QUINOLINE DERIVATIVES WITH AFFINITY FOR THE 5-HT2B RECEPTOR

The present invention relates to novel quinoline derivatives which bind to the 5-HT _2B receptor and are capable of interfering with the effects of 5-hydroxytryptamine (5-HT) at the 5-HT _2B receptor; to pharmaceutical compositions containing them; and to the use of such compounds in therapy.

5-Hydroxytryptamine (5-HT, or serotonin) is a neurotransmitter important to both the central nervous system (CNS) and as a pivotal mediator of sensory and motor function in the gastrointestinal (Gl) tract, McLean et al., TRENDS in Neurosciences (2006) Vol.30 No.1 , 9-13. When first isolated, serotonin was referred to as 'gut- stimulating factor', Erspamer et al., Nature (1952) Vol.169, 800-801 , and since that time has also become appreciated as a neurotransmitter in the brain. The cell surface receptors via which serotonin exerts its biological effects have been identified by molecular techniques. These receptors can be subdivided into at least 7 distinct subfamilies termed 5-HT ₁ , 5-HT _2, 5-HT ₃ , 5-HT ₄ , 5-HT ₅ , 5-HT ₆ and 5-HT ₇ based on gene sequence, pharmacological profile and signal transduction pathways, Robichaud et ai., Annual Reports in Medicinal Chemistry (2000) Vol.36, 11-20.

5-HT ₂ receptors are a family of seven transmembrane domain G-protein coupled receptors which can be subdivided into three distinct subtypes: 5-HT _2A , 5-HT ₂₆ and 5- HT _2C , sharing an overall amino acid homology of approximately 50% and the homology within the transmembrane domains being greater than 70%, J. E. Leysen, Current Drug Targets - CNS & Neurological Disorders (2004), Vol.3, 11-26.

5-HT _2B receptors are present on both smooth muscle and nerves and their activation by serotonin can cause direct smooth muscle contraction in the human ileum, Borman et ai, British Journal of Pharmacology (1995) Vol.114, 1525-1527. Furthermore, activation of 5-HT _2B receptors can enhance cholinergic-neuron- mediated contractions in human colon (Borman et al., British Journal of

Pharmacology (2002) Vol. 135, 1 144-1151 ; WO 2005/097113; and WO 02/056010). These studies suggest that blockade of 5-HT _2B receptors could reduce the prokinetic effects of serotonin on the Gl tract and thus 5-HT ₂₆ receptor antagonists may be of use in the treatment of functional bowel disorders such as Irritable Bowel Syndrome (IBS). Visceral pain is commonly associated with IBS, the most frequent features of which are recurrent abdominal pain and discomfort, altered bowel habits and a strong female predominance. The definition and criteria for IBS have been formalised by the Rome III criteria, Drossman et al., Journal of Gastrointestinal and Liver Diseases (2006) Vol.15(3), 237-241. The potential usefulness of 5-HT _2B receptors in the treatment of IBS is further supported by the observation that the 5-HT ₄ agonist

Tegaserod (Novartis) and the 5-HT ₃ antagonist Alosetron (GlaxoSmithKline), both of which are used for the management of IBS, also have affinity for the 5-HT ₂₆ receptor.

IBS may also be treated with gabapentin or the tricyclic antidepressant drug amitriptyline. A study by Poitras et al., Digestive Diseases and Sciences (2002), Vol.47(4), 914-920, suggested that amitriptyline was effective in decreasing the clinical symptomatology of IBS and that this clinical improvement was correlated to the modulation of visceral pain perception. Gabapentin, on the other hand, is widely used as a medication to relieve pain and has been shown to reduce rectal sensory thresholds through attenuating rectal sensitivity to distension and enhancing rectal compliance in diarrhoea-predominant IBS patients, Lee et al., Alimentary Pharmacology and Therapeutics (2005), Vol.22, 981-988.

There is also some evidence that 5-HT _2B receptor blockade is involved in migraine since the experimental drug tool m-chlorophenylpiperazine that triggers migraine in selected subjects has affinity for both the 5-HT _2B receptor and the 5-HT _2C receptor, Fozard et al., Naunyn-Schmiedeberg's Arch Pharmacol (1994) Vol. 250, 225-229, as do a number of migraine prophylactic drugs, Kalkman Life Sciences (1994) Vol.54(10), 641-644.

It has also been demonstrated in both humans and mice that pulmonary hypertension is associated with a substantial increase in 5-HT _2B receptor expression in pulmonary arteries. These data show that activation of 5-HT _2B receptors is a limiting step in the development of pulmonary hypertension (Launay, J. -M. et al., (2002) Nature Medicine 8(10), 1 129-1135).

As well as being implicated in migraine, studies in 5-HT _2C receptor-knockout mice suggest that the 5-HT _2C receptor may also be involved in obesity and epilepsy, Tecott et al. Nature (1995) Vol.374, 542-546. In addition, the 5-HT _2C receptor is closely involved in the weight gain that occurs following treatment with many antipsychotic drugs, for example clozapine and olanzapine, and is likely to contribute directly via the 5-HT _2C antagonistic effects of these drugs, Reynolds et al., Journal of Psychopharmacology (2006) Vol. 20, 15-18.

Animal models of human disease are commonly used to predict the effectiveness of a test pharmaceutical compound in treating humans. One such animal model is the intra rectal mustard oil model of visceral pain described by Laird et al. in Pain 2001 ; 92: 335-342. Mustard oil has been used in a variety of conscious and anaesthetised models to elicit pain or stimulate nociceptive pathways.

