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Synthesis and pharmacological studies on some 2–hydroxy haloalkylamines as

A thesis submitted in fulfillment of the Requirement of the University of Khartoum.

For: Master Degree of Pharmacy (M. Pharm.)

To:

DEPARTMENT OF FACULTY OF PHARMACY UNIVERSITY OF KHARTOUM

By: Amna Elhassan Hamad (B. Pharm., U. of K.)

Supervisor: Prof. Abdallah Awad Abdallah

Co – Supervisor: Dr. Osama Yousif Mohamed

December 2003

This work is dedicated to: My parents, brothers and sisters, for their support and supreme encouragement

To my family: Engineer Mohammed I. Hamad, for his encouragement and motivation

To my kids and daughters: Ahmed, Abubakr, Omer, Osama, Huda and Nuha. For their infinite patience & endurance during all time I spend away from them. ACKNOWLEDGEMENT

Initially I solely thank Almighty Allah for accomplishing this work.

I would like to express my thanks and gratitude to my supervisor Dr. Abdalla Awad Abdalla, former Professor of Medicinal Chemistry (CTS) for his courtesy and careful supervision and for his continuous guidance.

My sincere thanks and gratitude to my Co–Supervisor, Associated Professor Osama Yousif Mohammed, Department of Pharmacology (U. of K.) for his valuable advises throughout all stages of the study.

Special thanks and gratitude to Dr. Abdel Wahab Hassan Mohammed, Head Department of Pharmacology, Medicinal and Aromatic Plants Institute National Center for Research, for his valuable advises and helps.

And my appreciation and thanks giving to all staff members of Medicinal and Aromatic Plants Institute, especially Reem Hassan Ahmed (B. V. & MSc. V, U. of K.).

Thanks to all staff members of Pharmacology and Pharmaceutical Chemistry Departments, Faculty of Pharmacy, U. of K. for their co– operation.

My greatest and deepest gratitude to Ali Hassan Hamad B. Sc., U. of K., for his intangible support and encouragement. LIST OF CONTENTS

Subjects Page List of Contents: ………………………………………………….. I List of Tables: …………………………………………………….. IV List of Schemes: …………………………………………………… IV List of Figures: ………………………………………………….. V Abstract in Arabic: ………………………………………………. VI Abstract in English: ……………………………………………… VII

MEDICINAL CHEMISTRY

CHAPTER ONE: INTRODUCTION 1.1 and anti–histaminic : …….…………………… 1 1.1.1 Histamine: ………………………..……………….….. 1 1.1.1.1 Histamine releasing substances: …………… 2 1.1.1.2 Anti–: …………………………… 3 1.2 Pharmacology: ………………………………….……………. 3 1.3 Chemistry: …………………………..…………….…….….…. 5 1.3.1 Salt formation: ………………………………….…….. 5 1.3.2 Structure–activity relationships (SAR): ……....………. 5 1.4 Ethanolamine derivatives: ……………….……………………. 7 1.5 Ethylene diamine derivatives: ………………………………..... 9 1.6 Propylamine derivatives: …………………………………….... 12 1.7 Testing of : ………………………………….….. 14 1.8 Gastric secretion: ………………………………………….…… 14 1.9 Cardiovascular effect of histamine: ……………………....…… 14 1.10 Histamine receptors: ……………………………………….…... 15 1.11 Histamine : ……………………………………….……..16 1.12 Histamine antagonist: ………………………………………..... 16 1.13 Aim of the project: …………………………………………….. 18

CHAPTER TWO EXPERIMENTAL 2.1 Apparatus and materials: ………………………………………. 21 2.2 Methods: ……………………………………………………..…23 2.2.1 Preparation of the 2–(N,N dibenzylamino)–I–P– haloaryl–I– hydroxy–ethanes: ………………………………………23 2.2.1.1 Preparation of the bromoacetophenones from the acetophenones: ………………………………23 2.2.1.2 The preparation of the bromohydrins from the bromoacetophenone: …………..………..….. 26 2.2.1.3 Preparation of the 1–hydroxy–I–aryl–2–(N,N–diben– zyl amino) ethane: …………………………. 29 2.2.1.4 The preparation of 1–hydroxy–I–aryl–2–(N,N–diben– zylamino) ethanes hydrochloride salts: ….….. 32 2.2.2 Experimental discussion: ……………..……………….. 34 2.2.2.1 Bromination: ……………………………...… 34 2.2.2.2 Reduction: ………………………………….. 37 2.2.2.3 Coupling of bromohydrin with dibenzylamine 38

CHAPTER THREE DISCUSSION 3.1 Receptors: ……………………………………………..………..40 3.2 Agonist: ……………………………………………..…………. 41 3.3 Antagonist: …………………………………………….………. 41 3.4 Ligands: …….……………………………….…………..……...41

PHARMACOLOGY CHAPTER FOUR: INTRODUCTION 4.1 Histamine: ………………………...…….…….…………..…… 53 4.1.1 Histamine synthesis: ……………………………….….. 53 4.1.2 Histamine storage and release: ………….…………….. 54 4.1.3 Histamine metabolism: ……………………………….. 55 4.1.4 Histamine receptors: …………………………………... 56 4.1.5 Chemical structure of histamine and its agonist: ……… 57 4.1.6 Chemical structure of histamine antagonist: ………….. 57 4.1.7 Structure activity relationship of histamine agonist and antagonist: …………………………………………….. 58 4.2 Peptic ulcer: ……………………………………………………. 60 4.2.1 Peptic ulcer prevalence: ……………………………….. 60 4.2.2 Types of peptic ulcer: ………..…………………….…. 61 4.2.3 Peptic ulcer etiology: ………………………………….. 62 4.2.4 Ulcer treatment: ……………………………………….. 64 4.2.4.1 Surgical treatment: ……………….…………. 65 4.2.4.2 Medical treatment (Medicines): …………….. 65 4.2.4.2.1 Antacids: ……………………..…………. 66 4.2.4.2.2 Sucralfate: ……………………………….. 67 4.2.4.2.3 Bismuth chelate: ……..……………….…. 67 4.2.4.2.4 Prostaglandin analog: Misoprostol: …….. 67 4.2.4.2.5 Histamine H2 receptors antagonists e.g. cimetidi– ne, and : ………..….. 68 4.2.4.2.6 Proton pump inhibitors: ……………….... 69 4.2.4.2.7 Treatment of H pylori infection efficacy: .. 69 4.2.5 Efficacy: ………………………………………………. 70 4.2.6 Side effects: ………………………..…………………. 70 4.2.7 –drug interaction: ………..……………………… 71 4.2.8 Rationale: ………………………………………………72

CHAPTER FIVE: EXPERIMENTAL 5.1 Chemicals and their sources: ………………………………….. 73 5.2 Equipments and instruments: ………………………………….. 73 5.3 Animals: ……………………………………………………….. 74 5.4 Methods: ………………………………………………………..74 5.4.1 Contracting rat uterus: ……………………………….. 74 5.4.2 Evaluation of antiulcerogenic activity using experimentally induced ulcer in rat stomach and duodenum: …………. 75 5.4.3 Guinea pig ileum preparation: ……..………..………… 76 5.4.4 Guinea pig atrium preparation: ……………..……..…... 77 5.4.5 The principle of pA2 system: ………………..…..…….. 78 5.4.6 Determination of pA2 value of P–chlor, and P– bromoalkyl–amine acting on rat uterus: ………………. 79 5.4.7 Determination of pA2 value of 2–chloralkylamine and 2–bromoalkylamine acting on guinea pig ileum: … 79 5.5 Materials (Preparation of test solution and blank): ……………. 80

CHAPTER SIX: RESULTS & DISCUSSION 6.1 The antiulcerogenic activity of 2–chloroalkylamine, 2–bromoa lkyl– amine and cimetidine on experimentally aspirin–induced ulcers in rat: …………………………………………………………... 81 6.2 Effect on guinea pig atria: ……………………………………... 82 6.3 Effect on the contracting rat uterus: ……………………..…….. 82 6.4 Effect on isolated guinea pig ileum: ………………………...... 83 6.5 Median inhibitory concentration (IC50) of cimetidine, 2–chloro and 2– bromo–alkylamine on aspirin induced ulcer in rat (n = 5): ..…. 83 6.6 pA2 values for cimetidine, 2–chloro and 2–bromoalkylamine versus histamine acting on rat uterus: …….…………………………... 83 6.7 pA2 values for mepyramine, 2–chloro, and 2–bromoalkylamine versus histamine acting on guinea pig ileum: …………………………. 84 6.8 Results Discussion: …..……………………………..…………. 97

CHAPTER SEVEN : SUMMARY AND CONCLUSION: ……… 101

CHAPTER EIGHT: SUGGESTION FOR FUTURE WORK: …. 102 REFERENCES: ………………………………………………………………. 103 LIST OF TABLES

Tables Title Page

Tab. 1 Starting materials and specifications: ………..………………… 22

Tab. 2 Chemical nomenclature of the compounds synthesized: ……… 22

Tab. 3 Bromoacetophenones: ……………………..…………………... 25

Tab. 4 Bromohydrins: ……………………………..………………….. 28

Tab. 5 Aminoalcohols liquids: ……………..…………………………. 31

Tab. 6 Aminoalcohol Hcl salts: ……………..………………………… 33

Tab. 7 Effect of 2–chloro alkylamine on aspirin–induced ulcer in rat: .. 85

Tab. 8 Effect of 2–bromoalkylamine on asprinin–induced ulcer in rat: . 85

Tab. 9 Effect of cimetidine on asprinin–induced ulcer in rat: ………… 86

Tab. 10 The value of pA2 of cimetidine, P–chlor, and P–bromoalkylamine versus histamine acting on rat uterus: ……….………………… 86

Tab. 11 The value of pA2 of mepyramine, P–chloro and P–bromo alkylamine Versus histamine acting on guinea pig ileum: ………………… 87

Tab. 12 Median inhibitory concentration (IC50) of cimetidine, 2–chloroalky– lamine and 2–bromoalkylamine on aspirin–induced ulcer in the rat (n = 6): …………………………………………………………. 87

LIST OF SCHEMES

Scheme No. Title of Scheme PAGE Scheme 1 General scheme for the preparation of intermediates and the amino–hydroxy ethanes: ……………………………. 20 LIST OF FIGURES

Fig.No. Figures Title Page

Fig. 1 Chemical structure of histamine agonist and antagonist: … 16 Fig. 2 Examples of the structures of the of histamine Receptors: ………………………..……………..…………. 48 Fig. 3 The effect of an ideal competitive antagonist on the dose response curve for an agonist: …………….……………..…………. 49 Fig. 4 An outline of the mode of action of a competitive antagonist (6) with an antagonist: …………….………..………………… 50 Fig. 5 The effect of a non–competitive antagonist on the dose–response curve of agonist: ……………..………………..……………….. 51 Fig. 6 The effect of an ideal antagonist on the response of a : …………………………………………………... 52 Fig. 7 (a) No desensitization (b) Desensitization: ……………….. 52 Fig. 8 2–chloroalkylamine versus histamine acting on rat uterus: … 88 Fig. 9 2–bromoalkylamine versus histamine acting on rat uterus: … 89 Fig. 10 2–chloroalkylamine versus histamine acting on guinea pig atrium: …………………………………………………………. 90 Fig. 11 2–bromoalkylamine versus histamine acting on guinea pig atrium: …………………………………………………………. 91 Fig. 12 2–chloroalkylamine versus histamine acting on guinea pig ileum: …………………………………………………………...92 Fig. 13 2–bromoalkylamine versus histamine acting on guinea pig ileum: …………………………………………………………...93 Fig. 14 Cimetidine versus histamine acting on guinea pig atrium: ……. 94 Fig. 15 Cimetidine versus histamine acting on contracting rat uterus: … 95 Fig. 16 Irreversible blockade of 2–chloroalkylamine versus inhibitory effect of histamine on spontaneous contracting rat uterus: ……………… 96

ABSTRACT

The preparation of the 2–(N,N–dibenzylamino)–1–P–haloaryl–1– hydroxy ethane was carried out according to the synthetic routes shown in the general scheme of work. The methods used are known in the literature. The acetophenones (1) were first brominated to the phenacyl bromides (2) using molecular bromine in dry diethyl ether at room temperature. The acidic conditions necessary for the reactions were attained by the small amount of HBr usually present in the bromine liquid. However, depending on the reactivity of the acetophenone, the use of a catalyst is sometimes recommended. This has actually been the case in the preparation of some of the phenacyl bromides in this work by using anhydrous AlCl3. The phenacyl bromides using Na BH4. This reagent was chosen because of its specificity in reducing Keto groups and because of the mild conditions usually used with it. The reductions were carried out in aqueous methanol suspension at room temperature and sometimes at 50°C. In all the reactions the phenacyl bromides and the bromohydrins indicated sufficient purity to justify direct subsequent use.

