Synthesis and Radiolabeling of Modified Peptides Attached to Heterocyclic Rings and Their Possible Medical Applications

Menoufiya University Faculty of Science Chemistry Department

A thesis Submitted in Partial Fulfillment of the Requirements for the Master Degree in Science (Organic Chemistry)

Hesham Abd El-Aal Abd El-Moaty Shams El-Din B.Sc. in Chemistry (May 2007) Demonstrator in Labeled Compounds Department, Hot Laboratories Center, Atomic Energy Authority

Supervised by

Prof. Dr. Hamed Mohammed Prof. Dr. Kamel Abd El-Hameed Abd El-Bary Moustafa Professor of Organic Chemistry, Professor of Radiobiochemistry, Chemistry Department, Labeled Compounds Department, Faculty of Science, Hot Laboratories Center, Menoufiya University Atomic Energy Authority

Dr. Ibrahem Youssef Abd El-Ghaney Ass. Professor of Radiobiochemistry, Labeled Compounds Department, Hot Laboratories Center, Atomic Energy Authority

2012

﴿ ﴾ رةاء / ١١٣ Contents

Contents

Dedication I

Acknowledgment II

Notes III List of Figures IV List of Tables VI

Abbreviations VII Aim of the Work IX

Summary X

Chapter I 1. Introduction 1

1.1 Peptide Synthesis 2

1.1.1 Protection of Functional Groups 3

1.1.2 Peptide Bond Formation: Methods and Strategies 4

1.2 1,2,4-Triazoles 10

1.2.1 Synthesis of 1,2,4-Triazoles 10

1.2.2 Reactions of 1, 2, 4-Triazoles 14

1.3 Tetrazoles 16

1.3.1 Synthesis of Tetrazoles 16 1.3.2 Reactions of Tetrazoles 19

1.4 1,3,4-Oxadiazoles 22

1.4.1 Synthesis of 1,3,4-Oxadiazoles 22 1.4.2 Reactions of 1,3,4-Oxadiazoles 23 Contents

1.5 Radiolabeled compounds 24

1.5.1 Types of labeling 25 1.5.2 Radioiodination 26

2. Results and Discussion 34 Chapter II 2.2 Synthesis of compounds 34

2.2 Radioiodination of dipeptide derivatives 8 and 15 50

3. Experimental 62

Chapter III 3.1 Synthesis of compounds 62 3.2 Radioiodination of dipeptide derivatives 8 and 15 82

4. Biological Evaluation 89

Antimicrobial activity of the newly synthesized 4.1 89 Chapter IV Compounds Biodistribution studies of radiolabeled derivatives 4.2 91 in normal mice

References 95

Published papers

Arabic summary

أ اا

Dedication

To the spirit of my mother

To my father & my brothers

Acknowledgements

First of all, limitless gratitude to God, Most Merciful, who blesses my effort, shows me the way and enables me to present this work in an acceptable style. On the top my Parent and my brothersbrothers, none of the things I have done would be done if it was not for your endless support, patience and encouragement throughout my years of study. Thanks for pretending to be interested in what I do.

I would like to express my deeply heart thanks to Prof.Prof. Dr. Hamed Abd ElEl----BaryBary (Prof. of Organic Chemistry, Menoufiya University),, Prof. Dr. Kamel Abd ElEl----HameedHameed (Prof. of Radio-biochemistry, Atomic Energy Authority), Dr. Ibrahem Youssef (Ass. Prof. of Radio biochemistry, Atomic Energy Authority) and Dr. Khaled Sallam Salem (Ass. Prof. of Radiochemistry, Atomic Energy Authority) for giving me the honor of working under their supervision, formulating the objectives, supplying the equipments and facilities required to accomplish the work and above all for their unforgettable sincere encouragement.

I would like to express my sincere appreciation, regards and gratitude to ProfProf.. Dr. Adel Nassar ( (professor of Organic Chemistry, faculty of science, Menoufiya University) for his help, continuous advice and encouragement. I wish for him a great success and a good health all his life.

I would like to express my deepest thanks to Dr. SSShima Shima Basher (Botany department, faculty of science, Menoufiya University) for her help and support in the antimicrobial study. I wish for her great success and good health all her life.

I wish also to express my deep thanks to all who supporting me during the study and the investigations of all the stages of the thesis specially Prof. Dr. Mohamed Abd ElElEl-El ---MotalMotalMotaleeeeb,b, Dr. Wael Sh Sheeeendy,ndy, HHHassanHassan Medhat, Mangoud ElElEl-El ---WakeWakeWakeeeeellll,, Mohammed El El----GGGGiiiizawy,zawy, Heba Raslan, Samar Ramadan, Maram ZahraZahra,, Tayseer Zidan, Walaa Gado, Walaa Diab, Eman Mansour, Shaban Abd ElEl----Satar,Satar, Mohamed Fawzy, Ibrahim Katan, SSSayedSayed Gamal, Mostafa Ataf and Ahmed Galhoum for their real help, moral support and continuous encouragement.

Finally, I would like to express my thanks and gratitude to all staff members of the Atomic Energy Authority. Also, my deepest thanks all my friends and colleagues at the Faculty of Science, Menoufiya University for their notes and additions which made this work more valuable. I would like to express my hearty thanks and gratitude to all my friends for their help and moral support. I really would like to thank those who helped me even with a word of support, those who really affected and still affecting my life everywhere. With my best regards Hesham Abdel-Aal Shamsel-Din II

Notes

The present thesis is submitted to the Faculty of Science, Menoufiya University in partial fulfillment for the requirements of Master Degree of Science in Organic Chemistry. Besides the work carried out in this thesis, the candidate Hesham Abd El-Aal Abd El-Moaty Shams El-Din passed post graduate courses for one year in Organic Chemistry, these cover the following topics: 1- Advanced Physical Organic Chemistry. 2- Organo-Metallic Chemistry. 3- Nature Products Chemistry. 4- Polymer Chemistry. 5- Biochemistry. 6- Chromatography. 7- Dyes. 8- Electro-Organic Chemistry. 9- Carbohydrates. 10- Spectroscopy. 11- Statistics. 12- English language. 13- Computer Science. 14- Photochemistry.

Head of Chemistry Department Prof. Dr. Ahmed Abd El-Mageed

III List of Figure

List of Figures

Figure No. label Page Fig.(2.1) Variation of the labeling yield of 125 I-EHPIP as a function of different EHPIP amounts; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) EHPIP, 100 µg of CAT, at pH 7, the reaction mixtures were kept at room temperature for 15 min…………………………………………………………. 51 Fig. (2.2) Variation of the labeling yield of 125 I-EHPIP as a function of CAT amount; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) of CAT, 100 µg of EHPIP, at pH 7, the reaction mixtures were kept at room temperature for 15 min…………………………………………………………. 52 Fig. (2.3) Variation of the labeling yield of 125 I-EHPIP as a function of reaction time; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 100 µg of EHPIP, 150 µg of CAT, at pH 8, the reaction mixtures were kept at room temperature for different intervals of time………………………………….. 53 Fig.(2.4) Variation of the labeling yield of 125 I-EHPIP as a function of pH; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 100 µg of EHPIP, 150 µg of CAT, at different pH, the reaction mixtures were kept at room temperature for 15 min………………………………………………………… 54 Fig. (2.5) Variation of the labeling yield of 125 I-EHPIP as a function of reaction temperature; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 100 µg of EHPIP, 150 µg of CAT, at pH 8, the reaction mixtures were kept at different temperatures for 15 min…………………………………………………. 54 Fig.(2.6) In vitro stability of 125 I-EHPIP in refrigerator…………… 55 Fig.( 2.7) In vitro stability of 125 I-EHPIP at room temperature……... 55 Fig. (2.8) Variation of the labeling yield of 125 I-EHPTIP as a function of different EHPTIP amounts; reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) EHPTIP, 100 µg of CAT, at pH 7, the reaction mixtures were kept at room temperature for 15 min……………………………………………………… 57 Fig. (2.9) Variation of the labeling yield of 125 I-EHPTIP as a function of CAT amount; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) of CAT, 10 µg of EHPTIP, at pH 7,the reaction mixtures were kept at room temperature for 15 min………………………………………………………….. 58

IV List of Figure

Figure No. label Page Fig.(2.10) Variation of the labeling yield of 125 I-EHPTIP as a function of pH; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 10 µg of EHPTIP, 150 µg of CAT, at different pH, the reaction mixtures were kept at room temperature for 15 min………. 59 Fig. (2.11) Variation of the labeling yield of 125 I-EHPTIP as a function of reaction time; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 10 µg of EHPTIP, 150 µg of CAT, at pH 7, the reaction mixtures were kept at room temperature for different intervals of time………………………………….. 60 Fig. (2.12) Variation of the labeling yield of 125 I-EHPTIP as a function of reaction temperature; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 10 µg of EHPTIP, 150 µg of CAT, at pH 7,the reaction mixtures were kept at different temperatures for 45 min………………………………………………….. 60 Fig. (2.13) In vitro stability of 125 I-EHPTIP…………………………… 61 Fig. (3.1) Paper electrophoresis for 125 I-EHPIP, 125 I-EHPTIP and free iodide………………………………………………………. 85

V List of Tables

List of Tables

Table No. label Page

Table (1.1) Examples of radiolabeled compounds used as therapeutic agents………………………………………………………. 24 Table (1.2) Examples of radiolabeled compounds used as diagnostic agents...... 25 Table (1.3) Production methods and radioactive properties of 123 I, 125 I and 131 I………………………………………………………. 27 Table (3.1) Radio-chromatographic behavior of different radiochemical 125 125 species and their R f values for of I-EHPIP and I- EHPTIP in paper chromatography………………………….. 84 Table (3.2) Radio-chromatographic behavior of different radiochemical species for 125 I-EHPIP and 125 I-EHPTIP in paper electrophoresis……………………………………………… 85 Table (4.1) In vitro antimicrobial activity of the newly synthesized compounds (at 50 µg / mL of test solutions)……………….. 90-91 Table (4.2) Biodistribution of 125 I-EHPIP in normal mice at different time intervals post-injection. (% ID/gram± S.D, n= 5)……... 93 Table (4.3) Biodistribution of 125 I-EHPTIP in normal mice at different time intervals post-injection. (% ID/gram± S.D, n = 5)…… 94

VI Abbreviations

Abbreviations

1H-NMR Proton Nuclear magnetic resonance. Ac Actyl ADAM Dimethylamino methyl phenyl iodophenylamine ANOVA Analysis of variance aq. An aqueous solution Ar- Aryl BAc 2 Base-catalyzed ester hydrolysis (B= base catalyzed, AC= acyl-oxygen bond is broken, 2= the reaction order) Bn- Benzyl Boc- tert -Butyloxycarbonyl BOM N- benzyloxymethyl Bu- Butyl CAT Chloramine-T ( N-Chloro-p-toluene sulfonamide sodium salt). DCC N,N' -Dicyclohexyl carbodiimide DEAE Diethylaminoethyl DHPM 5-etoxycarbonyl-4-phenyl-6-methyl-3,4-dihydro-(1H)-pyrimidine-2-one DIPEA N,N -Diisopropylethylamine DMF Dimethylformamide DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid DPPA Diphenylphosphoryl azide E.coli Escherichia coli EC Electron-capture ECD Ethyl cysteinate dimmer EHPIP Ethyl 2-(3-(4-hydroxyphenyl)-2-(2-(pyridin-4-ylamino)acetamido)- propanamido)-3-(1H-indol-3-yl)propanoate. EHPTIP Ethyl 2-(3-(4-hydroxyphenyl)-2-(2-(4-phenyl-5-((pyridin-4-ylamino)- methyl)-4H-1,2,4-triazol-3-ylthio)acetamido)propanamido)-3-(1H-indol-3- yl)propanoate. Et Ethyl Fig. Figure Fmoc- Fluorenylmethyloxycarbonyl HATU 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HEDP Hydroxyethylidene diphosphonate HMPAO Hexamethylpropyleneamineoxime HOBt N-Hydroxybenzotriazole I.D. Injected dose ICl Iodine monochloride IR Infrared spectroscopy. KeV Kilo electron volt Log P Partition coefficient

VII Abbreviations

M.p. Melting point m/z Mass-to-charge ratio M+ Molecular ion MAA Macroaggregated albumin MBq Mega Becquerel MeV Mega electron volt MHz Megahertz Mol. Molecular MS Mass spectrometry n Straight-chain or Neutron NBS N-Bromosuccinimide N-SHPP N-succinimidyl-3(4-hydroxy-phenyl)-propionate OTf Trifluoromethanesulfonate P Level of significance Pd 2(dba) 3 Tris(dibenzylideneacetone)dipalladium PET Positron emission tomography Pr- Propyl ppm Part per million PTAD 4-Phenyl-1,2,4-triazoline-3,5-dione Rf Rate of flow (or Retention factor) RIA Radioimmunoassay S.D. Standard deviation SBSB Succinimidyl para-tri-n-butylstannyl benzoate SN1 Unimolecular nucleophilic substitution SN2 Bimolecular nucleophilic substitution SPECT Single photon emission computed tomography SPPS Solid-phase peptide synthesis t- or tert - Tertiary TBAF Tetra-n-butylammonium fluoride TFA Trifluoroacetic acid THF Tetrahydrofuran TMEDA Tetramethylethylenediamine TMS Tetramethylsilane Tol- Toluyl Ts- Toluene-p-sulphonyl V Volt δ Chemical shift α Alpha particles β Beta particles γ Gamma emission

VIII Aim of the work

Aim of the Work

Keeping in mind the pharmacological potential of heterocyclic rings as well as the advantage of biodegradability and biocompatibility of amino acids/peptides, in this thesis we were prompted for the following: 1. Synthesis of novel dipeptide derivatives coupled with different heterocyclic rings (pyridine, 1,2,4-triazol-pyridine, 1,3,4-oxadiazol- pyridine and tetrazol-pyridine rings). 2. Characterization of the synthesized compounds on the basis of their spectral data (IR, Mass and 1H-NMR spectra). 3. Study their antimicrobial activity as one of their expected biological activities. 4. Study the radioiodination of some synthesized dipeptide derivatives. 5. Study the biodistribution of the radiolabeled compounds in normal mice as preliminary studies for the possibility of using them as agents for imaging and treatment.

SUMMARY

Summary

Summary This thesis presents synthesis of novel dipeptide derivatives coupled with different heterocyclic rings (pyridine, 1,2,4-triazol-pyridine, 1,3,4- oxadiazol-pyridine and tetrazol-pyridine rings) via the azide coupling method. Then, the synthesized compounds were characterized by IR, mass and 1H-NMR spectra which all agreed with the assigned structures. Then, the synthesized compounds were screened for their antimicrobial activities and some of which were found to possess good or moderate activities against the test microorganisms. It is worth mention that the triazole derivatives 10 and 14 were effective against the growth of all tested microorganisms and the dipeptide derivatives 15 and 21 attached to 1,2,4-triazol-pyridine and 1,3,4-oxadiazol-pyridine rings, respectively were effective against both the tested gram positive and gram negative bacteria. On the other hand ; dipeptide derivatives 8 and 15 were radiolabeled with I-125 by the direct electrophilic substitution method under oxidative conditions in the presence of chloramine-T, then subjected to chromatography analysis (paper chromatography and paper electrophoresis) to determine the labeling yield and the radiochemical purity of the final product. The influence of various reaction parameters and conditions on radioiodination efficiency (such as the amount of oxidizing agent, concentration of substrate, pH of the reaction and the reaction time) were investigated and optimized in order to maximize the labeling yield. Additionally, the biodistribution of the radiolabeled derivatives in normal mice was studied and the preliminary results showed a good in vitro and in vivo stability of the radiolabeled derivatives and the possibility of using them as novel imaging agents.

X Summary

1. Synthesis of dipeptide derivatives coupled with different heterocyclic rings : The synthesis is carried as the following procedure: 1.1. Synthesis of different heterocyclic rings coupled with pyridine moiety: When 4-aminopyridine (1) was treated with ethyl-chloroacetate in ethanol and in presence of CH 3COONa.3H2O at reflux temperature, it afforded ethyl 2-(pyridin-4-ylamino) acetate (2) in 92 % yield. Treatment of 2 with hydrazine hydrate in absolute ethanol gives the corresponding hydrazide derivative 3 in 90 % yields. The utility of hydrazides as key intermediates for the synthesis of several series of heterocyclic compounds and the broad spectrum of biological activities of their cyclized products have been reported. Thus, our interest aroused in exploring the utility of hydrazide as versatile precursor for the synthesis of a variety of substituted heterocycles. Thus, the reaction of hydrazide derivative 3 with phenylisothiocyanate gave phenyl-thiosemicarbazide 9 which on treatment with

NaOH cyclized to triazole 10 . In addition to, refluxing equimolar mixture of the hydrazide derivative 3 with potassium hydroxide and excess of carbon- disulfide in ethanol afforded 5-([pyridin-4-ylamino]-methyl)-1,3,4-oxadiazole-2-thiol (16). When 4-aminopyridine (1) was treated with chloro-acetonitrile in DMF containing sodium hydride, it afforded 22 , which was subsequently cyclized using sodium azide and ammonium chloride to yield N-([2H- tetrazol-5-yl]-methyl)-pyridin-4-amine (23) in 86% yield (scheme I) .

XI Summary

Scheme I 1.2. Synthesis of hydrazide derivatives of different heterocyclic rings as key intermediates for the azide coupling method: The hydrazide derivatives 3, 12, 18 and 25 could be prepared from different heterocyclic rings 1, 10, 16 and 23 , respectively, by alkylation with ethyl-chloroactetate to give the corresponding ethyl-actetates 2, 11, 17 and 24 which were subsequently hydrazinolyzed by hydrazine hydrate (scheme- II) .

XII Summary

Scheme II 1.3. Coupling of amino acids by the azide coupling method: Treatment of hydrazide derivatives 3, 12, 18 and 25 with nitrous acid ° (NaNO 2 / HCl) at -5 C afforded the corresponding azides, which were isolated in ethyl acetate at low temperature (0 °C). Then, the azide solution in ethyl acetate reacted with amino acid ethyl esters hydrochloride, previously treated with triethylamine in ethyl acetate at low temperature, to produce the amino acid ester derivatives 4, 5, 6, 13, 19 and 26 in good yields. The dipeptide derivatives 8, 15, 21 and 28 were obtained by the azide coupling method in moderate yields. The dipeptide derivatives were prepared from L-tyrosine ethyl ester derivatives 4, 13, 19 and 26 after conversion to the corresponding hydrazides 7, 14, 20 and 27 , respectively, by refluxing with excess hydrazine hydrate in ethanol ( scheme III ).

XIII Summary

NH 2NH EtO 2 H, r eflux

Cl , H 2 NO Na

Scheme III

2. Radioiodination of dipeptide derivatives: 2.1. Radioiodination of dipeptide derivative coupled with pyridine moiety: The maximum labeling yield of 90 ± 2.80 % for 125 I-EHPIP was obtained when the reaction was carried out at 100 µg of the dipeptide derivative (EHPIP) (8), 10 µl of Na 125 I (~7.2 MBq), 150 µg of chloramine-T in phosphate buffer pH 8 and the reaction mixture was kept at room temperature for 5 min. (scheme IV ).

XIV Summary

Scheme IV 2.2. Radioiodination of dipeptide derivative coupled with 1,2,4-triazol-

pyridine moiety : The maximum labeling yield of 92.5 ± 2.0 % for 125 I-EHPTIP was obtained when the reaction was carried out at 10 µL (~7.2 MBq) of Na 125 I, 10 µg of the dipeptide derivative (EHPTIP) (15), 150 µg of CAT in phosphate buffer pH 7 and the reaction mixture was kept at 80 ˚C for 45 min. (scheme V).

Scheme V

CHAPTER I

INTRODUCTION Chapter І Introduction

1. Introduction

A number of very important physiological and biochemical functions of life are influenced by peptides. Peptides are involved as neurotransmitters, neuromodulators and hormones in receptor-mediated signal transduction. New therapeutic methods based on peptides for a series of diseases give rise to the hope that diseases, where peptides play a functional role, can be amenable to therapy [1]. Several technologies for their production are now available, among which chemical and enzymatic synthesis are especially relevant [2]. The incorporation of amino acids and peptides into the aromatic and heterocyclic congeners played a vital role in the development of compounds with potent bioactivities [3, 4]. Pyridine, a heterocyclic nucleus, played a vital role in the development of different medicinal agents and agrochemicals. This nucleus is present in many products such as drugs, vitamins and food. Pyridine derivatives continue to attract great interest due to the wide variety of interesting biological activities observed for these compounds such as anticancer, analgesic, antimicrobial and antidepressant [5, 6]. Additionally, heterocycles containing more than two heteroatoms have received considerable attention owing to their synthetic and effective biological importance. For example, several 1,2,4-triazole, 1,3,4-oxadiazole and tetrazole derivatives have been reported to possess biological activities such as antimicrobial, anti-tumor, antiviral, anti-malarial, analgesic, anti- inflammatory, anticonvulsant, hypoglycemic and other biological properties such as genotoxic studies and lipid peroxidation inhibitor [7-14]. On the other hand; the radioiodine plays a very important role in radiolabeled compounds used for medical imaging and therapy [15, 16].

- 1 - Chapter І Introduction

The most frequently used radioactive isotopes of iodine in nuclear medicine are 131 I, 125 I and 123 I. They have proven to be useful for labeling both large and small molecules. The radioiodinated compounds are widely used for the diagnosis and therapeutic treatment of human diseases e.g. 125 I- Iodothioguanine for cancer treatment [17], 123 I-Dimethylamino methyl phenyl- iodophenylamine ( 123 I -ADAM) for brain imaging [18] and 123 I-Iodocelecoxib for tumor imaging [19]. Thus, keeping in mind the pharmacological potential of heterocyclic rings as well as the advantage of biodegradability and biocompatibility of amino acids/peptides, in this thesis we prompt to prepare new dipeptides coupled with different heterocyclic rings and study their antimicrobial activity as one of their expected biological activities. Furthermore, study their radioiodination and biodistribution of the radiolabeled compounds in normal mice as preliminary studies for the possibility of using them as agents for imaging and treatment. 1.1. Peptide Synthesis : Peptide synthesis – the formation of each peptide bond – is therefore a three-step procedure (scheme 1.1 ) [1]:

In the first step, the preparation of a partially protected amino acid is required. The second step, the formation of the peptide bond. The N-protected amino acid must be activated at the carboxy function in order to be converted into a reactive intermediate. In the third reaction step, selective or total cleavage of the protecting groups is carried out.

- 2 - Chapter І Introduction

Scheme 1.1: The multi-step process of peptide synthesis. Y 1= amino-protecting group; Y 2 = carboxy protecting group; R 1, R 2 = amino acid side chains.

1.1.1. Protection of Functional Groups: Temporary and semi-permanent protecting groups are distinguished with respect to cleavage selectivity. A temporary protecting group must fulfill the following requirements [1]: Introduction of the protecting group leads to an amino acid derivative that is no longer present in a zwitterionic structure. Cleavage must occur without hampering the stability of semi- permanent protecting groups or peptide bonds. Racemization must not occur during all necessary operations. Stability and characterization of protected intermediates must be favorable. Solubility of the protected amino acid components must be favorable.

- 3 - Chapter І Introduction

1.1.2. Peptide Bond Formation: (Methods and Strategies) Peptide bond formation is a nucleophilic substitution reaction of an amino group (nucleophile) at a carboxy group involving a tetrahedral intermediate. Therefore, the carboxy component (X-R2 = OH) must be activated prior to peptide bond formation. Furthermore, the peptide coupling reaction must be performed under mild conditions and at room temperature. Activation of the carboxy component (an increase of the electrophilicity) is achieved by the introduction of electron-accepting moieties. Groups which exert either an inductive (-I) effect or mesomeric (-M) effect (or both) decrease the electron density at the C=O group, thereby favoring the nucleophilic attack of the amino component. The latter attacks with its nitrogen lone pair the electrophilic position of the carboxy group to give the tetrahedral zwitterionic intermediate. Peptide bond formation is then completed by dissociation of the leaving group (nucleofuge R 2X–) from the tetrahedral zwitterionic intermediate (scheme 1.2). The leaving group capacity (nucleofugicity) is another factor which influences the reaction rate. The variation of the leaving group XR 2 provides a broad spectrum of methods for peptide bond formation [20].

Scheme 1.2 1.1.2.1. Acyl Azides: The acyl azide method was introduced into peptide chemistry as early as 1902 by Theodor Curtius and hence is one of the oldest coupling methods. Besides free amino acids and peptides, which are coupled with an

- 4 - Chapter І Introduction

acyl azide in alkaline aqueous solution, amino acid esters can be used in organic solvents. In contrast to many other coupling methods, the addition of an auxiliary base or a second equivalent of the amino component in order to trap the hydrazoic acid is not necessary. Although the azide method has been considered the method with the lowest tendency towards racemization, an excess of base in the reaction is accompanied by considerable racemization. Hence, any contact with bases during the coupling reaction must be avoided. The coupling reaction should be performed at low temperature because of the inherent thermal instability of acyl azides. The starting materials for this method are the crystalline amino acid or peptide hydrazides, respectively that are easily accessible from the corresponding esters by hydrazinolysis. These hydrazides are transformed into the azides by N-nitrosation with one equivalent of sodium nitrite in hydrochloric acid at -10 ˚C and simultaneous water elimination ( scheme 1.3) [21]. Alternately, mixtures of acetic acid, tetrahydrofuran, or DMF with hydrochloric acid have been used [22, 23].

Scheme 1.3

1.1.2.2. Anhydrides : Carboxylic acids and inorganic acids, respectively, are used for the formation of mixed anhydrides. The regiochemistry of the aminolysis reaction of the mixed anhydride, which is initially formed from the carboxy

- 5 - Chapter І Introduction

component and the chloroformate, depends on the electrophilicity and/or the steric situation of the two competing carboxy groups. In the case of mixed anhydrides formed from a N-protected amino acid carboxylate (carboxy- component) and an alkyl chlorocarbonate (activating component, e.g., from alkyl chloroformate), the amine nucleophile predominantly attacks the carboxy group of the amino acid component with the formation of the desired peptide derivative and release of the activating component as free acid. On application of an alkyl chloroformate (R 1 = isobutyl, ethyl, etc.), the free monoalkyl carbonic acid formed is unstable and immediately decomposes into carbon dioxide and the corresponding alcohol (scheme- 1.4) [24].

Scheme 1.4: Mechanism of mixed anhydride formation and regiochemistry of aminolysis. Y = amino- protecting group; R 1 = side chain of the carboxy component; R 2= subsistent of the chlorocarbonate; 3 H2N-R = amino component; B = tertiary base.

The relatively high stability of mixed anhydrides towards hydrolysis allows for their purification by washing the organic phase containing the mixed anhydride with water. The stability of mixed anhydrides obtained

- 6 - Chapter І Introduction

with alkyl chloroformates also depends on the alkyl group present. Mixed anhydrides from Boc- and Fmoc-protected amino acids and isopropyl- chloroformate can be purified and isolated. These are much more stable compared to the derivatives obtained with ethyl or isobutyl chloroformate [25]. 1.1.2.3. Carbodiimides: Carbodiimides can be used for the condensation of a carboxy group and an amino group. Initially N,N'-dicyclohexyl carbodiimide (DCC) was used because it is relatively cheap and soluble in the used solvents in peptide synthesis. During peptide bond formation the carbodiimide is converted into the corresponding urea derivative, which in the case of N,N'-dicyclohexyl urea precipitates from the reaction solution. The reaction mechanism for carbodiimide-mediated peptide couplings have been established in extensive studies performed by several groups. The carboxylate anion adds to the protonated carbodiimide with the formation of a highly reactive O-acylisourea; this has not yet been isolated, but its existence is postulated by close analogy to stable compounds of this class. The O-acylisourea reacts with the amino component to produce the protected peptide and the urea derivative (reaction A). Alternatively, the O- acylisourea in equilibrium with the N-protonated form is attacked by a second carboxylate as the nucleophile forming the symmetrical amino acid anhydride together with N,N '-disubstituted urea (reaction C). The former reacts with the amino acid component to give the peptide derivative and the free amino acid (reaction D). An undesired side reaction is the base- catalyzed acyl migration from the isourea oxygen to nitrogen; this results in an N-acylurea that does not undergo further aminolysis (reaction B) (scheme-1.5). This O-N acyl migration is catalyzed not only by an excess of

- 7 - Chapter І Introduction

base, but also by the basic amino component or the carbodiimide. Polar solvents additionally favor this reaction pathway. The N-acyl urea formation mentioned above may be a prominent side reaction that can be suppressed by keeping the reaction at low temperatures, or by using nonpolar solvents in which the dicyclohexyl urea should dissolve [26-28].

Scheme 1.5: Mechanism of carbodiimide-mediated peptide couplings. R 1COOH = carboxy component 2 3 (amino acid or peptide); R = e.g. cyclohexyl (DCC); R -NH 2 = amino component (amino acid or peptide). 1.1.2.4. Active esters: Peptide bond formation by ester aminolysis proceeds analogously to ester saponification (X= O, S, Se) according to a BAc 2 mechanism. Nucleophilic attack of the amino group to the carboxy group leads, in the rate determining step of this reaction, to the tetrahedral intermediate, the formation of which is favored by electron-withdrawing groups XR 3 (scheme-

- 8 - Chapter І Introduction

1.6). The rate of peptide bond formation correlates with the leaving group capacity of XR 3. For example, a good leaving group is the conjugate base of a relatively strong acid and this is the case when the leaving group anion -

XR 3 is well-stabilized by inductive or mesomeric effects which facilitate cleavage of the (C=O)–XR 3 bond. The initial application of thiophenyl-, cyanomethyl-, 4-nitrophenyl- and pentafluorophenyl esters triggered further development of a broad variety of active esters [29, 30].

1.1.2.5. Acyl Halides : An obvious method of activating a carboxy group for amide formation at room temperature or below would utilize the acid chloride. However, amino acid chlorides have until rarely been used as they are reputed to be over-activated and prone to numerous side reactions, including racemization. Despite this, because the Fmoc-group is highly stable under acidic conditions and does not readily undergo S N1 or S N2 displacement reactions at the 9-fluorenylmethyl residue, the Fmoc-protected amino acids have emerged as ideal substrates for conversion into the corresponding acid chlorides. Consequently, these derivatives have experienced a renaissance in peptide synthesis. All common amino acids lacking polar side chains, as well as a number of that bearing benzyl- or allyl-based side-chain protection, can be converted to stable, crystalline acid chlorides. For chemical reasons, amino acids bearing tert -butyl-protected side chains cannot generally be accommodated. A hindered base such as 2,6-di-tert -butylpyridine is used

- 9 - Chapter І Introduction

routinely as hydrogen chloride acceptor, as conversion to oxazolone is slow with such bases. Ammonium or potassium salts of N-hydroxybenzotriazole (HOBt) have also been applied as bases in solid-phase syntheses involving Fmoc amino acid chlorides. Fmoc amino acid chlorides have been used as stable, easily accessible derivatives for rapid coupling reactions, without any racemization. Fmoc-protected amino acid fluorides were found to be efficient carboxy-activated derivatives suited both for solution syntheses and for Solid-phase peptide synthesis (SPPS). The highly reactive Fmoc amino- acid fluorides are especially recommended for SPPS of complicated longer peptides and also mainly for the coupling of sterically hindered amino acid- building blocks [31]. 1.2. 1,2,4-Triazoles: 1.2.1. Synthesis of 1,2,4-Triazoles: 1,2,4-Triazoles are available via cyclodehydration reactions of N,N'- diacyl-hydrazines with amines, although the conditions are often quite vigorous (scheme 1.7 ) [32].

