92-4.83 (p, 1 H), 4.13-4.04 (m, 1 H), 3.60 (s, 2 H), 2.88 (d, 2 H), 2.24 (t, 2 H), 2.10-2.05 (m, 2 H), 1.75-1.61 (m, 8 H), ¹³C-NMR (75 MHz, CDCl₃):δ 165.2, 162.6, 145.9, 140.3, 137.3, 130.5, 129.3, 128.1, 127.8, 126.7, 123.4, 123.3, 122.8, 109.8, 95.6, 74.7, 63.0, 53.0, 51.3, 46.4, 32.8, 22.5 MS (ES): 551.1 [M + H] ⁺

Reference example 27 Hydrolysis of the trichloroethyl ester from Example 26

Following the procedure outlined in Example 4, the trichloroethyl ester from example 26 (0.42 g, 0.76 mmol) was converted to the title compound as a white solid (0.25 g, 78.9 %). ¹H-NMR (300 MHz, CD₃OD):δ 8.22 (d, 1 H), 8.04 (d, 1 H), 7.66 (d, 1 H), 7.48-7.41 (m, 3 H), 7.26 (t, 1 H), 5.08-4.99 (m, 1 H), 4.10-4.03 (m, 1H), 3.13 (d, 2 H), 2.53 (t, 2 H), 2.08-2.04 (m, 2 H), 1.92-1.85 (m, 2 H), 1.61 (d, 2 H) ¹H-NMR (75 MHz, CD₃OD):δ 163.5, 140.4, 136.7, 129.7, 126.6, 123.0, 122.6, 122.0, 109.9, 61.7, 52.2, 51.1, 46.0, 30.6, 21.3 MS (ES): 419.1 [M + H]⁺

Reference example 28 Preparation of intermediate 4-aminomethyl-1-(tert-butoxycarbonyl) piperidine

Benzaldehyde (8.73 g, 82.3 mmol) was added all at once to a stirred solution of 4-aminomethylpiperidine (9.42 g, 82.3 mmol) in toluene (100 ml). The mixture was heated under reflux for 4 h with a Dean-Stark trap attached to collect the water. The reaction mixture was cooled to room temperature and di-tert-butyldicarbonate (19.75 g, 90.5 mmol) was added in divided portions under continuously stirring. The mixture was stirred overnight, evaporated in vacuo and the residue stirred vigorously with aqueous 1 N KHSO₄ (100 ml) at room temperature for 4 h. The mixture was extracted with Et₂O (3 x 100 ml) and then the aqueous layer was made strongly basic with NaOH. The aqueous layer was extracted with CH₂Cl₂ (3 x 100 ml). The combined extracts were dried with Na₂SO₄, filtered and the solvent evaporated in vacuo to leave the product as an oil (15.4 g, 86.5 %). ¹H-NMR (200 MHz, DMSO- _d6 )δ 4.04-4.01 (m, 2 H), 2,60 (t, 2 H), 2.50 (d, 2 H), 1.62 (d, 2 H), 1.32 (s, 9 H), 1.31-1.28 (m, 1 H), 1.06 (br s, 2 H), 1.03-0.93 (m, 2 H)

Reference example 29 Synthesis of 4-amino-N-(tert-butoxycarbonyl)piperidin-4-ylmethyl]-5-chloro-2-methoxybenzamide

A mixture of 4-aminomethyl-1-(tert-butoxycarbonyl) piperidine (10.0 g, 46.7 mmol), 4-amino-5-chloro-2-methoxybenzoic acid (9.41 g, 46.7 mmol) and NEt₃ (6.80 ml, 46.7 mmol) in DMF (100 ml) were added 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) (9.39 g, 46.7 mmol) and 1-hydroxybenzotriazole (HOBT) (6.62 g, 46.7 mmol) at 0 °C. The reaction mixture was stirred to room temperature overnight and concentrated in vacuo. The resulting residue was added H₂O (100 ml) and extracted with EtOAc. The combined organic extracts were washed with aqueous K₂CO₃ and dried over Na₂SO₄. The solvent was removed in vacuo and the residue separated with flash chromatography (SiO₂, EtOAc) to give the expected product as a white solid (11.91 g, 64.1 %). ¹H-NMR (200 MHz, CDCl₃):δ 8.06 (s, 1 H), 7.77 (t, 1 H), 6.33 (s, 1 H), 4.64 (s, 2 H), 4.08 (d, 2 H), 3.86 (s, 3 H), 3.30 (t, 2 H), 2.67 (t, 2 H), 1.78-1.66 (m, 3 H), 1.43 (s, 9 H), 1.24-1.11 (m, 2 H)

Reference example 30 Preparation of intermediate 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamide hydrochloride

A stirred solution of 4-amino-N-(tert-butoxycarbonyl)piperidin-4-ylmethyl]-5-chloro-2-methoxybenzamide (1.70 g, 4.3 mmol) in 1,4-dioxane (30 ml) at 0 °C was added 4 M HCl in 1,4-dioxane (10 ml) in portions. The reaction mixture was stirred to room temperature for 4 h, evaporated in vacuo and the residue recrystallized from acetone to leave the product as a red solid (0.89 g, 61.8 %). ¹H-NMR (300 MHz, DMSO- _d6 ):δ 9.28 (br s, 1 H), 9.04 (br s, 1 H), 8.00 (t, 1 H), 7.64 (s, 1 H), 7.31 (br s, 4 H), 6.58 (s, 1 H), 3.81 (s, 3 H), 3.21-3.16 (m, 4 H), 2.82-2.71 (q, 2 H), 1.80-1.71 (m, 3 H), 1.45-.34 (m, 2 H)

