Synthesis and pharmacological evaluation of N-aryl sulfonamides as 5-HT
4receptor 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
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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 2.1.2. Remodelling
2.1.3. Treatment 2.1.4. β-blockers
2.1.5. ACE inhibitors and ARBs 2.1.6. Aldosterone
14.
16.
16.
16.
17.
19.
2.2 Pharmacology and function of serotonin (5-HT) 20.
2.2.1. Synthesis, release and storage of serotonin 2.2.2. The signalling mechanism of the 5-HT
4receptor 2.2.3. Function of serotonin in the heart
21.
21.
22.
2.3 Chemical background 23.
2.3.1. The pharmacophore model of the 5-HT
4receptor 2.3.2. The search for selective 5-HT
4receptor ligands
23.
24.
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
4.1.2. Synthesis of indole sulfonamides (5-8)
4.1.3. Synthesis of benzodioxane sulfonamides (13-19) 4.1.4. Synthesis of piboserod sulfonamides (26-33) 4.1.5. The log of distribution coefficients (logD
Oct7.4)
30.
33.
36.
38.
42.
4.2 Interpretation of pharmacological data 43.
4.2.1. Analysis of binding curves for the compounds 4.2.2. Analysis of adenylyl cyclase curves for the
selected compounds
4.2.3. Analysis of inverse agonist effect
43.
48.
53.
4
4.3 Structure-affinity relationship (SAFIR) 54.
4.3.1. Comparing derivatives with same sulphonamide side-chain group.
4.3.2. Comparing derivatives with increasing side-chain length.
4.3.3. Comparing electron donating and electron withdrawing sulfonamide side-chain groups.
55.
61.
63.
5. CONCLUSIONS 64.
6. MATERIALS AND METHODS 65.
6.1 Experimental chemistry
6.1.1. General procedures for preparations of intermediates 20 and 21
6.1.2. Preparation of benzyl protected piperidine amines 6.1.3. Hydrogenolysis of benzyl protected piperidine
amines
6.1.4. Preparation of arylic nitro compounds 6.1.5. Reduction to arylic amine compounds
6.1.6. General procedure for the synthesis of aryl sulfonamides
65.
65.
66.
67.
68.
69.
70.
6.2 Experimental pharmacology
6.2.1. Radioligand binding assay (Protocol) 6.2.2. Adenylyl cyclase assay (Protocol)
75.
75.
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.
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© Mirusha Navaratnarajah 2013
Synthesis and pharmacological evaluation of N-aryl sulfonamides as 5-HT4 receptor antagonists.
http://www.duo.uio.no/
Printed: Reprosentralen, Universitetet i Oslo
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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
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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 determine h5-HT4(b) receptor binding affinity, in a [3H]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.
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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
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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 wide variety of disorders as migraine (5-HT1 antagonists), irritable bowel syndrome1 (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.
1IBS 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).
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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
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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.
16 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,
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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
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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).
19 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).
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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 signalling pathway
Cardiovascular response
5-HT1 A, B, D, E, F Metabotropic, GPCR,
Inhibits AC, cAMP
Renal vascular dilation?,
vasoconstriction, cerebral arteriolar dilation, vascular nerve endings
5-HT2 A, B, C Metabotropic,
GPCR
Stimulates PLC,
IP3
Vasoconstriction, platelet aggregation, vasodilation.
5-HT3 A, B Ionotropic,
ligand operated
Ion channel Reflex bradycardia, pain
5-HT4 (short, long) Metabotropic, GPCR
Stimulates AC, cAMP
Cardio stimulation, pulmonary vein dilation
5-HT5 A, B Metabotropic,
GPCR
Unknown Unknown
5-HT6 Metabotropic,
GPCR
Stimulates AC, cAMP
Unknown
5-HT7 a, b, d Metabotropic,
GPCR
Stimulates AC, cAMP
Vascular relaxation
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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).
22 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).
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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
24
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
2Bioisosteres are defined as substituents which have similar chemical and physical properties, and expected to mediate the same biological response in vivo (40).
25
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,
26
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).
Figure 2: Chemical structure of serotonin, 5-HT3 and 5-HT4 receptor ligands (39).
YM-53389 RS17017
Serotonin (5-HT) Metoclopramide Renzapride (BRL 24924)
Zacopride Cisapride (Propulsid
X = O SB204070 X = NH SB205800
X = O SB207058 X = NH SB207266
27
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)
28
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).
29
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 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
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.
30
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).
Figure 6a Figure 6b
x HCl
GR113808 DDL6001 (SB207266)
Figure 6: Structure of commercially available 5-HT4 ligand lead compounds used a) GR113808 and b) DDL 6001 (SB207266, Piboserod®)
31
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.
PG =
Ar = Ar = PG =
a b
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.
32
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 characterized by HPLC and 1H/13C 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.
33 4.1.2. Synthesis of indole sulfonamides (5-8)
The synthesis of indole-3-carboxylic acid derivatives 5–8 are shown in Scheme 2.
c
e
b 1
d 3 4
5-8
2 a
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
34
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.
35
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.
36
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.
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
b
R = Benzyl R = BOC
CO2
Imidazole
Imidazole
Scheme 4: Reagents and conditions: a) 1,4-Benzodioxane-5-carboxylic acid, CDI, DCM, room temperature; b) Protected 4-piperidinylmethanamine, DCM, reflux.
a b
e c d
9 10
11 12
13-19
37
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.
38 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.
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
20 21
23 b
d
24 22
f a c
25
26-33
e
g
39
14. 12
. 15.
18.
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.
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.
40 12.
14.
13.
17.
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 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.
41
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.
42 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):
( )
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.
α - the degree of dissociation of the compound in water calculated from ionization constants.