Field of the Invention

The invention relates to novel compounds and compositions that are useful as selective P2-adrenoceptor (P2-AR) antagonists and their use in the treatment and prevention of disease.

Background

P-adrenoceptors (P-AR) are a family of G protein-coupled receptors (Pi, P2, and P3) that mediate physiological responses to epinephrine (adrenaline) and norepinephrine (noradrenaline). Pi and P2 are the dominant types of p-AR. P1-AR are mainly in the heart and P2-AR are mainly on blood vessels and in the airways. Therefore, P1-AR and P2-AR are associated with different physiological effects and accordingly different diseases.

Non-selective p-AR antagonists (“P-blockers”), such as propranolol and nadolol, inhibit both P1-AR and P2-AR, and can be used to treat effects resulting from P1-AR activation, such as anxiety, angina and hypertension, and effects resulting from P2-AR activation such as physiological tremor, and migraine. Selective P1-AR antagonists, such as atenolol and metoprolol, inhibit P1-AR to a greater extent than P2-AR and are used to treat cardiovascular conditions, for example hypertension, chronic stable angina, and post-myocardial infarction. Selective P2-AR antagonists inhibit P2-AR to a greater extent than P1-AR. No selective P2-AR antagonist has ever been marketed for clinical use to date and currently, propranolol is often used in place of a selective P2-AR antagonist. A disadvantage of the use of propranolol as a P2-AR antagonist is that P1-AR antagonism is associated with various side-effects.

ICI 118,551 has been shown to be a highly selective P2-AR antagonist but has never been marketed.

Receptor selectivity is generally sought because adverse effects are often attributed to off-target drug actions. Thus, it is desirable to improve selectivity to increase affinity for a target receptor and decrease affinity for off-target receptors. Additionally, with increased selectivity there is a reduced risk of drug-drug interactions, which are a common cause of side-effects and can reduce pharmacological activity of one of the drugs. Generally, high selectivity and potency also allow doses to be minimised, reducing the cost of medication and increasing the ease of administration. In recent years, it has been found that the P2-AR signaling pathways are linked with certain diseases and thus that a selective P2-AR antagonist could be beneficial in their treatment. Those diseases include a tumour, such as a vascular tumour, cancer, paraganglioma and tuberous sclerosis; a vascular tumour, such as infantile haemangioma, von Hippel-Lindau disease, angiosarcoma, and glioma; cancer, such as soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, and lymphoblastic leukaemia; a vascular abnormality such as hereditary haemorrhagic telangiectasia, and cerebral cavernous malformations; respiratory disease, such as asthma and chronic obstructive pulmonary disease; cardiovascular disease, such as chronic heart failure and takotsubo syndrome; and other indications such as physiological tremor and migraine.

A selective P2-AR antagonist could be beneficial in the treatment of those conditions. Thus, there is an unmet need for a highly selective P2-AR antagonist.

EP 0003664 and GB 2165835 describe alkanolamine derivatives as selective P2-AR antagonists.

ICI 118,551 (DL-erythro-3-isopropylamino-1-(7-methyl-4-indanyloxy)-2-but anol hydrochloride), which is considered to be the ‘gold-standard’ selective P2-AR antagonist, is reported to have 100 times greater selectivity for the P2- over the P1-AR (Kawakami K et al, British Journal of Pharmacology 2006; 147: 642-652; Mauriege P, et al. Journal of Lipid Research 1988; 29: 587-601).

ICI 118,551 has the structure:

In preclinical trials, when compared with propranolol, ICI 118,551 was found to be more potent at the P2-AR than the P1-AR in vitro and in vivo. ICI 118,551 was also found to be highly selective for the P2-AR. Nor did it affect other systems, such as the central nervous system, reproductive system and gastro-intestinal system. In clinical trials in healthy subjects, ICI 118,551 proved to be a highly selective P2-AR antagonist at doses of 2.5 to 40 mg. However, at higher doses, ICI 118,551 became increasingly active at the P1-AR.

In phase 2 studies of patients with hypertension, anxiety, physiological tremor, general neurotic syndrome, flight phobia stress, and neuroleptic-induced akathisia, ICI 118,551 inhibited tremor and neuroleptic-induced akathisia similar to propranolol, had minimal effect on flight phobia stress, and had no effect on the other conditions. ICI 118,551 was not developed further and has never been marketed for clinical use.

Upon administration, multiple different metabolites of ICI 118,551 are formed (French, K.H. Journal of Liquid Chromatography 1989; 12:861-873). Of those metabolites, three are known to be selective P2-AR antagonists, but it is generally understood that they are less selective than ICI 118,551 (Fitzgerald, J.D. Cardiovascular Drugs and Therapy 1991 ; 5(3): 561-576).

Therefore, there remains an unmet need for a selective P2-AR antagonist for clinical use.

Surprisingly, compounds of Formula (I) or (II), or pharmaceutically acceptable salts thereof, have been found to act as selective antagonists of P2-AR over P1-AR:

The compound of Formula (I) has SSR stereochemistry, while the compound of Formula (II) has SSS stereochemistry, as shown above.

In addition, these compounds have been shown to be more selective for the p-AR over other receptors when compared to ICI 118,551 whilst being more or equally as potent as ICI 118,551.

Summary of the Invention

In a first aspect, there is provided a compound of Formula (I) or (II): or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound is of Formula (I). In any of the above embodiments, the pharmaceutically acceptable salt may be the hydrochloride salt.

In a preferred embodiment, the compound of Formula (I) or (II) has an achiral purity of at least 85%, preferably at least 90%, more preferably at least 92%, and even more preferably at least 95%.

In a preferred embodiment, the compound of Formula (I) or (II) has an enantiomeric excess (ee) and diastereomeric excess (de) of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

In a second aspect, there is provided a composition comprising: a) compound of Formula (I) or (II): or a pharmaceutically acceptable salt thereof; and b) a pharmaceutically acceptable carrier or excipient.

In a preferred embodiment, the compound of Formula (I) or (II) has an achiral purity of at least 85%, preferably at least 90%, more preferably at least 92%, and even more preferably at least 95%. In a preferred embodiment, the compound of Formula (I) or (II) has an enantiomeric excess (ee) and diastereomeric excess (de) of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

In a preferred embodiment, the compound is of Formula (I). In any of the above embodiments, the pharmaceutically acceptable salt may be the hydrochloride salt. In any of the above embodiments, the composition may further comprise an additional active agent.

In any of the above embodiments, the pharmaceutical composition may be provided as an oral, buccal, sublingual, subcutaneous, intravenous, intramuscular, intranasal, inhalable, rectal, or topical dosage form.

In a third aspect, there is provided the compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any embodiment of the second aspect of the invention, for use as a medicament.

In a fourth aspect, there is provided the compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any embodiment of the second aspect of the invention, for use in the treatment or prevention of a disorder associated with P2-AR signalling pathways.

In some embodiments, the disorder associated with P2-AR signalling pathways is a tumour, vascular abnormality, respiratory disease, cardiovascular disease, physiological tremor, or migraine.

In some embodiments, the tumour is a vascular tumour, cancer, paraganglioma, or tuberous sclerosis, preferably a vascular tumour or cancer. In some embodiments, the vascular tumour is infantile haemangioma, von Hippel-Lindau disease, angiosarcoma, or glioma, preferably infantile haemangioma or von Hippel-Lindau disease. In some embodiments, cancer is soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, or lymphoblastic leukaemia. In some embodiments, the vascular abnormality is hereditary haemorrhagic telangiectasia, or cerebral carvernous malformations, preferably hereditary haemorrhagic telangiectasia. In some embodiments, the respiratory disease is asthma or chronic obstructive pulmonary disease, preferably asthma. In some embodiments, the cardiovascular disease is chronic heart failure or takotsubo syndrome. Preferably, the disorder associated with P2-AR signalling pathways is a vascular tumour, such as infantile haemangioma, von Hippel-Lindau disease, angiosarcoma or glioma; a vascular abnormality, such as hereditary haemorrhagic telangiectasia; or cancer, such as breast, pancreatic, or ovarian cancer.

More preferably, the disorder associated with P2-AR signalling pathways is a vascular tumour, such as infantile haemangioma, von Hippel-Lindau disease, angiosarcoma or glioma; or a vascular abnormality, such as hereditary haemorrhagic telangiectasia.

In embodiments where the disorder is asthma, the compound or composition may be used to treat or prevent P2-AR downregulation resulting from administration of a long acting P2-AR agonist (LABA), ultra-long acting P2-AR agonist (ultra-LABA) or overuse of a short acting P2- AR agonist (SABA). Where the disorder is asthma, the compound or composition may be administered to a subject suffering from P2-AR downregulation resulting from administration of a LABA, ultra-LABA or overuse of a SABA. The LABA may be salmeterol and/or formoterol, the ultra-LABA may be olodaterol and/or vilanterol, and the SABA may be salbutamol and/or terbutaline.

In a fifth aspect, there is provided the compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any embodiment of the second aspect of the invention, for use as a selective P2-AR antagonist.

In embodiments, the compound or pharmaceutical composition for use of any of the embodiments of the third to fifth aspects may be administered orally, buccally, sublingually, subcutaneously, intravenously, intramuscularly, intranasally, via inhalation, rectally, or topically.

In a sixth aspect, there is provided a method of treating or preventing a disorder associated with P2-AR signalling pathways, the method comprising administering to a subject the compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any embodiment of the second aspect of the invention. In embodiments, the disorder associated with P2-AR signalling pathways may be as defined for the fourth aspect. In embodiments, the disorder associated with P2-AR signalling pathways is a tumour, vascular abnormality, respiratory disease, cardiovascular disease, physiological tremor, or migraine. The disorder associated with P2-AR signalling pathways may be as defined in the fourth aspect. In a seventh aspect, there is provided a use of the compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any embodiment of the second aspect of the invention, for the manufacture of a medicament for the treatment or prevention of a disorder associated with P2-AR signalling pathways. In embodiments, the disorder associated with P2-AR signalling pathways may be as defined for the fourth aspect. In embodiments, the disorder associated with P2-AR signalling pathways is a tumour, vascular abnormality, respiratory disease, cardiovascular disease, physiological tremor, or migraine. The disorder associated with P2-AR signalling pathways may be as defined in the fourth aspect.

In an eighth aspect, there is provided a use of the compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any embodiment of the second aspect of the invention, as a selective P2-AR antagonist.

Detailed Description

As used herein, the term "comprises" means "includes, but is not limited to."

Compounds of Formula (I) or (II): or pharmaceutically acceptable salts thereof, are P2-AR antagonists. These will also be referred to as compounds according to the present invention herein.

P2-AR signaling pathways are linked with certain disorders and a compound of the present invention could be beneficial in the treatment of these disorders.

