Design, Synthesis, Molecular Docking and Biological

Design, Synthesis, Molecular Docking and Biological Investigation of New Hydroxamic Acid, Amide and Hydrazide Derivatives of Fluoroquinolones. A thesis submitted by

Mohammed Abdullah Ali Abdullah (B.Sc.Pharm. Sci., 2011) Faculty of Pharmacy, Minia University In the partial fulfillment of the requirements for the Master Degree of Pharmaceutical Sciences (Pharmaceutical Medicinal Chemistry)

Supervisors Prof. Dr. Gamal El-Din A. A. Abuo-Rahma Prof. of Pharm. Med. Chem. Faculty of Pharmacy Minia University Dr. Heba Ahmed Hassan Lecturer of Pharm. Med. Chem. Faculty of Pharmacy Minia University

Dr. El-Shimaa M. N. Abdelhafez Lecturer of Pharm. Med. Chem. Faculty of Pharmacy Minia University

Med. Chem. Dept. Faculty of Pharmacy Minia University, Minia, Egypt (2016)

‫) ﺳورة اﻷﻋراف اﻵ ﺔ‪(43 :‬‬

Dedicated to my parents and my sister

Acknowledgement In the name of Allah, the beneficent, the merciful First of all, deepest and humble gratitude to Almighty Allah, for each grace and favor in my life, and for providing me the health, knowledge and blessings to accomplish this work. It is a pleasure to express my sincere appreciation and deep gratitude to Prof. Dr. Gamal EL-Din A. A. Abuo-Rahma, Professor of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Minia University, for his kind supervision, guidance, continuous help, encouragement, giving his precious time and continuous support. Everlasting thanks are expressed to Dr. Heba Ahmed Hassan, Lecturer of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Minia University, for her sincere advices to help to complete this work. My sincere appreciation to Dr. El Shimaa Mohamed Naguib, Lecturer of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Minia University, for her helpful cooperation and support all over the period of research. Great thanks are also expressed to Prof. Takehiko Yoshimitsu, Professor of Pharmaceutical Chemistry , Graduate School of Pharmaceutical Sciences, Osaka University, Japan and for Dr. Gamal Abdeltawab Lecturer of Medicinal Chemistry, Mina University for the valuable cooperation and performing HRMS spectra, and also to Dr. Abdelhamid El-Sawy, Ph.D candidate at University of Connecticut, Storrs, Connecticut, USA for performing other HRMS spectra. My grateful thanks for Dr. Rehab Mahmoud Abd-Elbaky, Associate Professor of microbiology immunology at department of microbiology and immunology, Faculty of

Pharmacy, Minia university for her great effort in performing antibacterial and urease inhibition investigations. I would like also to express my thanks to all staff members of the Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, and every one helped me for complete of this work. Mohammed Abdullah Ali Minia 2016

List of abbreviations AHA AIDS DCM DMSO DNA FDA FT-IR HA HCV HIV HRMS MALDI-TOF MDR-TB MIC MOE MRSA NCI NMR PDB ppm SAR TB TEA TMS TNF TOPO I TOPO II TOPO IV UTIs UV

Acetohydroxamic Acid Acquired Immunodeficiency Syndrome Dichloromethane Dimethyl Sulphoxide Deoxyribonucleic acid Food and Drug Administration Fourier Transfer Infra-red Hydroxamic acid Hepatitis C Virus Human Immunodeficiency Virus High Resolution Mass Spectroscopy Matrix-assisted laser desorption/ionization-time of flight spectrometer Multi Drug Resistant Tuberculosis Minimum Inhibitory Concentration Molecular Operating Environment Multi-Resistant Staphylococcus aureus National Cancer Institute Nuclear Magnetic Resonance Protein Data Bank Part Per Million Structure Activity Relationship Tuberculosis Triethyl Amine Tetramethylsilane Tumor Necrosis Factor Topoisomerase I Topoisomerase II Topoisomerase IV Urinary Tract Infections Ultraviolet

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Table of content Page number Summary…………………………………………………………………………... viii 1. INTRODUCTION……………………………………………………. 1 1.1. Quinolones. ………………………………………………………………............ 1 1.1.1. Mechanism of action of quinolones.…………………………..………………... 4 1.1.2. Biological importance of quinolones…………………………..…..…..…...…... 5 1.1.2.1. Antibacterial Activity.………………………………………………………... 5 1.1.2.2. Anticancer activity…………………………………......................................... 8 1.1.2.3. Antiviral activity……………………………………........................................ 11 1.1.2.4. Antimycobacterial activity…………………………......................................... 11 1.2. Urease inhibitors………………………………………………………………… 13 1.2.1. Classification of Urease inhibitors…………………….……….……….………. 16 1.2.1.1. Hydroxamic acid derivatives………………...……….……….………………. 16 1.2.1.2. Phosphoramide derivatives………………….………….………….…………. 18 1.2.1.3. Thiol derivatives……………………………...……………………………….. 18 1.2.1.4. Metal complexes…………………………….................................................... 19 1.2.1.5. Fluoroquinolones as urease inhibitors…………………………………............ 20 1.2.1.6. Other urease inhibitors…………………………………………………........... 20 2. AIM OF THE WORK…………………………………………………................ 22 3. RESULTS AND DISCUSSION…………………………………………………. 25 3.1. Chemistry………………………………………………………………………… 25 3.1.1. Synthesis of hydroxamic acid 3a-e, amide 4a-e, and hydrazide 6a-e derivatives 25 of Ciprofloxacin……………………………………………………………………….. 3.1.1.1. Synthesis of N-4 (substituted) piprazinyl ciprofloxacin hydroxamic acids 3a-e…28 3.1.1.2. Synthesis of N-4 (substituted) piprazinyl ciprofloxacin amides 4a-e……….... 34 3.1.1.3. Synthesis of N-4 (substituted) piprazinyl ciprofloxacin hydrazides 6a-e….…. 39 3.1.2. Synthesis of levofloxacin hydroxamic acid, amide and hydrazide derivatives 8, 46 9 and 11………………………………………………………………………………... 3.1.2.1. Synthesis of levofloxacin hydroxamic acid 8………………………………… 47 3.1.2.2. Synthesis of levofloxacin amide derivative 9………………………………… 48 3.1.2.3. Synthesis of levofloxacin hydrazide derivative 9…………………………….. 51 3.2. Biology……………………………………………………………………………. 54 3.2.1 Anticancer screening…………………………………………………………….. 54 3.2.1.1. In vitro one-dose assay……………………………..………..………..…….. 54

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3.2.2. Antibacterial Screening………………………………………..………………... 3.2.3. Urease inhibition activity……………………………………………………….. 3.2.3.1. Molecular docking studies (H. Pylori urease)……………...………...………. 4. EXPERIMENTAL…………………………………………………………… 4.1. Chemistry………………………………………………………………………… 4.1.2. General procedure for synthesis N-4-acylated piprazinyl ciprofloxacin 1a-c.… 4.1.3. Preparation of ciprofloxacin chalcone derivatives 1e-d……...……...……...…. 4.1.3.1. General Procedure for synthesis Chalcones A1 and A2…………………........ 4.1.3.2. General procedure for the synthesis of 2-bromo-N-{4-[3- arylacryloyl]phenyl and 3,4,5 trimethoxy phenyl]} acetamides B1 and B2……….……….……….……… 4.1.3.3. Synthesis of Ciprofloxacin-chalcones derivatives 1d-e….…………………… 4.1.4. General procedure for synthesis of hydroxamic acids derivatives 3a-e and 8…. 4.1.5. General procedure for synthesis of fluoroquinolone amides 4a-e and 9……….. 4.1.6. General procedure for synthesis of fluoroquinolone hydrazides 6a-e ……….. 4.1.6.1. Synthesis of fluoroquinolone methyl esters 5a-e and 10…………………… 4.1.6.2. Synthesis of fluoroquinolone hydrazide derivatives 6a-e and 11…………….. 4.2. Biological experiments…………………………………………...……...……..... 4.2.1. Anticancer screening…………………………...………………………….......... 4.2.2. Antibacterial Activity…………………………..……………..……………..….. 4.2.3. Urease inhibitory activity……………………...……………………...………… 4.3. Protocol of Docking studies……………………………………...……………… 5. REFERENCES………………………………………………………………….

Arabic Summary………………………..………………………...

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61 63 65 73 73 75 76 76 76 77 78 82 87 87 90 95 95 96 97 97 99 1

List of Figures Figure No.

Page No.

1

Mechanism of action of quinolones…………………………………………...……..

4

2

Binding model of quinolones………………………………………………………...

5

3

General structure of quinolones. …………………………………….………………

6

4

Hydrolysis of urea by urease enzyme………………………………...……………...

13

5

Urease Active site……………………………………………………………………

14

6

The structures of three well-characterized ureases…………………………………

15

7

The target hydroxamic, amide and hydrazide derivatives of fluoroquinolones……...

24

8

1

31

9

13

C NMR (100 MHz) (CDCl3)spectrum of compound 3c. ……….............................

32

10

HRMS spectrum of compound 3c.…………………..................................................

33

11

1

35

12

13

C NMR spectrum (75 MHz) (CDCl3) for compound 4b………………………….

37

13

HRMS spectrum for compound 4b………………………………..............................

38

14

1

41

16

13

C NMR (100 MHz) (CDCl3) spectrum of compound 5b………………………….

42

16

HRMS spectrum for compound 5b………………………………..............................

41

17

1

44

18

13

C NMR (100 MHz) (CDCl3) spectrum of compound 6a……………......................

44

19

HRMS spectrum of compound 6a………………………………...............................

45

20

1

49

21

13

C NMR spectrum (75 MHz) (CDCl3) of compound 9……………...…...................

49

22

HRMS spectrum of compound 9……………………………….................................

50

23

1

52

24

13

C NMR (125 MHz) (CDCl3) spectrum of compound 11……….…………….......

52

25

HRMS spectrum of compound 11……………………………………………….......

53

26

One dose growth (%) and mean graph of compound 3b…………….........................

56

27 28

One dose growth (%) and mean graph of compound 4c……………......................... 2D Diagram of compounds LXIV and LXV...……………………………………...

57

29

2D Diagram of compounds LXIV and LXV ………………………….....................

60

H NMR (400 MHz) (DMSO) spectrum of compound 3c..……………………….....

H NMR spectrum (300 MHz) (CDCl3) for compound 4b…………………………

H NMR (400 MHz) (CDCl3) spectrum for Compound 5b………………………....

H NMR (400 MHz) (CDCl3) spectrum for compound 6a……………......................

H NMR spectrum (300 MHz) (CDCl3) of compound 9………………………….....

H NMR (500 MHz) (CDCl3) spectrum of compound 11…………….……….........

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59

30

3D of AHA Docked into H.pylori urease……………………………….…......….....

65

31

2D and 3D docking modes of compound 8 with H.pylori urease……….…......….....

66

32

2D and 3D docking modes of levofloxacin with H.pylori urease…………......…......

67

33 34 35

2D and 3D docking modes of compound 3a with H.pylori urease…......…......…...... 2D and 3D docking modes of N-acetyl ciprofloxacin 1a with H.pylori urease……... 2D and 3D docking modes of ciprofloxacin with H.pylori urease….......…......….....

67 68 68

36

2D and 3D docking modes of 3d with H.pylori urease……………………......….....

69

37

2D and 3D docking modes of 6a with H.pylori urease……………………......…......

69

38

2D and 3D docking modes of 4a with H.pylori urease……………………......…......

70

39

2D diagram and 3D structure of 4c with H.pylori urease…………………......…......

70

40

2D diagram and 3D structure of 9 with H.pylori urease……………......…......…......

71

41

2D diagram and 3D structure of 4b with H.pylori urease…………………......…......

71

42

2D diagram and 3D structure of 4d with H.pylori urease…………………......…......

71

43

2D diagram and 3D structure of 4e with H.pylori urease…………………......…......

72

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List of Schemes Scheme No. 1 2 3 4 5 6 7 8 9 10 11 12 13

Page No. Formation of ciprofloxacin un-isolated mixed anhydrides 2a-e and synthesis of ciprofloxacin derivatives 3a-e, 4a-e and 6a-e………………… Synthesis of compounds 1a-c……………………………….......…………. Synthesis of compounds 1d and 1e………………………………………... Formation of ciprofloxacin un-isolated mixed anhydrides 2a–e…………... Reaction mechanism for formation of mixed anhydride………..…………. Synthesis of ciprofloxacin hydroxamic acid derivatives 3a-e……………... Reaction mechanism for hydroxamic acid preparation……………………. Synthesis of ciprofloxacin amide acid derivatives 4a-e……….................... Reaction mechanism for formation of amides 4a-e……………………….. Synthesis of ciprofloxacin methyl ester derivatives 5a-e………………….. Reaction mechanism of formation methyl ester derivatives 5a-e…………. Reaction mechanism of preparation hydrazide derivatives 6a-e…………... Formation of levofloxacin un-isolated mixed anhydrides 7a and synthesis of levofloxacin derivatives 7-11……………………………………………

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25 26 27 27 28 29 29 34 34 39 39 40 46

List of Tables Table No. 1 2 3 4 5 6

Mean growth percentage for the tested compounds of one dose screening.... Docking scores of compounds LXIV, LXV, 3b and 4c on topoisomerase II Anti-Proteus mirabilis of the tested compounds…………………………….. IC50 of urease inhibition of the tested compounds……………........................ Docking scores of the docked compounds…………………………………... Different binding mode of hydroxamic, hydrazide, amide derivatives of fluoroquinolones and AHA………………………………………………………

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Page No. 55 59 61 63 66 72

Summary

Summary

This study is concerned with the design and synthesis of some new hydroxamic acid, amide and hydrazide derivatives of both ciprofloxacin and levofloxacin and evaluation of their anticancer, anti-Proteus mirabilis and urease inhibitory activity supported by their molecular docking studies. The thesis combines four main sections: introduction, aim of the work, results and discussion and experimental section in addition to references. The first part, introduction: Introduction gives a general view about quinolone and fluoroquinolone derivatives, their generations, their various biological activities and mechanism of action. In addition, it discusses structure activity relationships of antibacterial fluoroquinolones. Furthermore, this section includes general account about urease enzyme, its role in pathogenicity, urease enzyme crystal structure of the active site, and various classes of urease inhibitors. The second part, aim of the work: This section outlined the goal of this work including design and synthesis of new hydroxamic acids, amide and hydrazide derivatives of ciprofloxacin and levofloxacin and their biological evaluation as anticancer, anti-Proteus mirabilis and urease inhibitory activity. Furthermore, the docking studies of the newly synthesized compounds was evaluated with H.pylori urease enzyme to prove their urease inhibitory activity. The third part, Results and Discussion This section includes detailed explanation of the results and data obtained from different stages of synthesis, structural elucidation and biological evaluation of the target compounds (3a-e, 4a-e, 5a-e, 6a-e and 8-11). This section is subdivided into three main parts: The first section: Chemistry section, which includes description of the different methods used for synthesis and structural elucidations of the target compounds by different spectral analysis including IR, 1H NMR,

13

C NMR and HRMS spectroscopy. In this thesis we

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Summary

reported, 24 reported compounds were prepared in addition to 22 new compound. Chemical nomenclatures of the new compounds as follows: Hydroxamic acid derivatives • • • • •

•

7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-hydroxy-4oxoquinoline-3-carboxamide 3a 7-(4-Benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-hydroxy-4oxoquinoline-3-carboxamide 3b 7-(4-(3,4,5)-Trimethoxybenzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydroN-hydroxy-4-oxoquinoline-3-carboxamide 3c 7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-1cyclopropyl-6-fluoro-1,4-dihydro-N-hydroxy-4-oxoquinoline-3-carboxamide 3d 7-(4-((4-((E)-3-(3,4,5-TrimetoxyPhenylacryloyl) phenylcarbamoyl) methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-hydroxy-4oxoquinoline-3-carboxamide 3e (S)-9-Fluoro-3,7-dihydro-N-hydroxy-3-methyl-10-(4-methyl piperazin-1-yl)-7-oxo2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide 8

N-methyl amide derivatives •

7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4oxoquinoline-3-carboxamide 4a

•

7-(4-Benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4oxoquinoline-3-carboxamide 4b

•

7-(4-(3,4,5)-Trimetoxybenzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydroN-methyl-4-oxoquinoline-3-carboxamide 4c

•

7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-1cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4-oxoquinoline-3- carboxamide 4d

•

7-(4-((4-((E)-3-(3,4,5-TrimetoxyPhenylacryloyl) phenylcarbamoyl) methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4oxoquinoline-3-carboxamide 4e

•

(S)-9-Fluoro-3,7-dihydro-N,3-dimethyl-10-(4-methylpiperazin-1-yl)-7-oxo-2H[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide 9

Methyl ester derivatives • •

Methyl 7-(4-benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3-carboxylate 5b Methyl 7-(4-3,4,5-trimethoxybenzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4dihydro-4-oxoquinoline-3-carboxylate 5c

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Summary

• •

Methyl 7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl) piperazin-1-yl)1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3- carboxylate 5d Methyl 7-(4-((4-((E)-3-(3,4,5-trimetoxyPhenylacryloyl)phenylcarbamoyl)methyl) piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3- carboxylate 5e

Hydrazide derivatives •

7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3carbohydrazide 6a

•

7-(4-Benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3carbohydrazide 6b

•

7-(4-(3,4,5)-Trimethoxypiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3-carbohydrazide 6c

•

7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-1cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3- carbohydrazide 6d

•

7-(4-((4-((E)-3-(3,4,5-TrimetoxyPhenylacryloyl)phenylcarbamoyl)methyl) piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3carbohydrazide 6e

•

(S)-9-Fluoro-3,7-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2H[1,4]oxazino[2,3,4-ij]quinoline-6-carbohydrazide 11

The second part of results and discussion: Biology section, this section is subdivided into three parts: Evaluation of anticancer activity: Anticancer activity was evaluated in vitro at the National Cancer Institute, Bethesda, MD, USA. Among the synthesized derivatives compounds 3a, 4a, 3b, 4b, 3c, 4c, 8 and 9 were selected according to the protocol of drug evaluation branch of the National Cancer Institute. The selected compounds were screened against sixty different cell lines (NCI-60 cell line panel). Screening results revealed that the selected compounds have no anticancer activity. Evaluation of anti-Proteus mirabilis activity: Anti-Proteus mirabilis activity of the target compounds 3a-e, 4a-e, and 6a-e and 811 were evaluated using P. mirabilis strain which was isolated from the urine of patients suffering from urinary tract infection. Isolation and identification were performed

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Summary

according to standard procedures of Well Diffusion method. Some of the tested compounds showed better activity than the reference fluoroquinolones. Evaluation of urease inhibitory activity: Urease inhibitory activity of the target compounds 3a-e, 4a-e, and 6a-e and 8-11 were evaluated using indophenol method. Some of the tested compounds experienced better activity than the reference acetohydroxamic acid. The third section: Molecular docking section, this section includes the results obtained from molecular docking of selected examples of the synthesized compounds on H. pylori urease enzyme. Results showed good fitting of the docked compounds on urease enzyme that agreed with biological screening results obtained. The fourth part, experimental This part outlines the detailed explanations of different experiments used, this part is subdivided into three sections: The first section: Chemistry section, this section described the different procedures used for synthesis of the target compounds 3a-e, 4a-e, 6a-e and 8-11. It also presented all detailed spectroscopic and analytical data of the synthesized compounds. The second section: Biology section, this part outlined the method and procedure used to investigate the anticancer activity, anti-Proteus mirabilis investigation and urease inhibitory activity of the synthesized compounds 3a-e, 4a-e, 6a-e and 8-11. The third section: Molecular docking, this section described the methodology and software used for molecular modeling of the selected compounds to investigate their conformation and binding mode with H.pylori urease enzyme (PDB: 1E9Y). Publications: 1. Abdullah, M. A., El-Baky, R. M. A., Hassan, H. A., Abdelhafez, E.-S. M. & AbuoRahma, G. E.-D. A. Fluoroquinolones as Urease Inhibitors: Anti-Proteus mirabilis Activity and Molecular Docking Studies. American Journal of Microbiological Research 4, 81–84 (2016).

