Selective β2-adrenergic receptor antagonist

Abstract

The present invention relates to compounds and compositions useful as selective β2-adrenergic receptor (β2-AR) antagonists, and their use in the treatment and prevention of disease.

Description

[Technical Field]

[0001] The present invention relates to novel compounds and compositions useful as selective β2 adrenergic receptor (β2-AR) antagonists, and their use in the treatment and prevention of disease.

[0002] background β-Adrenergic receptors (β-ARs) are a family of G protein-coupled receptors (β1, β2, and β3) that mediate physiological responses to epinephrine (adrenaline) and norepinephrine (noradrenaline). β1 and β2 are the major types of β-ARs. β1-ARs are primarily found in the heart, while β2-ARs are primarily found in blood vessels and airways. Therefore, β1-ARs and β2-ARs are associated with different physiological effects and, consequently, different diseases.

[0003] Nonselective β-AR antagonists ("β-blockers"), such as propranolol and nadolol, inhibit both β1-AR and β2-AR and can be used to treat conditions resulting from β1-AR activation, such as anxiety, angina, and hypertension, as well as conditions resulting from β2-AR activation, such as physiological tremor and migraine. Selective β1-AR antagonists, such as atenolol and metoprolol, inhibit β1-AR more strongly than β2-AR and are used to treat cardiovascular diseases such as hypertension, chronic stable angina, and post-myocardial infarction. Selective β2-AR antagonists inhibit β2-AR more strongly than β1-AR. To date, no selective β2-AR antagonists have been marketed for clinical use, and propranolol is now commonly used instead. A drawback of using propranolol as a β2-AR antagonist is that β1-AR antagonism is associated with various side effects.

[0004] ICI 118,551 has been shown to be a highly selective β2-AR antagonist but has never been commercially available.

[0005] Receptor selectivity is commonly sought because side effects are often due to off-target drug effects. Therefore, improving selectivity is desirable, increasing affinity for target receptors and decreasing affinity for off-target receptors. Furthermore, improved selectivity reduces the risk of drug-drug interactions, which are a common cause of side effects and can reduce the pharmacological activity of one drug. In general, high selectivity and efficacy allow for minimal dosage, leading to reduced drug costs and easier administration.

[0006] In recent years, the β2-AR signaling pathway has been implicated in certain diseases, for which selective β2-AR antagonists may be effective in treating them. These diseases include vascular tumors, cancers, paragangliomas, and tuberous sclerosis; vascular tumors such as infantile hemangiomas, von Hippel-Lindau disease, angiosarcomas, and gliomas; cancers such as soft tissue sarcomas, melanomas, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, and lymphoblastic leukemia; vascular abnormalities such as hereditary hemorrhagic telangiectasia and cerebral cavernous malformations; respiratory diseases such as asthma and chronic obstructive pulmonary disease; cardiovascular diseases such as chronic heart failure and takotsubo syndrome; and other conditions such as physiological tremor and migraine.

[0007] Selective β2-AR antagonists may be beneficial in the treatment of these diseases. Thus, there is an unmet need for highly selective β2-AR antagonists.

[0008] EP0003664 and GB2165835 describe alkanolamine derivatives as selective β2-AR antagonists.

[0009] ICI 118,551 (DL-erythro-3-isopropylamino-1-(7-methyl-4-indanyloxy)-2-butanol hydrochloride) is considered the "gold standard" of selective β2-AR antagonists, and has been reported to be 100-fold more selective for β2-AR than β1-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). The structure of ICI 118,551 is as follows:

[0010] [ka]

[0011] In preclinical studies, ICI 118,551 was shown to be more potent at the β2-AR than the β1-AR in vitro and in vivo compared with propranolol, and was highly selective for the β2-AR, without affecting other organ systems, including the central nervous system, reproductive system, and gastrointestinal system.

[0012] In clinical trials in healthy subjects, ICI 118,551 proved to be a highly selective β2-AR antagonist at doses of 2.5 to 40 mg, but at higher doses, ICI 118,551 exhibited increased activity at β1-AR.

[0013] In a phase 2 study in patients with hypertension, anxiety, physiological tremor, generalized nervousness, fear of flying stress, and neuroleptic-induced akathisia, ICI 118,551 suppressed tremor and neuroleptic-induced akathisia similarly to propranolol, had minimal effect on fear of flying stress, and had no effect on other symptoms. ICI 118,551 was not subsequently developed or marketed for clinical use. Following administration, ICI 118,551 produces several distinct metabolites (French, KH Journal of Liquid Chromatography 1989; 12:861-873). Three of these metabolites are known to be selective β2-AR antagonists, although they are generally recognized to be less selective than ICI 118,551 (Fitzgerald, JD Cardiovascular Drugs and Therapy 1991; 5(3): 561-576). Summary of the Invention [Problem to be solved by the invention]

[0014] Thus, there remains an unmet need for selective β2-AR antagonists for clinical use.

[0015] Surprisingly, it has been found that compounds of formula (I) or (II), or pharmaceutically acceptable salts thereof, act as selective antagonists of the β2-AR over the β1-AR.

[0016] [ka]

[0017] As shown above, compounds of formula (I) have SSR stereochemistry and compounds of formula (II) have SSS stereochemistry.

[0018] Furthermore, these compounds have been shown to be more selective for the β-AR over other receptors compared to ICI 118,551, while still being as potent or more potent than ICI 118,551. [Means for solving the problem]

[0019] Summary of the Invention In a first aspect, there is provided a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof:

[0020] [ka]

[0021] In a preferred embodiment, the compound has formula (I): In any of the above embodiments, the pharmaceutically acceptable salt may be the hydrochloride salt.

[0022] 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%, even more preferably at least 95%.

[0023] In a preferred embodiment, the compounds of formula (I) or (II) have 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%.

[0024] In a second aspect, a composition is provided comprising: a) a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof:

[0025] [ka]

[0026] ; and b) A pharmaceutically acceptable carrier or excipient.

[0027] 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%, even more preferably at least 95%.

[0028] In a preferred embodiment, the compounds of formula (I) or (II) have 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%.

[0029] In a preferred embodiment, the compound is represented by formula (I). In any of the above embodiments, the pharmaceutically acceptable salt may be a hydrochloride salt. In any of the above embodiments, the composition may further comprise an additional active agent.

[0030] In any of the above embodiments, the pharmaceutical composition may be provided in an oral, buccal, sublingual, subcutaneous, intravenous, intramuscular, intranasal, inhalation, rectal, or topical dosage form.

[0031] In a third aspect, a compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any of the embodiments of the second aspect of the invention, is provided for use as a medicament.

[0032] In a fourth aspect, there is provided a compound according to any of the embodiments of the first aspect of the invention, or a pharmaceutical composition according to any of the embodiments of the second aspect of the invention, for use in the treatment or prevention of a disorder associated with the β2-AR signaling pathway.

[0033] In some embodiments, the disorder associated with the β2-AR signaling pathway is a tumor, a vascular abnormality, a respiratory disease, a cardiovascular disease, physiological tremor, or a migraine.

[0034] In some embodiments, the tumor is a vascular tumor, carcinoma, paraganglioma, or tuberous sclerosis, preferably a vascular tumor or carcinoma. In some embodiments, the vascular tumor is infantile hemangioma, von Hippel-Lindau disease, angiosarcoma, or glioma, preferably infantile hemangioma or von Hippel-Lindau disease. In some embodiments, the cancer is soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, or lymphoblastic leukemia. In some embodiments, the vascular abnormality is hereditary hemorrhagic telangiectasia or cerebral cavernous malformation, preferably hereditary hemorrhagic 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.

[0035] Preferably, the disorder associated with the β2-AR signaling pathway is a vascular tumor such as infantile hemangioma, von Hippel-Lindau disease, angiosarcoma or glioma, a vascular abnormality such as hereditary hemorrhagic telangiectasia, or a cancer such as breast cancer, pancreatic cancer, or ovarian cancer.

[0036] More preferably, the disorder associated with the β2-AR signaling pathway is a vascular tumor such as infantile hemangioma, von Hippel-Lindau disease, angiosarcoma or glioma, or a vascular abnormality such as hereditary hemorrhagic telangiectasia.

[0037] In the embodiment where the disease is asthma, the compound or composition can be used to treat or prevent the β2-AR downregulation caused by the excessive use of long-acting β2-AR agonists (LABA), very long-acting β2-AR agonists (ultra-LABA) or short-acting β2-AR agonists (SABA).When the disease is asthma, the compound or composition can be administered to the subject suffering from the β2-AR downregulation caused by the excessive use of LABA, ultra-LABA or SABA.The LABA can be salmeterol and / or formoterol, the ultra-LABA can be olodaterol and / or vilanterol, and the SABA can be salbutamol and / or terbutaline.

[0038] In a fifth aspect, there is provided a compound according to any embodiment 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 β2-AR antagonist.

[0039] In embodiments, the compound or pharmaceutical composition for use in any of the embodiments of the third to fifth aspects may be administered orally, bucally, sublingually, subcutaneously, intravenously, intramuscularly, intranasally, by inhalation, rectally, or topically.

[0040] In a sixth aspect, a method for treating or preventing a disease associated with the β2-AR signaling pathway is provided, the method comprising administering to a subject a compound according to any one of the embodiments of the first aspect of the present invention or a pharmaceutical composition according to any one of the embodiments of the second aspect of the present invention. In an embodiment, the disease associated with the β2-AR signaling pathway may be as defined in the fourth aspect. In an embodiment, the disease associated with the β2-AR signaling pathway is a tumor, a vascular abnormality, a respiratory disease, a cardiovascular disease, physiological tremor, or migraine. The disease associated with the β2-AR signaling pathway may be as defined in the fourth aspect.

[0041] In a seventh aspect, there is provided a compound according to any one of the first and second aspects of the present invention, or a pharmaceutical composition according to any one of the second and second aspects of the present invention, for use in the manufacture of a medicament for the treatment or prevention of a disease associated with the β2-AR signaling pathway. In an embodiment, the disease associated with the β2-AR signaling pathway may be as defined in the fourth aspect. In an embodiment, the disease associated with the β2-AR signaling pathway is tumor, vascular abnormality, respiratory disease, cardiovascular disease, physiological tremor, or migraine. The disease associated with the β2-AR signaling pathway may be as defined in the fourth aspect.

[0042] In an eighth aspect, there is provided the use of a compound according to any embodiment 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 β2+-AR antagonist.

[0043] Detailed Description As used herein, the term "including" means "including but not limited to."

[0044] The compounds of formula (I) or (II) or pharmaceutically acceptable salts thereof are β2-AR antagonists.

[0045] [ka]

[0046] These are also referred to herein as compounds according to the invention.

[0047] The β2-AR signaling pathway has been implicated in certain disorders and the compounds of the present invention may be beneficial in treating these disorders.

[0048] Diseases associated with the β2-AR signaling pathway tumor Tumors treatable with the β2-AR antagonists described in the present invention include vascular tumors, cancer, paraganglioma, and tuberous sclerosis. Preferably, the tumor is a vascular tumor or cancer. Vascular tumors include infantile hemangioma, von Hippel-Lindau disease, angiosarcoma, and glioma. Preferably, the vascular tumor is infantile hemangioma 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 leukemia.