The bromohydrins were condensed with dibenzylamine (4) in dry benzene to give the amino alcohol (5). This is SN2 reaction and the choice of dry benzene has been the result of several trials of solvents of varying degrees of polarity. The use of heat was found essential for the increase of the yield of the amino alcohols. The condensation reactions were carried out in refluxing benzene.

Due to the high boiling point of dibenzylamine and its similar solubility properties to those of the amino alcohols, the latter were isolated from the reaction mixtures by preparative TLC. The amino alcohols were obtained as yellow liquids. The amino alcohols were used in the pharmacological screenings as their hyarochloride salts. These were obtained by adding calculated amounts of ethanolic HCl to the solutions of the amino alcohols free bases in absolute ethanol and precipitating the hydrochloride salts with diethyl ether. Two 2–hydroxyhaloalkylamine have been prepared according to well– documented synthesis routes in literature. The two compounds thus obtained were investigated pharmacologically to show their anti–ulcer activity. Both studied compounds blocked the relaxing effect of histamine in rat uterus. Both studied compounds antagonized histamine effect completely in guinea pig heart and brought the heart to its normal. In rat stomach, both compounds reduce ulcer lesions.

All the above actions of the studied were found to be through H2 receptors blocking activity. In addition the two compounds showed H1 antihistaminic activity as they blocked histamine effect in guinea pig ileum. ﺨﻼﺼﺔ ﺍﻷﻁﺭﻭﺤﺔ

ﻟﻘﺩ ﺘﻡ ﺘﺤﻀﻴﺭ ﻤﺠﻤﻭﻋﺔ ﻤﻥ ﺍﻷﻤﻴﻨﺎﺕ ﺜﻼﺜﻴﺔ ﺍﻟﺘﻜﺎﻓﺅ ﻁ ﺒ ﻘ ﺎﹰ ﻟﻁﺭﻕ ﺍﻟﺘﺤﻀﻴﺭ ﺍﻟﻤﻌﻤﻠﻴﺔ ﺍﻟﻤﻌﺭﻭﻓﺔ ﻭﺍﻟﻤﻭﺜﻘﺔ ﻓﻲ ﻜﺘﺏ ﺍﻟﻜﻴﻤﻴﺎﺀ . ﺘﻡ ﺍﻟﺘﺤﻀﻴﺭ ﻓﻲ ﺃﺭﺒﻌﺔ ﻤﺭﺍﺤل :

ﻓﻲ ﺍﻟﻤﺭﺤﻠﺔ ﺍﻷﻭﻟﻰ : ﺃﻀﻴﻑ ﺍﻟﺒﺭﻭﻡ ﻟﻤﺸﺘﻘﺎﺕ ﺍﻷﺴﻴﺘﻭﻓﻴﻨﻭﻥ . ﻭﻓﻲ ﺍﻟﻤﺭﺤﻠﺔ ﺍﻟﺜﺎﻨﻴﺔ : ﺘﻡ ﺍﺨﺘﺯﺍل ﻤﺠﻤﻭﻋﺔ ﺍﻟﻜﻴﺘﻭﻥ ﻟﻴﻨﺘﺞ ﺍﻟﻜﺤﻭل . ﻭﻓﻲ ﺍﻟﻤﺭﺤﻠﺔ ﺍﻟﺜﺎﻟﺜﺔ : ﺘﻡ ﺭﺒﻁ ﺍﻟﻜﺤﻭل ﻤﻊ ﺍﻷﻤﻴﻥ (ﺜﻨﺎﺌﻲ ﺍﻟﺒﻨﺯﻴل) ﻤ ﻌ ﻁ ﻴ ﺎﹰ ﺴ ﺎ ﺌ ﻼﹰ ﺃ ﺼ ﻔ ﺭ ﺍﹰ ﻭﺍﻟﺫﻱ ﺒﺩﻭﺭﻩ ﺘﺤﻭل ﻓﻲ ﺍﻟﻤﺭﺤﻠﺔ ﺍﻷﺨﻴﺭﺓ ﺇﻟﻰ ﻤﻠﺢ ﺍﻟﻤﺭﻜﺏ (ﻫﻴﺩﺭﻭﻜﻠﻭﺭﻴﺩ) ﺒﺘﻔﺎﻋﻠﻪ ﻤﻊ ﺤﺎﻤﺽ ﺍﻟﻬﻴﺩﺭﻭﻜﻠﻭﺭﻴﻙ ﺍﻹﻴﺜﺎﻨﻭﻟﻲ . ﻭﻫﻲ ﺍﻟﺼﻭﺭﺓ ﺍﻟﻌﺎﻤﺔ ﻻﺴﺘﻌﻤﺎل ﺍﻟﻤﺭﻜﺏ ﻓﻲ ﺍﻟﻤﺴﺢ ﺍﻟﻔﺎﺭﻤﺎﻜﻭﻟﻭﺠﻲ ﻭﺍﻟﻤﻌﻤﻠﻲ .

ﻓﻲ ﻜل ﻤﺭﺤﻠﺔ ﻤﻥ ﺍﻟﻤﺭﺍﺤل ﺃﺨﺘﻴﺭ ﺍﻟﻤﺫﻴﺏ ﺍﻟﻤﻨﺎﺴﺏ ﻭﺩﺭﺠﺔ ﺍﻟﺤﺭﺍﺭﺓ ﺍﻟﻤﻨﺎﺴﺒﺔ ﻭﺘﻡ ﺍﻟﺘﺄﻜﺩ ﻤﻥ ﻨﻘﺎﺀ ﺍﻟﻤﺭﻜﺏ ﺒﻔﺤﺹ ﻨﻭﺍﺘﺞ ﺘﺤﻠﻴﻠﻪ ﺍﻟﻜﺭﻭﻤﺎﺘﻭﺠﺭﺍﻓﻲ ﺒﻭﺍﺴﻁﺔ ﺍﻷﺸﻌﺔ ﻓﻭﻕ ﺍﻟﺒﻨﻔﺴﺠﻴﺔ .

ﺍﻟﻤﺭﻜﺒﺎﺕ ﺍﻟﺘﻲ ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻴﻬﺎ ﺒﺎﻟﻁﺭﻕ ﺍﻟﺴﺎﺒﻘﺔ ﺘﻡ ﺍﺨﺘﺒﺎﺭﻫﺎ ﻋﻠﻰ ﺃﻨﺴﺠﺔ ﻤﻌﺯﻭﻟﺔ ﻤﻥ ﺤﻴﻭﺍﻨﺎﺕ ﻤﻌﻤﻠﻴﺔ ﻟﻤﻌﺭﻓﺔ ﺁﺜﺎﺭﻫﺎ ﺍﻟﻔﺎﺭﻤﺎﻜﻭﻟﻭﺠﻴﺔ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﻘﺭﺤﺔ ﻋﻠﻰ ﺃﻋﻀﺎﺀ ﺃﺨﺫﺕ ﻤﻥ ﺤﻴﻭﺍﻨﺎﺕ ﻤﻌﻤﻠﻴﺔ ﻫﻲ ﻤﻌﺩﺓ ﺍﻟﺠﺭﺫ ﻭﺭﺤﻡ ﺍﻟﺠﺭﺫ ﻭﻗﻠﺏ ﺨﻨﺯﻴﺭ ) ، ﻭﻗﺩ H2ﻏﺎﻨﺎ . ﻭﻓﻴﻬﺎ ﺃﺜﺭﺕ ﺍﻟﻤﺭﻜﺒﺎﺕ ﻤﻥ ﺨﻼل ﻤﺴﺘﻘﺒﻼﺕ ﺍﻟﻬﺴﺘﺎﻤﻴﻥ ﺍﻟﺜﺎﻨﻴﺔ ( ﺨﻔﻀﺕ ﺁﺜﺎﺭ ﺍﻟﻘﺭﺤﺔ ﺍﻟﻤﺴﺘﺤﺩﺜﺔ ﻓﻲ ﻤﻌﺩﺓ ﺍﻟﺠﺭﺫ ، ﻭﺃﺒﻁﻠﺕ ﻤﻔﻌﻭل ﺍﻟﻬﺴﺘﺎﻤﻴﻥ ﻓﻲ ﺭﺤﻡ ﺍﻟﺠﺭﺫ ، ﻭﻗﻠﺏ ﺨﻨﺯﻴﺭ ﻏﺎﻨﺎ ، ﻜﻤﺎ ﺃﻨﻬﺎ ﺃﺒﻁﻠﺕ ﻤﻔﻌﻭل ﺍﻟﻬﺴﺘﺎﻤﻴﻥ ﻓﻲ ﺃﻤﻌﺎﺀ ) .H1ﺨﻨﺯﻴﺭ ﻏﺎﻨﺎ ﻤﻥ ﺨﻼل ﺘﺄﺜﻴﺭﻫﺎ ﻋﻠﻰ ﻤﺴﺘﻘﺒﻼﺕ ﺍﻟﻬﺴﺘﺎﻤﻴﻥ ﺍﻷﻭﻟﻲ ( CHAPTER ONE

INTRODUCTION

1.1 Histamine and Antihistaminic Drugs:

1.1.1 Histamine:

Histamine is found throughout the body, especially in the skin, intestinal

mucosa, and lungs.

H

CH2 C H

NN NH2

Histamine

Heparin: consists of a building block of glucuronic acid and glucosamine units with a sulfonic acid group on nitrogen and two sulfonic acid groups on oxygen. Molecular weight is approximately 10,000 to 13,000.

CO2H CH OH O 2 H H O H H H H O O OH H H OH 2SO H H OH + 3 H NHSO H 3 Heparin Histamine: is bound to heparin via an electrostatic bond. If all the “bound” histamine were released at once, death would result.

Histamine is formed by the decarboxylation of the amino acid histidine as shown

below.

H

CH2 CH2 NH2 CH2 C CO2H CO2 NNH NNH NH2

Histidine Histamine

Histamine exists in two tautomeric forms:

CH CH NH CH2 CH2 NH2 2 2 2 NNH H NN

Which is the pharmacological active form at all 's (O. Le

Roy Saierni, 1976).

1.1.1.1 Histamine–releasing substances:

Include d–tubocuraime morphine, snake and bee Venoms, surface–active agents (Tween's), and lobster.

Because histamine is so widespread, it has been accused of causing rhinitis,

dermatitis, asthma, anaphylaxis, and sever headaches. Pharmacologically, smooth

muscle is contracted, bronchioles are constricted, and salivary, gastric and pancreatic

gland, secretions are activated by histamine (O. Le Roy Saierni, 1976).

1.1.2 Anti–histamines:

Antihistamine in therapeutic doses do not effect the binding of histamine or

the release of histamine. Antihistamines have a single action: they compete with histamine for intracellular receptors, there are by decreasing the physiological action of histamine. The “antihistaminic moiety” that is, the chemical moiety responsible for the competition with histamine, is:

Structure emphasizes relationship with imidazole CC X N portion of histamine R or Structure emphasizes relationship with side chain C CRX of histamine N

When: X = C = propylamines (chlorpheniramine)

X = O = Ethanolamines ()

X = N = Ethylenediamines ().

1.2 Pharmacology:

There is very little difference in the competitive inhibition of one as compared to another. However, there are considerable differences in the nature of the secondary responses (side effects) to the antihistamines.

Ethanolamines (Diphenhydramine) and ethylene–diamines (Promethazine) can produce sedation and sleep; however, most of the propylamines (Chlorpheniramine) have minimal effects of this type. The ethanolomines and ethylenediamines have some activity. The sedative qualities of antihistamines facilitate their use for motion sickness, nausea and vomiting of pregnancy, insomnia, and surgical premeditation other uses unrelated to the sedative effect include treatment of seasonal “hay fever” serum sickness, and in combination with analgesic agents for colds. Antihisttamines provide only symptomatic relief from the common cold; they don not the underlying disorder, they are of little or no value for bronchial asthma (Rang H.P., et al., 1999).