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Summary

2. “New fluoroquinolone hydroxamic acids as antibacterial and urease inhibitors: Design, synthesis and molecular docking studies” 251st ACS National Meeting, San Diego, CA, March, 2016 (Poster Presentation).

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1. Introduction

Introduction

1. Introduction 1.1. Quinolones Quinolones constitutes an important class of synthetic broad-spectrum antibacterial agents, they have been the center of considerable scientific and clinical interest since their discovery. Nalidixic acid I, a 1, 8-naphthyridine derivative, is the prototype of this group which is developed in 1962 by Lescher and colleagues

[1,2]

during investigations of an

antimalarial agent, chloroquine.[3,4] Although nalidixic acid showed a moderate activity against Gram-negative bacteria and low absorption, it is being marketed for treatment of UTIs.[4,5] Consecutive structural modifications for quinolones were performed to develop the first fluoroquinolone derivative norfloxacin in 1978 by the Japanese Kyroin company and approved by FDA in 1986.[6] It has a broader spectrum and better pharmacokinetic profile against Gram-negative and some Gram-positive bacteria with antibacterial activities 1000 times more potent than those observed with the nalidixic acid.[7] This potent action is attributable to introduction of a fluorine atom at C-6 position and the piperazinyl group at C-7 position, which are essential for inhibition of a target enzyme DNA gyrase and better bacterial cell-wall penetration. After the discovery of norfloxacin various SAR studies have been performed to develop new fluoroquinolones compounds with better solubility, antimicrobial activity, prolonged serum half-life, less adverse effect profiles and both oral and parenteral routes of administration.[8-10] Because of their potent activity, the novel synthetic antibacterial fluoroquinolones begin a new era in the curing diverse infectious diseases and today they are beneficial for clinical use. Clinically, fluoroquinolones have been used to treat a variety of infections due to their broad spectrum of activity such as respiratory infections, gonorrhea, mycobacterial infections [11] and enteric infections.[12] Fluoroquinolone agents has classified into different four generations according to their structure modification, spectrum of activity and clinical applications.[13,14] Nalidixic acid I and cinoxacin II represented the first generation of fluoroquinolones, which were the first used fluoroquinolones but now their clinical use is limited to treatment of uncomplicated UTIs.[15,16]

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Introduction

Second generation of fluoroquinolones are widely used in urinary and respiratory tract infections nowadays, they include norfolxacin

III, ciprofloxacin IV, enoxacin V,

lomefloxacin VI.[14] The introduction of C-6 fluorine atom enhanced the spectrum of activity while introduction of C-7 piprazinyl moiety showed potent anti-pseudomonal activity.[14,17] Moreover, ciprofloxacin has good bone penetration, the orally administered ciprofloxacin is a useful alternative to parenterally introduced antibiotics for the treatment of osteomyelitis.[18] O

O

O F

HO N

O F

HO N

N

N NH

NH III O

IV

O

O F

HO N

N

O F

HO

N

N

N

NH

F

V

NH

VI

The third generation of this class currently includes levofloxacin VII, sparfloxacin VIII, and grepafloxacin IX which have a broad activity against Gram-positive bacteria, specially

penicillin

resistant

strains

of

S.

pneumonia,

mycoplasma

pneumoniae and chlamydia pneumoniae.[19,20] Furthermore, they are used in treatment of acute sinusitis and acute exacerbations of chronic bronchitis.[20]

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Introduction

Moxifloxacin X, gatifloxacin XI and trovafloxacin XII represent the fourth generation of fluoroquinolones, which are characterized by a methoxy group at position 8. Gatifloxacin XI is removed from market due to its hypoglycemic effect .[21] Moreover, moxifloxacin X is proved to be the most active quinolone tested against Mycobacterium tuberculosis (MIC90 0.25 mg/L), Mycobacterium avium-intracellulare (MIC90 1.0 mg/L), Mycobacterium kansasii (MIC90 0.06 mg/L) and Mycobacterium fortuitum (MIC90 1 mg/L).[22] Although trovafloxacin has a significant antimycobacteial activity as well as maintaining Gram-positive and Gram-negative activity,[23] it was withdrawn from market due to its high potential for inducing serious fatal liver damage.[24] O

O

O

H F

HO

F

HO

N

N

O

H

N

N O

O

NH

H HN X

XI O

O F

HO N

N

H

N

F H F XII

-3-

NH2

Introduction 1.1.1.

Mechanism of action of quinolones

Fluoroquinolones perform their antibacterial and anticancer action by inhibition of DNA replication process by disturbing the normal function of DNA topoisomerase in different ways targeting and suppressing type II bacterial Topoisomerases, DNA gyrase and Topoisomerase IV.[25] They bind to the enzyme-DNA complex and form a ternary complex then block the process of DNA replication, fig.1.[25,26] There two models describes fluoroquinolones binding to the DNA-enzyme complex one of them shows that fluoroquinolones bind to the enzyme via magnesium ion which is necessary for their antibacterial action and the other model shows that four fluoroquinolone molecules bound as two pairs of noncovalently associated drug dimers in a single-stranded DNA bubble opened up by topoisomerase action, fig. 2.[8]

Fig.1: Mechanism of action of quinolones

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Introduction Chelate to DNA Mg2+ ion Stacking with another quinolone molecule R5 R6 enzyme interaction domain R7

Hydrogen bonding to DNA bases O

O

Mg2+ R5

O

O

R6

OH

OH X

R7

N R1

X

N R1

stacking with DNA bases

Self-association

Fig.2: Binding model of quinolones. On the other hand, fluoroquinolones are found to stabilize the enzyme-DNA cleavage complex, hinder the process of relegation and induce formation of cleavage complexes by the enzyme.[27-29] Both mechanisms can increase the number of DNA breaks more than normal. Accumulation of DNA breaks in addition to the blocked replication forks lead to inhibition of cell division and even cell death by induction of apoptosis in mammalian cells.[30] On the other side, bacterial cell death seems to be due to accumulation of several mutations due to the resultant stimuli of replication errors.[31,32] Recently, it is reported that the poisoning of gyrase enzyme by quinolones is followed by the formation of reactive oxygen species, which play an important role in DNA damage and bacterial cell death.[33]

1.1.2. Biological importance of quinolones 1.1.2.1. Antibacterial Activity The ability of fluoroquinolones to target type II topoisomerases gives various advantages over other antibiotics due to various reasons. First, type II topoisomerases are involved in DNA replication, which is an important cell process that is prevalent in all types of bacteria. Second, there is a unique difference between type II topoisomerases of human and bacteria that gives selectivity to target bacterial cells over human cells. Third, the action of inhibitory target is bactericidal. Finally, fluoroquinolones have the ability to potentially inhibit two unique bacterial enzymes, thus reducing the possibility of formation of resistant mutants. [34-36]

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Introduction SAR studies have enabled the recognition of features that lead to specific changes. All fluoroquinolones are structurally related to nalidixic acid I. As shown in fig. 3, fluoroquinolones have 1,4-dihydropyridine core structure, where positions 2, 3, and 4 are essential for activity and should not be altered because they are the binding pharmacophore with DNA gyrase enzyme. [2] Hydrogen moiety at position 2 is optimal for activity and the 3-carboxylic and carbonyl group at position 4 are indispensable for activity. On the other hand, the other four positions 5, 6, 7 and 8 can receive a wide range of potential substituents.[38]

R5 F

6

R7

7

O

5

4

X 8

1

N R1

O 3

2

OH R2

Fig. 3: General structure of quinolones Position 1: Substituent at this position participates in the DNA-enzyme binding complex that binds to the hydrophobic part of DNA major grove.[39] Both the cyclopropyl and 2,4difluorophenyl groups are the most potent substituent towards antibacterial activity,[40],[41] but the 2,4-difluorophenyl is favored against anaerobic bacteria.[2] Position 2: It was found that hydrogen is the optimal for the site.[35] Bulky group at this position diminishes the antibacterial activity because it hinders the interaction between quinolones and the target enzyme.[41,42] Position 3 and Position 4: They are considered crucial for binding to DNA-gyrase complex. Hence, the 3-carboxylate and 4-carbonyl groups are considered essential for antimicrobial activity. [43] Position 5: Substitution on this position has the capacity to alter the planarity and configuration of quinolones hence it changes cell penetration and binding mode of the drug into the target.[39] Bulky substituents at this position markedly reduce activity while other substituents result in improved activity, such as amino and methyl substituents in case of sparfloxacin X and grepafloxacin XI, respectively.[44, 45]

-6-

Introduction Position 6: The introduction of a C-6 fluorine atom is the best substitution here as it markedly enhances the antimicrobial spectrum

[43,44]

and improves both the DNA gyrase

complex binding and cell penetration.[46] Position 7: It is one of the most influential ring positions that have a vital impact on quinolone biological activity.[2] The substituent at this position has a hydrophobic interaction with DNA molecules as well as a hydrogen bonding with DNA gyrase or topoisomerase IV.[2,34] In addition, the substituent at this position has a remarkable effect on the pharmacokinetics.[40,47] Generally, five or six membered ring nitrogen heterocycles has a good effect on antibacterial activity and physicochemical properties, especially aminopyrrolidines and piperazines moieties.[2] Position 8: Addition of methoxy group at C-8 positon demonstrates a potent activity against Gram-positive bacteria like in moxifloxacin X and gatifloxacin XI.[48] In addition, it also increases binding to DNA gyrase and topoisiomerase[49], enhances the antimycobacterial activity [50,51] and reduces the phototoxicity.[52] To enhance the antibacterial activity of fluoroquinolones various substitution were introduced

to

the

N-4-piprazinyl.

For

example,

a

recent

study

showed

2-

aminobenzothiazoles derivatives of N-4-piprazinyl ciprofloxacin derivatives XIII, XIV and XV with potent activity against Gram-positive and Gram-negative bacteria more than its parent ciprofloxacin. [53]

-7-

Introduction Moreover, the introduction of functionalized thienylethyl oxime moieties into N4-piprazine ring of levofloxacin, compounds XVI and XVII, improve the antibacterial activity against both Gram-positive and Gram-negative bacteria.[54]

Another example, introduction of 3,5 dichlorophenethyl-N-4 piperazinyl oxime moiety into the N-7-piprazine of ciprofloxacin, compound XVIII, enhances the antibacterial activity against multi drug resistant S. aureus (MRSA).[55] O

O F

HO N

N

N

OCH3 Cl

N

Cl

XVIII

1.1.2.2. Anticancer activity DNA topoisomerase enzymes are vital for cell growth and proliferation for eukaryotics.[56-58] Furthermore, a structure similarity has been noticed between human and bacteria topoisomerases.[59] Human topoisomerases are possible targets for several chemotherapeutic agents, camptothecin XIX is an example for well-known topoisomerase type I inhibitors that prevent religation of cleaved DNA strands and trigger apoptosis preferentially during cell division.[60,61] In addition, the indenoisoquinoline derivative (NSC 7247760) XX is a promising topoisomerase I inhibitor under clinical trials.[62] Moreover, topoisomerases type II is well known as potential enzymatic targets for anticancer candidates. The known topoisomerase II inhibitor, doxorubicin XXI

[23,63]

can

obstruct the catalytic function of topoisomerases II either by ambushing the enzyme in

-8-

Introduction complex with crackled DNA via avoiding the process of relegation and by enhancing the formation of cleavable complexes.[64]

Introduction of substituted oxime derivatives into N-4 piprazinyl of ciprofloxacin and norfloxacin is reported to enhance their anticancer activity. Ciprofloxacin and norfloxacin

were

functionalized

at

the

N-4piperazinyl

with

2-(furan-2-yl)-2-

oxyiminoethyl XXII and 2-(thiophen-2-yl)-2-oxyiminoethyl moieties XXIII. The synthesized compounds showed anticancer activity more than the reference, the known topoisomerase II inhibitor anticancer drug etoposide.[65]

X = O, -NOH, -NOCH3 and –NO=CHPh; R = Ethyl or cyclopropyl.

The oxime derivatives of methylene-γ-butyrolactone and hydroxyiminoethyl derivatives of norfloxacin showed significant cytotoxic and antibacterial activities. Compounds XXIV and XXV has potent cytotoxic derivatives while compound XXVI has potent antibacterial one.[66]

-9-

Introduction

A series of N-4-piperazinyl ciprofloxacin chalcone derivatives was synthesized and evaluated for in vitro anticancer activity. Compound XXVII was the most potent derivative in this group. Some of the tested compounds showed remarkable inhibitory activity against both topoisomerase I and II enzymes comparable to that of camptothecin and etoposide as topoisomerase I and II inhibitors, respectively. [47]

H3CO

OCH3 OCH3 O

O

F O

O

N

OH N

N

N H

XXVII

A series of tetracyclic fluoroquinolones were synthesized and screened against various cancer cell lines. Compound XXVIII showed a potent anticancer activity against breast cancer cell lines more than the reference drug ellipticine XXIX.[42]

-10-

Introduction 1.1.2.3. Antiviral activity Some quinolone derivatives have potential antiviral activity.[67] Elvitegravir XXX (also known as GS-9137), a quinolone derivative, has significant HIV-1 integrase inhibitory action and reached advanced stages of clinical trials for treatment of AIDS.[6870]

Moreover, a series of 6-aminoquinolones were developed with several modifications

of N-1, C-3 and C-7 positions as anti-HCV agents. In particular, compound XXXI emerged as one of the most potent NS5B inhibitors and the most selective anti-HCV derivative within this series.[71] F

O

O

O

Cl

O

H2 N

O

OH O

N

N N

N

Cl

N Cl

OH XXXI

XXX

1.1.2.4. Antimycobacterial activity Fluoroquinolones reduce the total treatment duration of tuberculosis (TB) in comparison with the known antimycobacterial agent, rifampicin, lowest

MIC

against

M.Tuberculosis.[73]

Among

the

[72]

since they have the

commercially

available

fluoroquinolones, sparfloxacin has the most inhibi.tory activity against M.Tuberculosis DNA gyrase enzyme.[61] Moreover, ciprofloxacin shows a good antimycobacterial activity against M.ulcerans.[74] A clinical study showed that moxifloxacin X and gatifloxacin XI are effective in four months regimen for treatment of tuberculosis.[75,76] On the other hand, levofloxacin VII is considered the most favorable fluoroquinolone in TB management due to its long term safety profile.[77] Senthilkumar et al., has reported a variety of fluoroquinolone derivatives with diverse substituents at N-1 and C-7 positions.[78] Some of these compounds experienced moderate to good activity against M. tuberculosis and multi drug resistant tuberculosis MDR-TB. Compounds XXXII and XXXIII have excellent in vitro activity for M. tuberculosis and MDR-TB.[79,80]

-11-

Introduction O F N

O

O OH

N

O2 N O

OH

N

N

N

O

N(C2H5)2

XXXIII

XXXII

-12-

O

Introduction

1.2. Urease inhibitors Urea was the first organic molecule synthesized in laboratory by Wöhler reaction from ammonium cyanate.[81] In 1926, James B. Summer, at Cornell University was the first scientist who described that urease is a protein by testing its crystallized form[82] He was the first one to explain that a pure protein can function as an enzyme and he was awarded the Nobel prize in chemistry in 1946.[83] The structure of urease was first solved by P. A. Karplus in 1995.[84] Urease enzyme or urea aminohydrolase (E.C.3.5.1.5) is a multi-subunit nickel dependent metalloenzyme that catalyzes the hydrolysis of urea at a rate approximately 1014 times the rate of the un-catalyzed reaction[85] to ammonia and carbamic acid. The latter compound instinctively decomposes to yield another molecule of ammonia and carbonic acid, which is the final step of urea catabolism as shown in fig 4.[86] O

H2N

NH2

+

H2O

Urease

NH3 +

H2N

COOH

Urea H2N

COOH + 2H2O

NH3 + H2CO3

In aqueous media: H2CO3 2NH3 + 2H2O

H+

+ HCO3

2NH4+ +

2OH-

Fig.4: Hydrolysis of urea by urease enzyme Urease is widely distributed among different types of bacteria.[87-89] Several of these microorganisms utilize urea as a source of nitrogen.[90,91] Hydrolysis of urea leads to sudden pH elevation due to increasing ammonia concentration which has bad effects on human health.[92] Urea splitting bacteria belongs to both symbiotic natural flora or to pathogens.[93] Urease is well known as the major bacterial virulence factors during urinary tract infections caused by pathogenic bacteria.[93] This feature is characteristic to pathogenic bacteria Staphylococcus strains.[94] Moreover, ureolytic activity is detected in

-13-

Introduction Helicobacter sp. obtained from gastritis patients.[95,96] In addition, Proteus mirabilis is a widely known ureolytic human’s pathogen.[97] Urease produced by some microorganisms is implicated in the pathogenesis of a number of diseases.[98] Ureolytic activity lead to several implications such as formation of infection stones, which are generated by supersaturation of soluble polyvalent ions such as Ca2+ and Mg2+, when the pH increases from 6.5 to 9.0 due to urease activity.[99] In humans, Proteus mirabilis is the most common organism implicated in stone formation.[100] Moreover, urease enzyme is responsible for pyelonephritis, where Proteus mirabilis is the primary source of this disease in humans.[97] In addition, the ammonia liberated from ureolysis causes the alkalinisation of urine and may account, in part, for the necrosis of kidney tissue.[101] Until now crystallographic structure for limited number of bacterial ureases has been reported. However, amino acid sequence of all investigated ureases is analogically maintained, which shows presence of two Ni ions connected by the carboxylate group of the carbamylated lysine and coordinated by some surrounding histidine and aspartic residues that implies a common catalytic pathway fig. 5.[102]

Fig.5: Urease active site

-14-

Introduction Number of urease subunits is varied according to the sources of microorganism. Some bacterial ureases consist of three subunits, one big (α, 60–76 kDa) and two small (β, 8–21 kDa and γ, 6–14 kDa) such as Klebsiella aerogenes

[84]

and Bacillus pasteurii

[103]

ureases, fig. 6. On the other hand, it was reported four subunits for Staphylococcus saprophyticus

[104]

(αβγ)4 and five subunits (αβγ)5 of Staphylococcus leei urease.[105]

Conversely, the ureases of Helicobacter species consists of two subunits. [107-109]

Fig. 6:The structures of three well-characterized ureases. (A) K. aerogenes (PDB:1fwj), (B) H. pylori (PDB:1e9z), (C) Jack bean urease (PDB 3LA4).[106] Although this difference in number of subunits, a homology is showed in amino acid sequences. This proves that all ureases are evolutionary variants of same ancestral enzyme. In order of importance, it is known that the active site of urease from all origins is located in the α subunits. [110] A wide range of naturally isolated and synthetic urease inhibitors has been investigated in the literature through pharmacological importance in the bacterial infections. It has attracted the attention of the scientific community to explore new resourceful urease inhibitors. The urease inhibitors have been investigated for the treatment of bacterial infections and as well for the immoderate urea breakdown in soil, hence studies on the potent and specific inhibitors have been an in-action area of research. Taking the consideration of the great potential of urease in medicine, many classes of urease inhibitor have been designed in the latest years [70,111,112]

-15-

Introduction 1.2.1. Classification of Urease inhibitors Although, a variety of urease inhibitors have been demonstrated in the past, such as hydroxamic acid derivatives,