[0049] cancer Studies using the selective β2-AR antagonist ICI 118,551, the selective β1-AR antagonist atenolol, and the nonselective β-AR antagonist propranolol have demonstrated that the mechanism of tumor growth is β2-AR, not β1-AR. Furthermore, there is growing evidence that cancer metastasizes via neural pathways, and that β2-ARs present on blood vessels are a mechanism of tumor spread. Therefore, inhibiting β2-ARs with β2-AR antagonists may slow or prevent the spread of tumors, including cancer.

[0050] The role of β2-AR activation in promoting metastasis and cancer progression has been demonstrated in many tumor 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 leukemia (Lamkin et al., Brain Behav Immun 2012;26:635-641), and triple-negative brain metastasis cells (Choy et al., Oncol Rep 2016;35:3135-3142). Thus, this mechanism appears to be present in many tumor types (Pimental et al., In Jandial R, editor, Metastatic Cancer: Clinical and Biological Perspectives. Austin, Texas USA: Landes Bioscience 2013;169-179).

[0051] Selective β1-AR antagonists such as atenolol did not produce similar results, suggesting that the effects on tumor progression and metastasis are the result of β2-AR antagonism (Barron et al., J Clin Oncol 2011; 29: 2635-2644).

[0052] Examples of cancer include soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, and lymphoblastic leukemia.

[0053] Because β2-AR antagonism has been demonstrated to be superior to β1-AR antagonism, selective β2-AR antagonists are believed to be useful in the prevention and treatment of cancer.

[0054] Vascular tumors The compounds or compositions of the present invention can treat several types of vascular tumors, including infantile hemangioma, von Hippel-Lindau disease, angiosarcoma, and glioma. Preferably, the vascular tumor is infantile hemangioma or von Hippel-Lindau disease.

[0055] Infantile hemangioma Infantile hemangiomas are pediatric vascular tumors. Most are not present at birth. They usually appear between 4 and 6 weeks of age, then undergo periods of growth, stabilization, and involution. Infantile hemangiomas can occur in the skin, subcutaneous tissue, or any organ in the body. They most commonly occur on the skin of the head and neck and typically appear as bright red-blue lesions that can be raised or flat. These tumors are benign, and most resolve without treatment. However, they can be associated with debilitating or life-threatening complications, such as respiratory failure and congestive heart failure. Visible lesions can cause psychological problems for children and parents. Approximately 10% are complex and require referral to a specialist.

[0056] Propranolol is currently approved by the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) for the treatment of infantile hemangiomas, and numerous studies have demonstrated its efficacy in treating infantile hemangiomas (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, a comparison of propranolol with nadolol, which does not cross the blood-brain barrier, showed that nadolol was more effective in treating infantile hemangiomas (Pope et al., Br J Dermatol 2013; 168: 222-224). Propranolol and nadolol are nonselective β1- and β2-AR antagonists. However, nadolol is more selective for β2-AR, suggesting that selective β2-AR antagonism may be beneficial in the treatment of infantile hemangiomas.

[0057] The pathogenesis of infantile hemangiomas is not fully understood, but neovascularization and angiogenesis are likely involved. In vitro studies using von Hippel-Lindau hemangiomas cells and studies using a mouse model of oxygen-induced retinopathy provide evidence that the effects of propranolol on infantile hemangiomas are mediated by β2-AR, as detailed herein.

[0058] One proposed hypothesis for the pathogenesis of infantile hemangiomas is fetal or neonatal hypoxia. Hypoxia is a condition in which insufficient oxygen reaches tissues. Oxygen delivery is regulated by hypoxia-inducible factors (HIFs) and an oxygen-sensing mechanism that involves hydroxylation of HIFs by a series of dioxygenases. Hypoxia suppresses these processes, and HIFs activate a large transcriptional cascade of proteins, including erythropoietin (EPO), vascular endothelial growth factor (VEGF), the entire glycolytic enzyme array, and many others. Together, these proteins enhance cellular resistance to hypoxia and ischemia (Bishop and Ratcliffe 2014).

[0059] von Hippel-Lindau disease Von Hippel-Lindau disease (VHL) is a rare autosomal dominant disorder caused by germline mutations in the tumor suppressor gene VHL on the short arm of chromosome 3. A variety of benign and malignant vascular tumors develop throughout life, including hemangioblastomas of the retina and central nervous system, renal carcinoma, pheochromocytoma, paraganglioma, endolymphatic sac, pancreatic cystadenomas and neuroendocrine tumors, and cystadenomas of the epididymis and broad ligament. Von Hippel-Lindau disease is a long-term, debilitating, and life-threatening condition that often leads to early death. Patients with VHL primarily undergo surgery to remove tumors in affected organs and laser therapy to treat ocular symptoms.

[0060] In January 2017, the European Medicines Agency (EMA) granted orphan drug designation to propranolol for the treatment of von Hippel-Lindau disease. Patients with von Hippel-Lindau disease demonstrated a stabilization of the number and size of retinal hemangioblastomas after one year of daily propranolol administration. This correlated with a gradual decrease in plasma biomarker levels of VEGF and miR210, similar to that observed in the general population (Gonzalez-Rodriguez et al., BMJ Open Ophthalmol 2019;4:e000203. doi:10.1136 / bmjophth-2018-000203). Furthermore, propranolol has been shown to act via the β2-AR rather than the β1-AR, despite the expression of both receptors in hemangioma stem cells (Munabi et al., Stem Cells Transl Med 2016;5:45-55). However, propranolol is a nonselective β1-AR and β2-AR antagonist, causing hypotension and bradycardia, making it a disadvantage in treating normotensive patients with von Hippel-Lindau syndrome. The effects of propranolol on blood pressure and heart rate are primarily mediated through β1-AR.

[0061] The selective β2-AR antagonist ICI 118,551 inhibited pro-angiogenic factors and reduced pathological angiogenesis, whereas atenolol, a selective β1-AR antagonist, did not. These effects of ICI 118,551 were confirmed to be due to its action on β2-AR (Martini et al., J Neurochem 2011; 119: 1317-1329). Furthermore, ICI 118,551 has been shown to have minimal effects on resting blood pressure and heart rate, and to have no effect on healthy vascular endothelial cells (McCafrey et al., J Cardiovasc Pharmacol 1988; 11: 543-551). In primary cultures of von Hippel-Lindau hemangioblastoma, ICI 118,551 reduced cell viability and halted or substantially delayed key angiogenic processes, cell migration and tube formation; under hypoxic conditions, it inhibited the nuclear import of HIF-1α in primary cultures of hemangioblastoma cells and human endothelial cells (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 inhibited proliferation, angiogenesis, and inflammation in vHL-ccRCC cancer cells. In two in vivo xenograft models of vHL-ccRCC, ICI 118,551 slowed tumor progression by inhibiting proliferation (Albihana et al., Orphanet J Rare Dis 2015;10:118). This suggests that selective β2-AR antagonists may be effective in treating von Hippel-Lindau disease.

[0062] Angiosarcoma Angiosarcoma is a rare malignant vascular endothelial cell tumor of vascular or lymphatic origin, with a high rate of local recurrence and metastasis. It can occur throughout the body, but in Caucasians, it primarily occurs in the head and neck, particularly the scalp. Only 10% of cases arise in deep soft tissues, with the remainder occurring in parenchymal organs such as the breast, bone, spleen, and liver.

[0063] Angiosarcoma is extremely difficult to treat. Treatment typically involves a combination of cytotoxic therapy, surgery, and radiation therapy. Tissue samples from angiosarcoma patients showed strong β2-AR expression and weak to moderate β1-AR expression (Porcelli et al., Sci Rep 2020;10:10465). Patient-derived soft tissue sarcoma cells and tissues with high β2-AR expression responded favorably to propranolol and chemotherapy (docetaxel), suggesting that β2-AR expression levels may be associated with chemotherapy responsiveness in some tumors. This study further strengthens evidence that β2-AR plays an important role in mechanisms of resistance to standard chemotherapy and that antagonism of this receptor improves tumor response.

[0064] In vitro studies of angiosarcoma cells using the β2-AR-selective ICI 118,551 and the nonselective β1-AR and β2-AR antagonist propranolol demonstrate that they reduce tumor cell viability by inhibiting mitogenic signaling pathways and MAPK mediators downstream of β2-AR (Amaya et al., Oncoscience 2018;5:109-119). The similar reduction in cell viability induced by ICI 118,551 and propranolol suggests that their mechanisms of action are via antagonism of the same β2-AR signaling pathway. Studies using propranolol have shown that most treated patients achieved at least partial remission (Wagner et al., J Exp Pharmacol 2018:10:51-58). Therefore, selective β2-AR antagonists are likely to achieve similar efficacy with fewer side effects attributable to β1-AR binding.

[0065] vascular abnormality The compound or composition of the present invention can also treat some types of vascular abnormalities that may be life-threatening and / or cause serious debilitation.These include hereditary hemorrhagic telangiectasia or cerebral cavernous malformation.Preferably, the vascular abnormality is hereditary hemorrhagic telangiectasia.

[0066] Hereditary hemorrhagic telangiectasia Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant disorder characterized by telangiectasia of blood vessels in the skin and mucous membranes, and arteriovenous malformations in internal organs, including the lungs, liver, gastrointestinal tract, brain, and spinal cord. Patients with HHT are at increased risk for brain abscesses, stroke, migraines, and bleeding, primarily from the nose (nosebleeds), lungs, and gastrointestinal tract, which can lead to iron deficiency and anemia. HHT is a disabling and life-threatening condition that affects both children and adults.

[0067] There are currently no safe and effective first-line treatments for hereditary hemorrhagic telangiectasia. The topical and / or oral administration of propranolol, a nonselective β-AR antagonist, has attracted attention as an antiangiogenic therapy. Propranolol has been shown to reduce the severity, duration, and frequency of epistaxis in multiple 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). In peripheral vascular endothelial cells, β2-AR is the predominant β-AR. Activation of β2-AR on blood vessels leads to stimulation of VEGF, which regulates angiogenesis (Kritharis et al., Haematologica 2018; 103: 1433-1443). Therefore, the therapeutic effect of propranolol in hereditary hemorrhagic telangiectasia, as well as other vascular diseases such as infantile hemangioma and von Hippel-Lindau disease, is likely mediated by inhibition of β2-AR.

[0068] Therefore, selective β2-AR antagonists are of interest for the treatment of hereditary hemorrhagic telangiectasia, as they avoid the hypotension and bradycardia side effects of propranolol.

[0069] respiratory disease Respiratory diseases include asthma, chronic obstructive pulmonary disease (COPD), occupational lung disease, and pulmonary hypertension. As a respiratory disease, asthma is preferred. Asthma is characterized by airway hyperresponsiveness, airway inflammation, airway expiratory limitation, mucus hypersecretion, and airway remodeling. Symptoms of an asthma attack include shortness of breath, wheezing, coughing, chest tightness, and dyspnea. The goal of asthma management is to reverse or prevent acute bronchoconstriction and inflammation associated with β2-AR signaling.

[0070] Most asthma patients successfully control their asthma symptoms with short-acting β2-AR agonists (SABAs) such as salbutamol and terbutaline, inhaled corticosteroids (ICS), long-acting β2-AR agonists (LABAs) such as salmeterol and formoterol, and ultra-long-acting β2-AR agonists (ultra-LABAs) such as olodaterol and vilanterol. However, approximately 10% of patients develop resistance to ICS or LABA therapy (Chung KF, J Intern Med 2016; 279: 192-204). Long-term use of LABAs alone can lead to tolerance, increasing the risk of asthma exacerbations and death (Nelson et al., Chest 2006; 129: 15-26; Salpeter et al., Ann Intern Med 2002; 137: 715-25). Resistance correlates with desensitization and downregulation of β2-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., AJ Med 1994; 97: 29-37; Grove et al., Thorax 1995; 50: 134-138; Aziz et al., Chest 1999; 115: 623-8).