Additionally, condensations of amino-guanidine with esters, or an orthoformate, give the 3-amino-1,2,4-triazoles (scheme 1.8 ) [33].

- 10 - Chapter І Introduction

A copper-catalyzed reaction under an atmosphere of air provides 1,2,4-triazole derivatives by sequential N-C and N-N bond-forming oxidative coupling reactions (scheme 1.9). Starting materials and the copper catalyst are readily available and inexpensive. A wide range of functional groups is tolerated [34].

Scheme 1.9 A highly regioselective one-pot process provides rapid access to highly diverse 1,3,5-trisubstituted-1,2,4-triazoles from the reaction of carboxylic acids, primary amidines and mono-substituted hydrazines (scheme 1.10) [35].

Scheme 1.10 An effective 1,3-dipolar cycloaddition for the synthesis of 1,3,5- trisubstituted-1,2,4-triazole derivatives by the reaction of oximes with hydrazonoyl hydrochlorides using triethylamine as a base gave the desired 1,3,5-trisubstituted-1,2,4-triazoles in good yields (scheme 1.11). The reaction was applicable to aliphatic, cyclic aliphatic, aromatic and heterocyclic oxime substrates [36].

Scheme 1.11

- 11 - Chapter І Introduction

A series of new oxamide-derived amidine reagents can be accessed in excellent yield with minimal purification necessary. A subsequent reaction of these reagents with various hydrazine hydrochloride salts efficiently generates 1,5-disubstituted-1,2,4-triazole compounds in good yields (scheme-1.12). Both aromatic and aliphatic hydrazines react readily with the [37] amidine reagents under very mild reaction conditions .

Scheme 1.12

3-N,N '-Dialkylamino-1,2,4-triazoles can be prepared from S-methyl-isothioureas and acyl-hydrazides in good yields (scheme 1.13). The reaction conditions are relatively mild and tolerate a broad range of functional groups [38].

Scheme 1.13

A mild, one-pot cyanoimidation of aldehydes using cyanamide as a nitrogen source and NBS as an oxidant was achieved in high yields without the addition of a catalyst. Subsequently, the substituted N- cyanobenimidate products may also undergo a cyclization reaction to give 1,2,4-triazole derivatives in high yields (scheme 1.14) [39].

- 12 - Chapter І Introduction

Scheme 1.14

An efficient and convenient synthesis of [1,2,4]triazolo[4,3- a]pyridines involves a palladium-catalyzed addition of hydrazides to 2- chloropyridine, which occurs chemo-selectively at the terminal nitrogen atom of the hydrazide, followed by dehydration in acetic acid under microwave irradiation (scheme 1.15) [40].

Scheme 1.15

The reaction of hydrazide with phenyl-isothiocynate gave phenylthiosemicarbazide which on treatment with NaOH cyclised to triazole (scheme 1.16) [41, 42].

Scheme 1.16 Stirring of hydrazide derivatives with carbon disulphide in the presence of alcoholic KOH gives potassium carbothioate salt which on refluxing with hydrazine hydrate cyclised to triazole (scheme 1.17) [43-45].

Scheme 1.17

- 13 - Chapter І Introduction

1.2.2. Reactions of 1,2,4-Triazole s: N-Alkylations and -acylations generally occur at N-1, reflecting the higher nucleophilicity of N–N systems, however 4-alkyl derivatives can be prepared via quaternization of 1-acetyl-1,2,4-triazole or the acrylonitrile or crotononitrile adducts (note that N-1 and N-2 are equivalent until substitution occurs) (scheme 1.18) [46]. In alkylations of 1,2,4-triazole, generally a ratio of (9:1, N-1:N-3) isomers is present in the crude product, but the minor product is very easily removed; even simple distillation completely separates the isomers, illustrating the very large difference in physical properties between isomers [47]. Direct hydroxymethylation of 3,4– disubstituted 1,2,4-triazoles occurs at C-5 by heating with formalin or paraformaldehyde [48].

Scheme 1.18 Bromination occurs readily in alkaline solution giving 3,5-dibromo- 1,2,4-triazole; the 3–monochloro derivative can be obtained by thermal rearrangement of the N-chloro isomer; an analogous N →C 1,5-sigmatropic shift (scheme 1.19 ) [49].

Scheme 1.19 Secondary radical Minisci alkylations of 1-alkyl -1,2,4-triazoles take place at C-5 and primary radical substitutions are known in an intramolecular sense (schemes 1.20) [50].

- 14 - Chapter І Introduction

Schemes 1.20 C-Lithiations can be easily effected on N-1-protected 1,2,4-triazoles, the resulting 5-lithium derivatives being much more stable than C-lithiated- 1,2,3-triazoles. 5-Silylation can even be achieved using triethylamine with trimethylsilyl bromide (scheme 1.21) [51].

Scheme 1.21 The oxidative desulphurization of 1,2,4-triazole thiones is a common reaction to other electron-deficient nitrogen heterocycles; nitric acid is the oxidant in the example below. The process involves loss of sulfur dioxide from an intermediate sulfinic acid (scheme 1.22) [52].

Scheme 1.22 4-Phenyl-1,2,4-triazoline-3,5-dione (often PTAD or ‘Cookson's reagent’) is a highly reactive dienophile; it has often been used to trap unstable dienes or characterize dienes as adducts [53]. Two examples of its reactivity are shown below in scheme 1.23.

- 15 - Chapter І Introduction

Scheme 1.23 1.3. Tetrazoles: 1.3.1. Synthesis of Tetrazoles: Tetrazoles are usually prepared by the reaction of an azide with a nitrile, or an activated amide; tri-n-butyltin-azide and trimethylsilyl-azide are more convenient and safer reagents than azide anion in some cases; copper- (I) oxide catalysis in the trimethylsilyl azide protocol is very efficient for the production of N-unsubstituted tetrazoles [54] and arylsulfonyl cyanides react with organic azides very efficiently giving rise to 1- substituted 5- arylsulfonyl-tetrazoles [55]. Zinc bromide can be used to catalyze the reaction between sodium azide and nitriles in hot water [56-59]. Intramolecular examples involving cyanamides proceed in hot DMF [60]. (Schemes 1.24)

H N DMF , 120 C N RCN + NaN3 R R= Ar, Bn N N

Schemes 1.24 5-Substituted tetrazoles were prepared in very good yields and short reaction times by treatment of nitriles with sodium azide and

- 16 - Chapter І Introduction

triethylammonium chloride in nitrobenzene in a microwave reactor (scheme- 1.25). Even sterically hindered tetrazoles, as well as those deactivated by electron-donating groups, can be prepared [61].

Scheme 1.25 Related methods can be used to prepare 5-hetero-substituted compounds; isonitriles with N-halo-succinimides and azide give halo- derivatives [62] and aryl isothiocyanates with azide give the 5-thiols, which can be converted into 5-unsubstituted tetrazoles by oxidation with hydrogen- peroxide or chromium trioxide (scheme 1.26) [63].

N aq.NaN N N N 3 H O , NH OH Ph NC S 2 2 4 N 0 C HS N H N N Ph Ph Scheme 1.26 A series of 1-substituted 1H-1,2,3,4-tetrazole compounds have been synthesized in good yields from amines, triethyl-orthoformate and sodium- [64, 65] azide through the catalyzed reaction with Yb(OTf) 3 (scheme 1.27) .

Scheme 1.27 A series of primary alcohols and aldehydes were treated with iodine in ammonia water under microwave irradiation to give the intermediate nitriles, which without isolation underwent [2+3] cycloadditions with sodium azide and zinc bromide to afford the corresponding triazines and tetrazoles in high yields (scheme 1.28) [66].

Scheme 1.28

- 17 - Chapter І Introduction

The reaction of cyanogen azide and primary amines generates imidoyl-azides as intermediates in acetonitrile/water. After cyclization, these intermediates gave 1-substituted aminotetrazoles in good yield (scheme- [67] 1.29 ) .

Scheme 1.29 A versatile and highly efficient Zn(OTf) 2-catalyzed one-pot reaction of alkenes, NBS, nitriles and TMSN 3 gives various 1,5-disubstituted- tetrazoles containing an additional α-bromo functionality of the N-1-alkyl- substituent (scheme 1.30) [68].

Scheme 1.30 The reaction of 2-halopyridines with trimethylsilyl-azide in the presence of tetrabutylammonium-fluoride-hydrate gives tetrazolo-[1,5- a]pyridines (scheme 1.31). 8-bromotetrazolo-[1,5-a]pyridine is further trans- [69] formed into a variety of novel tetrazolo-[1,5-a]pyridine derivatives .

Scheme 1.31 Pyridine N-oxides were converted to tetrazolo[1,5-a]pyridines in good yield in the presence of sulfonyl or phosphoryl azides and pyridine by heating in the absence of solvent (scheme 1.32). Diphenyl-phosphorazidate (DPPA) was the most convenient reagent [70].

- 18 - Chapter І Introduction

Scheme 1.32

1.3.2. Reactions of Tetrazoles:

Due to the similarity of pKa of (NH) tetrazoles to that of carboxylic acids, tetrazoles have often been used as bioequivalent replacements for

CO 2H in pharmacologically active compounds. The acid replacement extends to the tetrazole analogue of proline, which is more soluble than proline itself, but retains its catalytic properties for condensation reactions. Tetrazoles are generally surprisingly stable, although tetrazole itself (M. p. = 158 ˚C, decomposes >180 ˚C) is classified as an explosive, at least for shipping purposes. Chloro-and alkylthio-tetrazoles decompose at somewhat lower temperatures. Salts of 5-nitrotetrazole are promising safer (less sensitive) and less toxic replacements for lead azide primers they are described as ‘green explosives’ because they generate non-toxic products [71]. Tetrazoles alkylate and acylate on N-1(4) and/or N-2(3), the regioselectivity depending, on the substituent at C-5. The formation of mixtures is the usual outcome for alkylation and this is a significant problem in medicinal chemistry. Although a range of specifically N-1-substituted tetrazoles are available by ring synthesis, the direct synthesis of N-2- substituted compounds is more problematic. In some cases, highly regioselective N-1-alkylation can be carried out by first introducing a bulky labile group at N-2, then quaternisation followed by cleavage of the 2- substituent. Both tri-n-butylstanyl and t-butyl have been used for this purpose (schemes 1.33) [72].

- 19 - Chapter І Introduction

N N MeI N N DMSO, CHCl3 NSnn-Bu3 N Me Me N N Me N N tBuOH N N Me SO 2 4 MeN N N N H2SO4 CHCl3, N MeOSO3 HCl, Ph Nt-Bu N Ph Nt-Bu N H N Ph Ph N N Me

Schemes 1.33 In addition to steric effects, the presence of an electron-withdrawing group at C-5 favors N-2-alkylation. The use of a benzyl-dithiocarbonate alkylating agent gives only the 2-benzyl isomer, in very high yield (scheme- 1.34) [73], while the methyl analogue gives a mixture, although still with very good N-2 selectivity (7:1) [74].

Scheme 1.34 The difference in physical and chemical properties between 1- and 2- substituted tetrazoles can be large. The relatively selective methylation (85:15, N-2:N-1) of 5-cyanotetrazole can be used to prepare pure 2-methyltetrazole by utilizing the very large difference in stabilities of 1- and 2- methyl-tetrazole-5-carboxylic acids. The resulting 1-methyltetrazole and 2- methyltetrazole-5-carboxylic acid can be easily separated, then the latter decarboxylated at 200 ˚C to give pure 2-methyl tetrazole (scheme 1.35) [75].

Scheme 1.35

- 20 - Chapter І Introduction

Remarkably, some C-electrophilic substitutions such as bromination, mercuration and even Mannich reactions, but not nitration, can be achieved though the mechanisms for these substitutions may not be of the conventional type (scheme 1.36).

Scheme 1.36 5-Lithiated-2-substituted-tetrazoles appear to be significantly more stable than the 1-isomers, as shown below, by a comparison of the two N- benzyloxymethyl(BOM)-protected isomers [76]. A tetrazole can also act as an ortho -directing group as in the lithiation of 5-phenyltetrazole [77]. A tetrazol- 5-ylether similarly directs ortho -metallation, but here the tetrazole migrates from the oxygen to the lithiated carbon [78]. Hypobromite oxidation of 5- benzylaminotetrazole provides a useful preparation of benzyl- isonitriles [79]. 1,3,4-Oxadiazoles are formed on heating tetrazoles with acylating agents, via rearrangement of the first formed 2-acyl derivatives [80]. (Schemes 1.37)

Schemes 1.37

- 21 - Chapter І Introduction

1.4. 1,3,4-Oxadiazoles: 1.4.1. Synthesis of 1,3,4-Oxadiazoles: 1,3,4-Oxadiazoles are available by cyclodehydration of N,N ′-diacyl- hydrazines or their equivalents [81], for example closure of N-acyl- thiosemicarbazides generates 2-amino-1,3,4-oxadiazoles [82]. Additionally, reaction of a hydrazide with a trialkyl-orthoalkanoate produces 1,3,4- oxadiazoles [83] or by treating a hydrazide with an acid with microwave heating [84] and a dehydrating agent as triphenylphosphine in carbon- tetrabromide [85]. Starting from a lower oxidation level, aldehyde N-acyl- hydrazones give the aromatic heterocycles on treatment with cerium (IV)- ammonium nitrate [86]. (Schemes 1.38)

O NH H N N 2 N t-Bu HN N N 2Tf2O , CH2Cl2 Ph N HC(OEt)3, H Ph t-Bu O Ph Ph O O O

Schemes 1.38 N-Isocyanimino-triphenyl-phosphorane, aldehydes and benzoic acids undergo a one-pot reaction under mild conditions to afford 2-aryl-5- hydroxyalkyl-1,3,4-oxadiazoles in good yields (scheme 1.39 ) [87].

Scheme 1.39 1.4.2. Reactions of 1,3,4-Oxadiazoles: As with the azoles, oxadiazoles are very weak bases due to the inductive effects of the extra heteroatoms. The important difference from

- 22 - Chapter І Introduction

other azoles is of course the absence of N-hydrogen, so that the N-anion- mediated reactions are not available. All these systems are susceptible to nucleophilic attack, particularly the oxadiazoles, which often undergo ring cleavage with aqueous acid or base unless both carbon positions are substituted (scheme 1.40) [88].

Scheme 1.40 Generally, amines can be diazotized and converted, for example, into halides, but in some cases the intermediate, N-nitroso compound, is stable and only then subsequently converted into a diazonium salt by treatment with strong acid (scheme 1.41), this may reflect the lower stability of a positively charged group attached to an electron-deficient ring [89].

Scheme 1.41

A simple and straight forward method for the direct carboxylation of aromatic heterocylces such as oxazoles, thiazoles and oxadiazoles using CO 2 as the C-1 source requires no metal catalyst and only Cs 2CO 3 as the base (scheme 1.42 ). A good functional group tolerance is achieved [90].

Scheme 1.42

- 23 - Chapter І Introduction

1.5. Radiolabeled compounds:

Labeled compounds of interest in the field of nuclear medicine will either be used in biochemical research, in medical diagnosis or therapy. Compounds labeled with β-emitting isotopes such as 14 C, 3H, 35 S and 32 p are principally used in biochemical research. Table (1.1) shows examples of some radiolabeled compounds used as therapeutic agents. Therapeutic applications of radiolabeled compounds have emerged from the concept that certain radionuclides possessing particulate emission such as alpha and beta radiations possess the ability to destroy diseased tissues. Table (1.1) Examples of radiolabeled compounds used as therapeutic agents. Compound Isotope Application Sodium iodide 131 I Thyroid carcinoma treatment [91] Iodothioguanine 125 I Cancer treatment [17] 188 Re-tin-colloid 188 Re Rheumatoid arthritis treatment [92]

186 Re-hydroxyethylidene 186 Re Treatment of osteosarcoma [93] diphosphonate ( HEDP)

Examples of some radiolabeled compounds used as diagnostic agents are shown in table (1.2). The radionuclides used for diagnostic nuclear medicine should have the following characteristics: They must have a short half-life from 10 seconds to 80 h; this is preferred because higher amounts can be given to the patients without significantly increasing the radiation dose. They emit slowly γ-rays, no particles, this is an ideal situation (e.g. 99mTc), and this would eliminate high local irradiation. The energies of the emitted γ-rays should be in the range of 100-300 KeV to be easily detected by present day gamma cameras.

- 24 - Chapter І Introduction

Table (1.2) Examples of radiolabeled compounds used as diagnostic agents . Compound Isotope Application Sodium iodide 123 I Thyroid uptake measurement [91] Dimethylamino-methyl-phenyl- 123 I Brain imaging [18] iodophenylamine (ADAM) 99mTc-gabapentin 99mTc Brain imaging [94] 99mTc-macroaggregated-albumin 99mTc Lung perfusion scan [95] (MAA) Iodocelecoxib 123 I Tumor imaging [19]

1.5.1. Types of labeling: Two types of labeling are recognized : 1.5.1.1.Isotopic labeling: In which the compound is labeled with an isotope of an element already present in the compound, so that the labeled compound is identical with the unlabeled compound as shown in (scheme 1.43). The majority of the labeled compounds are in this category . R R

I *I *I

Scheme 1.43 1.5.1.2. Non isotopic labeling: In which the compound is labeled with an isotope of a foreign element not present in the original compound, as shown in (scheme 1.44) for example labeling of albumin with 51 Cr and the labeling of proteins and peptides with radioactive iodine.

- 25 - Chapter І Introduction

R R H *I *I

Scheme 1.44

1.5.2. Radioiodination [91]: Iodination is used extensively for labeling the compounds of medical and biological interest. 1.5.2.1. Principles of Iodination: Iodination of a molecule is governed primarily by the oxidation state of iodine. In the oxidized form, iodine binds strongly to various molecules, whereas in the reduced form, it doesn’t bind. Commonly, iodine is available as NaI and I– oxidized to I + by various oxidizing agents. The free molecular iodine has the structure of I +, I– in aqueous solution. In either case the electrophilic species I + does not exist as a free species, but forms complexes with nucleophilic entities such as water or pyridine. + – – I2 + H 2O ↔H2OI + I I 2 + OH ↔HOI + I + The hydrated iodonium ion, H 2OI and hypoiodous acid, HOI, are believed to be the iodinating species in the iodination process. Iodination occurs by electrophilic substitution of a hydrogen ion by an iodonium ion in the molecule of interest, or by nucleophilic substitution (isotope exchange) where a radioactive iodine atom is exchanged with a stable iodine atom that is already present in the molecule. These reactions are represented as follows: • Nucleophilic substitution:

- 26 - Chapter І Introduction

• Electrophilic substitution:

1.5.2.2. Isotopes of iodine: Among the thirty two iodine radioactive isotopes, three of them are of special interest namely 123 I, 125 I and 131 I. Production methods and radioactive properties of these three isotopes of iodine are shown in table (1.3). Table (1.3) Production methods and radioactive properties of 123 I, 125 I and 131 I.

Radio- Main γ Half- life Mode of Production Application nuclide ray (keV) (T ) decay Method 1/2

123 I 159 13.2 h EC 123 Te (p,n) 123 I -SPECT 124 Xe(n, γγγ)125 Xe -RIA, 125 I 35.5 59.4 d EC E.C 125 I -Auger-electron therapy 130 Te(n, γγγ)131 Te 131 - β−β− I 364 8.02 d β ββ−− 131 I -Therapy

1.5.2.2.1. Iodine-123: It is increasingly used for diagnosis of thyroid function; it is a gamma emitter without beta radiation. Iodine can be produced by the cyclotron; by the following nuclear reaction: (p, 2n) 124 T 123 I 123 It also obtained from Xe as; (p, 5n) (B +, E.C) 127 I 123 Xe 123 I (p, 2n ) (B +, E.C) (B +, E.C) 124 Xe 123 Cs 123 Xe 123 I Among the numerous radioisotopes of iodine, 123 I is the most ideal radionuclide for in vivo applications. The advent of efficient high speed

- 27 - Chapter І Introduction

cameras, single photon emission computed tomography (SPECT) and positron emission tomography (PET), increased the labeling of pharmaceuticals by 123 I. The absence of β- particles emission, the relatively short half life of 13.2 hours and the convenient γ-energy of 159keV make 123 I an ideal radionuclide for diagnosis in nuclear medicine. 1.5.2.2.2. Iodine-125: 125 I can be produced in the reactor according to the following nuclear reaction [96]: (n, γγγ) (B+, E.C) 124 125 125 Xe gas Xe I Also it can be produced at cyclotron from 125 Te as; (p, n) (d, 2n) 125 Te 125 I or 125 Te 125 I 125 I emits gamma energy and Auger electron. Auger-emitters emit low-energy electrons with a short path length range of 0.06-14 m, which is too short to reach the DNA of a tumor cell unless the radionuclide is internalized into the cell [97]. 125 I is used mainly in a laboratory work [98]. The gamma energy of 35.5 keV is not strong enough to be detected outside the body. Its 60 day half-life permits the storage of the labeled materials for some time, subject to their chemical and radiochemical stability. 125 I is used for labeling proteins, hormones such as insulin, thyroid related hormones and steroids which find wide applications in radioimmunoassay [99]. 1.5.2.2.3. Iodine-131: 131 I was discovered by Livingood and Seaboreg in 1938. It can be produced in the reactor from (TeO 2) according to the f ollowing reaction; − − (n, γγγ) βββ−− βββ−− 130 Te 131 Te 131 I 131 Xe 25min 8.02d

- 28 - Chapter І Introduction

131 I can also be recovered from uranium ( 235 U) fission product. Its half-life of 8.02 days is sufficiently long to permit commercial distribution of labeled compounds but limits its use in vivo applications. The 131 I is used in high doses in the treatment of the thyroid cancer and may be also used in small doses in the treatment of the overactive thyroid. The β− emission of 606 keV of 131 I prohibits its use in situations where the radiation dose to the patient must be minimized but this medium energy beta emission can kill cells and it has proven to be useful in the therapy of thyroid cancer [100]. Its radiotherapeutic potential and its very good commercial availability make 131 I and 123 I radioisotopes widely used in the labeling of monoclonal antibodies . 1.5.2.3. Methods of Iodination [91]: There are several methods of iodination, and principles of the important ones only are described below; 1.5.2.3.1. Triiodide Method: The triiodide method essentially consists of adding radioiodine to the compound to be labeled in the presence of a mixture of iodine and potassium

131 131 131 iodide : I2 + KI + I2 + 2RH → R I + K I + RI + 2HI Where RH is an organic compound being labeled. In the case of protein labeling by this method, minimum denaturation of proteins occurs, but the yield is low, usually about 10–30 %. Because cold iodine is present, the specific activity of the labeled product is considerably diminished. 1.5.2.3.2. Iodine Monochloride Method: In the iodine monochloride (ICl) method, radioiodine is first equilibrated with stable 127 I in iodine monochloride in dilute HCl, and then the mixture is added directly to the compound of interest for labeling at

- 29 - Chapter І Introduction

a specific pH and temperature. Yields of 50–80% can be achieved by this process. However, cold iodine of ICl can be introduced in the molecule, which lowers the specific activity of the labeled compound and the yield becomes unpredictable, depending on the amount of ICl added. 1.5.2.3.3. Chloramine-T Method: Chloramine-T is the sodium salt of N-monochloro-p-toluene- sulfonamide and is a mild oxidizing agent. In this method of iodination, the compound for labeling is added first then chloramine-T to a solution of 131 I-sodium iodide. Chloramine-T oxidizes iodide to a reactive iodine species, which then labels the compound. When high specific activity radioiodide is oxidized in situ , it generates electropositive iodine, but it is unlikely to form I 2 because there is so little radioiodine present that statistically it is not possible for two iodine atoms to join together at the amounts involved [16]. In addition, although CAT is a mild oxidizing agent, - + its effect is enough to oxidize all I into I without forming I 2. In the absence of cold iodine, high specific activity compounds can be obtained by this method and the labeling efficiency can be very high (~90 %). However, chloramine-T is a highly reactive substance and can cause denaturation of proteins. Sometimes milder oxidants such as sodium nitrite and sodium hypochlorite can be used instead of chloramine-T. This method is used in iodination of various compounds. 1.5.2.3.4. Electrolytic Method: Many proteins can be radioiodinated by the electrolytic method, which consists of the electrolysis of a mixture of radioiodide and the material to be labeled. In the electrolytic cell, the anode and cathode compartments are separated by a dialyzing bag that contains the cathode

- 30 - Chapter І Introduction

immersed in saline, whereas the anode compartment contains the electrolytic mixture. Electrolysis releases reactive iodine, which labels the compound. Slow and steady liberation of iodine causes uniform iodination of the compound, and in the absence of any carrier iodine, a labeling yield of almost 80% can be achieved. 1.5.2.3.5. Enzymatic Method: In enzymatic iodination, enzymes, such as lactoperoxidase and chloroperoxidase, and nanomolar quantities of H 2O2 are added to the iodination mixture containing radioiodine and the compound to be labeled. The hydrogen peroxide oxidizes iodide to form reactive iodine, which in turn iodinates the compound. Denaturation of proteins or alteration in organic molecules is minimal because only a low concentration of hydrogen peroxide is added. Yields of 60–85 % and high specific activity can be obtained by this method. This method is mild and useful in the iodination of many proteins and hormones. 1.5.2.3.6. Conjugation Method: In the conjugation method, initially N-succinimidyl-3(4-hydroxyphenyl)-propionate ( N-SHPP) is radioiodinated by the chloramine-T method and separated from the reaction mixture. The radioiodinated N-SHPP in dry benzene is available commercially. Proteins are labeled by this agent by allowing it to react with the protein molecule, resulting in an amide bond with lysine groups of the protein. The labeling yield is not very high, but the method allows iodination without alteration of protein molecules whose tyrosine moieties are susceptible to alteration, although in vivo dehalogenation is encountered in some instances.

- 31 - Chapter І Introduction

1.5.2.3.7. Demetallation Method: To improve the in vivo stability of iodinated proteins, various organometallic intermediates such as organothallium, organoborane and organostannane have been used to iodinate the aromatic ring of the precursor. The carbon-metal bond is cleaved by radioiodination in the presence of oxidizing agents such as chloramine-T and iodogen. organostannane [succinimidyl para-tri-n-butylstannyl benzoate (SBSB)] is most attractive because of the ease of preparation, stability and easy exchange reaction with radioiodine. SBSB is first radioiodinated by a suitable method whereby tributyl stannyl group is substituted by radioiodine. Protein is then coupled to SBSB by mixing the two at alkaline pH. Tamoxifen, vinyl-estradiol and phenyl-fatty acids are iodinated by this technique. 1.5.2.3.8. Iodogen Method: Proteins and cell membranes can be radioiodinated by the iodogen method. Iodogen or chloramide (1,3,4,6-tetrachloro-3a,6a-diphenyl- glycoluril) dissolved in methylene chloride is evaporated in tubes in order to obtain a uniform film coating inside the tube. The radioidide and protein are mixed together in the tube for 10–15 min, and the mixture is removed by decantation. Iodogen oxidizes iodide, and iodine then labels the protein. The unreacted iodide is separated by column chromatography of the mixture using Sephadex gel or DEAE ion exchange material. The denaturation of protein is minimal, because the reaction occurs on a solid phase of iodogen, which is poorly soluble in water. The labeling yield is of the order of 70- 80%.

- 32 - Chapter І Introduction

1.5.2.3.9. Iodo-bead Method: In the iodo-bead method, iodo-beads are used to iodinate various peptides and proteins containing a tyrosine moiety. Iodo-beads consist of the oxidant N-chlorobenzenesulfonamide immobilized on 2.8 mm diameter nonporous polystyrene spheres. These spheres are stable for at least 6 months if stored in an amber bottle at 4 ˚C. Radioiodination is carried out by simply adding five to six iodo-beads to a mixture of protein (~100 mg) and 131 I-sodium iodide in 0.5 ml of phosphate buffer solution contained in a capped polystyrene tube. The reaction is allowed for 15 min at room temperature. The iodination mixture can be removed by pipetting and iodinated protein is then separated by conventional techniques. This method has been claimed to be very successful with little denaturation of the protein. The labeling yield is almost 99 %.

- 33 -

CHAPTER II

RESULTS AND DISCUSSION Chapter II Results and Discussion

2. Results and Discussion Part (1) 2.1. Synthesis of compounds : 2.1.1. Synthesis of amino acid derivatives coupled with pyridine moiety: Owing to the pharmacological properties of pyridine derivatives, our attention was directed to employ newly synthesized derivatives of this ring system as starting materials for the azide method for the synthesis of peptides. 4-Aminopyridine (1) was treated with ethyl chloroacetate in ethanol and in presence of CH 3COONa.3H2O at reflux temperature to afford ethyl 2- (pyridin-4-ylamino)-acetate (2) in 92 % yield. The IR spectrum of this compound showed a characteristic absorption band in the carbonyl frequency region at 1750 cm -1 corresponding to the ester carbonyl group. The 1H-NMR spectrum showed a triplet at δ 1.223 and a quartet at δ 4.183 ppm corresponding to -OCH 2CH 3 group. The singlet at δ 3.901 ppm corresponds to N-CH 2 protons, while the NH group appeared as a broad singlet at δ 8.241 ppm. Treatment of compound 2 with hydrazine hydrate in absolute ethanol gives the corresponding hydrazide derivative 2-(pyridin-4-ylamino)- acetohydrazide (3) in 90 % yield ( scheme 2.1). The IR spectrum of this compound showed a characteristic absorption band in the carbonyl frequency region at 1675 cm -1 corresponding to the amide carbonyl group. The 1H-NMR spectrum of this hydrazide derivative showed the signals of

CONHNH 2 as a broad singlet at δ 4.347 and 9.655 ppm. The aromatic protons appeared as multiplet signals at δ 8.068-7.976 and 6.870-6.786 ppm.

- 34 - Chapter II Results and Discussion

Scheme 2.1 Synthesis of the target amino acid derivatives 4-6 were successfully achieved via the azide coupling method , which was reported to minimize the degree of racemization in amino acid coupling. Treatment of hydrazide 3 ˚ with nitrous acid (NaNO 2/HCl) at -5 C afforded the corresponding azide, which was isolated in ethyl acetate at low temperature (0 ˚C), to suppress its transformation to the isocyanate derivative by Curtius rearrangement. It is known that isocyanates are reactive towards the amino components in the coupling step leading to the formation of the undesired byproduct urea derivative [101]. Then, the azide solution in ethyl acetate reacted with amino acid ethyl esters hydrochloride, previously treated with triethylamine in ethyl acetate at low temperature, to produce the amino acid ester derivatives 4-6 in good yields ( scheme 2.2). Their chemical structures were confirmed by IR, mass and 1H-NMR spectra. Their IR spectrums showed the characteristic absorption bands for the ester carbonyl group and the amide carbonyl group. Their mass spectrums showed peaks corresponding to their molecular ion s. The 1H-NMR spectrum showed a signal for the NH proton of the peptide bond between δ 8.715 and 8.815 ppm, multiplet signals in the range δ 6.665- 7.682 ppm for the aromatic protons, a multiplet signal in the range δ 3.632- 3.662 ppm for the α-CH proton of the amino acids, a triplet signal for the three protons of -OCH 2CH 3 in the range δ 1.061-1.201 ppm and a quartet

- 35 - Chapter II Results and Discussion

signal in the range δ 3.994-4.064 ppm for the two protons of –OCH 2CH 3 of the ester groups.