Reference example 31 Alkylation of 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamide hydrochloride with ethyl 4-bromobutyrate

Following the method outlined in Example 7, 4-Amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamide hydrochloride (0.76 g, 1,98 mmol) was converted to the title compound as an oil (0.57 g, 69.9 %). ¹H-NMR (300 MHz, CDCl₃):δ 8.08 (s, 1 H), 7.74 (t, 1 H), 6.30 (s, 1 H), 4.49 (s, 2 H), 4.14-4.07 (q, 2 H), 3.87 (s, 3 H), 3.30 (t, 2 H), 2.90 (d, 2 H), 2.35-2.28 (m, 4 H), 1.94-1.69 (m, 7 H), 1.29-1.18 (m, 5 H) ¹³C-NMR (75 MHz, CDCl₃):δ 173.9, 164.9, 157.7, 147.0, 133.4, 112.9, 111.9, 98.2, 66.2, 58.3, 56.5, 53.8, 45.5, 36.5, 32.7, 30.4, 15.6 MS (ES): 411.9 [M + H]⁺ Conversion to the hydrochloride salt was effected with ethereal HCl.

Reference example 32 Alkylation of 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamide hydrochloride with diethyl 2-bromoethylphosphonate

Following the procedure outlined in Example 7, 4-Amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamide hydrochloride (1.53 g, 4.0 mmol) was converted to the title compound as an oil (1.12 g, 57.1 %). ¹H-NMR (300 MHz, CDCl₃):δ 7.99 (s, 1 H), 7.70 (t, 1 H), 6.29 (s, 1 H), 4.66 (s, 2 H), 4.08-3.97 (m, 4 H), 3.80 (s, 3 H), 3.24 (t, 2 H), 2.83 (d, 2 H), 2.58-2.53 (m, 2 H), 1.96-1.84 (m, 4 H), 1.66 (d, 2 H), 1.55-1.47 (m, 1 H), 1.30-1.22 (m, 8 H) ¹³C-NMR (75 MHz, CDCl₃):δ 165.0, 157.7, 147.6, 133.1, 112.3, 111.6, 98.1, 61.9, 56.4, 53.2, 52.0, 45.3, 36.4, 29.5, 24.9, 23.1, 16.8 MS    462.1 [M + H]⁺ Conversion to the hydrochloride salt was effected using ethereal HCl.

Reference example 33 Preparation of intermediate 1-benzyl-4-carbonylamide piperidine

A stirred suspension of isonipectamide (16.5 g, 0.13 mol) and K₂CO₃ (35.6 g, 0.26 mol) in EtOH (350 ml) was added benzylbromide (22.0 g, 0.13 mol) and heated under reflux for 3 h, cooled to room temperature and filtered. The filtrate was evaporated in vacuo and added H₂O (200 ml). The aqueous layer was extracted with CH₂Cl₂ (3x 150 ml), the organic layers combined and dried over Na₂SO₄ and filtered. The solvent was evaporated in vacuo to leave the product as a white solid (20.0 g, 71.0 %). ¹H-NMR (300 MHz, CDCl₃):δ 7.34-7.21 (m, 5 H), 6.36 (br s, 1 H), 5.80 (br s, 1 H), 3.49 (s, 2 H), 2.92 (d, 2 H), 2.14-1.99 (m, 1 H), 1.96 (t, 2 H), 1.85-1.72 (m, 4 H)

Reference example 34 Preparation of intermediate 1-benzyl-4-cyano-piperldine

1-Benzyl-4-carbonylamide piperidine (20.0 g, 91.7 mmol) was mixed with P₂O₅ (16.92, 119.2 mmol) and heated under argon at 180-200 °C for 3 h, cooled to room temperature and added H₂O (150 ml). The aqueous solution was basified by careful addition of K₂CO₃ and then extracted with EtOAc (3 x 150 ml). The organic extracts were dried over Na₂SO₄, filtered and the solvent evaporated in vacuo to leave a yellow oil (16.7 g, 90.9 %). ¹H-NMR (200 MHz, CDCl₃):δ 7.41-7.25 (m, 5 H), 3.53 (s, 2 H), 2.75-2.64 (m, 2 H), 2.40-2.34 (m, 2 H), 1.98-1.86 (m, 5 H)

Reference example 35 Preparation of intermediate 1-benzyl-4-aminomethylpiperidine

A suspension of LiAlH₄ (4.84 g, 0.128 mol) in dry Et₂O (40ml) under argon atmosphere at 0 °C was dropwise added a solution of 1- benzyl-4-cyano-piperidine (18.3 g, 91.5 mmol) in dry Et₂O (80 ml) and stirred to room temperature for 24 h. The reaction mixture was treated carefully with H₂O (10 ml), 10 % aqueous NaOH (10 ml) and H₂O (30 ml) to give a mineral precipitate. The precipitate was filtered through a pad of kiselguhr, washed with Et₂O and the filtrate evaporated in vacuo to leave the product as an oil (21.4 g, 82.3 %). ¹H-NMR (200 MHz, CDCl₃): δ 7.37-7.22 (m, 5 H), 6.42 (br s, 1 H), 5.84 (br s, 1 H), 3.51 (s, 2 H), 2.94 (d, 2 H), 2.16-1.67 (m, 7 H)

Reference example 36 Preparation of intermediate methyl 2-(3-chloropropoxy)indole-3-carboxylate

