Study of β-Lactamase Inhibitory Potential of Synthesized Ketophosph(on)ates and Phytochemicals from Apocynaceae and Compositae Families.
MUHAMMAD AKMAL KHAN
SESSION (2008-2012)
19-Ph.D.-GCU-Chem-04
DEPARTMENT OF CHEMISTRY
GOVERNMENT COLLEGE UNIVERSITY LAHORE.
i Study of β-Lactamase Inhibitory Potential of Synthesized Ketophosph(on)ates and Phytochemicals from Apocynaceae and Compositae Families.
Submitted to GC University, Lahore in partial fulfillment of the requirements for the award of degree of
DOCTOR OF PHILOSOPHY IN CHEMISTRY
By
MUHAMMAD AKMAL KHAN SESSION (2008-2012)
19-Ph.D.-GCU-Chem-04
DEPARTMENT OF CHEMISTRY
GOVERNMENT COLLEGE UNIVERSITY LAHORE.
ii DECLARATION
I, Muhammad Akmal Khan Roll No. 19-Ph.D.-GCU-Chem-04, student of Ph.D. in the subject of Chemistry, Session 2008-2012, hereby declare that the matter printed in the thesis titled “Study of beta-Lactamase Inhibitory Potential of Synthesized Ketophosph(on)ates and Phytochemicals from Apocynaceae and Compositae Families.” is my own research work and has not been printed, published and submitted as research work, thesis, publication or in any form in any University, Research Institution etc. in Pakistan or abroad.
Dated:------. ------Muhammad Akmal Khan
iii RESEARCH COMPLETION CERTIFICATE
Certified that the research work contained in this thesis; entitled “Study of β- Lactamase Inhibitory Potential of Synthesized Ketophosph(on)ates and Phytochemicals from Apocynaceae and Compositae Families” has been carried out and completed by Mr. Muhammad Akmal Khan; Roll No. 19-Ph.D.-GCU-Chem-04, under my supervision, during his Ph.D. (Chemistry) studies in the laboratories of the Department of Chemistry.
Dated: ------Supervisor Dr. Durre Shahwar Associate Professor, Department of Chemistry, Government College University, Lahore. Submitted through
------Prof. Dr. Islam Ullah Khan Chairman, Department of Chemistry, Government College University, Lahore. ------Controller of Examination, Government College University, Lahore.
iv CERTIFICATE OF EXAMINERS
Certified that the quantum and quality of the research work contained in this thesis entitled “Study of β-Lactamase Inhibitory Potential of Synthesized Ketophosph(on)ates and Phytochemicals from Apocynaceae and Compositae Families.” is adequate for the award of the degree of Doctor of Philosophy.
------Supervisor Dr. Durre Shahwar Associate Professor, Department of Chemistry, Government College University, Lahore. ------External Examiner Prof. Dr. C. M. Ashraf, Pakistan Representative, The Royal Society of Chemistry, UK
------Prof. Dr. Islam Ullah Khan Chairman, Department of Chemistry, Government College University, Lahore.
v Dedicated to All the Well-Wishers Of My Beloved Motherland “Islamic Republic of Pakistan”
vi ACKNOWLEDGEMENTS All praises to Almighty Allah, Who induced the man with aptitude, knowledge, sight to observe and mind to think. Peace and blessings of Allah almighty be upon the Holy Prophet, Hazrat Muhammad (Peace be upon Him) who exhorted his followers to seek for knowledge from cradle to grave. My joy knows no bound to express my cordial gratitude to my learned research adviser Dr. Durre Shahwar, Associate Professor, Department of Chemistry, GC University, Lahore. Her keen interest, scholarly guidance and encouragement were a great help throughout the course of this research work. I feel great pleasure in expressing my sincere gratitude and thanks to the most respected, honorable, Prof. Dr. Islam Ullah Khan, Chairperson, & Prof. Dr. M. Akram Kashmiri, Ex-Chairperson, Department of Chemistry, GC University Lahore, for providing all facilities to undertake this research work. I am also thankful to Dr. Athar Abbasi & Dr. Ahmad Adnan for their kind support and help. My cordial prays are for my beloved Father and Mother (late) who always remember me in their prayers. Their everlasting love, guidance and encouraging passion will remain with me Insha Allah till my last breath. All my family members; wife, brother and sisters are always a source of inspiration to me. I am thankful to them for their full support, encouragement and sincere prayers for my success. My heart-felt thanks are due to all my teachers, friends and those who contributed in this research work in any way, especially Brother Naeem Ahmad, Sami Ullah, Saif Ullah, Muhammad Asam, Safiq- Ur-Rehman, M. Nadeem Arshad Qadri, and Hafiz Muhammad Shafiq. I am also thankful for the support of my junior fellows especially Miss Afifa Saeed, Asma Yasmeen, Uzma Sana , Misbah Naz, Tania Khan and Mr. Muhammad Mansha. I express my feelings of gratitude to all the members of non-teaching staff of the Department, especially Mr. Shahzad Khan, Mr. Ejaz, Mr. Rahmat, Mr. Manzoor Mr. Farasat and Mr. Khalil for their constant help. Thanks and gratitude are due to Mr. Abdul Waheed (Chief Librarian), Mr. Qaiser, Miss Samreen and Mr. Ghulam Murtaza among the Main-Library staff. My worthy thanks are due to Mr. Shafiq Mughal (Treasurer), Mr. Waseem Shahzad (Deputy Treasurer), Mr. Qaisar Khan and Mr. Mubashar. I am also thankful to HEC (Higher Education Commission) for financial support under Indigenous PhD Fellowship Program. Throughout the course of my Ph.D I have had help from numerous people. The space here does not allow to thank everybody by name, I’m sorry. It is not my forgetfulness: I do remember smiling face of each & every one.
vii ABSTRACT Present work consists of study of β-Lactamase (BLase) inhibitory potential of phytochemicals from Apocynaceae & Compositae families and Ketophosph(on)ates. For this study; an easy, efficient & economical method was developed, used successfully and published. In this study, thirty two compounds were prepared and characterized by EIMS & 1H-NMR. These compounds comprise of: Diethyl (2-oxo-2-phenylethyl) phosphonate(1), Diethyl [(E)-1-benzoyl-2-phenylvinyl] phosphonate(2), Diethyl[(E)- 1-benzoyl-2-(2-hydroxyphenyl) vinyl] phosphonate(3), Diethyl [(1E,3E)-1-benzoyl-4- phenylbuta-1,3-dien-1-yl] phosphonate(4), Diethyl [(1E)-1-benzoyl-2-phenylprop-1- en-1-yl]phosphonate(5), Diethyl [(E)-1-benzoyl-2-(4-hydroxy-3-methoxyphenyl) vinyl] phosphonate(6), Diethyl benzyl phosphonate(7), Diethyl [2-(2-hydroxyphenyl)- 2-oxo-1-phenylethyl] phosphonate(8), Diethyl [(3E)-2-oxo-1,4-diphenylbut-3-en-1- yl] phosphonate(9), Diethyl(2-oxo-1,2-diphenylethyl) phosphonate(10), Diethyl (2,4- dioxo-1-phenylpentyl) phosphonate(11), Diethyl (2,4-dioxo-1,4-diphenylbutyl) phosphonate(12), Ethyl (diethoxyphosphoryl) acetate(13), Diethyl (oxiran-2- ylmethyl) phosphonate(14), Diethyl[2-(2-hydroxyphenyl)-1-oxiran-2-yl-2-oxoethyl] phosphonate(15), 6-Diethyl [(3E)-1-oxiran-2-yl-2-oxo-4-phenylbut-3-en-1-yl] phosphonate(16), Diethyl (1-oxiran-2-yl-2-oxo-2-phenylethyl) phosphonate(17), Diethyl (1-oxiran-2-yl-2,4-dioxopentyl) phosphonate(18), Diethyl (1-oxiran-2-yl-2,4- dioxo-4-phenylbutyl) phosphonate(19), Diethyl (2-nitrobenzyl) phosphonate(20), six derivatives (21-26) containing the Tetraethyl ethane-1,2-diylbis (phosphonate) motif, Diethyl methylphosphonate(27), Diethyl [2-(2-hydroxyphenyl)-2-oxoethyl] phosphonate(28), Diethyl [(3E)-2-oxo-4-phenylbut-3-en-1-yl] phosphonate(29), Diethyl (2-oxo-2-phenylethyl) phosphonate(30), Diethyl(2,4-dioxopentyl) phosphonate(31) and Diethyl (2,4-dioxo-4-phenylbutyl) phosphonate (32). Seventeen compounds showed BLase inhibition activity. The compounds 1, 12, 29 & 32 were found more active than Clavulanic acid (used as standard). Extraction and bioassay guided isolation of Cichorium intybus resulted in the purification and identification of ten compounds: [Lupeol (33), β-Sitosterol(34), p- hydroxy phenyl acetic acid(35), Isovanillic acid(36), Syringic acid(37), Vanillic acid(38), Esculetin(39), Scopoletin(40), Umbelliferone(41) and Kaempferol(42)]. Stigmasterol(43) was the only compound isolated from Ageratum conyzoides but six compounds: [Conessine(44), Kurchinin(45), Conimine(46), Kurchamine(47), Holaromine(48) & Kurchessine(49)] were extracted from Holarrhena antidysenterica. viii However two compounds: [Lupeol(32) & Campesterol(50)], were extracted from Carissa opaca while Quercetin(51) & Kaempferol(42) were isolated from Alstonia scholaris. β-Sitosterol(34), α-Amyrin(52) & Ursolic acid(53) came out of Calotropis procera. All the phytochemicals were subject to BLase inhibition study. In general these phytochemicals showed poor activity however Kurchamine(47), Holaromine(48) and Quercetine(51) were relatively more active in this respect.