No. R Amino acid 4 L-tyrosine

5 L-tryptophan

6 L-phenylalanine

Scheme 2.2 The dipeptide derivative 8 was prepared from L-tyrosine ethyl ester derivative 4 after conversion to the hydrazide derivative 7 by refluxing with excess hydrazine hydrate in ethanol. The 1H-NMR, mass and IR spectra of the dipeptide derivative 8 confirmed its structure. The dipeptide derivative 8 was obtained by the azide coupling method in 45 % yield (scheme 2.3). The IR spectrum of 8 showed two characteristic absorption bands for the ester carbonyl group at 1736 cm -1 and the amide carbonyl group at 1667cm -1. The 1H-NMR spectrum revealed two signals at δ 8.784 and δ 9.090 ppm for the two NH protons of the peptide bonds, a broad singlet at δ 5.186 ppm for OH group, a broad singlet at δ 10.938 ppm for NH group of the indole- ring, a triplet signal at δ 3.783 ppm for the α-CH protons of the two amino acids, a triplet at δ 1.197 and a quartet at δ 4.031 ppm

- 36 - Chapter II Results and Discussion

corresponding to -OCH 2CH 3 group. Its mass spectrum showed peak corresponding to its molecular ion at m/z 529 .

Scheme 2.3

2.1.2. Synthesis of amino acid derivatives coupled with 1,2,4-triazol- pyridine moiety: The synthesis of compounds containing 1,2,4-triazole rings in their structure has attracted widespread attention, mainly in connection with their wide range of pharmacological properties. The utility of hydrazides as key intermediates for the synthesis of several series of heterocyclic compounds and the broad spectrum of biological activities of their cyclized products have been reported [102]. Thus, our interest aroused in exploring the utility of hydrazides as versatile precursors for the synthesis of a variety of substituted heterocycles.

- 37 - Chapter II Results and Discussion

The reaction of hydrazide derivative 3 with phenyl-isothiocyanate gave phenylthiosemicarbazide 9 which on treatment with NaOH cyclized to triazole 10 (scheme 2.4). The structure of the 1,2,4-triazole thione derivative was proved by IR and mass spectra. The IR spectrum of this compound showed a characteristic absorption band for the C=S group at 1200 cm -1 and bands at 3223 and 3422 cm -1 for two NH groups. Its mass spectrum showed peak corresponding to its molecular ion at m/z 283 .

Scheme 2.4 The cyclization of phenylthiosemicarbazide 9 to the 1,2,4-triazole derivative 10 could be explained by the following mechanism [103]:

- 38 - Chapter II Results and Discussion

The hydrazide derivative 12 could be prepared from compound 10 by regioselective S-alkylation [104] with ethyl-chloroactetate to give compound 11 , which was subsequently hydrazinolyzed by hydrazine hydrate ( scheme- 2.5). The structure of compound 11 was proved by IR and 1H-NMR spectra. The IR spectrum of this thioester derivative showed the absorption band at 1738 cm -1 corresponding to the ester carbonyl group. The 1H-NMR spectrum showed a triplet at δ 1.211-1.258 ppm and a quartet at δ 4.243- 4.313ppm corresponding to OEt group. The hydrazide derivative was proved by IR and 1H-NMR spectra. The IR spectrum of this compound showed a characteristic absorption band at 1670 cm -1 corresponding to the amide 1 carbonyl group. The H-NMR spectrum showed the signals of CONHNH 2 as a broad singlet at δ 9.200 and 4.538 ppm. The aromatic protons appeared as multiplet signals in range δ 6.901-7.492 ppm.

Scheme2.5 Synthesis of ethyl-3-(4-hydroxyphenyl)-2-(2-[4-phenyl-5-([pyridin-4- ylamino]-methyl)-4H-1,2,4-triazol-3-ylthio]-acetamido)-propanoate (13 ) was successfully achieved from hydrazide derivative 12 via the azide coupling method in 60 % yield (scheme 2.6 ). Its chemical structure was confirmed by IR and 1H-NMR spectra. The IR spectrum of 13 showed two characteristic absorption bands for the ester carbonyl group at 1738 cm -1 and the amide carbonyl group at 1651cm -1. The 1H-NMR spectrum showed a signal at δ 8.500 ppm for

- 39 - Chapter II Results and Discussion

the NH proton of the peptide bond, multiplet signals between δ 6.661 and 7.410 ppm for the aromatic protons, a broad singlet at δ 5.115 ppm for OH group, a triplet signal at δ 3.516 ppm for the α-CH proton of the amino acid, a triplet signal at δ 1.116 ppm for the three protons of –OCH 2CH 3 and a quartet signal at δ 3.721 ppm for the two protons of –OCH 2CH 3 of the ester group.

Scheme 2.6

Refluxing of compound 13 with excess hydrazine hydrate in absolute ethanol gives the corresponding hydrazide derivative 14 . Then, the dipeptide derivative 15 was obtained from compound 14 via the azide coupling method in 50 % yield (scheme 2.7 ). The 1H-NMR, mass and IR spectra of 15 confirmed its structure. The IR spectrum of 15 showed two characteristic absorption bands for the ester carbonyl group at 1745 cm -1 and the amide carbonyl group at 1667cm -1. The 1H-NMR spectrum revealed two signals at δ 7.900 and 6.587ppm for the two NH protons of the peptide bonds, a broad singlet at δ 5.150 ppm for

- 40 - Chapter II Results and Discussion

OH group, broad signal at δ 10.883 ppm for NH group of the indole ring, a triplet signal at 3.662 ppm for the α-CH protons of the two amino acids, a triplet signal for the three protons of -OCH 2CH 3 at δ 1.073 ppm and a quartet signal at δ 3.990 ppm for the two protons of -OCH 2CH 3 of the ester group. Its mass spectrum showed peak corresponding to its molecular ion at m/z 718 .

2.1.3. Synthesis of amino acid derivatives coupled with 1,3,4-oxadiazol- pyridine moiety: Refluxing equimolar mixture of the hydrazide derivative 3 with potassium hydroxide and excess of carbon disulfide in ethanol afforded 5-([pyridin-4-ylamino] methyl)-1,3,4-oxadiazole-2-thiol (16) (scheme 2.8). Its IR spectrum showed the absence of the characteristic absorption band

- 41 - Chapter II Results and Discussion

corresponding to the amide carbonyl group and the characteristic absorption bands appeared at 1200 cm -1 and 3422 cm -1 for C=S and NH groups, respectively. The mass spectrum of 16 revealed the presence of the molecular ion peak at m/z 209 [M++1].

Scheme 2.8 The suggested mechanism for synthesis of 5-([pyridin-4-ylamino]- methyl)-1,3,4-oxadiazole-2-thiol 16 could be depicted by the following [105] mechanism :

Compound 16 was treated with ethyl-chloroacetate in DMF containing anhydrous K 2CO 3 to afford ethyl-2-(5-[(pyridin-4-ylamino)- methyl]-1,3,4-oxadiazol-2-ylthio)-acetate (17) in 92 % yield. The IR spectrum of compound 17 showed the absorption band for the ester carbonyl group at 1736 cm -1. The 1H-NMR spectrum showed a triplet at δ 1.239 ppm and a quartet at δ 4.191 ppm corresponding to -OEt group. The singlet at δ

- 42 - Chapter II Results and Discussion

4.310 ppm corresponds to SCH 2, while the NH group appeared as a broad singlet at δ 8.323 ppm. Treatment of 17 with hydrazine hydrate in absolute ethanol gives the corresponding hydrazide derivative 18 in 80 % yield ( scheme 2.9). The IR spectrum of this compound 18 showed a characteristic absorption band in the carbonyl frequency region at 1667 cm -1 corresponding to the amide carbonyl group and the characteristic absorption bands appeared at 3214, 3302 and 3459 cm -1 for NH groups. Its mass spectrum showed peak corresponding to its molecular ion at m/z 281 [M ++1].

N N N N N N HN ClCH2COOEt, HN K CO HN O 2 3 O NH NH 2 2 O SH DMF, r.t. S O S EtOH, reflux O

N N EtO N H2NHN 16 17 18

Scheme 2.9 Ethyl-3-(4-hydroxyphenyl)-2-(2-[5-([pyridin-4-ylamino]-methyl)-1,3,4- oxadiazol-2-ylthio]-acetamido)propanoate (19) was prepared from the hydrazide derivative 18 via the azide coupling method in 50 % yield (scheme 2.10 ).

- 43 - Chapter II Results and Discussion

N N N N HN HN

O NaNO2, HCl O S S O O

N H2NHN N N3 O 18 EtO o NH2 .HCl Et3N, EtOAc, 0 C, 24 hr . N N HO HN O H S N

O N O O 19 OH Scheme 2.10 The IR spectrum of 19 showed two characteristic absorption bands for the ester carbonyl group at 1750 cm -1 and the amide carbonyl group at 1626cm -1. The 1H-NMR spectrum showed a broad singlet at δ 8.833 ppm for the NH proton of the peptide bond, a broad singlet at δ 4.957 ppm for OH group, multiplet signals between δ 6.642 and 7.775 ppm for the aromatic protons, a triplet signal at δ 3.502 ppm for the α-CH proton of the amino acid, a triplet signal at δ 1.128 ppm for the three protons of –OCH 2CH 3 and quartet signal at δ 4.008 ppm for the two protons of –OCH 2CH 3 of the ester group. Its mass spectrum showed peak corresponding to its molecular ion at m/z 458 [M ++1]. The dipeptide derivative 21 was prepared from 19 after conversion to hydrazide 20 by refluxing with excess hydrazine hydrate in absolute ethanol. The dipeptide derivative 21 was obtained by the azide coupling method in 47 % yield (scheme 2.11 ). The IR spectrum of 21 showed two characteristic absorption bands for the ester carbonyl group at 1720 cm -1 and the amide carbonyl group at

- 44 - Chapter II Results and Discussion

1670cm -1. The 1H-NMR spectrum revealed two signals at δ 8.390 and 7.900ppm for the two NH protons of the peptide bonds, a broad singlet at δ5.203 ppm for OH group, a broad singlet at δ 10.890 ppm for NH group of the indole ring, a triplet signal at δ 3.655-3.698 ppm for the α-CH protons of the two amino acids, a triplet signal for the three protons of -OCH 2CH 3 at δ 1.083 ppm and a quartet signal at δ 3.994 ppm for the two protons of –

OCH 2CH 3 of the ester group. Its mass spectrum showed peak corresponding to its molecular ion at m/z 643.

- 45 - Chapter II Results and Discussion

2.1.4. Synthesis of amino acid derivatives coupled with tetrazol-pyridine moiety: Tetrazole and its derivatives have attracted much attention because of their unique structure and applications as antihypertensive, antialergic, antibiotic and anticonvulsant agents. Synthesis of tetrazole derivatives is obviously an important task in modern medicinal chemistry. The most common synthetic approach to prepare 5-substituted 1H-tetrazole derivatives involves the addition of azide salts to organic nitriles in a temperature range of typically 100–150 ˚C. Thus, 4-aminopyridine (1) was treated with chloro-acetonitrile in DMF containing sodium hydride to afford 2-(pyridin-4-ylamino)-acetonitrile (22), which was subsequently cyclized using sodium azide and ammonium- chloride to yield N-([2H-tetrazol-5-yl]-methyl)-pyridin-4-amine (23 ) in 86 % yield ( scheme 2.12 ). The 1H-NMR and mass spectra of 23 confirmed its structure. The 1H-NMR spectrum revealed two signals at δ 8.600 and 5.000ppm for the two NH protons. Its mass spectrum showed peak corresponding to its molecular ion at m/z 177 [M ++1].

Scheme 2.12 To a solution of 23 in DMF, sodium hydride was added and the mixture was stirred at room temperature for 2 h., then ethyl-chloroacetate was added to afford ethyl 2-(5-[(pyridin-4-ylamino) methyl]-2H-tetrazol-2- yl) acetate (24) in 74% yield. The IR spectrum of this compound showed a characteristic absorption band in the carbonyl frequency region at 1750 cm -1

- 46 - Chapter II Results and Discussion

corresponding to the ester carbonyl group. Its mass spectrum showed peak corresponding to its molecular ion at m/z 262. Treatment of compound 24 with hydrazine hydrate in absolute ethanol gives the corresponding hydrazide derivative 25 in 60 % yield ( scheme 2.13 ). The IR spectrum of this compound 25 showed a characteristic absorption band in at 1645 cm -1 corresponding to the amide carbonyl group and the characteristic absorption bands appeared at 3225, 3462 and 3550 cm - 1 for NH groups. Its mass spectrum showed peak corresponding to its molecular ion at m/z 248.

Scheme 2.13 Synthesis of ethyl-3-(4-hydroxyphenyl)-2-(2-[5-([pyridin-4-ylamino]- methyl)-2H-tetrazol-2-yl]-acetamido) propanoate (26) was achieved from hydrazide derivative 25 via the azide coupling method in 52 % yield (scheme 2.14 ). Its mass spectrum showed peak corresponding to its molecular ion at m/z 425. The IR spectrum of 26 showed two characteristic absorption bands for the ester carbonyl group at 1756 cm -1 and the amide carbonyl group at 1640 cm -1.

- 47 - Chapter II Results and Discussion

Scheme 2.14 Treatment of compound 26 with hydrazine hydrate in absolute ethanol gives the corresponding hydrazide derivative 27 in 75 % yield. Then, the dipeptide derivative 28 was prepared from the hydrazide derivative 27 by the azide coupling method in 48 % yield (scheme 2.15 ).

- 48 - Chapter II Results and Discussion

The IR spectrum of 28 showed two characteristic absorption bands for the ester carbonyl group at 1752 cm -1 and the amide carbonyl group at 1670cm -1. The 1H-NMR spectrum revealed two signals at δ 8.450 and 7.790ppm for the two NH protons of the peptide bonds, a broad singlet at δ 5.200 ppm for OH group, a broad singlet at δ 10.924 ppm for NH group of the indole ring, a triplet signal at 3.711 ppm for the α-CH protons of the two amino acids, a triplet signal for the three protons of -OCH 2CH 3 at δ 1.083 ppm and a quartet signal at δ 4.001 ppm for the two protons of –

OCH 2CH 3 of the ester group. Its mass spectrum showed peak corresponding to its molecular ion at m/z 611.

- 49 - Chapter II Results and Discussion

Part (2) 2.2. Radioiodination of dipeptide derivatives 8 and 15: 2.2.1. Radioiodination of dipeptide derivative coupled with pyridine moiety: Ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(pyridin-4-ylamino)-acetamido]- propanamido)-3-(1H-indol-3-yl) propanoate (EHPIP) (8) was labeled by 125 direct electrophilic substitution with I (t 1/2 = 60 days) under oxidative conditions in the presence of chloramine-T (CAT) (sodium salt of N- monochloro-p-toluene-sulfonamide) as shown in scheme 2.16.

Scheme 2.16

The radiochemical purity of the 125 I-EHPIP was determined using paper chromatography where radioiodide (I -) remained near the origin 125 (R f = 0.1), while the I-EHPIP moved with the solvent front (R f = 0.9). Radiochemical purity was further confirmed by paper electrophoresis where the free radioiodide and 125 I-EHPIP moved to different distances away from the spotting point towards the anode depending on the molecular weight of each one (distance from spotting point = 14 and 5 cm,

- 50 - Chapter II Results and Discussion

respectively). Results of labeling yield from the two separation methods are nearly the same. The influence of various reaction parameters and conditions on radioiodination efficiency, such as the amount of oxidizing agent (CAT), the amount of substrate, pH of the reaction and the reaction time were investigated and optimized in order to maximize the labeling yield. The level of significance was set at P < 0.05. The maximum labeling yield of 90 ± 2.80 % for 125 I-EHPIP was obtained at the optimum conditions. Effect of substrate amount: The dependence of the labeling yield on the amount of EHPIP is depicted in fig. (2.1), the reaction was performed at different EHPIP amounts (10-200 µg). The labeling yield of 125 I-EHPIP was small at low substrate amount where at 10 µg the labeling yield was 71 ± 2.90 % and by increasing EHPIP amount the labeling yield was increased where the maximum labeling yield (79 ± 3.00 %) was obtained at 100 µg.

1 0 0

% 1 2 5 I-E H P IP 8 0 % Free iodide

6 0

4 0 %Labeling yield %Labeling 2 0

0 0 50 100 150 200 EHPIP amount, µµµ g

Fig. (2.1) Variation of the labeling yield of 125 I-EHPIP as a function of different EHPIP amounts; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) EHPIP, 100 µg of CAT, at pH 7, the reaction mixtures were kept at room temperature for 15 min.

Effect of oxidizing agent: Radioiodination of EHPIP has been performed by using CAT as a mild oxidizing agent, transforming iodide (I -)

- 51 - Chapter II Results and Discussion

to an electropositive form of iodine (oxidative state I +), which allows a spontaneous electrophilic substitution on the highly activated phenolic- tyrosine residue at the ortho-position [106]. The effect of oxidizing agent amount (chloramine-T) on the labeling efficiency of 125 I-EHPIP is demonstrated in fig. (2.2).

100

% 125 I-EHPIP 80 % Free iodide

40 % Labeling yield Labeling % 20

0 0 50 100 150 200 250 CAT amount, µµµg

Fig. (2.2) Variation of the labeling yield of 125 I-EHPIP as a function of CAT amount; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) of CAT, 100 µg of EHPIP, at pH 7, the reaction mixtures were kept at room temperature for 15 min.

At low CAT amounts (10 µg), the labeling yield of 125 I-EHPIP was small and equal 68.9 ± 2.00 %. A high labeling yield of 78.7 ± 1.80 % was achieved by increasing the amount of CAT to 150 µg. Increasing the CAT amount above that value led to a decrease in the iodination yield where at 250 µg of CAT, the labeling yield was 51.5 ± 1.90 %. This decrease in labeling yield may be attributed to the formation of undesirable oxidative byproducts like chlorination, polymerization and denaturation of EHPIP [19]. The formation of these impurities may be attributed to the high reactivity and amount of CAT [107]. Consequently, the

- 52 - Chapter II Results and Discussion

optimum amount of CAT (150 µg) is highly recommended in order to avoid the formation of byproducts and to obtain a high labeling yield. Effect of reaction time: It is clear from fig. (2.3) that the labeling yield is slightly increased with increasing the reaction time from 10 Sec. to 60 min. The results indicate that the reaction is very fast. After 5 min the maximum labeling yield (90 ± 2.80 %) was obtained.

1 0 0

8 0

6 0 % 1 2 5 I-E H P IP % Free iodide

4 0 %Labeling yield %Labeling 2 0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 Reaction tim e, m inutes

Fig. (2.3) Variation of the labeling yield of 125 I-EHPIP as a function of reaction time; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 100 µg of EHPIP, 150 µg of CAT, at pH 8, the reaction mixtures were kept at room temperature for different intervals of time.

Effect of pH: The effect of pH of the reaction mixture on the labeling of EHPIP was studied by changing the pH values in the range from 2 up to 11 using buffers to adjust pH at the desired value, while other factors were kept constant. The labeling yield for each pH value of the reaction mixture was measured and the pH value at 8 the maximum labeling yield (90 ± 2.80 %) was obtained considered the optimum pH. Increasing in the pH of the medium above that pH 8 led to a slightly decreasing in the iodination yield where at pH 11, the labeling yield was 79.6 ± 1.00 % ( fig. (2.4)).

- 53 - Chapter II Results and Discussion

1 0 0

8 0

6 0 % 1 2 5 I-E H P IP % Free iodide

4 0 %Labeling yield %Labeling

2 0

0 1 2 3 4 5 6 7 8 9 101112 p H

Fig. (2.4) Variation of the labeling yield of 125 I-EHPIP as a function of pH; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 100 µg of EHPIP, 150 µg of CAT, at different pH, the reaction mixtures were kept at room temperature for 15 min .

Effect of reaction temperature: The influence of the temperature of the reaction mixture on the labeling yield of 125 I-EHPIP is shown in fig. - (2.5), the reaction was carried out at room temperature, 40, 60, 80 and 100 ˚C. The results of this study clearly show that reaction temperature has no significant effect on the labeling yield of 125 I-EHPIP.

1 0 0

8 0

6 0 % 1 2 5 I-E H P IP % Free iodide

4 0 %Labeling yield %Labeling 2 0

0 20 30 40 50 60 70 80 90 100 110 Tem perature, o C

Fig. (2.5) Variation of the labeling yield of 125 I-EHPIP as a function of reaction temperature; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 100 µg of EHPIP, 150 µg of CAT, at pH 8, the reaction mixtures were kept at different temperatures for 15 min.

Partition coefficient of 125 I-EHPIP: Lipophilicity and molecular weight of a compound influence its rate of clearance from the lung. The partition coefficient (Log P) equals 1.45 ± 0.01, showing that 125 I- EHPIP is relatively lipophilic.

- 54 - Chapter II Results and Discussion

In vitro stability of 125 I-EHPIP: In vitro stability of 125 I-EHPIP was studied in order to determine the suitable time for injection to avoid the formation of the undesired products that result from the radiolysis of the labeled compound. These undesired radioactive products might be accumulated in non-target organs. The results of stability showed that the 125 I-EHPIP is stable up to 48 h in refrigerator as shown in fig. (2.6) but is stable up to 12 h. at room temperature as shown in fig. (2.7), this may be due to the thermal decomposition of the 125 I-EHPIP.

100 100

80 80

60 60 % 125 I-EHPIP % 125 I-EHPIP % Free iodide % Free iodide

40 40 %Labeling yield %Labeling yield %Labeling 20 20

0 0 0 6 12 18 24 30 36 42 48 54 60 66 72 0 10 20 30 40 50 Time post iodination, hours Time post iodination, hours

Fig. (2.6) In vitro stability of 125 I-EHPIP Fig. (2.7) In vitro stability of 125 I-EHPIP in refrigerator. at room temperature .

- 55 - Chapter II Results and Discussion

2.2.2. Radioiodination of dipeptide derivative coupled with 1,2,4-triazol- pyridine moiety: Ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(4-phenyl-5-[(pyridin-4-ylamino)- methyl]-4H-1,2,4-triazol-3-ylthio)-acetamido]-propanamido)-3-(1H-indol-3- yl)-propanoate (EHPTIP) (15) was labeled by direct electrophilic substitution with 125 I under oxidative conditions in the presence of chloramine-T as shown in scheme- 2.17 .

Scheme 2.17

The radiochemical purity of the 125 I-EHPTIP was determined using paper chromatography where radioiodide (I -) remained near the origin 125 (R f = 0.1), while the I-EHPTIP moved with the solvent front (R f = 0.8). Radiochemical purity was further confirmed by paper electrophoresis where the free radioiodide and 125 I-EHPTIP moved to different distances away from the spotting point towards the anode (distance from spotting point = 14 and 10 cm, respectively). Results of labeling yield from the two separation methods are nearly the same.

- 56 - Chapter II Results and Discussion

The influence of various reaction parameters and conditions on radioiodination efficiency were investigated and optimized in order to maximize the labeling yield. The level of significance was set at P < 0.05. The maximum labeling yield of 92.5 ± 2.0 % for 125 I-EHPTIP was obtained at optimum conditions. Effect of substrate amount: The dependence of the labeling yield on the amount of EHPTIP is depicted in fig. (2.8), the reaction was performed at different EHPTIP amounts (5-250 µg).

1 0 0

8 0

1 2 5 6 0 % I-E H P T IP % Free iodide

4 0 %Labeling yield %Labeling 2 0

0 0 50 100 150 200 250 EHPTIP amount, µµµ g

Fig. (2.8) Variation of the labeling yield of 125 I-EHPTIP as a function of different EHPTIP amounts; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) EHPTIP, 100 µg of CAT, at pH 7, the reaction mixtures were kept at room temperature for 15 min .

The labeling yield of 125 I-EHPTIP was small at low substrate amount where at 5 µg the labeling yield was 77 ± 2.90 % and by increasing EHPTIP amount the labeling yield was increased where the labeling yield (84 ± 2.50 %) was obtained at 10 µg, after which no changes in yield occur; thus, 10 g was the optimum amount of the substrate. An increase in the labeling yield with increasing amount of the substrate is due to an increase in the interaction between the substrate and the generated iodonium ion to some extent, after which equilibrium takes place.

- 57 - Chapter II Results and Discussion

Effect of oxidizing agent: Radioiodination of EHPTIP has been performed by using CAT as an oxidizing agent which allows a direct electrophilic substitution on the highly activated phenolic tyrosine residue at the ortho-position [106]. The effect of oxidizing agent amount (chloramine-T) on the labeling efficiency of 125 I-EHPTIP is demonstrated in fig. (2.9), at low CAT amounts (10 µg), the labeling yield of 125 I-EHPTIP was small and equal 67.50 ± 2.00 %. A high labeling yield of 87 ± 2.30 % was achieved by increasing the amount of CAT to 150 µg. Increasing the CAT amount above that value led to a decrease in the iodination yield where at 200 µg of CAT, the labeling yield was 77.9 ± 2.00 %.

1 0 0

8 0

6 0 % 12 5 I-E H PT IP % Free iodide

4 0 %Labeling yield %Labeling 2 0

0 0 50 100 150 200 CAT amount, µµµg

Fig. (2.9) Variation of the labeling yield of 125 I-EHPTIP as a function of CAT mount; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, (x µg) of CAT, 10 µg of EHPTIP, at pH 7, the reaction mixtures were kept at room temperature for 15 min.

This decrease in labeling yield may be attributed to the formation of undesirable oxidative byproducts like chlorination, polymerization and denaturation of EHPTIP. The formation of these impurities may be attributed to the high reactivity and amount of CAT . Consequently, the optimum amount of CAT (150 µg) is highly recommended in order to avoid the formation of byproducts and to obtain a high labeling yield and purity.

- 58 - Chapter II Results and Discussion

Effect of pH: The effect of pH of the reaction mixture on the labeling of EHPTIP was studied by changing the pH values in the range from 3 up to 11 using buffers to adjust pH at the desired value, while other factors were kept constant. The labeling yield for each pH value of the reaction mixture was measured and the pH value at 7 the maximum labeling yield was obtained considered the optimum pH. Increasing in the pH of the medium above that pH 7 led to a significantly decreasing in the iodination yield where at pH 11, the labeling yield was 37 ± 1.00 % ( fig. (2.10)).

1 0 0 % 1 2 5 I-E H P T IP % Free iodide

8 0

6 0

4 0 %Labeling yield %Labeling

2 0

0 2 3 4 5 6 7 8 9 10 11 12 p H

Fig. (2.10) Variation of the labeling yield of 125 I-EHPTIP as a function of pH; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 10 µg of EHPTIP, 150 µg of CAT, at different pH, the reaction mixtures were kept at room temperature for 15 min.

Effect of reaction time: It is clear from fig. (2.11) that the labeling yield is significantly increased with increasing the reaction time from 10 Sec. to 60 min. After 45 min the maximum labeling yield (90 ± 2.30 %) was obtained.

- 59 - Chapter II Results and Discussion

1 0 0

8 0

6 0 % 1 2 5 I-E H P T IP % Free iodide

4 0 %Labeling yield %Labeling

2 0

0 10 20 30 40 50 60 Reaction tim e, m inutes

Fig. (2.11) Variation of the labeling yield of 125 I-EHPTIP as a function of reaction time; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 10 µg of EHPTIP, 150 µg of CAT, at pH 7, the reaction mixtures were kept at room temperature for different intervals of time.

Effect of reaction temperature: The influence of the temperature of the reaction mixture on the labeling yield of 125 I-EHPTIP is shown in fig. - (2.12), the reaction was carried out at room temperature, 40, 60, 80 and 100˚C. The results of this study clearly show that the yield is significantly increased with increasing the reaction temperature; where at higher temperature degrees (80 and 100 ˚C) the maximum labeling yield (92.5 ± 2.00 %) was obtained.

1 0 0

8 0

6 0 1 2 5 % I-E H P T IP % Free iodide

4 0 %Labeling yield %Labeling 2 0

0 20 30 40 50 60 70 80 90 100 110 Tem perature, o C

Fig. (2.12) Variation of the labeling yield of 125 I-EHPTIP as a function of reaction temperature; Reaction conditions: 10 µL (~7.2 MBq) Na 125 I, 10 µg of EHPTIP, 150 µg of CAT, at pH 7, the reaction mixtures were kept at different temperatures for 45 min.

- 60 - Chapter II Results and Discussion

Partition coefficient of 125 I-EHPTIP: The partition coefficient value 125 was 2.35 ± 0.11, showing that I-EHPTIP is relatively lipophilic . In vitro stability of 125 I-EHPTIP: In vitro stability of 125 I-EHPTIP was studied in order to determine the suitable time for injection to avoid the formation of the undesired products that result from the radiolysis of the labeled compound. These undesired radioactive products might be accumulated in non-target organs. The results of stability showed that the 125 I-EHPTIP is stable up to 48 h as shown in fig. (2.13).

1 0 0

8 0

6 0 % 1 2 5 I-E H P T IP % Free iodide

4 0 %Labeling yield %Labeling 2 0

0 0 6 12 18 24 30 36 42 48 54 60 66 72 Tim e post iodination, hours

Fig. (2.13) In vitro stability of 125 I-EHPTIP .

- 61 -

CHAPTER III

EXPERIMENTAL Chapter III Experimental

3. Experimental General All melting points were determined with a Kofler Block apparatus. The progress of the reactions was monitored by thin layer chromatography (TLC) using aluminum silica gel plates 60 F 245.The 1H-NMR spectra were recorded on a Varian Gemini-300 BB Spectrometer at 300 MHz with TMS as a standard, IR spectra were recorded on a Perking Elmer 1430 ratio recording infrared spectrophotometer with CDS data station using KBr Wafer technique, and Mass spectra were measured on a GC-MSQP 1000EX Shimadzu at the Micro Analytical Center, Cairo University, Cairo, Egypt. Chemicals and solutions were purchased from Merck Co. and they were in reactive grade. Na 125 I was purchased from Institute of Isotopes Co., Ltd. (IZOTOP) Budapest, Hungary.

Part (1) 3.1. Synthesis of compounds: Ethyl 2-(pyridin-4-ylamino) acetate (2):

O HN

N 2 To a solution of 4-aminopyridine (1) (0.94 g, 0.01 mol) in ethanol (25 ml) and in presence of CH 3COONa.3H2O (1.36 g, 0.01 mol), ethylchloro- acetate (1.23 g, 0.01 mol) was added and the reaction mixture was refluxed

- 62 - Chapter III Experimental

for 8 h. After the disappearance of the starting material, the mixture was cooled and poured onto crushed ice. The formed precipitate was filtered off, dried and recrystallized from ethanol. Its physical properties are white crystals (1.65 g, 92 %) and M.p. = 193-195 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1620 (C=N), 1750 (COO), 1 2996 (CH), 3268 (NH); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.241(1H, bs, NH), 8.146-8.065 (2H, m, ArH), 6.938-6.842 (2H, m, ArH), 4.183 (2H, q, J= 7.1Hz, OCH 2CH 3), 3.901 (2H, s, CH 2), 1.223 (3H, t, J = 7.1Hz,

OCH 2CH 3). 2-(Pyridin-4-ylamino) acetohydrazide (3):

3 To a solution of 2 (1.80 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 0.03 mol) was added and the reaction mixture was refluxed for 2 h, and then left to cool. The formed solid product was filtered off, dried and recrystallized from ethanol. Its physical properties are white crystals (1.49 g, 90 %) and M.p. = 261-263 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1654 (C=N), 1675 (CONH), 1 3027 (CH), 3151 (NH), 3257 (NH); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 9.655 (1H, bs, NH), 8.320 (1H, bs, NH), 8.068-7.976 (2H, m,

ArH), 6.870-6.786 (2H, m, ArH), 4.504 (2H, s, CH 2), 4.347 (2H, bs, NH 2).