Disorders associated with P2-AR signaling pathways

Tumour

Tumours that may be treated using a P2-AR antagonist described in the present invention include vascular tumours, cancer, paraganglioma, and tuberous sclerosis. Preferably, the tumour is a vascular tumour or cancer. Vascular tumours include infantile haemangioma, von Hippel-Lindau disease, angiosarcoma, and glioma. Preferably the vascular tumour is infantile haemangioma or von Hippel-Lindau disease. Preferably the cancer is soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, or lymphoblastic leukaemia.

Cancer

Studies using ICI 118,551 , a selective P2-AR antagonist, atenolol, a selective P1-AR antagonist, and propranolol, a non-selective p-AR antagonist, have shown that the P2-AR, and not P1-AR, is the mechanism for tumour growth. Furthermore, there is increasing evidence that cancers metastasise by the neural route and that P2-AR, which are present on blood vessels, is the mechanism by which tumours spread. Therefore, by blocking the P2-AR using a P2-AR antagonist, the spread of tumours, such as cancer, can be reduced or prevented.

The role of P2-AR activation in increasing metastases and cancer progression has been indicated across a number of tumour models including: ovarian cancer cells (Thaker et al., Nat Med 2006; 12: 939-944); prostate cancer cells (Palm et al., Int J Cancer 2006; 118: 2744-2749); acute lymphoblastic leukaemia (Lamkin et al., Brain Behav Immun 2012; 26: 635-641); and triple-negative brain metastatic cells (Choy et al., Oncol Rep 2016; 35: 3135- 3142). The mechanism therefore appears to be present in many tumour types (Pimental et al., In Jandial R, editor, Metastatic Cancer: Clinical and Biological Perspectives. Austin Texas USA: Landes Bioscience 2013; 169 - 179).

Similar results were not obtained with a selective P1-AR antagonist, such as atenolol, suggesting that the effects on tumour progression and metastases are a result of the P2-AR antagonism (Barron et al., J Clin Oncol 2011; 29: 2635-2644).

Examples of cancer include soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, or lymphoblastic leukaemia.

Given that the benefits of P2-AR antagonism over P1-AR antagonism have been demonstrated, it is believed that a selective P2-AR antagonist will aid in the prevention and treatment of cancer. Vascular tumour

Several types of vascular tumour may be treated by the compounds or compositions according to the present invention. These include infantile haemangioma, von Hippel-Lindau disease, angiosarcoma, and glioma. Preferably, the vascular tumour is infantile haemangioma or von Hippel-Lindau disease.

Infantile haemangioma

Infantile haemangioma are vascular tumours of childhood. Most are not present at birth. They usually appear during the first 4 to 6 weeks of life, after which they undergo periods of proliferation, stabilisation, and involution. Infantile haemangioma form on the skin, in tissue below the skin, or in an organ anywhere in the body. They are most common on the skin of the head and neck, where they may be raised or flat, and usually appear as bright red-blue lesions. The tumours are benign and most resolve without the need for treatment. But some are associated with complications that can be debilitating or life-threatening, such as respiratory failure or congestive heart failure. Visible lesions may trigger psychological problems in the child and parents. An estimated 10% are complex and require referral to a specialist.

Currently, propranolol is approved for the treatment of infantile haemangioma by the European Medicines Agency (EMA) and the USA Food and Drug Administration (FDA), with numerous studies demonstrating its efficacy in the treatment of infantile haemangioma (Leaute-Labreze et al., N Engl J Med 2015; 372, 735-746; Wedgeworth et al., Br J Dermatol 2016; 174: 594-601). Because propranolol crosses the blood-brain barrier, nadolol, which does not cross the blood-brain barrier, was compared with propranolol and was shown to be even more effective in the treatment of infantile haemangioma (Pope et al., Br J Dermatol 2013; 168: 222-224). Propranolol and nadolol are non-selective Pi- and P2-AR antagonists. However, nadolol is more selective for the P2-AR, suggesting that selective P2-AR antagonism may be beneficial in the treatment of infantile haemangioma.

The pathogenesis of infantile haemangioma is not well understood, but neovascularisation and angiogenesis are probably involved. Studies of von Hippel-Lindau haemangioma cells in vitro and a mouse model of oxygen-induced retinopathy provide supporting evidence that the effect of propranolol on infantile haemangioma is mediated via the P2-AR, as described in detail herein. A suggested hypothesis for the pathogenesis of infantile haemangioma is hypoxia during foetal development or in the neonatal period. Hypoxia is a deficiency in the amount of oxygen reaching the tissues. The supply of oxygen is controlled by hypoxia inducible factors (HIFs) and an oxygen sensing mechanism that involves hydroxylation of HI F by a set of dioxygenases. In hypoxia, those processes are suppressed, which causes HIF to activate a massive transcriptional cascade of encoding proteins, including erythropoietin (EPO), vascular endothelial growth factor (VEGF), the entire glycolytic enzyme sequence, and a host of other proteins, which together increase cellular resistance to hypoxia and ischaemia (Bishop and Ratcliffe 2014).

Von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is a rare autosomal dominant disease caused by germline mutations in the tumour suppressor gene VHL on the short arm of chromosome 3. Different, multiple, benign and malignant vascular tumours develop throughout life such as: retinal and central nervous system hemangioblastoma, renal cancer, pheochromocytoma, paraganglioma, endolymphatic sac tumour, pancreatic cystadenoma and neuroendocrine tumour, cystadenoma in the epididymis and broad ligament. Von Hippel-Lindau disease is a long-term debilitating and life-threatening disease that frequently leads to premature death. Patients with von Hippel-Lindau disease are treated mainly with surgery, to remove tumours in the affected organs, and with laser therapy, to treat eye symptoms.

The EMA designated propranolol an orphan medicinal product for treatment of von Hippel-Lindau disease in January 2017. It has been demonstrated that the number and size of retinal haemangioblastomas remained stable in von Hippel-Lindau patients treated with propranolol daily for 1 year. That correlated with a progressive reduction in plasma levels of the biomarkers VEGF and miR210, similar to those of the general population (Gonzalez-Rodriguez et al., BMJ Open Ophthalmol 2019; 4: e000203. doi:

10.1136/bmjophth-2018-000203). Furthermore, it has been shown that propranolol acts via the P2-AR, and not the P1-AR, although both receptors are expressed in haemangioma stem cells (Munabi et al., Stem Cells Transl Med 2016; 5: 45-55). However, propranolol is a non-selective Pi-and P2-AR antagonist and causes hypotension and bradycardia, which is a disadvantage when treating normotensive von Hippel-Lindau patients. The effects of propranolol on blood pressure and heart rate are mediated primarily via the P1-AR.

ICI 118,551, a selective P2-AR antagonist, has been shown to reduce levels of pro-angiogenic factors and reduce pathogenic neovascularization, whereas atenolol, a selective Pi-AR antagonist, did not do so. Those effects of ICI 118,551 were confirmed to be due to action at the P2-AR (Martini et al., J Neurochem 2011 ; 119: 1317-1329). It has also been demonstrated that ICI 118,551 has little or no effect on blood pressure and heart rate at rest, and no effect on healthy endothelial cells (McCafrey et al., J Cardiovasc Pharmacol 1988; 11 : 543-551). In primary cultures of von Hippel-Lindau hemangioblastoma, ICI 118,551 was found to: impair the viability of cells; halt or almost delay the main angiogenic processes of cell migration and tube formation; and prevent the nuclear internalization of HIF-1a in hemangioblastoma cells and human endothelial primary cultures under hypoxic conditions (Cuesta et al., Sci Rep 2019; 9: 10062). Similar effects were observed in von Hippel-Lindau clear cell renal carcinoma (ccRCC) cells and primary cultures, demonstrating that ICI 118,551 inhibits vHL-ccRCC cancer cell proliferation, angiogenesis and inflammation. In two xenograft models of vHL- ccRCC in vivo, ICI 118,551 slowed tumour progression by reducing growth (Albihana et al., Orphanet J Rare Dis 2015; 10: 118).

This suggests that a selective P2-AR antagonist may be useful in the treatment of von Hippel- Lindau disease.

Angiosarcoma

Angiosarcoma are a rare, aggressive, malignant, endothelial-cell tumour of vascular or lymphatic origin with a high rate of local recurrence and metastasis. They are found throughout the body but occur mainly in the head and neck of white individuals, especially the scalp. Only 10% occur in deep soft tissues, and the rest are found in parenchymal organs such as the breast, bone, spleen and liver.

Angiosarcoma are exceptionally difficult to treat. Treatment usually consists of a mixture of cytotoxic therapy, surgery, and radiation. Tissue samples from patients with angiosarcoma showed strong P2-AR expression, with only weak or moderate Pi-AR expression (Porcelli et al., Sci Rep 2020; 10: 10465). Patient-derived soft tissue sarcoma cells and tissues with higher P2-AR expression were found to respond better to the combination of propranolol with chemotherapy (docetaxel), suggesting that the P2-AR expression level may be linked to the responsiveness of some tumours to chemotherapy. It also adds to the evidence that P2-AR plays an important role in the mechanism for resistance to standard chemotherapy and that antagonism of this receptor improves the tumour response.

Studies of angiosarcoma cells in vitro using the P2-AR selective ICI 118,551 and the non- selective Pi-AR and P2-AR antagonist propranolol showed that they abrogate mitogenic signalling and reduce tumour cell viability through inhibition of MAPK mediators, which are downstream of P2-AR (Amaya et al., Oncoscience 2018; 5: 109-119). The similar reduction in cell viability induced by ICI 118,551 and propranolol suggests that the mechanism of action is through antagonism of the same P2-AR signalling pathways. Studies of propranolol have resulted in at least partial remission in most of the patients treated (Wagner et al., J Exp Pharmacol 2018: 10: 51-58). Therefore, a selective P2-AR antagonist is likely to achieve the same response with fewer side effects resulting from P1-AR binding.

Vascular abnormality

Several types of potentially life-threatening and/or seriously debilitating vascular abnormalities may also be treated by the compounds or compositions according to the present invention. These include hereditary haemorrhagic telangiectasia or cerebral carvernous malformations. Preferably, the vascular abnormality is hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia

Hereditary haemorrhagic telangiectasia is a rare autosomal-dominant disorder characterised by telangiectasia of blood vessels in the skin and mucous membranes, and arteriovenous malformations in visceral organs, such as the lung, liver, gastrointestinal tract, brain, and spinal cord. Patients with hereditary haemorrhagic telangiectasia have a higher risk of cerebral abscess, stroke, migraine, and bleeding, mainly from the nose (epistaxis), lung and gastrointestinal tract, which can result in iron deficiency and anaemia. Hereditary haemorrhagic telangiectasia affects children and adults and is disabling and life-threatening.

