Synthesis and Pharmacological Evaluation of N-Aryl Sulfonamides As 5-HT4 Receptor Antagonists

Synthesis and pharmacological evaluation of

N-aryl sulfonamides as 5-HT4 receptor antagonists

Thesis for the degree Master of Pharmacy

by Mirusha Navaratnarajah

Section of Medicinal Chemistry Department of Pharmaceutical Chemistry School of Pharmacy Faculty of Mathematics and Natural Sciences University of Oslo 2013

Drug Discovery Laboratory AS Oslo Innovation Center

K.G. Jebsen Cardiac Research Centre, Center for Heart Failure Research, and Department of Pharmacology Faculty of Medicine University of Oslo and Oslo University Hospital

TABLE OF CONTENTS

Acknowledgements 7. Summary 9. Abbreviations 11. 1. INTRODUCTION 13. 2. BACKGROUND 14. 2.1 Congestive heart failure 14.

2.1.1. Prevalence and symptoms of heart failure 14. 2.1.2. Remodelling 16. 2.1.3. Treatment 16. 2.1.4. β-blockers 16. 2.1.5. ACE inhibitors and ARBs 17. 2.1.6. Aldosterone 19.

2.2 Pharmacology and function of serotonin (5-HT) 20.

2.2.1. Synthesis, release and storage of serotonin 21. 2.2.2. The signalling mechanism of the 5-HT4 receptor 21. 2.2.3. Function of serotonin in the heart 22.

2.3 Chemical background 23.

2.3.1. The pharmacophore model of the 5-HT4 receptor 23. 2.3.2. The search for selective 5-HT receptor ligands 24. 4 2.4 hERG potassium ion channel 27.

2.5 Adenylyl cyclase (AC) 28.

3. AIMs 29. 4. RESULTS AND DISCUSSION 30. 4.1 Interpretation of chemistry data 30.

4.1.1. General synthesis strategy 30. 4.1.2. Synthesis of indole sulfonamides (5-8) 33. 4.1.3. Synthesis of benzodioxane sulfonamides (13-19) 36. 4.1.4. Synthesis of piboserod sulfonamides (26-33) 38. 4.1.5. The log of distribution coefficients (logD ) 42. Oct7.4 4.2 Interpretation of pharmacological data 43.

4.2.1. Analysis of binding curves for the compounds 43. 4.2.2. Analysis of adenylyl cyclase curves for the selected compounds 48. 4.2.3. Analysis of inverse agonist effect 53.

4.3 Structure-affinity relationship (SAFIR) 54.

4.3.1. Comparing derivatives with same sulphonamide side-chain group. 55.

4.3.2. Comparing derivatives with increasing side-chain length. 61. 4.3.3. Comparing electron donating and electron withdrawing sulfonamide side-chain groups. 63. 5. CONCLUSIONS 64. 6. MATERIALS AND METHODS 65. 6.1 Experimental chemistry 65. 6.1.1. General procedures for preparations of intermediates 20 and 21 65. 6.1.2. Preparation of benzyl protected piperidine amines 66. 6.1.3. Hydrogenolysis of benzyl protected piperidine amines 67. 6.1.4. Preparation of arylic nitro compounds 68. 6.1.5. Reduction to arylic amine compounds 69. 6.1.6. General procedure for the synthesis of aryl sulfonamides 70.

6.2 Experimental pharmacology 75. 6.2.1. Radioligand binding assay (Protocol) 75. 6.2.2. Adenylyl cyclase assay (Protocol) 77.

7. REFERENCES 80. APPENDIX A – Table of synthesised compounds. 84. APPENDIX B – 1H and 13C nuclear magnetic resonance spectre. 87. APPENDIX C – High-performance liquid chromatography (HPLC). 123. APPENDIX D – Manufactures: Instruments, chemicals and enzymes. 141.

2013

Synthesis and pharmacological evaluation of N-aryl sulfonamides as 5-HT4 receptor antagonists. http://www.duo.uio.no/

Printed: Reprosentralen, Universitetet i Oslo

Acknowledgements

I am proud to present my work during the period from August 2012 to May 2013 at the Department of Chemistry, University of Oslo, in cooperation with Drug Discovery Laboratory (DDL) AS. Additional support was provided by the K.G. Jebsen Cardiac Research Centre, Center for Heart Failure Research and the Department of Pharmacology, Faculty of Medicine, University of Oslo and Oslo University Hospital. This has been a highly inspiring and enlightening journey for me. I would like to thank all my encouraging supervisors for their guidance, help and support for completing this Thesis.

My sincere thanks to Professor Jo Klaveness for let me be a part of this exciting project with his excellent scientist group at DDL AS. I simply can´t say thanks enough for all your time and inspiring talks. My greatest thanks to Bjarne Brudeli at the DDL for all the encouraging support throughout the project and for helping me through the chemistry work and NMR interpretations. This work became easy with excellent guidance and valuable advices from you.

A special thanks to Professor Finn Olav Levy and his brilliant co-workers at the Department of Pharmacology for support and guiding me through this task. I am grateful to Kjetil Wessel Andressen, Ornella Manfra and Marie Dahl for all the support and help during some intensive time. I appreciate all the effort towards the final pharmacological evaluation of my synthesised compounds for this Thesis. Thanks for all the support, inspiration and enthusiasm! This has been very educational and uplifting for me as a student.

Last but not least, thanks to all my fellow students and employees at the Department of Pharmaceutical Chemistry and the Department of Pharmacology. Thanks for being there, helping and cheering during some frustrating times and sharing good memories. Your help and support will always be valued!

And to my family, thanks for understanding and giving me all the time to concentrate on my work. This is for you!

Oslo, May 2013

Mirusha Navaratnarajah

Summary

Serotonin (5-HT) is a naturally occurring excitatory neurotransmitter in the CNS and a locally acting vasoactive signalling molecule with diverse effects in the human cardiovascular system. 5-HT is suggested to be involved in the pathophysiological progression of heart failure (HF), due to previous studies indicating increased expression of 5-HT4 receptors in the left ventricle of failing hearts in both humans and animal models. In the human heart, stimulation of 5-HT4 receptors activates adenylyl cyclase (AC) and increases cAMP levels, thus activating a similar signalling pathway as the β-adrenoceptors, producing enhanced rate (chronotropic effect), force of contraction (inotropic effect) and hastening of contraction-relaxation cycle (lusitropic effect). New treatment of HF and atrial fibrillation with 5-HT4 receptor antagonists has been suggested to be beneficial by recent studies. However, further development of 5-HT4 receptor ligands will require further studies of efficacy, as well as elimination of the potential harmful risk of hERG potassium channel binding causing QT prolongation with increased risk of ventricular arrhythmia (TdP).

With the aim of developing new 5-HT4 antagonists for further drug development, we have synthesised 19 novel acidic N-aryl sulfonamides based on three aromatic ring systems: Indole-3-carboxylic acid sulfonamides 5-8, 1,4-benzodioxane-5-carboxylic acid sulfonamides 13-19, and 3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxylic acid (piboserod) sulfonamides 26-33. The new N-aryl sulfonamides were characterized by 1H/13C-NMR spectroscopy, HPLC analysis and the logarithmic distribution between phosphate buffer with pH 7.4 and n-octanol (log Doct7.4). The compounds were also pharmacologically evaluated to 3 determine h5-HT4(b) receptor binding affinity, in a [ H]GR113808 radioligand binding assay, and antagonist property, in an adenylyl cyclase assay. The reference compounds used were GR113808 and SB207266 (Piboserod®). The structure-affinity relationships were evaluated based on the various side-chain substituents introduced to the N-aryl sulfonamides.

The piboserod sulfonamides and the benzodioxan sulfonamides seem to have higher affinity for the 5-HT4 receptor compared to the indole sulfonamides. The replacement of the hydrogen bond donor NH- in the indole ring by a hydrogen bond acceptor oxygen in the oxazino[3,2-a] ring of piboserod and in the benzodioxan ring, could be favourable for obtaining increased affinity for the 5-HT4 receptor, as indicated in earlier studies. Incorporating various side-chain groups to the piboserod and benzodioxan derivatives seem to alter the affinity for the 5-HT4 receptor, but increasing the side-chain length could not reveal any significant changes to the affinity. Electron withdrawing or electron donating side-chain groups seem to reduce the affinity for the receptor, compared to other substituents. More studies should be initiated to reveal any influence on the hydrophobic pocket and to the affinity for the 5-HT4 receptor, by using various substituents on N-aryl sulphonamides. Future studies should examine the hERG potassium channel affinity of the novel acidic N-aryl sulphonamides as well, since this will be critical for further clinical development.

Abbreviations

5-HIAA 5-hydroxyindole acetic acid 5-HT 5-hydroxytryptamine AC Adenylyl cyclase ACE Angiotensin converting enzyme ANP Atrial natriuretic peptide ATP Adenosine – 5´triphosphate BBB Blood brain barrier BNP Brain natriuretic peptides cAMP 3´, 5´- cyclic adenosine monophosphate cGMP 3´, 5´- cyclic guanosine monophosphate CNS Central nervous system CRC Contraction-relaxation cycle EF Ejection fraction ESC European Society of Cardiology GC Guanylyl cyclase GDP Guanosine diphosphate GI Gastro-intestinal GPCRs G-protein-coupled receptors GTP Guanosine 5′-triphosphate hERG Human ether-á-go-go related gene HF Heart failure IBS Irritable bowel syndrome LVEF Left ventricular ejection fraction LVSD Left ventricular systolic dysfunction LVSD Left ventricular systolic dysfunction MAOA Monoamine oxidase A mRNA Messenger ribonucleic acid N.D. Not determined NO Nitric oxide NYHA New York Heart Association PDE Phosphodiesterase PG Preferred protection group PKA cAMP-dependent protein kinase (protein kinase A) RAS Renin angiotensin system SA Sinoatrial node SAFIR Structure-affinity relationship SAR Structure-activity relationship TdP Torsades de pointes

1. Introduction

Congestive heart failure (HF) is a common cardiovascular syndrome and is a major public health problem, especially in the western world. At this stage, the heart is unable to fulfil the circulatory requirements of the body. The main aim of today’s treatment of HF is to oppose activation and further progression of the compensatory mechanisms. Despite the improvement in current advanced treatment with several “blockbuster” drugs available, it fails to stop the progression of this complicated disease. Therefore it is always an utmost need for new therapeutic regimens of the HF treatment.

This is where the newly on-going studies towards 5-HT4 receptor antagonist are important. The functional cardio-excitatory neurotransmitter serotonin enhances contraction and hastens relaxation in human atria and failing human ventricle, through 5-HT4 receptor stimulation (3).

Both agonists and antagonist of the 5-HT4 receptor have been applied for therapeutic use in a 1 wide variety of disorders as migraine (5-HT1 antagonists), irritable bowel syndrome (IBS, 5-

HT4 agonists) and latest suggested for the treatment of HF and arrhythmia (4). Cisapride ® (Prepulsid ), a 5-HT4 receptor agonist, was withdrawn from the market in 2004 (in Norway) as it appeared to have significant affinity to the hERG potassium channel. This is a major risk of causing long QT-syndrome, ventricular fibrillation (torsades de pointes) and cardiac arrest ® (5-7). The potent and selective 5-HT4 receptor antagonist Piboserod has been evaluated in a 6-month treatment of chronic, symptomatic HF and revealed some promising improvement in left ventricular ejection fraction (LVEF), and suggested to be an alternative for patients intolerant to β-blocker treatment. However, the clinical benefit by blockade of myocardial serotonin receptors in HF remains still uncertain. Piboserod® could not reveal any significant changes in other efficacy parameters than LVEF and reported a higher number of adverse events in piboserod-treated patients compared to placebo (8). Thus, there is a rationale and a potential medical value to develop new selective 5-HT4 receptor antagonists for the treatment of HF with reduced risk of serious cardiovascular side-effects.

1 IBS is gastro-intestinal motility disorder, characterized as a hypersensitive gut-syndrome with changed peristaltic motility and altered bowel function, often as part of psychosocial disorders (4).

2. Background

2.1. Congestive heart failure

Congestive HF is a severe condition where the heart fails to maintain its pumping function, thus causing an inadequate blood supply to the tissues and deficiency of oxygen and nutrients occurs. The body responds to this by increasing the heart rate (chronotropic effect), force of contraction (inotropic effect), rate of cardiac relaxation (lusitropic effect), preload (central venous pressure) and number of contractile elements (hypertrophy) in the heart through activation of compensatory mechanisms. There are two main compensatory mechanisms which are activated during HF. Catecholamines are central neurotransmitters which are released from the sympathetic nerves and adrenal medulla, and responsible for cardio- stimulation. Activation of the adrenergic sympathetic nervous system provides an inotropic and chronotropic support for the heart to maintain the blood pressure. The RAS-system (Renin angiotensin system) is activated by the baroreceptors in the kidney sensing reduced blood pressure due to reduced blood flow and releases renin from juxtaglomerular cells in the kidney. Renin is an enzyme that catalyses the formation of angiotensin I from angiotensinogen, released from the liver. Angiotensin I is converted to angiotensin II catalysed by ACE (angiotensin converting enzyme), which is a target in the blood pressure and HF treatment. Angiotensin II mediates vasoconstriction and aldosterone mediates salt and water retention in the distal renal tubule, which contributes to a total increase in preload. This allows the heart to operate at elevated end-diastolic volumes. Angiotensin II and aldosterone are main targets of HF treatment today and contribute to the progression of the heart disease. Counterproductive activation over an extended period of time, propagate severe progression of the disease ((9)9).

2.1.1. Prevalence and symptoms of heart failure

The fact that the number of patients with chronic symptomatic HF is increasing is evident. The recent ESC guideline reports that about 1-2 % of the adult population in the western world is suffering from HF. The prevalence is higher (>10 %) and the prognosis is poorer in the elderly population above 70 years of age (10). The most important reason for this is that

the elderly population with highest risk of cardiovascular disease is rapidly rising and the rate of survival in those patients is positively improving with better treatment (11).

Patients with symptomatic HF are diagnosed according to the New York Heart Association (NYHA) – classification system (Table 1). The HF condition can also be asymptomatic for patients with left ventricular systolic dysfunction (LVSD) or other underlying cardiac abnormalities. The risk of sudden death is predicted to be higher in those with mild-to- moderate form of HF, with preserved LV function (12). LVSD is a primary reason for exacerbation of HF and the condition becomes symptomatic with elevated blood levels of natural vasodilators (ANP, atrial natriuretic peptide, and BNP, brain natriuretic peptides). The fraction ejected (EF) is found to be significantly reduced in more severe form of LVSD and is often used as a measurement for the progression of the disease (10). Based on the patient’s condition and the impairment in the heart’s ability to fill or empty blood, HF can further be categorized into acute or chronic, left- or right-sided, and systolic or diastolic. The typical clinical symptoms of HF are volume overload (oedema, breathlessness) and inadequate tissue perfusion (impaired exercise tolerance, fatigue and renal dysfunction) (9, 13).

Table 1: New York Heart Association (NYHA) functional classification of HF. Adapted from (10). Class Patient symptoms

Class I (Mild) No limitation of physical activity. Ordinary physical activity does not cause any symptoms. Class II (Mild) Slight limitation of physical activity. Comfortable at rest, but ordinary physical activity results in fatigue, rapid/ irregular heartbeat (palpitation) or shortness of breath (dyspnea). Class III (Moderate) Marked limitation of physical activity. Comfortable at rest, but less than ordinary physical activity results in fatigue, rapid/irregular heartbeat (palpitation) or shortness of breath (dyspnea). Class IV (Severe) Unable to carry out any physical activity without discomfort. Symptoms of fatigue, rapid/irregular heartbeat (palpitation) or shortness of breath (dyspnea) are present at rest. If any physical activity is undertaken, discomfort increases.

2.1.2. Remodelling

The occurrence of chronic HF is mainly caused by myocardial dysfunction. This may be initiated by several reasons as sustained myocardial stress caused by heart attack, coronary artery disease, peripheral vascular atherosclerosis, inflammation, hemodynamic overload or idiopathic dysfunction (13). “LA remodelling” is defined as “a time-dependent adaptive regulation of cardiac myocytes in order to maintain homeostasis against external stressors” (13). As a result of long-standing lack of treatment of hypertension and increasing tension on the ventricular wall, a progressive remodelling takes place by increasing cardiac muscle mass (hypertrophy, increase in the size of individual myocytes) and volume, which results in irreversible structural changes of the heart’s size, shape and function. This ultimately increases workload and oxygen consumption, causing insufficient oxygen supply to the myocardial tissues. A total reduction in the cardiac output triggers activation of compensatory mechanisms, which leads to a further remodelling and progression of the heart disease (14).