5-HT _2B receptor activation is also believed to influence mitogenic signalling. This activity is thought to underpin the reason why 5-HT ₂₆ receptor antagonists are likely to provide useful treatments of conditions associated with the development of fibrosis, including valvular heart diseases, pulmonary hypertension and lung and liver fibrosis. The evidence pointing to a link between the 5-HT ₂₆ receptor and the various cardiovascular diseases is summarised by Kaumann & Levy (2006, Pharmacology &

Therapeutics, 1 11 , 674-706) and specifically described by Jaffre et al (2004, Circulation, 110, 969-974) and Launay et al (2002, Nature Medicine, 8, 1129-1 135). The evidence pointing to a link with lung fibrosis is exemplified by Fabre et al (2008, Eur Respiratory Journal., doi 10.1 183/09031936.00126907), and that to liver fibrosis by Ruddell et al (2006, Am J Pathol, 169, 861-76), where high expression in hepatic stellate cells may be associated with liver fibrosis. Interestingly, 5-HT _2B receptors are also thought to limit the regenerative capacity of liver hepatocytes, suggesting that 5- HT _2B receptor antagonists will be useful in promoting liver regeneration in end stage liver disease (Ebrahimkhani et al, 2007, Hepatology, 46, S1 , 786A).

5-HT _2B receptor antagonists are also useful for the treatment of disorders where inappropriate release of 5-HT leads to tissue remodelling. For example, it has been demonstrated in both humans and mice that pulmonary hypertension is associated with a substantial increase in 5-HT _2B receptor expression in pulmonary arteries. These data show that activation of 5-HT ₂₆ receptors is a limiting step in the development of pulmonary arterial hypertension (Launay, J. -M. et al., (2002) Nature Medicine 8(10), 1129-1135) associated with, for example, hypoxic lung disease or hypoxemia. Treatment with 5-HT ₂₆ receptor antagonists would, therefore, be expected to inhibit both the vasconstrictor and vascular cell proliferative actions of 5- HT (Kaumann, AJ & Levy, F. O. (2006) Pharmacology & Therapeutics 111, 674-706). Additionally, 5-HT ₂₆ receptor antagonists are also useful in the treatment of conditions where there is cardiac hypertrophy (Jaffre, F., et al (2004) Circulation, 110, 969-974). In the liver, 5-HT exerts a negative influence on hepatic growth and function which can be prevented by 5-HT ₂₆ receptor antagonists, demonstrating their use in the treatment of liver degeneration (Ebrahimkhani, M., et al., 2007, 58th Ann Mtg Am Assoc for the study of Liver Diseases, Abstract 1238). Further, the ability of 5-HT ₂₆ receptor antagonists to reduce the profibrogenic activity of 5-HT extends the use of such compounds to disorders associated with fibrosis, such as cardiac valvulopathy (Hoffman, C, et al., 2006, Clinical Neuropharmacol., 29, 80-86).

Recent evidence suggesting that the function of the 5-HT transporter can be regulated by the 5-HT _2B receptor (Launay, J-M., et al., 2006, FASEB J., 20, 1843- 1854), provides a mechanism whereby 5-HT ₂₆ receptor antagonists can affect any disorder where 5-HT may be abnormally functioning.

Additional uses of 5-HT ₂₆ receptor antagonists include the treatments of narclopesy or other conditions where increased wakefulness is required (Kantor, S, et al., 2004, Br J Pharmacol., 142, 1332-1342).

The present invention provides compounds with affinity for the 5-HT _2B receptor and which are capable of interfering with the affects of serotonin at these receptors. In a first aspect there is provided a compound which is 8-[4-(1 ,1-dioxidotetrahydro-2H- thiopyran-4-yl)-1-piperazinyl]-2-methylquinoline:

or a pharmaceutically acceptable salt thereof (hereinafter referred to as compounds of formula (I)).

In particular, the compound is 8-[4-(1 ,1-dioxidotetrahydro-2H-thiopyran-4-yl)-1- piperazinyl]-2-methylquinoline (E1 ).

In a further embodiment, the compound is 8-[4-(1 ,1-dioxidotetrahydro-2H-thiopyran- 4-yl)-1-piperazinyl]-2-methylquinoline hydrochloride (E2).

In a yet further embodiment, the compound is 8-[4-(1 ,1-dioxidotetrahydro-2H- thiopyran-4-yl)-1-piperazinyl]-2-methylquinoline dihydrochloride (E3).

In one embodiment of the invention, a method of treatment of fibrosis or a fibrotic condition is provided, which method comprises the administration to the mammal in need of such treatment, an effective amount of a compound of formula (I).

In one embodiment of the invention, the mammal in need of such treatment is human. In a further embodiment of the invention, the human is female.

As used herein, the term "fibrosis" refers to the formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to a formation of fibrous tissue as a normal constituent of an organ or tissue. Similarly, the term "fibrotic condition" refers to a disease or condition mediated by fibrosis. Examples of types of fibrotic conditions and associated conditions include but are not limited to cardiovascular disorders (e.g. valvular heart diseases, conditions associated with cardiac hypertrophy and pulmonary hypertension), lung fibrosis (e.g. after chronic obstructive pulmonary disease), liver fibrosis (e.g. precirrhotic alcoholic liver disease, nonalcoholic steatohepatitis/ fatty liver disease and hepatitis C), cystic fibrosis (such as cystic fibrosis of the pancreas and lungs), injection fibrosis (such as fibrotic conditions which can occur as a complication of intramuscular injections, especially in children), fibrosis which follows organ transplant or other therapeutic regimens, such as anti-cancer treatment with radiation, schleroderma, endomyocardial fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myleofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, cirrhosis, diffuse parenchymal lung disease, post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia and rheumatoid

arthritis. In one embodiment, the fibrotic condition is a cardiovascular disorder (e.g. valvular heart diseases, conditions associated with cardiac hypertrophy and pulmonary hypertension), lung fibrosis (e.g. after chronic obstructive pulmonary disease) or liver fibrosis (e.g. precirrhotic alcoholic liver disease, nonalcoholic steatohepatitis/ fatty liver disease and hepatitis C).

Antagonists of the 5-HT _2B receptor may also be useful in preventing, treating or ameliorating Gl tract disorders, for example IBS. 5-HT _2B receptor antagonists may also be useful in preventing, treating or ameliorating pain, for example inflammatory pain or visceral pain. In addition, 5-HT ₂₆ receptor antagonists may be useful in preventing, treating or ameliorating certain CNS disorders including migraine. 5-HT _2B receptor antagonists may also be useful in preventing, treating or ameliorating certain cardiovascular disorders including pulmonary arterial hypertension.