There is no safe and effective front-line drug therapy for hereditary haemorrhagic telangiectasia. There is growing interest in the use of topical and/or oral propranolol, as a non-selective p-AR antagonist, as an anti-angiogenic therapy. Propranolol has been found to reduce the severity, duration and frequency of epistaxis in a number of open studies (Contis et al., Clin Otolaryngol 2017; 42: 911-917; Mei-Zahav et al., J Otolaryngol Head Neck Surg 2017; 46: 58; Esteban-Casado et al., Laryngoscope 2019; 129: 2216-2223). The P2-AR is the dominant p-AR on peripheral blood-vessel endothelium. Activation of the P2-AR on blood vessels leads to stimulation of VEGF, which regulates angiogenesis (Kritharis et al., Haematologica 2018; 103: 1433-1443). The therapeutic effect of propranolol in hereditary haemorrhagic telangiectasia is therefore likely via inhibition of P2-AR, as seen in other vascular diseases, such as infantile haemangioma and von Hippel-Lindau disease. Therefore, a selective P2-AR antagonist is of interest for the treatment of hereditary haemorrhagic telangiectasia, as this would avoid hypotension and bradycardia which are side effects of propranolol.

Respiratory disease

Respiratory diseases include asthma, chronic obstructive pulmonary disease (COPD), occupational lung diseases and pulmonary hypertension. Preferably, the respiratory disease is asthma.

Asthma is characterised by airway hyper-responsiveness, airway inflammation, variable airway expiratory limitation, hypersecretion of mucus and airway remodelling. During an asthma attack, patients have shortness of breath, wheezing, coughing, a tight chest, and difficulty in breathing. The aim of managing asthma is to reverse or prevent acute bronchoconstriction and inflammation, which are associated with P2-AR signalling.

Asthma symptoms are controlled in most asthmatics by short-acting P2-AR agonists (SABA), such as salbutamol and terbutaline, inhaled corticosteroids (ICS), long-acting P2-AR agonists (LABA) such as salmeterol and formoterol, and ultra-long acting P2-AR agonists (ultra-LABA), such as olodaterol and vilanterol. However, about 10% of patients are resistant to ICS or LABA therapy (Chung KF, J Intern Med 2016; 279: 192-204). Long-term use of LABA alone can lead to tolerance and increased risk of asthma exacerbation and even death (Nelson et al., Chest 2006; 129: 15-26; Salpeter et al., Ann Intern Med 2002; 137: 715-25). Tolerance correlates with desensitisation and down-regulation of the P2-AR (Callaerts-Vegh et al., Proc Natl Acad Sci USA 2004; 101 : 4948-4953; Finney et al., Br J Pharmacol 2001 ; 132: 1261- 1270; Newnham et al., A J Med 1994; 97: 29-37; Grove et al., Thorax 1995; 50: 134-138; Aziz et al., Chest 1999; 115: 623-8).

In preclinical studies, chronic dosing with nadolol or ICI 118,551 was found to reduce airway hypersensitivity, inflammation, and mucin content, and reverse pathological changes in the airway epithelium (Lin et al., Pul Pharmacol Ther 2008; 21 : 115-124; Nguyen et al., Proc Natl Acad Sci 2009; 106: 2435-40). Thus, a selective P2-AR antagonist may be used in the treatment of asthma.

Without being bound by theory, it is believed that chronic stimulation of the airway P2-AR through chronic use of a LABA or ultra-LABA leads to desensitisation and down regulation of the P2-AR and upregulation of deleterious signalling pathways. Similar effects are also observed following overuse of a SABA. Due to this downregulation of the P2-AR, subsequent administration of a SABA becomes less effective, or even ineffective. Another problem with the use of a LABA is saturation of the P2-AR. This means that subsequent administration of a SABA, for example to alleviate immediate symptoms, may be ineffective. Therefore, administration of a selective P2-AR antagonist has the potential to upregulate the P2-AR and control symptoms of severe asthma.

Examples of LABA are salmeterol and formoterol, ultra-LABA are olodaterol and vilanterol, and SABA are salbutamol and terbutaline.

Cardiovascular diseases

Cardiovascular diseases that may be treated using a P2-AR antagonist described in the present invention include chronic heart failure or takotsubo syndrome.

Chronic Heart Failure

Chronic heart failure is a complex syndrome characterised by decreased cardiac output, fluid retention and raised venous pressure.

Multiple p-AR antagonists have been approved for use in the treatment of chronic heart failure, all of which are non-selective P1-AR antagonists. However, more recently it has been found that P2-AR coupling pathways have an important role in chronic heart failure and thus a P2-AR antagonist may be used in the treatment of chronic heart failure (Black & Fitzgerald, Curr Pharm Des 2010; 16: 4148-4158).

All the p-AR antagonists currently used to treat chronic heart failure require the use of the time-consuming and complicated dose-titration that must be individually calibrated for each patient by a highly skilled physician. That involves starting with a very small dose and subsequently increasing the dose gradually over a period of 2-12 months until a therapeutically effective dose is reached. Dose-titration is required because the presently used p-AR antagonists all antagonise the P1-AR to at least some degree and consequently have unwanted side effects from blocking the signalling through this receptor that promotes heart contraction. Not wishing to be bound by theory, it is believed that during dose-titration, the Pi-AR activity in the failing heart increases sufficiently that the final dose of the p-AR antagonist does not cause significant inhibition of the signalling mediated by the Pi-AR population but does cause significant inhibition of the deleterious effects mediated by P2-AR. Consequently, by the time the final dose is reached, there is sufficient Pi-AR activity in the heart to cope with the Pi-AR blockade and to maintain heart function. Because P2-AR signalling is also inhibited, heart function improves. However, if a patient were to be given a final dose without the preceding dose-titration, the failing heart would not possess sufficient Pi-AR activity to cope with the Pi-AR blockade, and heart function would deteriorate.

Both Pi- and P2-AR are present in the healthy cardiac ventricular muscle, and are present in the ratio of about 3:1 (Hedberg et al., J Pharmacol Exp Ther 1980; 212(3): 503-8). Pi- and P2- AR activation increases cardiac output, both by raising heart rate and increasing the volume expelled with each beat (increased ejection fraction) and leads to the phosphorylation of the Pi- and P2-AR. It has recently been shown that phosphorylation of the P2-AR leads to the activation of alternative, or “non-classical” pathways (Lohse et al., Circ Res 2003; 93(10): 896-906; Zamah et al., J Biol Chem 2002; 277(34): 31249-56; Daaka et al., Nature 1997; 390(6655): 88-91 ; Hill & Baker, Br J Pharmacol 2003; 138(7): 1188-89; Hasseldine et al., Br J Pharmacol 2003; 138(7): 1358-66). This leads to the activation of several deleterious cellular pathways, which contribute to the development of heart failure by initiating apoptotic, fibrotic and inflammatory processes characteristic of the failing heart (Osadchii et al. Pflugers Arch 2006; 452(2): 155-63; Chandrasekar et al., Biochem Biophys Res Commun 2004; 319(2): 304-11 ; Sanz-Rosa et al., J Hypertens 2005; 23(6): 1167-72; Sanz-Rosa et al., Am J Physiol Heart Circ Physiol 2005; 288(1): H111-H115; Pace et al., Mol Biol Cell 1995;

6(12): 1685-95; Kouchi et al., Hypertension 2000; 36(1): 42-7).

As heart failure develops, the density of the Pi-AR is halved, whilst the density of the P2-AR remains the same or even increases (Bristow et al. Circ Res 1986; 59(3):297-309). Thus, in the failing heart, the decreased population of the Pi-AR continues to mediate heart contraction, while a proportionally increased population of the P2-AR mediates negative effects through the alternative pathways discussed above. These changes in the failing heart lead to decreased ejection fraction, early hypertrophy of ventricular muscle followed by apoptosis, inflammation and fibrotic damage to the heart tissue.

Therefore, it is believed that selective blocking of the P2-AR will mitigate the negative effects associated with activation of the alternative pathways discussed above without having the unwanted effect of inhibiting Pi-AR mediated heart contraction and may be used in the treatment of chronic heart failure. Additionally, a selective P2-AR antagonist should not require dose titration, and therefore is simpler and easier to administer. In addition, it is believed that a selective P2-AR antagonist may be administered at the therapeutic dose from the start, meaning that effective treatment begins sooner (Black & Fitzgerald, Curr Pharm Des 2010; 16: 4148-4158).

This is supported by studies which compare the non-selective p-AR antagonist carvedilol and metoprolol for the treatment of chronic heart failure in the Carvedilol or Metoprolol European Trial (COMET) (Poole-Wilson et al., Lancet 2003; 362: 7-13). These studies demonstrate that carvedilol, which is weakly selective for the P2-AR, was preferred over metoprolol, which is weakly selective for the P1-AR.

Takotsubo syndrome

Takotsubo syndrome, also known as takotsubo cardiomyopathy or ‘broken-heart’ syndrome, is a severe form of heart failure characterised by acute apical dysfunction in which the shape of the left ventricle (LV) changes. The main symptoms are acute chest pain and dyspnea, which are usually preceded by intense emotional or physical stress.

Several lines of evidence implicate epinephrine (adrenaline) in the pathophysiology of takotsubo syndrome (Wittstein et al., N Engl J Med 2005; 352: 539-548; Batisse-Lignier et al; Medicine 2015; 94: e2198; Nazir et al., Int J Cardiol 2017; 229: 67-70; Paur et al., Circulation. 2012; 126: 697-706). Studies of takotsubo syndrome support the concept that high levels of epinephrine trigger apical cardiodepression via a switch in signalling through the P2-AR from Gas-protein activated cardiostimulation to Gai-protein activated cardiodepression, an effect known as biased agonism. The ratio P2/P1-AR is higher at the apex compared with the base of the heart, which explains the larger contractile response to epinephrine by the apex and the resulting apical ballooning. Therefore, takotsubo syndrome has been linked to P2-AR activation (Paur et al., Circulation. 2012; 126: 697-706; Ali et al., Int J Cardiol 2019; 281 : 99-104).

Given the strong evidence of P2-AR involvement in takotsubo syndrome, it is believed that a selective P2-AR antagonist may be used in the treatment and prevention of takotsubo syndrome.

General Compounds of the present invention and pharmaceutical compositions comprising compounds according to the present invention are useful for the treatment or prevention of disorders associated with P2-AR signalling pathways. Preferably the pharmaceutical compositions and compounds of the present invention are used for the treatment of disorders associated with P2-AR signalling pathways. As discussed in detail above, such disorders include: a tumour, such as a vascular tumour, cancer, paraganglioma, and tuberous sclerosis; a vascular abnormality, such as hereditary haemorrhagic telangiectasia, and cerebral cavernous malformations; respiratory disease, such as asthma, and chronic obstructive pulmonary disease; cardiovascular disease, such as chronic heart failure and takotsubo syndrome; and other indications, such as physiological tremor and migraine. Without limitation, exemplary vascular tumours include infantile haemangioma, von Hippel-Lindau disease, angiosarcoma, and glioma. Without limitation, exemplary cancers include soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, and lymphoblastic leukaemia.

Compounds of Formula (I) and Formula (II) have also been found to show increased selectivity for the p-AR when compared to ICI 118,551. Increased selectivity for the p-AR allows these compounds to act as effective P2-AR antagonists without interacting with alternative pathways. This improves control over the pharmacology and also reduces drug-drug interactions and unwanted side-effects.