ix CONTENTS List of Tables (xv) List of Figures (xvi) List of Schemes (xvii) List of Abbreviations/acronyms (xviii) 1. Introduction 1-20 1.1. Antibiotics 1 Fig-1.1: Introductory Facts and Core structures of Lactam Antibiotics 1 Table-1.1:- Some Common Agents Used for Antimicrobial Chemotherapy 2 1.1.1:- Mechanism of β-Lactam Antibiotic Activity 3 Fig-1.2: A comparison between the structure of Penicillins with that of D-alanyl-D-alanine-Peptidoglycan 3 1.1.2:-Resistance towards Antibiotics 4 Fig1.3: Inactivation of Antibiotic Molecule through Hydrolysis by Enzyme 4 1.1.2.1:-Strategy To Combat Bacterial Resistance 5 1.1.2.2:-Classification of Beta-Lactamases (BLase) 5 Fig-1.4: Ambler’s classification of enzyme BLase. 6 Table 1.2- Bush Functional Grouping of BLase 8 1.1.2.2.1:-Extended Spectrum BLases (ESBLases) 9 1.1.2.2.2:- Class-C or group-1 (AmpC)-BLases 10 1.1.2.2.3:-Carbapenemases 10 TABLE-1.3. Main families of BLases of clinical importance 11 1.1.3:- Beta-Lactamase Inhibitors 12 1.1.3.1.Methods to Screen BLase Inhibitors 12 Figure-1.5Antibiotic susceptibility assay by Disk Diffusion Method(Kirby-Bauer) 14 Figure-1.6.ESBLase positive results by the Double Disc Synergy Test (DDST). 15 1.2 Ketophosph(on)ates 16 1.3 Plants 18 2:- Literature Review 21-32 FIG. 2.1. Increase in numbers of group 1, 2, and 3 BLases from 1970 to 2009 23 FIG: 2.2 The structural configuration of CENTA 26 Fig. 2.3 Clavulanic acid 27 3.- Experimental 33-100 3.1:- General experimental Conditions, Instruments and Chemicals. 33 3.2- Preparation of Ketophosph(on)ates 34-63 x 3.2.1:- Diethyl (2-oxo-2-phenylethyl)phosphonate 34 3.2.2:- Diethyl [(E)-1-benzoyl-2-phenylvinyl]phosphonate 35 3.2.3- Diethyl [(E)-1-benzoyl-2-(2-hydroxyphenyl)vinyl]phosphonate 35 3.2.4- Diethyl [(1E,3E)-1-benzoyl-4-phenylbuta-1,3-dien-1-yl]phosphonate 36 3.2.5- Diethyl [(1E)-1-benzoyl-2-phenylprop-1-en-1-yl]phosphonate 37 3.2.6- Diethyl [(E)-1-benzoyl-2-(4-hydroxy-3-methoxyphenyl)vinyl]phosphonate38 3.2.7- Diethyl benzylphosphonate 39 3.2.8- Diethyl [2-(2-hydroxyphenyl)-2-oxo-1-phenylethyl] phosphonate 40 3.2.9- Diethyl [(3E)-2-oxo-1,4-diphenylbut-3-en-1-yl] phosphonate 41 3.2.10- Diethyl (2-oxo-1,2-diphenylethyl)phosphonate 42 3.2.11- Diethyl (2,4-dioxo-1-phenylpentyl)phosphonate 43 3.2.12- Diethyl (2,4-dioxo-1,4-diphenylbutyl)phosphonate 44 3.2.13- Ethyl (diethoxyphosphoryl)acetate 45 3.2.14- Diethyl (oxiran-2-ylmethyl)phosphonate 46 3.2.15- Diethyl[2-(2-hydroxyphenyl)-1-oxiran-2-yl-2-oxoethyl] phosphonate 47 3.2.16- Diethyl[(3E)-1-oxiran-2-yl-2-oxo-4-phenylbut-3-en-1-yl] phosphonate 48 3.2.17- Diethyl (1-oxiran-2-yl-2-oxo-2-phenylethyl)phosphonate 49 3.2.18- Diethyl (1-oxiran-2-yl-2,4-dioxopentyl)phosphonate 50 3.2.19- Diethyl (1-oxiran-2-yl-2,4-dioxo-4-phenylbutyl) phosphonate 51 3.2.20- Diethyl (2-nitrobenzyl) phosphonate 52 3.2.21- Tetraethyl ethane-1,2-diylbis (phosphonate) 52 3.2.22- Derivative A of tetraethyl ethane-1,2-diylbis (phosphonate) 53 3.2.23- Derivative B of tetraethyl ethane-1,2-diylbis(phosphonate) 54 3.2.24- Derivative C of tetraethyl ethane-1,2-diylbis(phosphonate) 55 3.2.25- Derivative D of tetraethyl ethane-1,2-diylbis (phosphonate) 56 3.2.26- Derivative E of tetraethyl ethane-1,2-diylbis(phosphonate) 57 3.2.27- Diethyl methylphosphonate 58 3.2.28- Diethyl [2-(2-hydroxyphenyl)-2-oxoethyl] phosphonate 59 3.2.29- Diethyl [(3E)-2-oxo-4-phenylbut-3-en-1-yl]phosphonate 60 3.2.30- Diethyl (2-oxo-2-phenylethyl) phosphonate 61 3.2.31- Diethyl (2,4-dioxopentyl) phosphonate 62 3.2.32- Diethyl (2,4-dioxo-4-phenylbutyl) phosphonate 63 3.3:-Bioassay Guided Extraction & Isolation from Plants 64-100 3.3.1:- Compositae Family 64 3.3.1.1:- Cichorium intybus [Kasni] 64-72 xi Scheme-3.1Bioassay guided Extraction, Fractionation &Isolation from C. intybus.65 3.3.1.2:- Ageratum conyzoides 73-75 Scheme-3.2Bioassay guided Extraction, Fractionation, Isolation from A. conyzoides74 3.3.2Apocynaceae Family 76 3.3.2.1:- Hollarrhena antidysenterica (Kurchi bark) 76-83 Scheme-3.3:- Bioassay guided Extraction, Fractionation and Isolation from H. antidysenterica bark. 77 3.2.2.2:- Carissa opaca 84-87 Scheme-3.4Bioassay guided Extraction, Fractionation & Isolation from C. opaca. 85 3.3.2.3:- Alstonia scholaris 88-90 Scheme-3.5Bioassay guided Extraction, Fractionation & Isolation from A.scholaris89 3.3.3:- Asclepiadaceae Family 91 3.3.3.1:- Calotropis procera 91-95 Scheme-3.6Bioassay guided Extraction, Fractionation & Isolation from C.procera 92 3.4:- Β-Lactamase (BLase) Inhibition Assays 96 3.4.1:-Development of an Efficient Assay to Screen β-Lactamase Inhibitors from Plant Extracts 96 3.4.2:- Preliminary Screening for BLase Inhibition Potential 96 Table-3.1: Assay Scheme and Observations for BLase Inhibitory Potential 97 3.4.3:- Spectrophotometric Assay for Determining BLasé Inhibion Activity using Nitrocefin 98 Figure-3.1:- Pictorial Demonstration of Table 3.1 100 4.- Results and Discussion 101-150 4.1- Ketophosph(on)ates 101 4.1.1:- Diethyl (2-oxo-2-phenylethyl)phosphonate 101 4.1.2:- Diethyl [(E)-1-benzoyl-2-phenylvinyl]phosphonate 102 4.1.3- Diethyl [(E)-1-benzoyl-2-(2-hydroxyphenyl)vinyl]phosphonate 103 4.1.4- Diethyl [(1E,3E)-1-benzoyl-4-phenylbuta-1,3-dien-1-yl]phosphonate 104 4.1.5- Diethyl [(1E)-1-benzoyl-2-phenylprop-1-en-1-yl]phosphonate 105 4.1.6- Diethyl[(E)-1-benzoyl-2-(4-hydroxy-3-methoxyphenyl)vinyl]phosphonate 106 4.1.7- Diethyl benzylphosphonate 107 4.1.8- Diethyl [2-(2-hydroxyphenyl)-2-oxo-1-phenylethyl] phosphonate 108 4.1.9- Diethyl [(3E)-2-oxo-1,4-diphenylbut-3-en-1-yl] phosphonate 109 4.1.10- Diethyl (2-oxo-1,2-diphenylethyl)phosphonate 110 4.1.11- Diethyl (2,4-dioxo-1-phenylpentyl)phosphonate 111 xii 4.1.12- Diethyl (2,4-dioxo-1,4-diphenylbutyl)phosphonate 112 4.1.13- Ethyl (diethoxyphosphoryl)acetate 113 4.1.14- Diethyl (oxiran-2-ylmethyl)phosphonate 114 4.1.15- Diethyl[2-(2-hydroxyphenyl)-1-oxiran-2-yl-2-oxoethyl] phosphonate 115 4.1.16- Diethyl[(3E)-1-oxiran-2-yl-2-oxo-4-phenylbut-3-en-1-yl] phosphonate 116 4.1.17- Diethyl (1-oxiran-2-yl-2-oxo-2-phenylethyl)phosphonate 117 4.1.18- Diethyl (1-oxiran-2-yl-2,4-dioxopentyl)phosphonate 118 4.1.19- Diethyl (1-oxiran-2-yl-2,4-dioxo-4-phenylbutyl) phosphonate 119 4.1.20- Diethyl (2-nitrobenzyl) phosphonate 120 4.1.21- Tetraethyl ethane-1,2-diylbis (phosphonate) 121 4.1.22- Derivative A of tetraethyl ethane-1,2-diylbis (phosphonate) 122 4.1.23- Derivative B of tetraethyl ethane-1,2-diylbis(phosphonate) 123 4.1.24- Derivative C of tetraethyl ethane-1,2-diylbis(phosphonate) 124 4.1.25- Derivative D of tetraethyl ethane-1,2-diylbis (phosphonate) 125 4.1.26- Derivative E of tetraethyl ethane-1,2-diylbis(phosphonate) 126 4.1.27- Diethyl methylphosphonate 127 4.1.28- Diethyl [2-(2-hydroxyphenyl)-2-oxoethyl] phosphonate 128 4.1.29- Diethyl [(3E)-2-oxo-4-phenylbut-3-en-1-yl]phosphonate 129 4.1.30- Diethyl (2-oxo-2-phenylethyl) phosphonate 130 4.1.31- Diethyl (2,4-dioxopentyl) phosphonate 131 4.1.32- Diethyl (2,4-dioxo-4-phenylbutyl) phosphonate 132 4.1.33- β-Lactamase (BLase) Inhibition Assay Results: 133 Table 4.1: Preliminary Screening of Ketophosph(on)ates for β-Lactamase Inhibition Activity 133 Table 4.2: Spectrophotometric Screening of Ketophosph(on)ates for β-Lactamase Inhibition Activity 135 4.2 Bioassay Guided Extraction & Isolation 136 Table 4.3:- Compounds Isolated from Plants 136 4.2.1 Composiate family 137 4.2.1.1 Cichorium intybus 137 4.2.1.1.1 CIRCHNe-1 : Lupeol (33) 137 4.2.1.1.2 CIRCHNe-2 : β-Sitosterol (34) 137 4.2.1.1.3- CIRCHAc-1: p-hydroxy phenyl acetic acid (35) 137 4.2.1.1.4- CIRCHAc-2: Isovanillic acid (36) 138 4.2.1.1.5- CIRCHAc-3: Syringic acid (37) 138 xiii 4.2.1.1.6- CIRCHAc-4: Vanillic acid (38) 138 4.2.1.1.7- CIREANe-1: Esculetin (39) 138 4.2.1.1.8- CIREANe-2: Scopoletin (40) 139 4.2.1.1.9- CIREANe-3: Umbelliferone (41) 139 4.2.1.1.10- CIREANe-4: Kaempferol (42) 139 4.2.1.2 Ageratum conyzoides 140 4.2.1.2.1 AC-Hex-Fr-2: Stigmasterol (43) 140 4.2.2 Apocynaceae family 140 4.2.2.1: Hollarhena antidysenterica 140 4.2.2.1.1- HA-CH-1 : Conessine (44) 141 4.2.2.1.2- HA-CH-2: Isoconessimine / Kurchinine (45) 141 4.2.2.1.3- HA-CH-3: Conimine (46) 141 4.2.2.1.4- HA-EA-1 : Kurchamine (47) 142 4.2.2.1.5- HA-EA-2 : Holaromine (48) 142 4.2.2.1.6- HA-BU-1 : Kurchessine (49) 142 4.2.2.2- Carissa opaca 142 4.2.2.2.1- CO-Hex-Basic Ether-1: Lupeol (33) 143 4.2.2.2.2- CO-Hex-Basic Ether-2: Campesterol (50) 143 4.2.2.3: Alstonia scholaris 143 4.2.2.3.1- AS-EA-1: Quercetin (51) 144 4.2.2.3.2- AS-EA-2: Kaempferol (42) 144 4.2.3:- Asclepiadaceae Family 144 4.2.3.1: Calotropis procera 144 4.2.3.1.1- CP-DCM-Fr-3-2: β-Sitosterol (34) 145 4.2.3.1.2- CP-DCM-Fr-3-3A: α-Amyrin (52) 145 4.2.3.1.3- CP-DCM-Fr-3-3B: Ursolic acid (53) 145 4.2.4- β-Lactamase (BLase) Inhibition Assay – Results 146 Table4.4 Preliminary Screening of Plant Extracts for BLase Inhibition Activity146 Table 4.5: Spectrophotometric Screening of Isolated Compounds for β-Lactamase Inhibition Activity 148 4.3.- Achievements and conclusions 150 7.- References 151-163
xiv List of Tables
Table # Title Page #
1.1 Some Common Agents Used for Antimicrobial Chemotherapy 2 1.2 Bush Functional Grouping of Beta-Lactamases 8 1.3 Main families of BLases of clinical importance 11 3.1 Assay Scheme and Observations for BLase Inhibitory Potential 97 4.1 Preliminary Screening of Ketophosph(on)ates for βLactamase Inhibition Activity 133 4.2 Spectrophotometric Screening of Ketophosph(on)ates for βLactamase Inhibition Activity 135 4.3 Compounds Isolated from Plants 136 4.4 Preliminary Screening of Plant Extracts for β-Lactamase Inhibition Activity 146 4.5 Spectrophotometric Screening of Isolated Compounds for β- Lactamase Inhibition 148
xv List of Figures
Figure # Title Page #
1.1 Introductory Facts and Core structures of Lactam Antibiotics 1 1.2 A comparison between the structure of penicillins with that of D-alanyl-D-alanine-Peptidoglycan 3 1.3 Inactivation of Beta-Lactam Antibiotic Molecule through Hydrolysis by Enzyme 4 1.4 Ambler’s Classification of enzyme BLase. 6 1.5 Antibiotic susceptibility assay by Disk Diffusion Method 14 1.6 ESBLase positive results by the Double Disc Synergy Test 15 2.1 Increase in numbers of group 1,2,3 BLases from 1970 to 2009 23 2.2 The structural configuration of CENTA 26 2.3 Clavulanic acid 27 3.1 Nitrocefin ( a cephalosporinic β -lactam compound ) 98 3.2 Pictorial Demonstration of Table 3.1 100