[0071] Preclinical studies have shown that chronic administration of nadolol or ICI 118,551 reduces airway hyperresponsiveness, inflammation, and mucin content, and ameliorates 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). Therefore, selective β2-AR antagonists may be useful for the treatment of asthma.

[0072] Without being bound by theory, chronic stimulation of airway β2-ARs by chronic use of LABAs or ultra-LABAs is thought to lead to desensitization and downregulation of β2-ARs and upregulation of harmful signaling pathways. Similar effects have been observed after excessive use of SABAs. This downregulation of β2-ARs can reduce or even eliminate the effectiveness of subsequent SABA administration. Another problem associated with LABA use is β2-AR saturation. This means that subsequent administration of SABAs, for example, to relieve immediate symptoms, is ineffective. Therefore, administration of selective β2-AR antagonists may upregulate β2-ARs and control severe asthma symptoms.

[0073] Examples of LABAs include salmeterol and formoterol, examples of ultra-LABAs include olodaterol and vilanterol, and examples of SABAs include salbutamol and terbutaline.

[0074] cardiovascular disease Cardiovascular diseases that can be treated with the β2-AR antagonists described in this invention include chronic heart failure or Takotsubo syndrome.

[0075] chronic heart failure Chronic heart failure is a complex syndrome characterized by reduced cardiac output, fluid retention, and elevated venous pressure. Although several β-AR antagonists have been approved for the treatment of chronic heart failure, they are all nonselective β1-AR antagonists. However, recent studies have revealed that the β2-AR pathway plays an important role in chronic heart failure, and β2-AR antagonists may be used for the treatment of chronic heart failure (Black & Fitzgerald, Curr Pharm Des 2010; 16: 4148-4158).

[0076] All β-AR antagonists currently used to treat chronic heart failure require a complex, time-consuming dose-titration regimen that must be individually tailored for each patient by a highly skilled physician. This involves starting with a very low dose and then gradually increasing it over 2 to 12 months until a therapeutically effective dose is reached. Dose-titration is necessary because all currently used β-AR antagonists antagonize the β1-AR, at least to some extent, thereby inhibiting signaling through this receptor that promotes cardiac contraction, resulting in undesirable side effects.

[0077] Without being bound by theory, it is believed that during dose escalation, β1-AR activity in the failing heart increases sufficiently that the final dose of the β-AR antagonist does not significantly inhibit signaling through the β1-AR population, but significantly inhibits adverse effects mediated by β2-ARs. As a result, by the time the final dose is reached, β1-AR activity in the heart is sufficient to counteract β1-AR blockade and maintain cardiac function. Because β2-AR signaling is also inhibited, cardiac function improves. However, if the patient receives the final dose without prior dose escalation, the failing heart will not have sufficient β1-AR activity to counteract β1-AR blockade, and cardiac function will decline.

[0078] Healthy ventricular myocardium contains both β1-AR and β2-AR in a ratio of approximately 3:1 (Hedberg et al., J Pharmacol Exp Ther 1980; 212(3): 503-8). Activation of β1-AR and β2-AR increases cardiac output by increasing heart rate and cardiac output (increased ejection fraction), and induces phosphorylation of β1-AR and β2-AR. Recently, it has been shown that phosphorylation of β2-AR leads to activation of an alternative, or "non-classical," pathway (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 activates several deleterious cellular pathways, initiating the apoptotic, fibrotic, and inflammatory processes that characterize heart failure and contribute to its pathogenesis (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).

[0079] As heart failure progresses, the density of β1-ARs is halved, while the density of β2-ARs remains constant or even increases (Bristow et al. Circ Res 1986; 59(3):297-309). Thus, in heart failure, reduced β1-ARs continue to drive cardiac contraction, while proportionally increased β2-ARs induce deleterious effects via the alternative pathway described above. These changes in heart failure lead to a reduced ejection fraction, early ventricular hypertrophy, and subsequent apoptosis, inflammation, and fibrotic damage to cardiac tissue.

[0080] Therefore, selective β2-AR inhibition may mitigate the adverse effects associated with activation of the alternative pathway without the undesirable effect of inhibiting β1-AR-mediated cardiac contraction. Selective β2-AR antagonists are easier to administer for the treatment of chronic heart failure because they do not require dose titration. Furthermore, selective β2-AR antagonists can be administered at therapeutic doses from the start, allowing for earlier onset of effective treatment (Black & Fitzgerald, Curr Pharm Des 2010; 16: 4148-4158).

[0081] This is supported by the Carvedilol or Metoprolol European Trial (COMET) (Poole-Wilson et al., Lancet 2003; 362: 7-13), which compared the nonselective β-AR antagonists carvedilol and metoprolol in the treatment of chronic heart failure. These studies showed that carvedilol, which has weak selectivity for the β2-AR, is superior to metoprolol, which has weak selectivity for the β1-AR.

[0082] Takotsubo syndrome Takotsubo syndrome, also known as takotsubo cardiomyopathy or "broken heart syndrome," is a severe form of heart failure characterized by acute apical dysfunction resulting in changes in the shape of the left ventricle (LV). The main symptoms are acute chest pain and dyspnea, usually preceded by severe mental or physical stress.

[0083] Several lines of evidence suggest that epinephrine (adrenaline) is involved 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 concentrations of epinephrine cause apical cardiac inhibition (biased activation) by switching β2-AR-mediated signaling from Gαs-activated cardiac stimulation to Gαi-activated cardiac inhibition. The β2 / β1-AR ratio is higher at the apex than at the base, explaining the greater contractile response to adrenaline at the apex and the resulting apical swelling. Therefore, Takotsubo syndrome has been associated with β2-AR activation (Paur et al., Circulation. 2012; 126: 697-706; Ali et al., Int J Cardiol 2019; 281: 99-104).

[0084] Given the strong evidence implicating β2-AR in Takotsubo syndrome, it is believed that selective β2-AR antagonists may be used to treat and prevent Takotsubo syndrome.

[0085] general The compounds of the present invention and pharmaceutical compositions containing the compounds of the present invention are useful for treating or preventing diseases associated with the β2-AR signaling pathway. Preferably, the pharmaceutical compositions and compounds of the present invention are used to treat diseases associated with the β2-AR signaling pathway. As detailed above, such diseases include vascular tumors, cancers, paragangliomas, tumors such as tuberous sclerosis, hereditary hemorrhagic telangiectasia, cerebral cavernous malformations, respiratory diseases such as asthma and chronic obstructive pulmonary disease, cardiovascular diseases such as chronic heart failure and takotsubo syndrome, and other indications such as physiological tremors and migraines. Examples of vascular tumors include, but are not limited to, infantile hemangiomas, von Hippel-Lindau disease, angiosarcomas, and gliomas. Examples of cancers include, but are not limited to, soft tissue sarcomas, melanomas, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, and lymphoblastic leukemia.

[0086] The compounds of formula (I) and formula (II) have also been shown to have improved selectivity for the β-AR compared to ICI 118,551. This improved selectivity for the β-AR allows these compounds to act as effective β2-AR antagonists without interacting with alternative pathways, resulting in improved control of pharmacological action and reduced drug-drug interactions and unwanted side effects.

[0087] Both compounds of formula (I) and (II) have been shown to be selective antagonists for the β2-AR over the β1-AR and other receptors. In particular, compounds of formula (I) are most selective for the β2-AR over the β1-AR. As mentioned above, receptor selectivity is desirable because side effects are often due to off-target drug action. Furthermore, improved selectivity reduces the risk of drug-drug interactions. Therefore, improved selectivity also allows for minimal dosage, leading to reduced drug costs. Alternatively, higher doses can be administered with fewer side effects, enabling more effective treatment of diseases. It also improves ease of administration.

[0088] Preferably, the compound is a compound of formula (I), as this surprisingly has improved selectivity for the β2-AR over the β1-AR and better potency when compared to formula (II).

[0089] Compounds and Pharmaceutical Compositions When used to treat or prevent a disease, the compounds or compositions of the present invention can be administered in an "effective amount." By "effective amount" is meant a "therapeutically effective amount," i.e., an amount of compound sufficient, in a single or multiple administrations, to detectably reduce the severity of the disease, inhibit the progression of the disease, or provide a greater alleviation of disease symptoms than would be expected in the absence of treatment. The subject treated according to the therapeutic methods of the present invention is preferably a human.

[0090] The compounds or compositions of the present invention are useful for reducing the severity of the symptoms of the above-mentioned diseases to be treated.The compositions of the present invention are also useful for administration to patients who are susceptible to, at risk of, or suffering from any of the above-mentioned diseases.Compositions useful for preventing the above-mentioned diseases do not necessarily prevent the absolute occurrence of the disease in all cases, but when administered to patients who are susceptible to or at risk of the disease, they can prevent or delay the onset of the disease.A subject may have one or more of the above-mentioned diseases coexisting, and therefore, the pharmaceutical compositions of the present invention can simultaneously treat one or more of the above-mentioned diseases.

[0091] Preferably, the compounds of formula (I) or (II) have an achiral purity of at least 85%, preferably at least 90%, more preferably at least 92%, even more preferably at least 95%.

[0092] The compounds of formula (I) and (II) have three chiral centers that are clearly defined by stereochemistry.Preferably, the compounds of formula (I) and (II) are in optically pure form.These compounds can be present in optically pure form in the composition.

[0093] The optically pure forms of enantiomers referred to herein have an enantiomeric excess (ee) of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%, with a maximum ee of 100%. The ee can be assessed, for example, by chiral HPLC.

[0094] Preferably, the compound of formula (I) or formula (II), either alone or in a pharmaceutical composition according to the invention, has an ee of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

[0095] Optically pure forms of the diastereomers referred to herein have a diastereomeric excess (de) of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%, with a maximum de being 100%. De can be assessed, for example, by chiral HPLC.

[0096] Preferably, the compound of formula (I) or formula (II), either alone or in a pharmaceutical composition according to the invention, has a de of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

[0097] Preferably, the compound of formula (I) or formula (II), either alone or in a pharmaceutical composition according to the invention, has an ee and de of at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

[0098] Pharmaceutical compositions of the invention may include: a) a compound of formula (III):

[0099] [ka]

[0100] wherein the compound of formula (III) includes an isomer of formula (I) or an isomer of formula (II):

[0101] [ka]

[0102] wherein the compound of formula (III) contains 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) Pharmaceutically acceptable excipients. Preferably, the compound is an isomer of formula (I) and is present in the purity ranges described above.

[0103] The compounds disclosed herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as, for example, water, ethanol, and the like, and the present invention is intended to encompass both solvated and unsolvated forms.