2.1.3. Treatment

Insufficient treatment, progressive failure or acute heart rhythm disorder are main reason for mortality in the HF population. The aim of the treatment is to oppose the counterproductive compensatory mechanisms, improving left ventricular function and controlling the secondary effects that lead to the occurrence of symptoms, and thereby delaying the harmful progression of the HF syndrome. There is no standard appropriate treatment of HF that fits all patients; since it will depend on individual cases and on the patients’ conditions. Current recommended treatment is a combination of an ACE inhibitor or ARB, a β-blocker if tolerated, an aldosterone antagonist in most patients and a diuretic as needed to relieve symptoms of oedema and congestion in selected patients with continues symptoms of HF (10, 15, 16).

2.1.4. β-adrenoceptor antagonists (β-blockers)

Long-term treatment with β-blockers has shown clinical benefit in chronic symptomatic HF patients by improving the ventricular dysfunction and reversing the progression of the disease, apparently in those with lowest EF, as well as an arrhythmia protective role. This has been achieved by a significant reduction in the incidents of arrhythmia and acute vascular events,

hence reduced mortality. There have been observed a time-dependent reduction in preload (filling pressure), cardiac volumes, myocardial hypertrophy and increased EF. The short-term use of β-blockers seems to be unfavourable, so continuous treatment with β-blockers is crucial. RESOLVD pilot study (Randomized Evaluation of Strategies for Left Ventricular Dysfunction) also clarifies that β-blocker treatment is equally effective in combination with ACE inhibitors or an angiotensin receptor blocker (ARB). Studies also show that patients already getting a β-blocker will get a greater benefit from an ACE inhibitor. Patients’ receiving high doses of ACE inhibitors seem to preserve the beneficial effects of β-blockers. This supports the statement that β-blockers should be initiated for all stages of HF, and as early as possible to reduce the risk of adverse events. Further unanswered problems yet to be solved are the effectiveness of β-blockers in elderly patients and the efficacy in using between the commercially available β-blockers (β1 –selective and – non-selective inhibitors), where caution should be made for patients with cardiogenic shock and acute pulmonary oedema (17, 18).

The rationale for treatment targeting β-adrenoceptors in chronic HF is the realisation of the desensitization of β-adrenoceptors occurring in HF, and may reflect a naturally protective mechanism. It is estimated that about 50-60 % of the total β-adrenergic signalling transduction potential is lost in end-stage of the failing heart. Even the remaining signalling of

β1- and β2- adrenergic receptors is potentially harmful, so that further inhibition is important

(19, 20). Serotonin, through stimulation of 5-HT4 receptors, was found to activate the same intracellular signalling mechanism as the β-adrenoceptors and antagonism of these receptors could potentially be beneficial in those who are intolerant to β-blocker treatment (3). Another argument supporting the proposed new treatment of 5-HT4 receptor antagonist is that serotonin receptors are up regulated during the progression of HF syndrome and enhanced serotonin response has been seen during chronic β-blocker treatment (19).

2.1.5. ACE inhibitors and ARBs

Angiotensin converting enzyme (ACE) inhibitors are an appropriate treatment in patients, preferable in younger patients, with enlarged heart (systolic heart failure), low EF and fluid- retaining HF treated with diuretics. Evidence is lacking in elderly patients, with more

common diastolic HF (specified as normal sized to marginally enlarged heart). The advantageous effects of ACE inhibitors are a mild diuretic effect, kidney protective role, preservation of myocardium by modifying angiotensin II (as a growth factor) mediated left ventricle hypertrophy, increased bradykinin/ NO mediated vasodilation and diminishing angiotensin II stimulated release of vasoconstrictors (catecholamines). ACE inhibitors are also recommended for hypertensive patients for preventing HF. A crucial part of the HF treatment is to prevent activation of the RAS-system, which is responsible for the deleterious effects of angiotensin II. Further studies indicate escape of angiotensin II during long-term ACE- inhibition or an incomplete blockade. This reveals the utmost need for new treatment for HF.

By adding angiotensin-receptor-blockers (ARBs) to ACE inhibitors, a more complete AT1 blockade may be achieved and the bradykinin mediated beneficial response are expected to be preserved (21).

Initiating ACE inhibitor to the HF treatment has reported diminishing in coronary and stroke hospitalization events (reduced mortality and morbidity), improvement in symptoms and exercise capacity. Few side effects are reported with ACE inhibitors, except cough, probably due to accumulation of bradykinin in the lungs. The adverse effects are more common when the start dose is too high and not titrated upward according to the guidelines. ELITE (Evaluation of Losartan in the Elderly) studies suggest that ACE inhibitors should be prescribed first. But the fact that the RAS-system is not strongly activated until a diuretic is initiated, may question the benefit of using ACE-inhibitor alone. HOPE (The Heart Outcomes Prevention Evaluation) trials are the only reported long-term benefit studies of ACE inhibitors, in essentially asymptomatic HF. American Heart Association guidelines recommend the addition of ARBs to ACE inhibitors in patients with continuous symptoms of HF even though target doses of ACE inhibitors and β-blockers are given, or receiving monotherapy of ACE inhibitors and unable to tolerate β-blockers (15, 21).

2.1.6. Aldosterone

Latest guidelines have recommended the addition of an aldosterone receptor antagonist in the HF treatment. Aldosterone is released from the adrenal glands due to angiotensin II stimulation of the AT1-receptor and activation of the sympathetic nervous system. Aldosterone-elicited effects are water- and salt-retention, excretion of K+ and Mg2+, fibrosis, structural changes of the heart and further stimulation of sympathetic nervous system. RESOLVED (the Randomized Evaluation of Strategies for Left Ventricular Dysfunction) study have demonstrated that co-administration of ACE inhibitors and ARBs, or β-blockers can lead to escape of aldosterone (22). Serotonin is revealed to be actively involved in the release of aldosterone and proposed 5-HT4 receptor antagonists as a new target treatment for hyperaldoseronism (23). Recent ESC guidelines recommend the addition of an aldosterone receptor antagonist in addition to ACE inhibitor or ARB treatment (10). But triple RAS- inhibiting therapy of ACE inhibitor, ARB and aldosterone receptor antagonist are not recommended, because there is a lack of safety and efficacy documentations (21). Addition of an aldosterone receptor antagonist may be beneficial in some patients, but should be restricted to those with severe or progressive LVSD, with marginal serum creatinine and serum potassium levels (22).

2.2. Pharmacology and function of serotonin (5-HT) Serotonin is involved in the aetiology of diseases like migraine, anxiety, schizophrenia, social phobia, depression, eating disorders, panic disorders, hypertension and other cardiovascular disorders (arrhythmia), vomiting and irritable bowel syndrome (24). In the cardiovascular system, 5-HT elicits a multiplicity of physiological responses as shown in Table 2. Numerous receptor subtypes are involved in mediating diverse physiological responses in different species. Due to the multiplicity of receptor and effects, the development of novel, selective 5- HT receptor ligands is important for future treatment (25-27).

Table 2: 5-HT receptor mediated response in human cardiovascular system. Adapted from: (24).

Receptor Subtypes Receptor type Major Cardiovascular signalling response pathway

5-HT1 A, B, D, E, F Metabotropic, Inhibits AC, Renal vascular

GPCR, cAMP dilation?, vasoconstriction, cerebral arteriolar dilation, vascular nerve endings

5-HT2 A, B, C Metabotropic, Stimulates Vasoconstriction, GPCR PLC, platelet aggregation,

IP3 vasodilation.

5-HT3 A, B Ionotropic, Ion channel Reflex bradycardia, ligand operated pain

5-HT4 (short, long) Metabotropic, Stimulates Cardio stimulation, GPCR AC, pulmonary vein cAMP dilation

5-HT5 A, B Metabotropic, Unknown Unknown GPCR

5-HT6 Metabotropic, Stimulates Unknown GPCR AC, cAMP

5-HT7 a, b, d Metabotropic, Stimulates Vascular relaxation GPCR AC, cAMP

2.2.1. Synthesis, storage and release of serotonin Serotonin (5-hydroxytryptamine, 5-HT) is a well-known, naturally occurring vasoactive neurotransmitter and a locally acting signalling molecule found primarily in the brain, enterochromaffin tissues and blood platelets. The synthesis of serotonin occur mainly in the enterochromaffin cells of the intestine (> 95 %), but also in the raphe nuclei of the brain and in the neuroendothelial cells lining the lung. In these cells, the essential amino acid precursor tryptophan is enzymatically converted to 5-hydroxytryptamin. This is catalysed by the enzyme tryptophan hydroxylase that mediates a specific aromatic hydroxylation, followed by a decarboxylation by the enzyme amino acid decarboxylase. When 5-HT is synthesised, it is then released into the blood and actively stored in the blood platelets, therefore only a small amount of free circulating 5-HT is found under normal conditions. Serotonin mediates pharmacological and physiological role in the heart, intestine (gastrointestinal tract), CNS, urinary bladder, kidney and adrenal gland. The metabolism of serotonin occurs primarily in the lung, intestine and endothelial cells in the arteries by MAOA (monoamine oxidase A) and is released as 5-HIAA (5-hydroxyindole acetic acid) in the serotonergic synapse (28-30).

2.2.2. The signalling mechanism of the 5-HT4 receptor

At the present, seven major families of 5-HT receptors are characterized (5-HT1-5-HT7), coded by 14 different genes, which all except the 5-HT3 receptor belongs to the class of GPCRs (G-protein-coupled receptors). The signalling pathway is found to be similar for the

5-HT4 receptors in the rat (as well as in human and porcine) ventricles and in the human atria.

Serotonin binding to the 5-HT4 receptor activates Gs coupled G-protein, which stimulates adenylyl cyclase mediated increase in cAMP (cyclic adenosine monophosphate) levels. The subsequent increase in PKA (cAMP- dependent protein kinase) mediates phosphorylation of proteins involving in Ca2+ handling (as L-type Ca2+ channels, phospholamban, troponin I and intracellular Ca2+ receptor as ryanodine receptors), leading to increased Ca2+ availability and triggering myocardial contraction. However, this is considered as an energetically unfavourable way of increasing contractility. This explains the serotonin mediated effect of shortening of the contraction-relaxation cycle (CRC) in papillary muscle (positive lusitropic response), induction of a positive inotropic response (force of contraction) and a reinforced progress to atrial fibrillation (3, 31, 32).

2.2.3. Function of serotonin in the heart

5-HT stimulated activation of the 5-HT4 receptor enhances rate (chronotropic effect), force of contraction (inotropic effect) and hastens the contraction-relaxation cycle (lusitropic effect) in human atria, which may cause atrial fibrillation (arrhythmia) (29). Elevated levels of serotonin are a risk factor for HF and may be involved in a number of progressive pathways of the heart disease. Serotonin-enhanced vasoconstriction increase with age, but decrease in hypertension (33). Elevated blood levels of 5-HT has been associated with a diminished ability of platelets to bind serotonin, found in patients with pulmonary atrial hypertension (34). This may explain the reason for increased serotonin sensitivity in the elderly and hypertensive patients. Inhibition of 5-HT4 receptors was not suggested beneficial until the discovery of functional 5-HT4 receptors in human and porcine ventricle (35) and in post-infarcted rat cardiac ventricle (36), and not only in the cardiac atria as assumed in earlier discoveries. In failing rat and human hearts, functional ventricular 5-HT4 receptors are induced, thus opening for the possibility of that serotonin may play a pathophysiological role in the progression of

HF. In addition a 4-fold increase in 5-HT4 mRNA expression was detected in 20 failing human heart ventricles and also in failing rat hearts (19, 35).

Induction of ventricular serotonin effects in failing or infarcted heart is considered as a compensatory mechanism, like the adrenergic system, and suggested to mediate through the same signalling mechanism (cAMP/ AC). Although there are major similarities between 5-

HT4- and β1-mediated signalling, there are also differences. Recently, an enhancing effect on

5-HT4 receptor and β1-adrenoceptor mediated positive inotropic response by natriuretic peptides was discovered and studied in isolated cardiomyocytes from failing rat heart ventricle. The enhancing effect is mediated by the second messenger cGMP, which mediates a competitive inhibitory effect on PDE3 and reduces its breakdown of cAMP, leading to increased inotropic responses. PDE3 degrades both cAMP and cGMP, and suggested to have an important cardio-protective role by preventing progressive alteration in cardio-stimulation. The remarkable difference between the two receptor systems was found in the response to cGMP formed by soluble GC (guanylyl cyclase), which is stimulated by NO (nitric oxide). In contrast to the enhancement of the 5-HT4 -elicited inotropic response, the β1-receptor mediated inotropic response was inhibited through an unknown mechanism (36, 37).

2.3. Chemical background

2.3.1. The pharmacophore model of the 5-HT4 receptor

The well evaluated pharmacophore model of the 5-HT4 receptor is presented in Figure 1.

Figure 1: The pharmacophore model of the 5-HT4 receptor (38, 39).

A pharmacophore is defined as the chemical part of the molecule which is responsible for the active interaction with the receptor site and mediate response (40). The presence of a hydrophobic aromatic ring system, a hydrogen bond acceptor (e.g. carbonyl, ester, amid) and a basic group, acceptable for ligand binding in their protonated form, are essential parts of the pharmacophore model (39). Further, the model predicts the carbonyl group to be situated at 3.6 Å from the centroid of the aromatic ring and 5.4 Å from the basic nitrogen. The distance from the tertiary amine to the aromatic centroid is predicted to be 8.0 Å, respectively 3.6 – 4.0Å above the plane. The presence of a voluminous group substituted to the nitrogen atom is important for 5-HT4 receptor selectivity (38).

The 5-HT4 receptor ligands share some common structural parameters with the 5-HT3 receptor ligands and clarify the reason why a number of 5-HT3 antagonists were revealed to be 5-HT4 agonists or antagonists as well. The major structural difference between the 5-HT3 and 5-HT4 receptor, is that the 5-HT4 receptor is highly susceptible towards receptor ligands with voluminous substituents on the basic nitrogen atom in the piperidine ring. 5-HT3 receptor

ligands on the other side prefer reduced steric hindrance in this part, as zacopride and renzapride which consist of a tropane moiety. Molecular modelling studies led to the discovery the hydrophobic pocket of human 5-HT4 receptor (h5-HT4). The large, hydrophobic cavity was able to accommodate bulky substituents with hydrogen bonding property at the specific nitrogen anchoring point, without reducing receptor binding affinity (41).

The linker between the hydrogen bond acceptor and the basic nitrogen atom is liable in holding variable length. Ester and amides are bioisosteric groups2 which both have been used as hydrogen bond acceptors in the pharmacophore model, and should be in the same plan as the aromatic moiety. It is assumed that the difference in the conformational orientations of the ester and amides could influence the efficacy. Predominantly results showed that esters are expected to be more potent than amides, but esters are expected to have a poor bioavailability due to rapid hydrolysis by esterases in vivo. A contradictory study by Lopez-Rodriguez found that amides were more potent 5-HT4 receptor antagonists than the corresponding ester (39).

2.3.2. The search for selective 5-HT4 ligands

Early structure-activity relationship (SAR) studies revealed close structural similarities between 5-HT3 and 5-HT4 receptor ligands (Figure 2). This established a challenge for developing selective ligands for the 5-HT4 receptor. Diverse structural scaffolds are known to bind the 5-HT4 receptor: Serotonin analogues, benzimidazolones, benzamides, benzoic esters, aryl ketones and indole carboxylates or carboxamides (39).

Benzamide derivatives are the first structural scaffolds to be developed from 5-HT3 receptor antagonists to potential 5-HT4 receptor agonists or antagonist. Metoclopramide, a potential gastrointestinal motility stimulant, is a non-selective 5-HT4 receptor agonist and considered as the parent drug for several derivatives like renzapride, zacopride and cisapride. By introducing various linear, flexible amide side-chain substituents linked to the basic N-atom in the rigid framework (as quinolizidine and tropane moieties), more potent and selective 5-HT3

2 Bioisosteres are defined as substituents which have similar chemical and physical properties, and expected to mediate the same biological response in vivo (40).

receptor antagonist were developed, which later were explored to be potential 5-HT4 agonists.

This indicates occurrence of a voluminous binding site of 5-HT4 receptor and the later discovery of the hydrophobic pocket (41).

® Cisapride (Prepulsid ), a potent 5-HT4 receptor agonist, was discovered by alkylating the nitrogen atom of the piperidine ring with various voluminous substituents (as piperidine, OH, phenoxy, CN). Cisapride was the first derivative of the benzamide family to be tested in clinical studies for GI disorders and gained therapeutic use in the treatment of irritable bowel syndrome (IBS). Although, cisapride was withdrawn from the market in 2004 (in Norway) due to the discovery of a specific and high affinity blockade of the hERG potassium channel, which may cause the potential risk of QT prolongation and ventricular arrhythmias (torsades de pointes) (5, 7).

Bioisosteric replacement of the amide group with esters, revealed more potent 5-HT4 receptor ligands. By designing ester analogues (benzoates) of metoclopramide, higher affinity for the

5-HT4 receptor was obtained. The benzodioxan ester derivate SB204070 is a highly potent and selective 5-HT4 receptor antagonist, revealed in the guinea pig distal colon. The corresponding amide derivate (SB205800) was less potent. However, esters are rapidly hydrolyzed in vivo by esterases and obtained poor bioavailability. Therefore, aromatic ketones (as RS17017) and as well as 1,2,4-oxadiazole (YM-53389) derivatives have been prepared.