In one embodiment of the invention, a method of treatment of inflammatory pain in mammals is provided, which method comprises the administration to the mammal in need of such treatment, an effective amount of a compound of formula (I).

The cause of such inflammatory pain may be osteoarthritis or rheumatoid arthritis. There is therefore provided in one embodiment of the invention, a method of treatment of chronic articular pain associated with osteoarthritis or rheumatoid arthritis in mammals, which method comprises the administration to the mammal in need of such treatment, an effective amount of a compound of formula (I).

In another embodiment of the invention, a method of treatment of visceral pain in mammals is provided, which method comprises the administration to the mammal in need of such treatment, an effective amount of a compound of formula (I). In one embodiment of the invention, the visceral pain is associated with irritable bowel syndrome.

In another embodiment of the invention, a method of treatment of irritable bowel syndrome in mammals is provided, which method comprises the administration to the mammal in need of such treatment, an effective amount of a compound of formula

(I)-

In another embodiment of the invention, a method of treatment of headache (e.g. migraine) is provided, which method comprises the administration to the mammal in need of such treatment, an effective amount of a compound of formula (I).

As used herein, the term "pain" refers to any unpleasant sensation that is perceived by the individual and includes, but is not limited to, acute pain, chronic pain, somatic pain (originating from ligaments, tendons, bones, blood vessels or nerves), chronic articular pain, musculoskeletal pain, neuropathic pain, inflammatory pain, visceral

pain, pain associated with cancer, pain associated with migraine, tension headache and cluster headaches, pain associated with functional bowel disorders, lower back and neck pain, pain associated with sprains and strains, sympathetically maintained pain; myositis, pain associated with influenza or other viral infections such as the common cold, pain associated with rheumatic fever, pain associated with myocardial ischemia, post operative pain, cancer chemotherapy, headache, toothache and dysmenorrhea.

As used herein, the term "inflammatory pain" refers to any kind of pain that results from the inflammation of bodily tissues and includes, but is not limited to, inflammation resulting from soft tissue damage or infection.

As used herein, the term "neuropathic pain" refers to any kind of pain that results from injury or disease to the nerve tissue itself and includes, but is not limited to: diabetic neuropathy, sciatica, non-specific lower back pain, trigeminal neuralgia, multiple sclerosis pain, fibromyalgia, HIV-related neuropathy, post-herpetic neuralgia, trigeminal neuralgia, and pain resulting from physical trauma, amputation, phantom limb syndrome, spinal surgery, cancer, toxins or chronic inflammatory conditions. In addition, neuropathic pain conditions include pain associated with normally non- painful sensations such as "pins and needles" (paraesthesias and dysesthesias), increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static, thermal or cold allodynia), increased sensitivity to noxious stimuli (thermal, cold, mechanical hyperalgesia), continuing pain sensation after removal of the stimulation (hyperpathia) or an absence of or deficit in selective sensory pathways (hypoalgesia).

As used herein, the term "visceral pain" refers to any kind of pain that originates from the body's internal cavities or organs and includes, but is not limited to, pain that originates from the intestines.

As used herein, the term "Irritable Bowel Syndrome" (IBS) is defined according to the Rome III diagnostic criteria where the criteria are fulfilled for the last 3 months with symptom onset at least 6 months prior to diagnosis. The Rome III diagnostic criteria for IBS are as follows: - Recurrent abdominal pain or discomfort (where discomfort means an uncomfortable sensation not described as pain) at least 3 days per month in the last 3 months associated with 2 or more of the following:

1. Improvement with defecation;

2. Onset associated with a change in frequency of stool; 3. Onset associated with a change in form (appearance) of stool.

Other symptoms that are not essential but support the diagnosis of IBS include:

Abnormal stool frequency (greater than 3 bowel movements/day or less than 3 bowel movements/week);

Abnormal stool form (lumpy/hard or loose watery stool); - Abnormal stool passage (straining, urgency, or feeling of incomplete bowel movement): Passage of mucous; - Bloating or feeling of abdominal distension.

As used herein, the term "headache" refers to any unpleasant sensation that is localised to the individual's head and includes, but is not limited to, migraine, tension headache and cluster headaches.

The term "treatment" or "treating" as used herein includes the treatment of established disorders and also includes the prophylaxis thereof. The term "prophylaxis" is used herein to mean preventing symptoms in an already afflicted subject or preventing recurrence of symptoms in an afflicted subject and is not limited to complete prevention of an affliction.

Compounds of the present invention interact with the 5-HT _2B receptor and are capable of interfering with the effects of serotonin at the 5-HT _2B receptor. Such compounds may be competitive antagonists, inverse agonists, or negative allosteric modulators.

Compounds of formula (I) exhibit 100 fold selectivity for the 5-HT ₂₆ receptor over certain other serotonin receptors (e.g. 5-HT _2A and 5-HT _2C ).

Certain compounds of formula (I) may in some circumstances form acid addition salts thereof. It will be appreciated that for use in medicine compounds of formula (I) may be used as salts, in which case the salts should be pharmaceutically acceptable. Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse , J. Pharm. ScL, 1977, 66, 1-19. The term "pharmaceutically acceptable salts" includes salts prepared from pharmaceutically acceptable acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.

Examples of pharmaceutically acceptable salts include those formed from maleic, fumaric, benzoic, ascorbic, pamoic, succinic, hydrochloric, sulfuric, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and nitric acids.

The compounds of formula (I) may be prepared in crystalline or non-crystalline form, and, if crystalline, may optionally be solvated, e.g. as the hydrate. This invention includes within its scope stoichiometric solvates (e.g. hydrates) as well as compounds containing variable amounts of solvent (e.g. water).

The subject invention also includes isotopically-labeled compounds, which are identical to those recited in formula (I) and following, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, fluorine, such as 3H, 1 1 C, 14C and 18F.