xvi List of Schemes
Scheme # Title Page #
3.1 Bioassay guided Extraction, Fractionation and Isolation from 65 Cichorium intybus.
3.2 Bioassay guided Extraction, Fractionation and Isolation from 74 Ageratum conyzoides..
3.3 Bioassay guided Extraction, Fractionation and Isolation from 77 Holarrhena antidysenterica bark.
3.4 Bioassay guided Extraction, Fractionation and Isolation from 85 Carissa opaca.
3.5 Bioassay guided Extraction, Fractionation and Isolation from 89 Alstonia scholaris.
3.6 Bioassay guided Extraction, Fractionation and Isolation 92 from Calotropis procera.
xvii LIST OF ABBREVIATIONS / ACRONYMS
ESBLs Extended-spectrum β-lactams BLAs β-lactam antibiotics PBP Penicillin Binding Protein BLase β-Lactamase ESBLases Extended-spectrum beta-lactamases BLIP BLase-inhibitor protein MBLases metallo-BLases NMC Non-metalloenzyme carbapenemase SME Serratia marcescens enzyme IMI Imipenem hydrolyzing BLase GES Guyana extended spectrum NDM New Delhi MBLase PADAC Pyridinium-2-azo-p-dimethylaniline chromophore AMC Amoxicillin+clavulanic acid CRO Ceftriaxone CAZ Ceftazidim ATM Aztreonam CFM Ceftazidime DDST Double Disc Synergy Test MDR Multi drug resistant XDR Extremely drug resistant NTS Non typhoidal Salmonellae MDRCIs Multi-Drug Resistant Clinical Isolates ICP-AES Inductively coupled plasma-atomic emission spectroscopy NG Neisseria gonorrhoeae MIC Minimum inhibitory concentration CIEA Ethyl acetate extract of Cichorium intybus CIAE Aqueous extract of Cichorium intybus HAE Hydroalcoholic extract TLC Thin layer chromatography DMSO Dimethylsulfoxide
xviii I commence in the name of ALLAH, the most Beneficent, the ever Merciful.
xix Chapter-1: Introduction
1:- INTRODUCTION 1.1. ANTIBIOTICS Antibiotics are drugs used to treat infections caused by germs. The first antibiotic; named penicillin was accidently discovered by Sir Alexander Fleming in 1928, later it came to be a beta-lactam. In early 1940s, Dr. Florey Chain and Dr. Heatley (Oxford University) purified penicillin and proved it a cure against specific bacterial infections.
Cyclic amides are known as Lactams
N N N H O H O O H
α -lactam ring β-lactam ring γ-lactam ring δ-lactam ring ε-lactam ring
Monobactam Penam(e.g.. Penicillin) Cephem (oxacephem)
Carbapenem Backbone
Fig-1.1: Introductory Facts and Core structures of Lactam Antibiotics
Since their discovery to-date, many chemical/structural derivatives have been developed from penicillin to improve its efficacy and combat resistance in bacteria. These derivatives are generally known as extended-spectrum β-lactams (ESBLs) (e.g. cephalosporins, carbapenems and monobactams). Since their discovery over the last 60 years, this class of antibiotics has been the most widely used one in human and
1 Chapter-1: Introduction
animal medicine. It makes about 60% (by mass) of all of the antibiotics used. It was all because in general they are safe for human and animal consumption and work very well to treat bacterial infections with very few side effects observed.
Table-1.1:- Some Common Agents Used for Antimicrobial Chemotherapy
Classification Generic Names
Amoxicillin, Amoxicilli+Clavulanate, Ampicillin, Cloxacillin, Penicillin Nafcillin, Penicillin-G, Penicillin-V, Piperacillin, Piperacillin+ Tazobactam, Ticarcillin+Clavulanate
Carbapenems Imipenem, Meropenem, Ertapenum.
Aminoglycosides Amikacin, Gentamicin, Tobramycin
Cephalosporins Cefadroxil, Cefazolin, Cephalexin, Cephalothin 1stgeneration Cephalosporins nd Cefaclor, Cefonicid, Cefoxitin, Cefuroxime 2 generation Cephalosporins Cefixime, Cefotaxime, Ceftazidime, Ceftizoxime, Cefepime, rd 3 generation Ceftriaxone
Macrolides Azithromycin, Clarithromycin, Erythromycin
Ciprofloxacin, Norfloxacin, Levofloxacin, Gatifloxacin, Fluoroquinolones Moxifloxacin Antifungal Amphotericin B, Fluconazole, Itraconazole, Other antifungal Medications medications Antituberculous Ethambutol, Isoniazid, Pyrazinamide, Rifampin, Other Medications antituberculous medications
Tetracyclines Tetracycline, Doxycycline
Clindamycin, Chloramphenicol, Metronidazole, Nitrofuratoin, Others Rifampin, Sulfadiazine / Trimethoprim, Vancomycin, Others
2 Chapter-1: Introduction
1.1.1:- Mechanism of β-Lactam Antibiotic Activity β-lactam antibiotics (BLAs) are bactericidal agents which inhibit the enzyme (transpeptidase) responsible for bacterial cell wall. To avoid bursting like a balloon, the bacterial cell needs a tough cell wall around cell membrane. The β-lactam ring of BLAs is similar to bacterial D-alanyl- D-alanine/ peptidoglycan. The target of BLAs is D-alanyl-D-alanine carboxypepidases /transpeptidases (DD-peptidases) which in the final stages of bacterial cell wall synthesis catalyze the formation of peptide cross- links of peptidoglycan. Synthesis of cell wall takes place on the external surface of cytoplasmic membrane; so is easily approached by antibacterial agents. PBP (Penicillin binding proteins) bind penicilline rather than D-alanyl-D-alanine i.e. the BLAs inhibit DDpeptidases by forming a more stable covalent acyl-enzyme complex with DD-peptidase which has a longer half-life than the complex formed with peptidoglycan. In this way the construction of bacterial cell wall is disrupted i.e. bacteria can’t produce the cell wall and ultimately bursts to death[1,2,3]. The mammalian outer cell structure is quite different from that of bacteria, i.e. they have only cell membrane with no cell wall. This difference makes BLAs highly specific for bacteria with few side effects even at very high concentrations [4].
S OH N O CH3 HN HN CH3 O NH2 O NH2
Fig-1.2: The mimicry of beta-lactam antibiotics to D-alanyl-D-alanine (D-Ala-D- Ala)-Peptidoglycan/ Peptide Glycan / Peptidyl Transferase (PBP: Penicillin Binding Protein).
3 Chapter-1: Introduction
1.1.2:- Resistance towards Antibiotics The abuse, overuse and misuse of BLAs as medicine and animal growth promoters have made bacteria/germs to develop resistance against them. So sometimes these drugs can not kill the germs and these germs are termed as antibiotic resistant. This resistance may be due to lack of drug penetration (porin mutations), increased efflux pumps activity or due to release of hydrolyzing β-lactamases (β- lactamhydrolases; BLase) that cleave the endocyclic amide bond in beta-lactam ring making the antibiotic ineffective[4,5,6]. Still another resistance mechanism is conformational change of PBPs (the enzyme for peptidoglycan synthesis) which ultimately results a reduced affinity with antibiotic molecule. In 1978-1980, Haemophilus influenzae strains with resistance due to this mechanism were first time isolated in New Zealand and were named as BLase-Negative-Ampicillin resistant (BLANR) Haemophilus influenzae[7] As the bacteria developed resistance against some BLAs, chemists developed new antibiotic derivatives to cheat the bacterial recognition. These derivatives were called ESBLAs (Extended Spectrum Beta-Lactam Antibiotics e.g. cephalosporins, carbapenems and monobactams). But the bacteria proved much determined to fight back the human efforts/successes and it continually evolved and changed existing BLase enzymes to inactivate these improved derivatives. These enzymes that can break down ESBLAs are known as the extended- spectrum beta-lactamases (ESBLases).
Penicillin Hydrolyzed Penicillin/Penicilloic acid
Fig-1.3: Inactivation of Beta-Lactam Antibiotic Molecule through Hydrolysis by Enzyme (β- Lactamase)[8].
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The gene for BLase production is carried on plasmids which are easily disseminated from one bacteria to the other. Hence ESBLases arise due to mutations in genes for common plasmid-mediated BLases. This mutation results in more pathogens with increased hydrolytic ability of the BLases for oxyimino compounds. It is due to the mutational alterations in the configuration of enzyme near its active site that increases its affinity[9].
1.1.2.1:-Strategy to Combat Bacterial Resistance To combat this chaotic situation we have two choices. One way to address this problem is to develop new derivatives of existing molecules or look for new classes of antibacterial compounds. But the option one seems to be less appealing as we already have reached almost the brim. Second option is to use BLase-inhibitors in combination with existing BLAs. But unfortunately, we are seeing resistance to new derivatives of antibiotics as well as to enzyme inhibitors. So scientists have to keep one step ahead of the resistant species in this struggle between drug development and evolution of resistance in germs. A newer appealing strategy is the development and use of BLase-inhibitor protein (BLIP). So far it has been proved effective against class-A BLases such as TEM-1 and SHV-1[10].
1.1.2.2:- Classification of Beta-Lactamases (BLase)
Currently a wide range of bacteria is known for production of BLases. Some of them include Aeromonous hydrophilia, Citrobacter, Xanthomonas maltophilia, Enterobacter Bacillus anthracis, B cereu, B fragilis, E coli, Streptococcus, Bacteroides, Staphylococcus epidermidis. Pseudomonas aeruginosa, Providencia, Haemophilus, Acinetobacter, and Branhamella. Traditionally the classification of BLases has been based on either functional characteristics of enzymes[11] or their primary structure[12]. Consequently at present two classification schemes for BLases in use are: The Ambler’s molecular classification and Bush Functional classification.
5 Chapter-1: Introduction
AMBLER's classification of enzyme beta-Lactamase
class-B Metallo 3 {inactivation of all beta-Lactam} beta-Lactamase (carbapenmase)
class-C 1 (inactivation of cephalosporin) (cephasporinase)
beta-lactamase
class-D 2d (inactivation of cloxacillin) (OXA type) 2be (inactivation of 3rd generation cephem: ESBLs, Toho-1)
2br (resistance of beta-lactamase class-A Serine inhibitor) beta-Lactamase (penicillinase)
2f (inactivation of carbapenem)
Fig-1.4 Ambler’s Classification of enzyme BLase.