By a further modification of the o-methoxy group of the 5-HT4 receptor agonist RS17017, complete antagonist response was obtained (39).

From the indole carboxylate acid ester derivatives GR113808, a highly selective 5-HT4 receptor antagonist, was developed as the first ligand in this field to be introduced with a 4- piperidinylmethyl chain. Later work toward lead modification demonstrated that the N- substituent of the piperidine was an attractive position for modulation with steric tolerance (42). In the search for more orally potent compounds, SmithKlineBeecham presented the amide derivative SB207266 (Piboserod®). To mimic the benzodioxan ring in GR113808, an oxygen atom was introduced in position 2 of the indole ring, and added the oxazino, ® oxazepino, oxazolo groups to 5-HT4 ligand list (39). Piboserod , a fully reversible antagonist,

was tested in 137 HF patients for 24-weeks in a clinical phase II study (8). The study demonstrated that treatment with Piboserod® had a small, but significant improvement in LVEF compared to placebo. Treatment with Piboserod® was evaluated as safe when administered in addition to other standard therapies in patients with stable HF and with a trend towards a larger benefit in the small subset of patients not receiving β-blocker therapy (2.7 %; p=0.15). Further study of Piboserod® was not initiated due to increased number of adverse events, the relative small effect on the primary efficacy parameter (LVEF) and lack of significant effect on other parameters. At present, Piboserod® is approached for further study for the treatment of arrhythmia (43) and IBS (4).

Serotonin (5-HT) Metoclopramide Renzapride (BRL 24924)

Zacopride Cisapride (Propulsid

X = O SB204070 X = O SB207058 X = NH SB205800 X = NH SB207266

RS17017 YM-53389

Figure 2: Chemical structure of serotonin, 5-HT3 and 5-HT4 receptor ligands (39).

2.4. hERG potassium ion channel

The human ether-á-go-go related gene (hERG) encodes for a potassium ion channel, a tetrameric channel with six membrane-spanning domains, which has a crucial role in cardiac repolarization. Inhibition of this channel may give rise to prolongation of the QT interval in the cardiac action potential and ventricular arrhythmia, also known as torsades de pointes (TdP). Binding to this channel is quite non-specific, but because of a voluminous inner cavity there is a tendency of trapping larger drugs into this core. There are many drugs which are known to block this channel: Terfenadine, astemizole, fluoxetine and cisapride to mention some. Today, every new drug candidate has to be evaluated with respect to hERG affinity to assess any potential harmful side-effects in the early stage of lead discovery (44, 45).

Since hERG is a cationic potassium ion binding channel, drugs with a mono-covalent cationic charge are expected to have good affinity for the inner pore of the channel. Thus, drugs with a negative charged group are unfavourable for the electrostatic interaction with the pore and are unable to bind the hERG channel. A previously described strategy to reduce hERG channel affinity is to introduce a carboxylic acid group into the compound (44). Hence, by introducing a negatively charged acidic group to novel 5-HT4 receptor antagonists, inactivity towards hERG channel binding is to be expected. Also reduced penetration through blood brain barrier (BBB) and subsequent CNS distribution will be expected (45, 46).

Figure 3: Models of the hERG potassium channel. The hypothetical blockade by Fluoxetine is only attained in the open state of the channel. Adapted from: (1)

2.5. Adenylyl cyclase (AC)

Adenylyl cyclase (AC) is a membrane bound protein that converts ATP to cAMP (3´, 5´- cyclic adenosine monophosphate) by G-protein (GPCR) activation. The AC is activated by an intracellular signal cascade mediated by an agonist binding to the membrane-bound GPCR. The Gα subunit releases GDP and reduces its affinity for the Gα subunit by GTP binding. This results in a dissociation of the G-protein into βγ-subunit and an active α-subunit that interacts with the AC. Depending on whether the α-subunit stimulates (Gαs) or inhibits (Gαi), cAMP is formed by AC activation. The 5-HT4 receptor splice variants activate Gαs and thus stimulate the AC, thereby increase the cAMP formation. The second messenger cAMP activates protein kinase A (PKA), a kinase class of enzymes within the cell, which use ATP to phosphorylate proteins at specific Ser or Thr side-chains (47). The pharmacological signal transduction for AC activation is illustrated in Figure 4.

Figure 4: GPCR activation of adenylyl cyclase. Adapted from (2).

3. AIMs

Our goal is to synthesise novel, specific 5-HT4 receptor antagonists for the potential use in the treatment of HF with reduced affinity for the hERG potassium channel. In this Thesis we will synthesise three different N-aryl sulfonamide derivatives classified on the basis of the aromatic ring systems:

Indole-3-carboxylic 1,4-Benzodioxane-5-carboxylic 3,4-Dihydro-2H-[1,3]oxazino acid derivatives acid derivatives [3,2-a]indole-10-carboxylic acid (Piboserod) derivatives

We expect novel drug candidates with reduced risk of hERG channel causing serious side- effects as QT-prolongation (TdP) and less CNS side-effects, by introducing acidic N-aryl sulfonamides in the lateral side-chain. Replacement of the ester linkage group with an amide linkage group is expected to attain good oral bioavailability for our ligands.

Sub-aims of the Thesis:

Structure-affinity relationships (SAFIR) will be evaluated considered on the results presented. The first part of this Thesis is a chemical synthesis and characterization of a number of ligands, in cooperation with the Drug Discovery Laboratory AS, with the following aims:

 Synthesise chemical intermediates for the preparation of aryl sulfonamide derivatives.  Characterize new compounds with 1H/13C-NMR spectroscopy.  Determine the purity of new compounds with HPLC.  Determine the logarithmic distribution coefficient (n-octanol/ phosphate buffer pH 7.4).

The second part is a pharmacological evaluation of the synthesised ligands in association with the Department of pharmacology and the Center for Heart Failure Research at Rikshospitalet, with the following target aims:

 Determine binding property to the 5-HT4(b) receptor in a radioligand binding assay.  Determine antagonist response by inhibition of 5-HT (1µM)-stimulated AC activity.

4. Results and discussion

4.1. Interpretation of chemistry data

4.1.1. General synthesis strategy

The pharmacophore model for the 5-HT4 receptor features an aromatic ring system, a hydrogen bond acceptor group and a substituted nitrogen atom as described in Figure 1. We have varied the organic ring system and focused on three aromatic ring systems which are described to have promising 5-HT4 receptor affinity by previous studies (Section 3). The general figure for all the synthesised compounds is shown below in Figure 5.

Figure 5: General scaffold for N-aryl sulfonamides. The chemical structures of the lead compounds are shown in Figure 6. GR113808 (Figure 6a) represents an indole scaffold with an ester linkage group between the scaffold and the 4- piperidine ring. SB207266 (Piboserod®, Figure 6b) is a fused indole amide with an oxazino[3,2-a]ring. N-substituted-4-piperidinylmethyl group have shown improved affinity towards the 5-HT4 receptor (48). Both are selective antagonists with high affinity for the 5-

HT4 receptor. GR113808 is expected to have poor bioavailability due to a steric unhindered ester which undergo rabid breakdown by esterase in vivo. Replacement of the ester linkage with an amide linkage in SB207266 gave a long-acting drug after oral administration (49).

x HCl

GR113808 DDL6001 (SB207266)

Figure 6a Figure 6b

Figure 6: Structure of commercially available 5-HT4 ligand lead compounds used a) GR113808 and b) DDL 6001 (SB207266, Piboserod®)

The starting materials for indole-3-carboxylic acid and 1,4-benzodioxane-5-carboxylic acid derivatives are commercial available, while 3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10- carboxylic acid must be synthesised starting from methyl indole-3-carboxylate. The general synthetic approach for the synthesised compounds was to first couple the aromatic ring system with a protected 4-piperidinylmethanamine by a nucleophile reaction. The secondary nitrogen atom in 4-piperidinylmethanamine has to be protected before coupling with the aromatic carboxylic acids. This is to ensure selective coupling with the primary amine. The preferred protection group (PG) at the secondary amine in 4-piperidinylmethanamine was benzyl, but also the tert-butyl carbonate (BOC) group was successfully used in the synthesis of indole-3-carboxylic acid and 1,4-benzodioaxane-5-carboxylic acid derivatives, as shown in Scheme 1.

b a

Ar = Ar = PG = PG =

Scheme 1: Reagents and conditions: a) Indole-3-carboxylic acid/1,4-benzodioxane-5-carboxylic acid,

(COCl)2 or CH2Cl2, DMF/CDI, 1-BOC-4-(aminomethyl)piperidine/1-benzyl-4- piperidinylmethanamine, NEt3, room temperature; b) TFA/DCM, room temperature (25 °C).

After coupling the aromatic moiety with the protected 4-piperidinylmethanamine, the protection group was removed. Hydrogenolysis with palladium on charcoal as catalyst was used to remove the benzyl protection group. This is a clean and efficient method to remove benzyl groups. The BOC group can easily be removed by acidolysis. In the synthesis of piperidine amines, trifluoroacetic acid (TFA) was used in a solution of dichloromethane at room temperature. The piperidine amine derivatives were then alkylated with 4-nitrobenzyl bromide. The resulting nitro-compounds were then reduced to the corresponding aromatic amines with hydrogen using palladium on charcoal as catalyst. To avoid cleavage of the C-N bond of the benzyl group, the reaction time was only 30-60 minutes and the hydrogen pressure of only 1 bar was used. The catalyst was filtered off and the residue separated with column chromatography to give the aromatic amines.

Finally, the arylic sulfonamides were obtained by sulfonation of the aromatic amines with various sulfonyl chloride derivatives. The sulfonyl chlorides were commercially available, and sulfonyl chlorides with both substituted and straight alkyl chains as well as various aromatic groups were used. The final arylic sulfonamides had to be separated with column chromatography to obtain compounds with sufficient purity. All the new compounds were 1 13 characterized by HPLC and H/ C NMR. The lipophilicity was measured by log Doct7.4 for the compounds, as describes in Section 4.1.5. The synthesis of the novel 5-HT4 receptor ligands are shown in Scheme 2, 3 and 5.

Figure 7: Suggested critical steps in the sulfonation of N-aryl sulfonamides.

The critical points suggested for the sulfonation step, especially for the indole sulfonamides, are shown in Figure 7. The indole arylic amine compounds are susceptible for sulfonation at various labile nitrogen atoms. The sulfonation of the primary amine in the para-benzylic position is the preferred reaction. Incorporation of a less sterically hindered side-chain group to the halide is more likely to react with N-indole and the amide linker. Therefore we expect the sulfonation with methane sulfonic chloride to be more challenging than other reagents used, especially for the indole sulfonamides. The sulfonation with more bulky and sterically hindered side-chain groups is expected to be more favourable.

4.1.2. Synthesis of indole sulfonamides (5-8)

The synthesis of indole-3-carboxylic acid derivatives 5–8 are shown in Scheme 2.

b a

1 2

c d

3 4

5-8

Scheme 2: Reagents and conditions: a) (COCl)2 or CH2Cl2, DMF, (1-benzyl-4- piperidinyl)methanamine, NEt3, room temperature; b) H2, Pd-C 20%, MeOH, CH3CO2H, room temperature; c) 4-nitrobenzylbromide, K2CO3, acetone, reflux; d) H2, Pd-C 10%, MeOH, room temperature; e) RSO2Cl, CH2Cl2, pyridine, 0˚C  room temperature.

To prepare amides or esters from a carboxylic acid, it is normally needed to convert the carboxylic acid to a more reactive group like for instance an acid chloride, since halides are better leaving groups. This strategy was applied for the synthesis of indole-3-carboxylic acid derivatives in Scheme 2. The indole-3-carboxylic acid was transformed into the corresponding acid chloride with oxalyl chloride and DMF as catalyst, and was then added to a mixture of 1-benzyl-4-piperidinylmetanamine using triethylamine as a base to give the benzyl protected piperidine amine 1. The benzyl group was then removed by hydrogenolysis, using palladium on charcoal as catalyst to leave the piperidine amine 2. The piperdine amine 2 was alkylated with 4-nitrobenzyl bromide to give the nitro compound 3. The nitro group was reduced to the corresponding aromatic amine 4 with hydrogen and palladium on charcoal as catalyst. To avoid cleavage of the C-N bond of the benzyl amine, the reaction time was short and at a low hydrogen pressure (maximum 60 minutes at 1 bar). The catalyst was

filtered off and the residue separated with column chromatography. The appearance of the amine group can clearly be seen in Figure 8.

Figure 8a

Figure 8b

Figure 8: 1H NMR spectra of the nitro derivative 3 (Figure 8a) reduced to the corresponding aromatic amine 4 (Figure 8b). a) Before reduction: Absence of signal at δ 5 for the aryl amine is detected. b) After reduction: A detectable singlet at δ 5 that integrates for two hydrogen atoms of the amine, and at δ 11.5 is a singlet that integrates for the hydrogen atom in the indole ring. The amide hydrogen signal is a triplet at δ 8.0.

To a cooled solution of amine 4 in dichloromethane and pyridine, various sulfonyl chloride derivatives were added to give the final aryl sulfonamide compounds 5-8 after purification with column chromatography. The synthesised indole derivatives have characteristic signals at δ 11.5 for the indole amine proton, and at δ 10.6 for the sulfonamide proton, as shown in the NMR spectrum of benzyl derivative 8 in Figure 9.

Figure 9: 1H NMR spectrum of 8. The indole amine is a singlet at δ 11.5 and the sulfonamide is as a broad singlet at δ 10.63. The absence of the amine signal at approximately δ 5.0 indicates that the sulfonation reaction has succeeded.

4.1.3. Synthesis of benzodioxane sulfonamides (13-19)

The synthesis of 1,4-Benzodioxane-5-carboxylic acid derivatives 13-19 are shown in Scheme 3.

a b

9 10

c d

11 12

13-19

Scheme 3: Reagents and conditions: a) CDI, (1-benzyl-4-piperidinyl)methanamine, CH2Cl2, reflux; b)

H2, Pd-C 20%, CH3CO2H/MeOH, room temperature c) 4-nitrobenzylbromide, K2CO3, acetone, reflux; d) H2, Pd-C 10%, MeOH, room temperature; e) RSO2Cl, CH2Cl2, pyridine, 0˚C  room temperature.

Following the same methodology as for the indole sulfonamide derivatives, 1,4- benzodioxane-5-carboxylic acid was first activated with N,N’carbonyldiimidazole (CDI). CDI is a common coupling reagent for synthesis of ester and amides, and the reaction mechanism for this reaction is depicted in Scheme 4:

a CO2 Imidazole

R = Benzyl R = BOC

b Imidazole

Scheme 4: Reagents and conditions: a) 1,4-Benzodioxane-5-carboxylic acid, CDI, DCM, room temperature; b) Protected 4-piperidinylmethanamine, DCM, reflux.

The acylimidazole intermediate are not isolated and are used directly to prepare the benzyl protected intermediate 9. Further, the same synthetic strategy as for the indole derivatives were used for the N-aryl sulfonamides 13–19, and the final compounds were obtained after purification with column chromatography. The characteristic signals obtained for the 1,4- benzodioxane-5-carboxylic acid derivatives are shown in a proton NMR spectrum for the n- butyl derivative 14 in Figure 10.

Figure 10: 1H NMR spectra of 14. The sulfonamide hydrogen is a singlet at δ 9.71 and the amide is a triplet at δ 8.05. Typical for the benzodioxane aromatic ring are quartet (the doublets of doublets) at δ 4.3.

4.1.4. Synthesis of piboserod sulfonamides (26-33)

The synthesis of 3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxylic acid (piboserod) derivatives 26–33 are shown in Scheme 5.

a b c

20 21

d e

22 23

24 25

26-33

Scheme 5: Reagents and conditions: a) indole-3-carboxylic acid, 3-chloro-1-propanol, CH3SO3H,

NCS, DABCO, CH2Cl2, 0˚C; b) NaOH, PhMe/H2O, 60˚C; c) 1-benzyl-4-piperidinylmethylamine,

Al2Me6, PhMe, 0˚C  reflux; d) H2, Pd-C 20%, CH3CO2H/MeOH, room temperature; e) 4- nitrobenzylbromide, K2CO3, acetone, reflux; f) H2, Pd-C 10%, MeOH, room temperature; g) RSO2Cl,

CH2Cl2, pyridine, 0˚C  room temperature.

The aromatic ring system used in the piboserod derivatives was not commercial available, so we had to synthesis it from methyl indole-3-carboxylate as shown in Scheme 5. First, the methyl indole ester was treated with N-chlorosuccinimide (NCS) and the steric hindered 1,4- diazabicyclo[2.2.2]octane (DABCO) to create a reactive intermediate that was reacted with 3-chloro-1-propanol to give the chloride intermediate 20. Intermediate 20 was then heated in

refluxing aqueous NaOH in toluene to obtain the six-membered oxazino ring 21. The proton NMR spectrum of intermediate 20 and 21 are shown in Figure 11 and 12.

18.