A suspension of methyl indole-3-carboxylate (5.25 g, 30.0 mmol) and DABCO (1.84 g, 16.4 mmol) in dry CH₂Cl₂ (25 ml) was cooled to 0 °C under argon atmosphere, treated in one portion with NCS (4.41 g, 33.0 mmol) and the mixture stirred for 10 min. The resulting solution was added to a solution of 3-chloropropan-1-ol (3.12 g, 33.0 mmol) in dry CH₂Cl₂ (25 ml) containing anhydrous methane sulphonic acid (0.23 ml). The resulting suspension was stirred for 30 min and then washed with 10 % aqueous Na₂CO₃ solution (3 x 25 ml). The organic layer was dried over Na₂SO₄, filtered and concentrated in vacuo. The resulting oil was triturated with toluene (10 ml) at 0 °C for 1 h and the solid precipitate filtered, washed with a small amount of toluene and dried in vacuo to leave the product as an off-white solid (5,22 g, 65.0 %). ¹H-NMR (200 MHz, CDCl₃):δ 9.51 (s, 1 H), 8.04 (d, 1 H), 7.28-7.14 (m, 3 H), 4.49 (t, 2 H), 3.96 (s, 3 H), 3.67 (t, 2 H), 2,18-2.10 (m, 2 H)

Reference example 37 Preparation of intermediate methyl 3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxylate

Methyl 2-(3-chloropropoxy)indole-3-carboxylate (5.0 g, 18.7 mmol) was added to a stirred mixture of 5.4 M aqueous M NaOH (3.8 ml) and toluene (50 ml) and heated at 40 °C for 4 h. The aqueous layer was separated and the organic layer washed with H₂O (3 x 25 ml) while maintaining the temperature at 60 °C. The organic solvent was evaporated in vacuo to leave the product as a white solid (4.0 g, 93.2 %). ¹H-NMR (200 MHz, CDCl₃):δ 8.0 (dd, 1 H), 7.24-7.12 (m, 3 H), 4.50 (t, 2 H), 4.06 (t, 2 H), 3.91 (s, 3 H), 2.34-2.26 (m, 2 H)

Reference example 38 Preparation of intermediate 3,4-dihydro-N-[1-(phenylmethoxy)-4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide

Trimethylaluminium (2 M in toluene, 9 ml) was diluted with dry toluene (9 ml) and the solution cooled to 0 °C under argon atmosphere. 1-Benzyl-4-aminomethylpiperidine (from example 37) (3.37 g, 16.5 mmol) was added to the solution, followed by methyl 3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxylate (from example 39) (3.81 g, 16.5 mmol). The reaction mixture was heated under reflux for 5 h, cooled to room temperature and 10 % aqueous NaOH solution (40 ml) dropwise added. The toluene layer was washed with H₂O, brine and evaporated in vacuo to give an oil. The residue was purified by flash chromatography (SiO₂, CH₂Cl₂/MeOH (7 : 3)) to leave the product as an off white solid (3.52 g, 53.4 %). ¹H-NMR (300 MHz, DMSO- _d6 ): δ 8.34 (d, 1 H), 7.33-7.06 (m, 8 H), 6.53 (t, 1 H), 4.49 (t, 2 H), 4.04 (t, 2 H), 3.51 (s, 3 H), 3.34 (t, 2 H), 2.92 (d, 2 H), 2.36-2.28 (q, 2 H), 2.03-1.95 (m, 2 H), 1.78-1.62 (m, 3 H), 1.43-1.34 (m, 2 H)

Reference example 39 Preparation of intermediate 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide

A stirred solution of 3,4-dihydro-N-[1-(benzyl)-4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide (2.01 g, 5.0 mol) in EtOH (20 ml) was added hydrazine monohydrate (0.36 ml) and 10 % palladium on activated charcoal (M-type, 0.40 g) and heated under reflux for 2 h. The reaction mixture was cooled to room temperature, filtered through a pad of kiselguhr and the filtrate evaporated in vacuo to leave the expected product as a white solid (1.52 g, 97.3 %). ¹H-NMR (200 MHz, DMSO- _d6 ):δ 8.09-8.05 (m, 1 H), 7.31-7.27 (m, 1 H), 7.14-7.03 (m, 2 H), 6.81 (t, 1 H), 4.59 (t, 1 H), 4.23-4.17 (m, 1 H), 4.11 (t, 2 H), 3.17 (t, 2 H), 3.0 (d, 2 H), 2.56-2.45 (m, 2 H), 2.35-2.24 (m, 2 H), 1.66-1.61 (m, 3 H), 1.23-1.04 (m, 2 H)

Example 40 Alkylation of 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide with ethyl 4-bromobutyrate

Following the procedure outlined in Example 7, 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide (0.62 g, 2.0 mmol) was converted to the title compound as a colourless oil that crystallized upon standing (0.74 g, 86.5%). ¹H-NMR (300 MHz, CDCl₃): δ 8.33 (d, 1 H), 7.25-7.10 (m, 3 H), 6.56 (t, 1 H), 4.55 (t, 2 H), 4.14-4.10 (m, 4 H), 3.34 (t, 2 H), 2.98 (d, 2 H), 2.43-2.31 (m, 6 H), 2.01 (t, 2 H), 1.91-1.81 (m, 4 H), 1.73-1.66 (m, 1 H), 1.42-1.37 (m, 2 H), 1.26 (t, 3 H) ¹³C-NMR (75 MHz, CDCl₃):δ 173.4, 164.8, 149.2, 131.0, 125.6, 122.1, 121.0, 120.6, 107.4, 89.2, 66.8, 60.2, 57.8, 53.4, 44.2, 38.9, 36.1, 32.2, 29.7, 22.0, 21.2, 14.2 MS (ES): 450.1 [M + Na] ⁺ Conversion to the HCl-salt was effected with etheral HCl.. The precipitate was collected and recrystallized from acetone to leave the HCl-salt as a white crystalline solid.