The simplest grouping is by protein-sequences (nucleotide sequences with their deduced amino acid sequences, for genes that encode these enzymes) whereby on the basis of their conserved and distinguishing amino acid motifs they are classified into four groups. So on the basis of their primary structures and catalytic behaviour, BLases are classified into four groups: class A, B, C and D[12]. Class A, C and D BLases bear serine-residue/s in their active site. They resemble serine proteases and catalyze BLAs by forming an acyl-enzyme intermediate using their active-site embedded serine residue. Class A BLases are soluble enzymes. Class C BLases are
6 Chapter-1: Introduction
much similar to class A BLases but differ in that they are membrane bound. Class D BLases differ in primary sequence similarities from class A and C BLases, while class B BLases are quite different from the other three classes. They require divalent metal ion/s for their enzymatic activity hence known as metallo-BLases (MBLases) [12]. When isolated from their native source they have one/two Zn2+ ions bound to their active sites. Although they are isolated with Zn2+ embedded at their active site, yet other divalent metal cations (like Co2+, Cd2+, Mn2+, Hg2+ and Cu2+) support their catalytic activity to some extent. Penicillin and cephalosporin derivatives are the usual substrates for class B metallo-BLases. The same enzyme may differ greatly in its activity towards different substrates as well as enzymes from different classes catalyze same substrate to different extents e.g. 569/H/9-BLase-I (from B. cereus) is 8,800 times less active towards cephalosporin C than the metallo-BLase. Class B metallo-BLases can hydrolyze even those BLAs (such as carbapenems, cephamycins and imipenem) which are generally resistant to the serine BLases. Similarly they are also resistant to those BLase-inhibitors (like penem, penicillanic acid sulfone, 6-P- iodopenicillanic acid and clavulanic acid) which are generally active against other classes.
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Bush- Bush- Molecular Distinctive Inhibited by Defining Characteristic(s) Representative Jacoby Jacoby- Class substrate(s) enzyme(s) group Medeiros (subclass) CA or EDTA (2009) group TZB (1995) 1 1 C Cephalosporins No No Greater hydrolysis of E. coli, AmpC, cephalosporins than P99, ACT-1, benzylpenicillin, hydrolyzes CMY-2, FOX- Cephamycins 1, MIR-1 1e NIb C Cephalosporins No No Increased hydrolysis of GC-1, CMY- Ceftazidine and often other 37 oxymino β-lactams. 2a 2a A Penicillins Yes No Greater hydrolysis of PC1 benzylpenicillin than Cephalosporins 2b 2b A Penicillins, Yes No Similar hydrolysis of TEM-1, TEM- early benzylpenicillin and 2, SHV-1 Cephalosporins cephalosporins 2be 2be A Extended Yes No Increased hydrolysis of TEM-3, SHV- spectrum oxymino-β-lactams (cefotaxime, 2, CTX-M-15, cephalosporins, ceftrazidime, ceftriaxone, PER-1, VEB-1 monobactams cefepime, aztreonam) 2br 2br A Penicillins No No Resistance to Clavulanic acid, TEM-30, SHV- Sulbactam and tazobactam 10 2ber NI A Extended – No No Increased hydrolysis of TEM-50 spectrum oxymino-β-lactams combined cephalosporins, with resistance to Clavulanic monobactams acid, Sulbactam, and Tazobactam 2c 2c A Carbenicillin Yes No Increased hydrolysis of PSE-1, CARB- Carbenicillin 3 2ce NI A Carbenicillin, Yes No Increased hydrolysis of RTG-4 Cefepime Carbenicilin, Cefipime and Cefiprome 2d 2d D Cloxacillin Variable No Increased hydrolysis of OXA-1, OXA- Cloxacillin or Oxacillin 10 2de NI D Extended- Variable No Hydrlolyzes Cloxacillin or OXA-11, spectrum Oxacillin and Oxymino-β- OXA-15 Cephalosporins lactams 2df NI D Carbapenems Variable No Hydrolyzes Cloxacillin or OXA-23, Oxacillin and Carbapenems OXA-48 2e 2e A Extended- Yes No Hydrolyzes Cephalosporins, CepA spectrum Inhibited by Clavulanic acid but Cephalosporins not Aztreonam 2f 2f A Carbapenems Variable No Increased hydrolysis of SME-1 Carbapenems, Oxyimino-β- lactams, Cephamycins 3A 3 B (B1) Carbapenems No Yes Broad-spectrum hydrolysis IMP-1, VIM-1, including Carbapenems but not CcrA, IND-1 monobactams B (B3) L1, CAU-1, GOB-1, FEZ-1 3B 3 B (B2) Carbapenems No Yes Preferential hydrolysis of CphA, Sfh-1 Carbapenems NI 4 Unknown
CA, clavulanic acid; TZB, tazobactam. NI, not included. Table 1.2- Bush Functional Grouping of Beta-Lactamases (BLases) Table-1.2 includes an expanded version of the initially proposed scheme by Bush in 1989[13], then expanded in 1995[11]. Structural and functional classifications have been aligned as closely as possible in it.
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According to Bush classification the BLases are put into three main categories: Group 1 -cephalosporinases. Group 2 -serine β-Lactamases. Group 3 –MBLases (Metallo-β-Lactamases) Bush Classification is based on the ability to hydrolyze specific classes of BLAs and on the inactivation properties of the BLase-inhibitors such as tazobactam, clavulanic acid and sulbactam. Bush accounts substrate and inhibitor profiles in an attempt to group the BLases in ways that could be correlated with their phenotypes in clinical-isolates. A brief description of some of the groups/subgroups is as under: 1.1.2.2.1:- Extended Spectrum BLases (ESBLases) The emergence of bacterial species with ESBLases has created worldwide therapeutic/clinical problems in both human and veterinary medicine, as they can inactivate almost all BLAs[14,15,16]. They were observed 1st-time in Europe (1983) and then in USA (1989)[3,13,17]. They are a class of enzymes produced by some bacteria to make themselves resistant against certain ESBLAs. They hydrolyze all the members of oxyimino-cephalosporin, penicillin and monobactam families but not BLase inhibitors (carbapenems, cephamycins and related compounds). Predominantly they are derivatives of class A (e.g., TEM, SHV,CTX-M, and VEB families) Currently more than 157 TEM, 101 SHV, 65 CTX-M, and 5 VEB variants are known[18]. Generally BLase producers are typical gram negative organisms, like E. coli, K. pneumoniae and K. oxytoca but ESBLase production has also been observed in other organisms like Enterobacter, Salmonella Proteus, Pseudomonas, Serratia, Acinetobactor and Citrobacter species etc[10]. Following the first detection of ESBLases (ESBL, SHV-1) in Germany in 1983 in Klebsiella spp, TEM and SHV-ESBLases evolved greatly up to late 1990s and there were uninterrupted reports of outbreaks from France and Western Europe . Currently the whole world has become endemic area[17,19,20,21]. There are now more than 300 recognized ESBLases in a variety of gram-negative species conferring resistance to penicillins, monobactams, cephalosporins and even carbapenems. This emergence of enzymes has come out with poor patient outcomes, long hospital stays, increased use of antibiotics, so increased total health-care costs[3].
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1.1.2.2.2:- Class-C or group-1 (AmpC)-BLases They were first reported in the late 1970s. They are cephalosporinases and can hydrolyze all β-lactams except few. AmpC-BLases may be constitutive and inducible[22] and are further classified as: 2a- Chromosomal AmpC-BLases; They may be inducible or depressed and are found in Enterobacter spp., Providencia spp, C. freundii, M. morganii, Serratia spp[8]. Inducible cephalosporinase/AmpC-BLases are resistant to Tazobactam, Sulbactam and Clavulanic acid but are inhibited only by Aztreonam. 2b- Plasmid AmpC-BLases are not inducible and are encoded on highly mobile plasmids. They are able to hydrolyze monobactams, penicillins, cephamycins, extended and broad-spectrum cephalosporins and are resistant to BLase inhibitors but can not affect cefepime and carbapenems[23]. Transfer of AmpC-gene on plasmid allowed the dissemination to Proteus mirabilis, E. coli and Klebsiella spp., 1.1.2.2.3:- carbapenemases: When an Enterobacteriaceae produces an excess of ESBLase/ AmpC-BLase in combination with porin loss or acquires a carbapenemase, it becomes carbapenem resistant. According to their amino acid sequences carbapenemases fall into three groups. 3a- Class-A Carbapenemases are one of the two: • Chromosomal-Carbapenemases: NMC (non-metalloenzyme carbapenemase), SME (Serratia marcescens enzyme 1-3), IMI (imipenem hydrolyzing BLase 1-2) • Plasmid Carbapenemases: are not inhibited by EDTA, their substrates include penicillins, extend-spectrum cephalosporins, carbapenems and aztreonam. They include GES (Guyana extended spectrum 1-12) and KPCs. KPC 1-10 are more frequent in K.pneumoniae but are also found in Enterobacter spp. and Salmonella spp. 3b- Class-B Carbapenemases include Metallo-carbapenemases like IMP, NDM (New Delhi MBLase). After first MBLase report in 1960s from Bacillus cereus till now, 18 MBLases have been reported in several Gram negative bacteria. However they did not pose a serious threat of dissemination to other bacteria because they were chromosomally encoded. But the report of first plasmid-mediated MBLase (IMP-1 from Pseudomonas aeruginosa) from Japan (1991) and another type of acquired MBLase (VIM-1) from Italy (in 1999) has made the condition worrisome.
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3c- Class-D Carbapenemases include OXA carbapenemases. As new variants are frequently encountered, further divisions are made within these classes[24]. Enzyme Functional group No. of Representative enzymes familya of subgroup enzmesb,c
CMY 1, 1E 50 CMY-1 TO CMY-50
TEM 2b, 2be, 2br, 2ber 172 2b 12 TEM-1, TEM-2, TEM-13 2be 79 TEM-3, TEM-10, TEM-26 2br 36 TEM-30 (IRT-2), TEM-31 (IRT-1), TEM- 163 2ber 9 TEM-50 (CMT-1), TEM-158 (CMT-9)
SHV 2b, 2be, 2br 127 2b 30 SHV-1, SHV-11, SHV-89 2be 37 SHV-2, SHV-3, SHV-115 2br 5 SHV-10, SHV-72
CTX-M 2be 90 CTX-M-1, CTX-M-44 (Toho-1) to CTX-M- 92 PER 2be 5 PER-1 to PER-5 VEB 2be 7 VEB-1 to VEB-7 GES 2f 15d GES-2 to GES-7 (IBC-1) to GES-15 KPC 2f 9 KPC-2 to KPC-10 SME 2f 3 SME-1, SME-2, SME-3
OXA 2d, 2de, 2df 158 2d 5 OXA-1, OXA-2, OXA-10 2de 9 OXA-11, OXA-14, OXA-15 2df 48e OXA-23 (ARI-1), OXA-51, OXA-58
IMP 3a 26 IMP-1 to IMP-26 VIM 3a 23 VIM-1 to VIM—23 IND 3a 8 IND-1, IND-2, IND-2a, IND-3 to IND-7
TABLE 1.3. Main families of BLases of clinical importance [25]. a- numbers have been assigned based on primary aminoacid structures for these enzyme families (G. Jacoby & K. Bush, http://www.lahey.org/Studies/). b- Compiled through December 2009. c- The sum of the subgroups in each family does not always equal the total number of enzymes in each family, because some enzyme numbers have been withdrawn, and some enzymes have not been assigned a functional designation by the investigators who provided the amino acid sequence. d- GES-1, unlike other members of the GES family, has little detectable interaction with imipenem. e- 9 clusters of OXA carbapenemases with their individual members have been designated.
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1.1.3:- Beta-Lactamase (BLase) Inhibitors Several inhibitors for BLases have been described, such as: boronic acid derivatives and cloxacillin [26,27] for AmpC[28], EDTA and 2-mercaptoethanol against reversible inhibition of B. cereus 5/B/6 MBLase, penem, 6-P-iodopenicillanic acid, penicillanic acid sulfone and clavulanic acid for classes A, C & D (not Class B BLases). In the late 1990s, the structure and dynamics of metallo-BLases had been studied and a series of mercapto-acetic acid thiol esters and thiomandelic acid have been reported as metallo-BLase inhibitors. However, there is still a need to search, design and develop more effective inhibitors for metallo-BLases.