12 14. . 15.

Figure 11: 1H NMR spectrum of 20. The indole amine hydrogen is a singlet at δ 11.95 and the methyl ester as a singlet at δ 3.76. The triplets at δ 4.47 and 3.86 integrate each for the two hydrogen atoms of the chloropropane chain. Due to that oxygen (3.5) has a higher range of electronegativity than chlorine (3.0); we assume that the more downfield, deshieldet signal belongs to the hydrogen atoms closest to O-atom. The methylene protons in the middle of the oxazino group originate as a multiple at δ 2.23.

17.

12. 14. 13.

Figure 12: 1H NMR spectrum of 21. The methyl ester is detected as a singlet at δ 3.76. The cyclized oxazino ring is detected as triplets at δ 4.47 and δ 3.86, and the methylene protons in the middle of the oxazino ring as a multiple at δ 2.23. The absence of indole amine hydrogen signal confirms a succeeded cyclization.

Methyl ester 21 and 1-benzyl-4-piperidinylmethanamine were treated with trimethylaluminium (AlMe3) to give the amide intermediate 22. Following the same procedure as outlined in Scheme 2 and 3, acidic piboserod sulfonamides 26–33 were obtained after purification with column chromatography. The characteristic signals obtained for the piboserod derivatives are given in a proton NMR spectrum for the n-butyl sulfonamide 28 in Figure 13.

Figure 13: 1H NMR spectrum of 28. The sulfonamide hydrogen is detected as a singlet at δ 9.7, and the amide proton as a singlet at δ 6.79. The typical signals for the oxazino[3,2-a] ring are detected as a triplets at δ 4.56 and 4.13, and as a multiple at δ 2.28 as shown.

4.1.5. The log of distribution coefficients (logDOct7.4)

Lipophilicity is considered as an important drug property to be characterized in the early lead- optimization phases for a new drug. This influences both pharmacokinetic issues, as solubility and ADME properties (absorption, distribution, metabolism and excretion), and pharmacodynamics issues, as drug-receptor interactions. An administrated drug must be able to reach the site of actions and interact with the environments, both the lipophilic (e.g. cell membranes) and aqueous (e.g. cytoplasm). When the drug reaches the active site of actions, it needs to interact with the lipophilic membranes to activate a pharmacological response.

The log D value for the compounds are presented in Table 3 in Appendix A. Log D was determined as described in Section 6.1. based on the Hansch’s partition coefficient equation (40):

( )

α - the degree of dissociation of the compound in water calculated from ionization constants.

Several methods are used to determine lipophilicity. Ionisable compounds have pH – depended solubility, so the distribution coefficient (D) is preferred rather than log P (the log of partition coefficient), which are used for not ionisable compounds. It should be considered that ionization affects compounds to act more soluble in water than the structure appears to be. Acidic compounds have an increasing degree of protonation at higher pH value (at basic conditions) and lower the log D value. By increasing the alkyl side-chain length, we expect an increase in the lipophilicity. The Lipinski’s rule of five suggests that the log D should be less than 5, to attain good bioavailability. All of the synthesized compounds have a log Doct7.4 value within 0-5. However, amphoteric compounds are generally expected to have low bioavailability caused by reduced lipophilicity (46). More pharmacokinetic studies are needed to confirm anything.

4.2. Interpretation of pharmacological data

4.2.1. Analysis of binding curves for synthesised compounds

The synthesised ligands were tested in a competitive binding assay performed on stably transfected HEK293 (Human embryonic kidney) cells expressing h5-HT4(b) receptors (cell line 4b40), displacing the radioligand [3H]GR113808. The binding data was analysed by a non- linear regression method in Microsoft® Excel with the Solver add-in to determine IC50 values for the respective ligands, and the curves were created by the program GraphPad Prism 5.0.

The below equation was used to convert the respective IC50 values to Ki (and Kb) values (50).

The Ki value was analysed in the Cheng-Prusoff equation (51):

Ki refers to as an affinity constant of the unlabelled competitor for the receptor.

IC50 refers to the concentration of unlabelled ligand that

inhibits the binding of the radioactive ligand by 50 %. ( ) L refers to the concentration of the radiolabeled ligand

used.

Kd refers as an affinity constant for the radiolabeled

ligand.

As described in Section 6.2.1., we determined the affinities of the ligands by using constant concentration of the radiolabeled ligand 0.3 nM [3H]GR113808 and displaced this by increasing the concentration of competing ligand. Tritium-labelled GR113808 is a common used radioligand to characterize novel 5-HT4 receptor antagonists in vitro and is specific for the 5-HT4 receptor (27). The lead compounds GR113808 (Figure 6a) and SB207266 (Figure 6b) were included in all experiments (52). All of the experiments were prepared on membranes from the 4b40 cell line, which express 5-HT4(b) receptors at high levels, so maximum receptor binding could be determined. Only the ligands with highest purity in HPLC and NMR were selected for a pharmacological evaluation. The evaluated compounds with their respective pKi values are presented in Table 3 in Appendix A.

120 GR113808 DDL-6001 5 100 6 7 8 80 13 14 15 16 60 17 18 19 40 26 27 28 20 29 30

31 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 32 [ 33

0 -12 -10 -8 -6 -4 [antagonist] M Figure 14: Competitive binding curves of 0.3 nM [3H]GR113808 by all ligands and lead compounds at the h5-HT4(b) receptor (cell line 4b40).

10.0

9.5

9.0

i 8.5 pK 8.0

7.5 7 6 5 5 6 7 8 13 14 15 16 17 18 19 26 27 28 29 30 31 32 33

DDL6001 GR113808 Ligand

Figure 15: The affinity constant (pKi) for all ligands and lead compounds from the competitive binding analysis, presented in a box-plot (an = 2-6).

In figure 15 we see a large variation (about a shift in the log unit) in the estimated pKi values for the respective ligands and lead compounds in the binding assay. The pKi values were estimated by combining all the binding data, where the respective ligands were compared to

the respective DDL-6001, so we could exclude external variables. Even if the respective IC50 values for each experiment will differ, the respective pKi-values should theoretically be identical for all experiment. But we expect that the pKi-value will confidently differ by methodological considerations (e.g. different membrane preparation dilutions from stock).

Too high receptor density could give false prediction of Ki for the radioligand 3 ([ H]GR113808). Previous studies for GR113808 report pKi = 10.13 + 0.07 (46) and pKi of 9.5 in guinea-pig striatum (53), and determined to be 9.56 + 0.19 in this assay (Table 3 in

Appendix A). SB207266 (DDL-6001) is reported to have an approximately equal affinity (pKi

= 10.28 + 0.15) to the 5-HT4 receptor (46), and is determined to be 9.43 + 0.28 in this assay (Table 3 in Appendix A). Thus, we expect that the estimated affinities for the respective compound to be comparable to the reference drugs and should give reliable information worth discussing. The purpose of the displacement assay was to reveal whether the ligands had affinities equal or higher than the lead compounds GR113808 and SB207266.

Displacement curves for the indole-3-carboxylic acid sulfonamides (h5-HT4(b) receptor)

120 GR113808 DDL-6001 100 5 6 7 80 8

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 16: Competitive binding curves (0.3 nM [3H]GR113808 present) for the indole derivatives 5-8 compared to the references.

The displacement curves for the indole-3-carboxylic acid derivatives 5-8 are presented in Figure 16. In this particular assay, we see that all four derivatives of indole did not fully displace the radioligand at the concentrations used in our experiments. There are only a marginal difference seen for the cyclohexane derivative 7 (pKi = 6.27 + 0.28), the n-butyl derivate 6 (pKi = 6.29 + 0.33), the methyl derivative 5 (pKi = 5.99 + 0.29) and the benzyl derivative 8 (pKi = 5.95 + 0.30), and all showed consistently lower affinity for the 5-HT4 receptor than the lead compounds (pKi = 3.5 log units lower in Figure 15). Derivative 5-8 were not selected for the adenylyl cyclase evaluation.

Displacement curves for the 1,4-Benzodioxane-5-carboxylic acid sulfonamides

(h5-HT4(b) receptor)

120

GR113808 100 DDL-6001 13 14 80 15 16 17 60 18 19

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 17: Competitive binding curves (0.3 nM [3H]GR113808 present) for the benzodioxan derivatives 13-19 compared to the references.

The displacement profile for all of the 1,4-Benzodioxane-5-carboxylic acid derivatives 13-19 are presented in Figure 17. In this particular assay, the 4-(trifluoromethyl)benzyl derivative 19

(pKi = 8.80 + 0.62) and the cyclohexane derivative 16 (pKi = 8.87 + 0.58) performs the highest affinity for the h5-HT4(b) receptor, as good as the lead compounds. The average

estimated pKi values for 16 and 19 seems to be inconsistent with the result in Figure 17, hence we can not conclude which one has the highest affinity for the 5-HT4 receptor. The p-tolyl derivative 18 (pKi = 8.61 + 0.46) and the n-butyl derivative 14 (pKi = 8.62 + 0.16) perform high-affinity to the receptor, but not as good as the lead compounds. The cyclohexane derivative 16 (pKi = 8.87 + 0.58) has a higher receptor binding affinity than the benzyl derivative 17 (pKi = 8.26 + 0.42). The compound with the lowest affinity is the methyl derivative 13 (pKi = 8.52 + 0.07) and the isobutyl derivative 15 (pKi = 8.08 + 0.32), which show a similar binding curve in Figure 17.

Displacement curves for piboserod sulfonamides (h5-HT4(b) receptor)

120 GR113808 DDL-6001 26 100 27 28 29 80 30 31 32 60 33

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 18: Competitive binding curves (0.3 nM [3H]GR113808 present) for the 3,4-Dihydro-2H- [1,3]oxazino[3,2-a]indole-10-carboxylic acid (piboserod) derivatives 26-33 compared to the references.

Figure 18 shows the binding curves for the Piboserod derivatives 26-33. The respective assay shows that the n-butyl derivative 28 (pKi = 8.95 + 0.59) and the ethyl derivative 27 (pKi =

9.02 + 0.38) performs the highest affinity for the 5-HT4(b) receptor, or as good as the lead

compounds. Uncertainty is set to the benzyl derivative 31 with the estimated pKi value 9.12 + 0.36 and the binding curve presented in Figure 18. However, more data should be obtained to verify the potential high-affinity of 27, 28 and 31 for the 5-HT4 receptor. A notable reduction in the affinity for the receptor is seen for the 4-(trifluoromethyl)benzyl derivative 33 (pKi =

8.15 + 0.26) and the p-tolyl derivative 32 (pKi = 7.69 + 0.28), where 32 has the lowest affinity.

A comparable affinity is seen in Figure 18 for the methyl derivative 26 (pKi = 8.62 + 0.42), the ethyl derivative 27, the isobutyl derivative 29 (pKi = 8.36 + 0.38) and the cyclohexane derivative 30 (pKi = 8.69 + 0.54) and performs equally good binding as SB207266.

4.2.2. Analysis of adenylyl cyclase curves for selected compounds

We determined the 5-HT4(b) receptor antagonistic response for selected ligand, with respectively high receptor affinity and promising binding property in the analysis of the binding curves in Section 4.2.1., by comparing their ability to inhibit 5-HT (1µM)-stimulated adenylyl cyclase (AC) activity. Unfortunately, due to methodological issues, only the results from piboserod derivatives and compound 13 of benzodioxan sulfonamide derivative were obtained for the analysis. Out of six assays performed, only 3 were succeeded with good results. This was due to methodological issues with 5-HT (1µM), RS (regeneration system) and diluted membrane preparations. This was solved by doing a control experiment with new and old RS, 5-HT used in the initial experiments, forskolin (a direct activator of AC) and basal AC activity (cell line 4b13). The results showed clearly that the 5-HT was not optimal, but RS was working. So we made new 5-HT (1µM) in 10mM HCl, instead of concentrated HCl which seemed to lower the pH below the optimal for AC assays. The membrane preparations were not re-used, but we made sure to completely dissolve it in cold TE-buffer.

The AC assay was performed in HEK293 cells stably transfected with h5-HT4(b) receptors from the cell line 4b40 and 4b13 expressing high and low receptor levels, respectively. The cell line 4b13 expressing h5-HT4(b) receptor at low levels were used in the AC assay to increase the inhibition sensitivity of the method. The cell line 4b40, expressing higher receptor levels, were used in all experiments to validate if any of the ligands performed an inverse agonist effect in the absence of 5-HT (1µM), as described in Section 4.2.3.

The Kb values (also referred as Kd) were obtained by the same method as the pKi, described in Section 4.2.1, using the equation below (51):

a refers to the IC50 (antagonist concentration at ( ) ( ) 50% of maximal response) value of the

antagonist.

b refers to the EC50 (agonist concentration at 50% of maximal response) value of the agonist. x refers to the concentration of agonist.

The estimated pKb values of selected compounds 13, 26-31 and 33 from inhibition of AC activity are given in Figure 20, and the curves are presented in Figure 19.

GR113808 100 DDL 6001 13 26 80 27 28 29 30 60 31 33 40

cAMP formed (relative) formed cAMP 20

-12 -10 -8 -6 -4 -2 [Antagonist] M

Figure 19: Concentration-response curves of the inhibition of 5-HT (1 µM)-stimulated adenylyl

cyclase activity for selected ligands, via h5-HT4(b) receptor (cell line 4b13).

10 pK

13 22 23 24 26 27 30

GR113808DDL-6001

Figure 20: The pKb-values for selected ligands and references from the AC assay, presented in a box-plot (bn= 2-3).

The EC50 value of the agonist (serotonin, 5-HT) was measured in each experiment to obtain a correct estimation for the concentration used, and is a critical point for the AC assay. The 5-

HT (1 µM) mediated stimulation of AC activity for h5-HT4(b) receptor (cell line 4b13) is shown in Figure 21.

150 300 125 250 100 200 75 150

100 50 cAMP formed cAMP

50 (relative) formed cAMP 25 (pmol/mg pr protein/min) pr (pmol/mg 0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 [agonist] M [agonist] M

Figure 21a Figure 21b

Figure 21: Concentration-response curves of the 5-HT (1 µM) stimulation of adenylyl cyclase

activity, via h5-5HT4(b) receptors (cell line 4b13), presented in a) pmol cAMP/mg protein/min and b) relative. The basal AC activity was 56.76 pmol cAMP/mg protein/min and the maximum was 223.4 -08 pmol cAMP/mg protein/min. Respective pEC50 is 7.60 (EC50 = 2.48E M).

Adenylyl cyclase data for the piboserod sulfonamides

A C

100 100

80 80

60 60

40 40 GR113808 GR113808 20 DDL 6001 20 DDL 6001

26 29 cAMP formed (relative) formed cAMP 0 (relative) formed cAMP 0

-12 -10 -8 -6 -4 -12 -10 -8 -6 -4 [Antagonist] M [Antagonist] M

B D

100 100

80 80

60 60 40 GR113808 40 DDL 6001 GR113808 20 DDL 6001 28 20

33 cAMP formed (relative) formed cAMP 0 (relative) formed cAMP 0

-12 -10 -8 -6 -4 -12 -10 -8 -6 -4 [Antagonist] M [Antagonist] M

Figure 22: Concentration-response curves of the inhibition of 5-HT (1 µM)-stimulated adenylyl cyclase activity of the methyl derivative 26 (panel A), the n-butyl derivative 28 (panel B), the isobutyl derivative 29 (panel C) and the 4-(trifluoromethyl)benzyl derivative 33 (panel D), via h5-

HT4(b) receptor (cell line 4b13).

Figure 22 displays the inhibition activity on AC for the methyl derivative 26 (panel A, pKb =

9.35 + 0.04), the n-butyl derivative 28 (panel B, pKb = 9.67), the isobutyl derivative 29 (panel

C, pKb = 9.65 ± 0.25) and the 4-(trifluoromethyl)benzyl derivative 33 (panel D, pKb = 9.85). All compounds fully antagonize the AC activity, but with lower potencies than the reference compounds.

100

60 40

GR113808 20 DDL 6001 cAMP formed (relative) formed cAMP 27 0

-12 -10 -8 -6 -4 [Antagonist] M

Figure 23: Concentration-response curves of the inhibition of 5-HT (1 µM)-stimulated adenylyl cyclase activity of the ethyl derivative 27 (panel E), via h5-HT4(b) receptor (cell line 4b13).

G F

100 100 80 80 60 60 40 40 GR113808 GR113808 20 DDL 6001 20 DDL 6001

cAMP formed (relative) formed cAMP 31

cAMP formed (relative) formed cAMP 30 0 0

-12 -10 -8 -6 -4 -12 -10 -8 -6 -4 -2 [Antagonist] M [Antagonist] M Figure 24: Concentration-response curves of the inhibition of 5-HT (1 µM)-stimulated adenylyl cyclase activity of the cyclohexane derivative 30 (panel F) and the benzyl derivative 31 (panel G), via h-5HT4(b) receptor (cell line 4b13).