- 63 - Chapter III Experimental

General procedure for the azide coupling method : To a cold solution (-5 ˚C) of hydrazide (0.01 mol) in acetic acid (6 ml),

1 N HCl (3 ml) and water (25 ml), a solution of NaNO 2 (0.69 g, 0.01 mol) in cold water (3 ml) were added. The reaction mixture was stirred at -5 ˚C for 20 min. The syrup formed was extracted with cold ethyl acetate (30 ml), washed with cold 3 % NaHCO 3, H 2O and finally dried over anhydrous sodium sulphate; the azide was used without further purification in the next step. After stirring the reaction mixture of amino acid ester hydrochloride (0.01 mol) and triethylamine (0.2 ml) in ethyl acetate (30 ml) at 0 ˚C for 20 min, the formed triethylamine hydrochloride was filtered off. The previously prepared cold dried solution of the azide was added to the cold ethyl acetate solution of the amino acid ester. The mixture was kept 12 h in the refrigerator and then at room temperature for an additional 12 h.

The reaction mixture was washed with 0.1 N HCl, 5 % NaHCO 3 and water, then dried over anhydrous sodium sulphate. The solvent was evaporated in vacuum and the residue was crystallized from ethyl acetate: petroleum-ether (2:1) to give the desired product. Ethyl3-(4-hydroxyphenyl)-2-(2-[pyridin-4-ylamino] acetamido) propanoate (4):

- 64 - Chapter III Experimental

The compound (2.57 g, 75 %) was obtained from hydrazide 3 (1.66 g, 0.01 mol) via the azide coupling method. Its physical properties are pale yellow powder and M.p. = 98-100 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1620 (C=N), 1668 (CONH), 1750 (COO), 3300 (OH), 3437 (NH); MS (m/z (%)): 343 (50.24), 342 (4.39), 326 (8.29), 313 (5.85), 299 (0.49), 269 (62.93), 236 (11.71), 148 1 (14.15), 220 (100.00); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.815 (1H, bs, NH), 8.391 (1H, bs, NH), 7.493 (2H, d, J = 8.1Hz, ArH), 7.238- 7.187 (2H, m, ArH), 6.931 (2H, d, J = 8.1Hz, ArH), 6.665 (2H, d, J = 8.1Hz,

ArH), 5.100 (1H, bs, OH), 4.029 (2H, q, J= 7.0Hz, OCH 2CH 3), 3.815 (2H, s,

CH 2), 3.643 (1H, t , J = 6.6Hz, CH), 2.777 (2H, d, J = 6.3Hz, CH 2), 1.157 (3H, t, J = 7.0Hz, OCH 2CH 3). Ethyl 3-(1H-indol-3-yl)-2-(2-[pyridin-4-ylamino] acetamido) propanoate (5):

5 The compound (2.38 g, 65 %) was obtained from hydrazide 3 (1.66 g, 0.01 mol) via the azide coupling method. Its physical properties are pale brown crystals and M.p. = 107 ˚C. -1 Its spectral data; IR (KBr, νmax (cm )): 1665 (CONH), 1744 (COO), 2986 (CH), 3284 (NH); MS (m/z (%)): 366 (0.07), 252 (0.05), 293 (0.13), 274 (0.28), 250 (0.12), 236 (0.22), 216 (0.24), 130 (100.00); 1H-NMR (300

MHz, DMSO -d6, δ (ppm)): 10.828 (1H, bs, NH), 8.800 (1H, bs, NH), 8.382 (1H, bs, NH), 7.696 (2H, d, J = 8.1Hz, ArH), 7.238-7.187 (4H, m, ArH),

- 65 - Chapter III Experimental

6.865 (3H, m, ArH), 4.052 (2H, q, J = 6.9Hz, OCH 2CH 3), 3.799 (2H, s,

CH 2), 3.683 (1H, t, J = 6.3Hz, CH), 2.765 (2H, d, J = 6.3Hz, CH 2), 1.192 (3H, t, J = 6.9Hz, OCH 2CH 3). Ethyl-3-phenyl-2-(2-[pyridin-4-ylamino] acetamido) propanoate (6):

6 The compound (2.29 g, 70 %) was obtained from hydrazide 3 (1.66 g, 0.01 mol) via the azide coupling method. Its physical properties are pale yellow powder and M.p. = 200 ˚C. -1 Its spectral data; IR (KBr, νmax (cm )): 1663 (CONH), 1738 (COO), 2980 (CH), 3607 (NH); MS (m/z (%)): 327 (9.50), 326 (7.82), 311 (12.34), 254 (7.51), 220 (2.22), 179 (9.81), 91 (100.00); 1H-NMR (300 MHz, DMSO - d6, δ (ppm)): 8.715 (1H, bs, NH), 8.257 (1H, bs, NH), 7.506 (2H, d, J= 8.1Hz, ArH), 7.249-6.845 (5H, m, ArH), 6.678 (2H, d, J= 8.1Hz, ArH),

4.063 (2H, q, J= 6.9Hz, OCH 2CH 3), 3.815 (2H, s, CH 2), 3.653 (1H, t, J=

6.3Hz, CH), 2.668 (2H, d, J= 6.3Hz, CH 2), 1.178 (3H, t, J= 6.9Hz,

OCH 2CH 3). N-(1-Hydrazinyl-3-[4-hydroxyphenyl]-1-oxopropan-2-yl)-2-(pyridin-4-ylamino) acetamide (7):

- 66 - Chapter III Experimental

To a solution of 4 (3.43 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 0.03 mol) was added and the reaction mixture was refluxed for 2 h, then left to cool. The formed solid product was filtered off, dried and recrystallized from ethanol. Its physical properties are gray powder (2.46 g, 75 %) and M.p. = 110 ˚C. -1 Its spectral data; IR (KBr, νmax (cm )): 1651 (CONH), 3303 (OH), 3400 (NH), 3446 (NH); MS (m/z (%)): 328 (0.83), 326 (19.00), 312 (0.69), 1 236 (1.24), 220 (100.00); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.908 (1H, bs, NH), 8.606 (1H, bs, NH), 8.211 (1H, bs, NH), 7.606-7.534 (2H, m, ArH), 7.013-6.889 (4H, m, ArH), 6.701-6.544 (2H, m, ArH), 5.201 (1H, bs,

OH), 3.991 (2H, bs, NH 2), 3.815 (2H, s, CH 2), 3.252 (1H, t, J= 6.3Hz, CH),

2.734 (2H, d, J= 6.3Hz,CH 2). Ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(pyridin-4-ylamino)-acetamido] propanamido)-3-(1H-indol-3-yl) propanoate (8):

8 The black oily compound (2.38 g, 45 %) was obtained from hydrazide 7 (3.29 g, 0.01 mol) via the azide coupling method. -1 Its spectral data; IR (KBr , νmax (cm )): 1667 (CONH), 1736 (COO), 3145 (NH), 3214 (OH), 3400 (NH), 3497 (NH); MS (m/z (%)): 529 (3.16),

- 67 - Chapter III Experimental

501 (5.16), 272 (2.34), 270 (1.25), 259 (4.40), 231 (2.67), 136 (1.76), 1 130(100.00); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 10.938 (1H, bs, NH), 9.090 (1H, bs, NH), 8.784 (1H, bs, NH), 8.329 (1H, bs, NH), 8.052 (3H, d, J= 7.8Hz, ArH), 7.501-7.327 (4H, m, ArH), 7.147-6.979 (6H, m, ArH),

6.710 (1H, s, CH), 5.186 (1H, bs, OH), 4.030 (2H, q, J= 4.8Hz, OCH 2CH 3),

4.002 (2H, s, CH 2), 3.783 (2H, t, J= 6.6Hz, CH), 3.063 (4H, d, J= 6.6Hz,

CH 2), 1.197 (3H, t, J= 4.8Hz, OCH 2CH 3). N-Phenyl-2-(2-[pyridin-4-ylamino] acetyl) hydrazine-carbothioamide (9):

9 To a solution of hydrazide 3 (1.66 g, 0.01 mol) in ethanol (10 ml), phenylisothiocyanate (1.35 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 6 h. The product that separated on cooling was filtered off, washed with ethanol, and dried well to give the desired product (2.76 g, 92 %). Its physical properties are yellow crystals and M.p. = 205 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1657 (CONH), 1191 (C=S), 3042 (CH), 3173 (NH), 3471 (NH); MS (m/z (%)): 302 (4.91), 224 (4.16), 209 (44.53), 165 (5.39), 151 (3.9), 135 (4.64), 92 (46.40).

- 68 - Chapter III Experimental

4-Phenyl-5-([pyridin-4-ylamino] methyl)-4H-1,2,4-triazole-3-thiol (10):

N HN SH N N

N 10 A solution of 9 (3.01 g, 0.01 mol) in 2 N NaOH (50 ml) was heated under reflux for 4 h. The reaction mixture was cooled and acidified with 2 N HCl. The resulting precipitate was filtered off, washed with ethanol and recrystallized from ethanol. Its physical properties are white crystals (2.40 g, 85 %) and M.p. = 70-73 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1604 (C=N), 1200 (C=S), 3050 (CH), 3223 (NH), 3422 (NH); MS (m/z (%)): 283 (1.05), 190 (6.27), 107 (5.96), 93 (100.00). Ethyl 2-(4-phenyl-5-[(pyridin-4-ylamino)-methyl]-4H-1,2,4-triazol-3-ylthio) acetate (11):

To a solution of 10 (2.83 g, 0.01 mol) in DMF (10 ml), anhydrous

K2CO 3 (1.38 g, 0.01 mol) was added and the mixture was stirred at room temperature for 2 h. Then, ethyl-chloroacetate (1.85 g, 0.015 mol) was added and the reaction mixture was stirred at room temperature for 6 h.

- 69 - Chapter III Experimental

The resulting mixture was poured into crushed ice, and then the formed precipitate was filtered off, dried and recrystallized from ethanol. Its physical properties are yellow crystals (2.88 g, 78 %) and M.p . = 60 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1738 (COO), 1620 (C=N), 1 2986 (CH), 3465 (NH); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.673 (1H, bs, NH), 7.448 (2H, d, J= 8.1Hz, ArH), 7.332-7.042 (4H, m, ArH),

6.718 (3H, d, J= 7.5Hz, ArH), 4.278 (2H, q, J= 7.0Hz, OCH 2CH 3), 4.158

(2H, s, SCH 2), 3.771 (2H, s, CH 2), 1.235 (3H, t, J = 7.0Hz, OCH 2CH 3). 2-(4-Phenyl-5-[(pyridin-4-ylamino)-methyl]-4H-1,2,4-triazol-3- ylthio)aceto-hydrazide (12):

12 To a solution of 11 (3.69 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 0.03 mol) was added and the reaction mixture was refluxed for 6 h, and then left to cool. The formed solid product was filtered off, dried and recrystallized from ethanol. Its physical properties are white crystals (2.87 g, 81 %) and M.p. = 100 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1670 (CONH), 3025 (CH), 1 3161 (NH), 3262 (NH); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 9.200 (1H, bs, NH), 8.633 (1H, bs, NH), 7.493 (2H, d, J= 8.1Hz, ArH), 7.238-

7.187 (4H, m, ArH), 6.914 (3H, d, J= 8.1Hz, ArH), 4.538 (2H, bs, NH 2),

4.053 (2H, s, SCH 2), 3.821 (2H, s, CH 2).

- 70 - Chapter III Experimental

Ethyl 3-(4-hydroxyphenyl)-2-(2-[4-phenyl-5-([pyridin-4-ylamino] methyl)- 4H-1,2,4-triazol-3-ylthio] acetamido) propanoate (13):

The compound (3.19 g, 60 %) was obtained from hydrazide 12 (3.55 g, 0.01 mol) and L-tyrosine ethyl ester hydrochloride (2.45 g, 0.01 mol) via the azide coupling method. Its physical properties are pale yellow powder and M.p. = 110 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1607 (C=C), 1651 (CONH), 1 1738 (COO), 3384 (OH), 3427 (NH); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.500 (1H, bs, NH), 8.200 (1H, bs, NH), 7.410-7.150 (2H, m, ArH), 7.067-6.942 (4H, m, ArH), 6.661 (3H, d, J= 8.1Hz, ArH), 5.115 (1H, bs, OH), 4.398 (2H, s, SCH 2), 3.721 (2H, q, J= 6.6Hz, OCH 2CH 3), 3.856

(2H, s, CH 2), 3.516 (1H, t, J= 6.6Hz, CH), 2.717 (2H, d, J= 6.6Hz, CH 2),

1.116 (3H, t, J= 7.0Hz, OCH 2CH 3). N-(1-Hydrazinyl-3-[4-hydroxyphenyl]-1-oxopropan-2-yl)-2-(4-phenyl-5- [(pyridin-4-ylamino) methyl]-4H-1,2,4-triazol-3-ylthio) acetamide (14):

- 71 - Chapter III Experimental

To a solution of 13 (5.32 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 0.03 mol) was added and the reaction mixture was refluxed for 7 h, and then left to cool. The formed solid product was filtered off, dried and recrystallized from ethanol. Its physical properties are white crystals (3.63 g, 70 %) and M.p. = 90 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1615 (C=N), 1654 (CONH), 2987 (CH), 3280 (NH), 3305 (OH), 3420, 3440 (NH); MS (m/z (%)): 517 (82.68), 255 (80.31), 147 (81.10), 107 (16.54), 94 (85.04), 79 (11.02), 72 (88.98). Ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(4-phenyl-5-[(pyridin-4-ylamino) - methyl]-4H-1,2,4-triazol-3-ylthio) acetamido] propanamido)-3-(1H-indol- 3-yl) propanoate (15):

15 The black oily compound (3.59 g, 50 %) was obtained from hydrazide 14 (5.18 g, 0.01 mol) and L-tryptophan ethyl ester hydrochloride (2.68 g, 0.01 mol) via the azide coupling method. -1 Its spectral data; IR (KBr, νmax (cm )): 1610 (C=N), 1650, 1667 (CONH), 1745 (COO), 3145 (NH), 3310 (OH), 3497 (NH); MS (m/z (%)): 718 (14.25), 604 (13.84), 587 (4.07), 459 (2.71), 458 (17.10), 436 (15.47), 394 (17.10), 232 (100), 130 (60.11), 107 (6.24); 1H-NMR (300 MHz,

- 72 - Chapter III Experimental

DMSO -d6, δ (ppm)): 10.883 (1H, d, J = 8.4Hz, NH); 8.321 (1H, bs, NH), 7.900 (1H, bs, NH), 7.570-6.966 (17H, m, ArH), 6.943 (1H, s, CH), 6.587

(1H, bs, NH), 5.150 (1H, bs, OH), 4.200 (2H, s, SCH2), 3.990 (2H, q, J=

7.0Hz, OCH 2CH 3), 3.900 (2H, s, CH 2), 3.662 (2H, t, J= 6.3Hz, CH), 3.424-

2.995 (4H, m, CH 2), 1.073 (3H, t, J= 5.1Hz, OCH 2CH 3). 5-([Pyridin-4-ylamino] methyl)-1,3,4-oxadiazole-2-thiol (16):

16 To a solution of 3 (1.66 g, 0.01 mol) in ethanol (30 ml), a solution of potassium hydroxide (0.56 g, 0.01 mol) in water (2 ml) was added. Then, carbon disulphide (5 ml) was added and the reaction mixture was refluxed for 20 h. The solvent was evaporated, and then the residue was dissolved in water (20 ml), filtered off and acidified with dil. HCl. The precipitate was filtered off, washed with water and recrystallized from ethanol. Its physical properties are white crystals (1.73 g, 83 %) and M.p. = 215-220 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1200 (C=S), 1620 (C=N), 2922 (CH), 3422 (NH); MS (m/z (%)): 209 (83.08), 132 (100.00), 94 (17.74).

- 73 - Chapter III Experimental

Ethyl 2-(5-[(pyridin-4-ylamino) methyl]-1,3,4-oxadiazol-2-ylthio) acetate (17):

To a solution of 16 (2.08 g, 0.01 mol) in DMF (10 ml), anhydrous

K2CO 3 (1.38 g, 0.01 mol) was added and the mixture was stirred at room temperature for 2 h. Then, ethyl chloroacetate (1.23 g, 0.01 mol) was added and the reaction mixture was stirred at room temperature for 3 h. The resulting mixture was poured into crushed ice, and then the formed precipitate was filtered off, dried and recrystallized from ethanol. Its physical properties are yellow crystals (2.70 g, 92 %) and M.p. > 300 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1580 (C=N), 1736 (COO), 1 2986 (CH), 3269 (NH); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.323

(1H, bs, NH), 7.790-8.280 (4H, m, ArH), 4.310 (2H, s, SCH 2), 4.191 (2H, q,

J= 5.5 Hz, OCH 2CH 3), 3.900 (2H, s, CH 2), 1.239 (3H, t, J= 5.5Hz,

OCH 2CH 3). 2-(5-[(Pyridin-4-ylamino) methyl]-1,3,4-oxadiazol-2-ylthio) acetohydrazide (18):

18 To a solution of 17 (2.94 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 0.03 mol) was added and the reaction mixture was refluxed for 4 h, and then left to cool. The formed solid product was filtered

- 74 - Chapter III Experimental

off, dried and recrystallized from ethanol. Its physical properties are white crystals (2.24 g, 80%) and M.p. = 128-130 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1617 (C=N), 1667 (CONH), 2938 (CH), 3214, 3302, 3459 (NH); MS (m/z (%)): 281 (42.86), 223 (13.45), 107 (21.43), 94 (23.95), 63 (100.00). Ethyl 3-(4-hydroxyphenyl)-2-(2-[5-([pyridin-4-ylamino]-methyl)-1,3,4- oxadiazol-2-ylthio] acetamido) propanoate (19):

19 The compound (2.29 g, 50 %) was obtained from hydrazide 18 (2.80 g, 0.01 mol) and L-tyrosine ethyl ester hydrochloride (2.45 g, 0.01 mol) via the azide coupling method. Its physical properties are yellow powder and M.p. = 250-252 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1558 (C=N), 1626 (CONH), 1750 (COO), 2986 (CH), 3347 (OH), 3465 (NH); MS (m/z (%)): 458 (9.94), 427 (11.70), 293 (8.89), 281 (8.81), 250 (4.97), 208 (9.70), 95 (25.80); 1 H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.833 (1H, bs, NH), 8.559 (1H, bs, NH), 7.775-7.725 (2H, m, ArH), 6.945 (4H, d, J= 8.7Hz, ArH), 6.684-

6.642 (2H, m, ArH), 4.957 (1H, bs, OH), 4.357 (2H, s, SCH 2), 4.008 (2H, q,

J= 7.0Hz, OCH 2CH 3), 3.502 (1H, t, J= 6.6Hz, CH), 2.833-2.700 (2H, m,

CH2), 1.128 (3H, t, J= 6.9Hz, OCH 2CH 3).

- 75 - Chapter III Experimental

N-(1-Hydrazinyl-3-[4-hydroxyphenyl]-1-oxopropan-2-yl)-2-(5-[(pyridin-4- ylamino) methyl]-1,3,4-oxadiazol-2-ylthio) acetamide (20):

20 To a solution of 19 (4.57 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 0.03 mol) was added and the reaction mixture was refluxed for 2 h, and then left to cool. The solvent was evaporated in vacuum to give the black oily product (3.19 g, 72 %). -1 Its spectral data; IR (KBr , νmax (cm )): 1610 (C=N), 1643 (CONH), 2956 (CH), 3280 (NH), 3325 (OH), 3410, 3449 (NH); MS (m/z (%)): 443 (2.99), 426 (1.18), 3.86 (4.70), 350 (31.02), 95 (4.57), 79 (3.36). Ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(5-[(pyridin-4-ylamino)-methyl]-1,3,4- oxadiazol-2-ylthio) acetamido] propanamido)-3-(1H-indol-3-yl)-propanoate (21):

21 The yellow oily compound (3.02 g, 47 %) was obtained from hydrazide 20 (4.43 g, 0.01 mol) and L-tryptophan ethyl ester hydrochloride (2.68 g, 0.01 mol) via the azide coupling method. -1 Its spectral data; IR (KBr , νmax (cm )): 1615 (C=N), 1640, 1670 (CONH), 1720 (COO), 3135 (NH), 3300 (OH), 3450, 3497 (NH); MS (m/z

- 76 - Chapter III Experimental

(%)): 643 (1.38), 598 (1.07), 130 (100.00), 107 (7.23); 1H-NMR (300 MHz,

DMSO -d6, δ (ppm)): 10.890 (1H, s, NH), 8.390 (1H, bs, NH), 8.100 (1H, bs, NH), 7.900 (1H, bs, NH), 7.500-6.968 (12H, m, ArH), 6.690 (1H, s, CH),

5.203 (1H, bs, OH), 4.417 (2H, s, SCH 2), 4.200 (2H, s, CH 2), 3.994 (2H, q,

J=7.2Hz, OCH 2CH 3), 3.676 (2H, t, J=6.5Hz, CH), 3.103 (4H, d, J= 6.9Hz,

CH 2), 1.083 (3H, t, J= 7.2Hz, OCH 2CH 3). 2-(Pyridin-4-ylamino) acetonitrile (22):

HN CN

N 22 4-Aminopyridine (1) (0.94 g, 0.01 mol) and sodium hydride (0.5 g) were dissolved in DMF (10 ml) and the solution was stirred at room temperature for 2 h. Chloroacetonitrile (3 ml) was added and the reaction mixture was stirred for 24 h. The resulting mixture was poured into crushed ice, and then the formed precipitate was filtered off, washed with water and dried. Its physical properties are black ppt. (1.06 g, 80 %) and M.p. > 300 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1651 (C=N), 2210 (C ≡N), 2930 (CH), 3446 (NH). N-([2H-Tetrazol-5-yl]-methyl) pyridin-4-amine (23):

23 A mixture of compound 22 (1.33 g, 0.01 mol) sodium azide (0.57 g, 0.012 mol) and ammonium chloride (0.7 g, 0.012 mol) in DMF (10 ml) was

- 77 - Chapter III Experimental

heated under reflux for 24 h, then it was diluted with cold water (20 ml). The obtained precipitate was filtered off, washed with water, dried and recrystallized from ethanol. Its physical properties are black crystals (1.51 g, 86 %) and M.p. > 300 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1655 (C=N), 2931 (CH), 3440, 3518 (NH); MS (m/z (%)): 177 (13.52), 176 (7.54), 106 (16.37), 96 1 (3.06), 69 (1.78); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 8.600 (1H, bs,

NH), 7.391-7.000 (4H, m, ArH), 5.000 (1H, bs, NH), 3.994 (2H, s, CH 2). Ethyl 2-(5-[(pyridin-4-ylamino) methyl]-2H-tetrazol-2-yl) acetate (24):

24 To a solution of 23 (1.76 g, 0.01 mol) in DMF (10 ml), sodium hydride (0.5 g) was added and the mixture was stirred at room temperature for 2 h. Then, ethyl-chloroacetate (1.23 g, 0.01mol) was added and the reaction mixture was stirred at room temperature for 14 h. The resulting mixture was poured into crushed ice, and then the formed precipitate was filtered off, dried and recrystallized from ethanol. Its physical properties are brown crystals (1.94 g, 74 %) and M.p. > 300 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1644 (C=N), 1750 (COO), 2955 (CH), 3446 (NH); MS (m/z (%)): 262 (12.99), 247 (0.11), 235 (60.69), 189 (17.59), 175 (18.51), 170 (12.07), 155 (12.64), 107 (27.13).

- 78 - Chapter III Experimental

2-(5-[(Pyridin-4-ylamino) methyl]-2H-tetrazol-2-yl) acetohydrazide (25):

25 A mixture of ester 24 (2.62 g, 0.01 mol) and hydrazine hydrate (1.5 g, 0.03 mol) in absolute ethanol ( 20 ml) was refluxed for 5 h, the solvent was evaporated under vacuum and the obtained precipitate was recrystallized from ethanol to afford the corresponding hydrazide 25 (1.48 g, 60 %). Its physical properties are black crystals and M.p. > 300 ˚C. -1 Its spectral data; IR (KBr , νmax (cm )): 1620 (C=N), 1645 (CONH), 2948 (CH), 3225, 3462, 3550 (NH); MS (m/z (%)): 248 (15.99), 217 (8.11), 189 (12.30), 107 (21.62), 94 (17.59). Ethyl 3-(4-hydroxyphenyl)-2-(2-[5-([pyridin-4-ylamino]-methyl)-2H- tetrazol-2-yl] acetamido) propanoate (26):

26 The compound (2.21 g, 52 %) was obtained from hydrazide 25 (2.48 g, 0.01 mol) and L-tyrosine ethyl ester hydrochloride (2.45 g, 0.01 mol) via the azide coupling method. Its physical properties are yellow powder and M.p. = 110 ˚C.

- 79 - Chapter III Experimental

-1 Its spectral data; IR (KBr, νmax (cm )): 1626 (C=N), 1640 (CONH), 1756 (COO), 2998 (CH), 3335 (OH), 3400, 3455 (NH); MS (m/z (%)): 425 (10.73), 232 (13.69), 208 (15.73), 174 (23.80), 106 (38.71). N-(1-Hydrazinyl-3-[4-hydroxyphenyl]-1-oxopropan-2-yl)-2-(5-[(pyridin-4- ylamino)-methyl]-2H-tetrazol-2-yl)-acetamide (27):

27 To a solution of 26 (4.25 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 0.03 mol) was added and the reaction mixture was refluxed for 3 h, and then left to cool. The solvent was evaporated in vacuum to give the black oily product (3.08 g, 75 %). -1 Its spectral data; IR (KBr , νmax (cm )): 1630 (C=N), 1656 (CONH), 3000 (CH), 3190 (NH), 3310 (OH), 3420, 3492 (NH); MS (m/z (%)): 412 (36.59), 222 (47.04), 217 (42.51), 129 (100.00), 94 (4.53). Ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(5-[(pyridin-4-ylamino)-methyl]-2H- tetrazol-2-yl) acetamide] propanamido)-3-(1H-indol-3-yl) propanoate (28):

- 80 - Chapter III Experimental

The black oily compound (2.93 g, 48 %) was obtained from hydrazide 27 (4.11 g, 0.01 mol) and L-tryptophan ethyl ester hydrochloride (2.68 g, 0.01 mol) via the azide coupling method. -1 Its spectral data; IR (KBr , νmax (cm )): 1620 (C=N), 1653, 1670 (CONH), 1752 (COO), 3145 (NH), 3310 (OH), 3420, 3462, 3497 (NH); MS (m/z (%)): 611 (31.30), 584 (40.58), 538 (39.71), 395 (14.78), 380 (9.28), 1 315 (100.00); H-NMR (300 MHz, DMSO -d6, δ (ppm)): 10.924(1H, bs, NH), 8.450(1H, bs, NH), 8.120 (1H, bs, NH), 7.790 (1H, bs, NH), 7.505- 6.948 (12H, m, ArH); 6.600 (1H, s, CH), 5.200 (1H, bs, OH), 4.420 (2H, s,

CH 2), 4.001 (2H, q, J= 7.0Hz, OCH 2CH 3), 3.900 (2H, s, CH 2), 3.711 (2H, t,

J= 6.3Hz, CH), 3.031 (4H, d, J= 6.3Hz, CH 2), 1.083 (3H, t, J= 7.5Hz,

OCH 2CH 3) .

- 81 - Chapter III Experimental

Part (2) 3.2. Radioiodination of dipeptide derivatives 8 and 15: 3.2.1. Preparation of buffers [108]: • Citric acid buffer (pH= 2) It was prepared by adding 2.39 ml of 2M NaOH solution to 10 ml of 2M citric acid and volume was completed to 100 ml using distilled water. • Citric acid buffer (pH= 3) This buffer was prepared by adding 81.1 ml of 0.1M citric acid to 18.9 ml of

0.2M Na 2HPO 4. • Sodium acetate, acetic acid buffer (pH =5) This buffer was prepared by adding 14.8 ml of 0.2M acetic acid to 35.2 ml of 0.2M sodium acetate and volume was completed to 100 ml using distilled water. • Phosphate buffer (pH= 7) It was prepared by adding 30.5 ml of 0.2M disodium hydrogen phosphate solution (Na 2HPO 4) to 19.5 ml of 0.2M monosodium phosphate solution

(NaH 2PO 4) and volume was completed to 100 ml using distilled water. • Phosphate buffer (pH= 8) This buffer was prepared by adding 47.35 ml of 0.2M disodium hydrogen phosphate solution (Na 2HPO 4) to 2.65 ml of 0.2M monosodium phosphate solution (NaH 2PO 4) and volume was completed to 100 ml using distilled water. • Sodium bicarbonate, sodium hydroxide buffer (pH =10) This buffer was prepared by adding 50.0 ml of 0.05M sodium bicarbonate

(NaHCO 3) to 10.7 ml of 0.1M sodium hydroxide (NaOH) and volume was completed to 100 ml using distilled water.

- 82 - Chapter III Experimental

• Sodium bicarbonate, sodium hydroxide buffer (pH =11) This buffer was prepared by adding 50.0 ml of 0.05M sodium bicarbonate

(NaHCO 3) to 22.7 ml of 0.1M sodium hydroxide (NaOH) and volume was completed to 100 ml using distilled water. 3.2.2. Method of Radioiodination: 3.2.2.1. Radioiodination of ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(pyridin- 4-ylamino)-acetamido] propanamido)-3-(1H-indol-3-yl)- propanoate (EHPIP) (8) was prepared as follows: (1) In a reaction vial of 1.0 ml capacity with a Teflon septum and screw cap, 100 µl of EHPIP (1.9 mM) dissolved in distilled water were added. (2) Adding 150 µl of freshly prepared chloramine-T (4.4 mM) dissolved in distilled water. (3) Adding 150 µl of phosphate buffer pH 8. (4) Adding 10 µl of 125 I (7.2 MBq). (5) The reaction was done at room temperature. (6) After 5 min, the reaction was arrested by adding 450 µl of sodium- metabisulfite (5.3 mM) dissolved in distilled water. 3.2.2.2. Radioiodination of ethyl 2-(3-[4-hydroxyphenyl]-2-[2-(4- phenyl-5-[(pyridin-4-ylamino)-methyl]-4H-1,2,4-triazol-3- ylthio) acetamido] propanamido)-3-(1H-indol-3-yl) propanoate (EHPTIP) (15) was prepared as follows: (1) In a reaction vial of 1.0 ml capacity, 10 µl of EHPTIP (1.4 mM) dissolved in dimethyl-sulfoxide were added. (2) Adding 150 µl of freshly prepared chloramine-T (4.4 mM) dissolved in distilled water. (3) Adding 150 µl of phosphate buffer pH 7.

- 83 - Chapter III Experimental

(4) Adding 10 µl of 125 I (7.2 MBq). (5) The reaction was done at 80 ˚C. (6) After 45 min, the reaction was arrested by adding 450 µl of sodium- metabisulfite (5.3 mM) dissolved in distilled water. 3.2.3. Determination of the labeling yield and the radiochemical purity of 125 I-EHPIP and 125 I-EHPTIP: The labeling yield and radiochemical purity were measured by two methods: 3.2.3.1. Ascending paper chromatography: On Whatman paper sheet (1 cm width and 13 cm length), 1–2 l of the reaction mixture was placed 2 cm above the lower edge and allowed to evaporate spontaneously. For development a fresh mixture of chloroform: ethanol (9:1 v/v) was used as a mobile phase. After complete development, the paper sheet was removed, dried, and cut into strips, each strip is 1 cm width, and then each strip was counted in a well type γ-counter where - 125 radioiodide (I ) remained near the origin (R f = 0.1), while the I-EHPIP and 125 I-EHPTIP moved with the solvent front (R f = 0.9 and 0.8, respectively) as shown in table (3.1).