Recent reports show constant growth in variety of BLases (like in strains of Enterobacteriaceae, E. coli and K. pneumoniae) as well as metallo-BLases detection in an increasing number of pathogenic bacteria including Bacillus, Bacteroides, anthomomis, Croniontis, Pseudomonas, Sienotrophomouas, Klchsiella, Flavohacterium, Legionella, Enlcrohacter and Serratia. Current findings alarm the spreading of metallo-BLase genes among pathogenic bacteria. All this raises serious issues regarding the diagnosis and treatment in the future.
1.1.3.1. Methods to Screen BLase Inhibitors: A large-scale search for BLase inhibitors requires reliable, sensitive and flexible assays. A reasonable number of microbiological and/or instrumental assays/procedures have been reported for detection of presence and monitoring the activity/inhibition of BLases. But most of these methods are technically complicated and raise valid questions about interpretation while others require special reagents, not easily available. Rapid enzyme-based tests (chromogenic, iodometric or acidometric) and relatively slower growth based methods are in common use[29]. Acidometric and iodometric methods are two of the easy to perform and very cost effective methods[30, 31,32]. Acidometric methods are based on the pH change by the formation of acids such as penicilloic acid, While in iodometric methods iodine reacts with the acid produced on hydrolysis rather than BLA and the color of the starch iodine complex disappears[7]. The chromogenic method is the reference method where chromogenic cephalosporins {like Nitrocefin, CENTA, PADAC (Pyridinium-2-azo-p-dimethylaniline chromophore) and S1} are used. The color of
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the molecule changes due to cleavage of beta-lactam ring; an indication for BLase enzyme activity [30,33,34,35]. Both iodometric and acidometric methods can be performed in tubes, as strips or disks. Here is a brief description of one chromogenic cephalosporin (nitrocefin) method, three acidometric (strip, tube and disk) and three iodometric (tube, strip, and slide) methods that are/ could be used for BLase studies. In iodometric tube method, BLA (e.g. benzyl penicillin) is dissolved in phosphate buffer (pH 6). 100μL of the solution is taken to micro-titration plate. 3-4 colonies of bacteria are added to the solution. 30-60 minutes later , 20 μL of sterile 1% starch solution and iodine solution are added. Within 5 minutes disappearance of iodine colour indicates the presence and activity of BLase enzyme [30,36]. In iodometric strip method, antibiotic (e.g. penicillin) is dissolved in 0.2% starch solution. Whatman No-1 filter paper is soaked till saturation, dried and cut into strips (stored at -20°C until use). To carry out test strips are brought to room temperature in desiccator, moisturized with iodine and 2-3 colonies of bacteria are smeared. Colour change in 5 min means BLase is present and active [30]. Iodometric slide method using penicilline is performed as: Iodine solution is mixed with 150μL antibiotic solution (one million units of penicillin per mL of sterile distilled water). The mixture is dropped on the slide and bacterial colony is transferred to the solution. Having mixed with sterile loop 4% sterile starch solution is dropped. Fading away of purple color declares the bacteria to be BLase positive [30,37,38]. In acidometric tube method, Penicillin (or other BLA) is dissolved in phenol red solution and pH adjusted to 8.5 with NaOH. 100μL of this solution is taken to microtitration plate and bacterial colony transferred to this solution by loop. Violet color fading means bacteria is BLase positive [30,32]. For acidometric strip method, dissolve penicillin and bromocresol purple in NaOH solution. Saturate whatman No-1 filter papers with this and let them dry in air. Moisturize the strips with sterile distilled water and smear the bacteria on the strip with a loop. Within 5 minutes any change in purple color declares the bacteria to be BLase positive [30,36]. In acidometric disk method, Mix equal volumes of ethanol and penicillin solution (dissolve in NaOH solution and adjust to pH 9). Drop the mixture on to the disk and let it adsorb and dry. Then drop phenol red solution and wait for adsorption
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till dryness. Put a disk in 3 Mc-Farland turbid bacterial suspension. If colour of the disk turns yellow, the bacteria is BLase producer [30,39]. Chromogenic cephalosporin test is performed by moisturizing nitrocefin disk with sterile distilled water. Smear a loopful of bacteria on the disk. Appearance of red color on the disk in about one hour means bacteria is BLase positive [30,40]. The nitrocefin competition-assay is widely used to screen for BLase-inhibitors from plant extracts. Nitrocefin is a chromogenic BLase substrate that undergoes distinctive colour change from yellow (λmax 390 nm) to deep-red (λmax 486 nm) as the amide-bond in the BL-ring is hydrolyzed by BLase. These characteristics are suitable for convenient detection of BLase activities. Nitrocefin is commercially available but is prohibitively expensive (ca. $20,000/g) because of the circuitous routes to its synthesis. Though it has been used in competitive inhibiton studies in development work on BLase-resistant antibiotics using Direct Plate method, Slide method (avoid dessication), Broth method, Broken cell method, Paper disc spot method or spectrophtometric assay. Yet while working with nitrocefin one has to protect it from light to avoid degradation and store at -20 ºC. Stock solution could be stored at -20 ºC for only up to 2 weeks. Fig-1.5 & Fig-1.6 represent results of two real BLase inhibiton screeninig experiments for pure compounds. The results seem be good enough. But they produce much confusing output when used for screening of crude/semi purified plant extracts.
Figure-1.5.Antibiotic susceptibility assay by Disk Diffusion Method (Kirby-Bauer) In Fig-1.5, substantially wide inhibition zones around the disks show sensitivity of the test bacterium towards amoxicillin+clavulanic acid (AMC), ceftriaxone (CRO), and ceftazidim (CAZ), shown on left, middle & right respectively. While on the other hand no zones of inhibition around aztreonam (ATM) and
14 Chapter-1: Introduction
ceftazidime (CFM) disks (top & bottom respectively) show the bacterium-resistance towards aztreonam and ceftazidime [41].
Figure-1.6. ESBLase positive results by the Double Disc Synergy Test (DDST). In figure-1.6 the disk on right contained ceftriaxone(CFT) and that on left amoxicillin + clavulanic acid (AMC). There is clear extension of the inhibition-zone around CFT-disk in the direction of the disk having clavulanic acid. This enlargement of the inhibition-zone of the tested antibiotic (CFT) towards the AMC disk signifies a positive result, hence indicating the test organism is an ESBLase producer and clavulanic acid bahaved as enzyme inhibitor [41].
The above discussion makes it quite clear that preliminary screening of crude plant extracts is almost impossible using reported techniques. It is because the solution of the extracts is often brown, green or yellow and hinders the reasonable judgment of screening experiments. More over limited availability of purified BLase at large scale is another problem. Hence the scene demands development of some easy, efficient, economical and versatile assay to screen BLase inhibition activity.
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1.2 KETOPHOSPH(ON)ATES The effectiveness of β-Lactam antibiotics was compromised even at the beginning of their clinical application by the existence of BLases. BLases continue to represent a serious barrier to future clinical applications of the β-Lactams. All the commercially available BLase inhibitors contain a β -lactam ring and these compounds are active against the vast majority of Bush-Group-II enzymes (the most common BLases; produced by clinical-pathogens). Other compound classes have also been identified that have BLase-inhibitory activity. One such group is the phosphonates. Pratt has illustrated that phosphonate monoesters inhibit the Enterobacter cloacae P99 Group-1 serine β-lactamase by phosphonylation of the active-site serine. In addition, phosphonamidates have been shown to inhibit P99 via the same mechanism [42]. Phosphonics and analogues of naturally occurring phosphates have attracted considerable interest as potential regulators, mediators, or inhibitiors of metabolic processes. α-Ketophosphonates have a number of properties that make them attractive as reagents or intermediates for synthesis. These properties have stimulated a recent resurgence of interest in the chemistry of such compounds. For several years it has been known that the carbonyl of an α -ketophosphonate is activated towards attack by nucleophiles and that the carbon-phosphorus bond is readily cleaved. This property makes α-ketophosphonates potentially useful acylating agents, but also susceptible to hydrolysis and difficult to handle. The chemical properties of α- ketophosphonates are mainly determined by the phosphorus substituents [43]. Molecules with phosphonate functionalities are known to have significant importance in organic synthesis because of their anion stabilizing ability and their use in the construction of double bonds via the Horner-Wadsworth-Emmons reaction. Additionally, the phosphonates display a broad spectrum of activities including enzyme inhibitors such as the synthase HIV protease, rennin, phosphatasa, and PTPases; they also are antibacterial, antiviral, anti-fungal agents, and antitumor agents, herbicides, plant regulators, potent antibiotics , and in the antibody generation [44]. α-Ketophosphonates are fascinating and versatile compounds in organic synthesis. The chemical properties of α -ketophosphonates are mainly determined by the phosphorus substituents, but in general are a hybrid between ketones and
16 Chapter-1: Introduction
secondary amides. For instance, it is possible to derive hydrazones, imines, and oximes from the carbonyl function; to reduce α- ketophosphonates to the corresponding α-hydroxyphosphonates or use them in Wittig-Reactions. The C(O)_P bonds in these compounds are known to be sensitive towards hydrolysis. Therefore, handling ketophosphonates is not so easy and requires special precautions. The Michael–Arbuzov reaction is a general method for the preparation of α- ketophosphonates from acyl chlorides and trialkylphosphites. Oxidation of hydroxyphosphonates is another reaction for the preparation of ketophosphonates[45]. Chiral non-racemic α-hydroxy phosphonates are an important class of biologically active compounds that have a wide range of activities and are of interest in the design of drugs and bioregulators. They also serve as convenient precursors for a variety of biologically significant α-substituted phosphonates, in particular, chiral α- amino phosphonates[46].
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1.3. PLANTS Compositae (Asteraceae) is a very large and versatile family of Angiospermae. It has more than 23,000 reported species, consisting of 1,620 genera and 12 subfamilies. Its species are found from polar-regions to the tropics. The family bears a remarkable ecological and economical importance. Many of them are important in herbal medicine, especially in the areas with low or no access to modern medicine They are generally featured in scientific literature due to sesquiterpene lactones found in them[47,48]. In folk medicine Cichorium intybus has been reported as a treatment for sinus, gallstones, bruises, gastro-enteritis, and cuts as well as a general tonic. Major reported active classes of phytochemicals from Cichorium intybus are inulin, vitamins, minerals, fats, sesquiterpene lactones, mannitol and latex. Fructans have also been reported from roots. It also contains reduced tannins; though in low quantities. Fresh roots generally contain, , 68% inulin, 14% sucrose, 6% protein, 5% cellulose, , 4% ash, and 3% other compounds(w/w %). Dry root extracts contain about 98% inulin and 2% other compounds (W/W%) [49]. Cichorium intybus Linn. is well known for its toxicity to internal parasites. Though all parts contain volatile oils which are effective against intestinal worms, but majority of toxic constituents are concentrated in the root. The inulin [50] contained in it helps humans improving bowel function, weight loss and easing constipation [51] Many reports are there for its efficacy as antihepatotoxic agent [52-55]. Ageratum conyzoides Linn. is an annual herb that grows about 0.5–1 m high. It is native to tropical America, especially Brazil. It grows in dirty areas. Due to its toxic nature (tumors and liver lesions in humans) it has insecticide and nematicide properties. Lycopsamine and echinatine (pyrrolizidine alkaloids) are major active phytochemicals [56-60]. The Apocynaceae (dogbane family ) is a family of flowering plants, encompassing more than 150 genera, containing about 1,000 species of trees, shrubs, woody vines, herbs, and lianas,. According to AGP II, the family Asclepiadaceae has been merged in Apocynaceae [61]. Members in this family are found generally in tropical regions. Many of these species are poisonous and exude milky latex, if injured. Bushmen used latex from some plants of the family to poison arrows and the poisonous alkaloids from some species are important medicines; e.g. alkaloids;
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reserpine and rescinnamine from Indian Snakeroot are helpful for treatment of high blood pressure, alkaloids from are used in treating cancer, some compounds are good cardiac glycosides [62,63]. Hollarhena antidysenterica (Buch-Hem) wall. Ex. G. Don, is a small tree which is especially abundant in the sub-Himalayan tract. It is taken as one of the most valuable medicinal plants of India. For long times its seeds and bark have been used in the British Materia Medica. It finds its use in the treatment of dysentery, diarrhea, anemia, epilepsy, stomach pain and cholera. The tree is a rich source of alkaloids; connessimine, kurchine, conessine, conamine, conkurchine, conarrhinine, holarrhinene and isoconessimine [64,65]. Carissa opaca Stapf Ex. Haines is an evergreen shrub, growing up to 3.5 m high. It habitats drier parts of Pakistan, Burma, Sri Lanka and India (Himalayas up to 6000 ft) [66]. In phyto-therapy it finds its place as an effective hepatoprotector [67]. Disorders like respiratory dysfunction (such as asthma) are cured with decoction of its bark and leaves . In Pakistan its fruit and leaves are also used in treatment of cough and cardiac disorders. It also has shown antipyretic and aperients properties [68-70].