Toxic responses to antihistamines are more disturbing than dangerous. There are frequent reports of excessive central nervous system stimulation when large doses are employed. Severe gastrointestinal distress has also been noted. The anticholinergic activity may produce a dry mouth and blurred vision. Serious consequences can result from use of antihistamines prior to driving a car, operating machinery, climbing stairs and ladders, etc.

1.3 Chemistry:

1.3.1 Salt formation: Salts form reading at the basic aminonitrogen (this also the case with local anesthetic and ).

(+) H CH3 CH 3 (-) R N + HCl R N Cl CH CH3 3

Such salts yield free amines on treatment with base. In the antihistaminic field, HCL is the acid most widely used for the salt formation. However, organic acids such as succinic, fumaric, maleic and tartaric acids are also used.

There is a noticeable difference in the potency of the various salts of particular antihistamine. This may be due to differences in solubility or absorption. Some salts are more stable than others. Hydrochlorides are frequently hygroscopic.

1.3.2 Structure–activity relationships (SAR):

R X C CN x = O, N, or C

a. A tertiary (3°) amine is required. –N (CH3)2 is more

potent than –N(C2H5)2. b. When X = N or X = O, the length between them should

be –CH2CH2– when a phenothiazine is present, an isopropyl group can observed. c. Introduction of a chlorine or bromine in the para– position of the phenyl group results in increased activity. d. Dextrorotatory isomers display greater activity than Levorotatory isomers in these compounds that exhibit optical isomerism. The dextrorotatory isomer of chlorpheniramine has the S configuration and is marketed as polaramine.

Cl 2

H H H C N CH3 1 N C C 3 CH3 H H e. When X = O, the most effective group for R is benzhydryl, which is shown below:

H C = R f. When X = N, a heterocycllic ring is most effectively attached at the 2 position.

That is:

CH3 (CH ) CH CH N 2 N 2 2 N CH 3 R = Benzl or benzl-like moiety. The methlyene unit (CH2) may or may not be present. g. Hydrogenation of an aromatic ring (either one) results in loss of activity.

1.4 Ethanolamine derivatives: OCH2CH2N

A – 1

CH3

O CH2 CH2 N CH C 3 H

Diphenhydramine (Benadryl)

2-Dimethylaminoethyl benzhydryl ether.

It is a component of Benylin Expectorant and Ambenyl Expectorant.

A – 2 Br

CH3 HH H C N NC C

CH3 H H

Bromodiphenhydramine (Ambodryl) has bromine in the para position of the aryl ring and has increased activity. A – 3

CH3 N H H CH3 O N

C O CH2 CH2 N C Cl N (+) N CH3 CH3 O (-)

Dimenhydrinate (Dramamine)

It is the salt of diphenhydraminne prepared by using 8–chlorotheophylline as the

acid.

CH3 O N N C Cl N CH3 N H

The circled proton is acidic. After removal of the proton, the anion is resonance stabilized. The chlorine aids the acidity through electron withdrawal.

8–chlorotheophylline. It is the popular drug for motion sickness.

A – 4 CH3

O CH2 CH2 N

CH3 CH2

Phenyltoloxamine (Bristamin) 2–Benzylphenyl 2–diamethylaminoethyl ether. It is found in Sinutab and

Naldecon.

A – 5

2 CH N CH3 3 1 C OCH2CH2N CH3

Doxylamine (Decapryn)

1–Phenyl–1–(2–pyridyl)–1–(2–dimethylaminoethoyl) ethane.

1.5 Ethylene diamine derivatives: NCH2 CH2 N

A – 1

CH2 CH3

N CH2CH2 N CH N 3

Tripelenamine (Pyribenzamine; “PBZ”) N,N–dimethyl–N– benzyl–N– (2–Pyridyl) ethylenediamine.

A – 2 N CH3 S CH2 N CH CH N 2 2 2-Thenyl 3 4 CH3

CH2 5 2 S 1 S 2-Thienyl (Histadyl)

Methapyrilene (Histadyl):

N–N–dimethyl–N–(2–Pyridyl)–N–(2–thenyl) ethylenediamine:

It has a rapid onset of action and short duration of action. It is found in Co-pyronil and numerous over- the counter sleep aids.

A – 3

S

N CH3 ethylene-diamine portion CH2 CH N CH3 CH3 Promethazine (Phenergan)

10–(22–dimethylaminoproppyl) phenothiazine promethazine displays maximum antihistamine action in a staggered configuration as shown below: S

N

H CH3 H H N

CH3 CH3

Promethethazine was found to be antihistaminic in malaria research. Structural modification of 4,4-diaminodiphenylsulfone led to the discovery of promethazine as an antihistaminic agent. (“DDS”)

4,4–diaminodiphenylsulfone

Promethazine has tranquilizing properties and antiemetic effects; It is also used to potentiate sedative drugs. H2NSO2 NH2 A – 4

Ethylene diamine portion

H CH2 CH2

C N N CH3

CN2 CH2

Cyclizine (Marezine) 1–Benzhydryl–4–piperazine

Cyclizine is not used as an antihistamine, but rather for prophylaxis and treatment of motion sickness.

A – 5

H CH2 CH2

C N N CH2

CH2 CH2 CH3 Cl

Meclizme (Bonine, Antivert) 1–(P–chlorobenzhydryl)–4–(m–methylbenzyl) piperazine. It is a powerful antihistaminic agent, but it is used in motion sickness.

Vertigo, or nausea and vomiting associated with radiation sickness.

1.6 Propylamine derivatives:

CH2 CH2 CH2 N

N CH3 CH2 CH2 CH2 N CH3

Cl

A – 1 Chlorpheniramine (chlor-Trimeton, Teldrin).

1– (2-pyridyl) 1–p–chlorophenyl–3–dimethylaminopropane.

chlorpheniramine is a potent antihistamine. Removal of the P–chloro group yields (inhiston) and there is a twenty fold decrease in activity.

A – 2

N CH3 CH2 CH2 CH2 N CH3

Br

Brompheniramine (Dimetane) has a p–bromo group instead of a p–chloro group.

A – 3 23 4 C CH2 CH2 N 1 Cl CH2

Pyrrobutamine (Pyronil)

1–P–chlorophenyl–2–phenyl–4–pyrrolidino–2–butene. It has slow onset and long

duration of action.

A – 4

CH2 N CH3 C C H N

Triprolidinne (Actidil)

1– (P–tolyl) –1– (2–pyridyl) –3– pyrrolidino–1–propene.

The pyrrolidino and 2–pyridyl groups must be trans for maximum antihistaminic

activity.

Triprolidine is a constituent of Actifed and Actifed–c expectorant.

1.7 Testing of antihistamines:

Antihistamines may be tested using the following procedures: by measuring the minimum amount of drug that relaxes the histamine–induced spasm of guinea pig ileum (Magnus procedure), by determining the protection afforded the guinea pig against histamine inhalation, and by determining the protection afforded the guinea pig against injection (intrave– nous) of lethal doses of histamine (O. Le Roy Saierni, 1976).

1.8 Gastric Secretion:

Histamine stimulates the secretion of gastric acid by action

of H2 receptors; it causes contraction of the smooth muscles of the ileum, bronchi, bronchioles and relaxation of uterus (Rang H. P., et al., 1999).

1.9 Cardiovascular effect of histamine:

Histamine dilates blood vessels in man by an action of H1 receptors. It increases the rate and the output of the heart by action on cardiac H2 receptors. It also increases vascular permeability by action on H1 receptors.

Histamine is a transmitter in C.N.S as a mediator of type 1 hypersensitivity reactions such as urticaria and hay fever (H1 receptors).

1.10 Histamine receptors:

Histamine produces its action by an effect on specific

histamine receptors which are of three main types H1, H2, H3.

The main action of histamine through its receptors are:

A. H1 receptors: A–1 Responsible for contraction of most smooth muscles other than that of blood vessels.

A–2 Increase vascular permeability and vasodilatation.

B. H2 receptors:

B–1 Cardiac stimulation. B–2 Stimulation of gastric secretion.

C. H3 Receptors:

Occur at presynaptic sites and inhibit the release of a variety of neurotransmitters.

Drugs acting on receptors may be agonist or antagonists to histamine action.

1.11 Histamine agonist:

It initiates changes in the cell function producing effects of various types

1.12 Histamine antagonist:

A. It bind to receptors without initiating such changes.

B. Selective antagonists of H1, H2, H3 receptors are:

– Mepyraminne. – Cimetidine.

– H2 antagonist.

– . Figure (1):

Chemical structure of histamine agonists and antagonists:

NH

H2N C S(CH2)3N(CH3)2

NH2 CH2 C H Methylhistamine N N CH3

Chemical structure of histamine antagonists:

CH3O

CH2 CH3

N CH2 CH2 N CH3 N

Mepyramine

H N C N NN S

Thioperamide

H2C CH2 SCH2 CH2 NH CNHCH3 HN N NCN

Cimetidine

CH2SCH2 CH2 C NH2

SN NSO2NH2

N C(NH ) 2 2

Famotidine

CH2SCH2 CH2NH C NHCH3 CHNO SN 2

CH N(CH ) 2 3 2

CH2SCH2 CH2 NH C NHCH3 O CHNO2 CH N(CH ) 2 3 2 Ranitidine

1.13 Aim of the project:

It has been thought logical if the beneficiary effects of the rapid onset of action and the long duration of action of two adrenoreceptor–blocking agents can be combined in one molecule. This will form the proposal project of our work.

Since H2–receptors are responsible for cardiac stimulation and gastric acid secretion, the suggested compounds would potentate rapid onset and long duration of action as H2–antagonists. The compounds to be prepared are of the following general structure shown below:

CH2 R

N CH2 CH2

CH2 X R

The structural similarities are clearly shown when compared with the chemical structure of the selective antagonists of H1, H2 and H3 receptors such as mepyramine (See structure below).

CH3O CH2 CH3

N CH2 CH2 N

CH3 N

X CH2 CH CH2 N Y OH CH2 X=H Y=Br or Cl

CH2 HN CH2 HN CH2 CH2

Br CH CH2Br Cl CH CH2Br OH OH

NaBH4 NaBH4

Br C CH2Br Cl C CH2Br O O

Br C CH Br 3 + 2 Cl C CH3 + Br 2 O O

General Scheme For the Preparation of Intermediates and the Aminohydroxy ethanes.

CHAPTER TWO

EXPERIMENTAL

2.1 Apparatus and materials:

Quick fit glass apparatus was used throughout the synthesis. TLC was carried out using plates 5 × 20 cm and 20 ×

20cm for preparative, and silica gel GF 254 or PF 254. Melting points were measured using Gallenkamp melting point apparatus. Infrared spectra were obtained using Perkin Elmer

1310 infrared spectrophotometer. Elemental analysis (C.H.N) for aminohydroxyethanes and were carried out in Butterworth laboratories, Butterworth, England. All solvents and reagents used were of GRR grade. All compounds have been obtained by general procedures. So the general procedure for each step will be regarded with modifications or alterations given as notes preceding each procedure description or in the tables following each step description. For the products obtained in the different steps the physical description, m.p., TLC and IR spectra are

shown in tabular form.

Table 1: Starting materials and specifications:

Comp. Structure M.W. Physical M.P. °C B.P. Refractory I.R. CO 20 -1 No. state index nD abs. am

I Br CO CH3 199 Solid 51 – 52 – – 1700

II Cl CO CH3 155 Liquid – – – 1700

Source: Donated by the British council

Table 2: Chemical nomenclature of the compounds synthesised

Comp. X Y Z Chemical name No.

III H Br OH 2–(N,N–dibenzylamino)–1–P–bromoaryl–1–hydroxy ethane

IV Cl OH 2–(N,N–dibenzylamino)–1–P–chloroaryl–1–hydroxy ethane

2.2 Methods:

2.2.1 Preparation of the 2–(N,N–dibenzylamino)–1– P–haloaryl–1–hydroxy–ethanes: 2.2.1.1 Preparation of the Bromoacetophenones from the Acetophenones:

X X C CH 3 + Br 2 C CH2Br Y O Y O

Compounds I or II Compounds III or IV

Notes: i- In all the cases the acetophenon: bromine molar ratio was 1:1. ii- In some cases dry Alcl3 was used as catalyst as indicated in the table (III). iii- Modifications of the general procedure are shown in the table (III).