[113]

imidazoles

[114]

and phosphorodiamidates[115], a lot of

them are banned from using in vivo because of their toxicity or instability and another part of them had severe side effects. So, it is crucial to synthesis new potent urease inhibitors with good stability, bioavailability and low toxicity. The known urease inhibitors are either organic compounds such as hydroxamic acid analogs, phosphoramide compounds and thiol derivatives or metal complexes. 1.2.1.1. Hydroxamic acid derivatives Hydroxamic acid derivatives are among the well-studied urease inhibitors due to their ability to coordinates with the two nickel (II) atoms linked by a carbamate bridge in the active site.[102,116] Since the first determination of X-ray structure of acetohydroxamic acid enzyme complex was by Stemmler in 1995.[117] The hydroxamic acids (HAs) were categorized as potent and specific inhibitors of urease from various plants and bacterial origins by Kobashi and co-workers.[118-120] Acetohydroxamic acid (AHA) XXXIV is known as potent urease inhibitor with a weak antibacterial activity.[121,122] Stoichiometrically, two moles of hydroxamic acid can inhibit one mole of urease. [123]

Moreover, AHA shows a clinically significant growth inhibition of struvite stones in

the short term in patients infected with urea splitting bacteria.[124] AHA was approved by the FDA in 1983 to treat chronic urea-splitting urinary infections under the name of Lithostat® and in Europe it was introduced as Uronefrex®.[125] AHA has negative side effects such as inhibition of DNA synthesis, bone marrow depression, teratogenicity, hemolytic anemia, and liver dysfunction. [70,126] O HO

N H

CH3

XXXIV

During the last few decades, various HA derivatives of aliphatic, aromatic, amino acids, and dipeptidyl derivatives have been investigated, which has the ability to inhibit urease of Jack bean and Proteus mirabilis with low IC50 values (e.g., compounds XXXV-

-16-

Introduction XL).[127-130] N-aroylglycino-hydroxamic acid and glycoside hydroxamic acids series XL and XLI were well-studied, resulting in active compounds with IC50 values in the range of 0.5–2.0 µM and useful for treating urolithiasis and pyelonephritis, but most of them were mutagenic.[131],[132] O O

O N H

XXXV

OH XXXVI

NH2 O N H

N H

N H

OH O

XXXVII

O

OH N H

XXXVIII

O N H

OH

H N

HN OH

XXXIX

O N H

O

H N

O XL O

O HN OCH3 HN OH

OH

N H

OH

O O

OH OH

OH OH XLI

XLII

Another hydroxamic acid derivative of purine XLIII and indazole XLIV were reported with potent urease inhibitory activity. [116]

-17-

Introduction 1.2.1.2. Phosphoramide derivatives Phosphoramide compounds are another class of potent inhibitors of the urease enzyme, this class of inhibitors comprise of a large number of simple and complex compounds. In 1986, Andrews et al., reported that diamidophosphate derivatives XLVXLVII interacted with active site of jack bean urease and inhibit its action. Moreover, they showed potent urease inhibition activity. [133]

Mainly

the

less

complex

examples

include

phosphoramidates

and

diamidophosphate with substituted phenylphosphoradiamidates, and a range of N-acyl phosphoric triamidates that showed potent urease inhibition.[134] 1.2.1.3. Thiol derivatives Thiol derivatives are well-known with its strong inhibition action of the urease enzyme. This includes cysteamine XLVIII, which has a β-amino group with high affinity for the urease (Ki = 5 μM). The pH dependence studies demonstrate that the actual inhibitor is the thiolate anion. [135] H2N SH

XLVIII

It is reported that thiobarbiturates derivatives shows a great inhibitory activity against urease enzyme. Compound XLIX and L have IC50 of 8.61 μM and 7.45 μM, respectively. [136]

-18-

Introduction

Another thiourea based urease inhibitors LI and LII were reported with free anilino nitrogen of sulfanilamide has excellent urease inhibitor activity than the parent thiourea.[137] O Cl

S HN HN

LI

SO2NH2

Cl O S HN O 2N

HN

SO2NH2

LII

Moreover, thiourea phenyl derivatives LIII and LIV showed high percentage of urease inhibition more than thiourea.[138]

1.2.1.4. Metal Complexes A wide range of metal complexes investigated with good urease inhibitory activities are reported which includes heavy metal ions, such as Cu2+, Zn2+, Pd2+ and Cd2+.[139,140] It is reported that the relative efficacy of metal ions as urease inhibitors decrease in the following approximate order [141]

-19-

Introduction Hg2+>Ag+>Cu2+>Ni2+>Cd2+>Zn2+>Co2+>Fe3+>Pb2+>Mn2+ It is also reported that metal complexes of organotin(IV), vanadium(IV), bismuth(III), copper(II), and cadmium(II) has good urease inhibitory activities.

[113,142-145]

A Schiff based metal complex of manganese III compound LV is reported with significant urease inhibition than AHA.[146] Furthermore, Schiff base copper complexes compounds LVI and LVII were synthesized and tested against urease activity. They showed a potent inhibitory activity than the reference AHA. [147]

H3CO

N OHN2 Mn O O NCS OCH3

LV

O N Cu O N

O Br

N O Cu O N

Br

O

LVII

LVI

1.2.1.5. Fluoroquinolones as urease inhibitors Urease inhibition is another observed target for fluoroquinolones on urea splitting bacteria. Norfloxacin, in sub-inhibitory concentration 0.5 MIC, shows an inhibition on urease enzyme in vitro. It inhibits about 43.3 % of Proteus mirabilis urease activity.

[148]

Moreover, aluminum, arsenic, silver and lead metal complexes of sparfloxacin show a better urease inhibitory activity than thiourea.[149] Furthermore, silver moxifloxacin nanoparticles shows a remarkable activity against urease activity.[150] 1.2.1.5. Other urease inhibitors Other non hydroxamic non-thiols and non-metal complex have a potent urease inhibitory activity more than AHA. Metronidazole derivatives compounds LVIII and LIX show a percent of inhibition greater than AHA for H.pylori urease and their action is

-20-

Introduction attributed to the hydrogen bonding between oxygen of nitro group and His221 as described in molecular studies.[151]

Also some natural products were found to exhibit urease inhibitory activity. Quercetin LX, a flavonoid isolated from Lonicera japonica , demonsterated a potent urease inhibition more than AHA and its action is attributed to a hydrogen bond between 5-OH and Cys321, which is key residue in urease catalytic activity.[152] OH HO OH

O HO

5

O LX

OH

Furthermore, β-bowsellic acid derivative LXI isolated from Bowsellina Caterii, showed a stronger inhibitory activity agsainst jack bean urease (IC50=6.7 μM) than the reference thiourea (IC50 = 21.1 μM).[153]

-21-

2. Aim of the work

Aim of the work

2. Aim of the work Urease enzyme catalyzes hydrolysis of urea into carbamate and ammonia that is the final step of urea catabolism.[70] It is widely distributed among different types of bacteria such as Proteus mirabilis, which is a causing microorganism of urinary tract infections.[154,155] The role of bacterial urease as virulence factor of some bacterial pathogens is well established including formation of infection stones, pyelonephritis, peptic ulceration, hepatic encephalopathy and other diseases.[156] Development of infection stones, which is precipitated in alkaline media caused by ammonia as a result of urea breakdown is a urease-dependent process that is caused by Proteus and Kelibsiella species during urinary tract infection.[157,158] Moreover, urease plays a critical role in Helicobacter pylori acclimation in acidic environment of stomach in response to the acidic media of the periplasm Helicobacter pylori.[159,160] Although, urease inhibitors are essential to overcome the pathogenesis of bacterial urease, the currently reported compounds including the commercially-available

AHA[161]

and

other

hydroxamic

acid

derivatives,[115]

imidazoles,[116] phosphorodiamidates,[117] and are mostly unstable and may cause toxic side effects. Consequently, it is important to synthesize new potent urease inhibitors with good stability, bioavailability and low toxicity.[162] Hydroxamic acid derivatives has several biological activity due to their ability to chelate various metal ions bidentately and form a strong hydrogen bond.[163.164] Hence, they act as enzymatic inhibitors to different metal containing enzymes such as histone deacetylase,[165] TNF-α converting enzyme,[166] urease[167] and carbonic anhydrase, etc..,[168] they target a wide range of enzymes, So hydroxamic acid derivatives are used in treatment of variety of diseases such as cancer,[169] cardiovascular diseases,[170] Alzheimer’s,[171] malaria[172] ,etc... In addition, some hydroxamic acid derivatives are reported in the literature as effective antibacterial agents such as scorbic hydroxamic acid LXII and decanoyl hydroxamic acid LXIII. [173] O

O N H

OH

N H

LXII

LXIII

-22-

OH

Aim of the work Moreover, fluoroquinolone is a class of synthetic, broad-spectrum antibacterial agents used in treatment of urinary and respiratory tract infections.[174] In addition, norfloxacin[150] and some sparfloxacin metal complexes have a potent urease inhibition.[151] On the other hand, the substituent at the N-4 piperazinyl moiety of fluoroquinolones can reduce the zwitter ion formation properties.[40,55]

and hence has a remarkable effect on the physicochemical

Furthermore, recent reports have showed the potential activity of

fluoroquinolones as anticancer agents.[175] It is also reported that substitution at position 7 of fluoroquinolone is directly interacts with topoisomerase enzyme in enzyme-drug-DNA ternary complex, and shifts the activity of quinolones from antibacterial to anticancer.[176, 177]

Based on the above findings, this research aims to: 1- Synthesize of new ciprofloxacin and levofloxacin derivatives through replacing the C-3 carboxylic acids with hydroxamic acid, hydrazide and amide moieties. At the same time, the N-4-piprazinyl of ciprofloxacin was substituted with alkyl or acyl moieties, fig. 7. 2- Elucidate Structures of the synthesized compounds using different spectral analysis methods including IR, 1H NMR, 13C NMR and HRMS. 3- Screen of the anticancer activity of some of the final target compounds. 4- Screen of the anti-Proteus mirabilis activity of the final target compounds using Well diffusion method. 5- Screen of the urease inhibitory activity of the final target compounds using Indophenol method 6- Molecular modeling studies on representative examples of the target compounds along with H. Pylori urease (PDB:1E9Y) enzyme using Molecular Operation Environment program MOE® software.

-23-

Aim of the work O

O

O

F

R

F

X

N

N

N

N

O

3a-e: X=NHOH 4a-e: X=NHCH3 5a-e: X=NHNH2

8: X=NHOH 9: X=NHCH3 11: X=NHNH2

H3CO

O a: R =

X

N

N

O

b: R =

H3C

O

c: R =

O

H3CO H3CO

H N

OCH3

O

H N

e: R = H3CO

d: R =

H3CO

O

O

Fig.7: The target hydroxamic, amide and hydrazide derivatives of fluoroquinolones.

-24-

O

3. Results and discussion

3.1. Chemistry

Results and discussion: Chemistry 3.1. Chemistry 3.1.1. Synthesis of hydroxamic acid 3a-e, amide 4a-e, and hydrazide 6a-e derivatives of ciprofloxacin To prepare the target compounds of ciprofloxacin derivatives 3a-e, 4a-e and 6a-e scheme 1, it is necessary to prepare the starting carboxylic acid derivatives 1a-e as outlined in Schemes 2 and 3. O

O

F

N H N

N R

OH

N 2a-e NH2OH.HCl

4 hr O F

OH

N R

N

O Et

O

O

O

F

Cl

0-5 0C, TEA, CH2Cl2

N

CH2Cl2

O

O

N R

1a-e

O

O O

C2H5

CH3NH2

N

CH2Cl2, 4 hr

N

O

F

R

N

N H

CH3

N

N 3a-e

4hr

CH3OH

O

O

N R

O

O

F

F

CH3 NH2NH2, 80%

N

N

Ethanol, refluxr, 2hr

N

R

4a-e

N H

NH2

N

N

5a-e

O a: R = H3C

O

O

b: R =

H3CO c: R =

H3CO

O

H3CO H N d: R =

O

e: R = H3CO

OCH3

H3CO

O

H N

O

O

Scheme 1: Formation of ciprofloxacin un-isolated mixed anhydrides 2a-e and synthesis of ciprofloxacin derivatives 3a-e, 4a-e and 6a-e Treatment of ciprofloxacin in DCM with the respective acid chloride using TEA as a base in ice bath (0-5) oC afforded the acetylated derivative 1a-c in good yield. The prepared compounds were identified with their melting point as reported. [178]

-25-

Results and discussion: Chemistry O O F

O

O R

OH

N

N

O

F Cl , DCM

TEA, (0-5) oC, 6 hrs

HN

OH

N R

N

N O 1a-c H3CO

1a: R =

CH3

1b: R =

1c: R =

H3CO H3CO

Scheme 2: Synthesis of compounds 1a-c On the other hand, ciprofloxacin chalcone derivatives 1e-d were prepared as reported

[47]

in three steps, first reaction of p-aminoacetophenone with the respective

benzaldehyde in basic medium using Claisen-Schmidt condensation [180,181] that afforded the corresponding chalcone derivatives A1 and A2, Scheme 2. Acylation of chalcone derivatives was carried out with bromoacetyl bromide in presence of potassium carbonate to afford intermediates B1 and B2, Scheme 2. The prepared compounds B1 and B2 were identified with their reported melting point. Compound B1 (reported mp = 156-158 oC, found 155-157 oC) [47], compound B2 (reported mp =167-169 oC, found 166-168 oC).[47] Finally, ciprofloxacin was alkylated with derivatives B1 and B2 in acetonitrile in presence of TEA to obtain compounds 1e and 1d, respectively as showed in scheme 3. The structure of the prepared ciprofloxacin chalcone was identified using m.p., compound 1e (reported m.p = 270-273 oC, found 270-273 oC) [47], compound 1d (reported m.p = 273275oC, found 272-274oC).[47]

-26-

Results and discussion: Chemistry

+

H2N

O

NaOH

O

R Br

F

O HN

HN

N

OH N

Acetonitrile, TEA Reflux, 2 hrs

O Br

K2CO3, DCM

O A1, A2

O O

O B1,B2

Br

Ethanol 95 % R 2 hrs

H

R

H2N

O

O

F

O

OH

N

NH

R

O

N

N 1d, 1e

A1, B1: R = H A2, B2: R =3,4,5 -tri-OCH3

Scheme 3: Synthesis of compounds 1d and 1e The un-isolated ciprofloxacin mixed anhydride [182] derivatives 2a-e were prepared as a core step to prepare the target hydroxamic acids 3a-e, amides 4a-e and esters 5a-e. Mixed anhydride intermediate is formed by treating ciprofloxacin derivatives 1a-e in DCM with ethyl chloroformate in ice-bath (0-5) oC using TEA as a base

[182]

, Scheme 4. The

mechanism of this reaction is proposed by nucleophilic attack of the lone pair of electron of the carboxylate anion on the carbonyl of the ethyl chloroformate that lead to the cleavage of carbonyl chloride bond and forming the mixed anhydride as showed in Scheme 5.

Scheme 4: Formation of ciprofloxacin un-isolated mixed anhydrides 2a–e

-27-

Results and discussion: Chemistry O R

O

TEA OH

R

O O-

Cl

O

-TEA.HCl OEt

R

O O

OEt

Scheme 5: Reaction mechanism for formation of mixed anhydride 3.1.1.1. Synthesis of N-4 (substituted) piprazinyl ciprofloxacin hydroxamic acids 3a-e To prepare the target hydroxamic acid derivatives 3a-e, the un-isolated mixed anhydride of ciprofloxacin derivatives 2a-e was treated with solid hydroxylamine hydrochloride in ice-bath (0-5) oC and TEA as a base to obtain the hydroxamic acid derivatives [182] 3a-e, Scheme 6, the reaction mechanism is explained by nucleophilic attack of the lone pair of electrons on the carbonyl of mixed anhydride results in cleavage of oxygen carbonyl bond that forms the hydroxamic acid, Scheme7. The mixed anhydride method is widely used as one pot reaction for peptide synthesis.[183] Preparation of hydroxamic acid by mixed anhydride method is mild, efficient and provide good yield. In addition, the reaction occurs in one step in neutral medium. The mixed anhydride method is considered better than formation of hydroxamic acid by reaction of carboxylic esters with hydroxyl amine that occurs in alkaline medium. [184] The free N-4 secondary amine of piprazine moiety should be substituted to prevent formation of ciprofloxacin dimer.[185] The prepared hydroxamic acid requires special precautions and handling, it should be stored in refrigerator in dry conditions.

-28-

Results and discussion: Chemistry O

O

F

OH

N R

O F

N

TEA, ClCOOEt CH2Cl2, ( 0 - 5) oC

N

N R

O

O

H N

O

C2H5

N

2a-e

H3CO

O H3C

O

N

1a-e

a: R =

O

O

b: R =

O

c: R = H3CO H3CO

H N d: R =

O

OCH3 e: R = H3CO H3CO

O

O

Scheme 6: Synthesis of ciprofloxacin hydroxamic acid derivatives 3a-e

Scheme 7: Reaction mechanism for hydroxamic acid preparation The structures of newly prepared hydroxamic acid derivatives 3a-e were identified by various spectroscopic techniques such as IR, 1H NMR,

13

C NMR, and high resolution

mass spectroscopy. The IR spectroscopy showed a broad band at ~3300-3500 cm-1 of NHOH, in addition to carbonyl of CONH band between ~1690-1650 cm-1 and the C-4 carbonyl band at ~1622-1627 cm-1. In the aceylated derivatives, 3a-c the acyl carbonyl appeared between ~1670-1690 cm-1. Moreover, the carbonyl of acryloyl moiety at derivatives 3d-e appeared at ~1640-1650 cm-1. The 1H NMR spectrum of hydroxamic acid derivatives 3a-e (compound 3c as example, fig.8) showed disappearance of carboxylic proton at about δ~15 ppm and appearance of a singlet signal at δ ~ 9-10 ppm characteristic for OH and more deshielded signal of NH appeared at ~11-12 ppm. Moreover, The H-2 proton appeared as singlet at ~8.60 ppm that is more upfield than the H-2 of ciprofloxacin

-29-

Results and discussion: Chemistry by about ~ 0.3 ppm, this shift may be attributed to the decrease in efficiency of conjugation between the lone pair of N-1, the π electrons between C2-C3 and the carbonyl C-4 and hydroxamic acid carbonyl owing to the lower electronegativity of nitrogen than oxygen. The H-5 appeared as doublet signal at δ ~7.90 ppm with o-coupling with fluorine of JH-f = 12-14 Hz. Similarly, the doublet signal of H-8 appeared at ~7.50 with m-coupling with fluorine of J = 6-8 Hz. Also, the eight piprazinyl protons appeared as one or two multiplets between δ ~ 3.80-3.10 ppm and the multiplet signal of CH cycloproply appeared at δ ~4.20-4.10 ppm on using DMSO-d6 and at δ ~3.80-3.50 ppm in CDCl3. The two characteristic multiplets of cyclopropyl two methylenes appeared at δ ~1.30-1.20 ppm and δ ~1.20-1.10 ppm. Meanwhile, the characteristic singlet of CH3-N in acetylated compound 3a appeared at δ ~2.10 ppm and aromatic protons multiplet of compound 3b appeared at ~7.50 ppm. In addition, the characteristic singlet of the two equivalent aromatic protons of compound 3c appeared at δ ~6.60 ppm and the 9 protons of methoxy group appeared as two singlets at δ ~3.80 ppm and δ ~3.70 ppm. The 1H NMR of chalcone derivatives 3d and 3e showed a singlet signal at δ ~3.80 ppm corresponding to CH2-NHCO. Moreover, the coupling constant J of the acrylolyl CH=CH is 14-16 Hz that identify the E-isomer of the acryloly moiety. The

13

C NMR spectra of hydroxamic acid derivatives 3a-e (compound 3c as

example, fig.9) showed a signal at δ ~ 165 ppm corresponding to CO-NHOH and a signal at δ ~175 ppm of carbonyl characteristic for

C-4 carbonyl. The piprazinyl carbons

appeared as two or three or four signal between δ 50-41 ppm, The cyclopropyl moiety appeared as two signal one between δ ~36-34 ppm characteristic to the CH-N and the other appeared as one signal at δ 8.50 ppm corresponding to the two methylene carbons. In addition, the characteristic carbonyl signal of aceylated compounds 3a-c appeared at δ ~170 ppm. The carbon of the methoxy of compound 3c appeared at δ ~56 ppm. The CH2NHCO of compound 3d-e appeared at δ ~ 60 ppm. In addition, four carbonyl group appeared in chalcone spectrum; carbonyl of the acryloyl carbonyl in compound 3d-e appeared downfield shifted at δ~ 188-185 ppm, and the amidic carbonyl CONH appeared at ~168 ppm. The HRMS is used to confirm the structure of the hydroxamic acid derivatives, (HRMS for compound for 3c, [C27H29FN4O7]

-30-

+

[M]+ calculated: 563.1912,

Results and discussion: Chemistry Found: 563.1915 fig.10). The high resolution mass spectroscopy (MALDI-TOF) technique is used rather than other techniques to ensure appearance of the mild unstable hydroxamic acid moiety. [186]

Fig.8: 1H NMR (400 MHz) (DMSO) spectrum of compound 3c

-31-


Fig.9: 13C NMR (100 MHz) (CDCl3) spectrum of compound 3c.