Compounds of formula (I) that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically- labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H, 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. 1 1 C and 8F isotopes are particularly useful in PET (positron emission tomography). PET is useful in brain imaging. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances, lsotopically labeled compounds of formula (I) and following of this invention can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless explicitly stated otherwise.

According to a further aspect of the invention there is provided a compound of formula (I) for use in therapy.

According to a further aspect of the invention there is provided a compound of formula (I) for use in the treatment of fibrosis or a fibrotic condition, Gl tract disorders, for example IBS, pain, for example inflammatory pain or visceral pain, headache, for example migraine, cardiovascular disorders including pulmonary arterial hypertension. In one embodiment, there is provided a compound of formula (I) for use in the treatment of fibrosis or a fibrotic condition.

In order to use a compound of formula (I) for the treatment of humans and other mammals it is normally formulated in accordance with standard pharmaceutical

practice as a pharmaceutical composition. Therefore in another aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (I), for use in human or veterinary medicine.

In order to use the compounds of formula (I) in therapy, they will normally be formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice. The present invention also provides a pharmaceutical composition, which comprises a compound of formula (I), and optionally a pharmaceutically acceptable carrier.

A pharmaceutical composition of the invention, which may be prepared by admixture, suitably at ambient temperature and atmospheric pressure, is usually adapted for oral, parenteral or rectal administration and, as such, may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable or infusable solutions or suspensions or suppositories. Orally administrable compositions are generally preferred.

Tablets and capsules for oral administration may be in unit dose form, and may contain conventional excipients, such as binding agents, fillers, tabletting lubricants, disintegrants and acceptable wetting agents. The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and, if desired, conventional flavourings or colourants.

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

The composition may contain from 0.1% to 99% by weight, preferably from 10 to 60% by weight, of the active material, depending on the method of administration.

The dose of the compound used in the treatment of the disorders mentioned herein will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses may be 0.05 to 1000 mg, more suitably 0.05 to 200 mg; and such unit doses will preferably be administered once a day, although administration more than once a day may be required; and such therapy may extend for a number of weeks, months or even years. In addition, such therapy could be given on demand, prophylactically, or continuously over a period of time until the patient no longer requires treatment.

The present invention also provides a process for the preparation of a compound of formula (I) which process comprises:

(a) oxidation of a compound of formula (II)

; or

(b) reacting a compound of formula (III)

(III) wherein L ¹ represents a suitable leaving group such as chlorine or bromine, with a compound of formula (IV) .

(IV)

Process (a) may be carried out using a suitable oxidizing agent such as Oxone™ (potassium peroxymonosulfate) or magnesium monoperoxyphthalate.

Process (b) may be carried out by reaction of a compound of formula (III) with a compound of formula (IV) in the presence of suitable reagents such as tris(dibenzylideneacetone)dipalladium(0), 2-dicyclohexylphosphino-2'-(N,N- dimethylamino)biphenyl, sodium t-butoxide and 1 ,4-dioxane.

Compounds of formula (II) may be prepared in accordance with the following scheme:

Step (ii)

Scheme 1 wherein L ¹ is as defined above and P ¹ represents a suitable protecting group such as Boc.

Examples of protecting groups and the means for their removal can be found in T. W. Greene 'Protective Groups in Organic Synthesis' (J. Wiley and Sons, 1991 ). Suitable amine protecting groups include sulfonyl (e.g. tosyl), acyl (e.g. acetyl, 2',2',2'- trichloroethoxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl) and arylalkyl (e.g. benzyl), which may be removed by hydrolysis (e.g. using an acid such as hydrochloric acid in 1 ,4-dioxane or trifluoroacetic acid in dichloromethane) or reductively (e.g. hydrogenolysis of a benzyl group or reductive removal of a 2',2',2'- trichloroethoxycarbonyl group using zinc in acetic acid) as appropriate. Other suitable amine protecting groups include trifluoroacetyl (-COCF ₃ ) which may be removed by base catalysed hydrolysis (e.g. treatment with ammonia in methanol) or a solid phase resin bound benzyl group, such as a Merrifield resin bound 2,6-dimethoxybenzyl

group (Ellman linker), which may be removed by acid catalysed hydrolysis, for example with trifluoroacetic acid.

Step (i) typically comprises analogous conditions to those described above for process (b).

Step (ii) typically comprises a deprotection reaction in the presence of a strong acid such as hydrochloric acid, or TFA, and a suitable solvent such as methanol or DCM.

Step (iii) typically comprises reaction of a compound of formula (VII) with a compound of formula (VIII) in the presence of suitable reagents such as acetic acid, sodium triacetoxyborohydride and a suitable solvent such as 1 ,2-dichloroethane.

Compounds of formula (IV) may be prepared in accordance with the following scheme:

Scheme 2 wherein P ¹ is as defined above.

Step (i) typically comprises reacting a compound of formula (V) with a compound of formula (VIII) in an analogous manner to that described for Step (iii) in Scheme 1.

Step (ii) typically comprises an oxidation reaction in an analogous manner to that described for process (a) above.

Step (iii) typically comprises a deprotection reaction in an analogous manner to that described for Step (ii) in Scheme 1.

Compounds of formula (III), (V) and (VIII) are either known or may be prepared in accordance with known procedures.

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

Intermediate 1

8-Bromo-2-methylquinoline (D1)

Toluene (60 mL) and crotonaldehyde (5.01 ml_, 60.5 mmol) were added to a solution of 2-bromoaniline (5.2 g, 30.2 mmol) in aqueous 6M HCI (200 mL) at 100 ⁰ C for 2 hours. The mixture was then allowed to cool to room temperature. The aqueous layer was separated and neutralised with 6M NaOH solution (200 mL). After extraction with DCM (2 x 100 mL), the organic layers were combined, dried over MgSO ₄ , filtered and evaporated to afford a brown oil which was purified by flash chromatography using the Biotage SP4 (40+M) eluting with 0% to 30% EtOAc/40-60 petroleum ether. The product containing fractions were combined and concentrated in vacuo to afford 5.3 g of an orange solid which was repurified by flash chromatography using the Biotage SP4 (40+M) eluting with 0% to 10% Et ₂ O/40-60 petroleum ether. The product containing fractions were combined and concentrated in vacuo to yield the title compound as a white solid (3.914 g). 1 H NMR (CDCI ₃ , 400MHz): δ ppm 8.02 (2H, m), 7.74 (1 H, dd, J=8.0, 1.5 Hz), 7.33 (2H, m), 2.82 (3H, s). Mass Spectrum (ESI): Ci ₀ H ₈ BrN requires 221/223; found 222/224 (MH ⁺ ).