General method procedure:

Calculated quantity of anhydrous bromine was added drop wise to a stirred ice–

cooled solution of the acetophenone in the solvent solution as continued until the

colour of bromine has disappeared. The solvent was immediately removed under

reduced pressure at room temperature. The phenacyl bromides separated as while

solids. The solids were recrystallized from suitable solvents as shown in Table (III). The phenacyl bromide separated before the addition of the bromine was completed. The solids obtained were washed with 50% ethanol.

2.2.1.2 The preparation of the Bromohydrins from the

Bromoacetophenone:

X X NaBH4 C CH2Br CH CH2Br Methanol Y O Y OH compounds III , IV compounds V , VI

Notes: i- The bromoacetophenones: NaBH4 molar ratio was 1:2. ii- The reflux time was varied according to reactivity as indicated in table IV.

General method procedure:

A solution of the bromo-acetophenone in methanol or absolute ethanol was added drop wise to a stirred ice-cooled slurry of sodium borohydride (NaBH). In methanol or absolute ethanol. The reaction mixture was then refluxed on a water bath.

During the reflux the reaction was followed by TLC using silica gel GE254 and CHcl4 as mobile phase the solvent was removed under reduced pressure and water was added to destroy the unreacted NaBH4 and the bromohydrin was extracted with chloroform. The chloroform. The chloroform extracts were washed with water and dried over anhydrous sodium sulphate. Removal of the chloroform under reduced pressure left a pale yellow liquid which was used in the next step without further purification.

TLC was done which showed one spot under U.V light and iodine vapour. Also infrared spectroscopy showed the disappearance of the absorption band at 1700cm-1 and the appearance of the absorption band at 3500cm-1.

2.2.1.3 The preparation of 1–hydroxy–1–aryl–2–(N,N– dibenzylamino) ethane:

X CH2 X CH2 CH CH2Br + HN CH CH2 N Y OH CH 2 Y OH CH compounds V , VI 2 compounds VII , VIII

Notes: i- In all the cases the bromohydrin: dibenzylamine molar ratio was 1:2. ii- The reaction mixtures were heated on water bath at 80°C. iii- The stirring time was from 72 hours to two weeks as shown for the individual cases in table (V).

General method procedure:

To a stirring solution of dibenzylamine in dry benzene was added a solution of the bromohydrin in dry benzene at room temperature.

The reaction mixture was stirred in a water bath at 80 °C. TLC on silica gel

GF254 and CHCl3 as mobile phase was performed to follow the reaction under U.V. light and iodine vapour. The amino alcohols were separated by preparative TLC. The aminoalcohols layer, which were natively coloured, were taken in CHCl3. after removal of the CH Cl3 under reduced pressure the aminoalcohals were obtained as thick pale yellow liquids.

2.2.1.4 The Preparation of 1–hydroxy–1–aryl–2–CN,N– dibenzylamino) ethanes hydrochloride salts:

X CH2 X H CH2 CH CH2 N ethanolic HCl CH CH2 N Cl Y OH CH2 Y OH CH compounds VII , VIII 2 compounds IX , X

General method procedure:

To a concentrated solution of the amino alcohol in methanol was added ethanolic HCl in a 1:1 molar ratio. The hydrochloride salt of the aminoalcohol was precipitated from the ethanol/methanol mixture by the addition of petroleum ether and cooling in a refrigerator over night.

2.2.2 Eexperimental Discussion:

2.2.2.1 Bromination:

X X dry ether C CH3 + Br 2 C CH2Br + HBr dry ALCl3 Y O Y O X= H , Y = Br , Cl

Mechanism:

The carbonyl group affects the acidity of α–hydrogen in just the same way it affects the acidity of carboxylic acids: by helping to accommodate the negative change of the anion (Morrison and Bayed 1).

O O C C H + H C H C H

Like bases, acids speed up the halogenation of ketones (Morrison and Boyed 2a). Acids are not consumed and hence catalysed halogenation (Morrison and Boyed 2b).

In the bromination of acetophenones, the rate determining step is the formation of enol, which involves two steps: rapid reversible protonation of carbonyl oxygen (step 1), followed by the slow loss of an α–hydrogen (step 2) (Morrison and Boyed 2 c). X X rapid C CH (1) C CH3 3 + Br HBr Y O Y OH

X H X (2) C C H + Br slow C CH2 + HBr Y OH H Y OH enol

X X (3) C CH2 Br rapid + Br C CH2Br + Br Y OH Y OH

X X ( ) 4 C CH2Br + Br C CH2Br + HBr Y OH Y O

Once formed the enol reacts rapidly with the halogen. The unsaturated enol undergoes addition: the partially positive halogen attaches itself to α carbon to form action. The ion 1 is a protonated ketone, loss of the proton yields the product (Morrison and Boyed 2d). From this we postulate the mechanism of bromination of keton in the presence of Lewis acid (AlCl3 anhydrus) as follows: X H X X C C C C C (1) + H = C + H O Y Y O Y O

X X C C C H C (2) + enol Y O Y OH

X C X C (3) + Br 2 + ALCl3 C CH2Br + Br A LCl3 Y OH Y OH

X X

(4) C CH2Br C CH2Br Y OH Y OH

X X (5) C CH Br 2 + Br A LCl3 C CH2Br + HBrAL+ Cl3 Y OH Y O

Since the Lewis acid AL Cl3 was not consumed, it acts as a catalyst. It acts as a carrier for the bromine action Br in a complex form, or bromine will be polarized to Br; Br and the partially positive part will attach the carbon of the acetophenone. This postulation was consistent with the progression of the reaction.

There was a latent period of about 20 – 30 minutes between the addition of the first drop of bromine and the addition of the first drop of bromine and the commence of the reaction. After the reaction begins it lasts abruptly and the colour of bromine disappears at once. The latent period is for the formation of the enol. X X methanol C CH2Br + NaBH CH CH2Br 4 or ethanol Y O Y OH

2.2.2.2 Reduction:

H X H X C CH Br 2 + H B H Na C CH2Br YHO Y O BH3Na

H2O X H

C CH2Br + NaOB( OH)2 Y OH

Bromohydrine

The reaction is nucleophilic addition: the nucleophile is a hydride ion H-:, transferred from boron to the carbonyl carbon of the acetophenone (Peter Sykes) sodium borohydride is a mild selective agent. It reacts sufficiently slowly with water in neutral or alkaline solution, that reductions which are reasonably rapid can be carried out in water solution without appreciable hydrolysis of the reagent (Fieser and Fieser). (Infrared spectroscopy showed OH absorption band at 3500cm-1 which was broad, and the disappearance of the absorption band at 1700 cm-1 this indicates the reduction of the carbonyl group to hydroxyl group).

2.2.2.3 Coupling of bromohydrin with benzylamine:

X X CH2 CH2 CH CH Br 2 + HN CH CH2 N

Y OH CH2 Y OH CH2 compounds V , VI compounds VII , VIII

Mechanism:

Since the bromohydrin is a primary alkylhalide, the reaction is a nucleophilic aliphatic substitution bimolecular, SN2 reaction, in which a halide ion, which is an extremely weak base is displaced by an other stronger base i.e. the secondary amine, dibenzylamine. Therefore, the reaction depends on the concentration of both the bromohydrin (substrate) and the amine (nucleophile) (Peter Sykes).

Since the order of reactivity of alkylhalides towards E2 or E1 elimination is the same: 30 > 20 > 10, we do not expect an alkene formation since the bromohydrin is a primary alkyl halide (Morrison an Boyed 3a).

- R - - H R R H R R NH+ R C Br N C Br N C H Br - + R - H - H R R H

From this we can postulate our reaction to proceed as follows:

X

H CH2 CH C Br + HN 1 Y OH H CH2

X H H H CH2 CH C N 2 Y OH Br CH2

transition state X H CH2 CH C N 3 YCOH H H2

Dibenzyl–amine (nucleophile) attacks carbon from the rear away from the bromine atom with complete stereochemical inversion of configuration in a single step reaction, that is, bond making and bond breaking occur simultaneously as shown above (Morrison and Boyed 3b). TLC on silica gel GF254 and chloroform as the mobile phase gave three spots the intensity of which was taken as semiquantitative measure of the product yield. The three spots were seen under U.V. light and iodine vapour. The aminoalcohols were isolated using preparative TLC in which the three zones were naturally coloured and no detecting aid was needed.

It was noticed that substitution on the benzene ring with halogen atom increased the yield. CHAPTER THREE

DISCUSSION

Positive and negative biological responses are believed to be caused by the binding of a ligand to a receptor. Ligands are the endogenous and exogenous chemical agents that bind to a receptor. The endogenous ligands that bind to and activate a receptor or are known as first messengers. First messengers are classified as neurotransmitters, hormones and autocoids (Gareth Thomas, 2000).

3.1 Receptors:

Are glycoproteins that are found either embedded in the membranes or inside the nuclei of cells. They are classified into four superfamilies according to their general structures and mode of action. The mechanism by which the chemical message carried by the ligand is converted by the receptor and its associated biochemical system is known as signal tranduction. Signal tranduction releases small molecules, known as secondary messengers, which target specific proteins with the cell. This stimulation results in the initiation of a series of biochemical events leading to a biological response. 3.2 Agonist:

Are drugs that give the same response, as the endogenous ligand for a specific receptor.

3.3 Antagonist:

Are drugs that inhibit the response of a receptor. The binding of bacteria, viruses and toxins to a receptor may result in unwanted pharmacological effects (Frank D. King, 1998).

3.4 Ligands:

Bind to receptors by covalent bonding, ionic bonding

(electrostatic bonding), dipole–dipole interactions (including hydrogen bonding), charge–transfer bonding, hydrophobic bonding and Van der Waals forces. Covalent bonding is the strongest form of ligand–receptor bond and it is usually irreversible. Ionic bonding between ligands and receptors is string and normally reversible. It is effective over larger distances than the other forms of bonding (Gareth Thomas, 2000).

The binding of a first messenger or drug to a receptor is believed to cause a change in the conformation of that receptor. This change stimulates further biochemical changes that lead to a cellular response. The receptors of superfamily type 1 often required the binding of two endogenous ligand molecules for activation. These receptors control the opening and closing of ion channels (Gareth Thomas, 2000).

The receptors of superfamily type 2 have seven membrane-spanning subunits. These subunits form a pocket that is believed to be the binding site for endogenous ligands. The binding of a ligand to this type of receptor results in

attraction of a G–protein to an intracellular domain of the receptor. G–proteins are proteins consisting of α–, β– and γ– subunits.

They can freely diffuse through the cytoplasm. The

binding of a G–protein to the receptor is followed by a series of biological changes, which either results in the activation/ deactivation of an enzyme system or opening/closing of an .

Superfamily type 3 receptors have only one transmembrane subunit. Built into their extracellular structure is

the tyrosine–kinase enzyme system. The binding of a ligand to a receptors is believed to result in dimerisation of the receptors and also phosphorylation of the tyrosine residues in the tyrosine- kinase domain. Proteins with an SH2 domain are attracted to and bind via their SH2 domain to the phosphorylated tyrosine–kinase domain, which triggers a wide variety of biological activities. Superfamily type 4 receptors are found in the nuclei of living cells. These, receptors are large proteins with conversed central sections containing two loops of about 12 – 15 amino acid residues held in position by coordination with zinic ions (zinic fingers). The binding of a ligand to the hormone receptor site,

which lies on the C–terminal side of the central region, is

believed to expose a DNA–binding domain, which ultimately leads to the production of specific mRNA that controls the production of the protein required for a specific cell response.

The affinity of a ligand is a measure of the ease with which a ligand binds to a receptor. Clark’s occupancy theory, which envisaged the ligand–receptor system as being the form of

a dynamic equilibrium, uses the dissociation constant (KD) for

the ligand–receptor complex as a measure of the affinity of a

ligand for a receptor. The lower the value of KD, the higher the

affinity of the ligand for the receptor. KD values are usually

recorded as PD2 values, EC50 and ED50 are used to compare the relative effects of the analogues of a lead compound in drug development (Gareth Thomas, 2000).