Example 41 Alkylation of 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide with methyl 6-bromohexanoate

Following the procedure outlined in Example 7, 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide (0.31 g, 1.0 mmol) was converted to the title compound as a white solid (0.37 g, 84.5 %). ¹H-NMR (300 MHz, CDCl₃): δ 8.33 (d, 1 H), 7.23-7.09 (m, 3 H), 6.56 (t, 1 H), 4.54 (t, 2 H), 4.10 (t, 2 H), 3.67 (s, 3 H), 3.34 (t, 2 H), 2.97 (d, 2 H), 2.38-2.29 (m, 6 H), 1.96 (t, 2 H), 1.79 (d, 2 H), 1.70-1.30 (m, 10 H) ¹³C-NMR (75 MHz, CDCl₃):δ 174.1, 164.7, 149.1, 131.0, 125.5, 122.0, 121.0, 120.6, 107.4, 89.1, 66.8, 58.7, 53.5, 51.4, 44.3, 38.9, 36.2, 33.9, 29.8, 27.1, 26.4, 24.7, 21.2, MS (ES): 464.2 [M+ Na] ⁺

Reference example 42 Alkylation of 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide with 2,2,2-trichloroethyl 4-bromobutyrate

Following the procedure outlined in Example 7, 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide(0.31 g, 1.0 mmol) was converted to the title compound as a white solid (0.40 g, 75.8 %). ¹H-NMR (300 MHz, CDCl₃): δ 8.32 (d, 1 H), 7.27-7.14 (m, 3 H), 6.63 (t, 1 H), 4.77 (s, 2 H), 4.59 (t, 2 H), 4.15 (t, 2 H), 3.38 (t, 2 H), 3.22 (d, 2 H), 2.70 (t, 2 H), 2.59 (t, 2 H), 2.40-2.10 (m, 4 H), 2.05-1.96 (m, 2 H), 1.91-1.85 (m, 3 H), 1.77-1.60 (m, 2 H) ¹³C-NMR (75 MHz; CDCl₃):δ 171.7, 166.4, 149.8, 131.5, 125.9, 122.6, 121.2, 121.1, 107.9, 95.2, 89.4, 74.3, 67.3, 57.4, 53.4, 44.2, 39.4, 35.5, 31.8, 28.8, 21.6, 21.1 MS (ES): 553.2 [M + Na]⁺

Example 43 Hydrolysis of the ethyl ester from example 40

The ethyl ester from example 40 (0.51 g, 1.20 mmol) was added to a mixture of 2 M aqueous NaOH solution (1.2 ml) and MeOH (5 ml) and refluxed for 2 h. The reaction mixture was cooled to room temperature, concentrated in vacuo and dropwise added 10 % aqueous HCl to pH 2. The precipitate was filtered off, washed with water and dried in vacuo to a white crystalline solid (0.31 g, 72.9 %). ¹H-NMR (300 MHz, DMSO- _d6 ):δ 12.31 (br s, 1 H), 10.25 (br s, 1 H), 8.10-8.04 (m, 1 H), 7.32-7.26 (m, 1 H), 7.14-7.04 (m, 2 H), 6.96 (t, 1 H), 4.59 (t, 2 H), 4.15 (t, 2 H), 3.43-3-01 (m, 8 H), 2.38-2.28 (m, 4 H), 1.97-1.81 (m, 5 H), 1.68-1.55 (m, 2 H) ¹³C-NMR (75 MHz, DMSO- _d6 ): δ 174.3, 164.6, 150.6, 131.8, 126.1, 122.0, 120.7, 120.4, 109.3, 88.7, 67.9, 56.1, 52.4, 43.7, 34.9, 31.5, 27.7, 21.4, 19.7 MS (ES): 398.1 [M + H]⁺

Example 44 Alkylation of 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide with diethyl 2-bromoethylphosphonate

Following the procedure outlined in Example 7, 3,4-dihydro-N-[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide (0.29 g, 0.92 mmol) was converted to the title compound as a white solid (0.36 g, 82.9 %). ¹H-NMR (300 MHz, CDCl₃):δ 8.30 (d, 1 H), 7.23-7.10 (m, 3 H), 6.59 (t, 1 H), 4.56 (t, 2 H), 4.17-4.06 (m, 6 H), 3-35 (t, 2 H), 3.09 (d, 2 H), 2.86-2.78 (q, 2 H), 2.39-2.33 (m, 2 H), 2.27-2.08 (m, 4 H), 1.88-1.60 (m, 3 H), 1.58-1-50 (m, 2 H), 1.33 (t, 6 H) ¹³C-NMR (75 MHz, CDCl₃):δ 163.5, 149.8, 131.5, 125.9, 122.5, 121.3, 121.1, 107.9, 89.4, 67.3, 62.4, 62.3, 53.2, 52.1, 44.3, 39.4, 35.9, 29.3, 21.6, 16.9, 16.8 MS (ES): 500.1 [M + Na]⁺

Reference example 45 Preparation of intermediate N-[1-(benzyl)-4-piperidinyl]methyl]-1,4-benzodioxane-5-carboxamide