Table (3.1) Radiochromatographic behavior of different radiochemical species and their R f values for of 125I- EHPIP and 125 I- EHPTIP in paper chromatography .

System Solvents Radioactive Rf species Paper Mixture Free iodide 0.1 chromatography (chloroform: ethanol) (9:1 v/v) 125 I- EHPIP 0.9

125 I- EHPTIP 0.8

3.2.3.2. Paper electrophoresis: On Whatman paper sheet (2 cm width and 47 cm length), 1–2 l of the reaction mixture was placed 12 cm away from the cathode and allowed

- 84 - Chapter III Experimental

to evaporate spontaneously. Electrophoresis is carried out for 80 min. at a voltage of 300 volts using normal saline (0.9 % w/v NaCl solution) as an electrolyte source solution. After complete development, the paper was removed, dried, and cut into strips, each strip is 1 cm width, and then the strip was counted in a well type γ-counter. Free radioiodide, 125 I-EHPIP and 125 I-EHPTIP moved to different distances away from the spotting point towards the anode (distance from spotting point = 14, 5and 10 cm, respectively) as shown in table (3.2) and fig. (3.1). The percentage of labeling yield was estimated as the ratio of the radioactivity of iodocompound to the total activity multiplied by 100. % Labeling yield = activity of labeled product X 100 Total activity Table (3.2) Radio chromatographic behavior of different radiochemical species for 125 I-EHPIP and 125 I- EHPTIP in paper electrophoresis .

System Source of electrolytes Radioactive Distance from species spotting point Paper Normal saline Free iodide 14 Cm electrophoresis (0.9 % w/v NaCl ) 125 I-EHPTIP 10 Cm

125 I-EHPIP 5 Cm

Fig. (3.1) Paper electrophoresis for 125 I-EHPIP, 125 I-EHPTIP and free iodide

- 85 - Chapter III Experimental

3.2.4. Study of the factors affecting the percent labeling yield of 125 I- EHPIP and 125 I-EHPTIP: The various factors affecting the labeling yield of 125 I-EHPIP and 125 I- EHPTIP were studied. These factors include the following: substrate amount, chloramine-T (CAT) amount, pH, reaction time and temperature and in vitro stability. Experiment studying each factor was repeated three times and differences in the data were evaluated with one way ANOVA test. Results for level of significance (P) are reported and all the results are given as mean ± SEM. 3.2.4.1. Effect of substrate amount: This factor was studied to obtain the minimum amounts of EHPIP and EHPTIP required obtaining maximum labeling yield. Different amounts (5- 250 µg) of EHPIP and EHPTIP were used. The minimum amount at which the maximum labeling yield was obtained considered the optimum amount while the other factors were kept constant. 3.2.4.2. Effect of oxidizing agent (chloramine-T) amount: Studying the amount of chloramine- T which added to give maximum labeling yield is a very important factor because excess chloramine- T may lead to oxidation of the drug itself. To study the effect of chloramine- T on the labeling yield, different amounts of chloramine- T (10-250 µg) were used while the other factors were kept constant. 3.2.4.3. Effect of pH of reaction medium: The effect of pH of the reaction mixture on the labeling of EHPIP and EHPTIP was studied by changing the pH values in the range from 2 up to 11 using buffers to adjust pH at the desired value. The experiment was performed using 100 and 10 µg of EHPIP and EHPTIP, respectively.

- 86 - Chapter III Experimental

CAT amount used were 150 µg, while other factors were kept constant. The experiment was repeated using 100 µl of each buffer at different pH values. The labeling yield for each pH value of the reaction mixture was measured and the pH value at which the maximum labeling yield was obtained considered the optimum pH. 3.2.4.4. Effect of reaction time: The time of the labeling reaction was studied to determine the minimum period required to obtain the maximum labeling yield. Because insufficient reaction time may lead to incomplete labeling while prolonged time may lead to denaturation of some labile compounds. The reaction was performed at room temperature and allowed to proceed in the range from 10 Sec. to 60 min. To find the time at which maximum labeling yield was obtained the test was carried out at different reaction times while the other factors were kept constant. The experiment was repeated at different periods as mentioned before and the optimum time was determined depending on the labeling yield. 3.2.4.5. Effect of reaction temperature: The effect of the reaction temperature on the labeling process was studied in the range from ambient temperature (25 ˚C) up to 100 ˚C. The experiment was performed using 100 and 10 µg of EHPIP and EHPTIP, respectively, while 100 µl of buffer solution pH 8 and 7 respectively was added to adjust pH of the reaction and CAT amount used were 150 µg. Reaction time was 5 and 45 min, respectively. For each temperature the labeling yield was calculated to determine the appropriate temperature at which the maximum labeling yield will be obtained.

- 87 - Chapter III Experimental

3.2.4.6. In vitro stability of the iodocompounds: The reaction mixtures were prepared at the conditions which gave the highest labeling yield for each of the two compounds (EHPIP and EHPTIP). Mixtures were left at ambient temperature for 72 hours and 1–2 l samples were taken from each mixture at different time intervals ranged from 1 hour up to 72 hours. The labeling yield and purity of the samples were measured by paper chromatography and paper electrophoresis. 3.2.5. Determination of the partition coefficient for iodocompounds: The partition coefficient was determined by mixing the iodocompounds with equal volumes of 1-octanol and phosphate buffer (0.025 M at pH 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and then centrifuged at 5000 rpm for 5 min. Subsequently 100 l samples from the 1-octanol and aqueous layers were pipetted into other test tubes and counted in a well gamma counter. The measurement was repeated three times. The partition coefficient value was expressed as log P [109].

Log p oct./wat. = Log ([solute] octanol / [solute] deionized water )

- 88 -

CHAPTER IV

BIOLOGICAL EVALUATION Chapter IV Biological Evaluation

4. Biological Evaluation Part (1) 4.1. Antimicrobial activity of the newly synthesized compounds The antimicrobial activity of the newly synthesized compounds was assayed by the agar well diffusion method [110] against two bacterial colonies

[Escherichia coli (Gram negative) and faecal streptococcus (Gram- positive)] and one fungal culture ( Aspergillus flavus ). The bacteria and fungi were maintained on nutrient agar and Czapek’s-Dox agar media, respectively. Six-millimeter diameter wells were cut out in agar plates using a sterile cork-borer. Fifty µl of 1 mg/ml test solutions were transferred aseptically to the wells. Plates were incubated at 30 ˚C for 24 h. for bacteria and 72 h. of incubation at 28 ˚C for fungi. The antimicrobial activity was evaluated by measuring the inhibition zone formed around the wells in mm. Tetracyclin and Penicillin-G-sodium were used as standard antibacterial drugs, while Nystatin was used as a standard antifungal drug. Dimethylsulphoxide was used as negative control. Results are listed in table (4.1). It is worth mention that the triazole derivatives 10 and 14 were effective against the growth of all tested microorganisms and the dipeptide derivatives 15 and 21 attached to 1,2,4- triazol-pyridine and 1,3,4-oxadiazol-pyridine rings, respectively were effective against both the tested gram positive and gram negative bacteria. 4.1.1. Antibacterial activity: All derivatives were effective against Escherichia coli but not against faecal streptococcus except compounds 5, 10, 14, 15, 21 and 22 were effective against both the tested gram positive and gram negative bacteria.

- 89 - Chapter IV Biological Evaluation

4.1.2. Antifungal activity: Derivatives 9, 10, 14, 20 and 23 were inhibitory to the growth of Aspergillus flavus , while all derivatives were inhibitory to sporulation growth except compounds 15 and 21 were generally inefficient against Aspergillus flavus . Table (4.1) In vitro antimicrobial activity of the newly synthesized compounds (at 50 µg / ml of test solutions) . Antibacterial Antifungal Inhibition zone Inhibition Inhibition zone Compound of zone of of No. growth(mm) growth(mm) sporulation(mm) faecal E.coli Aspergillus flavus streptococcus Tetracycline 21 20 0 0 Penicillin G - 20 18 0 30 sodium Nystatin 0 0 14 0 DMSO 0 0 0 0 2 11 0 0 65 3 14 0 0 45 4 13 0 0 45 5 17 11 0 30 6 19 0 0 50 7 19 0 0 40 8 13 0 0 45 9 15 0 23 45 10 16 14 50 65 11 13 0 0 45 12 14 0 0 30 13 16 0 0 53 14 18 15 26 0 15 17 17 0 0 16 19 0 0 50 17 15 0 0 45 18 14 0 0 35

- 90 - Chapter IV Biological Evaluation

Antibacterial Antifungal Inhibition zone Inhibition Inhibition zone Compound of zone of of No. growth(mm) growth(mm) sporulation(mm) faecal E.coli Aspergillus flavus streptococcus 19 16 0 0 40 20 11 0 25 0 21 11 11 0 0 22 16 12 0 58 23 11 0 8 30 24 11 0 0 30 25 13 0 0 35 26 12 0 0 50 27 11 0 0 30 28 11 0 0 35

Part (2) 4.2. Biodistribution studies of radiolabeled derivatives in normal mice

The experimental procedures of the biological studies were done in accordance with the guidelines set out by the Egyptian Atomic Energy Authority and were approved by the animal ethics committee, Labeled Compound Department. Biodistribution studies of radiolabeled derivatives in normal Swiss albino mice of body mass 20–25 g (n = 5), were carried out at 15, 30, 60, 120 and 240 min. post-injection. Prior to the study, animals were housed in groups of five and provided with food and water. Aliquots of 10 µl containing 7.2 MBq of the radiolabeled compounds were injected in each mouse via the tail vein. Each mouse was weighed then anaesthetized by chloroform. Samples of fresh blood, bone and muscle were collected in pre-weighed vials and

- 91 - Chapter IV Biological Evaluation

counted. Blood, bone and muscles were assumed to be 7, 10 and 40 % of the total body weight, respectively [111]. Whole brains were removed and frozen for an hour. The stomach weight was measured empty. Organs and tissues were rinsed with saline, collected in plastic containers and weighed. The radioactivity of each sample as well as the background was counted in a well-type NaI (Tl) well crystal coupled to SR- 7 scalar rate meter. Percent injected dose per gram (% ID/g ± SD) in a population of five mice for each time point are reported. Data were evaluated with one way ANOVA test. Results for level of significance ( p) are reported and all the results are given as mean ± SEM. The level of significance was set at P < 0.05. 4.2.1. Biodistribution of 125 I-EHPIP The biodistribution pattern of 125 I-EHPIP is shown in table (4.2). The high uptake of radioactivity in kidney indicates that excretion of 125 I-EHPIP occurs mainly through the renal system. The low radioactivity located in the thyroid gland indicates that the 125 I-EHPIP is stable against in vivo deiodination . The biodistribution data showed substantial lung uptake of 68.83 ± 0.23 % at 15 min. post-injection which is better than lung uptake of the recently discovered radiopharmaceuticals as potential lung perfusion agents 99m 99m Tc(CO) 5I and Tc-DHPM which have maximum lung uptake of 12.8 and 10.12 % ID/g at 1 h. and 2 min. post injection, respectively [112, 113]. Furthermore, lung retention of 125 I-EHPIP 30 min post injection still high (15.87 ± 0.26 %) which is attributed to the lipophilicity and high molecular weight of 125 I-EHPIP (log p = 1.45 ± 0.01, Mol. Weight = 653 g).

- 92 - Chapter IV Biological Evaluation

The high affinity and retention of the radioiodinated EHPIP in the lungs are two essential properties for any lung perfusion scan radiopharmaceutical suggest the possibility of using it as a novel agent for the lung perfusion scan. The high uptake of 125 I-EHPIP in the stomach also suggests the possibility of using it as agent for the stomach imaging and treatment. Table (4.2) Biodistribution of 125 I-EHPIP in normal mice at different time intervals post- injection. (% ID/gram ± S.D, n= 5) Organs and % injected dose/gram at different time intervals (min) body fluids 15 Min 30 Min 60 Min 120 Min 240 Min Blood 6.23 ± 0.21 14.43 ± 0.1 7.13 ± 0.02 7.39 ± 0.09 7.38 ± 0.01 Bone 5.58 ± 0.07 17.66 ± 0.00 6.60 ± 0.08 3.07 ± 0.01 0.74 ± 0.05 Brain 1.91 ± 0.00 1.27 ± 0.19 1.00 ± 0.00 0.56 ± 0.02 0.74 ± 0.07 Heart 8.81 ± 0.03 16.54 ± 0.03 4.49 ± 0.06 4.89 ± 0.04 4.22 ± 0.00 Intestine 8.63 ± 0.07 14.13 ± 0.13 6.43 ± 0.25 19.79 ± 0.1 15.53 ± 0.3 Kidneys 16.57 ± 0.02 24.67 ± 0.00 8.91 ± 0.00 9.79 ± 0.00 9.63 ± 0.03 Liver 9.31 ± 0.05 10.97 ± 0.17 5.25 ± 0.12 6.00 ± 0.03 6.09 ± 0.07 Lungs 68.83 ± 0.23 15.87 ± 0.26 6.41 ± 0.06 5.94 ± 0.03 4.98 ± 0.08 Muscle 2.70 ± 0.03 2.91 ± 0.26 3.94 ± 0.03 2.89 ± 0.06 4.22 ± 0.03 Spleen 8.81 ± 0.01 8.26 ± 0.02 4.37 ± 0.17 13.07 ± 0.16 4.24 ± 0.05 Stomach 53.51 ± 0.21 68.03 ± 0.26 144.76 ± 0.25 176.63 ± 0.16 83.05 ± 0.18 Thyroid 0.29 ± 0.01 0.89 ± 0.01 1.54 ± 0.07 2.22 ± 0.01 1.19 ± 0.08

4.2.2. Biodistribution of 125 I-EHPTIP The biodistribution pattern of 125 I-EHPTIP is shown in table (4.3). The high uptake of radioactivity in kidney indicates that excretion of 125 I-EHPTIP occurs mainly through the renal system. The low radioactivity located in the thyroid gland indicates that the iodocompounds are stable against in vivo deiodination. The biodistribution data showed substantial uptake of 7.60 ± 0.01 % in the brain at 30 min. post-injection which is attributed to the its partition

- 93 - Chapter IV Biological Evaluation

coefficient value was 2.35 ± 0.11, showing that 125 I-EHPTIP is relatively lipophilic and can cross the blood-brain barrier since their partition coefficient values are within the range (1.0-2.5) [114]. Furthermore, retention in brain remained high up to 1 h. After this time, point radioactivity dropped to 1.32 ± 0.04 at 120 min post-injection. The maximum brain uptake of 125 I-EHPTIP (7.60 ± 0.01 %) is higher than that of currently used radiopharmaceuticals for brain imaging, 99mTc-ECD and 99mTc-HMPAO which have maximum brain uptake of 4.7 and 2.25 %, respectively [115, 116]. These preliminary results strongly suggest that the radioiodinated EHPTIP could be used as a novel agent for brain SPECT. The high uptake of 125 I-EHPTIP in the stomach also suggests the possibility of using it as agent for the stomach imaging and treatment. Table (4.3) Biodistribution of 125 I-EHPTIP in normal mice at different time intervals post-injection. (% ID/gram ± S.D, n = 5)

Organs and % injected dose/gram at different time intervals (min)

body fluids 15 Min 30 Min 60 Min 120 Min 240 Min Blood 10.73 ± 0.10 8.77 ± 0.09 8.54 ± 0.01 2.87 ± 0.00 2.86 ± 0.09 Bone 3.20 ± 0.04 2.78 ± 0.06 2.69 ± 0.09 2.33 ± 0.04 2.35 ± 0.00 Brain 1.27 ± 0.03 7.60 ± 0.01 6.66 ± 0.02 1.32 ± 0.04 0.81 ± 0.09 Heart 4.49 ± 0.03 3.17 ± 0.09 3.43 ± 0.01 3.25 ± 0.03 1.11 ± 0.01 Intestine 12.74 ± 0.61 20.95 ± 0.46 20.91 ± 0.21 19.84 ± 0.15 19.21 ± 1.12 Kidneys 23.61 ± 0.08 22.76 ± 0.01 22.65 ± 0.04 11.61 ± 0.03 4.76 ± 0.01 Liver 12.16 ± 0.19 10.70 ± 0.82 11.07 ± 0.08 6.79 ± 0.21 3.89 ± 0.06 Lungs 9.21 ± 0.07 9.33 ± 0.06 9.28 ± 0.05 4.47 ± 0.00 2.66 ± 0.01 Muscle 1.75 ± 0.09 3.94 ± 0.08 4.07 ± 0.00 3.38 ± 0.01 1.68 ± 0.09 Spleen 3.36 ± 0.02 3.70 ± 0.06 2.37 ± 0.07 2.52 ± 0.04 2.47 ± 0.05 Stomach 53.42 ± 0.22 58.60 ± 0.25 104.54 ± 0.21 94.15 ± 0.14 69.70 ± 0.04 Thyroid 0.10 ± 0.05 1.27 ± 0.03 1.78 ± 0.02 0.12 ± 0.01 0.11 ± 0.04

- 94 -

REFERENCES

References

1. Norbert, S.; Hans-Dieter, J., "Peptids: Chemistry and Biology", In: Wiley-VCH Verlag GmbH& Co.KGaA , Weinheim, 2002, 135-200. 2. Guzmán, F.; Barberis, S.; Illanes, A., Electron. J. Biotechnol. , 2007 , 10(2), 279. 3. Aboelmagd, A.; Ali, I.A.I.; Salem, E.M.S.; Abdel-Razik, M., Arkivoc , 2011 , ix, 337. 4. El Rayes, S.M.; Ali, I.A.I.; Fathalla, W., Arkivoc, 2008 , xi , 86. 5. Dahiya, R.; Kumar, A.; Yadav, R., Molecules , 2008 , 13 , 958. 6. Amr, A.G.; Mohamed, A.M.; Mohamed, S.F.; Abdel-Hafez, N.A.; Hammam, A.G., Biorg. Med. Chem., 2006 , 14 , 5481. 7. Bhatia, M.S.; Mulani, A.K.; Choudhari, P.B.; Ingale, K.B.; Bhatia, N.M., Int. J. Drug Discov., 2010 , 1, 1. 8. El Ashry, H.E.; Kassem A.A.K.; Abdel Hameed, M.H., Carbohydr. Res., 2009 , 344 , 725. 9. Liu, J.; Li, L.; Dai, H.; Liu, Z.; Fang, J., J. Organomet. Chem., 2006 , 691 , 2686. 10. Sztanke, M.; Tuzimski, T.; Rzymowska, J.; Pasternak, K.; Kandefer- Szerszen, M., Eur. J. Med. Chem., 2008 , 43 , 404. 11. Isloor, M.A.; Kalluraya, B.; Shetty, P., Eur. J. Med. Chem., 2009 , 44, 3784. 12. Bhaskar, V.H.; Mohite, P.B., J. Optoelectron. Biomed. Mater. , 2010 , 2, 291. 13. Akhter, M.; Husain, A.; Azad, B.; Ajmal, M., Eur. J. Med. Chem., 2009 , 44 , 2372.

- 95 - References

14. Kumar, H.; Javed, S.A.; Khan, S.A.; Amir, M., Eur. J. Med. Chem. , 2008 , 43 , 2688. 15. Sallam, Kh.M.; Mehany, N.L., J. Radioanal. Nucl. Chem. , 2009 , 281(3), 329. 16. Adam, M.J.; Wilbur, D.S., Chem. Soc. Rev. , 2005 , 34 , 153. 17. Amin, A.M.; Soliman, S.E.; Killa, H.M.; Abd El-Aziz, H. , J. Radioanal. Nucl. Chem. , 2010 , 283 , 273. 18. Newberg, A.B.; Plössl, K.; Mozley, P.D.; Stubbs, J.B.; Wintering, N.; Udeshi, M.; Alavi, A.; Kauppinen, T.; Kung, H.F., J. Nucl. Med. , 2004 , 45 , 834. 19. El-Azony, K.M., J. Radioanal. Nucl. Chem. , 2010 , 285(2), 315. 20. Goodman, M.; Felix, A.; Moroder, L.; Toniolo , C. , "Synthesis of Peptides and Peptidomimetics ", in: Houben-Weyl-Methoden der organischen Chemie , Vol. E22a, Stuttgart, 2002 . 21. El Rayes, S.M., Arkivoc, 2008 , xvi , 243. 22. Ali, I.A.I.; Fathalla, W.; El Rayes, S.M., Arkivoc , 2008, xiii , 179. 23. El Rayes, S.M., Molecules , 2010 , 15 , 6759. 24. Al-Salahi, R.A.; Al-Omar, M.A.; Amer, A.E., Molecules , 2010 , 15 , 6588. 25. Benoiton, N.L.; Lee, Y.C.; Chen, F.M.F., Int. J. Peptide Protein Res., 1993 , 42 , 278. 26. El-Henawy, A.A., J. Am. Sci ., 2010 , 6, 240. 27. Himaja, M.; Tesmine, J.; Ramana, M.V.; Ranjitha, A.; Munirajasekhar, D., Int. Res. J. Pharm. , 2010 , 1, 436. 28. Dahiya, R.; Mourya, R.; Agrawal, S.C., Afr. J. Pharm. Pharmacol. , 2010, 4, 214.

- 96 - References

29. Bieling, P.; Beringer, M.; Adio, S.; Rodnina1, M.V., Nat. Struct. Mol. Biol. , 2006 , 13(5), 423. 30. Arima, J.; Morimoto, M.; Usuki, H.; Mori, N.; Hatanaka, T., Appl. Environ. Microbiol ., 2010 , 76(2), 4109. 31. Wenschuh, H.; Beyermann, M.; Winter, R.; Bienert, M.; Ionescu, D.; Carpino, L.A., Tetrahedron Lett ., 1996 , 37 , 5438. 32. Bartlett, R.K.; Humphrey, I.R., J. Chem. Soc. C, 1967 , 1664. 33. Zhang, Q.; Peng, Y.; Wang, X.I.; Keenan, S.M.; Arora, S.; Welsh, W.J., J. Med. Chem. , 2007 , 50 , 749. 34. Ueda, S.; Nagasawa, H., J. Am. Chem. Soc. , 2009 , 131 , 15080. 35. Castanedo, G.M.; Seng, P.S.; Blaquiere, N.; Trapp, S.; Staben, S. T., J. Org. Chem. , 2011 , 76 , 1177. 36. Wang, L.Y.; Tseng, W.C.; Lin, H.Y.; Wong, F.F., Synlett , 2011 , 10 , 1467. 37. Xu, Y.; McLaughlin, M.; Bolton, E.N.; Reamer, R.A. , J. Org. Chem. , 2010 , 75 , 8666. 38. Batchelor, D.V.; Beal, D.M.; Brown, T.B.; Ellis, D.; Gordon, D.W.; Johnson, P.S.; Mason, H.J.; Ralph, M.J.; Underwood, T.J.; Wheeler, S., Synlett , 2009, 40(5), 2421. 39. Yin, P.; Ma, W.B.; Chen, Y.; Huang, W.C.; Deng, Y.; He, L., Org. Lett. , 2009 , 11 , 5482. 40. Reichelt, A.; Falsey, J.R.; Rzasa, R.M.; Thiel, O.R.; Achmatowicz, M.M.; Larsen, R.D.; Zhang, D., Org. Lett. , 2010 , 12 , 792. 41. Cretu, O.D.; Barbuceanu, S.F.; Saramet, G.; Draghici C., J. Serb. Chem. Soc. , 2010 , 75 , 1463.

- 97 - References

42. Sharba, A.H.K.; Al-Bayati, R.H.; Aouad, M.; Rezki, N. , Molecules, 2005 , 10, 1161. 43. Sahu, G.; Patil, U.K.; Singour, P.; Garg, G., Int. J. Ph. Sci., 2011 , 3, 974. 44. Joshi, S.D.; Vagdevi, H.M.; Vaidya, V.P.; Gadaginamath, G.S., Eur. J. Med. Chem. , 2008 , 43, 1989. 45. Deepak, S.; Sandeep, S.; Abhishek, k.; Nagendra, P.; Mittan, D.S., The Pharma Res. , 2009 , 1, 127. 46. Horvath, A., Synthesis , 1996, 27(2), 1183. 47. Bulger, P.G.; Cottrell, I.F.; Cowden, C.J.; Davies, A.J.; Dolling, U.H., Tetrahedron Lett. , 2000 , 41 , 1297. 48. Ivanova, N.V.; Sviridov, S.I.; Shorshnev, S.V.; Stepanov, A.E., Synthesis , 2006 , 37(20), 156. 49. Habraken, C.L.; Cohen-Fernandes, P., J. Chem. Soc., Chem. Commun. , 1972, 2, 37. 50. Hansen, K.B.; Springfield, S.A.; Desmond, R.; Devine, P.N.; Grabowski, E.J.J.; Reider, P.J., Tetrahedron Lett. , 2001 , 42 , 7353. 51. Zarudnitskii, E.V.; Pervak, I.I.; Merkulov, A.S.; Yurchenko, A.A.; Tolmachev, A.A.; Pinchuk, A.M., Synthesis , 2006 , 37(34), 1279. 52. Alajar í n, M.; Cabrera, J.; Pastor, A.; S á bnchez-Andrada, P.; Bautista, D., J. Org. Chem., 2008 , 73 , 963. 53. Debledes, O.; Campagne, J.M. , J. Am. Chem. Soc. , 2008 , 130 , 1562. 54. Jin, T.; Kitahara, F.; Kamijo, S.; Yamamoto, Y., Tetrahedron Lett. , 2008 , 49 , 2824. 55. Demko, Z.P.; Sharpless, K.B., Angew. Chem. Int. Ed. , 2002 , 41 , 2110.

- 98 - References

56. Das, B.; Reddy, C.R.; Kumar, D.N.; Krishnaiah, M.; Narender, R., Synlett , 2010 , 41(24), 391. 57. Mirzaei, Y.R.; Tabrizi, S.B.; Edjlali, L. , Acta Chim. Slov. , 2008 , 55, 554. 58. Mohite, P.B.; Pandhare, B.R.; Khanage, S.G.; Bhaskar, V.H., Digest J. Nanomater. Biostruct. , 2009 , 4, 803. 59. Modarresi-Alam, A.R.; Nasrollahzadeh, M., Turk. J. Chem. , 2009, 33 , 267. 60. Demko, Z.P.; Sharpless, K.B., Org. Lett. , 2001 , 3, 4091. 61. Roh, J.; Artamonova, T.V.; Vávrová, K.; Koldobskii, G.I.; Hrabálek, A., Synthesis , 2009 , 13 , 2175. 62. Collibee, W.L.; Nakajima, M.; Anselme, J.P., J. Org. Chem. , 1995 , 60 , 468. 63. Markgraf, J.H.; Sadighi, J.P., Heterocycles , 1995 , 40 , 583. 64. Su, W.K.; Hong, Z.; Shan, W.G.; Zhang, X.X., Eur. J. Org. Chem. , 2006 , 2006(12), 2723. 65. Varadaraji, D.; Suban, S.S.; Ramasamy, V.R.; Kubendiran, K.; Raguraman, J.S.; Nalilu, S.K.; Pati, H.N., Org. Commun ., 2010 , 3, 45. 66. Shie, J.J.; Fang, J.M., J. Org. Chem. , 2007 , 72 , 3141. 67. Joo, Y.H.; Shreeve, J.M., Org. Lett. , 2008, 10 , 4665. 68. Hajra, S.; Sinha, D.; Bhowmick, M., J. Org. Chem. , 2007 , 72 , 1852. 69. Laha, J.K.; Cuny, G.D., Synthesis , 2008, 24 , 4002. 70. Keith, J.M., J. Org. Chem. , 2006 , 71 , 9540. 71. Huynh, M.H.V.; Coburn, M.D.; Meyer, T.J.; Wetzler, M., PNAS , 2006 , 103(27), 10322.

- 99 - References

72. Koren, A.O.; Gaponik, P.N.; Ivashkevich, O.A.; Kovalyova, T.B., Mendeleev Commun. , 1995 , 26(26), 10. 73. Faur-é-Tromeur, M.; Zard, S.Z. , Tetrahedron Lett. , 1998, 39 , 7301. 74. Boivin, J.; Henriet, E.; Zard, S.Z., J. Am. Chem. Soc. , 1994 , 116 , 9739. 75. Moderhack, D.; Lembcke, A., Chem. Ztg. , 1984 , 108, 188. 76. Bookser, B.C., Tetrahedron Lett. , 2000 , 41 , 2805. 77. Flippin, L.A., Tetrahedron Lett. , 1991 , 32 , 6857. 78. Dankwardt, J.W., J. Org. Chem. , 1998 , 63 , 3753. 79. Hoffle, G.; Lange, B., Org. Synth., Coll., 1992 , VII , 27. 80. Jursic, B.S.; Zdravkovski, Z., Synth. Commun. , 1994 , 24 , 1575. 81. Dolman, S.J.; Gosselin, F.; O’Shea, P.D.; Davies, I.W., J. Org. Chem. , 2006 , 71 , 9548. 82. Chekler, E.L.P.; Elokdah, H.M.; Butera, J., Tetrahedron Lett. , 2008 , 49 , 6709. 83. Polshettiwar, V.; Varma, R.S., Tetrahedron Lett. , 2008 , 49 , 879. 84. Wang, Y.; Sauer, D.R.; Djuric, S.W., Tetrahedron Lett. , 2006 , 47 , 105. 85. Rajapakse, H.A.; Zhu, H.; Young, M.B.; Mott, B.T. , Tetrahedron Lett. , 2006 , 47 , 4827. 86. Dabiri, M.; Salehi, P.; Baghbanzadeh, M.; Bahramnejad, M., Tetrahedron Lett. , 2006 , 47 , 6983. 87. Adib, M.; Kesheh, M.R.; Ansari, S.; Bijanzadeh, H.R., Synlett , 2009 , 10 , 1575. 88. Joule, J.A.; Mills, K., "Heterocyclic Chemistry", in: John Wiley & Sons , 5th ed., United Kingdom, 2010 , 569 .

- 100 - References

89. Butler, R.N.; Lambe, T.M.; Tobin, J.C.; Scott, F.L., J. Chem. Soc., Perkin Trans. 1, 1973 , 1357. 90. Vechorkin, O.; Hirt, N.; Hu, X., Org. Lett. , 2010 , 12 , 3567. 91. Saha, G.B., "Fundamentals of Nuclear Pharmacy", In: Springer , 6th ed., New York, 2010 , 96-100.

92. Lee, E.B.; Shin, K.C.; Lee, Y.J.; Cheon, G.J.; Jeong, J.M.; Son, M.W.; Song, Y.W., Nucl. Med. Commun. , 2003 , 24 , 689.

93. Syed, R.; Bomanji, J.; Nagabhushan, N.; Kayani, I.; Groves, A.; Waddington, W.; Cassoni, A.; Ell, P.J., J. Nucl. Med. , 2006 , 47 , 1927.