Ligand dose–response relationships: are normally determined in vitro using isolated tissue. Plots of response against molar concentration are hyperbolic in shape, those of percentage maximum response against log molar concentration are usually sigmoid in shape.

Agonists with similar structure acting on the same receptor will often exhibit similar parallel plots of percentage maximum response against log molar concentration. In a series of compounds, agonists that exhibit the highest response are known as full agonists and those that exhibit a lower response are known as in that series. Most drugs are partial agonists (Gareth Thomas, 2000).

Antagonists do not trigger a response when they bind to a receptor. Their presence inhibits the action of an agonist.

Antagonists are classified as competitive and non– competitive antagonists. The binding of competitive antagonist to a receptor is reversible. Its action is dependent on the concentration of the agonist: the higher the concentration of the antagonist, the higher the concentration of the competitive agonist needed to obtain a maximum response. The binding of a non-competitive antagonist to a receptor is irreversible. Its action is independent of the concentration of agonist: the higher the concentration of the non–competitive agonist, the lower the maximum response of the agonist. The concentration at which an antagonist displaces half of the endogenous ligand from a receptor is known as the IC50 for that system. It is a measure of the affinity of the agonist for the receptor and has some use in drug development. Clark’s original theory has been modified by Ariens and Stephenson to account for the existence of agonists, partial agonists and antagonists. Independently, they proposed that the action of a receptor was two–stage process. The first was the binding of the ligand to the receptor and the second was the initiation of the biological response. This second

stage is governed by the ability of the ligand–receptor complex to produce a response. Ariens defined this ability to produce a response by the concepts of (α), whereas, Stephenson used efficacy (e). Ariens modified Clark’s equation E αD to the form: = for full agonists α = 1; for partial Emax K D + D agonist α has a value between 0 and 1; for antagonists α = 0. Stephenson introduced the concept that the binding of a ligand to the receptor produced stimulus (S) that was related to the response. The stimulus was related to both the affinity of the ligand for the receptor as well as its efficacy. Stephenson E e[D] modified Clark’s equation to the form: S = = to Emax K D + [D] account for the fact that a maximum response may be obtained when only a proportion of available receptors were occupied. For full antagonists e = 0, whereas for agonists and partial agonists e, has a positive value. The higher the value of e, the higher the response and the lower the dose required to obtain a maximum response. Stephenson also introduced the concept of transducer function to account for the fact that the magnitude of the response is not linearly related to the stimulus.

The rate theory proposed by Paton states that the stimulus only occurs when the ligand first occupies the receptor site. Stimulation does not continue even though the receptor site is still occupied. Further stimulation of the receptor can only occur when the ligand has disengaged from the receptor. The rate theory states that the rate of disengagement of the drug from the receptor governs its general type of activity. The order of disengagement rate is:

Agonists (very rapid) > partial agonists > antagonists (very slow).

Rate theory offers an explanation of desensitisation (tachyphylaxis). Desensitisation occurs when repeated exposure of a receptor to identical doses of a drug results in a reduction of the response to the drug. The rate theory states that it occurs because the rate of dissociation of an agonists is so slow that some of the receptors are still occupied and therefore unable to be stimulated when the next dose of the agonist is given (Gareth Thomas, 2000).

The two–state model of receptor action postulates that receptors exist in two form, the relaxed state (R) and the tensed state (T). Receptors in the R state can provide a stimulus but those in the T state are unable to produce a stimulus. In the absence of any ligands a population of a group of receptors of the same type exist as a dynamic equilibrium mixture of receptors in the R and T states. Stimulation occurs when a ligand binds to a receptor in the R state, but no stimulation occurs when ligands bind to receptors in the T state. Full agonists have a strong affinity for the R state. When they bind to a receptor I its R state the equilibrium moves to increase the number of receptors in the R state, which results in stimulation. Antagonists have strong affinity for the T state. When they bind to a receptor in its T state the equilibrium moves to increase the number of receptor in the T state, which inhibits stimulation. Partial agonists have an affinity for both states of the receptors, the degree of partial agonism depending on the position of equilibrium (Gareth Thomas, 2000).

Agonists often have structures that are similar to the corresponding ligand but the structures of antagonists can be quite different to that of the endogenous ligand for the receptor.

Receptor Histamine Endogenous ligand: NH2

HN N

Histamine Agonists: NH2

HN N

CH3

2-Methyl histamine

NH CH3 2

HN N

4-Methyl histamine

NH2 N

2-(2-Pyridyl)ethylamine

Figure 2: Examples of the structures of the agonists of histamine receptors

Concentration of X increasing

A

100%

X1 X2 X3

50%

Response (% max) 0%

EC50 New EC50

of drug A of drug A

Log agonist

Figure 3: The effect of an ideal competitive antagonist X on the dose–response curves for an agonist A. Plot A is the dose response curve for the agonist A in the absence of antagonist X. Plots X1 to X3 are the dose–response curves for the agonist A in the presence of three different constant concentrations (X1, X2 and X3) of the antagonist X. The value of the EC50 of A will depend on the concentration of the antagonist.

Receptor + Agonist Receptor - agonist Normal Response (a)

Antagonist + Receptor + Agonist

K1 Agonist + Receptor - Antagonist Receptor - Agonist + Antagonist

Reduced response (b)

Figure 4: An outline of the mode of action of a competitive antagonist: (a) with no antagonist and (b) with an antagonist

100

x1

x2

50

x3 Response % max

Increasing contraction of X

0

Log [agonist]

(a)

Antagonist

Allosteric site Receptor site

Distorted receptor site (b)

Figure 5: The effect of a non–competitive antagonist on the dose–response curves for a drug A. Plot A is the dose–response curve for the agonist A in the absence of the antagonist X. Plot X1 is the dose– response curve for the agonist A in the presence of a constant concentration (X1) of the antagonist X. The effect of increasing the constant concentration of X is shown in the succeeding plots (b) A presencentation of the general mode of action of a non–competitive antagonist.

Agonist’s normal response

(antagonist is absent)

Response

Antagonist, ideal case

0.0 (agonist is absent) Concentration of agonist

Figure 6: The effect of an ideal antagonist on the response of a receptor

Figure 7: (a) No desensitization (b) Desensitization.

The start of each peak corresponds to the administration of an identical dose of the agonist. X CH2 CH CH2 N Y OH CH2 X=H Y=Br or Cl

CH2 HN CH2 HN CH2 CH2

Br CH CH2Br Cl CH CH2Br OH OH

NaBH4 NaBH4

Br C CH2Br Cl C CH2Br O O

Br C CH Br 3 + 2 Cl C CH3 + Br 2 O O

General Scheme For the Preparation of Intermediates and the Aminohydroxy ethanse.

Table I: starting materials and specifications

Comp. Structure M.W. Physical M.P. B.P. Refractory I.R. CO 20 -1 No. state °C index nD abs. am I 199 solid 51-52 - - 1700 Br CO CH3

II 155 liquid - - - 1700 Cl CO CH3

Source: Donated by the British council

Table II: Chemical nomenclature of the compounds sythesised

Comp. X Y Z Chemical name No. III H Br OH 2-(N,N-dibenzylamino)-1-P-bromoaryl-1- hydroxy ethane IV Cl OH 2-(N,N-dibenzylamino)-1-P-chloroaryl-1- hydroxy ethane

CHAPTER FOUR

INTRODUCTION

4.1 Histamine:

Histamine is β–imidazolylethylamine derivative that is present in essentially all mammalian tissues. The major physiological actions of histamine are centered on the cardiovascular system, nonvascular smooth muscle, exocrine glands and the adrenal medulla. The actions of histamine that are of interest from both a pharmacologic and therapeutic point of view include:

1. Its important but limited role as a chemical mediator of hypersensitivity reactions. 2. A major role in the regulation of gastric acid secretion. 3. An emerging role as a neurotransmitter in the CNS. (Jack De Ruiter, 2001).

4.1.1 Histamine Synthesis:

Histamine is synthesized in cytoplasmic granules of its principle storage cells, mast cells and basophils. Histamine is formed from the naturally–occurring amino acid, S–histidine via the catalysis of either the pyridoxal phosphate–dependent enzyme histidine decarboxylase or aromatic amino acid decarboxylase. Histamine is found in almost all mammalian tissues in concentration ranging from 1 to > 100 mg/g. It is in particularly high concentration in skin, bronchial and intestinal mucosa. It is found in higher concentrations in cerebrospinal fluid than in plasma and other body fluids.

NH2 NH2 Histidine OH decarboxylase N N HN O HN

Histidine Histamine

4.1.2 Histamine storage and release:

Most histamine is synthesized and stored in mast cells and basophile granulocytes. Protein complexed with histamine is stored in secretory granules and released by exocytosis in response to a wide variety and immune (antigen and antibody) and non–immune (bacterial products, xenobiotics, physical effects and effects) stimuli. The release of histamine as one of the mediators of hypersensitivity reactions is initiated by the interaction of an antigen IgE complex with the membrane of a histamine–storage cell. Exocytotic release of histamine follows the degradation of histamine storage cells. Histamine is released from mast cells in the gastric mucosa by gastrin and acetylcholine.

4.1.3 Histamine Metabolism: Three principle ways for terminating the physiological effects of histamine.

1. Cellular uptake, Na+ dependent process.

2. Desensitization of cells; some H1 receptor– containing tissues exhibit a homogenous loss of sensitivity to the actions of histamine.

3. The most common pathway for terminating histamine action, involves enzymatic inactivation. The enzyme histamine N–methyltransferase which catalyzes the transfer of methyl group to the ring Nj (N–atom in position 1 in imidazole ring) nitrogen of histamine producing Nj methyl histamine (Jack De Ruiter, 2001) or metabolized to imidazole acetic acid monoamine oxidase (Walting, K.J., 1998.)

NH2

N HN Histamine

histamine N-methyl diamine -transferase oxidase

OH NH2

O N H N HN H H3C N

imidazole acetic acid N-Methyl histidine 4.1.4 Histamine Receptors:

Histamine, like many other transmitters, mediates responses via receptors which are divided into:

1. H1 receptors which are found in the smooth muscle of the intestines, bronchi and blood vessels.

2. H2 receptors which are found in gastric parietal cells and in the heart and central nervous systems.

3. H3 receptors which are found in brain and in the periphery and regulate histamine release.

4. H4 receptors via which histamine mediates signaling and chemotaxis of mast cells. This mechanism might be responsible for mast cells accumulation in allergic tissues (Hofstra, C.L., et al., 2003).

4.1.5 Chemical structure of histamine and its agonist:

NH2 CH2 CH2 NH2 CH2 C NH H HN N HN N CH3 NH2 C S(CH2)3N(CH3)2

Histamine Dimaprit α–methyl histamine

4.1.6 Chemical structure of histamine antagonists: H3CO

CH2 CH3 NCH2 CH2 N CH3

N

Mepyramine

N C NH H3CCH2 SCH2 CH2 NH C NHCH3 N N S HN N NCN

Thioperamide Cimetidine

CH2SCH2 CH2 CNH2 CH SCH CH NHC NHCH3 2 2 2 NSO2NH2 O SN CHNO2

CH2 N(CH3)2 NC(NH2)2

Ranitidine Famotidine

CH2SCH2 CH2NHC NHCH3 CHNO SN 2

CH2 N(CH3)2

Nizatidine

4.1.7 Structure Activity relationship of histamine agonist

and antagonist:

Histamine known as 4(5–)(2–aminoethyl)–imidazole, consists of an imidazole heterocycle and ethylamine side chain. The methylene groups of the aminoethyl side chain are designated as and α and β. The side chain is attached, via the β–CH2 group to the 4 position of an imidazole ring. The imidazole N at position 3 is designated as the pros (π) N whereas the N at position 1 is termed the tele (τ) N. The side chain N is distinguished as Nα.