[0104] The compounds disclosed herein can be provided as free compounds or as suitable salts or hydrates.Salts are pharmacologically acceptable, and salts and hydrates can be prepared by conventional methods, such as contacting the compounds of the present invention with an acid or base whose counterion does not interfere with the intended use of the compound.Examples of pharmacologically acceptable salts include hydrohalic acid salts, inorganic acid salts, organic carboxylic acid salts, organic sulfonic acid salts, amino acid salts, quaternary ammonium salts, alkali metal salts, alkaline earth metal salts, etc. The basic moieties may form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, with a variety of inorganic and organic acids, 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, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, and pamoate. The acidic moieties may form salts with a variety of pharmacologically acceptable cations, such as alkali metal and alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Compounds containing a basic or acidic moiety may form pharmaceutically acceptable salts with various amino acids. Preferably, the compounds disclosed herein are in the form of hydrochloride salts.

[0105] The present invention includes providing the compounds described herein in substantially amorphous or substantially crystalline form. "Substantially crystalline" and "substantially amorphous" refer to a compound that is at least a certain weight percent crystalline or amorphous, respectively. In some embodiments, substantially crystalline or substantially amorphous refers to a compound that is at least 70%, at least 80%, at least 90%, or at least 95%, crystalline or amorphous, respectively.

[0106] The pharmaceutical composition of the present invention comprises one or more pharmaceutically acceptable additives, such as pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, stabilizers, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts, buffers, coating agents, antioxidants. Suitable additives and the techniques for formulating pharmaceutical compositions are well known in the art (see, for example, Remington: The Science 2000).

[0107] Suitable excipients include, but are not limited to, pharmaceutical grade starch, mannitol, lactose, corn starch, magnesium stearate, stearic acid, alginic acid, sodium saccharin, talc, cellulose, cellulose derivatives (e.g., hydroxypropylmethylcellulose, carboxymethylcellulose), glucose, sucrose (or other sugars), sodium carbonate, calcium carbonate, magnesium carbonate, sodium phosphate, calcium phosphate, gelatin, agar, pectin, liquid paraffin oil, olive oil, alcohol, detergent, emulsifier, or water (preferably sterile).

[0108] The pharmaceutical composition may further comprise an adjuvant and / or one or more additional therapeutically active agents.

[0109] The pharmaceutical composition may be provided in unit dosage form, typically in a hermetically sealed container, or may be provided as part of a kit. Such a kit will typically (but not necessarily) include instructions for use. The kit may also include a number of unit dosage forms.

[0110] The pharmaceutical composition can be administered by any suitable route, such as inhalation, oral, buccal, sublingual, intranasal, rectal, topical, or parenteral administration. Here, parenteral administration includes subcutaneous, intramuscular, intravenous, intraperitoneal, and intradermal administration. Preferably, the pharmaceutical composition is provided in an oral, buccal, sublingual, subcutaneous, intravenous, intramuscular, intranasal, inhalation, rectal, or topical dosage form. Such compositions can be prepared by any method known in the pharmaceutical arts, for example, by mixing the active ingredient and excipients under sterile conditions.

[0111] Preferably, when treating infantile hemangiomas, the compound or composition is administered intracavitary or sublingually.

[0112] Pharmaceutical compositions suitable for oral administration can be provided as discrete units such as capsules, tablets, powders, granules, solutions, syrups, suspensions (aqueous or non-aqueous liquids, edible foams or whips, emulsions), etc. Compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, compositions are provided in unit dosage forms to facilitate accurate administration. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dosage for humans and other mammals, each unit containing a predetermined amount of active agent calculated to produce the desired therapeutic effect, along with appropriate pharmaceutical excipients. Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or salts thereof, etc. Suitable excipients for soft gelatin capsules include, for example, water or oils (e.g., vegetable oils, liquid paraffin oil, olive oil), waxes, fats, semisolid or liquid polyols, etc. Solutions and syrups are prepared using excipients such as water, polyols, and sugars. To prepare a suspension, an oil (eg, vegetable oil) can be used to prepare an oil-in-water or water-in-oil suspension.

[0113] Pharmaceutical compositions suitable for oral, buccal, or sublingual administration may take the form of fast-dissolving formulations. For example, the compound or composition may be micronized and lyophilized in a water-soluble matrix, such as gelatin or a mixture of saccharin and a polymer. This is commonly used in Zydis® formulations. This enhances bioavailability, making it particularly effective for pediatric administration.

[0114] Pharmaceutical compositions suitable for inhaled or intranasal administration include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators.

[0115] Pharmaceutical compositions suitable for parenteral administration (e.g., subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal) include aqueous and nonaqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation substantially isotonic with the patient's blood, as well as aqueous and nonaqueous sterile suspensions, which may contain suspending agents, thickening agents, and wetting agents. Additives that can be used in injection solutions include, for example, water, alcohols, polyols, glycerin, vegetable oils, and the like. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions contain suspending agents, such as cellulose derivatives, sodium alginate, tragacanth gum, and polyvinylpyrrolidone, and wetting agents such as lecithin. The compositions can be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials, and can be stored in a lyophilized (lyophilized) state, requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0116] In some embodiments, the pharmaceutical compositions of the present invention are depot formulations, which are formulated to provide controlled drug release into the bloodstream, for example, over weeks or months, depending on the exact formulation. Depot formulations include, for example, nanoparticle formulations of nanoparticles containing a compound of Formula (I) or (II) and one or more additives (e.g., surface stabilizers, bulking agents, or carriers), or formulations in which a compound of Formula (I) or (II) is encapsulated in micellar nanoparticles. Depot formulations are typically injected subcutaneously or intramuscularly to form a drug depot in the muscle or subcutaneously. Depot formulations are typically solid or oily.

[0117] When used to prevent or treat a disease, the compositions of the present invention can be administered in an "effective amount." When used as a monotherapy, "effective amount" refers to a "therapeutically effective amount," i.e., an amount of compound sufficient to detectably reduce the severity of the disease, prevent disease progression, or alleviate disease symptoms beyond levels expected in the absence of treatment, upon single or multiple administrations. When the present invention is used in combination with other agents, "effective amount" refers to an amount sufficient, when administered in single or multiple doses with the other agent, to detectably reduce the severity of the disease, prevent disease progression, or alleviate disease symptoms beyond levels expected in the absence of treatment or treatment with the second agent alone. The "effective amount" when used as a monotherapy may be the same as or different from the "effective amount" when used in combination with the second agent.

[0118] The dosage of the compounds of the present invention can vary over a wide range, depending on various factors, such as the disease or disorder being treated, the age, weight, and condition of the individual being treated, and the route of administration. The appropriate dosage is ultimately determined by a physician. Typically, however, the compounds of the present invention are administered in a daily dose (whether administered as a single dose or in divided doses) for each route of administration ranging from 0.001 to 5,000 mg / day, usually from 0.1 to 1,000 mg / day, more usually from 0.5 to 200 mg / day, and even more usually from 1 to 120 mg / day (e.g., 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg). Typical dosages, expressed as a dose per unit body weight, range from 0.01 pg / kg to 50 mg / kg, preferably from 10 pg / kg to 10 mg / kg, e.g., from 100 pg / kg to 2 mg / kg.

[0119] The compounds of the present invention may be administered alone or in combination with one or more additional therapeutically active agents (i.e., compounds different from the compounds of the present invention). Preferably, the compounds of the present invention and the additional therapeutically active agents are administered in therapeutically effective amounts.

[0120] The compounds of the invention can be administered simultaneously with other therapeutic agents, or before or after other therapeutic agents. The compounds of the invention can be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition.

[0121] As explained above, when used to treat or prevent asthma, these compounds may be used in combination with steroids, such as inhaled corticosteroids.

[0122] As mentioned above, the problem of using long-acting β2-AR agonist or ultra-long-acting β2-AR agonist in the treatment of asthma is the down-regulation of β2-AR.Similarly, the overuse of short-acting β2-AR agonist can also cause the down-regulation of β2-AR.This means that, for example, for the purpose of immediate symptom relief, the subsequent administration of fast-acting β2-AR agonist may be ineffective.Therefore, the administration of the compound of the present invention as selective β2-AR antagonist can up-regulate β2-AR and potentially control the symptoms of severe asthma.

[0123] In particular, the compounds of the present invention can be administered before the administration of a long-acting β2-AR or an ultra-long-acting β2-AR. This prevents β2-AR saturation, allowing the fast-acting β2-AR agonist to bind even after the administration of the long-acting β2-AR. The compounds of the present invention can be administered in combination with steroids. "In combination" includes, for example, separate administration by different administration routes, but is intended to be administered with sufficient temporal proximity so that the compounds of the present invention and steroids act at least partially simultaneously. Preferably, the compounds of the present invention and steroids are administered simultaneously, more preferably by the same administration route.

[0124] As mentioned above, it has been found that the combination of nonselective β-AR antagonist and anti-cancer therapy improves the clinical outcome in the treatment of angiosarcoma.Therefore, it is preferred that the compound and composition of the present invention be administered in combination with anti-cancer therapy, such as chemotherapy or radiotherapy, preferably chemotherapeutic agent, in the treatment of angiosarcoma.

[0125] Therefore, the pharmaceutical composition may further comprise one or more additional active agents. Alternatively, the compound or pharmaceutical composition of the present invention and the additional active agent may be provided separately, for example, in the form of a kit. The additional active agent may be a steroid such as an inhaled corticosteroid, a long-acting β2-AR, an ultra-long-acting β2-AR, or an anti-cancer agent such as a chemotherapeutic agent.

[0126] The following examples illustrate compounds of formula (I) and (II) according to the present invention, which are compared with a compound of formula (D).

[0127] [ka]

[0128] Formula (D) is the isolated 2S,3S isomer of ICI 118,551. These compounds are also compared to nadolol, a non-selective β-AR antagonist. These examples demonstrate that the compounds of the present invention are selective β2-AR antagonists and, compared to the compound of formula (D) (also referred to herein as compound (D)), are far more specific for β-AR than for other receptors (e.g., 5-HT transporters, 5-HT receptors, o-receptors, Na+ channels). Both compounds exhibit efficacy comparable to or greater than that of the compound of formula (D). In particular, the compound of formula (I) has the highest selectivity and potency for β2-AR (compared to β1-AR).

[0129] example The following examples of the present invention are provided to aid in the understanding of the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, reagents are commercially available or can be prepared according to literature procedures.

[0130] Abbreviation ATCC: American Type Culture Collection CDCl3: deuterated chloroform DCM: dichloromethane DIPEA: N,N'-diisopropylethylamine DMEM: Dulbecco's Modified Eagle's Medium DMF: N,N-dimethylformamide DMSO: dimethyl sulfoxide D6-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 EC 50 : concentration that produces half the maximum response I C 50 : concentration that causes half-maximal inhibition of the control agonist response

[0131] Example 1 - Synthesis of Compounds of Formula (I) and (II) Part A - Synthesis of (2S,3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methrindan-1-one (2S,3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methrindan-1-one was synthesized according to the following scheme:

[0132] [ka]

[0133] Preparation of 4-hydroxy-7-methylindan-1-one (A) A 10 L three-necked round-bottom flask equipped with an overhead stirrer, reflux condenser, and thermometer was charged with glacial acetic acid (0.9 L), 48% hydrogen bromide solution (4.5 L), and 4-methoxy-7-methylindalone (450 g, 2.55 mol). Stirring was initiated, and the vessel contents were heated to approximately 100 °C until all solids dissolved. Heating and stirring was continued for 3 h until no starting material remained as determined by TLC (eluent: DCM). The vessel contents were cooled to 30-40 °C and partitioned between ethyl acetate (4.0 L) and water (8.0 L). The biphasic solution was separated, and the aqueous layer was salted and re-extracted with ethyl acetate (4.0 L × 2). The combined organic layers were backwashed with 25% w / w brine solution (2 × 4.0 L), dried over sodium sulfate, filtered, and the filtrate stripped on a rotavapor. The residue was azeotroped with toluene (2 x 1.0 L) in a rotavapor (to remove residual acetic acid residues) and then cooled / crystallized. The recovered crude solid (468 g, 113%) was recrystallized from methanol (2.34 L, 5 volumes), filtered, washed on the funnel with cold methanol (2 x 300 ml), and dried to constant weight in a vacuum oven at 40 °C. Dry weight = 247 g, 60%. The GC achiral purity of this material was 98.5%. The mother liquor was concentrated in a rotavapor to give a second crop. Yield = 76 g, 18%. The GC achiral purity of this material was 99%. The total recovery of 4-hydroxy-7-methylindan-1-one (A) was 323 g, a 78% recovery. This mixture was used directly in the next step.