Both compounds of Formula (I) and (II) have been found to be selective P2-AR antagonists over the P1-AR and other receptors . In particular, the compound of Formula (I) is the most selective for P2-AR over P1-AR. As discussed above, receptor selectivity is preferable because adverse effects are often attributed to off-target drug actions. Additionally, with increased selectivity there is a reduced risk of drug-drug interactions. Thus, the increased selectivity also allows doses to be minimised, which reduces the cost of medication, or alternatively allows for larger doses to be administered with fewer side effects, which can allow for more effective treatment of diseases. It also increases the ease of administration.

Preferably the compound is of Formula (I), since this surprisingly demonstrates improved selectivity for P2-AR over P1-AR when compared to Formula (II) and good potency.

Compounds and pharmaceutical compositions

Compounds or compositions of the invention, when used for treating or preventing a disorder, may be administered in an "effective amount". By an "effective amount" it is meant a "therapeutically effective amount", namely an amount of compound sufficient, upon single dose or multiple dose administration, to cause a detectable decrease in disease severity, to prevent advancement of a disease or alleviate disease symptoms beyond that expected in the absence of treatment. A subject to be treated in accordance with a method of treatment of the invention is preferably a human subject.

Compounds or compositions of the invention are useful for reducing the severity of symptoms of any of the above disorders to be treated. Compositions of the invention are also useful for administration to patients susceptible to, at risk of, or suffering from, any of the above disorders. Compositions useful for prevention of the above disorders are not required to prevent absolute occurrence of the disorder in all cases but may prevent or delay onset of the disorder when administered to a patient susceptible to or at risk of the disorder. One or more of the above disorders may be present in a subject in combination and, accordingly, a pharmaceutical composition of the invention may treat one or more of the above disorders in combination.

Preferably, the compound of Formula (I) or (II) has an achiral purity of at least 85%, preferably at least 90%, more preferably at least 92%, and even more preferably at least 95%.

Compounds of Formula (I) and (II) have three chiral centres which have defined stereochemistry. Preferably, compounds of Formula (I) and (II) are in an optically pure form. The compounds may be present in the compositions in an optically pure form.

An optically pure form of an enantiomer as referred to herein has an enantiomeric excess (ee) of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%, the maximum ee being 100%. ee may be assessed, for example, by chiral HPLC.

Preferably the compound of Formula (I) or Formula (II), either in isolation or in a pharmaceutical composition according to the present invention, has an ee of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

An optically pure form of a diastereomer as referred to herein has a diastereomeric excess (de) of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%, the maximum de being 100%. de may be assessed, for example, by chiral HPLC.

Preferably the compound of Formula (I) or Formula (II), either in isolation or in a pharmaceutical composition according to the present invention, has a de of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

Preferably the compound of Formula (I) or Formula (II), either in isolation or in a pharmaceutical composition according to the present invention, has an ee and a de of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

The pharmaceutical composition of the present invention may comprise: a) a compound of Formula (III): wherein the compound of Formula (III) comprises the isomer of Formula (I) or the isomer of

Formula (II): wherein the compound of Formula (III) comprises the isomer of Formula (I) or the isomer of Formula (II) in an amount of at least 95 mol%, preferably at least 97.5 mol%, more preferably at least 99 mol%, and even more preferably at least 99.5 mol%; and b) a pharmaceutically acceptable excipient.

Preferably the compound is the isomer of Formula (I) and is present in the above purities. The compounds disclosed herein can exist in unsolvated as well as solvated forms, for example with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embraces both solvated and unsolvated forms.

The compounds disclosed herein may be provided as the free compound or as a suitable salt or hydrate thereof. Salts should be those that are pharmaceutically acceptable, and salts and hydrates can be prepared by conventional methods, such as contacting a compound of the invention with an acid or base whose counterpart ion does not interfere with the intended use of the compound. Examples of pharmaceutically acceptable salts include hydrohalogenates, inorganic acid salts, organic carboxylic acid salts, organic sulphonic acid salts, amino acid salt, quaternary ammonium salts, alkaline metal salts, alkaline earth metal salts and the like. Basic moieties may form non-toxic acid addition salts with various inorganic and organic acids, i.e. , salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate and pamoate salts. Acidic moieties may form salts with various pharmacologically acceptable cations, including alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Compounds that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids. Preferably, the compounds disclosed herein are in the form of a hydrochloride salt.

The invention includes the provision of compounds as described herein in substantially amorphous or substantially crystalline form. "Substantially crystalline" and "substantially amorphous" refers to a compound that may be at least a particular weight percent crystalline or amorphous, respectively. In some embodiments, substantially crystalline or substantially amorphous refers to compounds that are at least 70%, at least 80%, at least 90%, or at least 95% crystalline or amorphous, respectively.

A pharmaceutical composition of the invention comprises one or more pharmaceutically acceptable excipients, for example pharmaceutically acceptable carriers, diluents, preserving agents, solubilising agents, stabilising agents, disintegrating agents, binding agents, lubricating agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents and antioxidants. Suitable excipients and techniques for formulating pharmaceutical compositions are well known in the art (see, e.g. Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000). Suitable excipients include, without limitation, pharmaceutical grade starch, mannitol, lactose, corn starch, magnesium stearate, stearic acid, alginic acid, sodium saccharin, talcum, cellulose, cellulose derivatives (e.g. hydroxypropylmethylcellulose, carboxymethylcellulose) glucose, sucrose (or other sugar), sodium carbonate, calcium carbonate, magnesium carbonate, sodium phosphate, calcium phosphate, gelatin, agar, pectin, liquid paraffin oil, olive oil, alcohol, detergents, emulsifiers or water (preferably sterile).

A pharmaceutical composition may further comprise an adjuvant and/or one or more additional therapeutically active agent(s).

A pharmaceutical composition may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

A pharmaceutical composition may be adapted for administration by any appropriate route, for example by inhalation or by oral, buccal, sublingual, intranasal, rectal, topical, or parenteral administration, where parenteral administration includes subcutaneous, intramuscular, intravenous, intraperitoneal, and intradermal administration. Preferably, the pharmaceutical composition is provided as an oral, buccal, sublingual, subcutaneous, intravenous, intramuscular, intranasal, inhalable, rectal, or topical dosage form. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with an excipient(s) under sterile conditions.

Preferably, the compound or composition is administered buccally or sublingually when treating infantile haemangioma.

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups, or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions). The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example water or oils (e.g. vegetable oils, liquid paraffin oil or olive oil), waxes, fats, semi-solid, or liquid polyols etc. For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) may be used to provide oil-in-water or water-in-oil suspensions.

Pharmaceutical compositions adapted for oral, buccal or sublingual administration may be in the form of a fast-dissolving formulation. For example, the compound or composition may be micronised and freeze-dried in a water-soluble matrix such as gelatin or a saccharine and polymer mixture. This may be a Zydis® formulation. This increases bioavailability and is particularly effective when administering the pharmaceutical composition to children.

Pharmaceutical compositions adapted for administration by inhalation or intranasally include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurised aerosols, nebulizers, or insufflators.

Pharmaceutical compositions adapted for parenteral administration (e.g. subcutaneous, intramuscular, intravenous, intraperitoneal and intradermal) include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, thickening agents and wetting agents. Excipients which may be used for injectable solutions include, for example, water, alcohols, polyols, glycerine, and vegetable oils.

Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions may, for example, include suspending agents such as cellulose derivatives, sodium alginate, gum tragacanth, polyvinylpyrrolidone and wetting agents such as lecithin. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

In some embodiments, the pharmaceutical composition of the invention is a depot formulation, formulated to provide controlled drug release into the bloodstream over a period of, for example, weeks or months, depending on the exact formulation. A depot formulation may, for example, be a nanoparticulate formulation of nanoparticles comprising a compound of formula (I) or (II) and one or more excipients, for example, surface stabilisers, bulking agents or carriers, or a formulation comprising a compound of formula (I) or (II) enclosed in micellar nanoparticles. A depot formulation is usually injected subcutaneously or intramuscularly, to produce a reservoir of drug in the muscle or under the skin. A depot formulation is usually solid or oil-based.

Compositions of the invention, when used for preventing or treating a disorder, may be administered in an "effective amount". For use as a single monotherapy, by an "effective amount" it is meant a "therapeutically effective amount", namely an amount of compound sufficient, upon single dose or multiple dose administration, to cause a detectable decrease in disease severity, to prevent advancement of a disease or alleviate disease symptoms beyond that expected in the absence of treatment. In the case where the invention is used in combination with another agent by an "effective amount" is meant to be an amount sufficient in combination with another agent when given either by a single or multiple dose administration to cause a detectable decrease in disease severity, to prevent advancement of a disease or alleviate disease symptoms beyond that expected in the absence of treatment or treatment with the second agent when given alone. The "effective amount" when used as a monotherapy may or may not be the same as that in combination with a second agent.

Dosages of the substance of the present invention can vary between wide limits, depending upon a variety of factors including the disease or disorder to be treated, the age, weight and condition of the individual to be treated, the route of administration, etc. A physician will ultimately determine appropriate dosages to be used. Typically, however, the daily dosage (whether administered as a dose or multiple divided doses) adopted for each route of administration when a compound of the invention is administered will be 0.001 to 5,000 mg/day, usually 0.1 to 1 ,000 mg/day, more usually 0.5 to 200 mg/day, and even more usually 1 to 120 mg/day, for example 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg. A typical dose may be, expressed as dosage per unit body weight, between 0.01 pg/kg and 50 mg/kg, preferably between 10 pg/kg and 10 mg/kg, for example between 100 pg/kg and 2 mg/kg.

The compounds of the present invention may be administered alone, or may be administered in combination with one or more additional therapeutically active agent(s) (/.e. a different compound to compounds of the present invention). Preferably, the compound of the invention and the additional therapeutic agent are administered in a therapeutically effective amount. The compound of the present invention may be administered either simultaneously with, or before or after, the other therapeutic agent. The compound of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition.

As discussed above, these compounds may be used in combination with a steroid, such as an inhaled corticosteroid, when used to treat or prevent asthma.

As discussed above, a problem with the use of a long-acting P2-AR agonist or ultra-long- acting P2-AR agonist, for example in the treatment of asthma, is downregulation of the P2-AR. Similarly, overuse of a short acting P2-AR agonist may result in P2-AR downregulation. This means that subsequent administration of a fast-acting P2-AR agonist, for example to alleviate immediate symptoms, may be ineffective. Therefore, administration of the compounds according to the present invention, which are selective P2-AR antagonists, has the potential to upregulate the P2-AR and control symptoms of severe asthma.