β NH2 NH2

5 4 α π τ τ N 3 π N H H N N 1 2

Nτ-tautomer π N -tautomer Histamine is a basic organic compound capable of existing as a mixture of different ionic and uncharged tautomeric species as shown below: NH2

N H H N

Dication NH2 NH2

N H N N H N Nπ−Τautomer Nτ−Tautomer (Monoaction) (Monocation)

NH2

NH2

N H N N H N

NH2

N N

+ Structure relationship studies suggest that the α–NH3 monoaction is important for agonist activity at histamine receptors and that transient existence of the more lipophilic uncharged histamine species may control the translocation of cell membranes. Other studies support the proposal that the Nτ–H tautomer histamine is the pharmacophoric species at the

H1 receptor while a 1,3–tautomer system is important for

selective H2 receptor agonist (Jack De Ruiter, 2001). Histamine is a chiral molecule, however histamine receptors are known to exert a high degree of stereo selectivity toward chiral ligands. Molecular modeling and steric–activity relationship studies of the influence of conformational isomerism on the activity of histamine suggest the importance of trans–gauche rotameric structure in the receptor activities of this substance. Studies with conformationally–restricted histamine analogues suggest that, while the trans rotamer of histamine

possesses affinity for H1 and H2 histamine receptors, the gauche

conformer does not act at H2 sites (Jack De Ruiter, 2001).

H N N

N N

H H H3N H

H H H H NH3 H trans gauche 4.2 Peptic Ulcer:

2.1.1 Peptic ulcer prevalence:

Mankind has lived with peptic ulcers since ancient times, one out of every ten American males will probably develop peptic ulcer disease some time in life. In the U.S. alone 300,000 new cases of peptic ulcer are diagnosed each year (Sturdevant, 1976). In Sudan no statistics are available but a work done by Osman (1973) suggests an overall incidence of 4/1000/year. Among western populations the incidence of duodenal ulcer increased up to the age of 35 (Bodemar, 1978). H. pylori infects one half or more of the world population and causes chronic gastritis, peptic ulcer and probably gastric cancer as well, and although its prevalence is declining in most developed countries, it is still contributing to a significant proportion of peptic ulcer globally (Leung, W.K.J. and Graham, D.Y., 2001).

Since there were insufficient records of the incidence of peptic ulcer over long periods, the actual trend of incidence has been difficult to follow. In spite ulcer remains an economically crippling disease responsible for a big loss whether surgical or medical and causes an overall mortality of 10%. Majority of deaths occurring within the first few years of diagnosis (Bonnevie, 1978).

4.2.2 Types of peptic ulcer:

The term acid peptic disorder encompasses a variety of relatively specific medical conditions in which injury to stomach and duodenum by gastric acid (& activated pepsin) is thought to play an important role. These disorders include:

- Gastresophageal Reflux Disease (GERD).

- Benign peptic ulcers of stomach and duodenum.

- Ulcer secondary to the use of conventional nonsteroidal

antinflammatory drugs NSAIDs. - Ulcer due to the Zollinger Ellison syndrome (which is rare), which is due to a gastrin producing tumour.

- Malignant ulcer.

4.2.3 Peptic ulcer etiology:

The genesis of peptic ulcers involves infection of the gastric mucosa with H. pylori or by an imbalance between the mucosal damaging mechanism (acid, pepsin) and mucosal protecting mechanisms (bicarbonate, mucus local synthesis of

PGE2 and PGI) (Rang, H.B. et al., 1999).

The stomach secrets about 2.5 liters of gastric juice daily. The principal exocrine secretions are pepsinogens from the chief or peptic cells, and hydrochloric acid from the parietal cells, gastric mucosa and bicarbonate ions also are secreted.

The gastric acid secretion, which is a complex continuous process, is controlled by multiple central (neural) and peripheral (endocrine, paracrine) factors, is one of the aggravating factor for peptic, duodenal ulcer.

The most potent endogenous secratogogues that induce excessive, release of HCl, are acetylcholine, histamine and gastrin. Each of these has its own receptors located on the pariated cells. Also release may be due to basal activity of the parietal cells. Normally parietal cells secreate acid by the aid of membrane pump known as H+/K+ ATPase which extrudes H+ from inside cells and move K+ ions into the cells.

In addition, one of the ulcer causes is, dysfunction of the lower oesophageal sphincter of unknown origin is the main etiology.

Less common pathophysiological reason are disorder of oesophageal motility, delayed gastric emptying and bile reflux (Strobel, et al., 2001).

Helicobacter infection is a one of peptic ulcer causes. H. pylori is a gram negative spiral organism that colonizes and survives in the deep mucosa layer and attaches to the gastric surface cells. It is an important risk factor for peptic ulcer diseases and chronic gastromucosal damage. It causes chronic gastritis (non atrophic) which progresses to atrophic gastritis H. pylori in childhood may cause gastric cancer in late life (Saloh, et al., 2001). Nonsteroidal anti–inflammatory drugs are major cause; that contributing to significant proportion of peptic ulcer globally, since it disturbs mucosal protecting mechnism. Chronic NSAID users have a 2% to 4% of developing asymptomatic ulcer, gastrointestinal bleeding or even perforation (La Conte et al., 1999; Wolf et al., 1999).

Stress is one of ulcer causes which accelerates the trafficing of granulocytes from bone marrow to the gastric mucosa (Kawamura, T. et al., 2000). There is association between alcohol consumption and peptic ulcer since it damages the mucosal layer in the GIT exposing the tissue to the direct action of HCl.

Ulcer is found to increase with a number of smoked cigarettes and length of smoking history (Harrison et al., 1998).

Also there are dietary factors e.g. dietary deficiency of protein and vitamins, spicy and less content fibers food.

All of the above causes collectively play important part in the incidence of ulcer day after another.

4.2.4 Ulcer treatment:

Healing of peptic ulceration can be promoted dy general measures e.g. stopping alcohol and smoking, taking high fiber content diet rich with protein and vitamin, but commonly healing of peptic ulceration can be promoted by: 1- Surgical treatment.

2- Medical treatment (drugs).

4.2.4.1 Surgical treatment:

Many duodenal ulcer patients are being treated as the long term outcome of surgery proved better but many factors affect the choice of operation e.g operative mortality, risk of gastric carcinoma. As causal surgical therapy for disorder fundoplicalion has been developed 50 years ago. This technique uses wrap of gastric fundus around the distal esophagus as reflux barrier. Because of post operative complication after fundoplicalion medical therapy become the treatment of choice and improved by introduction of labroscopic (Strobel, et al., 2001).

4.2.4.2 Medical treatment (medicines):

The stomach protects itself from damage by gastric acid through several mechanisms such as the presence of intracellular tight junctions between the gastric epithelial cells, mucin layer overlying the gastric epithelial cells, also presence of prostaglandin in the gastric mucosa and secretion of bicarbonate ions into the mucin layer form unstirred gel layer. In addition prostaglandin E inhibits gastric acid secretion by direct effect on the parietal cells also prostaglandin E enhance mucosal blood flow and stimulates secretion of mucin and bicarbonate. So the imbalance between mucosal defence factors (bicarbonate, mucin and prosfaglandine …etc) and aggressive factors (HCl & pepsin) result in ulcer (Range, H.B., et al., 1999).

So healing of peptic ulcer is promoted either by:

1) Neutralizing secreated acid with antacids.

2) cytoprotective mechanism:

i- Sucralfate. ii- Bismuth chelate. iii- prostaglandin analog e.g. misoprostol

3) Decreasing the secretion of the acid by.

i- H2 . ii- Proton pump inhibitor. iii- Muscarinic antagonists.

4) Treatment of Helicobacler pylori infections.

4.2.4.2.1 Antacids:

These act by neutralizing gastric acid and thus raising gastric pH. The common antacid used are salt of magnesium and aluminum e.g. magnesium hydroxide, magnesium trisilicate, aluminum hydroxide gel and sodium bicarbonate which raise pH to 7.4. CO2 is liberated which stimulates gastrin secretion and can result in secondary acid secretion.

4.2.4.2.2 Sucralfate:

The sucralfate is octasulfate of sucrose to which aluminum hydroxide has been

added. In an acid environment it undergoes extensive cross linking and

polymerization to produce a viscous and sticky gel that adheres strongly to epithelial

cells, thus inhibits the hydrolysis of mucosal proteins by pepisin. Also sucralfate stimulates local production of prostaglandin and epithelial growth factor (EGF). The

adverse effect of sucralfate is constipation and it alters the bioavailability of other drugs since it forms viscous layer in the stomach. Sucralfate use has diminished in

recent years.

4.2.4.2.3 Bismuth chelate:

Coloidal bismuth subcitrate, tri potassium dicitrato bismuthate has a protective action seemed in coating the ulcer base, adsorbing pepsin, enhancing prostaglandin synthesis and stimulating bicarbonate secretion.

4.2.4.2.4 Prostaglandin analog; Misoprostol:

Misoprostol is cytopratective. It provides physical barrier over the surface of the ulcer. Prostaglandin PGE2 and PGI2 are the major prostaglandins synthesised by gastric mucosa. They inhibit acid production by binding to the EP3 receptor on the parietal cells. Also they can prevent gastric injury by cytoprotective effects such as stimulation of secretion of mucin and bicarbonate and improvement of mucosal blood flow. The most reported side effect of misoprostal is diarrhoea, abdominal pain and cramps also it is contraindicated during pregnancy since it causes abortion.

4.2.4.2.5 Histamine H2 receptors antagonists e.g. cimetidine, ranitidine and famotidine:

The discovery of histamine H2 receptor antagonist and their refinement was a major break through in the medical treatment of peptic ulcer, and with their development the medical therapy became the treatment of choice for peptic ulcer.

H2 receptor antagonist competitively inhibit histamine

action at all H2 receptors, but their main clinical use is for inhibiting of gastric acid secretion. They inhibit histamine– stimulated and gastrin–stimulated acid secretion and decrease acetylcholine–stimulated acid secretion. Pepsin secretion also falls with the reduction in volume of gastric juice.

These agents not only decrease both basal and food stimulated acid secretion by 90% or more, but promote healing of duodenal ulcer as shown by clinical trials

(Rang, H.B, et al., 1999).

4.2.4.2.6 Proton Pump Inhibitors:

These agents are most effective drugs used as antiulcer therapy. They act by irreversible inhibition of the H+/K+ Atpase (the proton pump) they inhibit both basal and stimulated gastric acid secretion. They inhibit cytochrome P450 enzyme, therefore decreasing the metabolism of some drugs e.g. warfarin, phenytion.

4.2.4.2.7 Treatment of H. pylori infection:

H. pylori is a Gram negative bacillus which has been associated with gastritis and subsequent development of gastric and duodenal ulcer, adenocarcinoma and gastric B cell lymphoma (Veldhuyzen and Lee, 1999). Also responsible for failure and relapse. Because of its role in pathogenesis of peptic ulcer in the majority of cases it has become standard measure to eradicate this infection in patients with gastric or duodenal ulcers (Graham, 1997; Chita etal., 2000). Since single antibiotic regimens are ineffective in treating H. pylori infection and led to resistance so eradication obtained by triple therapy regimen containing pH–dependent antibiotic e.g. amoxicillin or

clarithromycin + H2 receptor antagonist or proton pump inhibitor + metronidazole.

In researches triple therapy regimen containing clarithromycin + Amoxcillin + omeprazole for one week yielded a healing of ulcer by 94% (Wong, B.C. et al., 2000).

4.2.5 Efficacy:

Proton pump inhibitors promote more rapid relief of duodenal ulcer symptoms and more rapid healing than do H2 receptor antagonist (McFarland et al., 1990).

H2 receptor antagonists are effective in the short term healing of duodenal ulcer as compared with placebo e.g. four weak healing rate for cimetidine ranges from 45 – 84% (Binder H.J. et al., 1978), for ranitidine 69% to 79% (Hirschowitz, B.J. et al., 1986), for famotidine 67% to 77% (Gitin N, 1987). When treatment is extended to 6 – 8 week the healing rates rise to 82 – 95%.

Generally, H2 blockers relieve pain sooner.

4.2.6 Side effects:

Although there are adverse effects associated with drugs used to inhibit or neutralize gastric acid secretion including diarrhea, or constipation, headache dizzness fatigue, nausea or vomiting, anxiety, back pain also CNS effects e.g. delerium, confusion, lethergy, restlessness, seizures, slurred speech, halucination depression and facial twiching, but all have proved safe in extensive world–wide experience and adverse effects are readily reversed on withdrowal of treatment. 4.2.7 Drug–Durg Interaction:

All drugs used for suppression of gastric acid production potentially interfere with absorption of other drugs due to the comlexities of the relationship between drug absorption and gastric acid secretion.