-32-


Fig.10: HRMS (MALDI-TOF) spectrum of compound 3c.

-33-

Results and discussion: Chemistry 3.1.1.2 Synthesis of N-4 (substituted) piprazinyl ciprofloxacin amides 4a-e The un-isolated mixed anhydride of ciprofloxacin derivatives 2a-e was treated with methyl amine to afford the methyl amide derivative 4a-e as showed in Scheme 8. The mechanism of the reaction is proposed to be via nucleophilic attack of the lone pair of electrons of methyl amine on the carbonyl of mixed anhydride that results in cleavage of oxygen carbonyl bond to form the amide, Scheme 9.

Scheme 8: Synthesis of ciprofloxacin amide derivatives 4a-e O EtO

O O

CH3 N+ H

O R

R O

NH2CH3 EtO

O R

H

O CH3 + N EtO H

OH

O-

Scheme 9: Reaction mechanism for formation of amides 4a-e The structural formula of the newly prepared N-methyl amide derivatives 4a-e were identified by various spectroscopic techniques such as IR, 1H NMR, 13C-NMR and HRMS. The IR spectroscopy showed a broad band at ~3300 cm-1 characteristic for the amidic NH, and the amidic carbonyl band at ~1650 cm-1. The 1H NMR spectra of compounds 4a-e

-34-

Results and discussion: Chemistry (compound 4b as example, fig.11) showed the characteristic doublet quartet pattern of CH3-NH- with J = 4.8 Hz, where the amidic NH appeared as quartet at δ ~ 9.8-9.7 ppm and the methyl protons appeared as doublet at δ ~ 2.90 ppm. Moreover, the H-2 proton appeared as singlet at δ ~ 8.75 ppm. The H-5 appeared as doublet signal at δ ~7.90 ppm with o-coupling with fluorine with JH-f = 12-14 Hz. Similarly, the doublet signal of H-8 appeared at δ ~7.30 with m-coupling with JH-f = 6-8 Hz. In addition, the eight piprazinyl protons appeared as one or two multiplets between δ ~ 3.90 and 3.20 ppm and the characteristic multiplet signal of cycloproply CH appeared at δ ~ 4.20-4.10 ppm at DMSOd6 or at δ ~ 3.80-3.60 ppm in CDCl3. Moreover, the two characteristic multiplets of the two methylenes of cyclopropyl appeared at δ ~1.30-1.20 ppm and δ ~1.20-1.10 ppm. In compound 4a, the singlet signal of CH3-CO- appeared at δ ~2.10 ppm. The aromatic protons of compound 4b appeared as multiplets signals at δ ~7.40 ppm. The characteristic singlet signal of the two aromatic protons of compound 4c appeared at δ ~6.60 ppm and the protons of the three methoxy groups appeared as two singlets at δ ~3.85 ppm and δ ~3.70 ppm. The 1H NMR of chalcone derivatives 4d-e showed a singlet signal at δ ~3.40 ppm corresponding to CH2-NHCO. Moreover, the coupling constant J of the acrylolyl CH=CH is calculated to be 14-16 Hz that identify the E-isomer. The

13

C NMR spectrum of compounds 4a-e (compound 4b as example, fig.12)

showed a signal characteristic for CH3NH- at δ ~ 25 ppm, and another signal at about ~ 165 ppm that corresponds to the amidic carbonyl CO-NH-. In addition, the downfield shifted signal at δ ~ 175 ppm corresponds to C-4 carbonyl. Moreover, the piprazinyl carbons appear either four or two signals at about ~ δ 50 and ~ 45 ppm. The CH of cyclopropyl appeared at δ ~ 36-34 ppm and the cyclopropyl methylene appeared as one signal at δ ~ 8.50 ppm. In addition, the signal of carbonyl of aceylated compounds 4a-c appeared at δ ~ 168 ppm. The CH3CO- in compound 4a appeared at δ ~ 21 ppm and the methoxy groups of compound 4c signals appeared at δ ~ 60 and 56 ppm. Compounds 4d and 4e showed characteristic signal at δ ~ 61 ppm for -CH2NHCO. In addition, a characteristic signal of the acryloyl carbonyl appeared at δ ~ 188-185 ppm and the carbonyl of CONH-CH2 appeared at δ ~ 168 ppm. HRMS is used to confirm the structure of the amide derivatives. The HRMS (MALDI-TOF) for compound 4b fig. 13 showed that

-35-

Results and discussion: Chemistry the molecular ion peak for [C25H25FN4O3Na]+ [M+Na]+ calculated: 471.1803, found: 471.1809.

Fig.11: 1H NMR spectrum (300MHz) (CDCl3) for compound 4b.

-36-


Fig.12: 13C NMR spectrum (75 MHz) (CDCl3) for compound 4b.

-37-


Fig.13: The HRMS spectrum for compound 4b.

-38-

Results and discussion: Chemistry 3. Synthesis of N-4 (substituted) piprazinyl ciprofloxacin hydrazides 6a-e The target hydrazides 6a-e were prepared by treatment of the un-isolated mixed anhydride of ciprofloxacin derivatives 2a-e with methanol to form the methyl ester intermediates, Scheme 10. The reaction mechanism for ester formation is proposed to be via attacking the carbonyl of mixed anhydride with the lone pair of electrons of methanolOH that leads to cleavage of carbonyl oxygen bond and formation of methyl ester, Scheme 10. The methyl ester derivatives 5a-e were heated under reflux with hydrazine hydrate (80%) in ethanol to obtain the respective hydrazide derivatives 6a-e, Scheme 11. The reaction mechanism is proposed to be through attacking the carbonyl of ester by lone pair of electrons of hydrazide leading to cleavage of carbonyl methoxy bond to form the hydrazide, Scheme 12. The structural formula of the reported methyl ester 5a was identified by reported melting point. [187] O F N R

O

O

O O

O

F

C2H5

N

CH3OH

O

N

DCM, 4hrs R

N

O

O F

CH3 NH2NH2.H2O

N

Ethanol 95%

N

5a-e

2a-e

O

b: R =

a: R = H3C

R

N H

NH2

N

N

6a-e H3CO

O

N

O

O

c: R = H3CO H3CO

H N d: R =

O

e: R = H3CO

H3CO

O

OCH3

H N

O

O

Scheme 10: Synthesis of ciprofloxacin methyl ester derivatives 5a-e and its hydrazide derivatives 6a-e

Scheme 11: Reaction mechanism of formation of methyl ester derivatives 5a-e

-39-


O +

R

OCH3 NH2 NH2

-H

-

O

R

H N

NH2 OCH3

O -OCH3

R

N H

NH2

Scheme 12: Reaction mechanism of preparation of hydrazide derivatives 6a-e The structural formula of the newly prepared methyl esters 5a-e (compound 5b as example, fig. 14) were identified by various spectroscopic techniques such as IR, 1H NMR, 13

C NMR and high resolution mass spectroscopy. The IR spectroscopy showed a band at

~1730 cm-1 corresponding to the carbonyl ester. The 1H NMR spectra showed the characteristic methyl ester signal -COOCH3 as singlet at δ ~ 3.90 ppm. Moreover, The H-2 proton appeared as singlet between δ ~8.70-8.50 ppm. The H-5 appeared as doublet signal at δ ~ 8.00 ppm with o-coupling with fluorine and coupling constant JH-f

=

12-14 Hz.

Similarly, the doublet signal of H-8 appeared at δ ~7.30 with m-coupling with fluorine and coupling constant J = 6-8 Hz. The eight piprazinyl protons appeared as one or two multiplets between δ ~ 3.70 and 2.90 ppm and the multiplet signal of CH cycloproply at at δ ~3.70-3.60 ppm. In addition, the two characteristic multiplets of cyclopropyl two methylenes appear as two multiplets at δ ~ 1.60-1.30 ppm and δ ~1.20-1.10 ppm. The aromatic protons appear as multiplet signal in compound 5b at δ ~7.50-7.40 ppm. Moreover, the characteristic singlet signal of two aromatic protons of compound 5c appeared at δ ~6.69 ppm and the nine protons of methoxy groups appeared as two singlets at δ ~3.93 ppm and δ ~3.94 ppm. The 1H NMR spectrum of compounds 5d and 5e showed a singlet signal at δ ~3.90-3.80 ppm corresponding to CH2-NHCO. Moreover, the coupling constant (J) of the acrylolyl -CH=CH- is 14-16 Hz which identify the E-isomer of the acryloly moiety. The

13

C NMR spectrum of the methyl esters 5a-e (compound 5b as example, fig.

15) showed a signal of signal at δ ~ 65 or 60 ppm characteristic for the COOCH3 and the carbonyl signal of the ester appeared at δ ~ 165 ppm and the C-4 carbonyl appeared at δ ~ 175. Moreover, the piprazinyl carbons appeared either as two or four signals between δ 50 and 45 ppm, the cyclopropyl moiety appeared as two peaks one between δ ~35-34 ppm and

-40-

Results and discussion: Chemistry the other carbons of the two methylene appeared as one signal at δ ~8.50 ppm. In addition, other peaks characteristic for N-4 substituents piprazinyl is similar that of the previously discussed data in hydroxamic acids 3a-e and amide 4a-e. HRMS (MALDI-TOF) for compound 5b fig. 16 showed that the molecular ion peak for [C28H30FN3O7]+ [M]+ calculated: 450.1829, found: 450.1840.

Fig.14: 1H NMR (400 MHz) (CDCl3) spectrum for Compound 5b

-41-


Fig.15: 13C NMR (100 MHz) (CDCl3) spectrum of compound 5b

Fig.16: The HRMS (MALDI-TOF) spectrum for compound 5b

-42-

Results and discussion: Chemistry The structural formula of the newly prepared ciprofloxacin hydrazide derivatives 6a-e (compound 6a example, fig.17) was identified by various spectroscopic techniques such as IR, 1H NMR, 13C NMR and HRMS. The IR spectra showed a band at ~1622 cm-1 corresponding to C-4 carbonyl, a band at ~1650 cm-1 characteristic for the hydrazide carbonyl in addition to broad band at ~3400-3200 cm-1 of NH and NH2, respectively. The 1

H NMR spectra (compound 6a as example, fig. 16) showed the characteristic signal of

NH at δ ~10-11 ppm and a broad signal of NH2 at δ ~3.8-4.1 ppm. Moreover, The H-2 proton appeared as a downfield singlet at δ ~8.30 ppm. The H-5 appeared as doublet single at ~ δ 7.80 ppm with o-coupling with fluorine JH-f

=

12-14 Hz. Similarly, a doublet

corresponding to H-8 appeared at ~ 7.20 ppm with m-coupling with fluorine and the coupling constant is J = 6-8 Hz. The eight piprazinyl protons appeared as one or two multiplets between δ ~ 3.43-3.35 ppm and the multiplet signal of CH cycloproply at δ ~ 4.20-4.10 ppm at DMSO-d6 while at δ ~3.70-3.60 ppm in CDCl3. The two characteristic multiplets of two methylene of cyclopropyl appeared at δ ~1.35-1.30 ppm and δ ~1.20-1.10 ppm. The aromatic protons of compound 6b appeared as a multiplet at δ ~7.55 ppm. In addition, the spectrum of compound 6c showed a singlet corresponding to the two aromatic protons of compound 6c at δ ~ 6.69 ppm and 9 protons of methoxy group appeared as two singlets at δ ~3.93 ppm and δ ~3.94 ppm. Other peaks characteristic for N-4 piprazinyl substituent is similar to that of the previously mentioned in hydroxamic acids 3a-e, amide 4a-e and ester 5a-e. The

13

C NMR spectrum of hydrazides 6a-e (compound 6a as example, fig. 18) showed a

characteristic signal at δ ~ 165 ppm for hydrazide carbonyls and a signal at δ ~175 ppm for C-4 carbonyl. Moreover, the piprazinyl carbons appeared as four or two peaks at about δ 50, and 40 ppm. The cyclopropyl CH signal appeared between δ ~35-34 ppm and the two other carbons of methylene appeared as one signal at ~8.50 ppm. Other peaks characteristic for N-4 piprazinyl substituent is similar to that of the previously mentioned in hydroxamic acids 3a-e and amide 4a-e. HRMS (MALDI-TOF) compound 6a, fig. 19 showed that the molecular ion peak for [C19H22FN5O3+Na]+ [M+Na]+ 410.1606.

-43-

calculated: 410.1599, found:


Fig.17: 1H NMR (400 MHz) (CDCl3) spectrum of compound 6a

Fig.18: 13C NMR (100 MHz) (CDCl3) spectrum of compound 6a

-44-


Fig.19: The HRMS spectrum of compound 6a

-45-

Results and discussion: Chemistry 3.1.2. Synthesis of levofloxacin hydroxamic acid, amide and hydrazide derivatives 8, 9 and 11 The Mixed anhydride intermediate is formed by treating levofloxacin 7 in DCM with ethylchloroformate in ice-bath (0-5) oC using four equivalents of TEA as a base, Scheme 13. The mechanism of reaction is as previously described in formation of ciprofloxacin mixed anhydride 2a-e, Scheme 4. The un-isolated levofloxacin mixed anhydride [184] derivative 7a was prepared as a core step to prepare the target levofloxacin hydroxamic acid 8, amide 9 and ester 10, Scheme 13. O

O

F

N H

N

OH

N

N

O 8

NH2OH.HCl

O F

O

O OH

N

N

N

O

DCM, 4hrs

TEA, ClCOOEt

F

DCM, ( 0 - 5) oC

N

O O

O

C2H5

O

F

CH3NH2

N H

N

DCM, 4hrs

N

N

O

O

N

N

O

CH3

O 9

7a

7 DCM, 4hrs

CH3OH O O

F

O

N N

F

O

N O

CH3

NH2NH2.H2o Ethanol 95%

N H

N N

O NH2

N O 11

10

Scheme 13: Formation of levofloxacin un-isolated mixed anhydrides 7a and synthesis of levofloxacin derivatives 8-11

-46-

Results and discussion: Chemistry 3.1.2.1. Synthesis of levofloxacin hydroxamic acid 8 To prepare the levofloxacin hydoxamic acid derivative 8, the un-isolated mixed anhydride of levofloxacin 7a was treated with solid hydroxylamine hydrochloride in icebath (0-5) oC and TEA as a base, Scheme 11. The reaction mechanism is proposed to be via attacking the lone pair of electron of hydroxyl amine to the carbonyl of mixed anhydride leads to cleavage of C-O bond and formation of hydroxamic acid derivative, Scheme 6. The structural formula of the newly synthesized levofloxacin hydroxamic acid 8 was identified by various spectroscopic techniques such as IR, 1H NMR,

13

C NMR, and

high resolution mass spectroscopy. The IR spectroscopy showed a broad band at ~33003500 cm-1 corresponding to NHOH. In addition, the carbonyl of –CONH- band appeared between ~1690-1650 cm-1 and the C-7 carbonyl band appeared at ~1622-1627 cm-1. The 1

H NMR spectrum of levofloxacin hydroxamic acid 8 the NH-OH signal appeared at δ ~

11.68 ppm. Moreover, The H-5 proton appeared downfield shifted at δ ~8.73 ppm. The H8 appeared as doublet singlet at δ ~7.52 ppm with o-coupling with fluorine at JH-f = 12-14 Hz. In addition, two multiplet signals of the two protons C-2 appeared at at δ 4.84-4.51 ppm and 4.83-4.32. The eight piprazinyl protons appeared as two multiplets between δ 3.56-3.51 and 3.35-3.27 ppm. The C-3 proton appeared as multiplet at δ 3.05-3.01 ppm and the singlet signal of methyl proton of N-CH3 appeared at δ 2.97 ppm, the signal of CH3 oxazine appeared as doublet signal at δ 1.38 ppm with J = 8 Hz. The

13

C NMR of levofloxacin hydroxamic acid 8 showed a signal at δ 165 ppm

corresponding to the hydroxamic acid carbonyl CO-NHOH and a signal at δ 174 ppm for C-7 carbonyl, the C-3 signal appeared at δ 55 ppm. Moreover, CH3-N- appeared at δ ~46 ppm and the piprazine carbons appeared as two signals between δ 50 and 54 ppm and the C-2 carbon appeared at δ 68 ppm and the CH3- of oxazine appeared at δ 18.2 ppm. HRMS is used to confirm the structure of the levofloxacin hydroxamic acid.

-47-

Results and discussion: Chemistry 3.1.2.2. Synthesis of levofloxacin amide derivative 9 The un-isolated mixed anhydride of levofloxacin 7a was treated with methyl amine to afford the levofloxacin methyl amide 9 as shown in Scheme 11. The reaction mechanism is proposed via attack of the lone pair of electron of methyl amine to the carbonyl of mixed anhydride that leads to cleavage of the C-O bond of mixed anhydride, Scheme 7. The structural formula of the newly synthesized levofloxacin N-methyl amide 9 fig.20 was identified by various spectroscopic techniques such as IR, 1H NMR, 13C NMR and HRMS. The IR spectroscopy showed a band at 3300 cm-1 characteristics for amidic NH. In addition, the carbonyl of CONH-CH3 band appeared at 1655 cm-1 and the C-7 carbonyl band appeared at 1622 cm-1. The 1H NMR of levofloxacin N-methyl amide 9 showed a singlet signal at δ 9.86 ppm corresponding to CO-NH-CH3. Moreover, The H-5 proton appeared as singlet at δ 8.61 ppm and the H-8 appeared as doublet single at δ 7.68 ppm with o-coupling with fluorine (JH-f = 12.3 Hz). In addition, the two protons of CH2 appeared as two multiplet at δ 4.41-4.39 ppm and δ 4.37-4.36. Furthermore, the eight piprazinyl protons appeared as two multiplets at δ 3.41-3.31 ppm and δ 2.59-2.56 ppm. The C-3 proton appeared as multiplet at δ 4.30 -4.26 ppm and the doublet signal of the Nmethyl amide protons appeared at δ 2.97-2.95 ppm with J = 4.8 Hz. The -N-CH3 of piprazine appeared as singlet at δ 2.37 ppm and the oxazine CH3 appeared as doublet at δ 1.55-1.53 ppm. The

13

C NMR of levofloxacin N-methyl amide 9 fig.21 showed a signal at δ 165

ppm corresponding to the amide carbonyl at -CO-NH-CH3 and signal at δ 175 ppm corresponding to C-7 carbonyl, the signal of C-3 appeared at δ 55 ppm. In addition, the signal of piprazinyl N-CH3 appeared at δ 46 ppm and the piprazine carbons appeared as two signals at δ 50 and 54 ppm. Moreover, the C-2 carbon appeared at 68 ppm and the carbon of N-methyl amide appeared at δ 25.81 ppm. The oxazine methyl signal appeared at δ 18.2 ppm. HRMS (MALDI-TOF) confirmed the structure of the levofloxacin N-methyl amide 9, fig.22 which showed that the molecular ion peak for [C19H23FN4O3Na]+ [M+Na]+ calculated: 397.1646, found: 397.1650.