A suspension of 1,4-benzodioxan-5-carboxylic acid (1.80 g, 10.0 mmol) and 1,1'-carbonyldiimidazole (1.78 g, 11.0 mmol) in CH₃CN (100 ml) was stirred at room temperature for 2 h. 1-Benzyl-4-aminomethylpiperidine (from example 37) (2.04 g, 10.0 mmol) in CH₃CN (10 ml) was added to the mixture and stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, added EtOAc (200 ml) and washed with H₂O (3 x 50 ml). The organic layer was dried over Na₂SO₄ and evaporated in vacuo to a solid material. The residue was separated with flash chromatography (SiO₂, EtOAc : MeOH, 1: 1) to leave the product as a white solid (2.31 g, 63.1 %). ¹H-NMR (200 MHz, CDCl₃):δ 7.74 (dd, 1 H), 7.67 (t, 1 H), 7.34-7.15 (m, 5 H), 7.03-6.89 (m, 2 H), 4.43-4.39 (m, 2 H), 4.33-4.29 (m, 2 H), 3.52 (s, 2 H), 3.37 (t, 2 H), 2.93 (d, 2 H), 2.06-1.93 (m, 2 H), 1.77-1.50 (m, 3 H), 1.47-1.28 (m, 2 H)

Reference example 46 Preparation of intermediate N-[4-piperidinyl]methyl]-1,4-benzodioxane-5-carboxamide hydrochloride

Following the procedure outlined in example 6, N-[1-(benzyl)-4-piperldinyl]methyl]-1,4-benzodioxane-5-carboxamide (1.88 g, 5.13 mmol) was converted to the title compound as an white solid (1.36 g, 85.2%). ¹H-NMR (300 MHz, CDCl₃):δ 9.68 (br s, 1 H), 9.37 (br s, 1 H), 7.79 (t, 1 H), 7.71-7.68 (dd, 1 H), 7.03-6.91 (m, 2 H), 4.47-4.45 (m, 2 H), 4.34-4.31 (m, 2 H), 3.52 (d, 2 H), 3.40 (t, 2 H), 2.94-2.82 (q, 2 H), 2.12-1.69 (m, 5 H)

Reference example 47 Alkylation of N-[4-piperidinyl]methyl]-1,4-benzodioxane-5-carboxamide hydrochloride with ethyl 4-bromobutyrate

Following the procedure outlined in example 7, N-[4-piperidinyl]methyl]-1,4-benzodioxane-5-carboxamide hydrochloride (0.56 g, 2.0 mmol) was converted to the title compound as an oil (0.66 g, 85.3 %). ¹H-NMR (300 MHz, CDCl₃):δ 7.74-7.71 (dd, 1 H), 7.66 (t, 1 H), 7.01-6.90 (m, 2 H), 4.44-4.41 (m, 2 H), 4.33-4.30 (m, 2 H), 4.16-4.09 (q, 2 H), 3.35 (t, 2 H), 2.92 (d, 2 H), 2.38-2.30 (m, 4 H), 2.02-1.50 (m, 7 H), 1.38-1.27 (m, 2 H), 1.25 (t, 3 H) ¹³C-NMR (75 MHz, CDCl₃):δ 173.5, 164.8, 143.4, 141.8, 124.0, 122.2, 121.3, 120.5, 64.9, 63.5, 60.2, 57.9, 53.3, 45.2, 36.0, 32.3, 29.9, 22.2, 14.1 MS (ES): 413.2 [M + Na]⁺

Reference example 48 Alkylation of N-[4-piperidinyl]methyl]-1,4-benzodioxane-5-carboxamide hydrochloride with diethyl 2-bromoethylphosphonate

Following the procedure outlined in example 7, N-[4-piperidinyl]methyl]-1,4-benzodioxane-5-carboxamide hydrochloride (0.71 g, 2.5 mmol) was converted to the title compound as an white solid (0.88 g, 80.7 %). ¹H-NMR (300 MHz. CDCl₃):δ 7.74-7.71 (dd, 1 H), 7.66 (t, 1 H), 6.98-6.93 (m, 2 H), 4.43-4.41 (m, 2 H), 4.33-4.30 (m, 2 H), 4.12-4.05 (m, 6 H), 3.35 (t, 2 H), 2.92 (d, 2 H), 2.58-2.45 (m, 2 H), 2.08-1.90 (m, 4 H), 1.71-1.50 (m, 3 H), 1.36-1.31 (m, 6 H) ¹H-NMR (75 MHz. CDCl₃):δ 165.2, 143.9, 142.3, 135.7, 127.6, 124.5, 122.6, 121.7, 121.0, 65.3, 63.9, 62.0, 61.9, 53.3, 52.1, 45.5, 36.3, 30.3, 16.7 MS (ES): 463.2 [M + Na]⁺

Reference example 49 Preparation of intermediate N-[1-(benzyl)-4-piperidinyl]methyl]indole-3-carboxamide

Following the procedure outlined in example 3, indole-3-carboxylic acid (5.56 g, 31.0 mmol) was converted to the title compound as an oil (3.78 g, 35.0 %). ¹H-NMR (300 MHz, CDCl₃):δ 9.96 (s, 1 H), 7.96 (d, 1 H), 7.67 (s, 1 H), 7.44-7.22 (m, 8 H), 6.24 (t, 1 H), 3.51 (s, 2 H), 3.40 (t, 2 H), 2.92 (d, 2 H), 1.98 (t, 2 H), 1.78-1.67 (m, 3 H), 1.44-1.30 (m, 2 H)

Reference example 50 Preparation of intermediate N-[4-piperidinyl]methyl]indole-3-carboxamide

Following the procedure outlined in example 39, N-[(1-benzyl-4-piperidinyl)methyl)indole-3-carboxamide (1.50 g, 4.3 mol) was converted to the title compound as a white solid (1.07 g, 96.7 %). ¹H-NMR (300 MHz, DMSO- _d6 ):δ 11.56 (br s, 1 H), 8.15-8.12 (m, 1H), 8.03 (s, 1 H), 7.85 (t, 1 H), 7.41 (d, 1 H), 7.15-7.08 (m, 2 H), 3.12 (t, 2 H), 2.92 (d, 2 H), 2.55-2.49 (m, 1 H), 2.41 (t, 2 H), 1.64-1.60 (m, 3 H), 1.06-1.01 (m, 2 H)