Drugs that may have altered absorption when administered together with an H2 blocker include:

Furosemide, tetracycline, salicylate, diazepam, however clinically significant decreased absorption has been observed when Ketoconazole (Nisoral) is administered with cimetidine.

The most significant effect of H2 antagonist specifically cimetidine on other drugs is related to the cytochrome P–450 system which is largely responsible for the oxidative metabolism of many drugs.

When cimetidine binds to the cytochrome P450 system oxidative metabolism is impaired. Thus when drugs that depend on this system for elimination are administered with cimatidine serum levels of the drugs will increase if dosage is not reduced. Drugs that cimetidine reportedly affects in this manner include, theophylline warfarin, phenytoin, propranolol, chlordiazepoxide, metronidazole, and lidocaine.

Cimetidine also interferes with elimination of agent such as procainamide and N–acetylprocainamide by competitively inhibiting renal tubular secretion.

Ranitidine and famotidine have not been reported to bind significantly to the cytochrome P–450 system (Staiger et al., 1984; Powell, K et al., 1984).

4.2.8 Rationale:

As the incidence of ulcer increases day after the other and relapse still occurs when treatment seizes.

As all the discovered anti–ulcer drugs fails in treating ulcer completely and in preventing recurrence, in addition of many effects associated with the use of discovered anti–ulcer drugs. So clinical challenges still needed to synthesize or to discover a new anti–ulcer drug that assures excellent and complete healing results, fast relief of gastric pain, excellent safety profile and minimal side effect, maintenance of healing and patients, compliance.

Therefore, present work is a trial to satisfy all above demands. CHAPTER FIVE

EXPERIMENTAL

5.1 Chemicals and their sources: Histamine acid phosphate BDH (England) Oestradiol benzoate Alvetra (GMBH) (W. Germany) Propylene glycol 600 Sigma (USA) Aspirin (acetyl salicylic acid) Sigma (London) Ltd., Poole Dorset

5.2 Equipments and instruments:

- Surgical instruments, scissors, forceps, blades, syringes (1, 3, 5 ml), Needle and thread.

- Cylinder of gas mixture 95% oxygen 5% carbondioxide.

- Petri dishes.

- Isometric and isotonic transducer.

- Isolated tissue bath (Harvard).

- Harvard universal electronic recorder

- Magnifier (x 20). 2.2.3 Animals:

- Albino wister rats

- Guinea pigs

Which are bred on wheat, grain and grass in the animal house of the faculty of pharmacy U of K.

5.4 Methods:

5.4.1 Contracting Rat uterus:

This was prepared according to the method described by Vane, and Williams, (1973). Young female wister rats (100 – 150g) were brought into oestrous by injection with oestradiol benzoate 2.5mg/kg subcutaneously 40 hours prior to the experiments. The animal was killed by dislocating the neck and the abdomen was opened. The intestine was removed aside to expose the uterus. The two uterine horns were dissected and transferred to a Petri–dish containing Ringer Locke's solution. Fats and adventitia were removed. Each horn was opened longitudinally to form a sheet instead of narrow tube, and suspended in a 25ml organ bath containing Ringer Locke's solution at 37°C aerated with oxygen. The free end of the horn was attached with a thread to an isometric transducer (Harvard) and this was connected to Harvard oscillographic recorder. The other end attached to a fixed pin. The preparation was allowed to equilibrate for 45 minutes under 0.5 g resting tension before addition of drugs. Histamine was allowed 2 minutes contact time with the tissue while the drug under test were left fore 5 minutes contact time with the tissue.

5.4.2 Evaluation of antiulcerogenic activity using experimentally–induced ulcer in rat stomach and duodenum: This was prepared according to Selye and Szabo (1973), Robert et al., (1974) and Scarpignato, Tramacere and Zappia (1987), peptic ulcer can be induced in fasting male rats by specific H2 receptors stimulation through intravenous injection of a single large dose of an ulcerogenic agent. In this experiment ulcer was induced in the stomach of fasting rats by oral administration of aqueous suspension of acetyl salicylic acid (300 mg/kg). 30 male rats were housed in a suitable environment of lighting, temperature, food and water supply for a week to get acclimatized. Then the rats were been housed individually in cages. On the day before the experiment the rats were divided in groups of six per group and were marked. The rats were fasted from food for 24 hrs but free access to water was allowed. On the day of experiment the rats were weighed individually and a calculated doses of the test compound of 0.1, 1, 10, 100 mg/kg in a volume of 2 ml/kg was administered orally by oral gavage to four rats while the other two were given N. saline by oral gavage. 45 minutes later aqueous oral suspension of aspirin 300mg/kg is administered to all rats and rats were returned to their cages. Twenty–four hours later, rats were killed by dislocating the necks and the abdomens were opened to expose the stomach and duodenum. Both organs (stomach and duodenum) were removed and opened carefully (the stomach opened along the greater curvature and washed with saline interiorly clean. Using a high power magnifier, red spots (representing ulcer lesions were identified and counted. 5.4.3 Guinea Pig ileum preparation: This preparation was carried out for testing the compounds with potential H1 histamine receptor antagonistic effects. The animal was killed with a blow to the head or back of the neck. With the animal on its back a midline incision was made to open the abdominal cavity. The ileum was removed into container of cold Tyrode’s solution. Then it was washed internally to free food particles and was cut into several pieces, 2 or 3 cm in length. Then it was suspended in the tissue bath. (10 ml size filled with Tyrode’s, solution maintained at 37C° and aerated with 95% oxygen and 5% carbon dioxide. Different doses of histamine were applied to the tissue and the responses were recorded. Then the tissue was washed thoroughly with warm (37C°) Tyrode’s solution several times to remove histamine. The tissue was equilibrated for 30 min. with periodic washing (two times). Then the compound was added to the bath allowed in contact with the tissue for 20 minutes. Then a submaximal dose of agonist – histamine – was repeated and response was recorded again. 5.4.4 Guinea Pig atrium preparation:

This preparation was carried out as described by Emmanuel, and Thompson (1990). A large guinea pig was killed by dislocating its neck. With the animal on its back, a midline ventral incision from the neck region to the abdomen was made and the chest cavity was exposed using a pair of scissors. A cut was made through the ribs and the heart was quickly removed. The heart was transferred to a petri–dish containing ice–cold Ringer Locke's solution. The pericardium and fat tissues were carefully removed. The atria were separated from the ventricles carefully by cutting at the atrioventricular septum. A thread was attached to the tip of each atrium and the preparation was mounted on a tissue holder. The tissue was then transferred to 25ml organ bath filled with aerated Ringer Locke's solution maintained at 30°C. The upper thread was attached to the Harvard oscillographic recorder. About three readings of the spontaneous atrial rate were made before drugs addition to ensure that the preparation was steady.

5.4.5 The principle of pA2 system:

The pA2 system was devised by Schild in 1947 for expressing the potency of competitive drug antagonist, this parameter is equivalent to the affinity of the antagonist to the receptor. It is a measure of the potency with which an antagonist drug reduces the effect of an agonist in any one isolated preparation and is defined as the negative logarithm to the base ten of the molar concentration of an antagonist which reduces the effect of a multiple dose of an agonist to that of a single dose.

pA2 can be described and calculated mathematically from the following equation: Log10(DR–1) = log10A + log10Ka

Where A is the molar concentration of antagonist which is required to produce a dose ratio (DR = ratio of dose of agonist required to produce a given response in presence of antagonist compared to that in absence of antagonist).

Log10Ka (Ka = the association constant for the antagonist– receptor interaction) = pA2 value.

5.4.6 Determination of pA2 value of cimetidine p–chloro, and p–bromonalkylamine acting on rat uterus:

pA2 of the three compounds was determined by constructing a dose response curve to histamine using 4 doses which gave responses between 25% and maximum. A dose, which gives about 50% of maximum was selected (ED50). Then the dose was repeated to ensure that it is reproducible. A certain dose of each of the three compounds was added to the bath alone and left to equilibrate for 3 minutes, then twice the ED50 of histamine was added to the bath. Repeated four times until the original response in the absence of antagonist was restored, doubling the dose of antagonist each time until the response to

2ED50 became less than the original ED50 in the absence of antagonist. 5.4.7 Determination of pA2 value of mepyramine, 2–chloro– alkylamine and 2–bromoalkyamine acting on guinea pig ileum:

The same above method was followed.

Response obtained to 2ED50 of histamine was plotted

versus log10 concentration of each antagonist, and horizontal line corresponding to the response to ED50 in the absence of

antagonist. Where these two intersected correspond to the pA2 concentration of antagonist.

Materials: 5.5

Preparation of test solution and blank:

Fresh solutions of the test drugs which were in the form of hydrochloride salts were prepared for each experiment by the following method:

One mg of each drug was weighed in a small beaker and dissolved in (1 ml) of propylene glycol 600 and the solution was made to 10ml with distilled water to give a stock solution. A further 1:10 dilution was made to give final solution of 10 µg/ml. The pH of the resulting solution was 7. A blank solution was prepared similarly containinng propylene glycol. The physiological solution used in all in vitro preparations have the composition shown in the following table (quantities are in grams for 5 liters).

Table of: Physiological solution used: Tyrode’s Ringer Locke NaCl 40 45 KCl 1.0 2.1 MgSO4.7H2O – – MgCl2 0.5 – KH2PO4 – – NaH2PO4 0.25 – NaHCO3 5.0 1.0 CaCl2 1.32 1.6 D-Glucose 5.0 5.0 Aeration Air O2

CHAPTER SIX

RESULTS & DISCUSSION

6.1 The anti–ulcerogenic activity of 2– chloroalkylamine and 2–bromoalkylamine and cimetidine on experi–mentally aspirin–induced ulcers in rat: Ulcer and erosions were induced in the stomach of fasting rats by oral administration of aqueous aspirin suspension.

As shown in Table (7) 2–chloroalkylamine produced dose–dependent reduction of ulcer lesions (r = 0.9235, n = 4,

ED50 = 8.6mmol/L).

Also 2–bromoalkylamine produced dose–dependent reduction of ulcer lesions (r = 0.6619, n = 4, ED50 = 137.8mmol/L) (Table 8).

Cimetidine produced dose–dependent reduction of ulcer

lesions (r = .8999, n = 4, ED50 = 38.7mmol/L) (Table 9).

Cimetidine and 2–chloroalkylamine were more potent than 2– bromoalkylamine (Table 10).

6.2 Effect on guinea pig atria: Histamine in a concentration of 2µg ml-1 increased the heart atrial rate from 96 + 61B/min to (252 + 12B/min).

2–chloroalkylamine (20µg ml–1) antagonised the effect of histamine completely and brought the heart to the normal rate (96 + 61B/min) (Fig. 10).

2–bromoalkylamine (40µg ml–1) blocked completely the stimulatory action of histamine and brought the heart to its normal rate (72 + 6) B/min. (Fig. 11).

Cimetidine (10µg ml–1) completely blocked the stimulatory effect of histamine and returned the heart to the normal rate (96 + 6) B/min (Fig. 14).

6.3 Effect on the contracting rat uterus:

Injection of rat with oestrogen brings rat into oestrus and increases the sensitivity to the drugs (Robinson et al., 1954). 2–chloroalkylamine in a concentration of (5µg ml–1) partially blocked the relaxing effect of histamine. In a concentration of (10µg ml–1) it completely blocked the relaxing effect of histamine (Fig. 8). 2–bromoalkylamine in a concentration of 20 mgml–1 completely blocked the relaxing response of histamine (Fig. 9).

Cimetidine in a concentration of (9µg ml–1) completely blocked the relaxing response of histamine (Fig. 15).

6.4 Effect on isolated guinea pig ileum: Histamine in a dose of (800ng ml–1) produced contractile –1 response of guinea pig ileum through H1 receptor. (10µg ml ) of 2–chloroalkylamine blocked the contraction induced by histamine (Fig. 12). (20µg ml–1) of 2–bromoalkylamine completely blocked the contraction induced by (800ng ml–1) of histamine in guinea pig ileum (Fig. 13).