-48-


Fig.20: 1H NMR spectrum (300 MHz) (CDCl3) of compound 9

Fig.21: 13CNMR spectrum (75 MHz) (CDCl3) of compound 9

-49-


Fig.22: The HRMS (MALDI-TOF) spectrum of compound 9

-50-

Results and discussion: Chemistry 3.1.2.3. Synthesis of levofloxacin hydrazides 11 Levofloxacin methyl ester 10 was prepared by treatment of the un-isolated mixed anhydride of levofloxacin 7a with methanol. The product was identified by its reported melting point (reported [188] mp=223-224 °C, found 222-223 oC), Scheme 11. The methyl ester derivative 10 was heated under reflux with hydrazine hydrate (80 %) in ethanol to obtain the target hydrazide derivative 11, Scheme 11. The reaction mechanism is similar to that mentioned in formation of ciprofloxacin hydrazides 6a-e, Scheme 10. The structural formula of the newly prepared levofloxacin hydrazide 11 was identified by various spectroscopic techniques such as IR, 1H NMR,

13

C NMR, and

HRMS. The IR spectroscopy showed a broad band at 3430-3250 of NH and NH2. In addition, the carbonyl of CONHNH2 band appeared at 1654 cm-1 and the C-7 carbonyl band appeared at 1623 cm-1. The 1H NMR spectrum of levofloxacin hydrazide 11 showed a singlet signal at δ 10.77 ppm corresponding to -CONH-, fig.23. Moreover, the H-5 proton appeared as down field singlet at δ 8.53 ppm. The H-8 appeared as doublet at 7.58 ppm with o-coupling with fluorine with coupling constant JH-f = 12.5 Hz. In addition, the two CH2 protons of oxazine appeared as multiplets at δ 4.39-4.32 ppm and δ 4.32-4.30 ppm. The characteristic signal of NH2 of hydrazide appeared as broad peak at 4.15 ppm. Furthermore, the eight piprazinyl protons appeared as two multiplets between δ 3.37-3.29 ppm and δ 2.59-2.52 ppm. The characteristic piprazinyl N-CH3 appeared as singlet at δ 2.33 ppm while the signal of oxazine CH3 appeared as doublet at δ 1.55-1.54 ppm. The

13

C NMR of levofloxacin hydrazide 11 fig.24 showed a signal at 165 ppm

corresponding to hydrazide carbonyl and a signal at δ 174 ppm characteristic for C-2 carbonyl. Moreover, the C-3 appeared at δ 55 ppm while the carbon of CH3-N appeared at δ 46 ppm. Furthermore, the four piprazinyl carbons appeared as two signals at δ 50 and 54 ppm and the C-2 carbon appeared at δ 68 ppm. In addition, The CH3-oxazine signal appeared at δ 18.2 ppm. The structure of levofloxacin hydrazide 11 is confirmed using HRMS (MALDI-TOF), fig.25 which showed that the molecular ion peak for [C38H39FN4O8]+ [M]+ calculated: 699.2830 found: 699.2819. [188]

-51-


Fig.23: 1H NMR (500 MHz) (CDCl3) spectrum of compound 11

Fig.24: 13C NMR (125 MHz) (CDCl3) spectrum of compound 11

-52-


Fig.25: The HRMS spectrum of compound 11

-53-

3.2 Biology

Results and discussion: Biology 3.2. Biology 3.2.1. Anticancer screening Compounds 3a, 4a, 3b, 4b, 3c, 4c, 8 and 9 were selected by the National Cancer Institute, Bethesda, Maryland, United states for preliminary screening to be tested in vitro for their cytotoxicity. Those selected compounds were screened against a panel of sixty cell lines (NCI-60 cell line panel) derived from nine different types of cancers including leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostatic, and breast. 3.2.1.1 In vitro one-dose assay The selected compounds 3a, 4a, 3b, 4b, 3c, 4c, 8 and 9 were first tested at a single concentration of 10 μM. Screening results for each compound were recorded to express growth percentage of the cancer cells which were treated with the tested compounds and then compared to that of the untreated ones. The negative values indicate cancer cell death. Mean graph midpoint (MG_MID) (differential activity patterns, bar scale) were constructed for each cell line to facilitate visual scanning of data for potential NCI patterns bars of selectivity, depicting the deviation of the individual tumor cell lines from the overall mean value for all the cell lines tested. In the mean graph, the center point is the mean value of all growth percentages recorded for all cell lines tested. Bars that point to the left (positive values) denote resistance where the growth is greater than the average, while bars that point to the right (negative values) denote sensitivity where the growth is less than the average. Delta means the logarithm of the difference between the (MG_MID) and log GI50 of the most sensitive cell line, while the range is the logarithm of the difference between log GI50 values of the most resistant cell line and the most sensitive one. Results of in vitro one-dose screening are listed Table .1 and the screening results for compounds 3b and 4c is showed in fig. 26 and 27.

-54-

Results and discussion: Biology Table.1: Mean growth percentage for the tested compounds of one dose screening. Percentage of growth inhibition % 00.03 1.12 00.45 -2 2.87 1.63 0.16 00.05

Compound 3a 3b 3c 4a 4b 4c 8 11

-55-

Results and discussion: Biology O F

O N H

N

OH

N

N O

3b

Fig. 26: One-dose growth (%) and mean graph of compound 3b.

-56-

Results and discussion: Biology O F

OCH3 H3CO

O N H

CH3

N

N N

H3CO O

4c

Fig. 27: One-dose growth (%) and mean graph of compound 4c.

-57-

Results and discussion: Biology Cytotoxicity screening results of the selected compounds in tabel.1 shows their effect on different cell lines. Generally, the tested compounds don’t show effective cytotoxic activity on most of the tested cell lines. However, it is clear that the best mean growth is for compound 3b which tested against non-small cell lung cancer (A549/ATCC) exhibiting growth percentage = 79.25 % and compound 4c against breast cancer cell line (T-47D) with growth percentage = 79.02 %, fig 26 and 27. Generally, it could be conclude that replacing the carboxylic group with hydroxamic acid or amides diminish the cytotoxic activity of fluoroquinolones. [189] To study the binding difference between C-3 carboxylic acid of ciprofloxacin and its analogue C-3 hydroxamic acid and amide moiety. We performed a molecular docking study for the weak anticancer compounds 3b, 4c and two previously reported

[190]

ciprofloxacin derivatives LXIV and LXV with potent broad spectrum anticancer activity (GI50 for compound LXII = 1.65 to 9.82 µM and for LXIII= 0.199 to 0.842 µM). In this docking study, we used Topoisomerase II (PDB: 4fm9) enzyme utilizing MOE® program and docking scores were showed in Table 2.

O

O

O

O

F

HO

O F

HO N

N

O N

N N

N

N

O N

S

LXIV

LXV

-58-

N N

S

Results and discussion: Biology Table.2: Docking scores of compounds LXIV, LXV, 3b and 4c on topoisomerase II. Compound LXIV LXV 3b 4c

Docking score Kcal/mol -6.9377 -7.5154 -7.6433 -7.0884

Although the binding scores of the compounds in LXII, LXIII, 3b and 4c in table 2 indicate that all four compounds can bind spontaneously with the active site, it is obvious in fig 28 and 29 that the binding mode is different. The 2D diagrams of compounds LXIV and LXV showed that both of compounds coordinate with Mg+2101 by C-4 carbonyl and C-3 carbonyl of carboxylic acid in addition to hydrogen bond with DG C4. Compound LXIV has additional hydrogen bonding with Gln500 while compound LXV has additional hydrophobic interaction with Lys798 that may explain their potent anticancer activity.

Fig. 28: 2D Diagram of compounds LXIV and LXV.

-59-

Results and discussion: Biology On the other hand, 2D diagram of compounds 3b and 4c showed no coordination with Mg+2 which is essential for inhibition Topoisomerase II enzyme.

Fig. 29: 2D Diagram of compounds 3b and 4c.

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Results and discussion: Biology 3.2.2. Antibacterial Screening The synthesized compounds 3a-e, 4a-e, 6a-e and 8-11 were tested against Proteus mirabilis, the common causing bacteria of urinary tract infections (UTIs), and is compared with the reference fluoroquinolones levofloxacin and ciprofloxacin. Table 2 shows the anti-Proteus mirabilis activities of the synthesized compounds 3a-e, 4a-e, 6a-e and 8-11 and The P. mirabilis strain was isolated from the urine of patients suffering from urinary tract infection. The strain is negative for hemolysis, motile and urease positive. Isolation and identification were performed according to standard procedures of the Well Diffusion method. [191,192] The isolate was cultured on trypticase soy agar (TSA, Difco) slants for daily use and stored in a trypticase soy both medium (TSB, Difco) along with 15% glycerol, at –80oC for subsequent uses. Table 3: Anti-Proteus mirabilis of the tested compounds Compounds Levofloxacin Ciprofloxacin N-Acetyl Ciprofloxacin AHA 3a 3b 3c 3d 3e 4a 4b

MIC (µM) 3.2 15.62 12.5 614.8 125 18.3 2.25 10.25 7 19.25 11.6

Compounds 4c 4d 4e 6a 6b 6c 6d 6e 8 9 11

MIC (µM) 8 10.27 8.23 12.8 10.8 8.75 8.12 9.25 250 16.25 1.07

Table. 3 illusterates that some derivatives have moderate to good anti-Proteus miarbilis activity. Most of hydroxamic acid derivatives showed good to moderate antiProteus miarbilis activity except compound 3a and 8 which experienced have very weak activity. On the other side, all hydrazide derivatives 6a-e have better anti-Proteus miarbilis activity than their parent compounds, levofloxacin and ciprofloxacin. Levofloxacin hydrazide 11 has potent activity than levofloxacin and the hydrazide derivatives of Nacetyl ciprofloxacin 6a revealed potent anti-Proteus miarbilis activity than ciprofloxacin. Similarly, N-methyl amide derivatives 4a-e and N-(3,4,5-trimethoxy) piprazinyl ciprofloxacin derivatives 3c, 4c and 6c showed good antibacterial activity against Proteus

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Results and discussion: Biology mirabilis than the parent drugs. Furthermore, ciprofloxacin chalcone derivatives 3d, 3e, 4d, 4e, 6d, and 6e have comparable or more activity compared to ciprofloxacin. This may be explained by additional biological benefits due to the chalcone analogue moiety of N-4piprazine. In conclusion, it is obvious that two factors may affect the anti-Proteus mirabilis activity; first physicochemical properties that is related to N-4-piprazinyl substituents and ; second the binding to the active site of topoisomerases that is related to C-3 moiety.

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Results and discussion: Biology 3.2.3. Urease inhibition activity The synthesized compounds 3a-e, 4a-e, 6a-e, 8-11 and the parent ciprofloxacin and levofloxacin and AHA were tested in vitro to determine their urease inhibitory activity in their sub-MIC (1/32-1/2 X MIC). The test was performed on jack bean urease and the IC50 were determined using indophenol method

[155,193]

which depends on the color change

according to the liberation of ammonia. The absorbance was measured by UV spectroscopy at λ = 630 nm using this following equation [A = λbc], where c is the concentration of solution (mol/L) and b the Length of the UV cell. The IC50 values were recorded in the Table 4. Table 4: IC50 of urease inhibition of the tested compounds: Compounds Levofloxacin Ciprofloxacin N-Acetyl Ciprofloxacin AHA 3a 3b 3c 3d 3e 4a 4b

Urease IC50 (µM) 2.90 3.5 2.26 120 2.22 18.93 7.17 19.3 949.25 1.29 2495.80

Compounds

Urease IC50 (µM)

4c 4d 4e 6a 6b 6c 6d 6e 8 9 11

2.92 2413. 40 630.90 1.22 250.44 2.08 812.06 912.01 2.20 2.89 772.34

Results in Table 4 show that most of hydroxamic acid derivatives 3a-d have good inhibitory activity of urease more potent than AHA. In particular, compounds N-acetyl ciprofloxacin HA 3a and levofloxacin HA 8 have the most potent inhibitory activity within the tested compounds. Moreover, the amide derivatives 4a and 4c with N-acetyl and N(3,4,5-trimethoxybenzoyl) moieties, respectively and levofloxacin amide 9 experienced remarkable urease inhibitory activity over other tested amides. The hydrazide derivatives 6a and 6c have potent inhibitory activity however, most of hydrazide derivatives are weak urease inhibitors.

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Results and discussion: Biology It is obvious that, ciprofloxacin derivatives with bulky substituents at N-4-piprazine 3e, 4e-d and 6e-d exhibit weak urease inhibitory activity. In contrast, the acetyl and 3,4,5trimethoxy moieties at N-4-piprazinyl are remarkable factor in increasing ciprofloxacin derivatives activity towards urease inhibition. It is clearly noted that, some of the tested compounds show potent urease inhibitors with anti-Proteus mirabilis activity especially N-(3,4,5-trimethoxy benzoyl) ciprofloxacin hydroxamic acid 3c, its hydrazide analogues 6c and levofloxacin amide derivative 9. So, these compounds may be beneficial in treatment of bacterial infection with prevention of urease associated complications that require further stability, toxicological and physicochemical studies for its optimization.

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Molecular Docking Studies 3.2.3.1. Molecular docking studies (H. Pylori urease) MOE dock program was utilized for performing the molecular docking study for compounds 1a, 3a, 3d, 4a-e, 6a, 8 and 9 ciprofloxacin and levofloxacin. The selected compounds were docked into the binding pocket of the active site of urease (PDB: 1E9Y) to investigate the docking fitness scores of bioactive conformations and their specificity for urease enzyme. The docking reliability was validated using the known X-ray structure of H. Pylori urease in complex with AHA. The ligand AHA was extracted from the complex and re-docked to the binding site of H. Pylori urease. In fig.30, it is obvious that AHA carbonyl coordinates with Ni3001 and form hydrogen bonds with Asp362, Ala365 and His221. The docking scores (S) of the studied compounds are shown in Table 4. The tested ligands were found to bind strongly to urease as inferred by binding energy values range from -13.6027 to -5.9614 Kcal/mol.

Fig.30: 3D Structure of AHA docked into H.pylori urease

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Molecular Docking Studies Table 5: Docking scores of the docked compounds Compounds

Docking score (Kcal/mole)

Compounds

Levofloxacin Ciprofloxacin N-Acetyl Ciprofloxacin

-8.5373 -7.1972 -7.0112

4c 4d 4e

Docking score (Kcal/mole) -5.9614 -6.1994 -7.5296

AHA

-11.2821

6a

-7.8098

3a

-7.5232

6b

-6.9250

3b

-7.6260

6c

-6.6200

3c

-13.6060

6d

-7.6127

3d

-10.0820

6e

-9.1398

3e

-13.6027

8

-2.6647

4a

-7.7554

9

-5.9432

4b

-6.2220

11

-7.2164

For compound 8, the binding mode (fig. 31) shows that the oxygen of the carbonyl of the hydroxamic acid can coordinates with Ni3001 and form a hydrogen bond with His221. Moreover, the hydrogen atom of the NH group makes a hydrogen bonding with Ala365. Similarly, the OH group has a hydrogen bond with Asp362. Moreover, compound 8 binding formation agree with its biological results (IC50 = 2.20 µM) as it mimics binding mode of AHA.

Fig. 31: 2D Diagram and 3D structure of compound 8 with H.pylori urease

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Molecular Docking Studies The binding mode of levofloxacin shows coordination of the carbonyl oxygen of the carboxylic acid moiety with Ni3001 while the OH group makes a hydrogen bond with Gly279, as showed in (fig. 32). It is obvious that the carboxylic acid coordination with Ni3001 is responsible for inhibitory activity of levofloxacin against urease but it doesn’t have the same binding mode of levofloxacin hydroxamic acid 8 where it can bind with His221, Asp365, Ala365 in addition to coordination with Ni3001. Consequently, both levofloxacin and hydroxamic acid analogue are highly active urease inhibitors.

Fig.32: 2D Diagram and 3D structure of levofloxacin with H.pylori urease Docking results for compound 3a fig. 33 shows the same binding mode as AHA that support its potent urease inhibitory activity (IC50 = 2.22 µM). Compound 3a hydroxamic moiety forms hydrogen bonds with Ala365 and Asp362 while the carbonyl group of the hydroxamic acid coordinates with Ni3001 and hydrogen bond with His221.

Fig.33: 2D Diagram and 3D structure of compound 3a with H.pylori urease

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Molecular Docking Studies The binding mode for N-acetyl ciprofloxacin 1a illustrates that the carbonyl of carboxylic acid coordinates with Ni3001 and the hydroxyl group forms hydrogen bond with Asp362, as shown in fig. 34 and that explains its potent urease inhibitors more than AHA.

Fig.34: 2D Diagram and 3D structure ofN-acetyl ciprofloxacin 1a with H.pylori urease In addition, modelling configuration of ciprofloxacin shows that carbonyl group of carboxylic acid moiety has two coordination bonds with both Ni3001 and Ni3002 while the hydroxyl group forms a hydrogen bond with Met366 in addition to hydrophobic interaction with His221 as showed in fig. 35. Thus, that explains the potent urease inhibitory activity more than AHA.

Fig.35: 2D Diagram and 3D structure of ciprofloxacin withH.pylori urease

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Molecular Docking Studies The binding mode of compound 3d shows that the hydroxyl group forms two hydrogen bonding with Asp362 and Ala365 while the oxygen of the carbonyl coordinates with Ni3001 and has a hydrogen bonding with His221 as showed in fig. 36. The compound 3d has good binding conformation as like as AHA and that support it has potent inhibitory activity against urease (IC50 = 19.3 µM).

Fig. 36: 2D Diagram and 3D structure of 3d with H.pylori urease Compound 6a modelling configuration shows hydrogen bonding interaction with His221, Asp362, Als365 and Gly279. Moreover, it shows coordination with Ni3001 by oxygen of the carbonyl moiety and NH2 group, as showed in fig. 37. This good binding mode with urease support its potent inhibitory activity with IC50 = 1.22 µM and docking score = 7.8098.

Fig. 37: 2D Diagram and 3D structure of 6a with H.pylori urease

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Molecular Docking Studies The ciprofloxacin N-methyl amide derivatives 4a and 4c and levofloxacin amide 9 have potent urease inhibitory activity than AHA with IC50= 1.29, 2.92, 2.89 µM, respectively but with binding mode differs from AHA. For example, compound 4a binding mode fig. 38 shows that N-acetyl carbonyl coordinates with Ni3001 and His221 instead of C-3 carbonyl and this binding mode is explaining its inhibitory activity.

Fig.38: 2D Diagram and 3D structure of 4a with H.pylori urease Binding mode of compounds 4c and 9 show different binding modes from AHA but still have potent urease inhibitory activity. They demonstrate a hydrogen bond with Arg338 and a hydrophobic interaction with Al169, as showed in fig. 39 and 40.

Fig.39: 2D Diagram and 3D structure of 4c with H.pylori urease

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Molecular Docking Studies

Fig.40: 2D Diagram and 3D structure of 9 with H.pylori urease The other amide derivatives 4b, 4d and 4e fig. 41-43 has very weak urease inhibitory activity and their binding mode is different from other active amide derivatives 4c and 9. They don’t show binding with both Arg338 and Al196. So, it is obvious that binding with both Arg338 and Al196 is essential for amide derivatives to active as urease inhibitors.