Intermediate 2 1,1-Dimethylethyl-4-(2-methyl-8-quinolinyl)-1-piperazinecarb oxylate (D2)

METHOD 1

Three separate microwave reactions were carried out, each containing 8-bromo-2- methylquinoline (1.164 g, 5.24 mmol) (Intermediate 1 ), 1-Boc-piperazine (1.073 g, 5.77 mmol), tris(dibenzylideneacetone)dipalladium(0) (240 mg, 0.262 mmol), 2- dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl (309 mg, 0.786 mmol), sodium tert-butoxide (705 mg, 7.34 mmol) and degassed 1 ,4-dioxane (15 mL). Each vial was heated in the microwave at 120 ⁰ C for 15 minutes. The three reactions were combined and filtered through celite, washing with 1 ,4-dioxane. The filtrate was concentrated in vacuo to afford a sticky red oil which was purified by flash chromatography using the Biotage SP4 (65M) eluting with 0% to 30% EtOAc/40-60 petroleum ether. The product containing fractions were combined and concentrated in vacuo to afford the title compound as a yellow oil (4.19 g). 1 H NMR (CDCI ₃ , 400MHz): δ ppm 8.00 (1 H, d, J=8.5 Hz), 7.39 (2H, m), 7.26 (1 H, m), 7.08 (1 H, dd, J=7.0, 1.5 Hz), 3.77 (4H, t, J=5.0 Hz), 3.36 (4H, t, J=5.0 Hz), 2.74 (3H, s), 1.51 (9H, s). Mass Spectrum (ESI): Ci ₉ H ₂₅ N ₃ O ₂ requires 327; found 328 (MH ⁺ ).

METHOD 2

8-Chloro-2-methylquinoline (1.0 g, 5.63 mmol), 1 , 1 -dimethylethyl-1 - piperazinecarboxylate (1.153 g, 6.19 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.258 g, 0.281 mmol), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl (0.332 g, 0.844 mmol) and sodium tert-butoxide (0.757 g, 7.88 mmol) were weighed into three different microwave vials, each one with 1 ,4-dioxane (15 ml.) under argon. Each vial was heated in the microwave at 120 ⁰ C for 10 minutes. The three reaction mixtures were combined, filtered through celite washing with 1 ,4-dioxane, and concentrated in vacuo to afford a brown oil which was purified by flash chromatography using the Biotage SP4 (65M) eluting with 0% to 25% EtOAc/40-60 petroleum ether. The product containing fractions were combined and concentrated in vacuo to yield the title compound as an orange oil (5.428 g). 1 H NMR (CDCI ₃ , 400MHz): δ ppm 8.00 (1 H, d, J=8.0 Hz), 7.39 (2H, m), 7.26 (1 H, m), 7.08 (1 H, dd, J=7.0, 2.0 Hz), 3.77 (4H, t, J=5.0 Hz), 3.36 (4H, t, J=5.0 Hz), 2.74 (3H, s), 1.51 (9H, s). Mass Spectrum (ESI): Ci ₉ H ₂₅ N ₃ O ₂ requires 327; found 328 (MH ⁺ ).

METHOD 3 Palladium(ll) acetate (0.36 g, 1.6 mmol), BINAP (1.50 g, 2.41 mmol) and caesium carbonate (7.84 g, 24.07 mmol) were combined in 1 ,4-dioxane (100 ml.) under argon and the solution was sonicated for 45 minutes. N-Boc-piperazine (4.48 g, 24.07 mmol) and 8-chloro-2-methylquinoline (2.85 g, 16.04 mmol) were added and the reaction mixture was heated under reflux overnight. The reaction was then cooled and the solvent removed under reduced pressure. The residue was partitioned between ethyl acetate (150 ml.) and water (150 ml.) and the organic layer was separated and washed with water (200 ml.) and then brine (200 ml_). The organic layer was then dried (Na ₂ SO ₄ ), filtered and concentrated to give a brown oil. This was purified by flash column chromatography using a Biotage SP4 system. The crude material was loaded onto a 65i silica column and eluted with a gradient of 0-50% ethyl acetate/petroleum ether (40-60) as solvent. Evaporation of the required fractions provided the title compound as a yellow oil (2.33 g).

1 H NMR (CDCI ₃ , 400MHz): δ ppm 8.00 (1 H, d, J=8.5 Hz), 7.39 (2H, m), 7.26 (1 H, m), 7.09 (1 H, dd, J=7.0, 1.5 Hz), 3.77 (4H, t, J=5.0 Hz), 3.36 (4H, t, J=5.0 Hz), 2.74 (3H, s), 1.51 (9H, s).

Mass Spectrum (ESI): Ci ₉ H ₂₅ N ₃ O ₂ requires 327; found 328 (MH ⁺ ).

Intermediate 3

2-Methyl-8-(1-piperazinyl)quinoline hydrochloride (D3)

To a solution of 1 ,1-dimethylethyl 4-(2-methyl-8-quinolinyl)-1-piperazinecarboxylate (5.428 g, 16.58 mmol) (Intermediate 2) in methanol (10 ml.) was added hydrochloric acid (10 ml_, 40.0 mmol, 4M in 1 ,4-dioxane). The mixture was stirred at room temperature under argon overnight. The reaction mixture was concentrated in vacuo then dried in the vacuum oven to afford the title compound as a yellow powder (3.558 g)-

1 H NMR (CD ₃ OD, 400MHz): δ ppm 9.08 (1 H, d, J=8.5 Hz), 8.14 (2H, m), 8.02 (1 H, d, J=8.5 Hz), 7.95 (1 H, t, J=8.0 Hz), 3.66 (4H, m), 3.37 (4H, t, J=5.0 Hz), 3.18 (3H, s). Mass Spectrum (ESI): Ci ₄ H ₁₇ N ₃ requires 227; found 228 (MH ⁺ ).