[0134] Preparation of (2R,3R)-7-methyl-4-(3-methyloxiranylmethoxy)indan-1-one (B) In a 500 mL three-neck round-bottom flask equipped with an overhead stirrer, thermometer, and nitrogen bubbler, 4-hydroxy-7-methylindan-1-one (A) (18 g, 0.11 mol, 1.0 equiv.) was dissolved in DMF (300 mL). Stirring was initiated, and under a nitrogen atmosphere, cesium carbonate (53.7 g, 0.16 mol, 1.5 equiv.) was added in one portion. The reaction mixture was stirred for 15 minutes. (2R,3R)-toluene-4-sulfonic acid-3-methyloxiranylmethyl ester (30 g, 0.12 mol, 1.1 equiv.) was then added in one portion, and the contents of the vessel were heated to 50 °C. Heating and stirring were continued for 2 hours, at which point TLC (eluent: hexane:ethyl acetate 4:1) showed no remaining starting material. The contents of the vessel were cooled to 30-40 °C and quenched in water (2.0 L). Stirring was continued at room temperature for 30 minutes, followed by extraction with diethyl ether (400 ml × 3). The combined organic layers were backwashed with 10% w / w lithium chloride solution (400 ml × 2), dried over sodium sulfate, filtered, and stripped with rotavapor to give an orange oil that slowly crystallized on standing. Crude yield = 29 g, 113%. This crude material was purified by column chromatography using silica gel (600 g) and 20-25% ethyl acetate / hexane as eluent. The good column fractions were stripped to give a pale yellow oil that crystallized on standing. Crude (2R,3R)-7-methyl-4-(3-methyloxiranylmethoxy)indan-1-one (B) weighed 25 g, a yield of 97.8%. This material was used directly in the next step.

[0135] Preparation of (2S,3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methylindan-1-one (D) Step 1-Synthesis of (C)

[0136] [ka]

[0137] In a 2 L three-neck round-bottom flask equipped with a magnetic stirrer, reflux condenser, and thermometer, (2R,3R)-7-methyl-4-(3-methyloxiranylmethoxy)indan-1-one (B) (25 g, 0.107 mol, 1.0 equiv.) was dissolved in methanol (500 mL). The apparatus was set to reflux, and isopropylamine (31.6 g, 46 mL, 0.53 mol, 5.0 equiv.) was added in one portion. The contents of the vessel were heated under gentle reflux for 2 days, with additional isopropylamine (95 g total, 138 mL, 1.6 mol, 15 equiv.) added periodically during the heating cycle. After this time, TLC (eluent: hexane:ethyl acetate 2:1) confirmed no starting material remained, and the contents of the vessel were transferred to a rotavapor and distilled to yield an orange / brown oil. The crude yield of (C) was 42 g, 120%.

[0138] Step 2-Synthesis of (D) The oil from step 1 was redissolved in methanol (200 ml) and 1 M hydrochloric acid solution (500 ml) was added. The apparatus was set to reflux, and the vessel contents were heated to a gentle reflux (87 °C) for a total of 4 hours. HPLC confirmed that all of the imine (C) had hydrolyzed back to the ketone (D). Heating was then discontinued, the vessel contents were transferred to a rotavapor, and the methanol was removed under reduced pressure. The resulting aqueous residue was cooled to room temperature, and the pH was adjusted to 12-13 with 2 M sodium hydroxide solution (200 ml). The resulting cloudy aqueous solution was extracted with ethyl acetate (3 × 200 ml), and the combined organics were backwashed with water (2 × 200 ml), dried over sodium sulfate, filtered, and stripped on a rotavapor to give an orange / brown oil. The crude product weighed 32 g, representing a 102% yield. The crude oil was immediately dissolved in warm acetonitrile (75 ml) and allowed to cool and crystallize (seeded with the test material) in a refrigerator overnight. The resulting slurry was filtered and washed on the funnel with acetonitrile (30 ml x 2). The wet weight yield of (D) was 23.5 g, 75%. The wet solid was dried in a vacuum oven at 40 °C to constant weight. The dry weight yield of (D) was 17.7 g, 56.7%.

[0139] A solution of R-(-)-mandelic acid (7.4 g, 48.7 mmol, 1.0 equiv.) in ethanol (43 mL) was added to a stirred solution of (2S,3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methylindan-1-one (D) (14.2 g, 48.7 mmol, 1.0 equiv.) in ethanol (43 mL), resulting in the immediate precipitation of a colorless solid. The precipitated solid did not redissolve upon heating the reaction slurry to reflux. After stirring at room temperature for 2 hours, the slurry was filtered and the cake washed with ethanol (2 x 200 mL). The collected wet solid was partitioned between ethyl acetate (250 mL) and 2 M sodium hydroxide solution (250 mL). Stirring was continued for 10 minutes, and the layers were separated. The aqueous layer was back-extracted with ethyl acetate (50 mL), and the combined organic layers were washed with water (200 mL x 2), dried over sodium sulfate, filtered, and stripped to a colorless powder. The wet weight was 9.9 g. The recovered solid was dried in a vacuum oven overnight to a constant weight. The dry weight of (2S,3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methylindan-1-one (D) was 9.8 g. The material was sieved. 1 The H NMR spectrum (CDCl3) was consistent with the above structure.

[0140] Appearance: Off-white solid HPLC achiral purity: 99.8% HPLC chiral purity: 99.1% ee 1 HNMR: 400 MHz: (CDCl3): Consistent with structure CHN: Detected values: C: 70.05%, H: 8.70%, N: 4.90% Theoretical values: C: 70.07%, H: 8.65%, N: 4.81% Optical rotation: [α] 22 D (CHCl3) + 33.24° Residual ash: <0.1%

[0141] Part B - Synthesis of Compounds of Formula (I) and (II)

[0142] [ka]

[0143] A 25 mL round-bottom flask equipped with a magnetic stirrer, reflux condenser, and nitrogen bubbler was charged with DCM (6 mL) and (2S,3S)-4-(2-hydroxy-3-isopropylaminobutoxy)-7-methylindan-1-one (D) (2 g, 6.68 mmol, 1 equiv.). Stirring was initiated under a nitrogen atmosphere. Triethylamine (3.48 g, 34.4 mmol, 5 equiv.) was added in one portion, followed by the careful addition of formic acid (1.9 g, 41.2 mmol, 6 equiv.) over 2 min (5 mL). The apparatus was then set to reflux, and the appropriate catalyst, RuCl(p-cymene)[(R,R)-Ts-DIPEN] (21.8 mg, 0.0342 mmol, 0.005 equiv.) for compounds of Formula (I) and RuCl(p-cymene)[(S,S)-Ts-DIPEN] (21.8 mg, 0.0342 mmol, 0.005 equiv.) for compounds of Formula (II), was added. Heating to 35 °C was initiated. Stirring was continued at this temperature overnight. After this time, TLC (eluent: 10% methanol / DCM) revealed a large amount of starting material remaining, so additional triethylamine (1.4 g, 13.8 mmol, 2 equiv.), formic acid (0.63 g, 13.8 mmol, 2 equiv.), and the appropriate catalyst (21.8 mg, 0.0342 mmol, 0.005 equiv.) were added. After stirring at 35 °C for an additional 48 h, analysis of both reactions by TLC again revealed the presence of starting material (D) in both reactions. Additional triethylamine (1.4 g, 13.8 mmol, 2 equiv.), formic acid (0.63 g, 13.8 mmol, 2 equiv.), and the appropriate catalyst (21.8 mg, 0.0342 mmol, 0.005 equiv.) were added. Stirring was continued at 35 °C for an additional 24 h, after which both reactions were judged complete by TLC. The reaction mixture was partitioned between saturated sodium bicarbonate (50 mL) and DCM (50 mL). After stirring for 10 min, the layers were separated. The aqueous layer was back-extracted with DCM (50 mL), and the combined organics were back-washed with 10% w / w brine solution (2 × 50 mL), dried over sodium sulfate, filtered, and stripped on a rotary evaporator to give a crude pale beige solid (approximately 2 g each).

[0144] Each crude product was purified by flash column chromatography using 20% ​​methanol / DCM as the eluent to give approximately 1 g of purified solid, which was recrystallized from acetonitrile (5 ml) to give the final product. After drying in a fan oven at 35 °C for 48 hours, a total of 0.7 g (35%) of compound of formula (I) or (II) was recovered. 1 The HNMR spectra (D6-DMSO) were all consistent with the above structure.

[0145] Compounds of formula (I) Yield: 0.7g, 35% HPLC purity: 95.67% Chiral HPLC purity: 98.62% de(R) 1 HNMR: (D6-DMSO): Consistent with structure Appearance: Colorless solid

[0146] Compound of formula (II) Yield: 0.7g, 35% HPLC purity: 98.00% Chiral HPLC purity: 99.37% de(S) 1 HNMR: (D6-DMSO): Consistent with structure Appearance: Colorless solid

[0147] Chiral purity (de) is expressed in relation to the stereochemistry of the OH on the five-membered ring, with the remaining chiral centers both having S configuration.

[0148] Example 2 - Binding Assay The purpose of the binding assay is to compare the selectivity and specificity of compounds for the β2-AR among various receptors.

[0149] method Compounds were incubated in vitro with specific concentrations of receptor agonist / antagonist radioligands. The radioligands, cells, and conditions used are shown in Table 1. For each procedure, please refer to the cited references, which are incorporated herein by reference.

[0150] To confirm the accuracy of the assay, reference compounds were selected and used as controls. Atenolol and ICI 118,551 were used as reference samples for β1-AR and β2-AR, respectively. All values ​​were within the acceptable range.

[0151] Filtration assays were performed using the following general procedure. 1. Add the radioligand, receptor, and one or more other compounds (see below for each binding experiment) to the plate wells and incubate. The radioligand and incubation conditions are listed in Table 1 for each receptor. These conditions were used for all filtration assays. 2. Receptor-bound radioligand is "captured" onto the filter by aspiration, and unbound radioligand is removed. To minimize nonspecific binding, the filter is washed several times with an appropriate buffer. 3. Dry the filters and add liquid scintillation cocktail. 4. Count the filters in a microplate scintillation counter.

[0152] Binding experiments were carried out as follows. Total binding was measured using the general procedure for the filtration assay outlined above, with no additional compound added in step 1.

[0153] Total binding = membrane + signal from radioligand

[0154] Nonspecific binding was measured using the general procedure for the filtration assay outlined above. In step 1, the radioligand and receptor were incubated with increasing concentrations of unlabeled competitors. The unlabeled competitors and their concentrations for each receptor are listed in Table 1.