Fig.41: 2D Diagram and 3D structure of 4b with H.pylori urease

Fig.42: 2D Diagram and 3D structure of 4d with H.pylori urease

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Molecular Docking Studies

Fig.43: 2D Diagram and 3D structure of 4e with H.pylori urease Table 6: Different binding mode of hydroxamic, hydrazide, amide derivatives of fluoroquinolones and AHA Compound Fluoroquinolones hydroxamic acids

-

Fluoroquinolones hydrazides

-

Fluoroquinolones amides Fluoroquinolones carboxylic acids AHA

-

Most common binding mode Coordination with Ni3001 Hydrogen binding with Al365, His221 and Asp365 amino acids Coordination with Ni3001 Hydrogen binding with Al365, His221, Gly279 and Asp365 amino acids Hydrogen bonding with Arg338 and Al169 amino acids Coordination with Ni3001 Coordination with Ni3001 Hydrogen binding with Al365, His221 and Asp365 amino acids

In conclusion, it is clear that (as shown in Table 6) the hydroxamic acid and hydrazide moieties exhibit their urease inhibitory by binding mode resembles the binding mode of AHA in coordination with Ni3001 and forming hydrogen bonding with Al365, His221 and Asp365 while the amide derivatives show different binding mode differs AHA, they bind with Arg338 and Al169 to achieve their urease inhibitory activity.

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4. Experimental

Experimental

4. Experimental 4.1. Chemistry •

All chemicals used for the preparation of the target compounds of commercial grade and were purchased from Merck®, Sigma Aldrich®, SD Fine® and El Nasr® pharmaceutical chemicals companies.

•

Ciprofloxacin and levofloxacin were purchased from Medical Union Pharmaceutical (MUP) Abou Sultan - Ismailia, Egypt.

•

The reaction progress as well as product purity were monitored using TLC (Kieselgel 60 F254 precoated plates, E. Merck, Darmstadt, Germany), the spots were detected by exposure to UV lamp at λ 254 and 365 nm.

•

Melting points were determined on a Stuart® SMP10 melting point apparatus and were uncorrected.

•

IR spectra were recorded on a Nicolet® iS 5 FT-IR Spectrometer at Faculty of Pharmacy, Minia university. IR wave lengths (υ) are expressed as cm-1.

•

1

H NMR spectra were ran on both Bruker® Advance III (400 MHz) spectrophotometer

(Bruker AG, Switzerland) at faculty of Pharmacy Bani-Suief University Egypt and JEOL® (300 or 500 MHz) at Osaka University Japan. TMS was used as an internal standard while CDCl3 or DMSO-d6 as a solvent. Chemical shift (δ) values are expressed in parts per million (ppm) and coupling constants (J) in Hertz (Hz). The signals are designed as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad singlet. •

13

C NMR spectra were recorded on both Bruker® Advance III (100 MHz)

spectrophotometer (Bruker AG, Switzerland), faculty of Pharmacy Bani-Suief University Egypt or JEOL® (75 or 125 MHz) at Osaka University Japan with TMS as internal standard and DMSO-d6 or CDCl3 as a solvent. Chemical shifts (δ) values are expressed in parts per million (ppm). •

Chemical shifts (δ) values of solvent used are given in parts per million (ppm) relative to TMS, CDCl3 (7.29 for proton and 76.9 for carbon) or DMSO-d6 (2.50 for proton and 39.50 for carbon) and coupling constants (J) in Hz.

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Experimental •

HRMS were performed using MALDI-TOF JOEL® at the graduate school of pharmaceutical sciences Osaka University, Japan or DART AccuTOF® at the department of chemistry, University of Connecticut, Connecticut, USA.

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Experimental 4.1.2. General procedure for synthesis N-4-acylated piprazinyl ciprofloxacin derivatives 1a-c. Ciprofloxacin (3.31 gm, 10 mmol) was dissolved in 50 mL DCM in ice bath (0-5 o

C). To this suspension TEA (4.3 mL, 30 mmol) was added while stirring. of the respective

acid chloride (1.2 equivalent) was added dropwisely and stirring was continued for 6 hrs. The organic layer was washed with water (3 x 25 mL) and dried over anhydrous sodium sulphate, filtered off and evaporated under reduced pressure.[181]

4.1.2.1.7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline3-carboxylic acid 1a White powder, m.p. (reported = 253-255 oC, found 253-254 oC) [178] , Yield =84% mp. 4.1.2.1.7-(4-Benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3-carboxylic acid 1b White powder, m.p. (reported m.p = 273-275 oC, found 273-275 oC) [179] , Yield = 88%. 4.1.2.1.7-(4-(3,4,5)-Trimethoxybenzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4dihydro-4-oxoquinoline-3-carboxylic acid 3b White powder, m.p. (reported 278-280, found 277-279 oC). [179], Yield = 85%.

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Experimental 4.1.3. Preparation of ciprofloxacin chalcone derivatives 1d-e: 4.1.3.1. General procedure for synthesis of chalcones A1 and A2 A round-bottom flask was loaded with rectified ethanol (35 mL) and aq 4 M NaOH solution (60 mL). The flask was cooled in ice bath, and p-amino acetophenone (10 mmol, 1.2 gm) was added. To this solution, the corresponsive aromatic aldehyde (10 mmol) was added with stirring and the temperature was kept at 25 °C, the mixture was stirred vigorously for 2-3 hrs. The reaction mixture was kept at 2-4 °C overnight. [183] R

H2N

O A1:R= H A2:R= 3,4,5-OCH3

4.1.3.2. General procedure for the synthesis of 2-bromo-N-{4-[3- arylacryloyl]phenyl and 3,4,5 trimethoxyphenyl]}acetamides B1 and B2 To a stirred cooled mixture of chalcones A1 and A2 (6.30 mmol) in DCM (20 mL) and potassium carbonate (1.30 mmol) in 100 mL water, bromoacetyl bromide (1.856 g, 9.20 mmol) in 30 mL DCM was added in a dropwise manner with stirring for 2 hrs at (0-5) o

C in ice bath, and at room temperature overnight. The reaction mixture was extracted with

DCM (2 x 60 mL). The organic layer was washed with distilled water (2 x 40 mL), dried over anhydrous sodium sulphate, filtered, evaporated under vacuum and the residue was recrystallized from absolute ethanol. [47] R Br

O HN O B1:R=H B2:R=3,4,5-OCH3

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Experimental 4.1.3.2.1 2-Bromo-N-(4-((E)-3-phenylacryloyl)phenyl)acetamide B1 Pale yellow powder, m.p. (reported mp= 156-158 oC, found 155-157 oC). [47], Yield = 82%. 4.1.3.2.1 2-Bromo-N-(4-((E)-3-(3,4,5) trimethoxyphenylacryloyl)phenyl)acetamide Pale yellow powder, m.p. (reported mp=167-169 oC, found 166-168 oC).[47], Yield = 80%.

4.1.3.3. Synthesis of ciprofloxacin-chalcones derivatives 1d-e To a mixture of ciprofloxacin (0.662 g, 0.02 mol) and the corresponding chalcone derivative B1 and B2 (0.02 mol) in acetonitril (50 mL), TEA (0.404 gm, 0.04 mol) was added. The mixture was heated under reflux for 5 hr. The precipitate formed was filtered off while hot, washed with acetonitrile and dried under vacuum. [47]

F

O

N N

R

HN

O HO

N

O

O 1d:R= H 1e:R= 3,4,5-OCH3

4.1.3.3.1. 7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-1cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid 1d Pale yellow powder, m.p. (reported 270-273 oC, found: 270-273 oC). [47], Yield= 63%. 4.1.3.3.2. 7-(4-((4-((E)-3-(3,4,5,trimethoxyPhenyl)acryloyl) phenylcarbamoyl) methyl) piperazin-1-yl) -1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid 1e Pale yellow powder, m.p. (reported 273-275oC, found 272-274oC).[47], Yield= 52%.

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Experimental 4.1.4. General procedures for synthesis of hydroxamic acids derivatives 3a-e and 8 To a cooled stirred suspension of respective fluoroquinolone (10 mmol) in DCM (50 mL), TEA (20 mmol, 2.02 gm, 2.8 mL) and ethylchloroformate (1.08 gm, 98 mL, 10 mmol,) were added dropwisely. The mixture was stirred for further one hour at (05°C).[168] A powder of hydroxylamine hydrochloride (1.38 gm, 20 mmol) was added portion wise and stirring was continued for further four hours at room temperature. The organic layer was washed with water (3 x 25 mL) and dried over anhydrous sodium sulphate, filtered off and evaporated under reduced pressure. Crystallization from methanol/ethyl acetate afforded the target hydroxamic acid. [193] 4.1.4.1 7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-hydroxy-4oxoquinoline-3-carboxamide 3a O F

N H

N O

O OH

N

N 3a

Pale yellow powder, m.p: 259-260, Yield = 54 %, °C; IR (cm-1): 3500-3340 (NH-OH), 1650 (C=O), 1696 (CH3-C=O), 1650 (C=O), 1622 (C-4 C=O); 1H NMR 500 MHz (CDCl3) δ (ppm): 12.12 (1H, s, NH), 11.80 (1H, s, OH-), 8.69 (1H, s , H-2), 7.99 (1H, d, J

H-F

=

13.5 Hz, H-5), 7.32 (1H, d, J = 6.5 Hz H-8), 3.83-3.68 (5H, m, piprazinyl 4H+ CH cyclopropyl), 3.29-3.23 (4H, m, piperazine 4H), 2.10 (3H, s, CH3-CO), 1.45-1.28 (2H, m, CH2 of cyclopropyl), 1.23-1.16 (2H, m, CH2 of cyclopropyl); 13C NMR 125 MHz (CDCl3) δ (ppm): 174.81, 169.12, 165.37, 154.54, 152.07, 146.59, 144.49, 122.15, 112.99, 110.40, 105.09, 50.45, 49.55, 46.19, 34.79, 21.79, 8.28. HRMS (MALDI-TOF) for [C19H21FN4O4]+ [M]+ calculated: 389.1620, found: 389.1621.

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Experimental 4.1.4.2. 7-(4-Benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-Nhydroxy-4-oxoquinoline-3-carboxamide 3b O

O

F

N H

N

OH

N

N 3b

O

White powder, mp: 260-263, Yield = 64 %, °C; IR (cm-1): 3580-3440 (NHOH), 1650 (C=O), 1680 (Ar-C=O), 1622 (C-4 C=O); 1H NMR 400 Hz (DMSO-d6) δ 12.64 (1H, s, NH), 10.34 (1H, s, OH-), 8.58 (1H, s , H-2), 7.90 (1H, d, JH-F = 13.2 Hz, H-5), 7.56 (1H, d, J = 7.2 Hz, H-8), 7.44-7.48 (5H, m, Ar-H), 4.28-4.27 (1H, m, CH- cyclopropyl), 3.85-3.04 (8 H, m, piperazine 8H), 1.31-1.21 (4H, m, CH2 of cyclopropyl);

13

C NMR 125 Hz

(CDCl3) δ (ppm): 172.85, 170.28, 165.72, 148.21, 143.82, 137.77, 134.94, 129.83, 128.40, 126.84, 123.09, 112.87, 109.57, 105.21, 105.19, 51.73, 47.20, 34.46, 8.59. HRMS (MALDI-TOF) for [C24H23FN4O4]+ [M]+ calculated: 450.1698 found: 450.1831. 4.1.4.3. 7-(4-(3,4,5-Trimethoxybenzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4dihydro-N-hydroxy-4-oxoquinoline-3-carboxamide 3c O F

OCH3 H3CO

O N H

N

OH

N

N

H3CO

3c

O

White powder, mp: 265-267, Yield = 81 %, °C; IR (cm-1): 3580-3340 (NHOH), 1650 (C=O), 1690 (Ar-C=O), 1627 (C-4 C=O); 1H NMR 400 MHz (DMSO-d6) δ (ppm): 11.67 (1H, s, NH), 9.23 (1H, s, OH-), 8.60 (1H, s , H-2), 7.89 (1H, d, J H-F = 13.2 Hz, H-5), 7.53 (1H, d, J = 6.5 Hz, H-8), 6.75 (2H, s, Ar-H), 3.87 (6H, s, 2 OCH3), 3.86 (3H, s, OCH3), 3.81-3.77 (1H, m, CH- cyclopropyl), 3.63-3.17 (8H, m, piperazine 8H), 1.32-1.30 (2H, m, CH2 of cyclopropyl), 1.17-1.11 (2H, m, CH2 of cyclopropyl); 13C NMR 100 MHz (CDCl3) δ (ppm): 173.85, 170.36, 162.25, 154.67, 153.47, 146.09, 144.59, 139.70, 138.30, 130.41, 118.20, 112.98, 112.76, 105.26, 104.70, 60.94, 56.42, 49.50, 47.20, 35.12, 8.40. HRMS (MALDI-TOF) for [C27H29FN4O7] + [M]+ calculated: 563.1912 found: 563.1915.

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Experimental 4.1.4.4. 7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-1cyclopropyl-6-fluoro-1,4-dihydro-N-hydroxy-4-oxoquinoline-3-carboxamide 3d O

O

F

O O N H

N H

N

OH

N

N 3d

Pale yellow powder; m.p: 274-275°C; Yield = 67%; IR (cm-1) 3532-3343 (NHOH), 1695 (C=O), 1660 (C=O), 1640 (C=O), 1625 (C-4 C=O); 1H NMR 500 MHz, (CDCl3) δ (ppm): 12.11 (1H, s, -NH ), 9.36 (1H, s, -NH), 8.65 (1H, s, H-2), 8.02 (2H, d, J = 8.5 Hz, Ar-H), 7.93 (1H, d, J H-F = 13 Hz, H-5), 7.78 (1H, d, J 15.5 Hz, =CH), 7.74 (2H, d, J = 8.5 Hz, ArH), 7.63-7.62 (2H, m, H-8, =CH), 7.62-7.41 (5H, m, Ar-H), 3.48 (2H, s, -CH2-), 3.33-3.24 (m, 9H, CH cyclopropyl, 8H piperazinyl-H), 1.28-1.23 (2H, m, CH2), 1.22-1.16 (m, 2H, cyclopropyl-H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 188.80, 174.27, 168.66, 164.06, 154.36, 151.90, 148.91, 146.56, 144.75, 144.65, 143.54, 138.97, 138.79, 128.92, 128.84, 127.56, 127.08, 126.37, 121.00, 120.93, 119.79, 111.77, 111.54, 110.11, 106.40, 64.09, 52.85, 49.89, 41.16, 40.61, 35.45, 8.04; HRMS (MALDI-TOF) [C34H32FN5O5+Na]+ [M+Na]+ calculated: 632.2280, found: 632.2270. 4.1.4.5. 7-(4-((4-((E)-3-(3,4,5-Trimetoxyphenylacryloyl)phenylcarbamoyl)methyl) piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-hydroxy-4-oxoquinoline-3carboxamide 3e O F

O H3CO

O

H3CO OCH3

N H

N

O N H

OH

N

N 3e

Yellow powder; m.p: 273-276°C; Yield = 67%; IR (cm-1) 3435-3323 (NHOH), 1680 (C=O), 1640 (C=O), 1650 (C=O), 1623 (C-4 C=O); 1H NMR 400 MHz, (DMSO-d6) δ (ppm): 11.68 (1H, s, -NH ), 10.22 (1H, s, -OH), 10.15 (1H, s, -NH), 8.60 (1H, s, H-2), 8.19 (1H, d, J H-F = 12 Hz, H-5), 7.93 (2H, d, J = 8.8 Hz, Ar-H), 7.89-7.87 (3H, m, Ar-H,

-80-

Experimental =CH), 7.79 (1H, d, J = 8.8 Hz, H-8), 7.54 (1H, d, J = 15.5 Hz, =CH), 6.99 (2H, s, Ar-H), 3.87 (2H, s, -CH2), 3.75 (3H, s, -OCH3), 3.70 (6H, s, 2 OCH3), 3.61-3.30 (9H, m, CH cyclopropyl, piperazinyl-8H), 1.30-1.23 (2H, m, cyclopropyl-H), 1.17-1.12 (2H, m, cyclopropyl-H);

13

C NMR δ 100 MHz, (DMSO-d6) δ (ppm): 187.94, 174.01, 169.37,

162.74, 154.42, 146.59, 144.84, 144.74, 144.40, 143.56, 140.40, 138.88, 138.57, 133.01, 132.31, 130.80, 130.30, 129.77, 122.40, 121.58, 119.25, 110.24, 106.97, 60.97, 60.61, 52.81, 49.89, 46.09, 35.57, 8.06; HRMS (MALDI-TOF):[C37H38FN5O8+Na]+ [M+Na]+ calculated: 722.2597, found: 722.2595. 4.1.4.6. (S)-9-Fluoro-3,7-dihydro-N-hydroxy-3-methyl-10-(4-methylpiperazin-1-yl)-7-

oxo-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide 8 O F

N H

N N

O OH

N O 8

Pale yellow powder, m.p.: 232-233, Yield = 81%; IR (cm-1): 3580-3300 (NHOH), 1623 (C-7 C=O); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 11.68 (1H, s, -NH-), 8.73 (1H, s, H-5), 7.52 (1H, d, JH-F = 12.28 Hz, H-8), 4.87-4.85 (1H, m, CH), 4.55 (1H, d, J = 8 Hz, CH-), 4.39 (1H, d, J = 8 Hz, -CH-), 3.56-3.27 (4H, m, piperazine 4H), 3.05-2.99 (4H, m, piperazine 4H), 2.99 (3H, s, CH3-N-piprazine), 1.37 (3H, d, J = 6.6 Hz, CH3-CH);

13

C

NMR 100 MHz (DMSO-d6) δ (ppm): 174.60, 165.50, 156.60, 154.63, 143.40, 139.30, 131.80, 122.03, 109.93, 104.91, 68.00,55.59, 54.88, 50.45, 46.30, 18.21. HRMS (MALDITOF) for [C18H21FN4O]+ [M+] calculated: 377.1620, found: 377.1624.

-81-

Experimental 4.1.5. General procedure for synthesis of fluoroquinolone amides 4a-e and 9 To a cooled stirred suspension respective fluoroquinolone (10 mmol) in DCM (50 mL), TEA (2.02 gm, 2.8 mL, 20 mmol) and ethylchloroformate (1.08 gm, 98 mL, 10 mmol) were added dropwisely. The mixture was stirred for further one hour at (0-5°C).[168] Excess amount of 50% methyl amine was added and stirred for further four hours at room temperature. The organic layer was washed with super saturated solution of sodium bicarbonate (2 x 25 mL) and then washed by distilled water (1 x 25mL) dried over anhydrous sodium sulphate, filtered off and evaporated under reduced pressure. The obtained residue was recrystallized from methanol/ethyl acetate. [195] 4.1.5.1.7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4oxoquinoline-3-carboxamide 4a O F

N H

N O

O CH3

N

N 4a

Pale yellow powder, m.p: 235-238 °C, Yield = 74 %,; IR (cm-1): 3300 (NH), 1650 (C=O), 1624 (C-4 C=O); 1H NMR 300 MHz (CDCl3) δ (ppm): 9.79 (1H, q, J = 3.6 Hz, NH-), 8.73 (1H, s , H-2), 7.93 (1H, d, JH-F = 12.9 Hz, H-5), 7.29 (1H, d, J =7.5 Hz, H-8), 3.92 3.18 (9H, m, CH- cyclopropyl 1 H, piperazine 8H), 2.92 (3H, d, J = 3.6 Hz, CH3-NH), 2.11 (3H, s, CH3-CO), 1.28-1.25 (2H, m, CH2 of cyclopropyl), 1.19-1.11 (2H, m, CH2 of cyclopropyl);

13

C NMR 75 MHz (CDCl3) δ (ppm): 175.18, 168.98, 165.34, 146.53 ,

144.25, 144.10, 138.19, 112.66, 112.34, 111.19, 104.96, 50.24, 49.36, 34.59, 25.74, 21.16, 8.11; HRMS (MALDI-TOF) for [C20H23FN4O3Na]+ [M+Na]+ calculated: 409.1646, found: 409.1652.