Intermediate 4

2 -M ethyl -8 -(1 -piperazinyl)quinoline (D4)

METHOD 1 2-Methyl-8-(1-piperazinyl)quinoline hydrochloride (2.558 g, 9.70 mmol) (Intermediate 3) was dissolved in methanol and loaded onto an SCX cartridge (50 g) which was then washed with methanol. The product was eluted using 2M NH ₃ /MeOH which was then concentrated in vacuo to afford the title compound as a yellow oil (1.804 g). 1 H NMR (CDCI ₃ , 400MHz): δ ppm 7.99 (1 H, d, J=8.5 Hz), 7.38 (2H, m), 7.24 (1 H, d, J=8.5 Hz), 7.10 (1 H, m), 3.39 (4H, m), 3.23 (4H, m), 2.74 (3H, s). Mass Spectrum (ESI): C ₁₄ H ₁₇ N ₃ requires 227; found 228 (MH ⁺ ).

METHOD 2

To a solution of 1 ,1-dimethylethyl 4-(2-methyl-8-quinolinyl)-1-piperazinecarboxylate (4.19 g, 12.80 mmol) (Intermediate 2) in methanol (10 ml.) was added hydrogen chloride (32.0 ml_, 128 mmol, 4M in 1 ,4-dioxane). This reaction was exothermic and as a consequence was cooled in ice for 10 minutes after addition. The mixture was stirred at room temperature under argon for 1 hour. The mixture was concentrated in vacuo then dried in the vacuum oven overnight to yield a yellow solid which was dissolved in methanol then loaded onto an SCX cartridge (50 g) which was then washed with methanol. The product was eluted using 2M NH ₃ /MeOH which was then concentrated in vacuo then dried overnight in the vacuum oven to afford the title compound as a yellow oil (2.504 g).

1 H NMR (CDCI ₃ , 400MHz): δ ppm 7.99 (1 H, d, J=8.5 Hz), 7.38 (2H, m), 7.24 (1 H, d, J=8.5 Hz), 7.10 (1 H, m), 3.39 (4H, m), 3.23 (4H, m), 2.74 (3H, s). Mass Spectrum (ESI): C ₁₄ H ₁₇ N ₃ requires 227; found 228 (MH ⁺ ).

METHOD 3

1 , 1 -dimethylethyl-4-(2-methyl-8-quinolinyl)-1 -piperazinecarboxylate (intermediate 2) (3.43 g, 10.5 mmol) was taken up in dichloromethane (80 mL) and cooled in an ice- bath. Trifluoroacetic acid (20 mL) was added slowly and the reaction was left to stir for 30 minutes before the ice-bath was removed. After a total of 90 minutes the solvent was removed under reduced pressure and the residue was taken up in dichloromethane (100 mL) and saturated NaHCC>3 (200 mL) was added slowly. The organic layer was separated and the aqueous was extracted with further dichloromethane (100 mL). The combined organics were dried (Na ₂ SO ₄ ), filtered and concentrated to leave an orange oil. This was purified by flash column chromatography using a Biotage SP4 system. The crude material was loaded onto a 40+M silica column and eluted with a gradient of 0-10% 2M methanolic ammonia in dichloromethane. Evaporation of the required fractions provided the title compound as a yellow foam (3.43 g). 1 H NMR (CDCI ₃ , 400MHz): δ ppm 7.99 (1 H, d, J=8.5 Hz), 7.38 (2H, m), 7.24 (1 H, d, J= 8.5 Hz), 7.10 (1 H, m), 3.45 (4H, m), 3.29 (4H, m), 2.74 (3H, s). Mass Spectrum (ESI): C ₁₄ H ₁₇ N ₃ requires 227; found 228 (MH ⁺ ).

Intermediate 5 2-Methyl-8-[4-(tetrahydro-2H-thiopyran-4-yl)-1 -piperazinyl]quinoline (D5)

METHOD 1

To a solution of 2-methyl-8-(1-piperazinyl)quinoline (4.308 g, 18.95 mmol) (Intermediate 4) in 1 ,2-dichloroethane (100 mL) was added molecular sieves (10 g), tetrahydro-4H-thiopyran-4-one (3.30 g, 28.4 mmol) and acetic acid (1.085 mL, 18.95 mmol). The mixture was stirred at room temperature for 30 minutes. Then the sodium triacetoxyborohydride (8.03 g, 37.9 mmol) was added carefully. The mixture was then stirred at room temperature under argon overnight. Saturated aqueous NaHCO ₃ (100 mL) was slowly added and the mixture was stirred for 20 minutes. The organic phase was isolated using a phase separation cartridge. It was then concentrated in vacuo to afford an orange oil which was purified by flash chromatography using the Biotage SP4 (65M) eluting with 0% to 5% (2M NH ₃ /MeOH)/DCM. The product containing fractions were combined, concentrated in vacuo then dried on the high vacuum for 30 minutes to afford the title compound as a yellow gum (6.496 g).

1 H NMR (CDCI ₃ , 400MHz): δ ppm 7.98 (1 H, d, J=8.5 Hz), 7.37 (2H, m), 7.24 (1 H, d, J=8.5 Hz), 7.09 (1 H, m), 3.44 (4H, bs), 2.94 (4H, t, J=5.0 Hz), 2.74 (7H, m), 2.46 (1 H, m), 2.26 (2H, m), 1.81 (2H, m). Mass Spectrum (ESI): C ₁₉ H ₂₅ N ₃ S requires 327; found 328 (MH ⁺ ).