Figure 23 presents the inhibition activity on AC for the ethyl derivative 27 (panel E). In the respective assay, 27 (pKb = 9.99) seem to be a more potent antagonist than GR113808 (pKb =

9.71). The average estimated pKb value for GR113808 is hence 10.13 ± 0.22. 27 is the only derivative tested to perform equal-to-better antagonist response as GR113808, and remains as a poorer antagonist than SB207266. However, more data is needed to confirm the potential

good antagonist response. In Figure 24 the inhibition curve for the cyclohexane derivative 30 (panel F) and the benzyl derivative 31 (panel G) are presented. The cyclohexane derivative 30

(pKb = 9.78 + 0.16) and the benzyl derivative 31 (pKb = 9.66) are equally good antagonist as the GR113808, but remains as poorer antagonists than SB207266. It seems like the compounds antagonize the 5-HT4 receptor regardless of the side-chain substituent.

4.2.3. Analysis of inverse agonist effect

The results from inverse agonist control test done parallel to the AC assay are presented in Figure 25. The relative values for the ligands are compared to the basal AC activity response (in per cent of basal AC activity). The selected ligands showed consistently lower AC response than the basal AC activity and hence appears as inverse agonists for the h5-HT4(b) receptor. However, more studies should be obtained to confirm whether or not the ligands are inverse agonists. The observations did not affect the AC assay performed.

100

AC activity (% of basal) of (% activity AC 0

13 22 23 24 26 27 28 30

GR113808DDL-6001

Figure 25: Results for the inverse agonist control test for selected ligands and reference compounds, presented in a box-plot, via h5-HT4(b) receptor (cell line 4b40).

4.3. Structure-affinity relationship (SAFIR)

The structure-affinity relationships for the N-aryl sulfonamides will be evaluated based on the chemical and pharmacological analysis of the data. By incorporating various side-chain groups to the aryl sulfonamide, we want to reveal their influence on the 5-HT4 receptor affinity and antagonist effect. Introducing various side-chain groups on the 5-HT4 receptor ligands, is earlier described to not affect the hydrophobic pocket and 5-HT4 receptor affinity is expected to be obtained (41). But this seems to be inconsistent with the results in this Thesis.

In the lead modification and optimisation of our compounds, following alterations were done. Amide linker was chosen as a hydrogen bond acceptor to obtain good oral bioavailability for our ligands; due to that previous 5-HT4 receptor antagonist with ester functionalities have shown limited bioavailability (39). Selectivity for the 5-HT4 receptor was obtained by incorporating 4-piperidine group to the aromatic amide group and linking voluminous substituents to the N-position, as described in previous studies (38, 42, 54). The N-position of the 4-piperidine ring was further incorporated with an acidic aryl sulfonamide, to attain inhibitory effect on hERG potassium channel binding, for our ligands. The functional aryl sulfonamide group is expected to be ionized at physiological pH (7.4) and will contribute to increased polarity and solubility. In previous article (44) strong inhibitory effect on hERG channel binding was seen by incorporating carboxylic acid groups into various sites of benzamidine analogues. We have replaced the carboxylic acids (pKa~5-6) with the acidic N- aryl sulfonamides (pKa~6-8).

This will give us novel aryl N-sulfonamide 5-HT4 receptor antagonist with reduced affinity towards the hERG potassium channel, thus reduced risk of serious side-effects as TdP, and less CNS adverse effects, due to reduced passage across BBB. However, amphoteric compounds are expected to have low bioavailability (46), which may be modified by adding substituents to the N-aryl sulfonamides with various side-chain length. The influences of the various incorporated side-chain groups (R-groups) will be considered in the following section.

4.3.1. Comparing derivatives with same sulfonamide side-chain (R-) group.

The following three aromatic ring systems were compared:

 Indole-3-carboxylic acid derivatives

 1,4-Benzodioxane-5-carboxylic acid derivatives

 3,4-Dihydro-2H-[1,3]oxazino [3,2-a]indole-10-carboxylic acid (Piboserod) derivatives

120

5 100 13

80 26

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 26: Comparing R= Methyl (-CH3) of aryl sulfonamide derivatives based on different aromatic moieties of the indole derivative 5, the benzodioxan derivative 13 and the piboserod derivative 26.

Figure 26 shows the affinity for the 5-HT4(b) receptor regardless of the methyl side-chain group for the indole, benzodioxan and piboserod aromatic moieties. The piboserod derivative

26 (pKi = 8.62 + 0.42) has a higher receptor affinity than the benzodioxan derivative 13 (pKi =

8.52 + 0.07). The indole derivative 5 (pKi = 5.99 + 0.29) has the lowest affinity and does not

completely displace the radioligand at this concentration. The AC data for the benzodioxan derivative 13 (pKb = 9.33 + 0.17) and the piboserod derivative 26 (pKb = 9.35 + 0.04) shows that they have promising antagonist effect for the h5-HT4(b) receptor.

120

100 6 14

80 28

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 27: Comparing R= n-butyl ( ) of aryl sulfonamide derivatives based on different aromatic moieties of the indole derivative 6, the benzodioxan derivative 14 and the piboserod derivative 28.

Figure 27 shows a similar binding profile as Figure 26. Regardless of similar n-butyl side- chain group, the piboserod aromatic derivative 28 (pKi = 8.95 ± 0.59) has a higher affinity than the benzodioxan derivative 14 (pKi = 8.62 ± 0.16). The indole aromatic derivate 6 (pKi =

6.29 ± 0.33) has the lowest affinity for the 5-HT4 receptor. AC assay was performed for the n- butyl derivative of piboserod 28 (pKb = 9.67) and shows good antagonistic effect for the 5-

HT4 receptor.

120

100 15 80 29

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 28: Comparing R= isobutyl ( ) of aryl sulfonamide derivatives based on different aromatic moieties of the benzodioxan derivative 15 and the piboserod derivative 29.

Figure 28 shows the receptor affinity for the benzodioxan derivative 15 and piboserod derivative 29 with the same isobutyl substituent. The piboserod derivative 29 (pKi = 8.36 +

0.38) performs higher affinity for the 5-HT4 receptor than the benzodioxan derivate 15 (pKi =

8.08 ± 0.32). AC assay performed for 29 (pKb = 9.65 ± 0.25) reveals good antagonist response.

120

100 7 16 80 30

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 29: Comparing R= cyclohexane ( ) of aryl sulfonamide derivatives based on different aromatic moieties of the indole derivative 7, the benzodioxan derivative 16 and the piboserod derivative 30.

Figure 29 shows that the cyclohexane substituent of the benzodioxan derivative 16 (pKi =

8.87 ± 0.58) and the piboserod derivative 30 (pKi = 8.69 ± 0.54) have an equal-to-small difference in receptor binding profile. The indole derivative 7 (pKi = 6.27 ± 0.28) has the lowest affinity for the 5-HT4 receptor. In AC assay the piboserod derivative 30 (pKb = 9.78 ±

0.16) reveals good antagonist response for the 5-HT4 receptor.

120

100 8 17 80 31

20 H]GR113808 bound (% of maximum) of (% bound H]GR113808

3 0 [

0 -12 -10 -8 -6 -4 [antagonist] M

Figure 30: Comparing R= benzyl ( ) of aryl sulfonamide derivatives based on different aromatic moieties of the indole derivative 8, the benzodioxan derivative 17 and the piboserod derivative 31.

Figure 30 shows a different binding profile than earlier seen in Figure 26-29. There seems to be a trend of benzyl side-chain group affecting the binding property of the 5-HT4 receptor.

The benzodioxan derivative 17 (pKi = 8.26 ± 0.42) has a much higher receptor affinity than the piboserod derivative 31 (pKi = 8.04) in the particular assay. Although, the estimated average pKi value for 31 is reported to be 9.12 ± 0.36. This can be explained by methodological differences (e.g. different membrane dilutions from stock). AC assay performed for 31 (pKb = 9.66) shows a good antagonist property. The indole derivative 8 has still the lowest affinity (pKi = 5.95 ± 0.30).

Based on the results from Figure 26, 27 and 28, we see a noticeable difference in the binding affinities for the new compounds based on the different aromatic ring systems. The influence of the side-chain substituents is hardly seen in the results. The trend seems to be that the piboserod derivatives has predominantly higher affinity to the 5-HT4 receptor than the

benzodioxan derivatives and the indole derivatives. Since the only difference is in the chemical structure of the aromatic ring system, this seems to be a contributing factor for the observed difference in the receptor affinity. The aromatic moiety was earlier describes to be a part of the pharmacophore model of the 5-HT4 receptor (Figure 1). The indole-3-carboxylic acid scaffold is earlier described to have poorer affinity for the 5-HT4 receptor compared to the benzodioxan and piboserod scaffold (39). The data can be explained by the favourable replacement of the hydrogen bond donor NH- in the indole ring by oxygen atoms, that can act as a hydrogen-bond acceptor in the oxazino[3,2-a] ring of piboserod and in the benzodioxan ring.

Figure 29 and 30 present inconsistent results from Figure 26-28. Introducing benzyl or cyclohexane as side-chain substituents seems to affect the affinity for the 5-HT4 receptor.

Replacement of the cyclohexane substituent in 30 (pKi = 8.69 ± 0.54, pKb = 9.78 ± 0.16) to a benzyl substituent in 31(pKi = 9.12 ± 0.36, pKb = 9.66) for the piboserod aromatic derivatives, gave a small increase in the affinity and a marginal-to-no reduction in the antagonist potency.

For the benzodioxan derivatives, the replacement of the cyclohexane substituent in 16 (pKi =

8.87 ± 0.58) to a benzyl substituent 17 (pKi = 8.26 ± 0.42) results in a marginal-to-no reduction in the affinity.

We expected an increase in the affinity by increasing the length of the side-chain substituents to the N-aryl sulfonamides (42). But there seems to be a trend of altered affinity by incorporating voluminous substituents. Perhaps the side –chain group could be a determinant part of holding the integrity of the pharmacophore (e.g. part of the auxophore), by being stabilised by the hydrophobic pocket, and is then expected to influence the bioactive conformation3 (40).Thus, we will further compare the side-chain derivatives with increasing hydrophobicity, to reveals if the 5-HT4 receptor affinity if affected by the influence of voluminous substituents to the N-aryl sulfonamides.

3 The bioactive conformation is the conformation of the ligand that most effectively binds to the receptor.

4.3.2. Comparing derivatives with increasing side-chain length.

7.0

6.5

6.0

5.5

5.0

5 6 7 8 (C1) (C4) (C6) (C7)

Figure 31: Ranging pKi values for the respective indole sulfonamide derivatives by increasing side- chain length (hydrophobicity).

10.0 9.5 9.0 8.5 8.0 7.5 7.0

13 14 15 16 17 18 19 (C1) (C4) (C4) (C6) (C7) (C7) (C8)

Figure 32: Ranging pKi values for the respective benzodioxan sulfonamide derivatives by increasing side-chain length (hydrophobicity).

11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0

26 27 28 29 30 31 32 33 (C1) (C2) (C4) (C4) (C6) (C7) (C7) (C8)

Figure 33: Ranging pKi values for the respective piboserod sulfonamide by increasing side-chain length (hydrophobicity).

In Figure 31-33 the pKi values for the aryl sulfonamide side-chain groups were aligned with increasing side-chain length and were plotted in Figure 16-18 based on the diverse aromatic scaffolds. For all the compounds, we only see a marginal difference in the obtained pKi values. The difference is more clearly viewed in Figure 16-18, but attention should be made since this is based on one particular assay. But there seems to be a trend of decrease in affinity for the piboserod derivatives by increasing the voluminous side-chain substituents (> 7 carbon atoms). However, the difference is too small to make any conclusion, so more data need to be attained. We assume that the binding property to the 5-HT4(b) receptor are unaffected by the increase in side-chain length for the aryl sulfonamide derivatives in this Thesis. A further increase in the side-chain length is also expected to cause some pharmacokinetic issues (e.g. absorption limitations).

Bioisoteric replacement of the n-butyl derivative with the isobutyl derivative, and of the cyclohexane derivative with the benzyl derivative, will be considered. Replacement of the straight chain n-butyl derivative with a branched isobutyl derivative seems to affect the 5-HT4 receptor affinity. The n-butyl derivatives 14 (pKi = 8.62 ± 0.16) and 28 (pKi = 8.95 ± 0.59) has a marginally higher affinity than the corresponding isobutyl derivatives 15 (pKi = 8.08 ±

0.32) and 29 (pKi = 8.36 ± 0.38). The pharmacokinetic attempted explanation for this is that chain branching is expected to lower the lipophilicity of the compound, than the corresponding straight alkyl chain. This is explained by that branched groups are bulky (larger molar volumes and shapes), so more water repellent (40). Another pharmacodynamics explanation for this is that the branching interferes with the hydrophobic pocket and the receptor binding property. Hence, this seems to not affect the antagonist response reported for n-butyl derivative 28 (pKb = 9.67) and the isobutyl derivative 29 (pKb = 9.65 ± 0.25). Replacement of the cyclohexane substituent with a benzyl substituent seems to reduce the affinity for the indole aromatic scaffold, unaffected the benzodioxan aromatic scaffold and increase the affinity for the piboserod scaffold. This can be explained by that the π-electrons in the benzyl substituent seem to affect the receptor affinity for the various aromatic rings.

Comparing the influence of the p-tolyl ( ) and the 4-(trifluoromethyl)benzyl

( ) substituents to the 5-HT4 receptor affinity, could perhaps reveal anything.

4.3.3. Comparing electron donating p-tolyl derivatives and electron withdrawing 4- (trifluoromethyl)benzyl derivatives as sulfonamide side-chain groups.

120 120

32 100 18 100 33 19 80 80

60 60

40 40

20 20

H]GR113808 bound (% of maximum) of (% bound H]GR113808

H]GR113808 bound (% of maximum) of (% bound H]GR113808 3

3 0 0

[ [

0 -12 -10 -8 -6 -4 0 -12 -10 -8 -6 -4 [antagonist] M [antagonist] M

Figure 34a Figure 34b

Figure 34: Comparing a) electron donating p-tolyl derivative 18 (pKi = 8.61 ± 0.46) and electron

withdrawing 4-(trifluoromethyl)benzyl derivative 19 (pKi = 8.80 ± 0.62) for the benzodioxan

aromatic ring versus b) electron donating p-tolyl derivative 32 (pKi= 7.69 ± 0.28) and electron

withdrawing 4-(trifluoromethyl)benzyl derivative 33 (pKi = 8.15 ± 0.26, pKb= 9.85) for the piboserod aromatic ring.

In Figure 34 the respective electron donating p-tolyl derivatives 18 and 32 or the respective electron withdrawing 4-(trifluoromethyl)benzyl derivatives 19 and 33 were compared within the same aromatic ring. The 4-(trifluoromethyl)benzyl derivative 19 (pKi = 8.80 ± 0.62) has a slightly higher affinity than the p-tolyl derivative 18 (pKi = 8.61 ± 0.46) for the benzodioxan sulfonamides. The 4-(trifluoromethyl)benzyl derivative 33 (pKi = 8.15 ± 0.26) has a higher affinity than p-tolyl derivative 32 (pKi = 7.69 ± 0.28) for the piboserod sulfonamides. In Figure 34b a higher difference in the affinity constant is noted for compound 32 and 33, than compound 18 and 19, which seem to by unaffected. The trend seems to be that the electron withdrawing 4-(trifluoromethyl)benzyl substituent seems to perform a higher affinity, than the electron donating p-tolyl substituent. This can be explained by an influence on the acidic property of the aryl sulfonamide group. The 4-(trifluoromethyl)benzyl group is electron withdrawing group that “withdraws” the electrons from the aryl sulfonamide group, making the -N–H bound weaker and more liable to be deprotonated. Hence the acidic property of the sulfonamide is expected to increase.

5. CONCLUSION

In this Thesis, structure-affinity relationships were evaluated for the following synthesised 19 novel N-aryl sulfonamides: Indole-3-carboxylic acid derivatives 5-8, 1,4-Benzodioxane-5- carboxylic acid derivatives 13-19, and 3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10- carboxylic acid (Piboserod) derivatives 26-33 (Table 3). The compounds were characterized 1 13 by H/ C-NMR spectroscopy, determined by HPLC analysis and the log Doct7.4, and pharmacological evaluated to determine specific affinity for the h5-HT4(b) receptor in a radioligand binding assay and antagonist response in an adenylyl cyclase assay.

There seems to be a trend that the piboserod sulfonamides have higher affinity for the 5-HT4 receptor, and with promising antagonist response, compared to the benzodioxan sulfonamides.

The indole sulfonamides showed consistently lower affinity for the 5-HT4 receptor. This could be explained by the favourable replacement of the hydrogen bond donor NH- in the indole ring by oxygen atoms, that can act as a hydrogen-bond acceptor in the oxazino[3,2-a] ring of piboserod and in the benzodioxan ring. We evaluated the influence of incorporating various side-chain substituents to the N-aryl sulfonamide for the 5-HT4 receptor affinity. However, increasing the side-chain length (hydrophobicity) could not reveal any significant difference in the estimated affinity constants (pKi values), so we assume that the binding property to the 5-HT4(b) receptor is unaffected by an increase in the side-chain length for N- aryl sulfonamides in this Thesis. We also evaluated the effect of introducing an electron withdrawing 4-(trifluoromethyl)benzyl group or an electron donating p-tolyl group. Even though the electron withdrawing group seems to be more favourable than the electron donating group, they both seem to reduce the affinity for the 5-HT4 receptor compared to the other substituents.