Reference example 51 Alkylation of N-[4-piperidinyl]methyl]indole-3-carboxamide with ethyl 4-bromobutyrate

Following the procedure outlined in example 9, N-[4-piperidinyl]methyl]indole-3-carboxamide (0.24 g, 0.94 mol) was converted to the title compound as a white solid (0.16 g, 47.1 %). ¹H-NMR (300 MHz, CDCl₃):δ 9.70 ( br s, 1 H), 8.00-7.95 (m, 1 H), 7.78 (s, 1 H), 7.49-7.44 (m, 1 H), 7.30-7.25 (m, 2 H), 6.33 (t, 1 H), 4.20-4.09 (q, 2 H), 3.40 (t, 2 H), 2.97 (d, 2 H), 2.44-2.31 (m, 4 H), 1.99-1.76 (m, 7 H), 1.43-1.24 (m, 5 H) ¹³C-NMR (75 MHz, CDCl₃):δ 173.4, 165.7, 136.4, 128.1, 124.7, 122.7, 121.4, 119.8, 112.1, 60.3, 57.8, 53.2, 44.8, 36.1, 32.2, 29.7, 21.9, 14.2 MS (ES): 394.1 [M + Na]⁺

Reference example 52 Alkylation of N-[4-piperidinyl]methyl]indole-3-carboxamide with 2,2,2-trichloroethyl 4-bromobutyrate

Following the procedure outlined in example 7, N-[4-piperidinyl]methyl]indole-3-carboxamide (0.94 g, 3.65 mmol) was converted to the title compound as a white solid (0.84 g, 48.4 %). ¹H-NMR (300 MHz, CDCl₃):δ 10.08 (br s, 1 H), 7.95-7.92 (m, 1 H), 7.72 (s, 1 H), 7.43 7.40 (m, 1 H), 7.23-7.20 (m, 2 H), 6.31 (t, 1 H), 4.15 (s, 2 H), 3-36 (t, 2 H), 2.90 (d, 2 H), 2.36-2.17 (m, 4 H), 1.89 (t, 2 H), 1.83-1.65 (m, 5 H), 1.36-1.32 (m, 2 H) ¹³C-NMR (75 MHz, CDCl₃):δ 173.9, 166.0, 136.5, 128.4, 124.6, 122.6, 121.4, 119.6, 112.2, 111.7, 99.7, 57.8, 53.2, 51.5, 44.9, 36.1, 32.0, 30.8, 29.7, 21.9 MS (ES): 497.2 [M + Na]⁺

Reference example 53 Hydrolysis of the trichloroethyl ester from example 52

Following the procedure outlined in example 4, the trichloroethyl ester from example 52 (0.47 g, 1.0 mmol) was converted to the title compound as a white solid (0.21 g, 61.1 %). ¹H-NMR (300 MHz, DMSO- _d6 ):

• 11.63 (s, 1 H), 8.13 (d, 1 H), 8.05 (d, 1 H), 7.97 (t, 1 H), 7.41 (d, 1 H), 7.15-7.05 (m, 2 H), 3.14 (t, 2 H), 3.02 (d, 2 H), 2.50 (t, 2 H), 2.26 (t, 2 H), 2.17 (t, 2 H), 1.75-1.53 (m, 5 H), 1.31-1.21 (m, 2 H)
• 13C-NMR (75 MHz, DMSO-d6):δ 174.4, 164.6, 136.0, 127.5, 126.1, 121.6, 120.9, 120.1, 111.7, 110.5, 57.0, 52.2, 43.6, 35.5, 33.4, 28.7, 20.9
• MS (ES): 366.2 [M + Na]+

Reference example 54 - Tegaserod Preparation of the primary amine 2,2,2-trichloroethyl 5-aminopentanoate

A stirred solution of 2,2,2-trichloroethyl 5-bromopentanoate (prepared by the same method as in example 1) in acetone is added potassium phtalimide and stirred overnight. The reaction mixture is filtered and the solvent evaporated in vacuo. The residue is added EtOAc and washed with H₂O. The organic layer is dried over Na₂SO₄, filtered and evaporated in vacuo to leave the title compound. Standard hydrazinolysis in EtOH gives the primary amine (see scheme 1).

Reference example 55 - Tegaserod Preparation of the monoalkylated amine N'-2,2,2-(trichloroethyl pentanoate)-N'-aminoguanidine

A suspension of thiosemicarbamide is added MeI in EtOH and heated at 60 °C for ½ h and cooled to room temperature. The resulting suspension is filtered and the filtrate washed with Et₂O to leave S-methyl isothiosemicarbazide hydroiodide. S-methyl isothiosemicarbazide hydroiodide is used in the next step without any further purification. A solution of this compound in MeOH is added 2,2,2-trichloroethyl 5-aminopentanoate (from example 54) and heated under reflux overnight. The reaction mixture is cooled to room temperature and the solvent evaporated in vacuo to leave the title compound. The amine is used in the next step without any further purification (scheme 2).