Alstonia is a genus having about 30 species; represented in Pakistan by only two cultivated species; Alstonia macrophylla Wall. Ex. G.Don and Alstonia scholaris (Linn.) R.Br. Alstonia scholaris (also known as Echites scholaris Linn) is an evergreen tropical shrub growing up to 17-20 m and is native to Pakistan, India, Sri Lanka, South East Asia, Africa, Northern Australia, Solomon Islands, Nepal, Thailand, Burma, Malaysia and Southern China. Its bark if injured secrets white milky latex; rich in poisonous alkaloids. In ethnopharmacy its bark is taken as a cure against/for curing coughs, sores, fever, snakebite, dysentery, bowl disorder, toothache, rheumatism, bacterial infection and malarial fever [71-80].
According to APG II, the family Asclepiadaceae is now treated as a subfamily (subfamily Asclepiadoideae) in the Apocynaceae. There are 348 genera, with about 2,900 species. It is represented in Pakistan by 23 genera and 41 species and is mainly distributed in the tropical and subtropical regions of the world [81,82]. Calotropis procera (Ait.) R.Br. (giant milkweed, locally known as aak) is an erect shrub, up to 2.4 m high. in sub-continent. It is used as herbal medicine to cure a variety of health disorders including asthma, cold, cough, diseases of skin, spleen,
19 Chapter-1: Introduction
liver & abdomen, leprosy, rheumatism, piles, ulcers, diarrhea and heart diseases [83- 88]. Flavonoids, alkaloids, glycosides, saponins, sterols and tannins have been reported from the plant [89,90].
Keeping in view the current scenario regarding antibiotics and resistant strains of microbes, the AIMS & OBJECTIVES of this study emerge as follows: i- to develop an esay, efficient and economical assay for preliminary screening of β-Lactamase (BLase) inhibition activity. ii- to prepare specified series of Ketophosph(on)ates which have not been so far evaluated, for BLase inhibition activity; to the best of our knowledge. iii- to screen a vast range of plants, herbs and shrubs. The selection of candidate plants is based on smart survey of ethnobotanical literature.
20 Chapter-2: Literature Review
2:- LITERATURE REVIEW
Mirela Flonta and Ariana Almas (2011) have described the increased number of MDR phenotypes (multi drug resistant) along with the emergence of XDR phenotypes (extremely drug resistant – which are susceptible only to colistin and tigecycline). For reliable clinical practices they urged the importance of correct detection and correct reporting of different mechanisms for resistance: ESBLases, class C BLases and carbapenemases [91].
Jabeen et al.2010 have reported an increase in isolation of ESBLase producing multidrug resistant nontyphoidal Salmonellae in Pakistan. They conducted retrospective analysis of laboratory data (1990-2006) to study resistance towards quinolone and cephalosporins (3rd generation) amongst NTS (non typhoidal Salmonellae) from Pakistan. 1967 NTS isolates were analyzed showing an alarming rise not only in number of ciprofloxacin resistance (from 23% in 2002 to 50.5% in 2006) but rise in tolerance level (i.e. increase in MIC values from 0.6 to 1.3 μg/mL) as well. ESBLase production was found in 98.7% isolates and ceftriaxone resistant NTS also increased. These isolates showed high resistance towards amikacin (44%), amoxicillin clavulanic-acid (57%), gentamicin (69%) and piperacillin tazobactam (30%). Surprisingly no resistance towards carbapenem was seen. To be worried about, in Salmonella typhimurium and in invasive isolates from children (less than one year) ceftriaxone resistance was significantly higher, They concluded this hike in quinolone and ceftriaxone NTS as a serious threat to public health. Continuous surveillance and use of proper screening tests for laboratory detection are strongly recommended [92].
Mamza et. al. (2010) have reported the occurrence of BLase producing E. coli and S. aureus in healthy as well as sick poultary chicks (broilers and layers). The two most important bacterial disease causing agents among poultry industry were found BLase positive with high level of resistance towards all twelve antibiotics. This is an implication for humans when consuming as well as handling chicken because of a risk of transferable MDR (multiple drug resistance). The enzymes found there, were designated as TEM-BLase, SHV-BLase, metallo-BLASÉ, CTX-M BLase, CMY- 21 Chapter-2: Literature Review
BLase (first described in 2006) and among carbapenems following enzymes were idenfied IPM, VIM and KPC-BLase. The following drugs were used: Ampicillin, cephalexin, penicillin-G, chloramphenicol, amoxicillin, ciprofloxacin, doxycycline, cefuroxime, gentamicin, tetracycline, erythromycin and tylosin [93].
Ashish Chauhan (2010) and Kumarasamy et. al. (2010) in their separate reports have described a serious threat about future of BLAs i.e. NDM-1(New Delhi metallo-BLase) which is an enzyme that has made bacteria resistant to a wide range of BLAs. It can inactivate even carbapenem family of antibiotics which has been usually seen as the most powerful against Gram negative bacilli. So they are rightly regarded as similar to but more resistant than ESBLases and bacteria with NDM-1 are often referred to as Superbugs. Because these bacteria are usually susceptible to only polymyxins and tigecycline so infections caused by them are difficult to treat. In December 2009, NDM-1 was first identified in a patient hospitalized in New Delhi infected by Klebsiella pneumoniae .Soon after that it was reported from Pakistan, Bangla Desh, United Kingdom and some other countries. Some repots also show the first time detection of IMP-1(the first metallo-BLase, conferring carbapenem resistance), from a Pseudomonas aeruginosa strain in Japan. Though currently gram negative bacteria such as E. coli and K. pneumoniae are known to produce NDM-1 but through horizontal gene transfer mechanism the gene for it can spread to other strains of bacteria. They have advised judicious and appropriate use of antibiotics to reduce the risk of NDM-1 spread. Antibiotic policies should be regularly formulated, monitored and modified as per need [94,95]. Shahla Mansouri and Samaneh Abbasi (2010) have reported prevalence of MDRCIs (Multi-Drug Resistant Clinical Isolates) of ESBLase Producing Enterobacteriaceae. Ten commonly used antimicrobials (Amoxicillin, Cephalexin, Ceftazidime, Ceftizoxime, Nalidixic acid, Ciprofloxacin, Tetracycline, Gentamicin, Trimethoprim-sulfamethoxazole) were tested against Gram-negative bacteria isolates from indoor and outdoor patients from three hospitals in southeast Iran. 500 isolates (found resistant to ≥3 antibiotics from different classes), were subjected to MICs and ESBLase- prevalence determination by agar dilution and double disc synergy methods respectively. Most frequent resistance was observed towards amoxicillin, trimethoprim/sulfamethoxazole, and tetracycline. Imipenem and ceftizoxime (with
22 Chapter-2: Literature Review
99.8% & 83% susceptibilities) were found the most active agents. A total of 53.8% of isolates were ESBLase producers. E. coli in outdoor and K. pneumoniae in indoor patient samples were most common. Indoor patient samples showed higher rate of resistance towards most antibiotics and ESBLase production. The high prevalence of ESBLase producers and isolates found resistant to 9 out of 10 drugs is distressing. Moreover presence of a single imipenem-resistant isolate is alarming as resistant organisms may be transferred from patient to patient and between different strains of the same or different species in hospitals. So this and other carbapenems be used with caution [96].
Bush and Jacoby (2010) have reported a review regarding the proliferation rate of certain functional classes of BLases from 1970-2009. This review also includes updated functional classification of BLases, along with their correlation with the broad based molecular classification. This updated system includes group-1 (class C)-cephalosporinases; group-2 (classes A and D)-broadspectrum, inhibitor-resistant, and ESBLses and serine-carbapenemases; and group 3 MBLases. On the basis of specific attributes of individual enzymes several subgroups with in each of the major groups have been introduced to describe a new BLase, a list of attributes has also been suggested [97].
FIG.-2.1 Increase in numbers of group 1, 2, and 3 BLases from 1970 to 2009. Group-1(class C cephalosporinases), group-2(class A and class D), and group-3 (class-B MBLases) are shown in black, blue and red respectively.
23 Chapter-2: Literature Review
Llarrull et. al. (2007) have reviewed and analyzed the issues like: current status of bacterial resistance proliferation, efforts about understanding of resistance mechanisms, enzyme inhibitor developement, improvement in existing antibiotics and other efforts to keep ahead of this problem. The study concludes to make serious efforts for development of new effective enzyme inhibitors because the diversity of BLase subgroups precdicts it almost impossible to have a single inhibitor for all BLases until we could search some common mechanistic features for all or large groups of BLases [98].
Butt, et. al. (2005) reported case of a sixty year old patient admitted (January 2003) to a tertiary care hospital of Rawalpindi, Pakistan. It was first metallo-BLase identification from Pakistan. This emergence of plasmid-mediated MBLase resistance in Pakistan is a matter of grave concern because it rapidly spreads to other Gram negative bacteria hence seriously restricting our therapeutic options. So the authors have advised a two-fold strategy to avoid the fast spread of MBLase: use of carbapenems be restricted and judicious as well, and facilities for early detection of MBLase in clinical isolates, be provided [99].
SUNG-KUN KIM (2002) in his doctoral thesis has emphasized to take serious attempts towards understanding the chemical reaction mechanism and then design new antibiotics and new BLase inhibitors. So far scientific community has been successful in keeping-ahead of the spread of drug resistance in bacteria, by slightly altering structures of existing BLAs. As a result, e.g. Cephalosporin family has passed through four generations. But as a fact choices for chemical manipulation of the existing antibiotic molecules are limited. Therefore it is very important to design new BLase inhibitors to be used in combination with existing antibiotics. It is only possible through detailed understanding of function of BLases [100].
SUNG-KUN KIM (2002) in his doctoral research work has employed the rational and combinatorial approaches to explore the inhibition of Bacillus cereus 5/B/6 metallo-BLase which catalyzes the hydrolysis of nearly all BLAs. His rational drug design approach (by chemical synthesis) to produce new metallo-BLase inhibitors was successful. His successful combinatorial chemistry approach (SELEX)
24 Chapter-2: Literature Review
led to an entirely new class of much efficient and specific metallo-BLase inhibitors [100]. Using ICP-AES (inductively coupled plasma-atomic emission spectroscopy) amount of metal (Zn2+) bound by metallo-BLase has been reported by SUNG-KUN KIM (2002). The stoichiometric study revealed that holo-enzyme has at least two Zn2+ ions and one extra Zn 2+ ion may be loosely held in some site other than active site which is not readily localized in crystal structure while apo-enzyme has no Zn2+ ion [100]. Rafael Llanes et. al. (2003) has reported a comparative study about four methods (acidimetric, inhibition, chromogenic & iodometric) for detection of BLase production by 70 isolates of Neisseria gonorrhoeae from Cuba. They found 100% correlation among all the four methods as well as between four methods and standardized penicillin-dilution-susceptibility test for BLase-Nonproducing Neisseria gonorrhoeae (NG). For BLase-producing NG strains, a perfect correlation was found between the chromogenic-method and penicillin-susceptibility testing except one and two strains which failed to give a +ve result for BLase with acidimetric/inhibition method and iodometric-method, respectively. A high concordance was observed between chromogenic method (a gold standard) and other BLase tests evaluated: Kappa Index (KI) = 0.98 for inhibition/acidimetric methods and KI = 0.97 for the iodometric method. Though all four methods were economical, accurate, reproducible, easily readable and easy to perform for screening yet they recommended the inhibition-method for testing BLase activity [101].