METHOD 2

In a carousel tube 2-methyl-8-(1-piperazinyl)quinoline (Intermediate 4) (250 mg, 1.100 mmol), tetrahydro-4H-thiopyran-4-one (192 mg, 1.650 mmol) and acetic acid (0.063 ml, 1.10 mmol) were dissolved in 1 ,2-dichloroethane (6 ml). The reaction mixture was stirred at room temperature for 30 min and then sodium triacetoxyborohydride (466 mg, 2.20 mmol) was added. The reaction was stirred at room temperature over the weekend. Saturated NaHCO ₃ (6 ml.) was added and the reaction was stirred for -20 minutes. The organic layer was separated using a phase separation cartridge and the solvent was removed under reduced pressure. This provided the title compound as a yellow oil (350 mg) pure enough for use in the next step.

1 H NMR (CDCI ₃ , 400MHz): δ ppm 7.98 (1 H, d, J=8.5 Hz), 7.37 (2H, m), 7.24 (1 H, d, J=8.5 Hz), 7.09 (1 H, m), 3.44 (4H, bs), 2.94 (4H, m), 2.74 (7H, m), 2.46 (1 H, m), 2.26 (2H, m), 1.81 (m, 2H).

Mass Spectrum (ESI): C ₁₉ H ₂₅ N ₃ S requires 327; found 328 (MH ⁺ ).

Example 1

8-[4-(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-1 -piperazinyl]-2-methylquinoline

METHOD 1

A solution of 2-methyl-8-[4-(tetrahydro-2H-thiopyran-4-yl)-1-piperazinyl]q uinoline (4.684 g, 14.30 mmol) (Intermediate 5) in methanol (95 ml.) and water (95 ml.) was treated with oxone (26.4 g, 42.9 mmol) and the mixture was stirred at 45 ⁰ C for 2.5 hours. The mixture was allowed to cool to room temperature then quenched by adding slowly 10% aqueous sodium sulfite (95 ml_). The mixture was stirred for 30 minutes (until no over-oxidised product could be seen by LC/MS). The methanol was removed in vacuo then the remaining mixture was diluted with water (400 ml.) and washed with DCM (3x400 ml_). The aqueous layer was saturated with solid NaCI then washed with EtOAc (3x250 ml_). Further ethyl acetate (-200 ml.) was then added to the aqueous layer and it was basified by adding solid sodium carbonate. The basic mixture was stirred for 10 minutes during which time a solid formed at the organic/aqueous interface. This solid was isolated by filtration and dried in the vacuum oven. The filtrate was transferred to a separating funnel and the ethyl acetate layer was isolated, dried over MgSO ₄ , filtered and concentrated in vacuo to afford a first batch of the crude product as a pale yellow solid. The solid initially

isolated by filtration was suspended in ethyl acetate (100 mL) and shaken vigorously with water (30 mL). The ethyl acetate layer was isolated, dried over MgSO ₄ , filtered and concentrated in vacuo to afford a second batch of crude product as a white solid. Toluene was heated to boiling point, 40 mL of this was added to the first batch of crude product, this was reheated to boiling point and swirled until all the material had dissolved. This solution was then pipetted into the flask containing the second batch of crude product. The mixture was brought back to boiling point and a further 25 mL of hot toluene was added. The resultant mixture was brought to boiling and agitated until fully dissolved. The solution was concentrated with heating under a slow argon stream to -25 mL. The mixture was then allowed to cool to room temperature during which time crystals formed. The crystals were isolated by filtration, washing with cold toluene followed by 1 :1 40-60 petroleum ether/diethyl ether. The resultant solid was dried overnight in the vacuum oven to afford an off-white solid. This was then purified by flash chromatography using the Biotage SP4 (40+M) eluting with 0% to 5% (2M NH ₃ /MeOH)/DCM. The product containing fractions were combined and concentrated in vacuo to afford the title compound as an off-white solid (3.106 g).

1 H NMR (CDCI ₃ , 400MHz): δ ppm 8.00 (1 H, d, J=8.5 Hz), 7.39 (2H, m), 7.26 (1 H, d, J=7.1 Hz), 7.09 (1 H, dd, J=6.5, 2.0 Hz), 3.45 (4H, bs), 3.30 (2H, m), 2.96 (2H, m), 2.89 (4H, t, J=5.0 Hz), 2.73 (3H, s), 2.64 (1 H, m), 2.39 (2H, m), 2.25 (2H, m). Mass Spectrum (ESI): Ci ₉ H ₂₅ N ₃ O ₂ S requires 359; found 360 (MH ⁺ ).

METHOD 2

Magnesium monoperoxyphthalate (1.06 g, 2.137 mmol) was dissolved in dichloromethane (8 mL) and methanol (2 ml). 2-methyl-8-[4-(tetrahydro-2H-thiopyran- 4-yl)-1-piperazinyl]quinoline (Intermediate 5) (350 mg, 1.069 mmol) dissolved in dichloromethane (5 mL) was added dropwise over -20 minutes. The reaction was stirred at room temperature for 4 hours. 10% w/v aqueous sodium sulfite (10 mL) was added slowly. Dichloromethane (8 mL) was added followed by saturated NaHCO ₃ (4 mL). The mixture was stirred for 20 minutes and then the organic layer was separated. This was washed with water (15 mL) and then dried (Na ₂ SO ₄ ), filtered and concentrated to give the crude product. Purification was undertaken by flash column chromatograhpy using a Biotage SP4 system. The crude material was loaded onto a 25+M silica column and eluted with a gradient of 0-5% 2M NH ₃ /MeOH in dichloromethane as solvent. Evaporation of the solvent provided the title compound as a yellow solid (123 mg).

1 H NMR (CDCI ₃ , 400MHz): δ ppm 8.00 (1 H, d, J=8.5 Hz), 7.39 (2H, m), 7.26 (1 H, d, J=7.1 Hz), 7.09 (1 H, dd, J=6.5, 2.0 Hz), 3.45 (4H, bs), 3.30 (2H, m), 2.96 (2H, m), 2.89 (4H, t, J=5.0 Hz), 2.73 (3H, s), 2.64 (1 H, m), 2.39 (2H, m), 2.25 (2H, m). Mass Spectrum (ESI): C ₁₉ H ₂₅ N ₃ O ₂ S requires 359; found 360 (MH ⁺ ).