[0155] Nonspecific binding = signal from unlabeled competitor + membrane + radioligand

[0156] Control specific binding is the difference between total binding and non-specific binding.

[0157] Control specific binding amount = total binding amount - non-specific binding amount

[0158] The amount of binding measured was determined using the general procedure for the filtration assay outlined above. In step 1, the radioligand and receptor are incubated with the test compound in the plate wells. The concentrations of the test compound are listed in Table 2.

[0159] Measured binding = test compound + radioligand + signal from membrane

[0160] The amount of specific binding measured is the difference between the amount of binding measured and the amount of non-specific binding.

[0161] Measured specific binding = Measured binding - Non-specific binding

[0162] Specific methods for β1-AR binding assays Transfected HEK-293 cells were used. Cell membrane homogenates (5 μg of protein) were incubated with 0.3 nM [ 3 H]CGP 12177 at 22°C for 60 min. Nonspecific binding was determined in the presence of 50 pM alprenolol. After incubation, samples were rapidly filtered under vacuum through glass fiber filters (GF / B, Packard) presoaked with 0.3% PEI and washed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). After drying, the radioactivity was measured in a scintillation counter (Topcount, Packard) using scintillation cocktail (Microscint 0, Packard). The amount of specific binding measured is calculated and the results are expressed as percent inhibition of control specific binding. The standard reference compound was atenolol, which was tested at several concentrations in each experiment and its IC 50 A competition curve was obtained to calculate

[0163] Specific methods for β2-AR binding assays Transfected CHO cells were used. Cell membrane homogenates (32 μg of protein) were incubated with 0.3 nM [ 3 H]CGP 12177 at 22°C for 120 min. Nonspecific binding was determined in the presence of 50 μM alprenolol. After incubation, samples were rapidly filtered under vacuum through glass fiber filters (GF / B, Packard) presoaked with 0.3% PEI and washed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). After drying, the radioactivity was measured in a scintillation counter (Topcount, Packard) using scintillation cocktail (Microscint 0, Packard). The amount of specific binding measured is calculated and the results are expressed as percent inhibition of control specific binding. The standard reference compound was ICI 118551, which was tested at several concentrations in each experiment and had an IC 50 A competition curve was obtained to calculate

[0164] The results were analyzed as follows: A decrease in the measured specific binding level compared to the control specific binding level (usually expressed as percent inhibition) indicates that the test compound has bound to the receptor. The results are expressed as a percentage of the control specific binding level (see Table 3).

[0165] (measured specific binding amount / control specific binding amount) x 100

[0166] The percent inhibition of control specific binding is as follows:

[0167] 100 - ((measured specific binding amount / control specific binding amount) x 100)

[0168] An increase in percent inhibition of control specific binding indicates that the test compound has bound to the receptor.

[0169] Results with percent inhibition (or percent stimulation, in the case of assays performed under basal conditions) greater than 50% are considered to indicate a significant effect of the test compound. Results with percent inhibition (or stimulation) in the range of 25% to 50% suggest a weak to moderate effect. Results with percent inhibition (or stimulation) less than 25% are not considered significant and are likely due primarily to fluctuations in the signal around the control level.

[0170] Table 2 shows the results of the binding studies of Compound (D), Formula (I), Formula (II), and nadolol. Results showing significant effects are shown in bold.

[0171] [Table 1-1]

[0172] Table 1-2

[0173] Table 1-3

[0174] Table 1-4

[0175] Table 1-5

[0176] Table 1-6

[0177] Table 1-7

[0178] Table 1-8

[0179] Table 1-9

[0180] Table 1-10

[0181] Table 1-11

[0182] Table 1-12

[0183] Table 2-1

[0184] Table 2-2

[0185] Table 2-3

[0186] Table 2-4

[0187] Table 2-5

[0188] Table 2-6

[0189] Table 2-7

[0190] Table 2-8

[0191] Table 2-9

[0192] [Table 2-10]

[0193] [Table 2-11]

[0194] [Table 3]

[0195] Table 3 summarizes the results in terms of percent inhibition of control specific binding of β2-AR and β1-AR for compounds (D), formula (I), formula (II), and nadolol in Table 2.

[0196] These results suggest that compounds of formulas (I) and (II) and nadolol inhibit the β2-AR to a degree equal to or greater than that of compound (D). The control specific binding inhibition rate for formula (I) against the β1-AR was significantly lower (96.2%) than that of the other compounds, suggesting that formula (I) has the lowest activity against the β1-AR. The data indicate that both formulas (I) and (II) are more selective for the β2-AR than the β1-AR. Furthermore, formula (I) is shown to have the highest selectivity among the four compounds.

[0197] [Table 4]

[0198] Selectivity for β-AR is determined by the number of receptors other than β1-AR and β2-AR at which a compound exerts a significant effect.

[0199] Table 4 is an excerpt from the results of Table 2 and summarizes the number of receptors that showed weak to moderate and significant effects with Compound (D), Formula (I), Formula (II), and nadolol. Not only are Formulas (I) and (II) more selective for the β2-AR than other compounds, but data also suggest that they are more selective for the β1- and β2-AR than other receptors. Table 2 shows that Compound (D) exhibits potent potency across the seven receptors investigated in this study (β1-AR, β2-AR, 5-HT 1B , 5-HT 2B , σ-receptor, Na + Furthermore, nadolol has been shown to induce significant inhibition or stimulation of four receptors (β1-AR, β2-AR, 5-HT 1A , 5-HT 1B ) were found to have a significant effect. In comparison, formula (I) and formula (II) caused significant inhibition only at the β1- and β2-ARs. Selectivity is a very important property of a drug for a variety of reasons, including limiting drug-drug interactions, limiting side effects, and improving patient acceptability.

[0200] conclusion The data show that, among the compounds tested, formula (I) is the most selective for β2-AR, and together with formula (II) it has the highest selectivity for β-AR. The results of 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) have fewer drug-drug interactions and side effects in vivo than compound (D) or nadolol.

[0201] Example 3 - Functional Assay The purpose of the cellular and nuclear functional assays is to compare the potency and affinity of antagonists at the β1-AR and β2-AR.

[0202] method cAMP detection overview: In functional screening, the regulation of Gi- and Gs-coupled G protein-coupled receptors (GPCRs) is typically monitored by detecting the intracellular signaling molecule 3',5'-cyclic adenosine monophosphate (cAMP). cAMP production is controlled by adenylate cyclase, an enzyme that is stimulated or inhibited as a result of direct interaction with G protein α subunits. The role of adenylate cyclase is to convert ATP into cAMP and inorganic pyrophosphate. When Gs-coupled GPCRs are activated, Gαs positively stimulates adenylate cyclase activity, increasing cAMP production. On the other hand, stimulation of Gi-coupled GPCRs negatively regulates adenylate cyclase and decreases cAMP levels.

[0203] Because the endogenous cAMP concentration in cells is low, models measuring Gi-coupled receptor activity require initial stimulation to detect the inhibition of cAMP production after receptor stimulation. Furthermore, to suppress the spontaneous degradation of cAMP to AMP catalyzed by phosphodiesterase (PDE) enzymes, PDE inhibitors (e.g., IBMX, Rolipram) are present during the assay. The cAMP concentration is estimated by the FRET method described below.

[0204] cAMP production measurement (GsPCR receptor) In the fluorescence resonance energy transfer (FRET) method, intracellular native cAMP binds to an anti-cAMP antibody labeled with europium cryptate (donor) in the presence of a competing cAMP (acceptor) labeled with a modified allophyocyanin dye d2. Therefore, energy transfer is inversely proportional to the cellular cAMP concentration in the sample. A standard curve is used to convert raw data to cAMP concentrations in the sample. Measurements are performed at λex = 337 nm, λem = 620 nm (donor emission peak), and λem = 665 nm (acceptor emission peak). Signals are normalized by calculating the fluorescence ratio (665 nm / 620 nm) to correct for the effects of colored compounds, serum, and other media optical properties.

[0205] The FRET method overcomes most of the problems of autofluorescence arising from unbound fluorophores.

[0206] β1-AR Functional Assay Method: Transfected HEK-293 cells were suspended in HBSS buffer (Invitrogen) supplemented with 20 mM HEPES (pH 7.4) and 500 μM IBMX. 3 The cells were dispensed into a microplate at a density of 1000 cells / well and pre-incubated in the presence of HBSS (basal control), test compound, or reference antagonist for 5 minutes at room temperature, followed by addition of the reference antagonist isoproterenol at a final concentration of 3 nM.

[0207] For basal control measurements, separate assay wells contained no isoproterenol. After 30 min at room temperature, the cells were lysed and a fluorescent acceptor (D2-labeled cAMP) and a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) were added. After 60 min at room temperature, fluorescence transfer was measured at λex = 337 nm, λem = 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 the signal measured at 620 nm (ratio). Results are expressed as percent inhibition of the control response to 3 nM isoproterenol. The standard reference antagonist was atenolol, which was tested at several concentrations in each experiment to generate concentration-response curves and determine its IC 50 The value was calculated.

[0208] β2-AR Functional Assay Method: The transfected CHO cells were suspended in HBSS buffer (Invitrogen) supplemented with 20 mM HEPES (pH 7.4) and 500 μM IBMX, and 10 4Cells were distributed into microplates at a density of 100 cells / well and pre-incubated for 5 minutes at room temperature in the presence of HBSS (basal control), test compound, or reference antagonist. The reference agonist isoproterenol was then added to a final concentration of 10 nM. For basal control measurements, no isoproterenol was added to other assay wells. After 30 min at room temperature, the cells were lysed and a fluorescent acceptor (D2-labeled cAMP) and a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) were added. After 60 min at room temperature, fluorescence transfer was measured at λex = 337 nm, λem = 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 the signal measured at 620 nm (ratio). Results are expressed as percent inhibition of the control response to 10 nM isoproterenol.

[0209] The standard reference antagonist was ICI 118551, which was tested at several concentrations in each experiment to generate concentration-response curves and determine its IC 50 The value was calculated.

[0210] These experiments were performed using the conditions defined below in Table 5. These experiments were performed using the antagonists listed below in Table 6. IC 50 and E.C. 50 Multiple concentrations of each compound were tested to determine the . These concentrations ranged from 1.0x10 -09 M to 3.0x10 -05 It was in the range of M.

[0211] [Table 5]

[0212] 1 Frielle, T. et al. (1987), Proc. Natl. Acad. Sci. USA, 84 : 7920-7924 2 Baker, JG (2005), Brit. J. Pharmacol., 144:317-322.

[0213] References are made to the literature citations describing each procedure, which are incorporated herein by reference. The results were analyzed as follows: Results are expressed as a percent (%) of the control agonist response:

[0214] (measured response / control response) x 100

[0215] As percent (%) inhibition of control agonist response:

[0216] 100 - ((measured response / control response) x 100)

[0217] A graph of concentration versus % inhibition of the control agonist response was plotted, and nonlinear regression analysis of the resulting curve yielded the EC 50 value (concentration producing half-maximal response) and IC 50 The value (concentration producing half-maximal inhibition of the control agonist response) was determined.

[0218] Dissociation constant (K B ) was calculated using the modified Cheng Prusoff formula:

[0219] K B =IC 50 / (1+(A / EC 50A ))

[0220] where A = concentration of control agonist in the assay, EC 50A = EC of the reference agonist 50 value.