94. Amin, A.M.; Abou-Zid, K.; Bayoumi, N.A.; Abd EL-hamid, M., J. Radioanal. Nucl. Chem. , 2010 , 283 , 55. 95. Fukuchi, K.; Hayashida, K.; Nakanishi, N.; Inubushi, M.; Kyotani, S.; Nagaya,N.; Ishida, Y., J. Nucl. Med ., 2002 , 43 , 757. 96. International Atomic Energy Agency, "Manual for Reactor produced Radioisotopes", in: IAEA-TECDOC-1340, Vienna, 2003 , 1. 97. Behr, T.M.; Behe, M.; Lohr, M.; Sgouros, G.; Angerstein, C.; Wehrmann, E.; Nebendahl, K.; Becker, W., Eur. J. Nucl. Med ., 2000 , 27 , 753. 98. Nestor, M.; Persson, M.; Cheng, J.; Tolmachev, V.; van Dongen, G.; Anniko, M.; Kairemo, K., Bioconjug. Chem ., 2003 , 14 , 805. 99. Michael, R.Z.; Acharan, S.N., Int. J. Appl. Radiat. Isot ., 1987 , 38 , 1051. 100. Salvatori, M.; Indovina, L.; Mansi, L., Curr. Radiopharm., 2008 , 1(3), 251.

- 101 - References

101. Bodansky, M. "Principles of Peptide Synthesis", in: Springer , 2nd ed., Berlin, 1993 , 20. 102. Abdel-Aziz, H.A.; Hamdy, N.A.; Farag, A.M.; Fakhr I.M.I. , J. Chin. Chem. Soc., 2007 , 54 , 1573. 103. aramet, I.; Dr ăghici, C.; B ărcu Ńean, C.; R ădulescu, V.; Loloiu, T.; Banciu, M.D., Rev. Roum. Chim. , 2002 , 47 , 139. 104. Fathalla, W.; Čajan, M.; Pazdera, P., Molecules , 2001 , 6, 557. 105. Salimon, J.; Salih, N.; Yousif, E.; Hameed, A.; Kreem , A., Arab. J. Chem. , 2010 , 3(4), 205. 106. Coenen, H.H.; Mertens, J.; Maziere, B. "Radioionidation Reactions for Radiopharmaceuticals. Compendium for Effective Synthesis Strategies" , in: Springer , Netherlands, 2006 , 64. 107. Tolmachev, V.; Bruskin, A.; Sivaev, I.; Lundqvist, H.; Sjo¨berg, S., Radiochim. Acta. , 2002, 90 , 229. 108. Perrin, D.D.; Dempsey, B., "Buffers for pH and metal ion control", in: John Wiley & Sons , New York, 1974 , 123. 109. Liu, F.E.I.; Youfeng, H.E.; Luo, Z., "Study of 99mTC Labelled Way Analogues for 5-Ht1a Receptor Imaging", in: IAEA-TECDOC-426, International Atomic Energy Agency, Austria, 2004 , 37. 110. Perez, C.; Pauli, M.; Bazerque, P., Acta Biol. Med. Exp. , 1990 , 15 , 113. 111. Rhodes, B.A., Semin J. Nucl. Med. , 1974, 4, 281. 112. Miroslavov, A.E.; Gorshkov, N.I.; Lumpov, A. L.; Yalfimov, A. N.; Suglobov, D.N.; Ellis, B.L.; Braddock, R.; Smith, A.M.; Prescott, M.C.; Lawson, R.S.; Sharma, H.L., Nucl. Med. Biol. , 2009, 36 , 73.

- 102 - References

113. De, K.; Chandra, S.; Sarkar, B.; Ganguly, S.; Misra, M., J. Radioanal. Nuc. Chem., 2010 , 283 , 621. 114. Moretti, J.L.; Caglar, M.; Weinmann, P., J. Nucl. Med ., 1995 , 36 , 359. 115. Neirinckx, R.D.; Canning, L.R.; Piper, I.M.; Nowotnik, D.P.; Pickett, R.D.; Holmes R.A.; Volkert, W.A.; Forster, A.M.; Weisner, P.S.; Marriott, J.A.; Chaplin, S.B., J. Nucl. Med. , 1987, 28 , 191. 116. Motaleb, M.A.; El-Kolaly, M.T.; Rashed, H.M.; Abd El-Bary, A., J. Radioanal. Nucl. Chem., 2011 , 289 , 915.

- 103 -

PUPLISHED PAPERS

View Letter http://www.editorialmanager.com/jrnc/viewLetter.asp?id=67148&lsid={...

View Letter

Date: 30 Aug 2012 To: "hesham shamseldin" [email protected] From: "Journal of Radioanalytical & Nuclear Chemistry (JRNC)" [email protected] Subject: Your Submission JRNC-D-12-03131R2

Dear Dr. Hesham Shamseldin,

We are pleased to inform you that your manuscript, "Synthesis, Radioiodination and Biological Evaluation of Novel Dipeptide Attached to Triazole-pyridine Moiety", has been accepted for publication in Journal of Radioanalytical and Nuclear Chemistry.

You will receive an email from Springer in due course with regards to the following items:

1. Offprints 2. Colour figures 3. Transfer of Copyright

Please remember to quote the manuscript number, JRNC-D-12-03131R2, whenever inquiring about your manuscript.

With best regards, Journals Editorial Office Springer

1 of 1 8/31/2012 11:23 PM View Letter http://www.editorialmanager.com/jrnc/viewLetter.asp?id=67822&lsid...

View Letter

Date: 13 Sep 2012 To: "Khaled Mohamed Sallam" [email protected] From: "Journal of Radioanalytical & Nuclear Chemistry (JRNC)" [email protected] Subject: Your Submission JRNC-D-12-03086R1

Dear Dr. Khaled Mohamed Sallam,

We are pleased to inform you that your manuscript, "Synthesis and radioiodination of new dipeptide coupled with biologically active pyridine moiety", has been accepted for publication in Journal of Radioanalytical and Nuclear Chemistry.

You will receive an email from Springer in due course with regards to the following items:

1. Offprints 2. Colour figures 3. Transfer of Copyright

Please remember to quote the manuscript number, JRNC-D-12-03086R1, whenever inquiring about your manuscript.

With best regards, Journals Editorial Office Springer

ص of 1 02/10/2012 12:19 1 J Radioanal Nucl Chem DOI 10.1007/s10967-012-2237-5

Synthesis, radioiodination and biological evaluation of novel dipeptide attached to triazole-pyridine moiety

I. Y. Abdel-Ghany • K. A. Moustafa • H. M. Abdel-Bary • H. A. Shamsel-Din

Received: 5 June 2012 Ó Akade´miai Kiado´, Budapest, Hungary 2012

Abstract A new dipeptide derivative, ethyl 2-(3-(4-hydroxy- Introduction phenyl)-2-(2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H- 1,2,4-triazol-3-ylthio)acetamido)propanamido)-3-(1H-indol During the past decades, compounds bearing nitrogen -3-yl)propanoate (EHPTIP) was successfully synthe- containing heterocyclic rings have received much attention sized and radiolabeled with 125I by the direct electrophilic due to their increased chemotherapeutic value and brain- substitution method. The non radiolabeled compound penetrating abilities in the development of novel antimi- (EHPTIP) was tested as an antimicrobial agent and the crobials, anthelmintics [1], anti-depressant [2] and agents radiolabeled derivative was tested as a new imaging agent. used in diagnosis of alzheimer’s disease [3]. Thus, pyridine The study results showed a good antimicrobial activity derivatives continue to attract great interest due to the wide of EHPTIP and a good in vitro and in vivo stability of variety of interesting biological activities observed for 125I-EHPTIP. The biodistribution of the radiolabeled these compounds, such as anticancer, analgesic, antimi- compound showed a high brain uptake of 7.60 ± 0.01 crobial, and antidepressant activities [4–11]. Additionally, injected activity/g tissue organ at 30 min post-injection and the chemistry of 1,2,4-triazoles and their fused heterocyclic retention in brain remained high up to 1 h, whereas the derivatives have received considerable attention owing clearance from the normal mice appeared to proceed via to their synthesis and effective biological importance. the renal system. Such brain uptake is better than that of For example, a large number of 1,2,4-triazole-containing currently used radiopharmaceuticals for brain imaging ring system have been incorporated into a wide variety (99mTc-ECD and 99mTc-HMPAO). As a conclusion, EHP- of therapeutically interesting drug candidates including TIP is a newly synthesized dipeptide with a good antimi- anti-septic, analgesic, anti-biotic, anti-allergic, anti- crobial activity and the radioiodinated EHPTIP which is inflammatory, diuretic, fungicidal, insecticidal, herbicidal, labeled with 123I could be used as a novel agent for brain anti-bacterial, anti-viral, anti-depressant, anti-microbial, SPECT. anti-tumor, antihypertensive and anti-migraine [12–17]. Furthermore, the literature contains several reports on Keywords Antimicrobial activity Radioiodination the incorporation of amino acids and peptides into the Pyridine Triazole Peptide aromatic and heterocyclic congeners played a vital role in the development of compounds with potent bioactivities [18–24]. On the other hand; radioiodination of organic compounds and biomolecules have been the subject of interest I. Y. Abdel-Ghany K. A. Moustafa H. A. Shamsel-Din (&) Labeled Compounds Department, Hot Laboratories Center, of many investigators [25, 26]. Radioiodinated compounds Atomic Energy Authority, P.O. Box 13759, Cairo, Egypt are widely used for the diagnosis and therapeutic treatment e-mail: [email protected] of human diseases e.g. 125I-iodothioguanine for cancer treatment [27], 123I-iodocelecoxib for tumor imaging [28] H. M. Abdel-Bary 123 Chemistry Department, Faculty of Science, Minoufia Univeristy, and I-dimethylamino methyl phenyl iodophenylamine Shabin El-Kom, Minoufia, Egypt (123I-ADAM) for brain imaging [29]. Specially, 123 I. Y. Abdel-Ghany et al.

-1 radiolabeled compounds for functional brain imaging (KBr) mmax,cm : 1657 (CONH), 1191 (C=S), 3042(CH), should possess in vivo and in vitro stability, be able 3173(NH), 3471(NH);MS: m/z(%): 302(4.91), 224(4.16), to penetrate intact blood–brain barriers and be lipophilic 209(44.53), 165(5.39), 151(3.9), 135(4.64), 92(46.4).

(log poctanol/water in the range 1.0–2.5) [30]. Thus, keeping in mind the pharmacological potential of 4-Phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazole- pyridine/triazole as well as the advantage of biodegrad- 3-thiol (3) A solution of 2 (3.01 g, 0.01 mol) in ability and biocompatibility of amino acids/peptides, in this 2 N NaOH(50 ml) was heated under reflux for 4 h. The study we were prompted to prepare new dipeptide coupled reaction mixture was cooled and acidified with 2 N HCl. The with triazole-pyridine moiety and study its antimicrobial resulting precipitate was filtered off, washed with ethanol activity as one of its expected biological activities. Fur- and recrystallized from ethanol. White crystals (2.40 g, thermore, study its radioiodination and biodistribution of 85 %); Mp: 70–73 °C; IR (KBr) m ,cm-1:1604(C=N), the radiolabeled compound in normal mice as preliminary max 1200(C=S), 3050(CH), 3223(NH), 3422(NH); MS: m/z(%): studies for the possibility of using it as a novel imaging 283(1.05),190(6.27), 107(5.96), 93(100). agent.

Ethyl 2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4- Experimental triazol-3-ylthio)acetate (4) To a solution of 3 (2.83 g,

0.01 mol) in DMF (10 ml), anhydrous K2CO3 (1.38 g, Materials 0.01 mol) was added and the mixture was stirred at room temperature for 2 h. Ethyl- chloroacetate (1.85 g, Chemicals and solutions were purchased from Merck Co. 0.015 mol) was then added and the reaction mixture was and they were of reactive grade. 125I was purchased from stirred at room temperature for 6 h. The resulting mixture Institute of Isotopes Co., Ltd. (IZOTOP) Budapest, Hungary. was poured into crushed ice, and the formed precipitate was ﬁltered off, dried and recrystallized from ethanol. Yellow -1 Instrumentations crystals (2.88 g, 78 %); Mp: 60 °C; IR (KBr) mmax,cm : 1738(COO), 1620(C=N), 2986(CH), 3465(NH); 1H-NMR:

All melting points were determined with a Kofler Block (300 MHz, DMSO-d6, d ppm): 8.673(1H,bs,NH); 7.448(2H, apparatus. The progress of the reactions was monitored by d, J = 8.1 Hz,ArH); 7.332–7.042(4H,m,ArH); 6.818(3H,d, thin layer chromatography (TLC) using aluminum silica J = 7.5 Hz,ArH); 4.278(2H,q, J = 7.2 Hz,OCH2CH3); 1 gel plates 60 F 245. The H-NMR spectra were recorded 4.158(2H,s,SCH2); 3.771(2H,s,CH2); 1.235(3H,t,J= on a Varian Gemini 200 NMR Spectrometer at 300 MHz 7.2 Hz, OCH2CH3). with TMS as a standard, IR spectra were recorded on a Perking Elmer 1430 ratio recording infrared spectropho- 2-(4-Phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazol- tometer with CDS data station using KBr Wafer technique, 3-ylthio)acetohydrazide (5) To a solution of 4 (3.69 g, and Mass spectra were measured on a GC-MSQP 1000EX 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate Shimadzu at micro analytical laboratory, Cairo University, (1.5 g, 0.03 mol) was added and the reaction mixture was Cairo, Egypt. refluxed for 6 h, and then left to cool. The formed solid product was filtered off, dried, and recrystallized from Procedure ethanol. White crystals (2.87 g, 81 %); Mp: 100 °C; IR -1 (KBr) mmax,cm : 1670(CONH), 3025(CH), 3161(NH), Synthesis 1 3262(NH); H-NMR (300 MHz, DMSO-d6,d ppm): 9.200(1H,bs,NH); 8.633(1H,bs,NH); 7.492(2H, d, J = Synthesis of a new dipeptide derivative was successfully 8.1 Hz, ArH); 7.238–7.187(4H, m, ArH); 6.901(3H,d, J = obtained via the azide coupling method as shown in 8.1 Hz,ArH); 4.538(2H,bs,NH ); 4.053(2H,s,SCH ); scheme (1) 2 2 3.821(2H, s, CH2). N-phenyl-2-(2-(pyridin-4-ylamino)acetyl)hydrazinecarbothioa- mide (2) To a solution of hydrazide 1 (1.66 g, 0.01 mol) General procedure for azide coupling method; preparation in ethanol (10 ml), phenylisothiocyanate (1.35 g, 0.01 mol) of compounds 8, 11 To a cold solution (-5 °C) of were added. The reaction mixture was heated under reflux hydrazide (0.01 mol) in acetic acid (6 ml), 1 N HCl (3 ml) for 6 h. The product that separated on cooling was filtered and water (25 ml) was added a solution of NaNO2 (0.69 g, off, washed with ethanol, and dried well to give the desired 0.01 mol) in cold water (3 ml). The reaction mixture was product. Yellow crystals (2.76 g, 92 %); Mp: 205 °C;IR stirred at -5 °C for 20 min. The white syrup formed was 123 Novel dipeptide attached to triazole-pyridine moiety

H N N NHNH2 N NH HN NH HN N HN N

O O Ph N BrCH2COOEt, N OEt PhNCS S N NaOH SH K2CO3 S H Ph Ph EtOH, reflux DMF, r.t. O N N N N 1 234

NH2NH2 EtOH, reflux

N N N HN N HN N HN N H N NHNH2 N N N N3 NH CHRCOOEt.HCl 7a NaNO ,HCl S S 2 S 2 Ph Ph Ph Et N, EtOAc, 0oC, 24 hr O O 3 O N N OOEt OH N 8 65

NH2NH2 EtOH, reflux

N N HN N HN N H H N N NaNO2,HCl N N S S Ph Ph O O N ONHNH2 OH N ON3 OH 9 10

NH2CHRCOOEt.HCl 7b o Et3N, EtOAc, 0 C, 24 hr

7 R N HN N H a (L- Tyr) N N S Ph OH O N ONH OH

11 O b (L- Trp)

N OEt H

Scheme 1 Synthesis of the target amino acid derivatives via the azide coupling method

extracted with cold ethyl acetate (30 ml), washed with cold Ethyl 3-(4-hydroxyphenyl)-2-(2-(4-phenyl-5-((pyridin-4-yla-

3 % NaHCO3,H2O and finally dried over anhydrous mino)methyl)-4H-1,2,4-triazol-3-ylthio)acetamido)propano- sodium sulphate; the azide was used without further puri- ate (8) Pale yellow powder (3.19 g, 60 %); Mp: 110 °C; IR -1 fication in the next step. After stirring the reaction mixture (KBr) mmax,cm: 1607(C=C), 1651(CONH), 1738 of amino acid ester hydrochloride (0.01 mol), triethyl- (COO), 3384(OH), 3427(NH); 1H-NMR(300 MHz, DMSO- amine (0.2 ml) in ethyl acetate (30 ml) at 0 °C for 20 min, d6, d ppm): 8.500(1H,bs,NH); 8.200(1H,bs,NH); 7.410–7. the formed triethylamine hydrochloride was filtered off. 150(2H,m,ArH); 7.067–6.942(4H,m, ArH); 6.661(3H,d,J = The previously prepared cold dried solution of the azide 8.1 Hz,ArH); 5.115(1H,bs,OH); 4.398(2H,s,SCH2); 4.020 was added to the cold ethyl acetate solution of the amino (2H,q,J = 6.6 Hz,OCH2CH3); 3.856(2H,s,CH2); 3.457(1H, acid ester. The mixture was kept 12 h in the refrigerator t,J = 6.6 Hz,CH); 2.717(2H,d,J = 6.6 Hz,CH2); 1.115 (3H, and then at room temperature for an additional 12 h. The t,J = 6.9 Hz,OCH2CH3). reaction mixture was washed with 0.1 N HCl, water, 5 %

NaHCO3, water and then dried over anhydrous sodium N-(1-hydrazinyl-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)-2- sulphate. The solvent was evaporated in vacuum and the (4-phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazol-3- residue was crystallized from ethyl acetate: petroleum ether ylthio)acetamide (9) To a solution of 8 (5.32 g, 0.01 mol) (2:1) to give the desired product. in absolute ethanol (30 ml), hydrazine hydrate (1.5 g,

123 I. Y. Abdel-Ghany et al.

0.03 mol) was added and the reaction mixture was refluxed above the lower edge and allowed to evaporate spontane- for 7 h, and then left to cool. The formed solid product was ously. Electrophoresis is carried out for 80 min at voltage filtered off, dried, and recrystallized from ethanol. White of 300 V using normal saline (0.9 % w/v NaCl solution) as -1 crystals (3.63 g, 70 %); Mp: 90 °C; IR(KBr)mmax,cm : electrolytes source solution. After complete development, 1615(C=N), 1654(CONH), 2987(CH), 3280(NH), the paper was removed, dried, and cut into strips, each strip 3305(OH), 3420, 3440(NH); MS: m/z (%): 517(82.68), 255 is 1 cm width, and then the strip was counted in a well type (80.31), 147(81.10), 107(16.54), 94 (85.04), 79 (11.02), c-counter. The percentage of radiochemical yield was 72(88.98). estimated as the ratio of the radioactivity of 125I-EHPTIP to the total activity multiplied by 100. Ethyl 2-(3-(4-hydroxyphenyl)-2-(2-(4-phenyl-5-((pyridin-4- ylamino)methyl)-4H-1,2,4-triazol-3-ylthio)acetamido)prop- Determination of in vitro stability of 125I-EHPTIP The anamido)-3-(1H-indol-3-yl)propanoate (11) Black oil reaction mixture was left at ambient temperature for 72 h -1 (3.59 g, 50 %); IR (KBr) mmax,cm : 1610(C=N), 1650, and 1–2 lL samples were taken from it at different time 1667(CONH), 1745(COO), 3145(NH), 3310(OH), 3497 intervals. The radiochemical yield and purity of the sam- (NH);MS: m/z (%):718(14.25), 604(13.84), 587(4.07), ples were measured by paper chromatography and paper 459(2.71), 458(17.10), 436(15.47), 394(17.10), 232(100), electrophoresis. 1 130(60.11), 107(6.24); H-NMR (300 MHz, DMSO-d6, d ppm): 10.883(1H, d, J = 8.4 Hz,NH); 8.321(1H,bs,NH); Determination of the partition coefficient for 125I-EHP- 7.900(1H,bs,NH);7.570–6.966(17H,m,ArH); 6.943(1H,s, TIP The partition coefficient was determined by mixing CH); 6.587(1H,bs,NH); 5.150(1H,bs,OH); 4.200(2H,s,SCH ); 2 125I –EHPTIP with equal volumes of 1-octanol and phos- 3.990(2H,q,J = 7.2 Hz, OCH CH );3.900(2H,s,CH );3.662 2 3 2 phate buffer (0.025 M at pH 7.4) in a centrifuge tube. The (2H,t,J = 6.3 Hz,CH);3.424–2.995(4H,m,CH ); 1.079(3H,t,J = 2 mixture was vortexed at room temperature for 1 min and 4.8 Hz,OCH CH ). 2 3 then centrifuged at 5,000 rpm for 5 min. Subsequently 100 lL samples from the 1-octanol and aqueous layers Radioiodination of ethyl 2-(3-(4-hydroxyphenyl)-2-(2-(4- were pipetted into other test tubes and counted in a gamma phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazol-3- counter. The measurement was repeated three times. The ylthio)acetamido)propanamido)-3-(1H-indol-3- partition coefficient value was expressed as log p [31]. yl)propanoate (EHPTIP) 11 ÀÁ Log poct:=wat: ¼ Log½ solute octanol=½solute deionizedwater In a reaction vial of 1.0 ml capacity with a Teflon septum and screw cap, 10 ll of EHPTIP (1.4 mM) dissolved in Biodistribution studies of 125I-EHPTIP in normal mice dimethyl sulfoxide (DMSO) were added then 10 llof The experimental procedures of the biological studies were Na125I (7.2 MBq), 150 ll of freshly prepared chloramine-T done in accordance with the guidelines set out by the (4.4 mM) in phosphate buffer pH 7 were added. The Egyptian Atomic Energy Authority and were approved by reaction vial was left at 80 °C for 45 min. The reaction was the animal ethics committee, Labeled Compound Depart- quenched by adding 450 ll of sodium metabisulfite ment. Biodistribution studies of 125I –EHPTIP in normal (5.3 mM) then subjected to chromatography analysis to Swiss albino mice of body mass 20–25 g (n = 5), were determine the radiochemical yield and the radiochemical carried out at 15, 30, 60, 120 and 240 min post-injection. purity of the final product. Prior to the study, animals were housed in groups of five and provided with food and water. Aliquots of 10 lL Quality control Radiochemical yield and purity of 125I- containing 3.7 MBq of the 125I–EHPTIP were injected into EHPTIP were determined by paper chromatographic each mice via the tail vein. Each mice was weighed then method using strips of Whatman paper. On paper sheet anaesthetized by chloroform. Samples of fresh blood, bone (1 cm width and 13 cm length), 1–2 lL of the reaction and muscle were collected in pre-weighed vials and mixture was placed 2 cm above the lower edge and counted. Blood, bone and muscles were assumed to be 7, allowed to evaporate spontaneously. For development a 10 and 40 % of the total body weight, respectively [32]. fresh mixture of chloroform: ethanol (9:1 v/v) was used. Whole brains were removed and frozen for an hour. After complete development, the paper sheet was removed, Organs and tissues were rinsed with saline, collected in dried, and cut into strips, each strip is 1 cm width, and then plastic containers and weighed. The radioactivity of each each strip was counted in a well type c-counter. Radio- sample as well as the background was counted in a well- chemical yield was further confirmed by paper electro- type NaI (Tl) well crystal coupled to SR-7 scalar rate phoresis. On Whatman paper sheet (2 cm width and 47 cm meter. Percent injected dose per gram (% ID/g ± SD) in a length), 1–2 lL of the reaction mixture was placed 12 cm population of five mice for each time point are reported. 123 Novel dipeptide attached to triazole-pyridine moiety

Data were evaluated with one way ANOVA test. Results peptide bonds, and triplet signal at 3.662 ppm for the a-CH for P are reported and all the results are given as protons of the two amino acids, in addition to several mean ± SEM. The level of signiﬁcance was set at other signals corresponding to protons of the individual P \ 0.05. side chains, (see the Experimental part).

Radioiodination of EHPTIP Results and discussion During radioiodination of the dipeptide derivative 11,a Synthesis very large percentage of the radioiodine will be present in the highly activated phenolic tyrosine residue at the ortho- As triazole applications in drug constructions were attrac- position as shown in scheme (2) and this behavior is agrees ted scientist attention; we extended our studying for this with that reported in radioiodination of similar com- heterocycle via synthesis of novel triazole-pyridine deriv- pounds[41]. atives. Reaction of hydrazide 1 with phenylisothiocyanate The radiochemical purity of the 125I-EHPTIP was gave phenylthiosemicarbazide 2 which on treatment with determined using paper chromatography where radioiodide NaOH cyclised to triazole 3. The hydrazide 5 could be (I-) remained near the origin (Rf = 0–0.1), while the prepared from 3 by regioselective S-alkylation [33] with 125I-EHPTIP moved with the solvent front (Rf = 0.8). ethylchloroactetate to give the corresponding ester 4, which Radiochemical purity was further conﬁrmed by paper was subsequently hydrazinolyzed by hydrazine hydrate. electrophoresis where the free radioiodide and 125I-EHP- Synthesis of ethyl 3-(4-hydroxyphenyl)-2-(2-(4-phenyl- TIP moved to different distances away from the spotting 5-((pyridin-4-ylamino) methyl)-4H-1,2,4-triazol-3-ylthio)- point towards the anode (distance from spotting point = 14 acetamido)propanoate 8 was successfully achieved via the and 10 cm, respectively) depending on the molecular azide coupling method [34–36, 39], which was reported to weight of each one. Results of radiochemical yield from minimize the degree of racemization in amino acid cou- the two separation methods (paper chromatography and pling [37, 38]. Treatment of hydrazide 5 with nitrous acid paper electrophoresis) are nearly the same. The inﬂuence

(NaNO2/HCl) at -5 °C afforded the corresponding azide of various reaction parameters and conditions on radio- 6, which was isolated in ethyl acetate at low temperature iodination efficiency were investigated and optimized in (0 °C), to suppress its transformation to the isocyanate order to maximize the radiochemical yield. derivative by Curtius rearrangement. It is known that isocyanates are reactive towards the amino components in the Effect of substrate concentration coupling step leading to the formation of the undesired byproduct urea derivative [40]. The azide solution in ethyl The dependence of radiochemical yield on the amount of acetate reacted with L-tyrosine ethyl ester hydrochloride EHPTIP is depicted in Fig. 1. The reaction was performed 7a, previously treated with triethylamine in ethyl acetate at at different EHPTIP concentrations (5–250 lg). The low temperature, to produce ethyl 3-(4-hydroxyphenyl)-2- radiochemical yield of 125I-EHPTIP was small at low (2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-tria- substrate concentration where at 5 lg the labeling yield zol-3-ylthio)acetamido)propanoate 8 in moderate yield, was 77 ± 2.90 % and by increasing EHPTIP concentration and its chemical structure was confirmed by IR and the labeling yield was increased where the labeling yield 1H- NMR spectra. The 1H- NMR spectra showed a signal (84 ± 2.50 %) was obtained at 10 lg. At EHPTIP con- at 8.5 ppm for the NH proton of the peptide bond, multi- centrations higher than the optimum amount, the labeling plet signals between 6.661 and 7.410 ppm for the aromatic yield was constant which may be attributed to the fact that protons, triplet signal at 3.457 ppm for the a-CH proton of 10 lg of EHPTIP is enough to capture the entire generated the amino acid, triplet signal at 1.115 ppm for the three iodonium ion as a result the yield reached maximum value protons of –OCH2CH3 and quartat signal at 4.02 ppm for at this concentration. the two protons of –OCH2CH3 of the ester group. The dipeptide derivative 11 was prepared from 8 after con- Effect of oxidizing agent version to hydrazide 9 by boiling with excess hydrazine hydrate in ethanol. The 1H- NMR, mass and IR spectra of The effect of oxidizing agent amount (chloramine-T) on 11 confirmed its structure as shown from the data reported the labeling efficiency of 125I-EHPTIP is demonstrated in in the experimental part. The dipeptide derivative 11 was Fig. 2. Radioiodination of EHPTIP has been performed by obtained by the azide-coupling method in 50 % yield using CAT as a mild oxidizing agent, transforming iodide (Scheme 1). The 1H- NMR spectra revealed two signals at (I-) to an electropositive form of iodine (oxidative state 7.900 and 6.587 ppm for the two NH protons of the I?), which allows a spontaneous electrophilic substitution 123 I. Y. Abdel-Ghany et al.

N N N HN HN N H H N N N N 125I S S Ph Ph O O O NH OH N N O NH OH O 11 Na125 I / CAT O

pH 7 OEt OEt

EHPTIP (11) 125 NH NH I-EHPTIP

Scheme 2 Radioiodination of EHPTIP

100 100

80 80

60 % 125I-EHPTIP % 125 I-EHPTIP 60 % Free iodide % Free iodide 40 40

%Labeling yield 20 %Labeling yield 20 0 0 50 100 150 200 0 CAT concentration, µg 0 50 100 150 200 250 EHPTIP concentration, µg Fig. 2 Variation of the radiochemical yield of 125I-EHPTIP as a function of CAT. Reaction conditions 10 lL(*3.7 MBq) Na125I, Fig. 1 Variation of the radiochemical yield of 125I-EHPTIP as a (9lg) of CAT, 10 lg of EHPTIP, at pH 7, the reaction mixtures were function of different EHPTIP amounts. Reaction conditions 10 lL kept at room temperature for 15 min (*3.7 MBq) Na125I, (9lg) EHPTIP, 100 lg of CAT, at pH 7, the reaction mixtures were kept at room temperature for 15 min formation of by-products and to obtain high labeling yield and purity. on aromatic ring with a good leaving group such as H?. Effect of pH When high speciﬁc activity radioiodide is oxidized in situ, it generates electropositive iodine, but it is unlikely to form The effect of pH of the reaction mixture on the labeling of I2 because there is so little radioiodine present that statis- EHPTIP was studied by changing the pH values in the tically it is not possible for two iodine atoms to join range from 3 up to 11 using buffers to adjust pH at the together at the concentrations involved [26]. In addition, desired value as shown in Fig. 3. The experiment was although CAT is a mild oxidizing agent, its effect is performed using 10 lg of EHPTIP. CAT amount used were - ? enough to oxidize all I into I without forming I2. 150 lg, while other factors were kept constant. The At low CAT amounts (10 lg), the radiochemical yield experiment was repeated using 150 lL of each buffer at of 125I-EHPTIP was small and equal 67.50 ± 2.00 %. A different pH values. The labeling yield for each pH value high radiochemical yield of 87 ± 2.30 % was achieved by of the reaction mixture was measured and the pH value at 7 increasing the amount of CAT to 150 lg. Increasing the the maximum labeling yield was obtained considered the CAT concentration above that value led to a decrease in the optimum pH. Increasing in the pH of the medium above iodination yield where at 200 lg of CAT, the labeling yield that pH 7 led to a slightly decreasing in the iodination yield was 77.9 ± 2.0 %. This decrease in labeling yield may be where at pH 11, the labeling yield was 37 ± 1.0 %. attributed to the formation of undesirable oxidative byproducts like chlorination, polymerization and denatur- Effect of reaction time ation of EHPTIP [28]. The formation of these impurities may be attributed to the high reactivity and concentration The labeling yield is strongly dependent on reaction time in of CAT [42].Consequently, the optimum concentration of the range from 10 S to 60 min. It is clear from Fig. 4 that CAT (150 lg) is highly recommended in order to avoid the the yield is signiﬁcantly increased with increasing the 123 Novel dipeptide attached to triazole-pyridine moiety

100 % 125 I-EHPTIP 100 % Free iodide 80 80 60

60 125 40 % I-EHPTIP % Free iodide 20 40 %Labeling yield

0 %Labeling yield 234567891011 20 pH 0 125 Fig. 3 Variation of the radiochemical yield of I-EHPTIP as a 20 30 40 50 60 70 80 90 100 110 125 function of pH. Reaction conditions 10 lL(*3.7 MBq) Na I, Temperature, °C 10 lg of EHPTIP, 150 lg of CAT, at different pH, the reaction mixtures were kept at room temperature for 15 min Fig. 5 Variation of the radiochemical yield of 125I-EHPTIP as a function of reaction temperature. Reaction conditions 10 lL (*3.7 MBq) Na125I, 10 lg of EHPTIP, 150 lg of CAT, at pH 7, 100 the reaction mixtures were kept at different temperatures for 45 min

80 100 60 % 125I-EHPTIP % Free iodide 80 40 60 % 125 I-EHPTIP % Free iodide %Labeling yield 20 40

20 0 %Labeling yield 0 102030405060 0 Reaction time, minutes 12 18 24 30 36 42 48 54 60 66 72 Time post iodination, hours Fig. 4 Variation of the radiochemical yield of 125I-EHPTIP as a function of reaction time. Reaction conditions 10 lL(*3.7 MBq) Fig. 6 In vitro stability of 125I-EHPTIP Na125I, 10 lg of EHPTIP, 150 lg of CAT, at pH 7, the reaction mixtures were kept at room temperature for different intervals of time

radiolysis of the labeled compound. These undesired reaction time. The results indicate that the reaction is very radioactive products might be accumulated in non-target fast. After 45 min the maximum radiochemical yield organs. The results of stability showed that the 125I-EHP- (90 ± 2.3 %) was obtained. TIP is stable up to 48 h as shown in Fig. 6.