6.5 Median inhibitory concentration (IC50) of

cimetidine, 2–chloroalkylamine and 2–bromoalkyl– amine on aspirin–induced ulcer in the rat (n = 5):

The values of IC50 of cimetidine 2–chloroalkylamine, and 2–bromoalklyamine were found to be 38.7mmol/L, 8.6mmol/L and 137.8mmol/L respectively (Table 12).

6.6 pA2 values for cimetidine 2–chloroalkylamine and 2– bromoalkylamine versus histamine acting on rat uterus:

pA2 values of cimetidine, 2–chloroalkylamine and 2– bromoalkylamine were found to be 6, 4.8 and 3.3 respectively (Table 10).

6.7 pA2 values for mepyramine 2–chloroalkylamine and 2– bromoalkylamine versus histamine acting on guinea pig ileum: pA2 values of mepyramine, 2–chloroalkylamine, and 2– bromoalkylamine were found to be 8, 4.17 and 2.1 respectively (Table 11). Table 7: Effect of 2–chloroalkylamine on aspirin–induced ulcer in the rat

Number of ulcer lesions Treatment (mean ± SEM)

Aspirin 300 mg/kg (control) 20.5 ± 0.67

2–chloroalkylamine 100 mg/kg + aspirin 300 mg/kg 1.5 ± 0.72**

2–chloroalkylamine 10 mg/kg + aspirin 300 mg/kg 4.33 ± 0.87**

2–chloroalkylamine 1 mg/kg + aspirin 300 mg/kg 15.66 ± 1.4*

2–chloroalkylamine 0.1 mg/kg + aspirin 300 mg/kg 20.0 ± 0.58* n = 6 * = P < 0.05 ** = P < 0.001 Table 8: Effect of 2–bromoalkylamine on aspirin– induced ulcer in the rat Number of ulcer lesions Treatment (mean ± SEM)

Aspirin 300 mg/kg (control) 21.15 ± 1.3

2- bromoalkylamine 240 mg/kg + aspirin 300 mg/kg 3.33 ± 0.99**

2- bromoalkylamine 24 mg/kg + aspirin 300 mg/kg 18.16 ± 0.29*

2- bromoalkylamine 2.4 mg/kg + aspirin 300 mg/kg 19.6 ± 1.52*

2- bromoalkylamine 0.24 mg/kg + aspirin 300 mg/kg 20.0 ± 1.15* n = 6 * = P< 0.05 ** = P < 0.001

Table 9: Effect of cimetidine on aspirin–induced ulcer in the rat

Number of ulcer lesions Treatment (mean ± SEM) Aspirin 300 mg/kg (control) 21.3 ± 1.33 cimetidine 240 mg/kg + aspirin 300 mg/kg 1.3 ± 0.49** cimetidine 24 mg/kg + aspirin 300 mg/kg 2.3 ± 0.93** cimetidine 2.4 mg/kg + aspirin 300 mg/kg 15.0 ± 0.93* cimetidine 0.24 mg/kg + aspirin 300 mg/kg 19.3 ± 0.71* n = 6 * = P< 0.05 ** = P < 0.001

Table 10: The value of pA2 of cimetidine, P–chloroalkylamine and P–bromo–alkylamine versus histamine acting on rat uterus:

Compound pA2 value

Cimetidine 6

2–chloroalkylamine 4.8

2–bromoalkylamine 3.3

Table 11: The value of pA2 of mepyramine, P– chloroalkylamine and P–bromoalkylamine versus histamine acting on guinea pig ileum Compound pA2 value

Mepyramine 8

2-chloroalkylamine 4.17

2-bromoalkylamine 2.1

Table 12: Median inhibitory concentration (IC50) of cimetidine, 2– chloroalkylamine, and 2–bromoalkylamine on aspirin induced ulcer in the rat (n=6)

Drug IC50 (mmol/L)

cimetidine 38.7

20–chloroalkylamine 8.6

2–bromoalkylamine 137.8

6.8 Results Discussion: The two compounds under test in the concentration employed blocked remarkably the effect of histamine in all experiments performed whether in right atrium of guinea pig, contracting rat uterus or rat stomach. This indicates that, these compounds possess H2 receptor blocking activity although their chemical structure is different from that of all known H2 blockers. The compounds under test have benzene ring instead of imidazole ring, which is not essential for H2 blocking activity as it is sometimes substituted by furan ring in ranitidine. All H2 blockers ranitidine, st cimetidine, famotidine and nizatidine have SCH2 group attached to the 1 carbon of the side chain, compounds under test have OH group in the same position. In addition they have as like all H2 blocker a tertiary N – atom to which the side chain is attached. The most structurally similar drugs to the compounds under test is dibenamine and phenoxybenzamine which are highly effective and specific blockers of excilatory response to stimuli on α adrenoceptors (Nickerson et al., 1949) found that the active species, elhyleneiminium is formed by cyclization of the β–haloalkylamine. In contrast compounds under test have a tertiary N–atom to which attached β– hydroxyl group and unsaturated ring system with p–position halo substituent that has two types of effects on the molecule. 1. Electron withdrawing group, which destabilized the ion formed.

2. Electron releasing group, which by resonance stabilizes ion formed and then this ion attaches to negatively charged portion of the receptor by covalent bond and then alkylated the receptor by rearrangement and this is most likely effect of p–halogen role.

Four factors were found to be necessary for the blocking activity: a. Ease of formation of the active ion.

b. Its activity at the receptor when formed.

c. Its stability.

d. The stability of the complex formed with receptor.

In addition the compounds under–test blocked the histamine effect in guinea pig ileum through acting on H1 receptors and this mean that these compounds possess antihistaminic activity which is less than mepyramine. There is a big relationship between these compounds and antihistaminics in chemical structure:

pA2 value of cimetidine is more than 2–chloroalkylamine which on turn more than 2–bromoalkylamine pA2 value.

Although the value of pA2 of cimetidine acting on H2 receptors of rat uterus is greater than that of 2–chloroalkylamine, the IC50 of 2–chloroalkylamine is less than IC50 of cimetidine, so the antiulcerogenic action of 2–chloroalkylamine may be in addition to H2 blocking activity, to another unidentified mechanism which might be antagonism of muscarenic receptor or inhibition of proton pump. The action of 2 compounds under test is irreversible as it persists on adding more concentration of histamine in the same organ bath. This may mean a long duration of action and once daily administration. The p–chloro substituted derivative is more potent than p–bromo substituted derivative.

Although p–chloro substitution increased activity at H2–receptors, it also increased the activity at H1–receptors. These compounds were prepared as a racimic mixture of dextro and levo– rotatory isomers. One of which display greater activity than the other, so they may show more activity in separate form.

P–fluoro substitution is possible to add more activity. Substitution of H of OH with alkyl group (ether linkage) is possible to give more potent compound. –O–SCR OCR

CHAPTER SEVEN

SUMMARY AND CONCLUSION

The two compounds synthesized and tested biologically in vitro and vivo showed prolonged H2 receptors blocking activity and H1 receptors antihistaminic activity. Although the two compounds have the same chemical structure p–chloro substituted derivative possessed high potency than p–bromo substituted derivative. CHAPTER EIGHT

SUGGESTIONS FOR FUTURE WORK

1. Biological tests on the prepared amino alcohols compounds for the H2– antagonistic actions should be highly considered than the haloanalogues (invivo and invitro).

2. Since these compounds contain a chral center, they must exist as enantiomers. If these are resolved into their (+) and (–) isomers they may

show better H2–antagonistic activity.

3. As long as the N–atom on the side chain is important for their activities, the bulkier benzyl group surrounding it may hinder its attachment to its receptor. Substitution with smaller groups such as ethyl group may give better results.

4. The presence of electron rich groups between the benzene ring and the ethyl side chain seem to be essential for their activities.

5. If these suggestions are taken into consideration in synthesizing new agents, better results may be obtained.

Table 3: Bromoacetophenones

I.R. Yield of Re Comp. Physical M.P. Quantity of Yield CO Structure TLC bromo- No. state °C acetophenone % abs. acetophenone cm-1

Br White r III CO CH2Br 9 109 109 g 8.04 70% 1700 solid white r IV Cl CO CH2Br 9 95 7 g 7.27 91% 1700 solid

9 = TLC was carried out

Table 4: Bromohydrins

Amount of Comp. Physical I.R. OH Yield of Structure TLC Bromoacetophen No. state abs. cm-1 bromohydrin one

Br CH Yellow V CH2Br 3500 9 6 g 3.58 g OH oil

CH Yellow VI Cl CH2Br 3500 9 8 g 3.4 g OH oil

9 = TLC was carried out

Table 5: Aminoalcohols liquids

Comp. Physical I.R. OH Quantity of Structure M.W. TLC No. state abs. cm-1 Bromohydrrine A

CH 2 Brownish VII Br CH CH2 N 396 3500 9 3.28 g oil OH CH2

CH 2 Brownish VIII Cl CH CH2 N 352 3500 9 2.1 g oil OH CH2

9 = TLC was carried out

Table 6: Aminoalcohol HCl salts

Comp. Quantity of Yield of M. Structure M.Wt. No. Aminoalcohol liquid Aminoalcohol salt °C

Cl- + CH2 IX Br CH CH2 N 433 0..5 g 0.2 g 2.

OH CH2

Cl- + CH2 X Cl CH CH2 N 389 0.5 g 0.24 g 12

OH CH2

9 = TLC was carried out

Figure 8: Demonstrates the effect of 2–chloroalkylamine on the contracting rat uterus. 5µg ml–1 of 2–chloroalkylamine produced partial blockade of the relaxing response of 1µg ml–1 of histamine while 10µg ml–1 produced complete blockade of relaxing response of histamine.

Hist. = 1µg ml–1 histamine. Cl = 2–chloroalkylamine. W = Wash. N = Normal.

Fig. 9: Demonstrates the effect of the compound 2– bromoalkylamine on the contracting rat uterus. 20µg/ml–1 produced complete blockade of 1µg ml–1 histamine relaxing response.

Hist. = 1µg ml–1 histamine. W = Wash. B = 2–bromoalkylamine. N = Normal.

Figure 10: Shows the effect of 2–chloroalkylamine on guinea pig atria. Histamine in a concentration of 2µg ml–1 increased remarkably the rate and the force of contraction of the isolated guinea pig atrium. Compound 2–chloroalkylamine in a concentration of 20µg ml–1 blocked the stimulatory effect of 2µg ml–1 histamine.

N = Normal. W = Wash. Hist. = 2µg ml–1 histamine. Cl = 2–chloroalkylamine.

Fig. 11: Shows the effect of 2–bromoalkylamine on guinea pig atria. Histamine in a concentration of 2µg ml–1 increased remarkably the rate and the force of contraction of the isolated guinea pig atrium 40µg m1–1 of 2–bromoalkyamine blocked the stimulatory effect of 2µg ml–1 histamine. N = Normal. W = Wash. Hist. = 2µg ml–1 histamine. B = 2–bromoalkylamine.

Fig. 12: Demonstrates the effect of 2–chloroalkylamina in a dose of 10µg ml–1 on the contractile response induced by histamine in a dose of 800ng ml–1 in isolated guinea pig ileum. Hist. = Histamine. Cl = 2–chloroalkylamina. W = Wash. N = Normal.

Fig. 13: demonstrates the effect of 2–bromoalkylamine in a dose of 20µg ml–1 on contractile response induced by histamine in a dose of 800ng ml–1 in isolated guinea pig ileum. Hist. = Histamine. B = 2–bromoalkylamine. W = Wash. N = Normal.

Fig. 14: Shows the effect of cimetidine on guinea pig atria. Histamine in a concentration of 2µg ml–1 increased remarkably the rate and the force of contraction of the isolated guinea pig atrium 10µg ml–1 of cimetidine blocked the stimulatory effect of 2µg ml–1 histamine.

Hist. = Histamine. N = Normal. Cim. = Cimetidine. W = Wash.

Fig. 15: Demonstrates the effect of cimetidine on contracting rat uterus. 10µg ml–1 produced complete blockade of the relaxing response of histamine (1µg ml–1). Hist. = Histamine. N = Normal. Cim. = Cimetidine. W = Wash.

Fig. 16: Shows the irreversible blockade of 2–chloroalkylamine of the inhibitory effect induced by histamine on the spontaneous contracting rat uterus. Hist. = Histamine. N = Normal. Cl = 2– chloroalkylamine . W = Wash. REFERENCES

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