[0221] I C50 A lower value indicates that the molecule is a more potent agonist or antagonist for the receptor being evaluated. B A lower value indicates that the compound has a higher affinity for that receptor.

[0222] Potency and affinity

[0223] [Table 6]

[0224] Data from Kawakami K et al, British Journal of Pharmacology 2006; 147: 642-652

[0225] The results in Table 6 show that Compound (D) and Formula (I) have higher IC values ​​for β2-AR than ICI 118,551. 50 and K. B These results suggest that these compounds have high affinity for β2-AR and are more potent antagonists of β2-AR. 50 and K. B The comparable values ​​suggest that formula (I) has comparable potency and affinity to compound (D) for β2-AR. 50 and K. B This value is comparable to that of ICI 118,551. Nadolol was the least potent β2-AR antagonist of the compounds tested and also showed the lowest affinity for β2-AR.

[0226] This assay also measured the potency and affinity of the compounds for the β1-AR. Formula (I) had the lowest potency and affinity for the β1-AR, suggesting its highest selectivity for the β2-AR. On the other hand, compound (D) had higher potency and affinity for the β1-AR than both formulas (I) and (II).

[0227] Selectivity

[0228] [Table 7]

[0229] Data from Kawakami K et al, British Journal of Pharmacology 2006; 147: 642-652

[0230] To evaluate the selectivity of the compounds, the IC for β1-AR was calculated as shown in Table 7. 50 and IC for β2-AR 50 The ratio of K to β-AR was calculated. The results were all greater than 1, indicating that all compounds were more potent antagonists for β-AR. Similarly, as shown in Table 7, the K B and K for β2-AR B The ratio of β2-AR activity was calculated, which also indicates selectivity for β2-AR.

[0231] Formula (I) showed significantly higher ratios than the other compounds in both calculations, indicating much greater selectivity for the β2-AR. This selectivity is important because it suggests that Formula (I) activates the β2-AR at concentrations significantly lower than those that activate the β1-AR. This allows for greater control over pharmacological effects and potential side effects by adjusting dosage. ICI 118,551 is reported to be only 100-fold more selective for the β2-AR than the β1-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 of this assay suggest that Formula (I) is more potent and selective than the current "gold standard" and has a higher affinity for the β2-AR.

[0232] conclusion The data from this study demonstrate that of all the compounds tested, formula (I) is the most potent antagonist for β2-AR. This is due to the IC 50 This is also clear from the value of K B The values ​​also indicate that formula (I) has the highest affinity and selectivity for β2-AR, which means that the use of formula (I) as a β2-AR antagonist may potentially reduce β1-related side effects. Formula (II) is less selective for β1 versus β2 adrenergic receptors compared to Formula (I), but its potency and affinity are comparable to ICI 118,551. Binding studies have shown that both Formula (I) and Formula (II) are more selective for β-AR than other β-AR antagonists studied, making Formula (II) a promising compound.

[0233] Example 4 - Solubility The purpose 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 fed-state simulated intestinal fluid (FeSSIF), and the results are shown in Table 8. 0.1 M phosphate buffer (pH 7.4) was prepared by dissolving 14.2 g of disodium hydrogen phosphate in a 1 L reagent bottle and 6.8 g of potassium dihydrogen phosphate in a 0.5 L volumetric flask in 0.5 L of MilliQ water. The disodium hydrogen phosphate solution was heated to 37°C and the pH was adjusted to 7.4 with potassium dihydrogen phosphate.

[0234] FeSSIF pH 5.0 was made using a kit from Biorelevant ( https: / / Biorelevant.com ).

[0235] A 100 μM solution of Formula 1 in DMSO was prepared by diluting 5 μL of a 10 mM solution of Formula 1 in DMSO with 495 μL of DMSO. A five-point standard curve was prepared for each test compound in 75:25 DMSO:water, covering the Formula (I) concentration ranges of 5 μM, 500 nM, 50 nM, 5 nM, and 0.5 nM.

[0236] One milligram of solid compound was placed in a Cyprotex vial, and 1 ml of either 0.1 M phosphate buffer (pH 7.4) or FeSSIF buffer (pH 5.0) was added. The vial was then placed on a rotating incubator table at room temperature for 24 hours. After incubation, 200 μL of each solution was removed and filtered using a 96-well 0.4 μM MultiScreen HTS PCF polycarbonate centrifugal filter plate. The filtrate was serially diluted 1:4, 1:40, and 1:400 with 75:25 DMSO:water. Standards and samples were quantified using a standard Waters Acquity UPLC-MS / MS system. The aqueous solubility of each test compound was determined from the linear fit of the standard curve.

[0237] [Table 8]

[0238] The results in Table 8 demonstrate that formula (I) is soluble in biologically relevant media.

[0239] Example 5 - Caco2 cell permeability The purpose of the permeability study was to test Formula (I) and control compounds in the Caco2 permeability assay and assess intestinal absorption (P app (cm / s)) and drug efflux (rate).

[0240] method Caco-2 cells are widely used as an in vitro model to predict human drug absorption. The Caco-2 cell line is derived from a human colon carcinoma and spontaneously differentiates to form a monolayer of polarized intestinal epithelial cells in culture. Caco-2 cells were seeded into multiwell insert plates and allowed to form confluent monolayers 20 days before the start of the experiment. On day 20, test compounds were added to the apical side of the membrane, and the flux of compounds across the monolayer was monitored over a 2-hour period. To examine drug efflux, the transport of compounds from the basolateral to the apical compartment was also examined.

[0241] Caco-2 cells were obtained from ATCC and used at passage numbers 40 to 60. Cells were plated in Transwell plates at 1x10 5 pieces / cm 2 Cells were seeded at 100°C. The culture medium, DMEM (Sigma-Aldrich), was replaced every 2 to 3 days. On day 20, when a confluent monolayer was formed, permeability tests were performed. Cell culture and assay incubation were performed at 37°C, 5% CO2, and 95% relative humidity. On the day of the assay, monolayers were prepared by rinsing the apical and basolateral sides with Hanks Balanced Salt Solution (HBSS) at the desired pH, prewarmed to 37°C. Next, cells were incubated with HBSS at the desired pH in both the apical and basolateral compartments for 30 minutes to stabilize physiological parameters.

[0242] Dosing solutions were prepared by diluting test compounds in assay buffer to achieve the desired test compound incubation concentration (typically 10 μM). The final DMSO concentration was 1% v / v. The dosing solution also contained Lucifer Yellow, a fluorescent integrity marker. Analytical standards were prepared from DMSO dilutions of test compounds and transferred to buffer to maintain a DMSO concentration of 1% v / v. The assay buffer was supplemented with HBSS, pH 7.4.

[0243] To assess AB permeability, HBSS was removed from the apical compartment and replaced with the test compound dosing solution. The apical compartment insert was then placed in a companion plate containing fresh buffer (containing 1% v / v DMSO). To assess BA permeability, HBSS was removed from the companion plate and replaced with the test compound dosing solution. Fresh buffer (containing 1% v / v DMSO) was added to the apical compartment insert, which was then placed in the companion plate. After 120 min, the apical compartment insert and companion plate were separated, and apical and basolateral samples were diluted and analyzed.

[0244] The permeability of test compounds was assessed in duplicate. Compounds with known permeability properties were tested as controls on each assay plate. Test and control compounds were quantified by standard LC-MS / MS cassette analysis using a seven-point calibration curve with appropriate sample dilutions. The starting concentration (C0) was determined from the dosing solution, and experimental recovery was calculated from C0 and the concentrations in both the apical and basolateral compartments. The HPLC column used was a Phenomenex Lux® i-Amylose-3 column, 3 μM, 100 mm x 3 m, and analysis was performed on a Waters Xevo Triple Quad MS. Monolayer integrity throughout the experiment was confirmed by monitoring Lucifer Yellow permeation using fluorometry.

[0245] Permeability coefficient (P aPP ) was calculated from the following formula:

[0246] P app =(dQ / dt) / (C0×A)

[0247] where dQ / dt is the cell permeation rate of the drug, C0 is the donor concentration at time 0, and A is the area of ​​the cell monolayer. Permeability was measured in both directions across the cell monolayer, i.e., A2B (apical to basolateral) and B2A (basolateral to apical), and the efflux ratio was determined. The efflux ratio is P app (BA) / P app(AB) was calculated.

[0248] Antipyrine, atenolol, and talinolol were used as controls. Antipyrine was absorbed 97% after oral administration in humans. Atenolol was absorbed 50% after oral administration in humans. Talinolol is a substrate of P-glycoprotein and was used as a negative control.

[0249] P app A lower value indicates greater absorption. A lower efflux ratio indicates less efflux, and a ratio greater than 2 indicates active efflux.

[0250] result

[0251] [Table 9]

[0252] The results in Table 9 show that Formula (I) exhibits good absorption (permeability) in Caco-2 cells. It also shows that the active reflux of Formula (I) in Caco-2 cells is low. The compound of Formulation (I) exhibits P app It was positioned between atenolol and antipyrine in measurement and between atenolol and talinolol in efflux, with talinolol having the highest active efflux.

[0253] conclusion The data from this study show that formula (I) exhibits high permeability and low efflux in Caco-2 cells.

[0254] Example 6 - Hepatocyte Stability The purpose of the hepatocyte stability study was to determine the stability of the compound within hepatocytes.

[0255] method Cryopreserved pooled hepatocytes were purchased from IVT (human and dog) and Lonza (rat) and stored in liquid nitrogen prior to use. Williams E medium (Sigma-Aldrich) supplemented with 2 mM L-glutamine and 25 mM HEPES (Sigma-Aldrich) was preincubated with the test compound (final substrate concentration 3 pM, final DMSO concentration 0.25%) at 37°C, and then cryopreserved hepatocyte suspensions (final cell density 0.5 x 10 6 The reaction was initiated by the addition of viable cells / mL in Williams E medium supplemented with 2 mM L-glutamine and 25 mM HEPES. The final incubation volume was 500 μL. Samples were taken at six time points over the 60-minute experimental period, and the test compounds were analyzed by LC-MS / MS.

[0256] The reaction was stopped at the appropriate time point by transferring the incubation solution to acetonitrile and diluting it 1:1 (1:2 ratio). The termination plate was centrifuged at 3,000 rpm for 20 minutes at 4°C to precipitate the protein.

[0257] After protein precipitation, sample supernatants were mixed into cassettes containing up to four compounds, and internal standards were added. LC-MS / MS was performed on a Vion instrument using a Phenomenex Lux® i-Amylose-3 column (3 μm, 100 mm x 3 mm). The eluent was deionized water (10% A), methanol (90% B) containing 10 mM ammonium bicarbonate. Flow rate: 0.5 mL / min, total run time: 15 min.

[0258] Each species contained two quality control compounds (verapamil and raloxifene) along with appropriate solvent controls.

[0259] The In peak area ratio (compound peak area / internal standard peak area) was plotted against time to determine the elimination rate constant. The half-life (t 1 / 2 The intrinsic clearance (CLint) and intrinsic clearance (CLint) were calculated. The In peak area (compound peak area / internal standard peak area) was plotted against time, and the slope of the line was determined. The elimination rate constant (K) = (-slope), half-life (t 1 / 2)(min)=0.693 / K.