Reference example 56 - Tegaserod Synthesis of the tegaserod derivative

To a stirred solution of 5-methoxyindole-3-carboxaldehyde in MeOH is added N-2,2,2-(trichloroethyl pentanoate)-N'-aminoguanidine at room temperature. The solution is acidified with conc. aqueous HCl and stirred overnight. The solvent is evaporated in vacuo and added MeOH. The solution is added etheral HCl and the precipitate filtered off. The precipitate is recrystallized from MeOH/Et₂O to leave the HCl salt of the trichloroethyl ester. This compound is added to a suspension of Zn and a mixture of 1 M aqueous KH₂PO₄ and THF and stirred overnight. The suspension is filtered through a pad of kiselguhr and the solvent evaporated in vacuo. The residue is separated with flash chromatography to leave the title compound as a free acid.

Example 57 In vitro biological testing of hydrophilic 5-HT ₄ ligands in adenylyl cyclase assays Materials and methods Establishment of HEK293 cell lines stably expressing human 5-HT_4(b) receptors

The development of HEK293 cell lines stably expressing human 5-HT_4(b) receptors was described and published previously (Bach et al. 2001). Briefly, HEK293 cells (ATCC) were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were transfected with plasmid DNA (pcDNA3.1(-) containing human S-HT_4(b) receptor cDNA) using SuperFect Transfection Reagent (QIAGEN) according to the manufacturers protocol. Serial dilutions of transfected cells were plated in 96 well plates containing G418 (geneticin; Amersham) at 0.4 mg/ml, and isolated single colonies of cells transformed to the neomycin-resistant phenotype were expanded and tested for expression of serotonin receptors by measuring serotonin-stimulated adenylyl cyclase activity (Themmen et al. 1993). Transformed cells were always grown in the presence of G418 (0.4 mg/ml). For binding and adenylyl cyclase analysis, stable cell lines were grown and maintained in UltraCULTURE^™ general purpose serum-free medium (BioWhittaker, Walkersville, MD, USA), supplemented with L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml).

Membrane preparation for radioligand binding and adenylyl cyclase assay

Membranes were prepared from stably transfected HEK293 cells cultured on 150-mm cell culture dishes and grown to 80% confluence in serum-free medium (UltraCULTURE^™, BioWhittaker) with penicillin (10 U/ml) and 2 mM L-Glutamine (BioWhittaker). Cells were washed twice with 10 ml ice-cold HBSS, scraped with a rubber policeman in 10 ml ice-cord HBSS and collected by centrifugation at 800 g for 5 min at 4 °C. The cell pellet was resuspended in 1 ml/dish ice-cold STE buffer (27% (w/v) sucrose, 50 mM Tris-HCl, pH 7.5 at 20 °C, 5 mM EDTA) and homogenized with an Ultra-Turrax (IKA) homogenizer, using five 10 s bursts with 30 s cooling in ice-water between bursts. To remove nuclei, the homogenate was centrifuged at 300 g for 5 min at 4 °C and the supernatant was further centrifuged at 17000 g for 20 min at 4 °C and the supernatant removed. The crude membrane pellet was resuspended with ten strokes of tight fitting pestle B in a Dounce glass-glass homogenizer in 1 ml/dish ice-cold TE (50 mM Tris-HCl, pH 7.5 at RT, 5 mM EDTA). This procedure was repeated twice and the resuspended membranes were finally aliqouted and flash frozen in liquid nitrogen and stored at -70 °C until use.

Radioligand binding assay

Binding assays were performed in 96-well, round-bottom microtiter plates with total reaction volumes of 50-200 µl, containing the indicated concentration of [³H]GR113808 with or without competing unlabelled ligand in a binding buffer containing 50 mM Tris-HCl (pH 7.5 at RT), 1 mM EDTA, 5 mM EGTA, 2 mM MgCl₂, 1 mM ascorbate, 0.1 % BSA and 100 µM GTP. The plates were incubated at 23EC for 60 min and harvested onto UniFilter™-96 GF/C™ (Packard Instrument Co., Meriden, CT, USA), presoaked in 0.3% polyethyleneimine (Sigma), using a Packard FilterMate Universal Harvester with 96-well format, and washed 4-6 times with approximately 0.25 ml/well of ice-cold buffer, containing 50 mM Tris-HCl (pH 7.0 at RT) and 2 mM MgCl₂. The filters were dried and counted at approximately 40% efficiency in a Top-Count liquid scintillation counter (Packard), using 20 µl per filter well of Micro-Scint liquid scintillation cocktail (Packard).

Adenylyl cyclase assay

Adenylyl cyclase activity was measured by determining conversion of [α-³²P]ATP to [³²P]cAMP in membranes prepared in STE by homogenization of cells grown and washed as described above in a Dounce glass-glass homogenizer by 10 strokes with the tight-fitting pestle. Membranes were kept on ice prior to assay. Adenylyl cyclase activities were measured on 10-µl aliquots in a final volume of 50 µl in the presence of 0.1 mM [α-³²P]ATP (1-2 x 10⁶ cpm/assay), 4 mM MgCl₂, 20 µM GTP, 1 mM EDTA, 1 mM [³H]cAMP (ca. 10,000 cpm/assay), 1 µM 3-isobutyl-1-methyl xanthine (IBMX; Sigma), a nucleoside triphosphate regenerating system consisting of 20 mM creatine phosphate (Sigma), 0.2 mg/ml creatine phosphokinase (Sigma) and 40 U/ml myokinase (Sigma) and additives described in the text and figures. When forskolin (Calbiochem, La Jolla, CA, USA) was used the concentration was 100 µM. Incubations were for 20 min at 32EC. Cyclic AMP formed was quantified by the double column chromatography system of Salomon et al. (1974) as modified by Bockaert et al. (1976).

Analysis of binding and adenylyl cyclase data

Binding and adenylyl cyclase data were analyzed by non-linear regression using Microsoft Excel with the Solver add-in, using the below equations.