In particular, the compounds of the present invention may be administered prior to administration of a long-acting P2-AR or ultra-long-acting P2-AR. This prevents saturation of the P2-AR, allowing for a fast-acting P2-AR agonist to bind even after administration of a long- acting P2-AR. The compound of the present invention may be administered in combination with a steroid. By “in combination with” it is intended to include administration separately, for example by different routes, but close enough in time that both the compound of the present invention and the steroid act at least partially simultaneously. Preferably, the compound of the present invention and the steroid are administered at the same time, more preferably via the same route of administration.

As discussed above, it has been found that co-administration of the non-selective p-AR antagonists with anti-cancer therapy improved the clinical outcomes in the treatment of angiosarcoma. Therefore, the compounds and compositions of the present invention are preferably administered in combination with an anti-cancer therapy, such as a chemotherapy or radiotherapy, preferably a chemotherapeutic agent, when treating angiosarcoma.

Thus, the pharmaceutical composition may further comprise one or more additional active agent(s). Alternatively, the compound or pharmaceutical composition according to the present invention and an additional active agent may be provided separately, e.g. in the form of a kit. This additional active agent may be a steroid, such as an inhaled corticosteroid, long- acting P2-AR, ultra-long-acting P2-AR, or an anti-cancer therapy, such as a chemotherapeutic agent.

The following examples describe compounds of Formula (I) and (II) according to the present invention. These have been compared to a compound of Formula (D):

Formula (D) is the isolated 2S, 3S form of ICI 118,551. These compounds are also compared to nadolol, a non-selective p-AR antagonist. These examples demonstrate that the compounds according to the present invention are selective P2-AR antagonists and are much more specific to p-AR over other receptors (e.g. 5-HT transporter, 5-HT receptor, o- receptors, Na+ channels) when compared to the compound of Formula (D), also referred to herein as Compound (D). Both compounds are shown to be more or equally effective as the compound of Formula (D). In particular, the compound of Formula (I) demonstrates the highest selectivity (when compared to P1-AR) and potency for P2-AR.

Examples

The following examples of the invention are provided to aid understanding of the invention but should not be taken to limit the scope of the invention. Unless otherwise described, reagents may be commercially available or prepared according to procedures in the literature.

Abbreviations

ATCC American Type Culture Collection

CDC deuterated chloroform

DCM dichloromethane

DI PEA N, /V’-diisopropylethyl amine

DM EM Dulbecco’s Modified Eagle’s Medium

DMF /V,/V’-dimethylformamide DM SO dimethyl sulfoxide

De-DMSO deuterated dimethyl sulfoxide

GC gas chromatography

HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HPLC high performance liquid chromatography

LC-MS/MS liquid chromatography with tandem mass spectrometry

TLC thin layer chromatography ee enantiomeric excess de diastereomeric excess EC50 concentration producing a half-maximal response

IC50 concentration causing a half-maximal inhibition of the control agonist response

Example 1 - Synthesis of Compunds of Formula (I) and (II)

Part A - Synthesis of (2S, 3S)-4-(2-Hvdroxy-3-isopropylaminobutoxy)-7-methlindan-1- one

(2S, 3S)-4-(2-Hydroxy-3-isopropylaminobutoxy)-7-methlindan-1-one was synthesised according to the scheme below:

Preparation of 4-Hydroxy-7-methylindan-1-one (A)

To a 10L, 3-necked round-bottomed flask equipped with overhead stirrer, reflux condenser and thermometer was charged glacial acetic acid (0.9L), 48% hydrogen bromide solution (4.5L) and 4-methoxy-7-methylindalone (450g, 2.55mol). Stirring was commenced and the vessel contents were heated to ~100°C where all solids dissolved. Heating/stirring was continued for 3 hours until TLC [eluent: DCM] revealed no starting material remaining. The vessel contents were cooled to 30-40°C and then partitioned between ethyl acetate (4.0L) and water (8.0L). The bi-phasic solution was separated and the aqueous layer was salted and re-extracted with ethyl acetate (2 x 4.0L). The combined organic layers were back washed with 25%w/w brine solution (2 x 4.0L) dried over sodium sulphate, filtered and the filtrate stripped on the rotorvapor. The residue was azeotroped on the rotorvapor with toluene (2 x 1 ,0L) (to remove any residual acetic acid residues) and then allowed to cool/crystallise. The recovered crude solid (468g, 113%) was recrystallized from methanol (2.34L, 5 volumes), filtered, washed on the funnel with cold methanol (2 x 300ml) and dried in a vacuum oven at 40°C until constant weight. Dried weight = 247g, 60%. The GC achiral purity of this material was 98.5%. The mother liquors were concentrated on the rotorvapor and a second crop was obtained. Yield = 76g, 18%. The GC achiral purity of this material was 99%. The total overall recovery of 4- hydroxy-7-methylindan-1-one (A) was 323g, 78%. This combined material was used directly in the next stage.

Preparation of (2R, 3R)-7-Methyl-4-(3-methyloxiranylmethoxy)indan-1-one (B)

To a 500ml, 3-necked round-bottomed flask equipped with overhead stirrer, thermometer and nitrogen bubbler was dissolved a solution of 4-hydroxy-7-methylindan-1-one (A) (18g, 0.11 mol, 1.0 eq) in DMF (300ml). Stirring was commenced and under a nitrogen atmosphere caesium carbonate (53.7g, 0.16 mol, 1.5 equiv) was added in one portion and the reaction mixture stirred for 15 minutes. After this time (2R, 3R)-toluene-4-sulphonic acid-3- methyloxyranylmethyl ester (30g, 0.12 mol, 1.1 equiv) was added in one portion and the vessel contents were raised to 50°C. Heating/stirring was continued for 2 hours until TLC [eluent: 4:1 hexane:ethyl acetate] revealed no starting material remaining. The vessel contents were cooled to 30-40°C and then quenched into water (2.0L). Stirring was continued at ambient for 30 minutes and then extracted with diethyl ether (3 x 400ml). The combined organic layers were backwashed with 10% w/w lithium chloride solution (2 x 400ml), dried over sodium sulphate, filtered and stripped on the rotorvapor to an orange oil which slowly crystallised on standing. Weight of crude yield = 29g, 113%. This crude material was purified via column chromatography using silica gel (600g) and 20-25% ethyl acetate/hexane as eluent. The good column fractions were stripped to a pale yellow oil which crystallised on standing. Weight of crude (2R, 3R)-7-methyl-4-(3-methyloxiranylmethoxy)indan-1-one (B) = 25g, 97.8%. This material was used directly in the next stage.

Preparation of (2S, 3S)-4-(2-Hydroxy-3-isopropylaminobutoxy)-7-methylindan-1-one (D)

Step 1 - Synthesis of (C)

To a 2L, 3-necked round-bottomed flask equipped with magnetic stirrer, reflux condenser and thermometer was dissolved a solution of (2R, 3R)-7-methyl-4-(3- methyloxiranylmethoxy)indan-1-one (B) (25g, 0.107 mol, 1.0 eq) in methanol (500ml). The apparatus was set for reflux and isopropylamine (31 ,6g, 46ml, 0.53 mol, 5.0 equiv) was added in one portion. The vessel contents were heated at gentle reflux for a total of 2 days and additional aliquots of isopropylamine (total = 95g, 138ml, 1.6 mol, 15 equiv) were added periodically during the course of the heating cycle. After this time TLC [eluent: 2:1 hexane:ethyl acetate] revealed no starting material remaining and the vessel contents were transferred to a rotorvapor and stripped to a an orange/brown oil. Crude yield of (C) = 42g, 120%.

Step 2 - Synthesis of (D)

The oil from step 1 was re-dissolved in methanol (200ml) and 1 M hydrochloric acid solution (500ml) was added. The apparatus was set for reflux and the vessel contents heated to gentle reflux (87°C) for a total of 4 hours until HPLC revealed that all of the imine (C) had been hydrolysed back to the ketone (D). Heating was then discontinued and the vessel contents transferred to a rotorvapor where the methanol was removed under reduced pressure. The aqueous residue was then cooled to ambient and pH adjusted to 12-13 with 2 M sodium hydroxide solution (200ml). The resulting turbid aqueous solution was extracted with ethyl acetate (3 x 200ml) and the combined organics back washed with water (2 x 200ml), dried over sodium sulphate, filtered and stripped on the rotorvapor to an orange/brown oil. Weight of crude yield = 32g, 102%. The crude oil was immediately dissolved in warm acetonitrile (75ml) and allowed to cool/crystallise (with seeding of trial material) in the fridge overnight. The resulting slurry was filtered and washed on the funnel with acetonitrile (2 x 30ml). Damp weight yield of (D) = 23.5g, 75%. The damp solid was dried in a vacuum oven at 40°C until constant weight. Dried weight yield of (D) = 17.7g, 56.7%. A solution of R-(-)-mandelic acid (7.4g, 48.7mmol, 1.0 equiv) dissolved in ethanol (43ml) was added to a stirred solution of (2S, 3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methylindan- 1-one (D) (14.2g, 48.7mmol, 1.0 equiv) dissolved in ethanol (43ml) where a colourless solid immediately precipitated. Warming the reaction slurry to reflux did not re-dissolve the precipitated solid. After stirring at room temperature for two hours the slurry was filtered and the cake washed with ethanol (2 x 200ml). The recovered damp solid was partitioned between ethyl acetate (250ml) and 2 M sodium hydroxide solution (250ml). Stirring was continued for 10 minutes and the layers separated. The aqueous layer was back extracted with ethyl acetate (50ml) and the combined organic layers were washed with water (2 x 200ml), dried over sodium sulphate, filtered and stripped to a colourless powder. Damp weight = 9.9g. The recovered solid was dried in a vacuum oven overnight until constant weight. Dried weight of (2S, 3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methylindan-1-one (D) = 9.8g. The material was sieved. ¹ H NMR spectrum (CDC ) was consistent with the above structure.

Appearance: Off-white solid

HPLC Achiral Purity: 99.8%

HPLC Chiral Purity: 99.1 % ee

¹ H NMR: 400MHz: (CDCI3): Conforms to structure

CHN: Found: C: 70.05%, H: 8.70%, N: 4.90%.

Theory: C: 70.07%, H: 8.65%, N: 4.81%.