Hiroshi Udo (1995) has described the phosphorylation of Pkn2, the gene product produced in E. coil under a T7 promoter at both residues (serine & threonine). Phosphorylation at threonine residues, changes its molecular mass from 29 to 44 kD. This phosphorylated BLase becomes unable to be secreted into periplasmic space thus remains captured in the cytoplasmic and membrane fractions. Their data showed that only nascent BLase was phosphorylated and ultimately prevented the translocation of BLase across the membrane. 5 out of 20 threonine residues were found in phosphorylation site motifs [102].
25 Chapter-2: Literature Review
Jones et. al. 1982 characterized a newly synthesized, BLase-labile, chromogenic cephalosporin reagent (CENTA ) that changes color from light yellow (λ-max 340 nm) to chrome yellow (λ-max 405 nm) on hydrolysis of the beta-lactam ring. Structurally it resembled nitrocefin, cephaloridine, and cephalothin. Like Nitrocefin and PADAC, CENTA qualified as a diagnostic reagent for the studies of bacterial BLase inhibition studies, early rapid recognition of BLase-producing clinical isolates and measurements of cephalosporin serum concentrations [103].
O NH S OH N S S O O - + O HO O N O FIG: 2.2 The structural configuration of CENTA.
Reading et. al. (1977) have reported isolation of a novel, potent BLase inhibitor from Streptomyces clavuligerus. They have described the conditions for cultivation of S. clavuligerus as well as detection and isolation of clavulanic acid produced. Though clavulanic acid resembles the nucleus of penicillin yet it differs in having a beta-hydroxyethylidine substituent in the oxazolidine ring, bearing no acyl- amino side-chain and an oxygen despite sulfur atom. In their study addition of even very low concentrations of clavulanic acid reduced the MICs of cephaloridine and ampicillin against BLase-producing penicillin-resistant strains of E. coli, K. aerogenes, S. aureus and P.Mirabilis [104].
26 Chapter-2: Literature Review
Fig. 2.3 Clavulanic acid Abdou and Shaddy (2009) have described a full review covering last 20 years about versatile utility of bis-phophonates.The review has clearly declared these compounds as important precursors of the corresponding bisphosphonic acids showing remarkable pharmacologically interesting properties [105].
Perumal et. al. (2008) have described preparation of a series of aryl and arylmethyl β-aryl- β -ketophosphonates as potential BLase inhibitors. The compounds were quite effective against the class D OXA-1 β –Lactamase and class C BLase of Enterobacter cloacae but showed less activity against the class A TEM-2 enzyme and OXA-10 enzyme. Reduction of the keto group to form the corresponding beta- hydroxyphosphonates led to reduced inhibitory activity. Molecular modeling, based on the OXA-1 crystal structure, suggested interaction of the aryl groups with the hydrophobic elements of the enzyme’s active site and polar interaction of the keto and phosphonate groups with the active site residues Ser 115, Lys 212 and Thr 213 and with the non-conserved Ser 258. Analysis of binding free energies showed that the beta-aryl and phosphonate ester aryl groups interacted cooperatively within the OXA- 1 active site. As a whole their findings suggest that based on the ketophosphonate skeleton much potent inhibitors of class C and some class D BLases could be designed [106].
R. F. Pratt (1989) investigated and reported inhibition of a class C β- Lactamase of Enterobacter cloacae P99 by m-carboxyphenylphenylacetamidomethyl phosphonate (a phosphonate monoester). His study suggested that phosphonylation of an active site functional group makes enzyme to lose its activity. These findings
27 Chapter-2: Literature Review
indicate the discovery of a new class of antibiotics or at least an effective general class of beta-Lactamase inhibitors [107].
Cichorium intybus contains large amounts of fructans that have prebiotic- bifidogenic properties. Cavin et. al. (2002) reported enzyme inhibition effects of ethyl acetate extract (CIEA) of Cichorium intybus on the expression and activity of cyclooxygenase-2. In their study, they investigated the potential anti-inflammatory activity of CIEA. It remarkably inhibited prostaglandin E(2) (PGE(2)) production in human colon carcinoma HT29 cells treated with the pro-inflammatory agent TNF- alpha. They identified two independent mechanisms of action: (1) a drastic inhibition of the induction by TNF-alpha of cyclooxygenase-2 (COX-2) protein expression and (2) a direct inhibition of COX enzyme activities with a significantly higher selectivity for COX-2 activity. The inhibition of TNF-alpha-dependent induction of COX-2 expression was mediated by an inhibition of NF-kappa-B activation. A major sesquiterpene lactone of CIEA, the guaianolide 8-deoxylactucin, was identified as the key inhibitor of COX-2 protein expression present in CIEA. Altogether, the results strongly support Cichorium intybus as a rich source of functional food ingredient, combining prebiotic and anti-inflammatory properties [108].
On account of their health-promoting effects, now a days fructo- oligosaccharides and inulin (fructans) have got appreciable physiological and nutritional interest. Finke et. al. (2002 reported; fructans, fat, vitamins, minerals, latex mannitol, inulin and sesquiterpene lactones as major active components in Cichorium intybus. They performed extraction of fructans by continuous annular and fixed-bed conventional gel chromatography [109].
He et. al. (2002) reported the isolation and identification of twelve compounds from Cichorium intybus. Azelaic acid & daucosterol were isolated from it for the first time while 2,3,4,9-tetrahydro-1H-pyrido-(3,4-b)indole-3-carboxylic acid was reported first time from the genus Cichorium along with isolation and identification of α-amyrin, taraxerone, baurenyl acetate and β-sitosterol [110].
28 Chapter-2: Literature Review
Kim et. al. (1999) had reported the inhibition of mast cell-mediated immediate-type allergic reactions in vivo & in vitro. They investigated the effect of an aq. extract of C. intybus (CIAE) on mast cell-mediated immediate type allergic reactions. CIAE (0.1-1000 mg/ kg) dose-dependently inhibited systemic anaphylactic reaction induced by compound 48/80 in mice. Especially, CIAE inhibited compound 48/80-induced anaphylactic reaction 100% with the dose of 1000 mg/ kg [111].
Du et. al. (1998) had first time reported four compounds; baurenyl acetate, α-amyrin, β-sitosterol and taraxerone from Cichorium intybus. They investigated the chemical constituents of Cichorium intybus by means of solvent extraction, chromatography on silical gel and reported total seven compounds from its roots [112].
Dayie et. al. (2008) studied antimicrobial activity of Ageratum conyzoides against resistant and multiple resistant strains of Staphylococcus aureus NCTC 6571, Methicillin Resistant Staphylococcus aureus NCTC 12493 and clinical isolates of resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The plant extracts exhibited very good antibacterial activity against one or more of the strains tested [113].
Moreira et. al. (2007) studied and reported insecticidal activity of Ageratum conyzoides. Hexane fraction of the plant was found active against adults of R. dominica. Fractionation by silica gel column chromatography came out with isolation and purification of three compounds : 5,6,7,8,3', 4', 5'-heptamethoxyflavone, 5,6,7,8,3'-pentamethoxy-4', 5'-methylenedioxyflavone and coumarin[114]. Moura et. al. (2005) have reported antiinflammatory and chronic toxicity of hydroalcoholic extract (HAE) of Ageratum conyzoides leaves in rats. The group of rats treated with HAE (250 mg/kg body wt.; p.o.) had a 38.7% (p < 0.05) reduction in cotton-pellet granuloma. The development of chronically induced paw edema was also reduced significantly (p < 0.05) by the plant extract [115].
Rehman et. al. (2004) has described antimicrobial activity of compounds isolated from alkaline ethyl acetate extract of stem bark of Hollarhena
29 Chapter-2: Literature Review
antidysenterica. They isolated the alkaloid holarifine-24-ol from bark and tested its antibacterial and antifungal activity against ten microbes. The alkaloid was found active against all of them with smallest MIC as 20 microgram [116].
Siddiqui et. al. (2001) have described isolation and characterization of three compounds, pubadysone 11 alpha-hydroxy-18, 20-oxido-3-oxo-pregna-1, 4, 17 (20)- triene (1), puboestrene 3-acetoxy-17-oxo-1,3,5(10)-estratriene (2) and pubamide 3,18- dioxo-11 alpha-hydroxycona-1,4-diene (3 from the bark of Holarrhena pubescens (antidysenterica) [117].
Siddiqui et. al. (1993) have described isolation of an androstane derivative (17-dioxo-11-alpha hydroxyandrostane) and two steroidal alkaloids (4-dien-3-oxo-15 -alpha hydroxyl- N-demethylconanine & 4 -dien -3 -oxo conanine from the bark of Hollarhena antidysenterica [118].
Sahreen et. al. (2011) have reported Hepatoprotective effects of Carissa opaca (Apocynaceae) leaves. The methanolic extracts also possessed antioxidant activity other than hepatoprotective effects. They claim Carissa opaca as a strong candidate as a possible therapeutic alternative in hepatic disorders. The prevention against hepatotoxicity is attributed to their membrane stabilizing and antioxidant properties. The phytochemical analysis of Methanolic extract indicated the presence of flavonoids, tannins, alkaloids, phlobatannins, terpenoids, coumarins, anthraquinones and cardiac glycosides. Hyperoside, vitexin, isoquercetin, myricetin and kaempherol were also found present [119].
Sahreen et. al. (2010) evaluated antioxidant activity of fruits of Carissa opaca. Due to presence of large quantities of total phenolics and flavonoids antioxidant activity results were amazingly good in all the asays employed: , β-carotene and phosphomolybdate assay, iron chelating activity, hydrogen peroxide, ABTS, DPPH, superoxides, hydroxyl radical scavenging. So the fruit finds its place in functional foods [120]. Rai et. al. (2005) reported presence of twenty compounds, accounting 95% of essential oil extracted from flowers of Carissa opaca. Palmitic acid (82.5%) was
30 Chapter-2: Literature Review
major component followed by benzyl salicylate (6.0%), benzyl benzoate (4.6%) and (E,E)-α-farnesene (3.5%) [121].
Jain et. al. (2009) reported three alkaloids (picrinine, strictamine and nareline) first time from fruit pods of Alstonia scholaris. A new indole alkaloid, N- formylscholarine was also isolated and characterized by various spectral techniques [122].
Salim et. al. (2004) have reported isolation and identification of seven compounds from Alstonia scholaris: a new indole alkaloid, akuammiginone, and a new glycosidic indole alkaloid, echitamidine-N-oxide 19-O-beta-d-glucopyranoside, other than five known alkaloids; N(b)-demethylalstogustine N-oxide , akuammicine N-oxide, N(b)-demethylalstogustine echitaminic acid and echitamidine N-oxide [123].
Asolkar et. al. (1992) described in detail about Alstonia scholaris. According to their review and report Strictamine (a flower alkaloid) showed monoamine oxidase inhibitor activity, extracts from stem bark hypotensive and anticancer activity while leaves exhibit potential antibacterial activity against Mycobacterium tuberculosis, Shigella dysenteriae, Staphylococcus aureus, B. cereus, B. magaterium and Bacillus sbtilis.The major reported constituents are n-hexacosane, lupeol, ß-amyrin, palmitic and ursolic acids, strictanine and tetrahydroalastonine, picrinine, ß-sitosterol and betulin [124].
Murti et. al. (2011) experimented and reported antioxidant activity of bioactive extracts from Calotropis procera. Their results revealed antioxidant activity of fractions from methanolic extract (IC50 82± 5.23 mg/ml) comparable to that of ascorbic acid ((IC50 69.13± 4.08 mg/ml) [125].