Further studies should be initiated to reveal any influence on the hydrophobic pocket and to the affinity for the 5-HT4 receptor, by using various substituents on N-aryl sulphonamides. The hERG potassium channel affinity of the novel acidic N-aryl sulphonamide should be examined in future, for the potential use of 5-HT4 receptor antagonists in the treatment of HF or arrhythmia with reduced risk of QT prolongation and serious side-effects (as TdP).

6. Materials and methods

6.1. Experimental chemistry (46)

1H NMR spectra were recorded on a Bruker Spectrospin Avance spectrometer at 200, 300 and 400 MHz for 1H and 50, 75 and 100 MHz for 13C. Chemical shifts are reported in parts per million relative to residual CHCl3 (δH = 7.26 ppm, δC = 77.0 ppm) or CHD2SOCD3 (δH = 2.50 ppm, δC = 39.43 ppm). Coupling constants (J) are reported in hertz (Hz). The following abbreviations are used to describe peak patterns when appropriate: s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), m (multiplet) and b (broad). Analytical thin-layer chromatography (TLC) was run on Merck silica gel plates (Kieselgel 60 F-254) with UV-light or iodine detection. For flash chromatography, Fluka silica gel type 60 (size 200-400 mesh) was used. All solvents and reagents were of analytical or reagent grade and were obtained from commercial sources.

The distribution coefficients (log Doct7.4) were determined for the new compounds (5 mg of analyte) in a mixture of aqueous 1M phosphate buffer with pH 7.4 (1.0 ml) and n-octanol (1.0 ml) at ambient temperature. The mixture was vigorously shaken, allowed to reach equilibrium for 3 days at room temperature before 0.1 ml of the aqueous and n-octanol solution was removed. Sampled were taken out from the two solutions. Concentration of the respective compounds was determined by HPLC and presented in Table 3 in Appendix A.

6.1.1. General procedures for preparations of intermediates 20 and 21.

Methyl 2-(2-chloroethoxy)-1H-indole-3-carboxylate (20). A suspension of methyl indole-3- carboxylate (5.25 g, 30.0 mmol) and DABCO (1.84 g, 16.4 mmol) in dry CH2Cl2 (25 ml) was cooled to 0 ºC under argon atmosphere, treated in one portion with N-chlorosuccinimide (4.41 g, 33.0 mmol) and the mixture stirred for 10 min. The resulting solution was added to a solution of 3-chloropropan-1-ol (3.12 g, 33.0 mmol) in dry CH2Cl2 (25 ml) containing anhydrous methane sulfonic acid (0.23 ml). The resulting suspension was stirred for 30 minutes and then washed with 10 % aqueous Na2CO3 solution (3 x 25 ml). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The resulting oil was triturated

with toluene (10 ml) at 0 °C for 1 h and the solid precipitate filtered, washed with a small amount of toluene and dried in vacuo to leave the title compound 20 as an off-white solid 1 (5,22 g, 65 %). H NMR (CDCl3):  9.51 (s, 1H), 8.04 (d, 1H), 7.28‒7.14 (m, 3H), 4.49 (t, 2H), 3.96 (s, 3H), 3.67 (t, 2H), 2.18‒2.10 (m, 2H).

Methyl 3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxylate (21). Methyl 2-(3- chloropropoxy)indole-3-carboxylate (5.0 g, 18.7 mmol) was added to a stirred mixture of 5.4 M aqueous NaOH (3.8 ml) and toluene (50 ml) and heated at 40 ºC for 4 h. The aqueous layer was separated and the organic layer washed with H2O (3 x 25 ml) while maintaining the temperature at 60 ºC. The organic solvent was evaporated in vacuo to leave the product as a 1 white solid (4.0 g, 93.2 %). H-NMR (DMSO-d6):  7.88‒ 7.83 (m, 1H), 7.30‒7.26 (m, 1H), 7.14‒7.04 (m, 2H), 4.50 (t, J = 5.3 Hz, 2H), 4.10 (t, J = 6.1 Hz, 2H), 3.71 (s, 3H), 2.29‒2.18 (m, 2H).

6.1.2. General procedures for preparation of benzyl protected piperidine amine intermediates (1, 9, 22). N-[(1-benzyl-4-piperidinyl)methyl]-1H-indole-3-carboxamide (1). Oxalyl chloride (1.84 ml, 20.7 mmol) and DMF (one drop) was added to a stirred suspension of indole-3-carboxylic acid (2.90 g, 18.0 mmol) in CH2Cl2 (75 ml) and stirred at room temperature for 2 h, then concentrated in vacuo to leave the acid chloride as a yellow solid. This was dissolved in a mixture of CH2Cl2 (30 ml) and THF (10 ml) and added dropwise to a stirred solution of (1- benzyl-4-piperidinyl)methanamine (3.07 g, 15.0 mmol) and NEt3 (1.82 g, 18.0 mmol) in

CH2Cl2 (30 ml). The reaction mixture was stirred at room temperature overnight, treated with brine (25 ml) and 10% aqueous NaHCO3 solution (25 ml). The organic layer was dried over anhydrous NaSO4, filtered and evaporated to leave the title compound as a pale yellow solid 1 (3.80 g, 73%). H NMR (CDCl3): δ 9.96 (s, 1H), 7.97‒7.95 (m, 1H), 7.67 (d, J = 2.8 Hz, 1H), 7.44‒7.22 (m, 8H), 6.24 (t, J = 5.9 Hz, 1H), 3.51 (s, 2H), 3.40 (t, J = 5.9 Hz, 2H), 2.94‒2.91 (m, 2H), 1.98 (t, J = 10.9 Hz, 2 H), 1.78‒1.67 (m, 3H), 1.44‒1.30 (m, 2H).

N-[(1-benzyl-4-piperidinyl)methyl]-2,3-dihydro-1,4-benzodioxine-5-carboxamide (9). A solution of 1,4-benzodioxan-5-carboxylic acid (1.80g, 10.0 mmol) in CH2Cl2 (25 ml) was

added N,N-carbonyldiimidazole (1.62 g. 10.0 mmol) and stirred at room temperature for 1 h, then (1-benzyl-4-piperidinyl)methanamine (2.04 g, 10.0 mmol) was added and the reaction heated to 60°C overnight. The resulting reaction mixture was cooled to room temperature, washed with saturated aqueous NaHCO3 solution (2 x 20 ml) and water (20 ml). The organic layer was dried over anhydrous NaSO4, filtered and evaporated in vacuo to leave the title 1 compound 9 as white solid. H NMR (CDCl3): δ 8.03 (t, J = 5.7 Hz, 7.30 ‒7.15 (m, 6H), 6.99‒ 6.80 (m, 2H), 4.32‒4.23 (m, 4H), 3.41 (s, 2H), 3.13 (t, J = 6.3 Hz, 2H), 2.80‒2.75 (m, 2H), 1.92‒1.81 (m, 2H), 1.65‒1.45 (m, 3H), 1.26‒1.14 (m, 2H).

N-[(1-benzyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10- carboxamide (22). Trimethylaluminium (2 M in toluene, 9 ml) was diluted with dry toluene (9 ml) and the solution cooled to 0 ºC under argon atmosphere. 1-Benzyl-4- aminomethylpiperidine (3.37 g, 16.5 mmol) was added to the solution, followed by methyl ester 21 (3.81 g, 16.5 mmol). The reaction mixture was heated under reflux for 5 h, cooled to room temperature and 10 % aqueous NaOH solution (40 ml) dropwise added. The toluene layer was washed with H2O, brine and evaporated in vacuo to give oil. The residue was purified by column chromatography (SiO2, CH2Cl2/MeOH 7:3) to leave the title compound 22 1 as an off white solid (3.52 g, 53.4 %). H NMR (DMSO- d6):  8.05‒8.02 (m, 1H), 7.32-7.21 (m, 5H), 7.09‒7.01 (m, 2H), 6.53 (t, J = 5.7 Hz, 1H), 4.54 (t, J = 4.7 Hz, 2H), 4.10 (t, J = 5.8 Hz, 2H), 3.41 (s, 3H), 3.16 (t, J = 6.1 Hz, 2H), 2.80‒2.76 (m, 2H), 2.27‒2.24 (m, 2H), 1.90‒ 1.83 (m, 2H), 1.63‒1.47 (m, 3H), 1.23‒1.16 (m, 2H).

6.1.3. General procedure for hydrogenolysis of benzyl protected amines to prepare intermediates (2, 10, 23). N-(4-piperidinylmethyl)-1H-indole-3-carboxamide (2). A solution of benzyl amine 1 (3.80 g, 11.0 mmol) in a mixture of glacial acetic acid (10 ml) and MeOH (50 ml) was hydrogenated over 20% Pd/C (0.5 g) and 5 bar at room temperature for 48 h. The reaction mixture was filtered and the filtrate made alkaline with K2CO3 to pH 11. The aqueous mixture was extracted with CH2Cl2 (3 x 50 ml) and the organic layers combined and dried over anhydrous Na2SO4, filtered and evaporated in vacuo to leave the title compound 3 as a white 1 solid (2.21 g, 81 %). H-NMR (DMSO- d6):  11.56 (bs, 1 H), 8.15‒8.12 (m, 1H), 8.03 (s, 1H), 7.85 (t, J = 5.7 Hz, 1H), 7.43‒7.40 (m, 1H), 7.15‒7.08 (m, 2H), 3.12 (t, J = 6.0 Hz, 2H),

2.94‒2.90 (m, 2H), 2.55‒2.49 (m, 1H), 2.4‒2.36 (m, 2H), 1.64‒1.60 (m, 3H), 1.06‒1.01 (m, 2H).

N-(4-piperidinylmethyl)-2,3-dihydro-1,4-benzodioxine-5-carboxamide hydrochloride (10). The hydrogenolysis product was converted to the hydrochloride salt using ethereal hydrochloride solution, and the hydrochloride salt recrystallized to leave the title compound 1 as a white solid. H NMR (DMSO-d6): δ 9.16 (bs, 1H), 8.18 (t, J = 5.9 Hz, 1H), 7.15 (dd, J = 1.8 and 7.6, 1H), 6.95‒6.92 (m, 1H), 6.86‒6.81 (m, 1H), 4.34‒4.24 (m, 4H), 3.25‒3.12 (m, 4H), 2.81‒2.71 (m, 2H), 2.79‒2.75 (m, 2H), 1.46‒1.34 (m, 2H).

N-(4-piperidinylmethyl)-3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide (23). 1 White solid. H NMR (DMSO-d6): δ 8.09‒8.05 (m, 1H), 7.31‒7.26 (m, 1H), 7.11‒7.06 (m, 2H), 6.78 (t, J = 5.9 Hz, 1H), 4.59 (t, J = 5.1 Hz, 2H), 4.14 (t, J = 5.9 Hz, 2H), 3.16 (t, J = 6.1 Hz, 2H), 2.97‒2.91 (m, 2H), 2.42‒2.30 (m, 4H), 1.62‒1.58 (m, 3H), 1.11‒1.04 (m, 2H).

6.1.4. General procedure for preparation of arylic nitro intermediates (3, 11, 24). N-[[1-[(4-nitrophenyl)methyl]-4-piperidinyl]methyl]-1H-indole-3-carboxamide (3). 4- Nitrobenzylbromide (1.86 g. 8.60 mmol) was added to a mixture of piperidine amine 2 (2.21 g,

8.60 mmol) and K2CO3 (2.37 g, 17.2 mmol) in acetone and stirred at reflux for 24 h. The reaction mixture was allowed to cool to room temperature, filtered and the filtrate evaporated in vacuo. The residue was separated with column chromatography (SiO2, CH2Cl2/MeOH 9:1) 1 to leave the title compound 3 as a white solid (2.24 g, 67 %). H NMR (DMSO-d6): δ 11.50 (s, 1H), 8.19‒8.10 (m, 3H), 8.02 (d, J = Hz, 1H), 7.87 (t, J = Hz, 1H), 7.59‒7.55 (m, J=12Hz, 2H), 7.41‒7.38 (m, 1H), 7.13‒7.07 (m, 2H), 3.57 (s, 2H), 3.33 (s, 2H), 3.14 (t, J = 9Hz, 2H), 2.81‒2.75 (m, 2H), 2.01‒1.90 (m, 2H), 1.70‒1.50 (m, 3H), 1.29‒1.19 (m, 2H).

N-[[1-[(4-nitrophenyl)methyl]-4-piperidinyl]methyl]-2,3-dihydro-1,4-benzodioxine-5- 1 carboxamide (11). White solid. H NMR (DMSO-d6): δ 8.03 (t, J = 5.7 Hz, 1H), 7.30‒7.15 (m, 5 H), 6.96‒6.80 (m, 2H), 4.32‒4.23 (m, 4H), 3.41 (s, 2H), 3.13 (t, 2H), 2.80‒2.75 (m, 2H), 1.92‒1.81 (m, 2H), 1.65‒1.45 (m, 3H), 1.26‒1.14 (m, 2H).

N-[[1-[(4-nitrophenyl)methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H-[1,3]oxazino[3,2- 1 a]indole-10-carboxamide (24).White solid. H NMR (DMSO-d6): δ 8.14 (d, J = 8.7 Hz, 2H), 8.02‒7.98 (m, 1H), 7.54 (d, J = 8.7 Hz, 2H), 4.52 (t, J = 5.1 Hz, 2H), 4.08 (t, J = 5.9 Hz, 2H), 3.28 (s, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.77‒2.72 (m, 2H), 2.26‒2.21 (m, 2H), 1.97‒1.86 (m, 2H), 1.63‒1.50 (m, 3H), 1.22‒1.14 (m, 2H).

6.1.5. General procedure for reduction to prepare arylic amine intermediates (4, 12, 25). N-[[1-[(4-aminophenyl)methyl]-4-piperidinyl]methyl]-1H-indole-3-carboxamide (4). A solution of nitro compound 3 (2.24 g, 5.76 mmol) in MeOH (25 ml) was hydrogenated over 10% Pd-C (0.20 g) and 5 bar at room temperature for 2 h. The reaction mixture was filtered and the filtrate evaporated in vacuo. The residue was separated with column chromatography

(SiO2, CH2Cl2/MeOH 7:3) to leave the title compound as a white solid (1.51 g, 73%). 1 H NMR (DMSO-d6): δ 11.50 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 2.8 Hz, 1H), 7.85 (t, J = 5.5 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.15‒7.05 (m, 2H), 6.91 (d, J = 7.6 Hz, 2H), 6.49 (d, J = 8.7 Hz, 2H), 4.90 (s, 2H), 3.24 (s, 2H), 3.18‒3.11 (m, 3H), 2.80‒2.76 (m, 2H), 1.85‒ 1,78 (m, 2H), 1.67‒1.44 (m, 3H), 1.23‒1.10 (m, 2H).

N-[[1-[(4-aminophenyl)methyl]-4-piperidinyl]methyl]-2,3-dihydro-1,4-benzodioxine-5- 1 carboxamide (12) H NMR (DMSO-d6): δ 8.03 (t, J = 5.7 Hz, 1H), 7.19‒7.14 (m, 1H), 6.96‒ 6.80 (m, 4H), 6.48 (d, J = 8.2 Hz, 2H), 4.92 (s, 2H), 4.30‒4.24 (m, 4H), 3.25 (s, 2H), 3.15‒ 3.09 (m, 2H), 2.80‒2.75 (m, 2H), 1.85‒1.80 (m, 2H), 1.65‒1.49 (m, 3H), 1.22‒1.12 (m, 2H).