Effect of reaction temperature Partition coefﬁcient of 125I-EHPTIP

The influence of the temperature of the reaction mixture on The partition coefficient value was 2.35 ± 0.11, showing the radiochemical yield of 125I-EHPTIP is shown in Fig. 5. that 125I-EHPTIP is relatively lipophilic and can cross the The reaction was carried out at room temperature, 40, 60, blood–brain barrier. 80 and 100 °C. The results of this study clearly show that that the yield is significantly increased with increasing Biological evaluation the reaction temperature, where at higher temperature degrees (80 and 100 °C) the maximum labeling yield Antimicrobial activity of EHPTIP (92.5 ± 2.0 %) was obtained. During the synthesis process of new dipeptide derivative In vitro stability of 125I-EHPTIP (EHPTIP) 11, numbers of newly synthesized compounds were obtained. The antimicrobial activity of all newly In vitro stability of 125I-EHPTIP was studied in order to synthesized compounds was assayed by the agar well dif- determine the suitable time for injection to avoid the for- fusion method [43] against two bacterial colonies [Esche- mation of the undesired products that result from the richia coli (Gram negative) and faecal streptococcus 123 I. Y. Abdel-Ghany et al.

Table 1 In vitro antimicrobial Compound No. Antibacterial Antifungal activity of the newly synthesized compounds (At E. coli F. streptococcus A. ﬂavus 50 lg/mL of test solutions) Inhibition zone of growth (mm) Inhibition zone of Inhibition zone sporulation (mm) of growth (mm)

Tetracycline 21 20 0 0 Penicillin G–sodium 20 18 0 30 Nystatin 0 0 14 14 2 15 0 23 45 316145065 4130045 5140030 8160053 91815260 11 17 17 0 14

Table 2 Biodistribution of 125I-EHPTIP in normal mice at different time intervals post-injection. (% ID/gram ± SD, n = 5) Organs and body ﬂuids % Injected dose/gram at different time intervals (min) 15 (min) 30 (min) 60 (min) 120 (min) 240 (min)

Blood 10.73 ± 0.1 8.77 ± 0.09 8.54 ± 0.01 2.87 ± 0.00 2.86 ± 0.09 Kidneys 23.61 ± 0.08 22.76 ± 0.01 22.65 ± 0.04 11.61 ± 0.03 4.76 ± 0.01 Liver 12.16 ± 0.19 10.70 ± 0.82 11.07 ± 0.08 6.79 ± 0.21 3.89 ± 0.06 Spleen 3.36 ± 0.02 3.70 ± 0.06 2.37 ± 0.07 2.52 ± 0.04 2.47 ± 0.05 Intestine 12.74 ± 0.61 20.95 ± 0.46 20.91 ± 0.21 19.84 ± 0.15 19.21 ± 1.12 Stomach 53.42 ± 0.22 58.60 ± 0.25 104.54 ± 0.21 94.15 ± 0.14 69.70 ± 0.04 Lungs 9.21 ± 0.07 9.33 ± 0.06 9.28 ± 0.05 4.47 ± 0.00 2.66 ± 0.01 Heart 4.49 ± 0.03 3.17 ± 0.09 3.43 ± 0.01 3.25 ± 0.03 1.11 ± 0.01 Thyroid 0.10 ± 0.05 1.27 ± 0.03 1.78 ± 0.02 0.12 ± 0.01 0.11 ± 0.04 Muscle 1.75 ± 0.09 3.94 ± 0.08 4.07 ± 0.00 3.38 ± 0.01 1.68 ± 0.09 Bone 3.20 ± 0.04 2.78 ± 0.06 2.69 ± 0.09 2.33 ± 0.04 2.35 ± 0.00 Brain 1.27 ± 0.03 7.60 ± 0.01 6.66 ± 0.02 1.32 ± 0.04 0.81 ± 0.09

(Gram positive)] and one fungal culture (Aspergillus fla- E. coli but not against F. streptococcus except compounds vus).The bacteria and fungi were maintained on nutrient 3, 9, 11 were effective against both the gram positive and agar and Czapek’s-Dox agar media, respectively. Six-mil- gram negative bacteria tested. Derivatives 2, 3, 9 were limeter diameter wells were cut out in agar plates using a inhibitory to the growth of A. flavus, while all derivatives sterile cork-borer. Fifty lL of 1 mg/mL test solutions were were inhibitory to sporulation growth (Table 1). It is worth transferred aseptically to the wells. Plates were incubated mention that derivatives 3, 9 were effective against all at 30 oC for 24 h for bacteria and 72 h of incubation at tested microorganisms. 28 oC for fungi. The antimicrobial activity was evaluated by measuring the inhibition zone formed around the wells Biodistribution of 125I-EHPTIP in mm. Tetracyclin and Penicillin G sodium were used as standard antibacterial drugs, while Nystatin was used The biodistribution pattern of 125I-EHPTIP is shown in as standard antifungal drug. Dimethyl sulphoxide was used Table (2). 125I-EHPTIP was injected in normal mice via as solvent for tested compounds showed no inhibition intravenous route and was distributed all over the body zones. Results are listed in Table (1). organs and fluids. All radioactivity levels are expressed as This study showed that the dipeptide derivative 11 was average percentage of injected dose per gram (% ID/ effective against both the tested gram positive and gram g ± SD). The high uptake of radioactivity in kidney indi- negative bacteria. All derivatives were effective against cates that excretion of 125I-EHPTIP occurs mainly through

123 Novel dipeptide attached to triazole-pyridine moiety the renal system. The biodistribution data showed sub- 16. Qin X, Yu H, Liu J, Dai BG, Qin Z, Zhang X, Wang T, Fang J stantial uptake of 7.60 ± 0.01 (% ID/g ± SD) in the brain (2009) Arkivoc ii: 201–210 17. Khanmohammadi H, Abnosi HM, Hosseinzadeh A, Erfantalab M at 30 min post-injection and retention in brain remained (2008) Spectrochim Acta A 71:1474–1480 high up to 1 h. After this time, point radioactivity dropped 18. Poojary B, Belagali SL, Kumar KH, Holla BS (2003) Farmaco to 1.32 ± 0.04 at 120 min post-injection. The low radio- 58:569–572 activity located in the thyroid gland indicates that the 19. Levit GL, Anikina LV, Vikharev YB, Demin AM, Saﬁn VA, Matveeva TV, Krasnov VP (2002) Pharm Chem J 36:232–236 iodocompounds are stable against in vivo deiodination. The 20. Belagali SL, Kumar KH, Holla BS, Bhagwat A (2001) Indian J 125 maximum brain uptake of I-EHPTIP (7.60 ± 0.01) is Heterocycl Chem 10:249–252 higher than that of currently used radiopharmaceuticals for 21. Himaja M, Rajiv RMV, Poojary B, Satyanarayana D, Subrah- brain imaging, 99mTc-ECD and 99mTc-HMPAO which have manyam EV, Bhat KI (2003) Boll Chim Farm 142:450–453 22. Himaja M, Rajiv RMV (2002) Indian J Heterocycl Chem 12: maximum brain uptake of 4.7 and 2.25 %, respectively 121–124 [44, 45]. These preliminary results strongly suggest that the 23. Aboelmagd A, Ali IAI, Salem EMS, Abdel-Razik M (2011) radioiodinated EHPTIP which is labeled with 123I could be Arkivoc ix:337–353 used as a novel agent for brain SPECT. The high uptake of 24. El-rayes SM (2010) Molecules 15:6759–6772 125 25. Bolton R (2002) J Labelled Compds Radiopharm 45:485–528 I-EHPTIP in stomach also enforces us to do more 26. Adam MJ, Wilbur DS (2005) Chem Soc Rev 34:153–163 research about the possibility of using it as agent for 27. Amin AM, Soliman SE, Killa HM, Abd El-Aziz H (2010) stomach imaging and treatment comparing it with currently J Radioanal Nucl Chem 283:273–278 used agents for that purpose. 28. El-Azony KM (2010) J Radioanal Nucl Chem 285:315–320 29. Newberg AB, Plo¨ssl K, Mozley PD, Stubbs JB, Wintering N, Udeshi M, Alavi A, Kauppinen T, Kung HF (2004) J Nucl Med 45:834–841 30. Moretti JL, Caglar M, Weinmann P (1995) J Nucl Med 36: References 359–363 31. Liu FEI, Youfeng HE, Luo Z (2004) IAEA, Austria, Technical 1. Dahiya R, Kumar A, Yadav R (2008) Molecules 13:958–976 Reports Series No 426, pp 37–52 2. Siddiqui N, Andalip BS, Ali R, Afzal O, Akhtar MJ, Azad B, 32. Rhodes BA (1974) Semin J Nucl Med 4:281–293 ˇ Kumar R (2011) J Pharm Bioall Sci 3(20):194–212 33. Fathalla W, Cajan M, Pazdera P (2001) Molecules 6:557–573 3. Kumar JR (2010) Pharmacophore 1(3):167–177 34. Fathalla W, El Rayes SM, Ali IAI (2007) Arkivoc xvi:173–186 4. Amr AG, Mohamed AM, Mohamed SF, Abdel-Hafez NA, 35. Ali IAI, Fathalla W, El Rayes SM (2008) Arkivoc xiii:179–188 Hammam AG (2006) Bioorg Medicinal Chem 14:5481–5488 36. El Rayes SM (2008) Arkivoc xvi:243–254 5. Bart A, Jansen J, Zwan JV, Dulk H, Brouwer J, Reedijk J (2001) 37. Hofmann K, Johl A, Furlemeier AE, Koppeler HJ (1957) J Am J Med Chem 44:245–249 Chem Soc 79:1636–1641 6. Shinkai H, Ito T, Iida T, Kitao Y, Yamada H, Uchida I (2000) 38. Hofmann K, Thompson TA, Yajima H, Schwarts ET, Enouye H J Med Chem 43:4667–4677 (1960) J Am Chem Soc 82:3715–3720 7. Klimesova V, Svoboda M, Waisser K, Pour M, Kaustova J (1999) 39. El Rayes SM, Ali IAI, Fathalla W (2008) Arkivoc xi:86–95 Farmaco 54:666–672 40. Bodansky M (1993) Principles of peptide synthesis, 2nd edn. 8. Maria GM, Valeria F, Daniele Z, Luciano V, Elena BJ (2001) Springer, Berlin, p 20 Farmaco 56:587–592 41. Coenen HH, Mertens J, Maziere B (2006) Radioionidation 9. Goda FE, Abdel-Aziz AAM, Attef OA (2004) Bioorg Medicinal reactions for radiopharmaceuticals. Compendium for effective Chem 12:1845–1852 synthesis strategies, chap. 4. Springer, Dordrecht, p 64 10. Bhatia MS, Mulani AK, Choudhari PB, Ingale KB, Bhatia NM 42. Tolmachev V, Bruskin A, Sivaev I, Lundqvist H, Sjo¨berg S (2009) Int J Drug Discov 1:1–9 (2002) Radiochim. Acta 90:229–235 11. Muthal N, Ahirwar J, Ahriwar D, Masih P, Mahmdapure T, 43. Perez C, Pauli M, Bazerque P (1990) Acta Biol Med Exp 15: Sivakumar T (2010) Int J Pharm Tech Res 2(4):2450–2455 113–115 12. El Ashry HE, Kassem AAK, Abdel Hameed MH (2009) Carbo- 44. Neirinckx RD, Canning LR, Piper IM, Nowotnik DP, Pickett RD, hydr Res 344:725–733 Holmes RA, Volkert WA, Forster AM, Weisner PS, Marriott JA, 13. Sztanke M, Tuzimski T, Rzymowska J, Pasternak K, Kandefer- Chaplin SB (1987) J Nucl Med 28:191–202 Szerszen M (2008) Eur J Med Chem 43:404–419 45. Motaleb MA, El-Kolaly MT, Rashed HM, Abd El-Bary A (2011) 14. Foroughifar N, Mobinikhaledi A, Ebrahimi S, Moghanian H, J Radioanal Nucl Chem 289:915–921 Fard BAM, Kalhor M (2009) Tetrahedron Lett 50:836–839 15. Isloor MA, Kalluraya B, Shetty P (2009) Eur J Med Chem 44:3784–3787

123 J Radioanal Nucl Chem DOI 10.1007/s10967-012-2237-5

3 Synthesis, radioiodination and biological evaluation of novel 4 dipeptide attached to triazole-pyridine moiety

5 I. Y. Abdel-Ghany • K. A. Moustafa • 6 H. M. Abdel-Bary • H. A. Shamsel-Din

7 Received: 5 June 2012 8 Ó Akade´miai Kiado´, Budapest, Hungary 2012

Author Proof 9 Abstract A new dipeptide derivative, ethyl 2-(3-(4-hydroxy- Introduction 34 10 phenyl)-2-(2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H- 11 1,2,4-triazol-3-ylthio)acetamido)propanamido)-3-(1H-indol During the past decades, compounds bearing nitrogen 35 12 -3-yl)propanoate (EHPTIP) was successfully synthe- containing heterocyclicPROOF rings have received much attention 36 13 sized and radiolabeled with 125I by the direct electro- due to their increased chemotherapeutic value and brain- 37 14 philic substitution method. The non radiolabeled penetrating abilities in the development of novel antimi- 38 15 compound (EHPTIP) was tested as an antimicrobial crobials, anthelmintics [1], anti-depressant [2] and agents 39 16 agent and the radiolabeled derivative was tested as a new used in diagnosis of alzheimer’s disease [3]. Thus, pyridine 40 17 imaging agent. The study results showed a good antimi- derivatives continue to attract great interest due to the wide 41 18 crobial activity of EHPTIP and a good in vitro and in vivo variety of interesting biological activities observed for 42 19 stability of 125I-EHPTIP. The biodistribution of the theseED compounds, such as anticancer, analgesic, antimi- 43 20 radiolabeled compound showed a high brain uptake of crobial, and antidepressant activities [4–11]. Additionally, 44 21 7.60 ± 0.01 injected activity/g tissue organ at 30 min the chemistry of 1,2,4-triazoles and their fused heterocyclic 45 22 post-injection and retention in brain remained high up derivatives have received considerable attention owing to 46 23 to 1 h, whereas the clearance from the normal mice their synthesis and effective biological importance. For 47 24 appeared to proceed via the renal system. Such brain example, a large number of 1,2,4-triazole-containing ring 48 25 uptake is better than that of currently used radiophar- system have been incorporated into a wide variety of thera- 49 26 maceuticals for brain imaging (99mTc-ECD and 99mTc- peutically interesting drug candidates including anti-septic, 50 27 HMPAO). As a conclusion, EHPTIP is a newly synthesized analgesic, anti-biotic, anti-allergic, anti-inflammatory, diuretic, 51 28 dipeptide with a good antimicrobial activity and the radioio- fungicidal, insecticidal, herbicidal, anti-bacterial, anti-viral, 52 29 dinated EHPTIP which is labeled with 123I could be used as a anti-depressant, anti-microbial, anti-tumor, antihypertensive 53 30 novel agent for brain SPECT. and anti-migraine [12, 17]. 54 31 Furthermore, the literature contains several reports on 55 32 Keywords Antimicrobial activity Á Radioiodination Á the incorporation of amino acids and peptides into the 56 33 Pyridine Á Triazole Á Peptide aromatic and heterocyclic congeners played a vital role in 57 the development of compounds with potent bioactivities 58 [18–24]. 59 On the other hand; radioiodination of organic com- 60 61 & pounds and biomolecules have been the subject of interest A1 I. Y. Abdel-Ghany Á K. A. Moustafa Á H. A. Shamsel-Din ( ) 62 A2 Labeled Compounds Department, Hot Laboratories Center, of many investigators [25, 26]. Radioiodinated compounds A3 Atomic Energy Authority, P.O. Box 13759, Cairo, Egypt are widely used for the diagnosis and therapeutic treat- 63 A4 e-mail: [email protected] ment of human diseases e.g. 125I-iodothioguanine for can- 64 cer treatment [27], 123I-iodocelecoxib for tumor imaging 65 A5 H. M. Abdel-Bary 123 A6 Chemistry Department, Faculty of Science, Minoufia Univeristy, [28] and I-dimethylamino methyl phenyl iodophenyl- 66 UNCORRECT 123 A7 Shabin El-Kom, Minoufia, Egypt amine ( I-ADAM) for brain imaging [29]. Specially, 67

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK I. Y. Abdel-Ghany et al.

68 radiolabeled compounds for functional brain imaging desired product. Yellow crystals (2.76 g, 92 %); Mp: 110 -1 69 should possess in vivo and in vitro stability, be able to 205 °C;IR (KBr) mmax,cm : 1657 (CONH), 1191 (C=S), 111 70 penetrate intact blood–brain barriers and be lipophilic (log 3042(CH), 3173(NH), 3471(NH);MS: m/z(%): 302(4.91), 112

71 poctanol/water in the range 1.0–2.5) [30]. 224(4.16), 209(44.53), 165(5.39), 151(3.9), 135(4.64), 113 72 Thus, keeping in mind the pharmacological potential of 92(46.4). 114 73 pyridine/triazole as well as the advantage of biodegrad- 74 ability and biocompatibility of amino acids/peptides, in this 4-Phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazole- 115 75 study we were prompted to prepare new dipeptide coupled 3-thiol (3) A solution of 2 (3.01 g, 0.01 mol) in 116 76 with triazole-pyridine moiety and study its antimicrobial 2 N NaOH(50 ml) was heated under reflux for 4 h. The 117 77 activity as one of its expected biological activities. Fur- reaction mixture was cooled and acidified with 2 N HCl. 118 78 thermore, study its radioiodination and biodistribution of The resulting precipitate was filtered off, washed with 119 79 the radiolabeled compound in normal mice as preliminary ethanol and recrystallized from ethanol. White crystals 120 -1 80 studies for the possibility of using it as a novel imaging (2.40 g, 85 %); Mp: 70–73 °C; IR (KBr) mmax,cm : 121 81 agent. 1604(C=N), 1200(C=S), 3050(CH), 3223(NH), 3422(NH); 122 MS: m/z(%): 283(1.05),190(6.27), 107(5.96), 93(100). 123

Ethyl 2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4- 124 82 Experimental triazol-3-ylthio)acetate (4) To a solution of 3 (2.83 g, 125

Author Proof 0.01 mol) in DMF (10 ml), anhydrous K CO (1.38 g, 126 83 Materials 2 3 0.01 mol) was added and the mixture was stirred at room 127 temperature for 2 h. Ethyl- chloroacetate (1.85 g, 0.015 128 84 Chemicals and solutions were purchased from Merck Co. mol) was then added and the reaction mixture was stirred at 129 85 and they were of reactive grade. 125I was purchased from PROOF room temperature for 6 h. The resulting mixture was 130 86 Institute of Isotopes Co., Ltd. (IZOTOP) Budapest, Hungary. poured into crushed ice, and the formed precipitate was 131 filtered off, dried and recrystallized from ethanol. Yellow 132 87 Instrumentations crystals (2.88 g, 78 %); Mp: 60 °C; IR (KBr) mmax, 133 cm-1: 1738(COO), 1620(C=N), 2986(CH), 3465(NH); 1H- 134 88 All melting points were determined with a Kofler Block NMR: (300 MHz, DMSO-d , d ppm): 8.673(1H,bs,NH); 135 89 apparatus. The progress of the reactions was monitored by 6 7.448(2H,d,EDJ = 8.1 Hz,ArH); 7.332–7.042(4H,m,ArH); 136 90 thin layer chromatography (TLC) using aluminum silica 6.818(3H,d, J = 7.5 Hz,ArH); 4.278(2H,q, J = 7.2 Hz, 137 91 gel plates 60 F 245. The 1H-NMR spectra were recorded OCH CH ); 4.158(2H,s,SCH ); 3.771(2H,s,CH ); 1.235 138 92 on a Varian Gemini 200 NMR Spectrometer at 300 MHz 2 3 2 2 (3H,t,J= 7.2 Hz, OCH CH ). 139 93 with TMS as a standard, IR spectra were recorded on a 2 3 94 Perking Elmer 1430 ratio recording infrared spectropho- 2-(4-Phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-tria- 140 95 tometer with CDS data station using KBr Wafer technique, zol-3-ylthio)acetohydrazide (5) To a solution of 4 (3.69 141 96 and Mass spectra were measured on a GC-MSQP 1000EX g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate 142 97 Shimadzu at micro analytical laboratory, Cairo University, (1.5 g, 0.03 mol) was added and the reaction mixture was 143 98 Cairo, Egypt. refluxed for 6 h, and then left to cool. The formed solid 144 product was filtered off, dried, and recrystallized from 145 99 Procedure ethanol. White crystals (2.87 g, 81 %); Mp: 100 °C; IR 146 -1 (KBr) mmax,cm : 1670(CONH), 3025(CH), 3161(NH), 147 1 100 Synthesis 3262(NH); H-NMR (300 MHz, DMSO-d6,d ppm): 9.200 148 (1H,bs,NH); 8.633(1H,bs,NH); 7.492(2H, d, J = 8.1 Hz, 149 101 Synthesis of a new dipeptide derivative was successfully ArH); 7.238–7.187(4H, m, ArH); 6.901(3H,d,J = 8.1 Hz, 150

102 obtained via the azide coupling method as shown in ArH); 4.538(2H,bs,NH2); 4.053(2H,s,SCH2); 3.821(2H, s, 151 103 scheme (1) CH2). 152

104 N-phenyl-2-(2-(pyridin-4-ylamino)acetyl)hydrazinecarbothioa- General procedure for azide coupling method; preparation 153 105 mide (2) To a solution of hydrazide 1 (1.66 g, 0.01 mol) of compounds 8, 11 To a cold solution (-5 °C) of 154 106 in ethanol (10 ml), phenylisothiocyanate (1.35 g, 0.01 hydrazide (0.01 mol) in acetic acid (6 ml), 1 N HCl (3 ml) 155 107 mol) were added. The reaction mixture was heated under and water (25 ml) was added a solution of NaNO2 (0.69 g, 156 108 reﬂux for 6 h.UNCORRECT The product that separated on cooling was 0.01 mol) in cold water (3 ml). The reaction mixture was 157 109 ﬁltered off, washed with ethanol, and dried well to give the stirred at -5 °C for 20 min. The white syrup formed was 158

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK Novel dipeptide attached to triazole-pyridine moiety

H N N NHNH2 N NH HN NH HN N HN N

O O Ph N BrCH2COOEt, N OEt PhNCS S N NaOH SH K CO S H Ph 2 3 Ph EtOH, reflux DMF, r.t. O N N N N 1 234

NH2NH2 EtOH, reflux

N N N HN N HN N HN N H N NHNH2 N N N N3 NH CHRCOOEt.HCl 7a NaNO ,HCl S S 2 S 2 Ph Ph Ph o O O Et3N, EtOAc, 0 C, 24 hr O N N OOEt OH N 8 65

NH2NH2 EtOH, reflux

N N HN N HN N H H N N N N NaNO2,HCl S S

Author Proof Ph Ph O O N ONHNH2 OH N ON3 OH 9 10

NHPROOFCHRCOOEt.HCl 7b 2 o Et3N, EtOAc, 0 C, 24 hr

7 R N HN N H a N N (L- Tyr) S Ph OH O EDN ONH OH 11 O b (L- Trp)

N OEt H

Scheme 1 Synthesis of the target amino acid derivatives via the azide coupling method

159 extracted with cold ethyl acetate (30 ml), washed with cold Ethyl 3-(4-hydroxyphenyl)-2-(2-(4-phenyl-5-((pyridin-4-yla- 175

160 3 % NaHCO3,H2O and finally dried over anhydrous mino)methyl)-4H-1,2,4-triazol-3-ylthio)acetamido)propan- 176 161 sodium sulphate; the azide was used without further puri- oate (8) Pale yellow powder (3.19 g, 60 %); Mp: 110 177 -1 162 fication in the next step. After stirring the reaction mixture °C; IR (KBr) mmax,cm : 1607(C=C), 1651(CONH), 1738 178 163 of amino acid ester hydrochloride (0.01 mol), triethyl- (COO), 3384(OH), 3427(NH); 1H-NMR(300 MHz, DMSO- 179 164 amine (0.2 ml) in ethyl acetate (30 ml) at 0 °C for 20 min, d6, d ppm): 8.500(1H,bs,NH); 8.200(1H,bs,NH); 7.410– 180 165 the formed triethylamine hydrochloride was filtered off. 7.150(2H,m,ArH); 7.067–6.942(4H,m, ArH); 6.661(3H,d, 181 166 The previously prepared cold dried solution of the azide J = 8.1 Hz,ArH); 5.115(1H,bs,OH); 4.398(2H,s,SCH2); 182 167 was added to the cold ethyl acetate solution of the amino 4.020(2H,q,J = 6.6 Hz,OCH2CH3); 3.856(2H,s,CH2); 3.457 183 168 acid ester. The mixture was kept 12 h in the refrigerator (1H,t,J = 6.6 Hz,CH); 2.717(2H,d,J = 6.6 Hz,CH2); 1.115 184 169 and then at room temperature for an additional 12 h. The (3H,t,J = 6.9 Hz,OCH2CH3). 185 170 reaction mixture was washed with 0.1 N HCl, water, 5 %

171 NaHCO3, water and then dried over anhydrous sodium N-(1-hydrazinyl-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)- 186 172 sulphate. The solvent was evaporated in vacuum and the 2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazol- 187 173 residue was crystallized from ethyl acetate: petroleum ether 3-ylthio)acetamide (9) To a solution of 8 (5.32 g, 0.01 mol) 188 174 (2:1) to giveUNCORRECT the desired product. in absolute ethanol (30 ml), hydrazine hydrate (1.5 g, 189

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK I. Y. Abdel-Ghany et al.