Optical Rotation: [O] ²² D (CHC ) + 33.24°

Residual Ash: <0.1%

Part B - Synthesis of compounds of Formula (I) and

To a 25ml, round-bottomed flask equipped with magnetic stirrer, reflux condenser and nitrogen bubbler was charged DCM (6ml) and (2S, 3S)-4-(2-hydroxy-3- isopropylaminobutoxy)-7-methylindan-1-one (D) (2g, 6.68mmol, 1 equiv). A nitrogen atmosphere was established and stirring was commenced. Triethylamine (3.48g, 34.4mmol, 5 equiv) was added in one portion closely followed by formic acid (1.9g, 41.2mmol, 6 equiv), which was added cautiously over 2 minutes (5ml). The apparatus was then set for reflux and the appropriate catalyst RuCI(p-cymene)[(R,R)-Ts-DIPEN] for compound of formula (I) or RuCI(p-cymene)[(S,S)-Ts-DIPEN] for compound of formula (II) (21.8mg, 0.0342mmol, 0.005 equiv) was added. Heating to 35°C was commenced. Stirring at this temperature was continued overnight. After this time TLC [eluent: 10% methanol/DCM] revealed large amounts of starting material remaining and an additional charge of triethylamine (1.4g, 13.8mmol. 2 equiv), formic acid (0.63g, 13.8mmol, 2 equiv) and the appropriate catalyst (21.8mg, 0.0342mmol, 0.005 equiv) were added. Stirring at 35°C was continued for an additional 48 hours and then both reactions were analysed by TLC, which again revealed the presence of starting material (D) in both reactions. An additional charge of triethylamine (1.4g, 13.8mmol. 2 equiv), formic acid (0.63g, 13.8mmol, 2 equiv) and the appropriate catalyst (21.8mg, 0.0342mmol, 0.005 equiv) were added. Stirring at 35°C was continued for an additional 24 hours and then both reactions were deemed complete by TLC. The reaction mixtures were partitioned between saturated sodium bicarbonate (50ml) and DCM (50ml), stirred for 10 mins and layers separated. The aqueous layer was back extracted with DCM (50ml) and combined organics back washed with 10%w/w brine solution (2 x 50ml), dried over sodium sulphate, filtered and stripped on a rotary evaporator to a pale beige coloured crude solid. (~ 2g for each product). Each crude product was purified via flash column chromatography using 20% methanol/DCM as eluent to give ~ 1g of purified solid which was recrystallized from acetonitrile (5ml) to give the final product. After drying in a fan oven at 35°C over 48 hours a total of 0.7g, 35% of either the compound of Formula (I) or (II) was recovered. ¹ H NMR spectrum (De-DMSO) were both are consistent with the above structures.

Compound of Formula (I)

Yield: 0.7g, 35%

HPLC purity: 95.67%

Chiral HPLC purity: 98.62% de (R)

¹ H NMR: (De-DMSO): Consistent with structure.

Appearance: Colourless solid.

Compound of Formula (II)

Yield: 0.7g, 35%

HPLC purity: 98.00%

Chiral HPLC purity: 99.37% de (S)

¹ H NMR: (De-DMSO): Consistent with structure.

Appearance: Colourless solid.

Chiral purity (de) is quoted in relation to the stereochemistry of the OH on the five-membered ring, with the remaining chiral centres both having S confirmation. Example 2 - Binding Assay

The aim of the binding assay was to compare the selectivity and specificity of the compounds for the P2-AR amongst a range of receptors.

Methods

Compounds were incubated in vitro with specific concentrations of receptor agonists/antagonist radioligands. The specific radioligands, cells, and conditions used are shown in Table 1. Reference is made to citations describing each procedure, which are incorporated by reference herein.

Reference compounds were selected and used as controls to confirm the accuracy of the assay. Atenolol and ICI 118,551 were used as reference samples for the Pi- and P2-AR, respectively. All values were acceptable.

Filtration assays were carried out using the following general procedure:

1. Add and incubate radioligand, receptor and optionally one or more other compounds (detailed below for each binding experiment) in a plate well. The radioligand and incubation conditions are shown in Table 1 for each receptor. These conditions were used for all filtration assays.

2. Apply vacuum to “trap” receptor and bound radioligand onto filters and remove unbound radioligand. Wash several times with an appropriate buffer to minimize nonspecific binding.

3. Allow filters to dry. Add a liquid scintillation cocktail.

4. Count filters in microplate scintillation counter.

Binding experiments were carried out as follows:

• Total binding was determined using the filtration assay general procedure outlined above, where in step 1 no additional compounds were added. Total binding = the signal from the membrane + radioligand.

• Non-specific binding was determined using the filtration assay general procedure outlined above, where in step 1 the radioligand and receptor are incubated with a high concentration of unlabeled competitor. The unlabeled competitor and its concentration are shown in Table 1 for each receptor. Non-specific binding = the signal from the unlabeled competitor + membrane + radioligand. • Control specific binding is the difference between total binding and non-specific binding. Control specific binding = total binding - non-specific binding.

• Measured binding was determined using the filtration assay general procedure outlined above, where in step 1 the radioligand and receptor are incubated with the test compound in the plate well. The concentration of the test compound is shown in Table 2. Measured binding = the signal from the test compound + radioligand + membrane.

• Measured specific binding is the difference between measured binding and non-specific binding. Measured specific binding = measured binding - non-specific binding.

Specific method for/3i-AR binding assays Transfected H EK-293 cells were used.

Cell membrane homogenates (5 pg protein) were incubated for 60 min at 22°C with 0.3 nM [ ³ H]CGP 12177 in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCI (pH 7.4), 10 mM MgCI ₂ , 2 mM EDTA and 0.1% BSA.

Non-specific binding was determined in the presence of 50 pM alprenolol.

Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCI using a 96-sample cell harvester (Unifilter, Packard). The filters were dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).

Measured specific binding was calculated and the results are then expressed as a percent inhibition of the control specific binding.

The standard reference compound is atenolol, which was tested in each experiment at several concentrations to obtain a competition curve from which its IC50 was calculated.

Specific method for /3 ₂ -AR binding assays

Transfected CHO cells were used.

Cell membrane homogenates (32 pg protein) were incubated for 120 min at 22°C with 0.3 nM [ ³ H]CGP 12177 in the absence or presence of the test compound in a buffer containing 10 mM NaH ₂ PO ₄ /Na ₂ HPO ₄ (pH 7.4), 85 mM NaCI, 30 mM KCI, 1 mM MgSO ₄ , 5.5 mM glucose, 0.005% bacitracin and 0.1% BSA.

Non-specific binding was determined in the presence of 50 pM alprenolol.

Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris- HCI using a 96-sample cell harvester (Unifilter, Packard). The filters were dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). Measured specific binding was calculated and the results are then expressed as a percent inhibition of the control specific binding.

The standard reference compound is ICI 118551, which was tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.

The results were analysed as follows:

A decrease in the measured specific binding when compared to the control specific binding (usually expressed in % inhibition) indicates that the test compound binds to the receptor. The results are expressed as a percent (%) of control specific binding (shown in Table 3):

And as a percent (%) inhibition of control specific binding: measured specific binding 100 - ( - - — , , - - - - x 100) control specific binding

An increase in percent (%) inhibition of control specific binding indicates that the test compound binds to the receptor.

Results showing a % inhibition (or stimulation for assays run in basal conditions) higher than 50% are considered to represent significant effects of the test compounds. Results showing a % inhibition (or stimulation) between 25% and 50% are indicative of weak to moderate effects. Results showing a % inhibition (or stimulation) less than 25% are not considered significant and mostly attributable to variability of the signal around the control level.

Table 2 shows the results of the binding assay for compound (D), Formula (I), Formula (II) and nadolol. Results representing significant effects are in bold.

Table 1:

Table 2:

% Inhibition of Control Specific Binding

Table 3:

Table 3 is a summary of the results from Table 2 for % Inhibition of Control Specific Binding for P2-AR and P1-AR of compound (D), Formula (I), Formula (II) and nadolol.

These results suggest compounds of Formulas (I) and (II) and nadolol block the P2-AR to an equivalent or higher degree than compound (D). The % inhibition of control specific binding of the P1-AR for Formula (I) was significantly lower (96.2%) compared to the other compounds, suggesting Formula (I) is the least active at the P1-AR. From the data it can be seen that both Formula (I) and Formula (II) are more selective for the P2-AR than the P1-AR. Moreover, Formula (I) is the most selective out of the four compounds.

Selectivity

Table 4

Selectivity for p-AR is determined from the number of receptors other than P1-AR and P2-AR for which the compound has a significant effect. Table 4 is an extract of the results of Table 2 summarizing the number of receptors for which compound (D), Formula (I), Formula (II) and nadolol showed a weak to moderate effect and a significant effect.

Not only do Formula (I) and Formula (II) show greater selectivity for the P2-AR than the other compounds, but the data also suggests they have a higher selectivity for the Pi- and P2-AR over other receptors. Table 2 shows that compound (D) causes significant inhibition or stimulation at seven receptors explored in this study (P1-AR, P2-AR, 5-HTIB, 5-HT2B, O- receptor, Na ⁺ channel, and 5-HT transporter). Additionally, nadolol was found to have significant effects on four receptors (P1-AR, P2-AR, 5-HTIA, 5-HTIB). In comparison, Formula (I) and Formula (II) only caused significant inhibition at the Pi- and P2-AR. Selectivity is a very important property of drugs for numerous reasons including limiting drug-drug interactions, limiting side-effects, and increasing patient acceptance.

Conclusion

The data demonstrates that Formula (I) is the most selective for the P2-AR of the compounds tested, and along with Formula (II) has the highest selectivity for the p-AR. The results from the binding assay also show that both Formula (I) and Formula (II) are more selective than compound (D). These results suggest that Formula (I) and Formula (II) will have fewer drugdrug interactions and fewer side effects in vivo, than either compound (D) or nadolol.

Example 3 - Functional assay

The aim of the cellular and nuclear functional assay was to compare the potency and affinity of the antagonists at the Pi- and P2-AR.

Methods cAMP Detection Overview:

In functional screens, the modulation of Gi- and Gs-coupled G-protein coupled receptors (GPCR) is typically monitored by detecting the intracellular signaling molecule 3’,5’-cyclic adenosine monophosphate (cAMP). The production of cAMP is controlled by adenylate cyclases, enzymes that are stimulated or inhibited as a result of direct interaction with G- protein alpha subunits. The role of the adenylate cyclases is to convert ATP to cAMP and inorganic pyrophosphate. After Gs-coupled GPCR activation, Gas positively stimulates the activity of adenylate cyclase, resulting in increased production of cAMP. In contrast, the stimulation of Gi-coupled GPCR results in a negative regulation of the adenylate cyclase and a decrease of cAMP levels.

Because endogenous levels of cAMP are low in the cells, models measuring activity of Gi- coupled receptors must first be stimulated with a stimulus in order to detect an inhibition of cAMP production following receptor stimulation. In addition, to counteract the natural degradation of cAMP to AMP that is catalyzed by phosphodiesterase (PDE) enzymes, an inhibitor of PDE (e.g. IBMX, rolipram) is present during the assay. The cAMP concentration is estimated by a FRET method, as described below. cAMP Production Measurement (GsPCR Receptors):

In the fluorescence resonance energy transfer (FRET) method, the native cellular cAMP binds to anti-cAMP antibodies labeled with europium cryptate (donor) in the presence of competing cAMP labeled with a modified allophyocyanin dye d2 (the acceptor). Therefore, the energy transfer is inversely proportional to the concentration of the cellular cAMP in the sample. A standard curve is used to convert raw data to cAMP concentration in the sample. Measurements are performed at A _ex = 337nm, A _e m = 620nm (peak of emission of the donor) and Aem = 665nm (peak of emission of the acceptor). The calculation of the fluorescence ratio (665nm/620nm) normalizes the signal, correcting the results for effects due to the optical characteristics of the media caused by colored compounds, serum, etc.