Example 2

8-[4-(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-1 -piperazinyl]-2-methylquinoline hydrochloride (E2)

8-[4-(1 ,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-1-piperazinyl]-2-met hylquinoline (123 mg) (Example 1 ) was dissolved in dichloromethane (-0.5 ml.) and treated with 1 M HCI in diethyl ether (0.4 ml.) and further diethyl ether (-0.5 ml_). The resulting yellow solid was triturated and filtered to leave the HCI salt which was further purified by MDAP. Evaporation of the solvent provided the product (formate salt) which was dissolved in methanol (-0.5 ml.) and treated with 1 M HCI in diethyl ether (0.35 ml_). The resulting yellow solid was triturated and filtered to leave title compound as a yellow solid (50 mg).

1 H NMR (CD ₃ OD, 400MHz): δ ppm 9.08 (1 H, d, J=8.5 Hz), 8.16 (2H, m), 8.02 (1 H, d, J=8.5 Hz), 7.95 (1 H, t, J=8.0 Hz), 4.07 (2H, bt, J=10.5 Hz), 3.84 (1 H, m), 3.68 (2H, bd, J=12 Hz), 3.59 (2H, bt, J=12 Hz), 3.23 (3H, s), 2.74 (2H, m, partially obscured by residual DMSO), 3.50-3.25 (6H ,m), 2.46 (2H, bq, J=13.0 Hz).

Mass Spectrum (ESI): Ci ₉ H ₂₅ N ₃ O ₂ S requires 359; found 360 (MH ⁺ ).

Example 3

8-[4-(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-1 -piperazinyl]-2-methylquinoline dihydrochloride (E3)

8-[4-(1 ,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-1-piperazinyl]-2-met hylquinoline (3.1g) (Example 1 ) was dissolved in the minimum of DCM and treated with 1.1 eq 1 M HCI/Et ₂ O, further 1 M HCI/Et ₂ O was added until the solution was acidic, further Et ₂ O was added to precipitate the hydrochloride salt which was isolated by filtration washing with Et ₂ O followed by hexane. The resultant powder was dried on the high vacuum for 30 minutes then taken up in boiling ethanol (-250 ml.) with methanol (-25 ml.) to aid solubility. The solution was heated to remove the methanol, the product was then left to crystallise out of the mixture over the weekend. The solid was isolated by filtration, washing with cold ethanol followed by Et ₂ O then hexane

and then dried in the drying pistol at 60 ⁰ C for 3 days in total to afford the title compound as a pale yellow solid (2.26 g).

Compound was found by elemental analysis to be the dihydrochloride salt.

1 H NMR {DMSO-de, 500MHz): δ ppm 11.70 (1 H, bs), 8.26 (1 H, bs), 7.57 (1 H, d, J=6.9 Hz), 7.46 (2H, m), 7.20 (1 H, bs), 4.10 (2H, bs), 3.69 (1 H, t, J=10.5 Hz), 3.57 (2H, d, J=10.3 Hz), 3.51 (2H, m), 3.41 (2H, t, J=11.5 Hz), 3.33 (4H, m), 2.71 (3H, bs), 2.68 (2H, m), 2.25 (2H, m). Mass Spectrum (ESI): C ₁₉ H ₂₅ N ₃ O ₂ S requires 359; found 360 (MH ⁺ ).

Pharmacological data

Compounds of the invention may be tested for in vitro biological activity at the 5- HT _2A , 5-HT _2B and 5-HT _2C receptors in accordance with the following studies:

Cell culture

Adherent SH-SY5Y cells stably expressing the recombinant human 5-HT _2A , 5-HT _2B or 5-HT _2C were maintained in culture at 37°C under 5% CO ₂ in Dulbecco's Modification of Eagle's Medium supplemented with 10% dialysed foetal calf serum and 400 micrograms geneticin. The cloning of human 5-HT _2A and 5-HT _2C receptors (previously denoted as 5-HT ₂ and 5-HT _1c receptors respectively) is described by Saltzman et al., Biochemical and Biophysical Research Communications (1991 ) Vol.181 No.3, 1469- 1478. The cloning of the human 5-HT _2B receptors is described by Schmuck et al., FEBS Letters (1994) Vol.342, 85-90. SH-SY5Y cells are commercially available from the American Type Culture Collection (ATCC), catalogue number CRL-2266.

Measurement of [CaI using the FLIPR

SH-SY5Y cells, separately expressing 5-HT _2A , 5-HT _2B or 5-HT _2C receptors, were seeded into black walled clear-base 384-well plates at a density of 16,000 cells per well and cultured overnight at 37°C under 5% CO ₂ . Media was aspirated off and cells were then incubated with Tyrode's medium (in mM; NaCI 145, KCI 2.5, HEPES 10, Glucose 10, MgCI ₂ 1.2, CaCI ₂ 1.5) containing the cytoplasmic calcium indicator, Fluo- 4 in the acetylmethyl form (4 mM) and 25OuM Brilliant Black (Molecular Devices) at 37°C for 60 min. The loaded cells were then incubated for 30 min at 37°C with either buffer (agonist mode) or compound (antagonist mode). The plates were then placed into a FLIPR (Molecular Devices, UK) to monitor cell fluorescence (λex 488nm, λem 540nm) before and after the addition of various compounds (in agonist mode) or a pre-determined concentration of 5-HT (approximately 4xEC50) for testing in antagonist mode. Functional Ki values were calculated from IC50 values in accordance with the following equation:

fKi = IC50 i

1 + agonist cone (M)

EC50

The functional pKi (fpKi) values can then be calculated from the negative Iog10 of the fKi values.

The compounds of Examples 1-2 were tested in certain of the above assays and found to have the fpKi values shown in the following table:

Example 1 was also tested in the 5HT _2C assay and displayed a plC50 value < 5.0. Example 2 was tested in the 5HT _2A and 5HT _2C assays and displayed a plC50 value < 5.0.