[0260] Intrinsic clearance (CLint) (µL / min / million cells) = (V × 0.693) / (t 1 / 2 )

[0261] where V = incubation volume (µL) / cell number

[0262] result

[0263] [Table 10]

[0264] [Table 11]

[0265] Here, SE in Table 11 is CL int is the standard error associated with (intrinsic clearance). The results in Tables 10 and 11 demonstrate that Formula (I) is more stable than Compound (D) in rat, dog, and human hepatocytes. Compound (D) exhibited high intrinsic clearance and a short half-life across species. Formula (I) demonstrated low intrinsic clearance and a long half-life in rat, dog, and human hepatocytes, demonstrating this high stability.

[0266] conclusion The data from this study indicate that formula (I) is more stable than compound (D) in rat, human and dog hepatocytes.

[0267] Example 7 - Interconversion test in primary cultured hepatocytes The purpose of this study was to determine whether formula (I) could be converted to formula (II) in primary hepatocytes. The hepatocyte incubation, analytical conditions and equipment were the same as in Example 6. However, in Example 7, reference standards for both Formula (I) and Formula (II) were provided in the LC-MS / MS, and both were run simultaneously to achieve chromatographic separation.

[0268] LC-MS / MS analysis indicated that formula (II) was not present in the hepatocyte samples, suggesting that conversion from formula (I) to formula (II) had not occurred. Therefore, the results of this study demonstrated that formula (I) was not converted to formula (II) in rat, dog, and human hepatocytes.

[0269] Example 8 - hERG channel inhibition The purpose of this study was to determine the cardiotoxicity of Formula (I) through hERG channel inhibition, specifically to evaluate the potential of Formula (I) to inhibit the hERG channel.

[0270] method Cumulative concentration-response curves (3 concentrations / compound, N=5) were performed. Compounds were tested at 1, 10, and 100 μM in 0.5% DMSO as a solvent. A vehicle group was also tested in which the vehicle (0.5% DMSO) was administered as the test substance.

[0271] The effects of test compounds on the hERG cardiac ion channel expressed in mammalian cells (HEK-293) were evaluated using the electrophysiology platform QPatch HTX, an automated electrophysiology system that follows the general principles of conventional whole-cell patch clamp techniques. The rate of change of the hERG tail current was calculated, and the IC 50 The value (concentration of test compound that produces 50% inhibition) was calculated.

[0272] Experiments were performed using the QPatch HTX system (Sophion Biosciences A / S). Prior to conducting experiments in 48-well plates (QPIate, Sophion Biosciences A / S), the system was primed with the appropriate extracellular solution (NaCl, KCl, CaCl, MgCl, D-glucose, HEPES, pH 7.4) and intracellular solution (KCl, MgCl, EGTA, MgATP, HEPES, pH 7.2). All cell suspensions, buffers, and test compound solutions were stored at room temperature throughout the experiment.

[0273] The cells used were HEK293 cells stably transfected with hERG cDNA. Cells were automatically washed, resuspended, and placed into individual wells (recording chambers) of the QPIate. The QPatch system follows the general principles of conventional whole-cell patch clamp techniques. A high-resistance seal is formed between the patch electrode and an individual cell, followed by membrane rupture across the electrode tip, establishing the whole-cell patch clamp configuration. If the cells were deemed of poor quality, the experiment was terminated and the process was repeated on a separate plate, if necessary. Once a stable patch was obtained, recording began in voltage clamp mode.

[0274] A typical voltage profile was as follows: a 200 ms step from -80 mV to -50 mV, followed by a 4.8 s step to +20 mV, a 5 s step to -50 mV, and then a step to a holding potential of -80 mV. The step from -80 mV to the test command (+20 mV) elicits an outward current (i.e., current leaving the cell), while the step from the test command (+20 mV) to -50 mV elicits a tail current (a tail current represents a current decaying over time).

[0275] Compound dilutions were prepared by diluting the compound in DMSO (default 10 mM) and then diluting with extracellular buffer to the final concentrations tested. The perfusion solution contained 0.05% Pluronic F-68 and was stored at room temperature daily for the duration of the experiment. The voltage protocol was continuously run and recorded during the experiment. Cells were treated with vehicle (0.1% DMSO) for 3 minutes, after which test substances were added sequentially at three concentrations. Test substances were tested in triplicate at each concentration, with at least two cells tested. The standard combined exposure time was 5 minutes. One vehicle group was run per experiment. The positive control group received the vehicle (0.1% DMSO) as the test substance, followed by the positive control (E-4031).

[0276] The effect of the test substance on each cell was calculated by calculating the residual current (% control) compared to the vehicle-treated (0% inhibition) current using the average tail current amplitude recorded from four consecutive voltage pulses. Data were plotted, and IC50 values ​​were estimated from the concentration-response relationship.

[0277] result Table 12 - Formula (I)

[0278] [Table 12]

[0279] Table 13 - Vehicle

[0280] [Table 13]

[0281] Table 14-E-4031

[0282] [Table 14]

[0283] Formula (I) showed less than 50% inhibition at the highest concentration tested (100 μM), with a pIC 50 E-4031 was tested as a hERG pharmacological standard and the results were consistent with previous in-house data.

[0284] conclusion Table 12 above shows that formula (I) does not inhibit the hERG channel, so hERG channel activity is not an issue with formula (I).

[0285] Example 9 - AMES Test The aim of this study was to investigate the mutagenicity of formula (I) using the AMES test.

[0286] method Compounds were tested at concentrations of 62.5, 125, 250, 500, 1000, and 2000 μg / mL (the highest concentration in this dose-response was chosen based on water solubility). 2-Aminoanthracene, 2-nitrofluorene, and 4-nitroquinoline N-oxide were included as positive controls. The AMES assay was performed using a kit (product number: A10-210-S2-P) from Xenometrix, Inc. This kit contains bacteria and all necessary solutions. Approximately 10 million bacteria were exposed to triplicate concentrations of Formula (I) (6 concentrations), a negative control (vehicle), and a positive control for 90 minutes in medium containing a low concentration of histidine (enough to achieve approximately two-fold growth). The cultures were then diluted into the indicated medium. It lacked histidine and was dispensed into 48 wells of a 384-well plate (microplate format, MPF). The plates were incubated at 37°C for 48 hours, after which the cells that underwent reversion growth in the wells changed color. The number of wells in which growth was observed was counted and compared with the solvent control. A positive response was determined when the colony count increased by at least twofold above baseline (control mean + standard deviation) and a dose-response was observed. An unpaired, one-tailed Student's t-test was used to identify conditions that were significantly different from the control group.

[0287] Because S9 contains both microsomal and cytosolic fractions and because metabolism may be required for a chemical to exert its mutagenicity, S9 was included as a metabolic system in the Ames test. Where noted, S9 fractions from the livers of Aroclor 1254-treated rats were used in the incubations at a final concentration of 4.5%.

[0288] In this study, we used S. typhimurium TA98: hisD3052, rfa, uvrB / pKM101 (TA98) and S. typhimurium TA100: hisG45, rfa, uvrB / pKM101 (TA100). TA98 detects frameshift mutations, while TA100 detects base pair substitutions.

[0289] result

[0290] [Table 15]

[0291] The data in Table 15 show that Formula (I) was not mutagenic, with or without S9 metabolic activation. Formula (I) did not exhibit genotoxic effects on any of the strains used in this study. All positive controls behaved as expected in this experiment.

[0292] Embodiments of the present invention have been shown and described herein. Those skilled in the art will recognize that these embodiments are presented for purposes of illustration only, and are not limiting. It will be understood that changes in form and detail may be made without departing from the scope of the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention, and that the invention encompasses embodiments within the scope of the claims and their equivalents.

Claims

1. A compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof: 【Chemistry 1】

2. 2. The compound of claim 1, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof: 【Chemistry 2】

3. 3. The compound of claim 1 or claim 2, wherein the pharmaceutically acceptable salt is the hydrochloride salt.

4. A pharmaceutical composition comprising: a) a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof: and 【Transformation 3】 b) Pharmaceutically acceptable excipients.

5. 5. The pharmaceutical composition of claim 4, wherein the compounds of formula (I) or (II) have an enantiomeric and diastereomeric excess of at least 90%, preferably at least 95%, more preferably at least 98%.

6. 6. The pharmaceutical composition of claim 4 or claim 5, wherein the compound has formula (I) or a pharmaceutically acceptable salt thereof: 【Chemistry 4】

7. 7. The pharmaceutical composition of any one of claims 4 to 6, wherein the pharmaceutically acceptable salt is the hydrochloride salt.

8. 8. The pharmaceutical composition of any one of claims 4 to 7, further comprising an additional active agent.

9. 9. The pharmaceutical composition of any one of claims 4 to 8, provided as an oral, buccal, sublingual, subcutaneous, intravenous, intramuscular, intranasal, inhalation, rectal, or topical dosage form.

10. A compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 4 to 9 for use as a medicament.

11. β ２ -A compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 4 to 9 for use in the treatment or prevention of disorders associated with the adrenergic receptor signalling pathway.

12. β ２ 12. The compound or pharmaceutical composition for use according to claim 11, wherein the disorder associated with the adrenergic receptor signaling pathway is a tumor, a vascular abnormality, a respiratory disease, a cardiovascular disease, physiological tremor, or a migraine.

13. 13. The compound or pharmaceutical composition for use according to claim 12, wherein the respiratory disease is asthma or chronic obstructive pulmonary disease.

14. 13. The compound or pharmaceutical composition for use according to claim 12, wherein the cardiovascular disease is chronic heart failure or Takotsubo syndrome.

15. 13. The compound or pharmaceutical composition for use according to claim 12, wherein the tumor is a vascular tumor, a carcinoma, a paraganglioma, or tuberous sclerosis.

16. 16. The compound or pharmaceutical composition for use according to claim 15, wherein the vascular tumor is infantile hemangioma, von Hippel-Lindau disease, angiosarcoma, or glioma.

17. 16. The compound or pharmaceutical composition for use according to claim 15, wherein the cancer is soft tissue sarcoma, melanoma, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, ovarian cancer, or lymphoblastic leukemia.

18. 13. The compound or pharmaceutical composition for use according to claim 12, wherein the vascular abnormality is hereditary hemorrhagic telangiectasia or cerebral cavernous malformation.

19. Selective beta ２ -A compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 4 to 9 for use as an adrenoceptor antagonist.

20. 20. The compound or pharmaceutical composition of any one of claims 10 to 19, which is administered orally, bucally, sublingually, subcutaneously, intravenously, intramuscularly, intranasally, by inhalation, rectally, or topically.

21. β ２ -A method for treating or preventing a disorder associated with the adrenergic receptor signaling pathway, comprising administering to a subject a compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 4 to 9.

22. β ２ 22. The method of claim 21, wherein the disorder associated with the adrenoceptor signaling pathway is a tumor, a vascular abnormality, a respiratory disease, a cardiovascular disease, physiological tremor, or a migraine.

23. β ２ - Use of a compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 4 to 9 for the manufacture of a medicament for the treatment or prevention of a disease associated with the adrenoceptor signaling pathway.

24. β ２ 24. The use according to claim 23, wherein the disorder associated with the adrenoceptor signaling pathway is a tumor, a vascular abnormality, a respiratory disease, a cardiovascular disease, physiological tremor, or a migraine.

25. Selective beta ２ - Use of a compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 4 to 9 as an adrenoceptor antagonist.

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