N-[[1-[(4-aminophenyl)methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H-[1,3]oxazino[3,2- 1 a]indole-10-carboxamide (25). White solid. H NMR (DMSO-d6): δ 8.09‒8.05 (m, 1H), 7.30‒7.26 (m, 1H), 7.10‒7.06 (m, 2H), 6.93 (d, J = 8.2 Hz, 2H), 6.81‒6.75 (m, 1H), 6.52 (d, J = 8.2 Hz, 2H), 4.94 (s, 2H), 4.57 (t, J = 5.0 Hz, 2H), 4.16 (t, 2H), 3.27 (s, 2H), 2.84‒2.78 (m, 2H), 2.31‒2.26 (m, 2H),

6.1.6. General procedure for the synthesis of N-aryl sulfonamides. Indole-3-carboxylic acid sulphonamides (5-8) Preparation of N-[[1-[[4-(methanesulfonamido)phenyl]methyl]-4-piperidinyl]methyl]- 1H-indole-3-carboxamide hydrochloride (5) A solution of methanesulfonyl chloride (77 mg,

0.67 mmol) in CH2Cl2 (1.0 ml) was added to a cooled solution of amine 4 (0.20 g, 0.56 mmol) in CH2Cl2 (2.5 ml) and pyridine (2.5 ml). The reaction mixture was stirred to room temperature for 24 h and evaporated in vacuo. The residue was separated with column chromatography (SiO2, CH2Cl2/MeOH 7:3) to leave the title compound 5 as a white solid (0.08 g, 33%) and 90.9 % purity by HPLC. The hydrochloride salt was prepared by dissolving

5 in CH2Cl2 (2.5 ml) and adding HCl in Et2O. The mixture was stirred for 15 minutes and 1 evaporated in vacuo to leave the hydrochloride salt as a white solid. H NMR (DMSO-d6): δ 11.69 (s, 1H), 10.34 (bs, 1 H), 10.06 (bs, 1H), 8.20‒8.10 (m, 3H), 7.60‒7.44 (m, 3H), 7.31‒ 7.27 (m, 2H), 7.19‒7.07 (m, 2H), 4.23 (bs, 2H), 3.59 (s, 3H), 3.37‒3.31 (m, 2H), 3.20‒3.11 (m, 2H), 3.06 (s, 2H), 3.00‒2.83 (m, 2H), 2.60‒2.50 (m, 2H), 1.91‒1.80 (m, 3H), 1.70‒1.55 13 (m, 2H). C NMR (DMSO-d6): δ 164.8, 146.7, 141.7, 139.5, 136.1, 132.5, 127.8, 126.1, 124.8, 121.8, 121.0, 120.2, 119.1, 111.8, 110.5, 66.4, 51.2, 37.2, 34.2, 26.8.

N-[[1-[[4-(butylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H-indole-3- carboxamide (6) White solid (0.063 g, 33 %) with 80.3 % purity by HPLC. 1H NMR

(DMSO- d6): δ 11.59 (s, 1H), 9.91 (bs, 1H), 9.11 (bs, 1H), 8.16 –7.93 (m, 34H), 7.45 - 7.09 (m, 5H), 3.19 - 3.07 (m, 10H), 2.48 - 2.44 (m, 1H), 1.65-1.33 (m, 14H), 0.91-0.79 (m, 3H).

N-[[1-[[4-(cyclohexylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H-indole-3- 1 carboxamide (7). White solid with 73.8 % purity by HPLC. H NMR (DMSO-d6): δ 11.57 (s, 1H), 9.87 (bs, 1H), 8.13–8.11 (d, 1H), 8.04 (s, 1H), 7.96 (bs, 1H), 7.42–7.40 (m, 2H), 7.24– 7.22 (m, 1H), 7.15–7.06 (m, 2H), 3.33 (s, 2H), 3.15 (s, 2H), 2.01 (t, 3H), 1.77–1.57 (m, 8 H), 13 1.42–1.40 (m, 3H), 1.25–1.14 (m, 5H). C NMR (DMSO-d6): δ 164.7, 136.1, 127.6, 126.1, 121.7, 121.0, 120.2, 114.5, 111.7, 110.5, 59.2, 58.1, 27.8, 25.9, 25.6, 25.3, 24.7, 24.3.

N-[[1-[[4-(benzylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H-indole-3- 1 carboxamide (8). White solid with 96.7 % purity by HPLC. H NMR (DMSO-d6): δ 11.65 (s, 1H), 10.63 (s, 1H), 8.13‒8.06 (m, 3H), 7.81‒7.78 (m, 2H), 7.57‒7.53 (m, 3H), 7.42‒7.39 (m, 3H), 7.14‒7.09 (m, 4H), 3.95 (bs, 2H), 3.36 (s, 2H), 3.17‒3.13 (m, 6H), 1.81‒1.75 (m, 3H),

13 1.60‒1.38 (m, 2H). C NMR (DMSO-d6): δ 165.55, 146.33, 140.38, 136.92, 133.80, 130.12, 128.60, 127.50, 126.99, 122.55, 121.83, 121.02, 120.35, 117.66, 112.61, 111.33, 26.65.

1,4-Benzodioxane-5-carboxylic acid sulfonamides (13-19) N-[[1-[[4-(methanesulfonamido)phenyl]methyl]-4-piperidinyl]methyl]-2,3-dihydro-1,4- benzodioxine-5-carboxamide (13). White solid (0.04 g, 16.6 %) with 64.4 % purity by 1 HPLC. H NMR (DMSO-d6): δ 9.63 (s, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.42–7.11 (m, 5H), 6.95‒6.94 (m, 1H), 6.85-6.83 (m, 1H), 4.33‒4.26 (m, 4H), 3.39 (s, 2H), 3.16–3.13 (m, 2H), 2.96 (s, 3H), 2.79‒2.77 (m, 2H), 1.89 (bs, 2H), 1.66–1.62 (m, 2H), 1.50 (bs, 1H), 1.18–1.09 13 (m, 2H). C NMR (DMSO-d6): δ 169.9, 148.8, 146.7, 135.3, 134.9, 129.8, 129.1, 127.0, 125.7, 125.0, 124.3, 69.8, 68.8, 58.2, 57.8, 49.6, 40.7, 34.8, 17.2.

N-[[1-[[4-(butylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3-dihydro-1,4- benzodioxine-5-carboxamide (14). White solid with 59.7 % purity by HPLC. 1H NMR

(DMSO-d6): δ 9.71 (s, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.26–7.12 (m, 5H), 6.98–6.81 (m, 2H), 4.33–4.25 (m, 4H), 3.40‒3.33 (m, 3H), 3.14 (t, J = 6.1 Hz, 2H), 3.08–3.00 (m, 3H), 2.79‒2.77 (m, 2H), 1.90 (bs, 2H), 1.67–1.43 (m, 4H), 1.39–0.99 (m, 4H), 0.81 (t, J = 7.3 Hz, 3H). 13 C NMR (DMSO-d6): δ 164.9, 145.6, 143.7, 142.1, 130.6, 128.1, 122.5, 120.8, 115.0, 80.1, 65.1, 63.7, 63.0, 53.3, 45.5, 36.2, 30.2.

N-[[1-[[4-(isobutylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3-dihydro-1,4- benzodioxine-5-carboxamide (15). White solid (0.07 g, 12 %) with 66.6 % purity by HPLC. 1 H NMR (DMSO-d6): δ 9.72 (s, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.25–7.16 (m, 5H), 6.96–6.94 (m, 1H), 6.87–6.83 (m, 1H), 4.33–4.26 (m, 4H), 3.31 (s, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.96– 2.94 (m, 2H), 2.79‒2.77 (m, 2H), 1.90 (bs, 2H), 1.65–1.41 (m, 3H), 1.25 (bs, 2H), 0.98 (d, J = 13 6.7 Hz, 6H). C NMR (DMSO-d6): δ 164.6, 149.6, 143.5, 141.4, 124.5, 123.9, 121.7, 120.5, 119.0, 64.9, 64.4, 63.6, 58.2, 40.1, 24.2, 22.0, 15.1.

N-[[1-[[4-(cyclohexylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3-dihydro- 1,4-benzodioxine-5-carboxamide (16). White solid with 75.6 % purity by HPLC.1H NMR

(DMSO-d6): δ 9.70 (s, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.25–7.16 (m, 5H), 6.98–6.94 (m, 1H), 6.88–6.83 (m, 1H), 4.33–4.25 (m, 4H), 3.31 (s, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.98–2.80 (m, 13 3H), 2.03–1.99 (m, 2H), 1.77–1.37 (m, 11H), 1.25–1.09 (m, 5H). C NMR (DMSO-d6): δ

165.5, 144.4, 142.3, 136.6, 132.1, 130.8, 125.4, 122.7, 121.4, 120.0, 119.9, 65.3, 64.5, 59.8, 53.5, 49.5, 45.3, 36.4, 30.2, 26.8, 25.6, 25.2.

N-[[1-[[4-(benzylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3-dihydro-1,4- benzodioxine-5-carboxamide (17). White solid with 96.7 % purity by HPLC. 1H NMR

(CDCl3): δ 7.75–7.64 (m, 4H), 7.51–7.36 (m, 3H), 7.15 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 6.98–6.86 (m, 2H), 4.39–4.26 (m, 4H), 3.74–3.69 (m, 1H), 3.42 (s, 2H), 3.31 (t, J = 6.1 Hz, 2H), 2.86–2.82 (m, 2H), 1.98–1.91 (m, 2H), 1.66–1.58 (m, 3H), 1.40–1.32 (m, 2H). 13 C NMR (CDCl3): δ 164.9, 143.5, 141.9, 139.3, 132.9, 128.9, 127.2, 124.1, 122.2, 121.7, 121.3, 120.7, 67.0, 65.0, 63.6, 62.4, 53.2, 35.7, 30.9, 29.7, 25.6

N-[[1-[[4-(p-tolylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3-dihydro-1,4- benzodioxine-5-carboxamide (18). White solid with 97.2 % purity by HPLC. 1H NMR

(DMSO-d6): δ 10.28 (s, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 7.19–7.14 (m, 3H), 7.06–7.03 (m, 2H), 6.95–6.81 (m, 2H), 4.31–4.24 (m, 4H), 3.52 (bs, 2H), 3.12 (t, J = 5.9 Hz, 2H), 2.84 (bs, 2H), 2.30 (s, 3H), 2.09 (bs, 1H), 1.67–1.55 (m, 13 3H), 1.30–1.20 (m, 2H). C NMR (DMSO-d6): δ 164.7, 149.6, 143.5, 143.2, 141.4, 136.7, 130.2, 129.6, 126.7, 124.4, 121.8, 120.5, 119.7, 119.0, 64.4, 63.6, 52.2, 44.2, 35.1, 28.5, 20.9.

N-[[1-[[4-[[4-(trifluoromethyl)phenyl]methylsulfonylamino]phenyl]-methyl]-4- piperidinyl]methyl]-2,3-dihydro-1,4-benzodioxine-5-carboxamide (19). White solid with 1 96.6 % purity by HPLC. H NMR (DMSO-d6): δ 9.84 (s, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.1 Hz, 2H), 7.24–7.13 (m, 5H), 6.96–6.94 (m, 1H), 6.87–6.84 (m, 1H), 4.60 (s, 2H), 4.33–4.25 (m, 4H), 3.56‒3.34 (m, 1H), 3.31 (s, 2H), 3.15 (t, J = 6.1 Hz, 2H), 2.86–2.80 (m, 2H), 2.08–1.80 (m, 1H), 1.68–1.55 (m, 3H), 1.30–1.14 (m, 2H). 13C NMR

(DMSO-d6): δ 164.7, 143.6, 141.5, 137.2, 134.4, 131.8, 129.9, 129.0, 128.6, 126.0, 125.2, 124.4, 122.4, 121.9, 120.5, 119.1, 64.5, 63.6, 61.4, 56.5, 52.7, 44.5, 35.5, 30.6, 29.3.

3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxylic acid (piboserod) sulfonamides (26-33) N-[[1-[[4-(methanesulfonamido)phenyl]methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H- [1,3]oxazino[3,2-a]indole-10-carboxamide (26). White solid (0.16 g, 64.5 %) with 77.2 % 1 purity by HPLC. H NMR (DMSO-d6): δ 9.69 (s, 1H), 8.04–7.99 (m, 1H), 7.40‒7.02 (m, 7H), 6.84 (s, 1H), 4.54 (t, J = 5.1 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.31 (s, 2H), 3.16–3.14 (m, 2H), 13 2.98 (s, 3H), 2.31–2.23 (m, 2H), 1.69–1.05 (m, 5H). C NMR (DMSO-d6) δ 163.7, 149.8, 130.9, 125.2, 121.1, 119.9, 119.6, 108.4, 87.9, 67.1 54.9, 20.6.

N-[[1-[[4-(ethylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H- [1,3]oxazino[3,2-a]indole-10-carboxamide (27). White solid (0.19 g, 73.3 %) with 81.4 % 1 purity by HPLC. H NMR (DMSO-d6): δ 9.78 (bs, 1H), 8.05–8.02 (m, 1H), 7.27–7.18 (m, 9H), 7.07–6.98 (m, 5H), 6.81 (bs, 1H), 5.75 (s, 1H), 4.55 (t, J = 5.1 Hz, 2H), 4.12 (t, J = 6.1 Hz, 2H), 3.17–3.06 (m, 4H), 2.29–2.26 (m, 2H), 1.67 (m, 5H), 1.20–1.17 (m, 3H). 13C NMR

(DMSO-d6): δ 163.7, 149.7, 130.9, 128.9, 128.2, 125.2, 121.1, 119.9, 119.6, 119.37, 108.4, 87.9, 67.1, 54.9, 45.1, 20.6, 8.0.

N-[[1-[[4-(butylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H- [1,3]oxazino[3,2-a]indole-10-carboxamide (28). White solid with 90.4 % purity by HPLC. 1 H NMR (DMSO-d6): δ 9.71 (s, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.26–7.12 (m, 5H), 6.97–6.81 (m, 2H), 4.33–4.25 (m, 4H), 3.40 s, 1H), 3.33 (s, 2H), 3.14 (t, J = 6.1 Hz, 2H), 3.11–3.00 (m, 2H), 2.81–2.77 (m, 2H), 1.95–1.87 (m, 2H), 1.67–1.47 (m, 5H), 1.39–1.15 (m, 4H), 0.81 (t, J 13 = 7.3 Hz, 3H). C NMR (DMSO-d6): δ 163.2, 153.8, 140.0, 136.5, 131.2, 130.0, 128.5, 128.2, 126.8, 124.7, 121.8, 120.3, 119.2, 108.8, 85.0, 66.5, 63.0, 52.5, 48.6, 35.1, 28.3, 20.2.

N-[[1-[[4-(isobutylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H- [1,3]oxazino[3,2-a]indole-10-carboxamide (29). White solid with 91.4 % purity by HPLC. 1 H NMR (DMSO-d6): δ 9.73 (s, 1H), 8.07–8.04 (m, 1H), 7.25–7.16 (m, 5H), 6.96–6.82 (m, 2H), 4.33–4.26 (m, 4H), 3.42–3.33 (m, 3H), 3.15 (t, J = 6.1 Hz, 2H), 2.96–2.94 (m, 2H), 2.85–2.78 (m, 2H), 2.12–2.08 (m, 2H), 1.97–1.90 (m, 2H), 1.67–1.53 (m, 3H), 1.23–1.11 (m, 13 2H), 0.98 (d, J = Hz, 6H). C NMR (DMSO-d6): δ 164.6, 149.6, 143.5, 141.4, 124.5, 121.8, 120.5, 119.3, 119.0, 79.1, 64.4, 63.6, 58.2, 24.2, 22.0.

N-[[1-[[4-(cyclohexylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-3,4-dihydro- 1 2H-[1,3]oxazino[3,2-a]indole-10-carboxamide (30). White solid. H NMR (DMSO-d6): δ 9.98 (s, 1H), 8.02 (m, 1H), 7.49- 7.46 (m, 2H), 7.27- 7.24 (m, 2H), 7.08- 7.05 (m, 1H), 6.91 (m, 1H), 4.55 (t, J= 5.1 Hz, 2H), 4.16 - 4.13 (t, J = 6.1 Hz, 2H), 3.76 (m, 6H), 3.34- 3.30 (m, 2H), 3.18 – 3.03 (m, 3H), 2.87- 2.72 (m, 2H), 2.28 (s, 1H), 2.04 -2.02 (m, 2H), 2.00 -1.74 (m, 4H), 1.60 -1.43 (m, 4H), 1.39 -1.15 (m, 3H).

N-[[1-[[4-(benzylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H- [1,3]oxazino[3,2-a]indole-10-carboxamide (31). White solid with 94.1 % purity by HPLC. 1 H NMR (DMSO-d6): δ 7.83 (d, J = 7.8 Hz, 1H), 7.64–7.62 (m, 1H), 7.46–7.29 (m, 9H), 7.17–7.09 (m, 2H), 4.50 (t, J = 5.1 Hz, 2H), 4.11–4.04 (m, 4H), 3.55 (s, 2H), 3.17 (s, 2H), 2.91–2.88 (m, 2H), 2.26–2.23 (m, 2H), 2.06–2.02 (m, 2H), 1.75–1.60 (m, 3H), 1.40–1.30 (m, 13 2H). C NMR (DMSO-d6): δ 163.3, 153.8, 140.0, 139.7, 136.3, 131.2, 130.0, 128.5, 128.2, 126.8, 124.7, 121.8, 120.3, 119.2, 108.8, 85.0, 66.5, 61.6, 52.5, 48.6, 35.1, 28.3, 20.2.