The FRET method overcomes most of the problems of autofluorescence resulting from unbound fluorophores.

Method for /3i-AR functional assays:

Transfected HEK-293 cells were suspended in HBSS buffer (Invitrogen) complemented with 20 mM HEPES (pH 7.4) and 500 pM IBMX, then distributed in microplates at a density of 3.10 ³ cells/well and preincubated for 5 min at room temperature in the presence of HBSS (basal control), the test compound or the reference antagonist. Thereafter, the reference agonist isoproterenol was added at a final concentration of 3 nM.

For basal control measurements, separate assay wells did not contain isoproterenol. Following 30 min at room temperature, the cells were lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) were added.

After 60 min at room temperature, the fluorescence transfer was measured at Aex=337 nm and Aem=620 and 665 nm using a microplate reader (Envison, Perkin Elmer). The cAMP concentration was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio).

The results are expressed as a percent inhibition of the control response to 3 nM isoproterenol.

The standard reference antagonist is atenolol, which was tested in each experiment at several concentrations to generate a concentration-response curve from which its IC50 value was calculated.

Method for /3 ₂ -AR functional assays:

Transfected CHO cells were suspended in HBSS buffer (Invitrogen) complemented with 20 mM HEPES (pH 7.4) and 500 pM IBMX, then distributed in microplates at a density of 10 ⁴ cells/well and preincubated for 5 min at room temperature in the presence of HBSS (basal control), the test compound or the reference antagonist.

Thereafter, the reference agonist isoproterenol was added at a final concentration of 10 nM. For basal control measurements, separate assay wells did not contain isoproterenol. Following 30 min at room temperature, the cells were lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAM P antibody labeled with europium cryptate) were added.

After 60 min at room temperature, the fluorescence transfer was measured at Aex=337 nm and Aem=620 and 665 nm using a microplate reader (Envison, Perkin Elmer).

The cAMP concentration was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio).

The results are expressed as a percent inhibition of the control response to 10 nM isoproterenol.

The standard reference antagonist is ICI 118551 , which was tested in each experiment at several concentrations to generate a concentration-response curve from which its IC50 value was calculated.

These experiments were performed using the conditions defined in Table 5 below. These experiments were performed on the antagonists listed in Table 6 below. Experiments were performed with several concentrations of each compound to allow for IC50 and EC50 determination. These concentrations ranged from 1.0 x1O ^-09 M to 3.0 x10'° ⁵ M. Table 5

¹ Frielle, T. et al. (1987), Proc. Natl. Acad. Sci. U.S.A., 84 : 7920-7924.

² Baker, J.G. (2005), Brit. J. Pharmacol., 144 : 317-322. Reference is made to citations describing each procedure, which are incorporated by reference herein.

The results were analysed as follows:

The results are expressed as a percent (%) of control agonist response: measured response - ■ - - - x 100 control response

And as a percent (%) inhibition of control agonist response: measured response

100 - ( - ■ - - - x 100) control response

Graphs were plotted of concentration against % inhibition of control agonist response and

EC50 values (concentration producing a half-maximal response) and IC50 values (concentration causing a half-maximal inhibition of the control agonist response) were determined by non-linear regression analysis of the resulting curves.

The dissociation constants (KB) were calculated using the modified Cheng Prusoff equation:

Where A = concentration of control agonist in the assay, and ECSOA = EC50 value of the reference agonist.

Lower IC50 values demonstrate molecules are more potent agonists or antagonists at the receptor being evaluated. Lower KB values show compounds have higher affinities for that receptor.

Potency and affinity

Table 6 ^a Data from Kawakami K et al, British Journal of Pharmacology 2006; 147: 642-652

The results in Table 6 show compound (D) and Formula (I) have lower IC50 and KB values than ICI 118,551 for the P2-AR, suggesting they are more potent antagonists at the P2-AR in addition to having higher affinities for the P2-AR. Additionally, the results suggest Formula (I) has a similar potency and affinity to compound (D) at the P2-AR, as the values of IC50 and KB are comparable. Formula (II) has values of IC50 and KB at the P2-AR which are comparable to those of ICI 118,551. Nadolol was shown to be the least potent P2-AR antagonist and had the lowest affinity for the P2-AR out of the compounds tested.

The assay also measured the potency and affinity of the compounds for the P1-AR. Formula (I) has the lowest potency and affinity for the P1-AR which suggests it has the highest selectivity for the P2-AR. Alternatively, compound (D) has a higher potency and affinity at the P1-AR than both Formula (I) and Formula (II).

Selectivity Table 7

In order to evaluate the selectivity of the compounds, the ratio of ICso for the Pi-AR compared to IC50 for the P2-AR was calculated, as shown in Table 7, and the results were all larger than 1 showing all the compounds are more potent antagonists at the P2-AR. Similarly, the ratio of KB at the Pi-AR compared to KB at the P2-AR was calculated, as shown in Table 7, again showing selectivity for the P2-AR.

Formula (I) gave significantly larger ratios than the other compounds from both calculations, showing it has a much higher selectivity to the P2-AR. This selectivity is important as it suggests Formula (I) will activate the P2-AR at concentrations significantly lower than those that will activate the Pi-AR. This will allow more control over the pharmacology as well as the potential side effects through the dosage. ICI 118,551 is quoted to have only 100 times greater selectivity for the P2- over the Pi-AR (Kawakami K et al, British Journal of Pharmacology 2006; 147: 642-652; Mauriege P, et al. Journal of Lipid Research 1988; 29: 587-601). Therefore, the results from this assay suggest Formula (I) is more potent, selective and has a higher affinity to the P2-AR than the current ‘gold standard’.

Conclusion

The data from this study shows Formula (I) is the most potent antagonist at the P2-AR out of all the compounds tested. This is seen in the IC50 values. Additionally, the KB values show Formula (I) has the highest affinity and selectivity for the P2-AR. This means it may be possible to limit Pi-related side effects by using Formula (I) as a P2-AR antagonist.

While Formula (II) is less selective for the P2 over the Pi -adrenoreceptor than Formula (I), the potency and affinity are still comparable to ICI 118,551. Combined with the results from the binding study, which shows that both Formula (I) and Formula (II) have higher selectivity for the p-AR than the other p-AR antagonists studied, Formula (II) is still a promising compound. Example 4 - Solubility

The aim of the solubility assay was to determine the solubility of Formula (I) in biologically relevant media.

The solubility of Formula (I) was measured in phosphate buffer and in fed state simulated intestinal fluid (FeSSIF). The results are shown in Table 8.

The 0.1 M phosphate buffer pH 7.4 was made by weighing out disodium hydrogen orthophosphate (14.2g) into a 1 L reagent bottle & potassium dihydrogen orthophosphate (6.8g) into a 0.5 L volumetric flask, and dissolving each in MilliQ water (0.5 L). The disodium hydrogen orthophosphate solution was warmed to 37 °C and adjusted to pH to 7.4 using the potassium dihydrogen orthophosphate.

FeSSIF pH 5.0 was made using a kit from Biorelevant (https://Biorelevant.com).

A 100 pM solution of Formula 1 in DMSO was made by diluting 5 pL of a 10 mM solution of Formula 1 in DMSO in 495 pL of DMSO. A 5 point standard curve of each test compound was made in 75:25 DMSO: water to give a range of concentrations of Formula (I); 5 pM, 500 nM, 50 nM, 5 nM and 0.5 nM.

1 ml of either 0.1 M phosphate buffer (pH 7.4) or FeSSIF buffer (pH 5.0) was added to 1 mg of solid compound in a vial supplied by Cyprotex and placed on a rotary incubator table at room temperature for 24 hr. Following incubation 200 pL of each of the incubated solutions was removed and filtered using a 96- well 0.4 pM MultiScreen HTS PCF Polycarbonate centrifugal filter plate. The filtrate was serially diluted 1 in 4, 1 in 40 and 1 in 400 fold into 75:25 DMSO:water. The standards and samples were quantified using a standard Waters Acquity UPLC-MS/MS system. The aqueous solubility of each test compound was quantified from a linear fit of the standard curve.

Table 8

The results in Table 8 demonstrate that Formula (I) Is soluble in biologically relevant media. Example 5 - Permeability in Caco2 cells

The aim of the permeability study was to test the Formula (I) and control compounds in a Caco2 permeability assay to model intestinal absorption (P _app (cm/s)) and drug-efflux (ratio).

Method

Caco-2 cells are widely used as an in vitro model for predicting human drug absorption. The Caco-2 cell line is derived from a human colorectal carcinoma, and when cultured, the cells spontaneously differentiate into monolayers of polarized enterocytes.

The Caco-2 cells were seeded on multiwell-insert plates and form a confluent monolayer over 20 days prior to the experiment. On day 20, the test compound was added to the apical side of the membrane and the flux of the compound across the monolayer was monitored over a 2-hour period. To study drug efflux, it was also necessary to investigate transport of the compound from the basolateral compartment to the apical compartment.

Caco-2 cells were obtained from the ATCC and used between passage numbers 40 - 60. The cells were seeded onto Transwell plates at 1 x 10 ⁵ cells/cm ² . The culture medium, DM EM (Sigma-Aldrich), was changed every two or three days. On day 20 the permeability study was performed, once a confluent monolayer was formed. Cell culture and assay incubations were carried out at 37 °C in an atmosphere of 5 % CO2 with a relative humidity of 95%. On the day of the assay, the monolayers were prepared by rinsing the apical and basolateral compartment with Hanks Balanced Salt Solution (HBSS) (Sigma-Aldrich) at the desired pH, warmed to 37 °C. Cells were then incubated with HBSS at the desired pH, in both apical and basolateral compartments for 30 minutes, to stabilise physiological parameters.

The dosing solutions were prepared by diluting test compound with assay buffer to give the desired test compound incubation concentration (typically 10 pM). The final DMSO concentration was 1% v/v. The fluorescent integrity marker lucifer yellow was also included in the dosing solution. Analytical standards were prepared from test compound DMSO dilutions and transferred to a buffer, maintaining a 1 % v/v DMSO concentration. The assay buffer was supplemented HBSS pH 7.4. For assessment of A-B permeability, HBSS was removed from the apical compartment and replaced with test compound dosing solution. The apical compartment insert was then placed into a companion plate containing fresh buffer (containing 1 % v/v DMSO). For assessment of B-A permeability, HBSS was removed from the companion plate and replaced with test compound dosing solution. Fresh buffer (containing 1 % v/v DMSO) was added to the apical compartment insert, which was then placed into the companion plate. At 120 min the apical compartment inserts and the companion plates were separated and apical and basolateral samples diluted for analysis.

Test compound permeability was assessed in duplicate. Compounds of known permeability characteristics were run as controls on each assay plate.