-82-

Experimental 4.1.5.2.

7-(4-Benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4-

oxoquinoline-3-carboxamide 4b O

O F

N H

CH3

N

N N O

4b

Pale yellow powder, mp: 231-233 °C, Yield = 64 %; IR (cm-1): 3300 (NH), 1650 (ArC=O), 1624 (C-4 C=O); 1H NMR 300 MHz (CDCl3) δ (ppm): 9.81 (1H, q, J = 3.3 Hz, NH-), 8.79 (1H, s , H-2), 8.02 (1H, d, J H-F =13.2 Hz, H-5), 7.42-7.40 (5H, m, Ar-H), 7.32 (1H, d, J =7.2 Hz, H-8), 3.98 -3.26 (9H, m, CH- cyclopropyl, piperazine 8H), 2.96 (3H, d, J =3.3 Hz, CH3-NH), 1.34-1.32 (2H, m, CH2 of cyclopropyl), 1.28-1.13 (2H, m, CH2 of cyclopropyl);

13

C NMR 75 MHz (CDCl3)

δ (ppm): 175.31, 170.44, 165.38, 146.64,

144.33, 144.19, 138.29, 135.11, 130.01, 128.57, 128.57, 112.83, 112.52, 111.41, 105.04, 50.24, 49.36 , 34.58, 25.78 8.14; HRMS (MALDI-TOF) for [C25H25FN4O3Na]+ [M+Na]+ calculated: 471.1803, found: 471.1809. 4.1.5.3. 7-(4-(3,4,5-Trimetoxybenzoylpiperazin-1-yl)) -1-cyclopropyl -6-fluoro-1,4dihydro-N-methyl-4-oxoquinoline-3-carboxamide 4c O F

OCH3 H3CO

O N H

CH3

N

N N

H3CO

4c

O

White powder, mp: 233-235 °C, Yield 73 %; IR (cm-1): 3300 (NH), 1643 (Ar-C=O), 1650 (C=O), 1622 (C=O); 1H NMR 300 MHz (CDCl3) δ (ppm): 9.78 (1H, q, J = 3.3 Hz, NH-), 8.75 (1H, s , H-2), 7.98 (1H, d, JH-F =12.6 Hz, H-5), 7.28 (1H, d, J = 6.6 Hz, H-8), 6.61 (2H, s, Ar-H), 4.1-4.2 (1H, m, cyclopropyl), 3.82 (6H, s, -OCH3), 3.80 (3H, s, 2-OCH3), 3.23-3.22 (4H, m, piprazine 4H), 2.92-2.91 (4H, m, piperazine 4H), 2.92 (3H, d, J = 3.3 Hz, CH3-NH), 1.30-1.26 (2H, m, CH2 of cyclopropyl), 1.23-1.10 (2H, m, CH2 of cyclopropyl); 13C NMR 75 MHz (CDCl3) δ (ppm): 175.29, 170.25, 165.42, 153.33, 146.68, 144.30, 144.16, 139.42, 130.39, 122.24, 112.59, 111.40, 105.06, 104.42, 60.85, 56.24,

-83-

Experimental 49.40, 47.36, 34.61, 25.80, 8.15; HRMS (MALDI-TOF) [C25H25FN4O3]+ [M]+ calculated: 539.2300, found: 539.2318. 4.1.5.4.7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-1cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4-oxoquinoline-3- carboxamide 4d O

O

F

O O N H

N

N H

CH3

N

N 4d

Yellow powder; m.p: 259-260 °C; Yield = 68%; IR(cm-1) 3400 (NH), 1685 (chalcone C=O), 1660 (amide C=O), 1645 (acetamide C=O), 1622(C-4 C=O); 1H NMR (300 MHz, CDCl3) δ (ppm): 9.89 (1H, q, J = 4.8 Hz, -NH), 9.31 (1H, s, -NH), 8.80 (1H, s, H-2), 8.047.99 (3H, m, Ar-H, H-5), 7.82-7.71 (3H, m, Ar-H, =CH), 7.63-7.62 (2H, m, =CH, H-8), 7.54-7.32 (5H, m, Ar-H), 3.48-3.44 (5H, m, piperazinyl 4 H, CH cyclopropyl), 3.28 (2H, s, CH2-CONH), 2.98 (3H, d, J = 4.8 Hz, CH3-N), 2.87-2.79 (4H, m, piperazinyl 4-H), 1.341.31 (m, 2H, cyclopropyl-H), 1.20-1.16 (m, 2H, cyclopropyl-H);

13

C NMR (75 MHz,

CDCl3) δ 188.78, 175.32, 168.21, 165.52, 146.63, 144.62, 141.44, 138.32, 134.75, 133.85, 130.53, 129.93, 128.90, 128.37, 121.79, 121.51, 120.69, 118.79, 112.82, 111.31, 104.73, 103.62, 61.94, 53.20, 49.90, 34.62, 25.83, 8.15 ; HRMS (MALDI-TOF) [C34H33FN6O4]+ [M]+ calculated: 631.2440, found: 631.2441.

-84-

Experimental 4.1.5.5.

7-(4-((4-((E)-3-(3,4,5-Trimetoxyphenylacryloyl)phenylcarbamoyl)methyl)

piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-N-methyl-4-oxoquinoline-3carboxamide 4e O F

O H3CO

O

H3CO

N H

N

CH3

N

N

N H

OCH3

O

4e

Pale yellow powder; m.p: 260-263 °C; Yield = 68%; IR (cm-1) 3400 (NH), 1690 (chalcone C=O), 1685 (acetamide C=O),1660 (amide C=O); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.88 (1H, bs, -NH), 9.86 (1H, s, -NH), 8.08 (1H, s, H-2), 8.07-8.04 (3H, m, Ar-H, H-5), 7.77-7.71 (3H, m, =CH, Ar-H), 7.43-7.35 (2H, m, H-8, =CH), 6.88 (2H, s, Ar-H), 3.96 (6H, s, -OCH3), 3.94 (3H, s, -OCH3), 3.88 (2H, s, -CH3), 3.50-3.43 (9H, m, CH cyclopropyl, 8H piperazinyl), 3.35 (2H, s, CH2), 2.94 (3H, d, J=3.4 N-CH3), 1.36-1.33 (m, 2H, cyclopropyl-H), 1.26-1.19 (m, 2H, cyclopropyl-H); 13C NMR (100 MHz, DMSO-d6) δ 188.89, 175.41, 175.41, 165.53, 154.9, 151.93,148.90, 146.72, 144.33, 144.23, 138.43, 134.08, 130.88, 130.36, 130.00, 122.26, 121.07, 118.91, 112.95, 112.53, 111.53, 106.57, 104.88, 61.91, 56.27, 49.90, 34.68, 34.68, 25.88, 8.25; HRMS (MALDI-TOF) for [C38H40FN5O7+Na]+ [M+Na]+ calculated: 720.2804, found: 720.2804. 4.1.5.6. (S)-9-Fluoro-3,7-dihydro-N,3-dimethyl-10-(4-methylpiperazin-1-yl)-7-oxo-2H[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide 9 O F

N H

N N

O CH3

N O 9

Pale yellow powder, mp : 219-220 oC, Yield 66%; IR (cm-1): 3300 (NH), 1622 (-C=O); 1H NMR (300 MHz) (CDCl3) δ (ppm): 9.86 (1H, q, J = 4.5, NH), 8.61 (1H, s, H-5), 7.68 (1H, d, J

H-F

= 12.3 Hz, H-8), 4.41-4.36 (2H, m, -C-2-H-, C-3-H), 4.28 (1H, d, J = 9 Hz -CH-),

-85-

Experimental 3.41-3.31 (4H, m, piperazine), 2.97 (3H, d, J = 4.5, CH3-NH-CO), 2.59-2.56 (4H, m, piperazine), 2.37 (3H, s, CH3-N pirazinyl), 1.54 (3H, d, J = 6.9 Hz, CH3-oxazine);

13

C

NMR 75 MHz , δ ppm, 175.23, 165.51, 154.03, 143.66, 139.4, 131.69, 131.50, 124.26, 105.19, 104.87, 68.10, 55.53, 54.75, 50.35, 46.17, 25.81, 18.21; HRMS (MALDI-TOF) for [C19H23FN4O3Na]+ [M+Na]+ calculated: 397.1646, found: 397.1650.

-86-

Experimental 4.1.6. General procedure for synthesis of fluoroquinolone hydrazides 6a-e: 4.1.6.1 Synthesis of fluoroquinolone methyl esters 5a-e and 10 To a cooled stirred suspension of the respective fluoroquinolone, (10 mmol) in DCM (50 mL), TEA (2.02 gm, 2.8 mL, 20 mmol) and ethylchloroformate (1.08 gm, 98 mL, 10 mmol) were added dropwisely. The mixture was stirred for further one hour at (0-5°C).[168] Highly pure methyl alcohol was added portion wise and stirring was continued for further four hours at room temperature. The organic layer was washed with super saturated solution of sodium bicarbonate (2 x 25 mL) and then washed by distilled water (1 x 25) dried over anhydrous sodium sulphate, filtered off and evaporated under reduced pressure. Residue is recrystallized from methanol/ethyl acetate. [196] 4.1.6.1.1 Methyl 7-(4-N-acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3-carboxylate 5a O

O

F

O

N

CH3

N

N 5a

O

White powder, mp: 241-239 oC (reported =240-242 oC)[189] , Yield= 78%. 4.1.6.1.2 Methyl 7-(4-N-benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3-carboxylate 5b O F

O O

N

CH3

N

N O

5b

White powder, m.p.: 243-240 °C, Yield 75 %; IR (cm-1): 1733(ester C=O), 1685 (ArC=O), 1624(C-4 C=O); 1H NMR 400 MHz (CDCl3) δ (ppm): 8.57 (1H, s , H-2), 8.05 (1H, d, J H-F = 12.8 Hz, H-5), 7.52-7.41 (5H, m, Ar-H), 7.30 (1H, d, J = 8 Hz, H-8), 3.93 (3H, s,

-87-

Experimental COOCH3), 3.71-3.67 (1H, m, CH- cyclopropyl), 3.49-3.31 (8H, m, piperazine 8H), 1.351.30 (2H, m, CH2 of cyclopropyl), 1.16-1.10 (2H, m, CH2 of cyclopropyl); 13C NMR 100 MHz (CDCl3) 172.95, 170.56, 166.39, 154.64, 152.17, , 148.54, 135.23, 130.10, 128.66, 127.19, 123.51, 113.64, 110.13, 105.48, 104.58, 61.92, 52.20, 50.84, 49.80, 35.39, 8.28. HRMS (MALDI-TOF) for [C28H30FN3O7]+ [M]+ calculated: 450.1829, found: 450.1840. 4.1.6.1.3 Methyl 7-(4-(3,4,5-trimethoxy)benzoylpiperazin-1-yl)-1-cyclopropyl-6fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate 5c O

O

F

OCH3 H3CO

O

N

CH3

N

N

H3CO

5c

O

White powder, mp: 247-245 °C, Yield 71 % ; IR (cm-1): 1730 (ester C=O), 1688 (ArC=O), 1623 (C-4 C=O); 1H NMR 400 MHz (CDCl3) δ (ppm): 8.59 (1H, s , H-2), 8.05 (1H, d, JH-F = 12 Hz, H-5), 7.39 (1H, d, J = 12.8 Hz H-8), 6.69 (2H, s, Ar-H), 3.96 (3H, s, COOCH3), 3.93 (6H, s, 2 OCH3), 3.94 (3H, s, OCH3), 3.70-3.72 (1H, m, CHCyclopropyl), 3.58-3.34 (8 H, m, Piperazine 8H), 1.44-1.40 (2H, m, CH2 of Cyclopropyl), 1.22-1.16 (2H, m, CH2 of Cyclopropyl);

13

C NMR 100 MHz (CDCl3) 172.70, 170.33,

166.47, 154.68, 153.44, 152.20, 148.47, 139.58, 138.42, 130.48, 122.98, 113.54, 113.31, 105.48, 104.58, 60.94, 56.37, 52.34, 50.11, 45.78, 35.37, 8.30. HRMS (MALDI-TOF) for [C28H30FN3O7]+ [M]+ calculated: 540.2416 found: 540.2144.

-88-

Experimental 4.1.6.1.4 Methyl 7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl) piperazin1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3- carboxylate 5d O

O

F

O O N H

O

N

CH3

N

N 5d

Pale yellow powder; m.p.: 245-243°C; Yield 80%; 1H NMR (400 MHz, CDCl3) δ (ppm): 9.32 (1H, s, NH), 8.85 (1H, s, H-2), 8.09 (2H, d, J = 13.6 Hz, H-5), 7.99 (1H, d, J

H-F

=

8.16 Hz, H-8), 7.86-7.76 (4H, m, Ar-H), 7.33-7.24 (6H, m, Ar-H, =CH), 7.26 (1H, d, J = 16 Hz, =CH), 3.94 (3H, s, COOCH3), 3.88 (2H, s, CH2), 3.43-3.33 (9H, m, CH cyclopropyl, 8H, piperazinyl), 1.33-1.32 (2H, m, cyclopropyl-H), 1.22-1.16 (2H, m, cyclopropyl-H);13C NMR (100 MHz, CDCl3) δ (ppm): 188.89, 173.09, 171.30, 166.29, 148.46, 145.20, 144.68, 141.57, 138.00, 134.91, 134.00, 130.60, 130.01, 129.00, 128.46, 122.10, 121.69, 118.91, 116.50, 113.42, 110.17, 104.99, 61.92, 53.27, 52.14, 49.90, 34.57, 8.22; HRMS (MALDI-TOF) for [C35H33FN4O5]+ [M]+ calculated:609.2513, found: 609.2526. 4.1.6.1.5 Methyl 7-(4-((4-((E)-3-(3,4,5-trimetoxyphenylacryloyl))phenylcarbamoyl) methyl) piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3carboxylate 5e O F

O H3CO

N

O

H3CO OCH3

N H

O O

CH3

N

N 5e

Pale yellow powder; m.p.: 250-253°C; Yield 85%; IR(cm-1) δ 1H NMR (400 MHz, CDCl3) δ (ppm): 9.16 (1H, s, NH), 8.78 (s, 1H, H-2), 8.03 (1H, d, J H-F =13.6, H-5), 7.76-7.62 (6H, m, Ar-H, CH=CH), 7.36-7.34 (1H, s, J = 8 Hz, H-8), 6.96 (2H, s, Ar-H) 3.94 (2H, s, -CH2) 3.91-3.88 (12H, m, 3-OCH3, COOCH3) 3.43-2.35 (9H, m, CH cyclopropyl, 8H,

-89-

Experimental piperazinyl), 1.33-1.32 (2H, m, cyclopropyl-H), 1.22-1.16 (2H, m, cyclopropyl-H);

13

C

NMR δ (100 MHz, CDCl3) (ppm): 188.94, 175.35, 168.40, 166.23, 153.53, 148.49, 144.91, 141.49, 134.34, 130.03, 121.09, 118.89, 105.72, 104.88, 62.01, 61.03, 56.28, 53.33, 52.16, 50.88, 50.03, 34.54, 8.21; HRMS (MALDI-TOF) [C38H39FN4O8]+ [M]+ calculated: 699.2830 found: 699.2819. 4.1.6.1.6.Methyl (S)-9-fluoro-3,7-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo2H-[1,4]oxazino[2,3,4-ij]quinoline-6- carboxylate 10 O F

O O

N N

CH3

N O 11

Pale yellow powder, m.p.: 222-223 oC (reported mp= 223-224 °C) [190], Yield= (88%). 4.1.7.2. Synthesis of fluoroquinolone hydrazide derivatives 6a-e and 11 To 50 mL of ethanol in a round flask (10 mmol) of fluoroquinolones ester was added then (0.5 gm, 10 mmol) of hydrazine hydrate 80 % was added and the mixture was refluxed for two hours. The mixture was evaporated under vacuum and crystalized from ethyl acetate. [196] 4.1.7.2.1.7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3-carbohydrazide 6a O F

O N H

N

NH2

N

N O

6a

Pale yellow powder, m.p.: 243-245 °C, Yield = 84 %; IR (cm-1): 3385-3200 (NH-NH2), 1690 (C=O), 1650 (hydrazide C=O), 1620 (C-4 C=O); 1H NMR 400 MHz (CDCl3) δ (ppm): 10.83 (1H, s, NH-), 8.79 (1H, s , H-2), 8.01 (1H, d, J =15.2 Hz, H-5), 7.32 (1H, d, J

-90-

Experimental H-F

= 6.9 Hz, H-8), 3.87 (2H , b , -NH2), 3.72 -3.25 (9H, m, CH- cyclopropyl H, piperazine

8H), 2.18 (3H, s, CH3-CO), 1.37-1.20 (4H, m, CH2 of cyclopropyl);

13

C NMR 125 MHz

(CDCl3) 174.81, 169.09, 165.73, 154.54, 152.07, 146.59, 144.49, 122.15, 112.99, 110.40, 105.09, 50.45, 46.19, 34.79, 21.32, 8.28; HRMS (MALDI-TOF) [C19H22FN5O3+Na]+ [M+Na]+ calculated: 410.1599, found: 410.1606. 4.1.7.2.2.7-(4-Benzoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3-carbohydrazide 6b O F

O N H

N

NH2

N

N O

6b

White powder, mp: 258-260 °C, Yield = 64 %; IR (cm-1): 3300-3200 (NH-NH2), 1690 (ArC=O), 1650 (hydrazide C=O), 1622 (C-4 C=O); 1H NMR 500 MHz (CDCl3) δ (ppm): 10.74 (1H, s, NH-), 8.73 (1H, s, H-2), 7.97 (1H, d, J H-F=14.4 Hz, H-5), 7.43 (5H, m, ArH), 7.31 (1H, d, J = 6.6 Hz, H-8), 4.14 (2H , b, -NH2) , 3.68 -3.22 (9H, m, CH- cyclopropyl 1 H, piperazine 8H), 1.39-1.09 (4H, m, CH2 and CH2 of cyclopropyl). 13C NMR 125 MHz (CDCl3) 174.66, 170.27, 165.27, 154.36, 146.36, 138.13, 135.04, 129.98, 128.52, 127.02, 122.03, 112.76, 112.76, 110.28, 104.98, 50.45, 46.19, 34.62, 8.12; HRMS (MALDI-TOF) [C24H24FN5O3+Na] + [M]+ calculated: 472.1755, found: 472.1757.

-91-

Experimental 4.1.7.2.3. 7-(4-(3,4,5-Trimethoxy piperazin-1-yl))-1-cyclopropyl-6-fluoro-1,4-dihydro4-oxoquinoline-3-carbohydrazide 6c O F

OCH3 H3CO

O N H

N

NH2

N

N

H3CO

6c

O

White powder, mp: 260-263 °C, Yield = 74 %; IR (cm-1): 3400-3200 (NH-NH2), 1670 (ArC=O), 1660 (hydrazide C=O), 1623 (C-4 C=O); 1H NMR 300 MHz (CDCl3) δ (ppm): 10.70 (1H, s, -NH-), 8.70 (1H, s , H-2), 7.90 (1H, d, JH-F =12.9 Hz, H-5), 7.28 (1H, d, J = 6.9 Hz, H-8), 6.63 (2H, s, Ar-H), 4.11 (2H , b, -NH2) , 3.83 (6H, s, OCH3), 3.82 (3H, s, OCH3), 3.82-3.24 (9H, m, CH- cyclopropyl 1 H, piperazine 8H), 1.33-1.13 (4H, m, CH2 and CH2 of cyclopropyl).