190 0.03 mol) was added and the reaction mixture was refluxed above the lower edge and allowed to evaporate spontane- 239 191 for 7 h, and then left to cool. The formed solid product was ously. Electrophoresis is carried out for 80 min at voltage 240 192 filtered off, dried, and recrystallized from ethanol. White of 300 V using normal saline (0.9 % w/v NaCl solution) as 241 -1 193 crystals (3.63 g, 70 %); Mp: 90 °C; IR(KBr)mmax,cm : electrolytes source solution. After complete development, 242 194 1615(C=N), 1654(CONH), 2987(CH), 3280(NH), 3305 the paper was removed, dried, and cut into strips, each strip 243 195 (OH), 3420, 3440(NH); MS: m/z (%): 517(82.68), 255 is 1 cm width, and then the strip was counted in a well type 244 196 (80.31), 147(81.10), 107(16.54), 94 (85.04), 79 (11.02), c-counter. The percentage of radiochemical yield was 245 197 72(88.98). estimated as the ratio of the radioactivity of 125I-EHPTIP to 246 the total activity multiplied by 100. 247 198 Ethyl 2-(3-(4-hydroxyphenyl)-2-(2-(4-phenyl-5-((pyridin-4- 199 ylamino)methyl)-4H-1,2,4-triazol-3-ylthio)acetamido)prop- Determination of in vitro stability of 125I-EHPTIP The 248 200 anamido)-3-(1H-indol-3-yl)propanoate (11) Black oil reaction mixture was left at ambient temperature for 72 h 249 -1 201 (3.59 g, 50 %); IR (KBr) mmax,cm : 1610(C=N), 1650, and 1–2 lL samples were taken from it at different time 250 202 1667(CONH), 1745(COO), 3145(NH), 3310(OH), 3497 intervals. The radiochemical yield and purity of the sam- 251 203 (NH);MS: m/z (%):718(14.25), 604(13.84), 587(4.07), 459 ples were measured by paper chromatography and paper 252 204 (2.71), 458(17.10), 436(15.47), 394(17.10), 232(100), 130 electrophoresis. 253 1 205 (60.11), 107(6.24); H-NMR (300 MHz, DMSO-d6, d 206 ppm): 10.883(1H, d, J = 8.4 Hz,NH); 8.321(1H,bs,NH); Determination of the partition coefficient for 125I-EHP- 254

Author Proof 207 7.900(1H,bs,NH);7.570–6.966(17H,m,ArH); 6.943(1H,s,CH); TIP The partition coefficient was determined by mixing 255 125 208 6.587(1H,bs,NH); 5.150(1H,bs,OH); 4.200(2H,s,SCH2); I –EHPTIP with equal volumes of 1-octanol and phos- 256 209 3.990(2H,q,J = 7.2 Hz, OCH2CH3);3.900(2H,s,CH2);3.662 phate buffer (0.025 M at pH 7.4) in a centrifuge tube. The 257 210 = 258 (2H,t,J 6.3 Hz,CH);3.424–2.995(4H,m,CH2); 1.079(3H,t, mixture was vortexedPROOF at room temperature for 1 min and 211 J = 4.8 Hz,OCH2CH3). then centrifuged at 5,000 rpm for 5 min. Subsequently 259 100 lL samples from the 1-octanol and aqueous layers 260 212 Radioiodination of ethyl 2-(3-(4-hydroxyphenyl)-2-(2-(4- were pipetted into other test tubes and counted in a gamma 261 213 phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazol-3- counter. The measurement was repeated three times. The 262 214 ylthio)acetamido)propanamido)-3-(1H-indol-3- partition coefficient value was expressed as log p [31]. 263 215 yl)propanoate (EHPTIP) 11 Log p Log solute solute EDoct:=wat: ¼ ÀÁ½octanol=½deionizedwater 216 In a reaction vial of 1.0 ml capacity with a Teflon septum Biodistribution studies of 125I-EHPTIP in normal mice 266265265 217 and screw cap, 10 ll of EHPTIP (1.4 mM) dissolved in The experimental procedures of the biological studies were 267 218 dimethyl sulfoxide (DMSO) were added then 10 llof done in accordance with the guidelines set out by the 268 219 Na125I (7.2 MBq), 150 ll of freshly prepared chloramine-T Egyptian Atomic Energy Authority and were approved by 269 220 (4.4 mM) in phosphate buffer pH 7 were added. The the animal ethics committee, Labeled Compound Depart- 270 221 reaction vial was left at 80 °C for 45 min. The reaction was ment. Biodistribution studies of 125I –EHPTIP in normal 271 222 quenched by adding 450 ll of sodium metabisulfite Swiss albino mice of body mass 20–25 g (n = 5), were 272 223 (5.3 mM) then subjected to chromatography analysis to carried out at 15, 30, 60, 120 and 240 min post-injection. 273 224 determine the radiochemical yield and the radiochemical Prior to the study, animals were housed in groups of five 274 225 purity of the final product. and provided with food and water. Aliquots of 10 lL 275 containing 3.7 MBq of the 125I–EHPTIP were injected into 276 226 Quality control Radiochemical yield and purity of 125I- each mice via the tail vein. Each mice was weighed then 277 227 EHPTIP were determined by paper chromatographic anaesthetized by chloroform. Samples of fresh blood, bone 278 228 method using strips of Whatman paper. On paper sheet and muscle were collected in pre-weighed vials and 279 229 (1 cm width and 13 cm length), 1–2 lL of the reaction counted. Blood, bone and muscles were assumed to be 7, 280 230 mixture was placed 2 cm above the lower edge and 10 and 40 % of the total body weight, respectively [32]. 281 231 allowed to evaporate spontaneously. For development a Whole brains were removed and frozen for an hour. 282 232 fresh mixture of chloroform: ethanol (9:1 v/v) was used. Organs and tissues were rinsed with saline, collected in 283 233 After complete development, the paper sheet was removed, plastic containers and weighed. The radioactivity of each 284 234 dried, and cut into strips, each strip is 1 cm width, and then sample as well as the background was counted in a well- 285 235 each strip was counted in a well type c-counter. Radio- type NaI (Tl) well crystal coupled to SR-7 scalar rate 286 236 chemical yield was further confirmed by paper electro- meter. Percent injected dose per gram (% ID/g ± SD) in a 287 237 phoresis. On WhatmanUNCORRECT paper sheet (2 cm width and 47 cm population of five mice for each time point are reported. 288 238 length), 1–2 lL of the reaction mixture was placed 12 cm Data were evaluated with one way ANOVA test. Results 289

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK Novel dipeptide attached to triazole-pyridine moiety

290 for P are reported and all the results are given as mean ± amino acids, in addition to several other signals corre- 339 291 SEM. The level of signiﬁcance was set at P \ 0.05. sponding to protons of the individual side chains, (see 340 the Experimental part). 341

292 Results and discussion Radioiodination of EHPTIP 342

293 Synthesis During radioiodination of the dipeptide derivative 11,a 343 very large percentage of the radioiodine will be present in 344 294 As triazole applications in drug constructions were attrac- the highly activated phenolic tyrosine residue at the ortho- 345 295 ted scientist attention; we extended our studying for this position as shown in scheme (2) and this behavior is agrees 346 296 heterocycle via synthesis of novel triazole-pyridine deriv- with that reported in radioiodination of similar com- 347 297 atives. Reaction of hydrazide 1 with phenylisothiocyanate pounds[41]. 348 298 gave phenylthiosemicarbazide 2 which on treatment with The radiochemical purity of the 125I-EHPTIP was 349 299 NaOH cyclised to triazole 3. The hydrazide 5 could be determined using paper chromatography where radioio- 350 300 prepared from 3 by regioselective S-alkylation [33] with dide (I-) remained near the origin (Rf = 0–0.1), while the 351 301 ethylchloroactetate to give the corresponding ester 4, which 125I-EHPTIP moved with the solvent front (Rf = 0.8). 352 302 was subsequently hydrazinolyzed by hydrazine hydrate. Radiochemical purity was further conﬁrmed by paper 353 303 Synthesis of ethyl 3-(4-hydroxyphenyl)-2-(2-(4-phenyl- electrophoresis where the free radioiodide and 125I-EHP- 354

Author Proof 304 5-((pyridin-4-ylamino) methyl)-4H-1,2,4-triazol-3-ylthio) TIP moved to different distances away from the spotting 355 305 acetamido)propanoate 8 was successfully achieved via the point towards the anode (distance from spotting point = 14 356 306 azide coupling method [34–36, 39], which was reported to and 10 cm, respectively) depending on the molecular 357 307 minimize the degree of racemization in amino acid cou- weight of each one.PROOF Results of radiochemical yield from 358 308 pling [37, 38]. Treatment of hydrazide 5 with nitrous acid the two separation methods (paper chromatography and 359

309 (NaNO2/HCl) at -5 °C afforded the corresponding azide paper electrophoresis) are nearly the same. The influence of 360 310 6, which was isolated in ethyl acetate at low temperature various reaction parameters and conditions on radio- 361 311 (0 °C), to suppress its transformation to the isocyanate iodination efficiency were investigated and optimized in 362 312 derivative by Curtius rearrangement. It is known that iso- order to maximize the radiochemical yield. 363 313 cyanates are reactive towards the amino components in the 314 coupling step leading to the formation of the undesired EffectED of substrate concentration 364 315 byproduct urea derivative [40]. The azide solution in ethyl 316 acetate reacted with L-tyrosine ethyl ester hydrochloride The dependence of radiochemical yield on the amount of 365 317 7a, previously treated with triethylamine in ethyl acetate at EHPTIP is depicted in Fig. 1. The reaction was performed 366 318 low temperature, to produce ethyl 3-(4-hydroxyphenyl)-2- at different EHPTIP concentrations (5–250 lg). The 367 319 (2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-tria- radiochemical yield of 125I-EHPTIP was small at low 368 320 zol-3-ylthio)acetamido)propanoate 8 in moderate yield, substrate concentration where at 5 lg the labeling yield 369 321 and its chemical structure was confirmed by IR and 1H- was 77 ± 2.90 % and by increasing EHPTIP concentration 370 322 NMR spectra. The 1H- NMR spectra showed a signal at the labeling yield was increased where the labeling yield 371 323 8.5 ppm for the NH proton of the peptide bond, multi- (84 ± 2.50 %) was obtained at 10 lg. At EHPTIP con- 372 324 plet signals between 6.661 and 7.410 ppm for the aro- centrations higher than the optimum amount, the labeling 373 325 matic protons, triplet signal at 3.457 ppm for the a-CH yield was constant which may be attributed to the fact that 374 326 proton of the amino acid, triplet signal at 1.115 ppm for 10 lg of EHPTIP is enough to capture the entire generated 375

327 the three protons of –OCH2CH3 and quartat signal at iodonium ion as a result the yield reached maximum value 376 328 4.02 ppm for the two protons of –OCH2CH3 of the ester at this concentration. 377 329 group. The dipeptide derivative 11 was prepared from 8 330 after conversion to hydrazide 9 by boiling with excess Effect of oxidizing agent 378 331 hydrazine hydrate in ethanol. The 1H- NMR, mass and 332 IR spectra of 11 conﬁrmed its structure as shown from The effect of oxidizing agent amount (chloramine-T) on 379 333 the data reported in the experimental part. The dipeptide the labeling efﬁciency of 125I-EHPTIP is demonstrated in 380 334 derivative 11 was obtained by the azide-coupling Fig. 2. Radioiodination of EHPTIP has been performed by 381 335 method in 50 % yield (Scheme 1). The 1H- NMR using CAT as a mild oxidizing agent, transforming iodide 382 336 spectra revealed two signals at 7.900 and 6.587 ppm for (I-) to an electropositive form of iodine (oxidative state 383 337 the two NH protons of the peptide bonds, and triplet I?), which allows a spontaneous electrophilic substitution 384 UNCORRECT ? 338 signal at 3.662 ppm for the a-CH protons of the two on aromatic ring with a good leaving group such as H . 385

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK I. Y. Abdel-Ghany et al.

N N N HN HN N H H N N N N 125I S S Ph Ph O O O NH OH N N O NH OH

O O 11 Na125 I / CAT H 7 OEt p OEt

EHPTIP (11) 125 NH NH I-EHPTIP

Scheme 2 Radioiodination of EHPTIP

100 100

80 80

60 % 125I-EHPTIP 125 60 % I-EHPTIP % Free iodide % Free iodide 40 Author Proof 40 20 %Labeling yield

%Labeling yield 20 0 0 50PROOF 100 150 200 0 CAT concentration, µg 0 50 100 150 200 250 Fig. 2 Variation of the radiochemical yield of 125I-EHPTIP as a EHPTIP concentration, µg function of CAT. Reaction conditions 10 lL(*3.7 MBq) Na125I, (9lg) of CAT, 10 lg of EHPTIP, at pH 7, the reaction mixtures were Fig. 1 Variation of the radiochemical yield of 125I-EHPTIP as a kept at room temperature for 15 min function of different EHPTIP amounts. Reaction conditions 10 lL (*3.7 MBq) Na125I, (9lg) EHPTIP, 100 lg of CAT, at pH 7, the ED reaction mixtures were kept at room temperature for 15 min Effect of pH 408

386 When high speciﬁc activity radioiodide is oxidized in situ, The effect of pH of the reaction mixture on the labeling of 409 387 it generates electropositive iodine, but it is unlikely to form EHPTIP was studied by changing the pH values in the 410 388 I2 because there is so little radioiodine present that statis- range from 3 up to 11 using buffers to adjust pH at the 411 389 tically it is not possible for two iodine atoms to join desired value as shown in Fig. 3. The experiment was 412 390 together at the concentrations involved [26]. In addition, performed using 10 lg of EHPTIP. CAT amount used were 413 391 although CAT is a mild oxidizing agent, its effect is 150 lg, while other factors were kept constant. The 414 - ? 392 enough to oxidize all I into I without forming I2. experiment was repeated using 150 lL of each buffer at 415 393 At low CAT amounts (10 lg), the radiochemical yield different pH values. The labeling yield for each pH value 416 394 of 125I-EHPTIP was small and equal 67.50 ± 2.00 %. of the reaction mixture was measured and the pH value at 7 417 395 A high radiochemical yield of 87 ± 2.30 % was achieved the maximum labeling yield was obtained considered the 418 396 by increasing the amount of CAT to 150 lg. Increasing the optimum pH. Increasing in the pH of the medium above 419 397 CAT concentration above that value led to a decrease in the that pH 7 led to a slightly decreasing in the iodination yield 420 398 iodination yield where at 200 lg of CAT, the labeling where at pH 11, the labeling yield was 37 ± 1.0 %. 421 399 yield was 77.9 ± 2.0 %. This decrease in labeling yield 400 may be attributed to the formation of undesirable oxidative Effect of reaction time 422 401 byproducts like chlorination, polymerization and denatur- 402 ation of EHPTIP [28]. The formation of these impurities The labeling yield is strongly dependent on reaction time in 423 403 may be attributed to the high reactivity and concentration the range from 10 S to 60 min. It is clear from Fig. 4 that the 424 404 of CAT [42].Consequently, the optimum concentration of yield is signiﬁcantly increased with increasing the reaction 425 405 CAT (150 lg) is highly recommended in order to avoid the time. The results indicate that the reaction is very fast. After 426 406 formation of by-products and to obtain high labeling yield 45 min the maximum radiochemical yield (90 ± 2.3 %) 427 407 and purity. UNCORRECTwas obtained. 428

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK Novel dipeptide attached to triazole-pyridine moiety

100 % 125 I-EHPTIP 100 % Free iodide 80 80 60

60 125 40 % I-EHPTIP % Free iodide 20 40 %Labeling yield

0 234567891011 %Labeling yield 20 pH 0 Fig. 3 Variation of the radiochemical yield of 125I-EHPTIP as a 20 30 40 50 60 70 80 90 100 110 * 125 function of pH. Reaction conditions 10 lL( 3.7 MBq) Na I, Temperature, °C 10 lg of EHPTIP, 150 lg of CAT, at different pH, the reaction mixtures were kept at room temperature for 15 min Fig. 5 Variation of the radiochemical yield of 125I-EHPTIP as a function of reaction temperature. Reaction conditions 10 lL (*3.7 MBq) Na125I, 10 lg of EHPTIP, 150 lg of CAT, at pH 7, 100 the reaction mixtures were kept at different temperatures for 45 min

Author Proof 100 60 % 125I-EHPTIP % Free iodide 80 40 60 % 125 I-EHPTIPPROOF % Free iodide %Labeling yield 20 40

20 0 %Labeling yield 0 102030405060 0 Reaction time, minutes 12 18 24 30 36 42 48 54 60 66 72 Time post iodination, hours Fig. 4 Variation of the radiochemical yield of 125I-EHPTIP as a ED function of reaction time. Reaction conditions 10 lL(*3.7 MBq) Fig. 6 In vitro stability of 125I-EHPTIP Na125I, 10 lg of EHPTIP, 150 lg of CAT, at pH 7, the reaction mixtures were kept at room temperature for different intervals of time

Partition coefficient of 125I-EHPTIP 446 429 Effect of reaction temperature The partition coefficient value was 2.35 ± 0.11, showing 447 430 The influence of the temperature of the reaction mixture on that 125I-EHPTIP is relatively lipophilic and can cross the 448 431 the radiochemical yield of 125I-EHPTIP is shown in Fig. 5. blood–brain barrier. 449 432 The reaction was carried out at room temperature, 40, 60, 433 80 and 100 °C. The results of this study clearly show that Biological evaluation 450 434 that the yield is significantly increased with increasing the 435 reaction temperature, where at higher temperature degrees Antimicrobial activity of EHPTIP 451 436 (80 and 100 °C) the maximum labeling yield (92.5 ± 2.0 %) 437 was obtained. During the synthesis process of new dipeptide derivative 452 (EHPTIP) 11, numbers of newly synthesized compounds 453 438 In vitro stability of 125I-EHPTIP were obtained. The antimicrobial activity of all newly 454 synthesized compounds was assayed by the agar well dif- 455 439 In vitro stability of 125I-EHPTIP was studied in order to fusion method [43] against two bacterial colonies [Esche- 456 440 determine the suitable time for injection to avoid the for- richia coli (Gram negative) and faecal streptococcus 457 441 mation of the undesired products that result from the (Gram positive)] and one fungal culture (Aspergillus fla- 458 442 radiolysis of the labeled compound. These undesired vus).The bacteria and fungi were maintained on nutrient 459 443 radioactive products might be accumulated in non-target agar and Czapek’s-Dox agar media, respectively. Six-mil- 460 444 organs. The resultsUNCORRECT of stability showed that the 125I-EHP- limeter diameter wells were cut out in agar plates using a 461 445 TIP is stable up to 48 h as shown in Fig. 6. sterile cork-borer. Fifty lL of 1 mg/mL test solutions were 462

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK I. Y. Abdel-Ghany et al.

Tetracycline 21 20 0 0 Penicillin G–sodium 20 18 0 30 Nystatin 0 0 14 14 2 15 0 23 45 316145065 4130045 5140030 8160053 91815260 11 17 17 0 14

Table 2 Biodistribution of 125I-EHPTIP in normal mice at different time intervals post-injection. (% ID/gram ± SD, n = 5) Author Proof Organs and body ﬂuids % Injected dose/gram at different time intervals (min) 15 (min) 30 (min) 60 (min)PROOF 120 (min) 240 (min) Blood 10.73 ± 0.1 8.77 ± 0.09 8.54 ± 0.01 2.87 ± 0.00 2.86 ± 0.09 Kidneys 23.61 ± 0.08 22.76 ± 0.01 22.65 ± 0.04 11.61 ± 0.03 4.76 ± 0.01 Liver 12.16 ± 0.19 10.70 ± 0.82 11.07 ± 0.08 6.79 ± 0.21 3.89 ± 0.06 Spleen 3.36 ± 0.02 3.70 ± 0.06 2.37 ± 0.07 2.52 ± 0.04 2.47 ± 0.05 Intestine 12.74 ± 0.61 20.95 ± 0.46 20.91 ± 0.21 19.84 ± 0.15 19.21 ± 1.12 Stomach 53.42 ± 0.22 58.60 ± 0.25 104.54 ± 0.21 94.15 ± 0.14 69.70 ± 0.04 Lungs 9.21 ± 0.07 9.33 ± 0.06ED 9.28 ± 0.05 4.47 ± 0.00 2.66 ± 0.01 Heart 4.49 ± 0.03 3.17 ± 0.09 3.43 ± 0.01 3.25 ± 0.03 1.11 ± 0.01 Thyroid 0.10 ± 0.05 1.27 ± 0.03 1.78 ± 0.02 0.12 ± 0.01 0.11 ± 0.04 Muscle 1.75 ± 0.09 3.94 ± 0.08 4.07 ± 0.00 3.38 ± 0.01 1.68 ± 0.09 Bone 3.20 ± 0.04 2.78 ± 0.06 2.69 ± 0.09 2.33 ± 0.04 2.35 ± 0.00 Brain 1.27 ± 0.03 7.60 ± 0.01 6.66 ± 0.02 1.32 ± 0.04 0.81 ± 0.09

463 transferred aseptically to the wells. Plates were incubated mention that derivatives 3, 9 were effective against all 480 464 at 30 oC for 24 h for bacteria and 72 h of incubation at tested microorganisms. 481 465 28 oC for fungi. The antimicrobial activity was evaluated 466 by measuring the inhibition zone formed around the wells Biodistribution of 125I-EHPTIP 482 467 in mm. Tetracyclin and Penicillin G sodium were used 468 as standard antibacterial drugs, while Nystatin was used as The biodistribution pattern of 125I-EHPTIP is shown in Table 483 469 standard antifungal drug. Dimethyl sulphoxide was used as (2). 125I-EHPTIP was injected in normal mice via intrave- 484 470 solvent for tested compounds showed no inhibition zones. nous route and was distributed all over the body organs and 485 471 Results are listed in Table (1). ﬂuids. All radioactivity levels are expressed as average 486 472 This study showed that the dipeptide derivative 11 was percentage of injected dose per gram (% ID/g ± SD). The 487 473 effective against both the tested gram positive and gram high uptake of radioactivity in kidney indicates that excre- 488 474 negative bacteria. All derivatives were effective against tion of 125I-EHPTIP occurs mainly through the renal system. 489 475 E. coli but not against F. streptococcus except compounds The biodistribution data showed substantial uptake of 490 476 3, 9, 11 were effective against both the gram positive and 7.60 ± 0.01 (% ID/g ± SD) in the brain at 30 min post- 491 477 gram negative bacteria tested. Derivatives 2, 3, 9 were injection and retention in brain remained high up to 1 h. 492 478 inhibitory toUNCORRECT the growth of A. ﬂavus, while all derivatives After this time, point radioactivity dropped to 1.32 ± 0.04 at 493 479 were inhibitory to sporulation growth (Table 1). It is worth 120 min post-injection. The low radioactivity located in the 494

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK Novel dipeptide attached to triazole-pyridine moiety

495 thyroid gland indicates that the iodocompounds are stable 17. Khanmohammadi H, Abnosi HM, Hosseinzadeh A, Erfantalab M 539 496 (2008) Spectrochim Acta A 71:1474–1480 540 against in vivo deiodination. The maximum brain uptake of 541 497 125 ± 18. Poojary B, Belagali SL, Kumar KH, Holla BS (2003) Farmaco I-EHPTIP (7.60 0.01) is higher than that of currently 58:569–572 542 99m 498 used radiopharmaceuticals for brain imaging, Tc-ECD 19. Levit GL, Anikina LV, Vikharev YB, Demin AM, Saﬁn VA, 543 499 and 99mTc-HMPAO which have maximum brain uptake of Matveeva TV, Krasnov VP (2002) Pharm Chem J 36:232–236 544 500 20. Belagali SL, Kumar KH, Holla BS, Bhagwat A (2001) Indian J 545 4.7 and 2.25 %, respectively [44, 45]. These preliminary 546 501 Heterocycl Chem 10:249–252 results strongly suggest that the radioiodinated EHPTIP 21. Himaja M, Rajiv RMV, Poojary B, Satyanarayana D, Subrah- 547 123 502 which is labeled with I could be used as a novel agent for manyam EV, Bhat KI (2003) Boll Chim Farm 142:450–453 548 503 brain SPECT. The high uptake of 125I-EHPTIP in stomach 22. Himaja M, Rajiv RMV (2002) Indian J Heterocycl Chem 12: 549 504 121–124 550 also enforces us to do more research about the possibility of 551 505 23. Aboelmagd A, Ali IAI, Salem EMS, Abdel-Razik M (2011) using it as agent for stomach imaging and treatment com- Arkivoc ix:337–353 552 506 paring it with currently used agents for that purpose. 24. El-rayes SM (2010) Molecules 15:6759–6772 553 507 25. Bolton R (2002) J Labelled Compds Radiopharm 45:485–528 554 26. Adam MJ, Wilbur DS (2005) Chem Soc Rev 34:153–163 555 27. Amin AM, Soliman SE, Killa HM, Abd El-Aziz H (2010) J 556 557 508 Radioanal Nucl Chem 283:273–278 References 28. El-Azony KM (2010) J Radioanal Nucl Chem 285:315–320 558 29. Newberg AB, Plo¨ssl K, Mozley PD, Stubbs JB, Wintering N, 559 509 1. Dahiya R, Kumar A, Yadav R (2008) Molecules 13:958–976 Udeshi M, Alavi A, Kauppinen T, Kung HF (2004) J Nucl Med 560 510 2. Siddiqui N, Andalip BS, Ali R, Afzal O, Akhtar MJ, Azad B, 45:834–841 561 Author Proof 511 Kumar R (2011) J Pharm Bioall Sci 3(20):194–212 30. Moretti JL, Caglar M, Weinmann P (1995) J Nucl Med 36: 562 512 3. Kumar JR (2010) Pharmacophore 1(3):167–177 359–363 563 513 4. Amr AG, Mohamed AM, Mohamed SF, Abdel-Hafez NA, 31. Liu FEI, Youfeng HE, Luo Z (2004) IAEA, Austria, Technical 564 514 Hammam AG (2006) Bioorg Medicinal Chem 14:5481–5488 Reports Series No 426, pp 37–52 565 515 5. Bart A, Jansen J, Zwan JV, Dulk H, Brouwer J, Reedijk J (2001) J 32. Rhodes BA (1974)PROOF Semin J Nucl Med 4:281–293 566 516 Med Chem 44:245–249 33. Fathalla W, Cˇ ajan M, Pazdera P (2001) Molecules 6:557–573 567 517 6. Shinkai H, Ito T, Iida T, Kitao Y, Yamada H, Uchida I (2000) J 34. Fathalla W, El Rayes SM, Ali IAI (2007) Arkivoc xvi:173–186 568 518 Med Chem 43:4667–4677 35. Ali IAI, Fathalla W, El Rayes SM (2008) Arkivoc xiii:179–188 569 519 7. Klimesova V, Svoboda M, Waisser K, Pour M, Kaustova J (1999) 36. El Rayes SM (2008) Arkivoc xvi:243–254 570 520 Farmaco 54:666–672 37. Hofmann K, Johl A, Furlemeier AE, Koppeler HJ (1957) J Am 571 521 8. Maria GM, Valeria F, Daniele Z, Luciano V, Elena BJ (2001) Chem Soc 79:1636–1641 572 522 Farmaco 56:587–592 38. Hofmann K, Thompson TA, Yajima H, Schwarts ET, Enouye H 573 523 9. Goda FE, Abdel-Aziz AAM, Attef OA (2004) Bioorg Medicinal (1960)ED J Am Chem Soc 82:3715–3720 574 524 Chem 12:1845–1852 39. El Rayes SM, Ali IAI, Fathalla W (2008) Arkivoc xi:86–95 575 525 10. Bhatia MS, Mulani AK, Choudhari PB, Ingale KB, Bhatia NM 40. Bodansky M (1993) Principles of peptide synthesis, 2nd edn. 576 526 (2009) Int J Drug Discov 1:1–9 Springer, Berlin, p 20 577 527 11. Muthal N, Ahirwar J, Ahriwar D, Masih P, Mahmdapure T, Si- 41. Coenen HH, Mertens J, Maziere B (2006) Radioionidation 578 528 vakumar T (2010) Int J Pharm Tech Res 2(4):2450–2455 reactions for radiopharmaceuticals. Compendium for effective 579 529 12. El Ashry HE, Kassem AAK, Abdel Hameed MH (2009) Carbo- synthesis strategies, chap. 4. Springer, Dordrecht, p 64 580 530 hydr Res 344:725–733 42. Tolmachev V, Bruskin A, Sivaev I, Lundqvist H, Sjo¨berg S 581 531 13. Sztanke M, Tuzimski T, Rzymowska J, Pasternak K, Kandefer- (2002) Radiochim. Acta 90:229–235 582 532 Szerszen M (2008) Eur J Med Chem 43:404–419 43. Perez C, Pauli M, Bazerque P (1990) Acta Biol Med Exp 15: 583 533 14. Foroughifar N, Mobinikhaledi A, Ebrahimi S, Moghanian H, 113–115 584 534 Fard BAM, Kalhor M (2009) Tetrahedron Lett 50:836–839 44. Neirinckx RD, Canning LR, Piper IM, Nowotnik DP, Pickett RD, 585 535 15. Isloor MA, Kalluraya B, Shetty P (2009) Eur J Med Chem 44: Holmes RA, Volkert WA, Forster AM, Weisner PS, Marriott JA, 586 536 3784–3787 Chaplin SB (1987) J Nucl Med 28:191–202 587 537 16. Qin X, Yu H, Liu J, Dai BG, Qin Z, Zhang X, Wang T, Fang J 45. New Drugs (2002) Australian prescriber 25: No 1 588 538 (2009) Arkivoc ii: 201–210 589

UNCORRECT

123

Journal : Large 10967 Dispatch : 5-9-2012 Pages : 9

Article No. : 2237 h LE h TYPESET MS Code : JRNC-D-12-03131 h44CP h DISK

ARABIC SUMMARY

اا

اا ولهارا ت ا اتاایة ا ت ﻥیة ا( یی ، اایزول یی ، ا وآدایزول یی او ا زول یی ) ایازدواجا زای و إﺙت اآتا ﺝة اﻥاويا ونواو اﺵااء . اطادوتآتاوأتاآت ﺝة ﺽاتااة . راﺵرة إأن تاایزول ١٠و ١٤ آﻥ ﺽ ﻥﺝ ا تا اة و ت ا اتا١٥ و ٢١ ات اایزول یی و ا وآدایزول یی ا آﻥ ﺽ آ ای إی و اام. ﻥﺡأﺥى ، ت ا اتا٨و ١٥ امید ١٢٥ وارأ –ت آآ ﺙی إﺝاء ا ا وﺝا ی ا ودرﺝ اء اﺵا آت ا . درا ﺙااایة ا دا ( آاآااد ،آااآ( آر أت ،) اس اروﺝ ا و زا) لإ أ . ﻥ ذ دراازیاي تاإﺵ اانا وأوﺽﻥارااو ﺙتاآتاداﺥوﺥرجا او ﺡ أ آا یﺝیة . .١ ﻡت ااتا ا تﻡﻥیة: ١١ ﻡت ااتا ا تﻡﻥیة: ی أ٤ یی )١( ای آروأ توﺝدﺥتادیم ا یرات وا یﻥ لدرﺝﺡارةانیا ی (٢ یی ٤ أی أ) ت ( )٢ ٩٢% . اآ( )٢ ارازیهراتا یﻥلای ارازی ا )٣( ٩٠% . اغ ة ارازی ات آ ر

أ اا

آت ﺡ ﻥیة و ا ذاتاﻥاﺝایة و. ء ذ ، اام ارازی ﺡتﻥ و . ارازی(٣ ) ا أیوﻥتی ﺙ رزی(٩ ) اي یهروآا دیماایزول( ١٠ ). ﺽإذ ، ﺥوي اتارازی(٣ ) وهروآاموزیدةاندایایﻥل ی (٥ [ یی ٤ یأ] ) ١٣٤ أ وآدایزول ٢ ﺙل( ١٦ ). ٤ أ یی(١ ) آ روأﻥیوهریادیم DMF ی اآ( ٢٢) ،ویN-([H٢ ازول ٥ ی] ) یی ٤ أ( ٢٣ ) اآ( ٢٢ ) وأزایادیموآریاﻥم ٨٦ %( أ ).

ﻡأ

ب اا

١٢ ﻡت ارازی تﻡﻥیة آﺱ ر یازدواج ا زای: یتارازی(٣ ، ١٢ ، ١٨ و ٢٥ ) ا ت ا ﻥ ا یة (١ ، ١٠ ، ١٦ و ٢٣) ،ا، امایآروأت ای أت ا (٢ ، ١١ ، ١٧ و ٢٤) ایی ا رازی ات ا ارازیهرات ( ب ).

ﻡب ١٣ ارطاﺡضاﻡ یازدواجا زای: ت ارازی ٣( ، ١٢ ، ١٨ و )٢٥ ﺡ اوز( ﻥیادیم / ﺡاروآری ) درﺝﺡارة ٥ ی ی ت ا زایا ،ای تا یدرﺝﺡارة . یل ازای ﺥت ای ای إ هروآری ا ( اً ای ی أ ﺥت ا ی درﺝﺡارة ) ت إ اتا ا (٤ ٥، ٦، ، ١٣ ، ١٩ و ٢٦ ) .ﺝ یالتااتا(٨ ، ١٥ ، ٢١و ٢٨ ) ی ازدواجا زای . ی ت ا ات ا ت ای إ وزی(٤ ، ١٣ ، ١٩و ٢٦ ) ی رازی اتا (٧ ، ١٤ ، ٢٠و ٢٧) ،ا، نزیدة ارازیهراتا یﻥل (. ت ).

ت اا

NH 2NH EtO 2 H, r eflux

Cl , H 2 NO Na

ﻡت .٢ ﻡ ت ااتا : ﻡ ا ا ا یی: أل I-EHPIP 125 آن ٩٠± ٢٨ % أ ﺝيا ب ١٠٠ وﺝام اا ( EHPIP ) )٨( و ١٠ وNa 125 I (~ ٧٢ ی) و ١٥٠ وﺝام ارأ –ت لاتاساروﺝ ٨ ویا درﺝﺡارةاة٥ د ( ث .)

ث اا

ﻡث ﻡ ا ا ا ١٢٤ ایزول یی: أ ل I-EHPTIP 125 آن ٩٢٥± ٢٠ % أ ﺝيا ب ١٠ وﺝ ام اا( EHPTIP () )١٥ و ١٠ وNa125 I (~ ٧٢ ی) و ١٥٠ و ﺝام ارأ –ت لاتاس اروﺝ٧ ویا درﺝﺡارة ٨٠ ی ة ٤٥ د ( ج ).

ﻡ ج

اا

﴿ ﴾ رةاء / ١١٣

ا آــــاـــم ـــاـــــء وااتاوات ﻥواا ﺭﺳﺎﻟﺔﻣﻘﺪﻣﺔﺇﻟﻰ اء آام ﻡا ﺱل ﻡت ال در ا ام " اءای " ﻣﻦ همالاﺵای ﺏریسماء( ﻡی ٢٠٠٧) ﻡﺏاآتا ﻡآاﻡارة هااری ﺗﺤﺖ ﺇﺷﺮﺍﻑ د.أ ./ ﺡـــــــــــــــاـــري د.أ ./ آاـــــ أﺱذاءای أﺱذاءایا اء اآتا آا م ــــﻡـــاـــــ ﻡآاﻡارة هااری د إ./ اهیﺱا أﺱذﻡاءایا اآتا ﻡآاﻡارة هااری

٢٠١٢