Test and control compounds were quantified by standard LC-MS/MS cassette analysis using a 7-point calibration with appropriate dilution of the samples. The starting concentration (Co) was determined from the dosing solution and the experimental recovery calculated from Co and both apical and basolateral compartment concentrations. The HPLC column was a Phenomenex Lux® i-Amylose-3 column 3 pm, 100 mm x 3 m and run on a Waters Xevo triple quad MS. The integrity of the monolayer throughout the experiment was checked by monitoring lucifer yellow permeation using fluorimetric analysis

The permeability coefficient (Pa _PP ) was calculated from the following equation:

Where dQ/dt is the rate of permeation of the drug across the cells, Co is the donor concentration at time zero and A is the area of the cell monolayer. Permeability was measured both ways across the cell monolayers: in the A2B (apical to basolateral) direction and B2A (basolateral to apical) direction to determine the efflux ratio. Efflux ratio was calculated as P _a pp(B-A)/P _a pp(A-B).

Antipyrine, atenolol, and talinolol were included as controls. Antipyrine shows a 97% human absorption following oral administration. Atenolol shows 50% human absorption following oral administration. Talinolol is a substrate of P-glycoprotein and was included as a negative control.

Lower values of P _app show greater absorption. Lower efflux ratios show lower efflux, and values over 2 have active efflux. Results

Table 9

The results in Table 9 show Formula (I) has good absorption (permeability in Caco2 cells). The results also show low active reflux of Formula (I) by Caco-2 cells. The compound of Formula (I) ranked between atenolol and antipyrine for both P _app measurements and between atenolol and talinolol for efflux. The highest active efflux was shown for talinolol.

Conclusion

The data from this study shows Formula (I) has high permeability and low efflux in Caco-2 cells.

Example 6 - Hepatocyte Stability

The aim of the hepatocyte stability study was to measure the stability of compounds in hepatocytes.

Methods

Cryopreserved pooled hepatocytes were purchased from IVT (human and dog) and Lonza (rat) and stored in liquid nitrogen prior to use.

Williams E media (Sigma-Aldrich) supplemented with 2 mM L-glutamine and 25 mM HEPES (Sigma-Aldrich) and test compound (final substrate concentration 3 pM; final DMSO concentration 0.25%) were pre-incubated at 37 °C prior to the addition of a suspension of cryopreserved hepatocytes (final cell density 0.5 x 10 ⁶ viable cells/mL in Williams E media supplemented with 2 mM L-glutamine and 25 mM HEPES) to initiate the reaction. The final incubation volume was 500 pL. Samples were removed at 6 time points over the course of a 60 min experiment and test compound was analysed by LC-MS/MS. The reactions were stopped by transferring incubate into acetonitrile and 1 :1 dilution at the appropriate time points, in a 1:2 ratio. The termination plates were centrifuged at 3,000 rpm for 20 min at 4 °C to precipitate the protein.

Following protein precipitation, the sample supernatants were combined in cassettes of up to 4 compounds and internal standards added. LC-MS/MS was done on the Vion instrument using a Phenomenex Lux® i-Amylose-3 Column: 3 pm, 100 mm x 3 mm. The eluents were: 10% A - Deionised water containing 10mM ammonium bicarbonate; 90% B - Methanol;

Flow rate: 0.5 mL/min. Total run time: 15 min.

Two quality control compounds (verapamil and raloxifene) were included with each species, alongside appropriate vehicle control.

From a plot of In peak area ratio (compound peak area/internal standard peak area) against time, the elimination rate constant was determined. Subsequently, half-life (f/2) and intrinsic clearance (CLint) were calculated using the equations below. The In peak area (compound peak area/internal standard peak area) was plotted against time and the gradient of the line determined). The elimination rate constant (K) = (-gradient). Half-life (ti/2) (min) = 0.693/K.

V x 0.693 Intrinsic Clearance (CLi _nt )(|iL/min/million cells) = - tl/2 where V = Incubation volume (pL) I number of cells.

Results

Table 10 Table 11

Where SE in Table 11 is the standard error associated with CLjnt (intrinsic clearance).

The results in Tables 10 and 11 shows that Formula (I) was more stable than Compound (D) in rat, dog, or human hepatocytes. Compound (D) had higher intrinsic clearance and lower half-life across the species. Formula (I) had lower intrinsic clearance and higher half-life stability in rat, dog, and human hepatocytes, demonstrating this higher stability.

Conclusion

The data from this study shows Formula (I) is more stable than Compound (D) in rat, human and dog hepatocytes.

Example 7 - Interconversion test in primary hepatocytes

The aim of this study was to determine whether the Formula (I) converted to Formula (II) in primary hepatocytes.

The hepatocyte incubation, analytical conditions and equipment were the same as in Example 6. However for Example 7, reference standards of both Formula (I) and Formula (II) were supplied for the LC-MS/MS and both run simultaneously to show that chromatographic separation was achieved.

LC-MS/MS showed no presence of Formula (II) in the hepatocyte samples, indicating no conversion of Formula (I) to Formula (II). Accordingly, the results of this study show that Formula (I) does not interconvert to Formula (II) in rat, dog, or human hepatocytes. Example 8 - hERG channel inhibition

The aim of this study was to measure cardiotoxicity of Formula (I) via hERG channel inhibition, specifically by assessing the potential of Formula (I) to inhibit the hERG channel.

Methods

A cumulative concentration-response-curve, 3 concentration/compound, N=5 was generated. The compound was tested at 1 , 10 and 100 pM with 0.5% DMSO as vehicle. A vehicle group, in which vehicle (0.5% DMSO) was dispensed as a test substance, was also tested.

The effect of test compounds on the hERG cardiac ion channel, expressed in mammalian cells (H EK-293), was assessed using the electrophysiology platform QPatch HTX. This was an automated electrophysiology system that follows the general principles of conventional whole-cell patch-clamping. The percent change in hERG tail-current was calculated and used to calculate an IC50 value (test compound concentration that produced 50 % inhibition).

The experiments were performed on a QPatch HTX system (Sophion Biosciences A/S). The system was primed with appropriate extracellular (NaCI, KCI, CaCh, MgCl2, D-glucose, HEPES, pH 7.4) and intracellular (KCI, MgCI ₂ , EGTA, MgATP, HEPES, pH 7.2) solutions prior to conducting the study in a 48-well plate (QPIate, Sophion Biosciences A/S). All cell suspensions, buffers and test compound solutions were at room temperature during the experiment.

The cells used were HEK293 cells stably transfected with hERG cDNA. The cells were automatically washed and resuspended before being positioned into each well (recording chamber) of the QPIate. The QPatch system followed the general principles of conventional whole-cell patch-clamping: a high resistance seal was formed between the patch electrode and an individual cell, the membrane across the electrode tip was then ruptured and the whole-cell patch-clamp configuration was established. If the quality of the cell was judged to be poor, the experiment was terminated at that point and if necessary, the process repeated on another plate. Once a stable patch was achieved, recording commenced in voltage-clamp mode.

The standard voltage profile was as follows: step from -80 mV to -50 mV for 200 ms, +20 mV for 4.8 s, step to -50 mV for 5 s then step to the holding potential of -80 mV. The step from - 80 mV to the test command (+20 mV) resulted in an outward current (i.e. current flows out of the cell) and the step from the test command (+20 mV) to -50 mV resulted in the tail current (the tail current represents deactivation of the current over time).

Compound dilutions were prepared by diluting a DMSO solution (default 10 mM), followed by dilution into extracellular buffer to the final concentrations tested. Perfusion solutions contained 0.05 % Pluronic F-68 and were stored at room temperature for the duration of the experiment each day.

The voltage protocol was run and recorded continuously during the experiment. Cells were treated with vehicle for 3 min (i.e. 0.1 % DMSO) followed by three increasing concentrations of the test substance. The test compound was applied in triplicate at each concentration and tested in at least 2 cells. The standard combined exposure time was 5 min. One vehicle group was run per experiment. In the positive control group, vehicle (0.1 % DMSO) was dispensed as a test substance, followed by addition of a positive control (E-4031).

The average of tail current amplitude values recorded from 4 sequential voltage pulses was used to calculate for each cell the effect of the test substance by calculating the residual current (% control) compared with vehicle pre-treatment (0 % inhibition). The data were plotted and an IC50 value was estimated from the concentration-response relationship.

Results

Table 12 - Formula (I)

Table 13 - Vehicle

Formula (I) showed <50% inhibition at the highest tested concentration (100 pM) and was classified as pICso <4. E-4031 was tested as hERG pharmacological standard and results were in line with historical in-house data.

Conclusion

Table 12 above shows that Formula (I) does not inhibit the hERG channel. hERG channel activity is therefore not of concern for Formula (I).

Example 9 - AMES test

The aim of this study was to study the mutagenic potential of Formula (I) using an AMES test.

Method

Compounds were tested at concentrations of 62.5, 125, 250, 500, 1000, 2000 pg/mL (the top concentration in this dose response was chosen based on aqueous solubility). 2- aminoanthracene, 2-nitrofluorene, and 4-nitroquinoline N-oxide were included as positive controls.

The AMES assay was performed using a kit from Xenometrix, part number: A10-210-S2-P, which contains the bacteria and all the required solutions. Approximately ten million bacteria were exposed in triplicate to Formula (I) (six concentrations), a negative control (vehicle) and a positive control for 90 minutes in medium containing a low concentration of histidine (sufficient for about 2 doublings). The cultures were then diluted into indicator medium lacking histidine, and dispensed into 48 wells of a 384 well plate (micro-plate format, MPF).

The plate was incubated for 48 hr at 37°C, and cells that have undergone a reversion grew in a well, resulting in a colour change in wells with growth. The number of wells showing growth were counted and compared to the vehicle control.

An increase in the number of colonies of at least two-fold over baseline (mean + SD of the vehicle control) and a dose response indicated a positive response. An unpaired, one-sided Student’s T-test was used to identify conditions that were significantly different from the vehicle control.

S9 contains both microsomal and cytosolic fractions and was included as a metabolising system in the Ames test, since chemical substances sometimes require metabolism in order to become mutagenic. Where indicated, S9 fraction from the livers of Aroclor 1254-treated rats was included in the incubation at a final concentration of 4.5%.

S. typhimurium 98 hisD3052, rfa, uvrB / pKM101 (TA98) and S. typhimurium MOO hisG45, rfa, uvrB I pKM101 (TA100) were used in this study. TA98 detects frame-shift mutations. TA100 detects base-pair substitutions.

Results

Table 15

The data in Table 15 shows that Formula (I) did not exhibit mutagenic potential in the absence or presence of S9 metabolic activation. Formula (I) did not exhibit any genotoxic effects against either strain used in this assay. The positive controls all behaved as expected in this experiment.

Embodiments of the invention have been shown and described herein. It will be apparent to those skilled in the art that such embodiments are provided by way of example only and are considered to be illustrative rather than restrictive. It will be appreciated that variations in form and detail can be made without departing from the scope of the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention and that embodiments within the scope of these claims and their equivalents are to be covered thereby.