13

C NMR 75 MHz (CDCl3) δ (ppm): 174.71, 170.23, 165.30,

153.33, 151.55, 146.42, 144.35, 139.42, 138.17, 130.37, 112.92, 112.62, 110.34, 105.03, 104.42,

60.85,

56.24,

50.45,

47.20,

34.66,

8.18;

HRMS

(MALDI-TOF)

[C24H24FN5O3+Na]+ [M+Na]+ calculated: 562.2072, found: 562.2072. 4.1.7.2.4. 7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-1cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3- carbohydrazide 6d O F

O O N H

N

O N H

NH2

N

N 6d

Yellow powder, m.p: 268-269°C; Yield = 68%; IR(cm-1) 3430-3240 (NH-NH2), 1690 (chalcone C=O), 1685 (acetamide C=O), 1660 (hydrazide C=O), 1622 (C-4 C=O); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.59 (1H, s, -NH), 9.89 (1H,s , -NH), 8.61 (s, 1H, H-2), 7.87-7.84 (1H, d, JH-F= 13.6, H-5), 7.69-7.67 (3H, m, Ar-H, =CH), 7.58 (2H, d, J = 8.8 Ar-H), 7.49-7.48 (2H, m, =CH, H-8), 7.34-7.26 (5H, m, Ar-H), 4.59 (2H, b, NH2), 3.72 (2H, s, -CH2-), 3.43-3.35 (m, 9H, -N-CH cyclopropyl, 8H piperazinyl), 1.32-1.30 (m, 2H,

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Experimental cyclopropyl), 1.22-1.10 (m, 2H, CH-cyclopropyl);

13

C NMR (100 MHz, DMSO-d6) δ

(ppm): 185.41, 174.31, 168.66, 164.03, 154.39, 151.93, 148.90, 146.61, 144.79, 143.55, 138.98, 138.84, 128.39, 127.57, 127.09, 121.04, 120.97, 119.79, 111.81, 111.59, 110.13, 106.50, 61.97, 52.85, 49.90, 35.48, 8.06 ; HRMS (MALDI-TOF) [C34H33FN6O4]+ [M]+ calculated: 631.2440, found: 631.2441. 4.1.7.2.5.

7-(4-((4-((E)-3-(3,4,5-Trimetoxyphenylacryloyl)phenylcarbamoyl)methyl)

piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carbohydrazide 6e O F

O H3CO

O

H3CO OCH3

O

N

N H

NH2

N

N

N H 6e

Pale yellow powder, m.p. : 270-273°C; Yield = 68%; IR (cm-1) 3440-3230 (NH-NH2), 1690 (chalcone C=O), 1640 (acetamide C=O), 1622( C-4 C=O); 1H NMR (500 MHz, CDCl3) δ (ppm): 9.87 (1H, t, J = 5 Hz, -NH), 9.32 (1H, s, -NH), 8.80 (s, 1H, H-2), 8.038.00 (3H, m, Ar-H, H-5), 7.73-7.71 (m, 3H, Ar-H, =CH), 7.38 (1H, d, J =15 Hz, =CH), 7.33 (1H, d, J = 7 Hz, H-8), 6.84 (2H, s, Ar-H), 4.89 (2H, b, NH2), 3.90 (6H, s, -OCH3), 3.88 (3H, s, -OCH3), 3.72 (2H, s, -CH2-), 3.46-2.87 (9H, m, CH cyclopropyl, 8H, piperazinyl), 1.33-1.32 (2H, m, cyclopropyl-H), 1.22-1.16 (2H, m, cyclopropyl-H);

13

C

NMR δ (125 MHz, DMSO-d6) δ (ppm): 188.88, 175.35, 168.23, 165.75, 153.40, 146.65, 144.83, 141.39, 140.35, 138.34, 133.93, 130.28, 129.93, 120.94, 118.81, 112.78, 111.32, 106.62, 105.58, 104.75, 102.43, 61.94, 60.94, 56.16, 53.22, 49.93, 34.63, 34.61, 8.16; HRMS for [C37H39FN6O7]+ [M]+ calculated: 721.2758, found: 721.2758.

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Experimental 4.1.7.2.6.

(S)-9-Fluoro-3,7-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2H-

[1,4]oxazino[2,3,4-ij]quinoline-6-carbohydrazide 11 O F

N H

N N

O NH2

N O 11

Pale yellow powder, m.p.: 213-215 oC, Yield = 86%; IR (cm-1): 3430-3254 (NH-NH2), 1650 (hydrazide C=O), 1623 (C-4 C=O);

1

H NMR (500 MHz) (CDCl3) δ (ppm): 10.77

(1H, s , NH), 8.53 (1H, s, H-5), 7.58 (1H, d, J H-F = 12.5 Hz, H-8), 4.39-4.37 (1H, m, -CH2), 4.32-4.30 (1H, m, CH), 4.15 (2H, b, NH2) 3.37-3.29 (4H, m, piperazine 4H), 3.10-2.52 (4H, s, piperazine 4 H), 2.34-2.34 (3H, s, CH3), 1.55 (3H, d, J = 6.6 Hz, CH3-CH);

13

C

NMR 125 MHz (CDCl3), δ (ppm): 174.60, 165.50, 156.60, 154.63, 143.40, 139.30, 131.80, 124.12, 122.03, 109.93 , 104.91, 68.04, 55.59, 54.88, 50.45, 46.30, 18.21; HRMS (MALDI-TOF): calculated for [C18H22FN5O3]+ [M]+ 376.1779, found: 376.1778.

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Experimental 4.2. Biological experiments 4.2.1. Anticancer screening Compounds 3a, 4a, 3b, 4b, 3c, 4c, 8 and 9 were selected by the NCI for in vitro anticancer screening. The NCI in vitro anticancer screen utilizes 60 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The methodology of the NCI anticancer screening has been described in detail elsewhere (http://www.dtp.nci.nih.gov). [197] The prepared compounds were added at single concentration 10-5M and the culture was incubated for 48 hrs. End point determinations were made with a protein binding dye, sulforhodamine B (SRB). All the selected compounds reduced the growth of most of the cancer cell lines to 32% or less, so they are called active and subsequently passed for further evaluation toward panel of approximately sixty cancer cell lines. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers. Results for each compound were reported as the growth percent of the treated cells which are evaluated spectrophotometrically and compared to that of the untreated control cells. Mean graph midpoint (MG–MID) (differential activity patterns, bar scale) were constructed for each cell line to facilitate visual scanning of data for potential NCI patterns of selectivity, with bars depicting the deviation of the individual tumor cell lines from the overall mean value for all the cells tested. In the mean graph the center point is the mean of all growth inhibition (GI) percentages over all cell lines. Bars that point to the right are (positive values) denote resistance where the inhibition is greater than the average, while bars that point to the left (negative values), denote sensitivity to the selected cell line where the inhibition is less than the average. The negative numbers indicate cancer cell death. Delta means the logarithm of the difference between the (MG MID) and the log GI50 of the most sensitive cell line.

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Experimental 4.2.2. Antibacterial Activity Bacterial strains, chemicals and media: A P. mirabilis strain was isolated from a urine sample obtained from a patient suffering from urinary tract infection. Isolation and identification were performed according to standard procedures.

[192, 193]

The isolate was

cultured on trypticase soy agar (TSA, Difco) slants for daily use and stored in a trypticase soy broth medium (TSB, Difco) along with 15% glycerol, at –80oC for subsequent uses. 4.2.2.1. Evaluation of the antibacterial effect of the tested compounds and the determination of their MIC using broth microdilution method: The antimicrobial activity of the tested compounds against P. mirabilis was performed using a microdilution method according to procedures recommended by the Clinical Laboratory Standards Institute (CLSI, 2011). Briefly, 2-fold serial dilutions of the compounds were prepared in sterile Mueller Hinton Broth (MHB, Oxoid) for a testing concentration range of 0.244 -500µM while the standard urease inhibitor (AHA) was tested at concentrations of (0.0032 - 1mM). Then 100 μL from each dilution was transferred into the well of a microtiter plate and inoculated with 5 μL of standardized (1.5 × 107 CFU/mL) cell suspension. Plates were incubated at 37oC overnight and the lowest concentration of the tested compounds that prevented visible growth was recorded as the MIC.

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Experimental 4.2.3. Urease inhibitory activity In vitro inhibitory studies on urease were determined using indophenols method. It measures the liberation of ammonia from the reaction according to the following. [198] 1- The assay mixture, containing 50 μl (2 mg/mL) of enzyme and 100 μl of different concentration of the tested agents, was added to 850 μl of 25mM urea and preincubated for 0.5 h in water bath at 37°C. The urease reaction was stopped after 0.5 hr incubation. 2- After pre-incubation, 500 μl of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 500 μl of alkali reagent (1% w/v NaOH and 0.075% active chloride NaOCl) were added to 100 μl of incubation mixture and kept at 37°C for 0.5 hr. 3- The absorbance (A) was measured at λ = 625 nm using the following equation A = λbc, where c is the concentration of solution (mol/L), b the Length of the UV cell. [155, 194] 4- All experiments were performed in triplicate in a final volume of 1 mL, and AHA was used as a standard urease inhibitor. The concentration of each compound (relative IC50) that provokes an inhibition halfway between the minimum and maximum response was determined by monitoring the inhibition effect of various concentrations of compounds in the assay. 4.3. Protocol of Docking studies The automated docking simulation study was performed utilizing the Molecular Operating Environment (MOE®) version 2014.09, at Assiut University Faculty of Pharmacy, Chemical Computing Group Inc., and Montreal, Canada. The X-ray crystallographic structure of the target urease (1E9Y) was obtained from Protein data bank. The target compounds were constructed into a 3D model using the builder interface of the MOE® program. After checking their structures and the formal charges on atoms by 2D depiction, the following steps were carried out: 1.

The target compounds were subjected to a conformational search.

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Experimental 2.

All conformers were subjected to energy minimization, all the minimizations were performed with MOE until a RMSD gradient of 0.01 Kcal/mole and RMS distance of 0.1 Å with MMFF94X force-field and the partial charges were automatically calculated.

The enzyme was prepared for docking studies by: 1.

Hydrogen atoms were added to the system with their standard geometry.

2.

The atoms connection and type were checked for any errors with automatic correction.

3.

Selection of the receptor and its atoms potential were fixed.

MOE® Alpha Site Finder was used for the active site search in the enzyme structure using all default items. Dummy atoms were created from the obtained alpha Spheres.

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‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬

‫الملخص العربي‬

‫الملخص العربي‬ ‫تتناول هذه الدراسة تهتم بتصميم وتشييد لمشتقات جديدة من هيدروكساميك واميد وهيدرازيد‬ ‫للسيبروفولكساسن والليفوفلوكساسين وتقييم فاعليتهم البيولوجية كمضادات للسرطان ومضادات‬ ‫لميكروب البورتيوس ميرابالس وقدرتهم على تثبيط انزيم اليورياز مدعمة بدراسة النمذجة الجزيئية‬ ‫للمركبات المشيدة‪.‬‬ ‫الرسالة مقسمة ألربع اقسام رئيسية‪ :‬المقدمة الهدف من الرسالة النتائج والمناقشة باالضافة إلى‬ ‫المراجع ملخص الرسالة‪.‬‬ ‫‪-1‬المقدمة‪:‬‬ ‫تتناول المقدمة نبذة عامة عن مشتقات الكينولون وتأثيراتها الحيوية المختلفة وميكانيكية عمل هذه‬ ‫المركبات‪ ،‬باإلضافة إلى عالقة التركيب الكيميائي لمشتقات الكينولون بنشاطها كمضادات للبكتريا‬ ‫والسرطان‪ .‬باإلضافة إلى نبذة عامة عن انزيم اليورياز ‪ ،‬والتركيب البنائي البللوري للموقع النشط‬ ‫لالنزيم‪.‬‬ ‫‪-2‬الهدف من الدراسة‪:‬‬ ‫يشمل هذا الجزء الهدف من هذا الدراسة بما في ذلك تشييد مشتقات حمض الهيدروكساميك ‪،‬‬ ‫الهيدرازايدات واألميد للسيبروفلوكساسين ولليفوفلوكساسين والتقييم البيولوجي ضد بكتريا بروتيوس‬ ‫ميرابالس ونشاط تثبيط انزيم اليورياز‪ .‬باألضافة لدراسات النمذجة الجزيئية للمركبات التى تم‬ ‫تحضيرها على انزيم اليورياز للهيليكوباكتر بيلورى‪.‬‬ ‫‪ -3‬النتائج والمناقشة‪:‬‬ ‫يتضمن هذا الجزء شرح مفصل للنتائج والبيانات التي تم الحصول عليها من مراحل مختلفة من‬ ‫التحضير‪ ،‬والتوضيح الهيكلي والتقييم البيولوجي للمركبات المستهدفة (‪-3‬أد‪4 ،‬أ‪-‬د‪5 ،‬أ‪-‬د‪6 ،‬أ‪-‬د و‬ ‫‪)11-8‬‬ ‫وينقسم هذا الجزء لثالثة أجزاء‪:‬‬ ‫الجزء األول ‪:‬الجزء الكيميائي‪ ،‬والذي يشتمل على شرح الطرق المختلفة المستخدمة لتحضير‬ ‫المركبات الوسيطة والنهائية وإثبات التركيب الكيميائي لهذه المركبات بوسائل التحليل المختلفة‬ ‫كجهاز األشعة تحت الحمراء وجهاز الرنين النووي المغناطيسي لعنصري الهيدروجين‬ ‫والكربون وجهاز مطياف الكتلة عالي اإلستبانة‪ .‬وقد تم في هذه الرسالة تحضير المركبات الوسطية‬ ‫‪-2‬أد و ‪ 10‬وكذلك تشييد المركبات النهائية ‪3‬أ‪-‬د و ‪4‬أ‪-‬د و ‪5‬أ‪-‬د و ‪6‬أ‪-‬د‪.‬‬ ‫الجزء الثاني من النتائج والمناقشة ‪:‬جزء االختبارات البيولوجية‪ ،‬وينقسم إلى ثالثة أجزاء فرعية‪:‬‬ ‫أ‪ -‬تقييم المركبات المشيدة كمضادات للسرطان‪:‬‬ ‫حيث تم تقييم قدرة المركبات كمضادات للسرطان بالمعهد القومي للسرطان بالواليات المتحدة‬ ‫األمريكية‪ .‬ومن بين المركبات المحضرة تم اختيار المركبات ‪3‬أ‪4 ،‬أ ‪3 ،‬ب ‪4 ،‬ب‪3 ،‬ج‪ 4،‬ج‪ 8 ،‬و‬ ‫‪.9‬‬ ‫‪1‬‬

‫الملخص العربي‬

‫طبقا للنظام المتبع من المعهد القومي للسرطان ‪.‬وقد تم اختبار كل من هذه المركبات على ستين‬ ‫نوع من الخاليا السرطانية المختلفة ‪.‬وقد أوضحت النتائج أن المركبات المختبرة لم يكن لها تأثير‬ ‫على الخاليا السرطانية‪.‬‬ ‫ب‪-‬تقييم المركبات كمضادات لبكتريا البروتياس ميرابيالس‪:‬‬ ‫حيث تم تقييم قدرة المركبات المستهدفة (‪3‬أ‪ -‬د‪4 ،‬أ‪ -‬د‪5 ،‬أ‪ -‬د‪6 ،‬أ‪ -‬د و ‪ )11-8‬كمضادات لبكتيريا‬ ‫البروتياس ميرابالس التي تم فصلها من بول المرضي المصابين بعدوى المجاري البولية‪ .‬أجريت‬ ‫طريقة عزل والتعرف على البكتريا د وفقا لإلجراءات القياسية من طريقة ‪.Well Diffusion‬‬ ‫أظهرت بعض المركبات اخضعت لالختبار فعالية أفضل من السيبروفلوكساسين والليفوفلوكساسين‪.‬‬ ‫ج‪-‬تقييم النشاط المثبط النزيم اليورياز‪:‬‬ ‫حيث تم تقييم قدرة المركبات المستهدفة على تثبيط انزيم اليورياز (‪3‬أ‪ -‬د‪4 ،‬أ‪ -‬د‪5 ،‬أ‪ -‬د‪6 ،‬أ‪ -‬د و‬ ‫‪ )11-8‬باستخدام طريقة االندوفينول‪ .‬قد أظهرت بعض المركبات المختبرة فاعلية اقوى من‬ ‫األسيتوهيدروكساميك أسيد‪.‬‬ ‫الجزء الثالث ‪:‬دراسة النمذجة الجزيئية للمركبات ‪ ،‬ويحتوي هذا الجزء على النتائج التي تم‬ ‫الحصول عليها من‬ ‫دراسة اتحاد بعض المركبات المحضرة مع إنزيم اليورياز للهيلويكوباكتر بايلوري ‪.‬وقد‬ ‫أوضحت النتائج إتحاد جيد بين هذه المركبات وانزيم اليورياز والذي يتفق مع النتائج التي تم‬ ‫الحصول عليها من تقييم الفاعلية البيولوجية لهذه المركبات‪.‬‬ ‫‪-4‬الجزء التجريبي‪:‬‬ ‫يستعرض هذا الجزء الشرح التفصيلي للتجارب العملية المستخدمة‪ ،‬ويحتوي هذا الجزء على ثالثة‬ ‫أجزاء ‪:‬‬ ‫الجزء األول ‪:‬التجارب الكيميائية‪ ،‬ويتضمن التجارب العملية المختلفة المستخدمة لتحضير‬ ‫المركبات المستهدفة (‪3‬أ‪ -‬د‪4 ،‬أ‪ -‬د‪5 ،‬أ‪ -‬د‪6 ،‬أ‪ -‬د و ‪ )11-8‬كما يحتوي على النتائج المفصلة‬ ‫للتحاليل المختلفة التي تم إجراؤها للمركبات المحضرة‪.‬‬ ‫الجزء الثاني‪ :‬االختبارات البيولوجية‪ ،‬وبوضح هذا الجزء الطرق والخطوات المتبعة التي اجريت‬ ‫لتقييم المركبات المخلقة (‪-3‬أد‪4 ،‬أ‪ -‬د‪5 ،‬أ‪ -‬د‪6 ،‬أ‪ -‬د و ‪ )11-8‬كمضادات للسرطان و كمضادات‬ ‫لبكتريا البروتياس ميراباليس و مثبطات النزيم اليورياز‪.‬‬ ‫الجزء الثالث‪ :‬النمذجة الجزيئية‪ ،‬ويحتوى هذا الجزء يوضح الطريقة المتبعة والبرامج المستخدمة‬ ‫لدراسة النمذجة الجزيئية للمركبات المختارة‪.‬‬ ‫‪2‬‬

‫تصميم وتشييد والنمذجة الجزيئية والفاعلية البيولوجية‬ ‫لمشتقات جديدة من هيدروكساميك واميد وهيدرازيد‬ ‫الفلوروكينولون‬ ‫رسالة مقدمة من‬

‫الصيدلي‪ /‬محمد عبدهللا علي عبدهللا‬ ‫(بكالوريوس العلوم الصيدلية‪)2011-‬‬ ‫(كلية الصيدلة‪-‬جامعة المنيا)‬ ‫لالستيفاء الجزئي لدرجة الماجستير في العلوم الصيدلية‬ ‫(كيمياء صيدلية طبية)‬

‫تحت إشراف‬

‫أ‪.‬د ‪ /‬جمال الدين علي أحمد حسن أبورحمه‬ ‫أستاذ الكيمياء الطبية الصيدلية‬ ‫كلية الصيدلة جامعة المنيا‬

‫د‪ /‬هبة أحمد حسن‬

‫د‪ /‬الشيماء محمد نجيب عبدالحافظ‬

‫مدرس الكيمياء الطبية الصيدلية‬ ‫كلية الصيدلة جامعة المنيا‬

‫مدرس الكيمياء الطبية الصيدلية‬ ‫كلية الصيدلة جامعة المنيا‬ ‫قسم الكيمياء الطبية‬ ‫كلية الصيدلة‬ ‫جامعة المنيا‬ ‫المنيا‪ -‬مصر‬ ‫(‪)2016‬‬