Competitive binding assays - The data were fit to the equation $Y=a+\left(b,-,a\right)/\left(1,+,x,/,c\right)$ where a is non-specific binding, b is total binding in the absence of competitor, c is IC ₅₀, and x is the concentration of competitor. Where relevant, relative binding data were obtained by recalculating the data using a=0 and b=100.

Activation of adenylyl cyclase - The data were fit to the equation $Y=a+\left(b,-,a\right)x/\left(c,+,x\right)$ where a is basal adenylyl cyclase activity, b is maximal adenylyl cyclase activity stimulated by the agonist, c is EC ₅₀, and x is the concentration of agonist.

IC ₅₀ values from competitive binding assays were converted to K _b values by the method of Cheng and Prusoff (1973).

Protein measurements

The protein concentrations in the membrane preparations were measured with the Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA) using bovine serum albumin (BSA) as standard.

Radiochemicals

[³H] GR113808 (84 Ci/mmol), [α-³²P]ATP (400 Ci/mmol) and [³H]cAMP (30-50 Ci/mmol) were from Amersham (Buckinghamshire, England).

Compounds

5-Hydroxytryptamine hydrochloride (5-HT, serotonin) was from Sigma (St. Louis, MO, USA). GR113808 (1-methyl-1H-indole-3-carboxylic acid, [1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl ester) maleate was from Tocris (Avonmouth, UK). The other compounds tested were synthesized by Drug Discovery Laboratories AS (DDL) (Oslo, Norway).

Standards

Results of in vitro biological testing of new 5-HT₄ ligands in adenylyl cyclase and binding assays, organised per compound (Table 1)

GR113808	9.98, 9.87, 9.77, 9.65, 9.82, 9.75	Antagonist	9.94 - 10.31 - 10.21 - 10.71
SB207266 (reference) (piboserod)	9.88 , 9.77	Antagonist	10.76
DDL-6002	9.89	Antagonist	10.55 - 10.66
DDL-6003	9.54	Antagonist	9.73 - 10.36
DDL-6004	9.00	Antagonist	8.76 - 9.79
DDL-6005	6.56	Antagonist	6.90
DDL-6006	n.d.	Unknown	5.71
DDL-6011	6.55	Antagonist	6.42
DDL-6013	8.49	Weak partial agonist	7.78
DDL-6014	9.95	Weak partial agonist	8.53
DDL-6015	9.17	Antagonist	8.70
DDL-6016	8.55	Weak partial agonist	8.49
DDL-6021	6.24	Partial agonist	7.50
DDL-6022	6.62 - 8.31	Partial agonist	7.89
DDL-6023	5.49 - 6.95	Partial agonist	6.64
DDL-6024	n.d.	n.d.	6.35
DDL-6025	n.d.	n.d	6.23
DDL-6032	8.36	Partial agonist	8.19
DDL-6040	9.72	Antagonist	10.65
DDL-6041	9.95	Antagonist	10.29
DDL-6042	10.14	Antagonist	10.81
DDL-6043	10.14	Antagonist	10.48
DDL-6044	9.16	Antagonist	9.55
DDL-6045	8.47	Antagonist	8.84

n.d.: not determined

Claims (5)

1. A compound of formula IV-P

   wherein L is absent or selected from the group consisting of straight chain or branched optionally substituted C_1-10-alkyl, optionally substituted C_2-10-alkenyl, optionally substituted C_2-10-alkynyl, C_1-10-alkylamine, C_1-10-alkoxy, C_2-10-alkenyloxy, C_2-10-alkynyloxy, C_1-10-alkoxycarbonyl, C_2-10-alkenyloxycarbonyl, C_2-10-alkynyloxycarbonyl; and A is selected from the group consisting of -C(O)-OR¹, -OP(O)OR²OR², -P(O)OR²OR², - SO₂OR², SO₃H, OSO₃H, and PO₃H; wherein R¹ and R² are independently selected from the group consisting of H, M, C_1-15-alkyl, C_3-8-cycloalkyl, aryl, and R^1,2 wherein R^1,2 is R'-O-C(O)-R", R'-O-C(O)-O-R", R'-C(O)-O-R", wherein R' and R" are independently selected from the group consisting of C_1-15-alkyl, C_3-8-cycloalkyl and aryl;

   R¹³ is selected from the group consisting of H, halogen, NH₂, and C_1-6-alkyl; and

   R¹⁶ is selected from the group consisting of H, halogen, OH, O-C_1-6-alkyl, and C_1-6-alkyl.
2. Use of a compound according to claim 1, or a composition comprising said compound or a salt of said compound for the preparation of a medicament for the treatment of a cardiovascular disorder.
3. Use of a compound according to claim 1, or a composition comprising said compound or a salt of said compound for the preparation of a medicament for the treatment of a gastrointestinal disorder or lower urinary tract disorder.
4. The use according to claim 2, wherein the cardiovascular disorder is selected from the group consisting of tachycardia, bradycardia, cardioexcitation, cardiodepression, arrhythmia, fibrillation, atrial fibrillation, Paroxysmal Supraventricular Tachycardia (PSVT), thromboembolisms and VTE.
5. The use according to claim 3, wherein the gastrointestinal disorder is selected from the group consisting of irrital bowel syndrome; gastrointestinal hypomotility disorders; gastroesophageal reflux, such as heartburn or mild oesophagitis; functional or nonulcer dyspepsia; gastroparesis; nausea and vomiting; early satiety in the elderly; paraneoplastic of HIV-associated gastroparesis; drug-induced delays in gastric emptying and functional bowel obstructions, such as bowel obstructions caused by pancreatic cancer or drugs; and emesis.