N-[[1-[[4-(p-tolylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-3,4-dihydro-2H- [1,3]oxazino[3,2-a]indole-10-carboxamide (32). White solid with 92 % purity by HPLC. 1 H NMR (DMSO-d6): δ 9.82 (s, 1H), 8.04–8.02 (m, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.27–7.21 (m, 3H), 7.14–7.12 (m, 2H), 7.06–7.04 (m, 2H), 6.77 (t, J = 5.2 Hz, 1H), 4.56 (t, J = 5.4 Hz, 2H), 4.12 (t, J = 6.1 Hz, 2H), 3.30 (s, 2H), 3.18 (t, J = , 2H), 2.82– 2.79 (m, 2H), 2.27–2.25 (m, 2H), 1.92–1.88 (m, 2H), 1.65–1.45 (m, 3H), 1.27–1.18 (m, 2H). 13 C NMR (DMSO-d6): δ 163.7, 149.8, 149.6, 145.7, 143.3, 137.6, 136.7, 130.9, 129.7, 128.0, 126.7, 125.5, 125.2, 121.1, 119.9, 119.6, 119.3, 108.4, 87.8, 67.1, 64.9, 56.0, 48.6, 20.9, 20.7, 20.6, 15.1.

N-[[1-[[4-[[4-(trifluoromethyl)phenyl]methylsulfonylamino]phenyl]methyl]-4- piperidinyl]methyl]-3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide (33). 1 White solid with 75 % purity by HPLC. H NMR (DMSO-d6): δ 10.1 (br s, 1H), 8.06–8.04 (m, 1H), 7.66–7.64 (m, 2H), 7.33–7.15 (m, 4H), 7.12–7.02 (m, 4H), 6.81 (t, J = 5.2 Hz, 1H), 4.56 (t, J = 5.4 Hz, 2H), 4.13 (t, J = 6.1 Hz, 2H), 3.60 (br s, 2H), 3.18 (s, 3H), 2.89 (br s, 2H), 13 2.35–2.23 (m, 5H), 1.68–1.56 (m, 3H), 1.27–1.23 (m, 2H). C NMR (DMSO-d6): 163.7, 149.7, 144.6, 143.2, 137.7, 136.8, 131.0, 130.6, 129.7, 129.3, 128.2, 126.7, 125.5, 123.9, 122.4, 121.2, 120.1, 108.3, 88.0, 67.2, 56.2, 54.9, 48.5, 20.9, 20.7, 20.5, 18.6.

6.2. Experimental pharmacology (50)

6.2.1. Radioligand binding assay

The term radioligand is defined as a radioactively labelled drug that associates with any target receptor, transporter or enzyme of interest. The radioligand should be carefully chosen and should have high affinity for the receptor (Kd < 100 nM). By using a radioactive drug, we can measure amount drug bound to a receptor of interest, in presence of a competitively binding ligand. Thus we can measure specific receptor affinity and 5-HT4 receptor expression densities in membranes from transiently transfected HEK293 cells, in this experiment.

Competition binding assay

We performed a competition binding assay, to determine specific ligand affinity for the 5-HT4 receptor. By using a single detectable radioligand concentration, we measured unlabelled ligand/ competitor binding property at different concentrations. The radioligand used for this assay was [3H]GR113808 with the specific activity of 81 – 82.1 Ci/mmol. The ligands were compared to the two references GR113808 and DDL-6001 (SB207266, Piboserod®), which both are highly potent and selective antagonist of the 5-HT4 receptor. The experiment was performed in 96-well, round-bottom microtiter plates with the total substance volume of 100 µl. The highest ligand concentration used was 1-100 µM. This was diluted in a 1:2 dilution in

21 wells. GTP was added to the assay mixture, to make sure that the GPCR (5-HT4) exist in the low affinity state, and avoiding unpredictable conformations changes between high (preferred for agonist binding assay) and low affinity state.

Content of prep. solutions Amount in well 10 X Binding buffer 10 µl GTP (100 µM final) 10 µl 3 [ H]GR113808 10 µl dH2O 50 µl Membranes 20 µl Total 100 µl

Protocol:

1. Prepare the solutions ready.  Make the assay mix of 10X binding buffer (1X in final), 98 mM GTP (100 µl final) and 246.9 nM [3H]GR113808, hot ligand (0,3 nM final) in the ratio 1:1:1.  Make the ligand and reference solutions ready (1 µM in final).

2. Add 50 µl dH2O to each well 1-24 (columns 1-3) except the first well in the serial dilution. 3. Add 100 µl of each ligand (unlabelled, cold competitor) to the first well of each serial dilution. Make a 1:2 dilution by taking out 50 µl from first well, then mixing this with

the dH20 in the next well, and transfer further 50 µl from well 2 to 3. Continue this process to well 21. Discard 50 µl of the mixed content in well 21. 4. Add 30 µl of the assay mixture in each well. For determination of actual concentration of radioligand, add 7 x 30 µl of the assay mixture in scintillation vials filled with 3 ml of scintillation fluid (Ultima Gold XR, Packard). The samples were counted in a Wallac WinspectralTM 1414 Liquid Scintillation Counter (Perkin ElmerTM).

5. Prepare the 5-HT4(b) membrane solution from 4L40 cell line, diluted in TE (50 nM Tris-HCl, pH 7.5 at 20 °C, 1 mM EDTA) in 17 (or lower) dilution of stock. 6. Add 20 µl of the membrane solution in each well. Measure protein on 4 X 20 µl of the membrane preparation. 7. Shake the plate for 30 seconds on a Vortex. Incubate the plates at room temperature for 1 hour. 8. Harvest membranes onto Whatman GF-2 filter pre-soaked in 0.3 % polyethyleneimine, with a Packard Cell Harvest (Packard Instrument Co.). Wash membranes 4-6 times

with cold (+4 °C) washing buffer (Tris-HCl, pH 7.0 at 25 °C, 2 mM MgCl2). 9. Dry the filter at 37 °C for 1 hour. 10. Add 20 µl MicroScint scintillation fluids (Packard Instrument Co.) to each well. Cover the plates with transparent paper on the top and white paper on the bottom. 11. Count the filter plate in a Packard TopCount Scintillation Counter (Packard Instrument Co.).

6.2.2. Adenylyl cyclase assay

In this assay method, the adenylyl cyclase activity (antagonist property) was measured by determining the formation of [32P]cAMP from [α32P]ATP in membrane preparations. This is a highly sensitive assay method which has been developed by sequential chromatography on Dowex 50 cation exchange resin and on neutral aluminium oxide (Alumina) columns, and is able to detect small amounts of cyclic AMP formed at low enzyme concentrations (55). As an internal standard [3H]cAMP (~10 000 cpm/reaction) was used to monitor the recovery of each sample through the columns and to eliminate individual differences between the columns. The respective ligands, with the highest concentrations used 1-100 µM (final concentration in 50 µl), were evaluated in a 21-points concentration/ dose-response curve. The amount of radiolabeled cAMP is determined in the present of 5-HT (endogenous stimulator of cAMP) and our ligand, which are expected to antagonize the 5-HT effect and thereby block the cAMP stimulation. The labelled cAMP level is expected to decrease in the present of a 5-HT4 receptor antagonist. Membranes used in the experiments were 4b40 and 4b13, with respectively high and low 5-HT4 receptor levels.

Assay content

Content of prep. solutions Amount in well Crude membrane preparation 10 µl Additives (hormones) 20 µl Assay mixture (IM) 20 µl Total 50 µl

Incubation mixture (IM): (Tris-HCl, EDTA, cAMP and [3H]cAMP) ATP, [32P]ATP, GTP, Mg2+, ATP-regenerating system (RS) and IBMX (3-isobutyl-1-methylxanthine).

 Total concentration of ATP and [α32P]ATP: Around 0.1 mM.  Specific activity of ATP and [α32P]ATP: 200 cpm/ pmol (calculated for each assay). 32  Half-life (T1/2) for radiolabeled [α P]ATP is 14.3 days.  EDTA is included to permit linear cAMP accumulation during extended periods of time.  GTP is added as substrate for activation of the GPCR to obtain hormonal stimulation of AC.

 Mg-ATP is an allosteric activator of AC. Mg2+ is added in surplus over EDTA to obtain enzyme activity.  ATP regenerating system (RS) contains: Myokinase (40 U/ml), creatine phosphokinase (0.2 mg/ml) and creatine phosphate (20 mM). RS is added to diminish any ATPase alteration of substrate available for the enzyme in the crude membrane preparation.  IBMX (phosphodiesterase inhibitor, 1 mM) along with unlabelled cAMP (1mM) is included in the assay mixture to prevent any phosphodiesterase catalysed breakdown of the generated [32P]cAMP formed and [3H]cAMP added.

Protocol:

1. Preparation of assay mixture. Mix the following reagents in a tube kept on ice:

Content Stock concentration Final Volume per concentration reaction in 50 µl IM 10X 1X 5.0 µl (Incubation (250 mM Tris-HCl, pH 7.6 mix) at 20 ᵒC; 10 mM cAMP; 10 mM EDTA; [3H]cAMP ~2000 cmp/µl) RS 10X 1X 5.0 µl (Regenerating (200 mM creatine system) phosphate; 2mg/ml creatine phosphokinase; 400 U/ml myokinase) GTP 10 mM 20 µM 0.1 µl MgCl2 100 mM 4 mM 2.0 µl IBMX 25 mM 1 mM 2.0 µl ATP/ [32P]ATP 1 mM 0.1 mM 5.0 µl (106 cpm/ 5 µl) dH2O 0.9 µl Total: 20 µl

Add 5 µl of the IM to two counting vials filled with 3.5 ml scintillation fluid and 3.5 ml imidazole-HCl buffer for determining of actual concentration of [3H]cAMP. 32 Add 10 µl of the ATP/ [ P]ATP to two tubes (1 and 2) with 990 µl dH2O. Transfer 5 µl from each tube to two counting vials filled with 3.5 ml scintillation fluid and 3.5 ml imidazole-HCL buffer. Measure the OD of the two solutions 1 and 2.

2. Preparation of additives:

Perform a serial dilution (1:2 or 1:3) of 10 µM/ 1 µM of 5-HT/ 5-CT in dH2O to achieve 21 different concentrations. Dilute the other additives to the correct

concentration (i.e. FSK, NaF, Iso, PgE1, clozapine) in dH2O. 3. Add 20 µl of additives to reaction tubes placed on ice (use 20 µl, 0.1% BSA/ 1mM ascorbic acid as a blank). 4. Add 10 µl of membrane preparations to each reaction tube (10 µl TE as blank) on ice. Dispense 4 x 10 µl of each membrane preparation into separate wells for protein quantification. 5. Add 20 µl of assay mixture to each tube. 6. START REACTION: Mix by vortex and transfer the reaction tubes to shake water bath at 32 °C. Incubate for 20 minutes. 7. STOP REACTION: Add 100 µl of STOP solution (40 mM ATP, 10 mM cAMP, 1% SDS).

8. Add 850 µl of dH2O to the reaction tubes and pour the adenylyl cyclase reactions into

Dowex columns (Pre-equilibrated with ~10 ml of dH2O and completely drained). Let the columns drain.

9. Wash the columns twice with 2 ml dH2O and place the rack of Dowex columns on top of the Alumina columns (pre-equilibrated with ~10 ml imidazole-HCl buffer). Elute

reaction product from Dowex columns into Alumina columns with 4 ml dH2O. 10. Wash the alumina columns with 1.8 ml imidazole-HCl buffer. Place the rack of the Alumina columns on top of rack of scintillation vials containing 3.5 ml scintillation fluid (Ultima Gold XR, Packard). Elute the reaction product from Alumina columns into the scintillation vials with 3.5 ml imidazole-HCl buffer to the Alumina columns. 11. Cap the vials, shake them well and count the sample (Wallac WinspectralTM 1414 Liquid Scintillation Counter, PerkinElmerTM).

Procedure for regenerating the columns:

 The Dowex columns are regenerated by sequential washing with ~10 ml each of 2

M NaOH, dH2O, 2 M HCl, and 3 X H2O respectively. The Dowex columns can be

stored dry and need only to be re-equilibrated with dH2O before use.  Alumina columns are regenerated by washed with ~10 ml imidazole-HCl buffer

before use and stored in dH2O after use.

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Appendix A: Table of synthesised and pharmacological evaluated compounds

Table 3.

a b No. pKi±SEM pKb±SEM Ar R Log Molecular a b DOct7.4 n = 2-6 n = 2-3 formula

GR113808 9.56±0.19 10.13±0.22 Figure 6a N.D. C H N O S (6) (3) 19 27 3 4

SB207266 9.43±0.28 10.48±0.08 Figure 6b N.D. C22H31N3O2 (6) (3)

5 CH 5.99±0.29 N.D. 3 0.5 C23H28N4O3S (2)

6 6.29±0.33 N.D. 1.4 C26H34N4O3S (2)

6.27±0.28 N.D. 7 2.5 C28H36N4O3S (3)

5.95±0.30 N.D. 8 1.2 C29H32N4O3S (3)

13 CH 8.52±0.07 9.33±0.17 3 0.5 C23H29N3O5S (3) (2)

14 8.62±0.16 N.D. 1.7 C26H35N3O5S (4)

15 8.08±0.32 N.D. 2.0 C26H35N3O5S (3)

16 8.87±0.58 N.D. 2.5 C28H37N3O5S (3)

8.26±0.42 N.D. 17 2.3 C29H33N3O5S (4)

8.61±0.46 N.D. 18 1.9 C29H33N3O5S (4)

8.80±0.62 N.D. 19 N.D. C30H33N3O5SF3 (4)

8.62±0.42 9.35±0.04 26 CH3 1.0 C26H32N4O4S (5) (2)

1.1 9.02±0.38 9.99 27 C29H38N4O4S (5)

8.95±0.59 9.67 28 2.3 C H N O S (4) 29 38 4 4

29 8.36±0.38 9.65±0.25 2.1 C29H38N4O4S (6) (3)

8.69±0.54 9.78±0.16 30 N.D. C30H38N4O4S (3) (2)

31 9.12±0.36 9.66 1.6 C32H36N4O4S (4)

32 7.69±0.28 N.D. 2.5 C32H36N4O4S (5)

33 8.15±0.26 9.85 N.D. C33H36N4O4SF3 (5)

Appendix B: NMR-spectra of synthesised compounds

Indole-3-carboxylic acid sulfonamides (5-8)

1H NMR: N-[[1-[[4-(methanesulfonamido)phenyl]methyl]-4-piperidinyl]methyl]-1H- indole-3-carboxamide hydrochloride (5)

Appendix B: NMR-spectra of synthesised compounds: Indole sulfonamides (5-8)

13C NMR: N-[[1-[[4-(methanesulfonamido)phenyl]methyl]-4-piperidinyl]methyl]-1H- indole-3-carboxamide hydrochloride (5)

Appendix B: NMR-spectra of synthesised compounds: Indole sulfonamides (5-8)

1H NMR: N-[[1-[[4-(butylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H- indole-3-carboxamide (6)

Appendix B: NMR-spectra of synthesised compounds: Indole sulfonamides (5-8)

1H NMR: N-[[1-[[4-(cyclohexylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H- indole-3-carboxamide (7)

Appendix B: NMR-spectra of synthesised compounds: Indole sulfonamides (5-8)

13C NMR: N-[[1-[[4-(cyclohexylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H- indole-3-carboxamide (7)

Appendix B: NMR-spectra of synthesised compounds: Indole sulfonamides (5-8)

1H NMR: N-[[1-[[4-(benzylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H- indole-3-carboxamide (8)

Appendix B: NMR-spectra of synthesised compounds: Indole sulfonamides (5-8)

13C NMR: N-[[1-[[4-(benzylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-1H- indole-3-carboxamide (8)

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

1,4-Benzodioxane-5-carboxylic acid sulfonamides (13-19)

1H NMR: N-[[1-[[4-(methanesulfonamido)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (13)

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

13C NMR: N-[[1-[[4-(methanesulfonamido)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (13)

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

1H NMR: N-[[1-[[4-(butylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (14)

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

13C NMR: N-[[1-[[4-(butylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (14)

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

1H NMR: N-[[1-[[4-(isobutylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (15)

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

13C NMR: N-[[1-[[4-(isobutylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (15)

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

1H NMR: N-[[1-[[4-(cyclohexylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (16)

100

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

13C NMR: N-[[1-[[4-(cyclohexylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (16)

101

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

1H NMR: N-[[1-[[4-(benzylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (17)

102

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

13C NMR: N-[[1-[[4-(benzylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (17)

103

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

1H NMR: N-[[1-[[4-(p-tolylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (18)

104

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

13C NMR: N-[[1-[[4-(p-tolylsulfonylamino)phenyl]methyl]-4-piperidinyl]methyl]-2,3- dihydro-1,4-benzodioxine-5-carboxamide (18)

105

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

1H NMR: N-[[1-[[4-[[4-(trifluoromethyl)phenyl]methylsulfonylamino]phenyl]-methyl]-4- piperidinyl]methyl]-2,3-dihydro-1,4-benzodioxine-5-carboxamide (19)

106

Appendix B: NMR-spectra of synthesised compounds: Benzodioxane sulfonamides (13-19)

13C NMR: N-[[1-[[4-[[4-(trifluoromethyl)phenyl]methylsulfonylamino]phenyl]-methyl]- 4-piperidinyl]methyl]-2,3-dihydro-1,4-benzodioxine-5-carboxamide (19)

107