Remya et. al. (2010) investigated and described antimicrobial and anti HIV-1 activity of bioactives from leaves and roots of Calotropis procera. They found effective the aqueous extract (hot) against growth of HIV, Inhibition of p24 antigen′s expression and against four different bacteria [126].
31 Chapter-2: Literature Review
Sangraula et. al. (2002) investigated anti-inflamatory activity of latex from C. procera. They used various chronic and acute models of inflammation; UV-induced erythema, vascular permeability, cotton pellet granuloma, carrageenan air pouch inflammation, carrageenan-induced oedema, and Freund's adjuvant-induced oedema. Oral administration of dry-latex from the plant reduced inflammation in all of the models. Its anti-inflammatory activity was comparable with general anti- inflammatory drugs [127].
32 Chapter-3: Experimental
3:- Experimental 3.1:- General experimental Conditions, Instruments and Chemicals.
UV-Visible spectra and absorbance of samples for different assays (β- Lactamase inhibition assay using Nitrocefin) were recorded on Shimadzu UV-1700 Pharma Spec (Shimadzu Corporation, Japan). 1H-NMR of isolated and synthesized
compounds were recorded in deuterated solvents (CDCl3 and CD3OD) at 400 MHz on Bruker AM 500 FT-NMR spectrometer. HREIMS spectra were recorded on JMX- HX-110 spectrometer. Column chromatography was performed on silica gel of E. Merck (type 60, 70-230 mesh size). Thin layer chromatography was performed on
silica gel-60-HF254 (E. Merck Darmstadt, Germany) preparatory plates (20x20 cm, 1.0 mm). Solvent system development and purity monitoring of the compounds was done
by TLC on pre-coated aluminum sheets (Silica Gel-60 F254, 0.2 mm, Merck, Germany). Depending upon nature; spots were visualized either under UV radiation {Model:CX- 20/FE offer both LW (UV-A/365nm) and SW (UV-C/254nm), UV irradiance by SPECTROLINE UV Equipment U.S.A.} or by any suitable spraying reagents. Dragondorff’s Reagent and Ceric-sulphate reagents were generally used as spraying agents. GCMS analysis was performed by GCMS-QP 2010 (Shimadzu) equipped with reverse phase capillary column DB-5ms ( 30 m long, 0.25 mm internal diameter and film thickness of 0.25 μm). Generally the chemicals were purchased from Merck, Fluka and Sigma Aldrich and used without further purification. Solvents used were of analytical grade (E. Merck and Panreac). Clavulanic acid was kindly provided by Schazu Laboratories Lahore.
33 Chapter-3: Experimental
3.2:- Preparation of Ketophosph(on)ates
3.2.1- Preparation of diethyl(2-oxo-2-phenylethyl)phosphonate(1-a) (1) Simple keto- and ester-substituted phosphonates are prepared by the Arbusov reaction - alkylation of a trialkyl phosphite with an alkyl bromide or iodide [128]. The procedure has been used as template to prepare compound (1) which was further derivatized to afford five compounds 2-6, using Jiwane et. al. (2009) procedure. It is a mild, high-yielding and general procedure for the condensation of active methylene with aldehydes and ketones. It provides products in high yields within minutes. The reaction procedure is operationally simple and amenable to large-scale preparations [129]. The procedural details of the methods and respective spectral data for the compounds 1-6 are described as follows: 0.02 moles of 2-bromoacetophenone were added to 0.02 moles of triethyl phosphate in a 50 ml round bottom flask. The mixture was stirred at room temperature for 15-20 minutes and monitored with TLC (n-Hexane : ethylacetate 1 : 1). The reaction mixture was put to column chromatography over silica gel. Solvent system swept from n-Hexane towards Ethylacetate. Fifteen fractions were collected and the target compound was found present in fraction thirteen
O O O Br H5C2O P OCH P OCH 2 5 + 2 5 OC2H5 H5C2O (1) Spectral Data: EIMS m/z (rel. int. %): [M]+ 256(4), 146(11), 123(7), 120(14), 106(8), 105(100), 91(7), 78(7), 77(36), 51(11), 29(11),
1 H-NMR (CD3OD, 400 MHz) δ: 8.23( 2H, d, J =7.6 Hz., CH aromatic, H-2,6), 7.49( 2H, t, J=7.6, 7.1Hz, CH-aromatic, H-3,5), 8.05 (1H, t, J=7.12, Hz, CH-aromatic, H-
4), 3.56 ( 2H, s, CH2, H-8), 4.11-3.55 ( 4H, m, J=7.15, CH2, H-9-11), 1.3 (6H, t,
J=7.15,, CH3, H-10,12) 34 Chapter-3: Experimental
3.2.2-Preparation of diethyl [(E)-1-benzoyl-2-phenylvinyl]phosphonate(1a-1) (2) The compound (1) was derivatized to prepare compound (2) using Jiwane et. al.(2009) procedure [129]. The procedural details of the method and respective spectral data for the compound (2) are described as follows: 0.001 moles of 1a (0.256 g), 0.001 moles of benzaldehyde (0.106 g) and 0.002 moles of sodium acetate (0.164 g) were taken in 3 ml of acetic acid in a 25 ml round bottom flask. The mixture was refluxed for 1 hour and monitored with TLC (n- Hexane : ethylacetate 1 : 1). 30 ml of ice water was added to reaction mixture and extracted twice with ethyl acetate (2 X 50 mL). The ethyl acetate layer was separated, further washed with water and dried to get 1a-1.
O O O O O P P OC H H 2 5 OC 2 H 5 OC H 2 5 + OC 2 H 5 Sodium acetate
(2) Spectral Data: EIMS m/z (rel. int. %): [M]+ 344(2), 254(1), 239(1), 207(13), 130(3), 119(10),105(80), 102(20), 90(5), 77(100), 51(5), 29(8),
1 H-NMR (CD3OD, 400 MHz) δ: 8.07(2H, d, J=7.9 Hz, CH-aromatic, H-2,6), 7.5 (2H, t, J=7.9, 7.1 Hz, CH-aromatic, H-3.5), 7.62 (1H, t, J=7.12, Hz, CH-aromatic, H-4),
4.1(4H, m, J=7.1 HzCH2, H-9, H), 1.3 (6H,t,J=7.1, Hz, CH3, H-10,12), 7.79 (1H, s,
CH, H-13 ), 7.72(2H, d, J=7.7Hz,CH-aromatic, H-2’,6’), 7.5 (2H,t, J=7.1,7.7 Hz, CH- aromatic, H-3’,5’), 7.55 (1H, , J=7.1Hz, CH-aromatic, H-4’)
3.2.3- Preparation of diethyl [(E)-1-benzoyl-2-(2-hydroxyphenyl) vinyl] phosphonate (1a-2) (3) The compound (1) was derivatized to prepare compound (3) using Jiwane et. al. (2009) procedure [129]. The procedural details of the method and respective spectral data for the compound (3) are described as follows:
35 Chapter-3: Experimental
0.001 moles of 1a(0.256 g), 0.001 moles of salicylaldehyde/ 2- hydroxybenzaldehyde (0.122 g) and 0.002 moles of sodium acetate (0.164 g) were taken in 3 ml of acetic acid in a 25 ml round bottom flask. The mixture was refluxed for 3 hours and monitored with TLC (n-Hexane : ethylacetate 3 : 2). 30 ml of ice water was added to reaction mixture and extracted twice with ethyl acetate (2 x 50 mL). The ethyl acetate layer was separated, further washed with water and dried to get 1a-2.
O O O O O P P OC H H 2 5 OC 2 H 5 OC H 2 5 + OC 2 H 5 Sodium acetate O H O H
(3) Spectral Data: EIMS m/z (rel. int. %): [M]+ 360(2), 255(1), 254(1), 118(20), 117(9), 105(79), 93(1), 77(100), 51(4), 1 H-NMR (CD3OD, 400 MHz) δ: 8.07 (2H, d, J=7.9 Hz, CH-aromatic, H-2,6), 7.5 ( 2H,t, J=7.9,7,1 Hz, CH-aromatic, H-3,5), 7.6 (1H,,t, J=7.1, Hz, CH-aromatic, H-4),
4.1 (4H, m, J=7.1 Hz, CH2, H-9,11), 1.3 (6H, t, J=7.1 Hz, CH3, H10, 12), 7.91 (1H, s, H-13 ), 6.7 (1H, d, J=7.3 Hz, CH-aromatic, H-3’), 7.78 (1H, t, J=7.3,7.6 Hz, CH- aromatic, H-4’), 7.14 (1H, t, J=7.6, 7.9 Hz, CH-aromatic, H-5’), 7.7 (1H, d, J=7.9 Hz, CH-aromatic, H-6’).
3.2.4- Preparation of diethyl [(1E,3E)-1-benzoyl-4-phenylbuta-1,3-dien-1-yl] phosphonate. (1a-3) (4) The compound (1) was derivatized to prepare compound (4) using Jiwane et. al. (2009) procedure [129]. The procedural details of the method and respective spectral data for the compound (4) are described as follows: 0.001 moles of 1a(0.256 g), 0.001 moles of cinnamaldehyde/ (2E)-3- phenylprop-2-enal (0.132 g) and 0.002 moles of sodium acetate (0.164 g) were taken in 3 ml of acetic acid in a 25 ml round bottom flask. The mixture was refluxed for 1 hour and monitored with TLC (n-Hexane : ethylacetate 60 : 40). 30 ml of ice water
36 Chapter-3: Experimental
was added to reaction mixture and extracted twice with ethyl acetate (2 x 50 mL). The ethyl acetate layer was separated, further washed with water and dried to get 1a-3.
H O
O O O O P P OC H 2 5 OC 2 H 5 OC H 2 5 + OC 2 H 5 Sodium acetate
(4) Spectral Data: EIMS m/z (rel. int. %): [M]+ 370(3), 293(1), 265(1), 235(16), 234(100), 233(35), 156(20), 137(1), 128(20), 127(50), 105(28), 79(1), 77(45), 50(5),
1 H-NMR (CD3OD, 400 MHz) δ: 8.04 (2H, d, J=7.9 Hz, CH-aromatic, H-2,6), 7.5 (2H, t, J=7.9, 7.1 Hz, CH-aromatic, H-3,5), 7.62(1H,t, J=7.12, Hz, CH-aromatic, H-
4), 4.1 (4H, m, J=7.1 Hz, CH2, H-9, 11 ), 1.3 (6H, t, J=7.1 Hz, CH3, H-10,12), 7.3 (1H, d, J=11.3 Hz, CH, H-13), 7.01(1H, t, J=11.3, 13.2 Hz, CH, H-14), 6.9 (1H, d, J=13.2 Hz, CH, H-15), 7.7 (2H, d, J=7.7 Hz, CH-aromatic, H-2’,6’), 7.26 (2H, t, J=7.7, 7.1 Hz, CH-aromatic, H-3’,5’), 7.25 (1H, d, 7.1 Hz, CH-aromatic, H-4’).
3.2.5- Preparation of diethyl [(1E)-1-benzoyl-2-phenylprop-1-en-1-yl] phosphonate. (1a-4) (5) The compound (1) was derivatized to prepare compound (5) using Jiwane et. al. (2009) procedure [129]. The procedural details of the method and respective spectral data for the compound (5) are described as follows: 0.001 moles of 1a(0.256 g), 0.001 moles of acetophenone/ 1-phenylethanone (0.12 g) and 0.002 moles of sodium acetate (0.164 g) were taken in 3 ml of acetic acid in a 25 ml round bottom flask. The mixture was refluxed for 2 hours and monitored with TLC (n-Hexane : ethylacetate 60 : 40). 30 ml of ice water was added to reaction mixture and extracted twice with ethyl acetate (2x50 mL). The ethyl acetate layer was separated, further washed with water and dried to get (5)
37 Chapter-3: Experimental
O O O O O P P OC H CH 2 5 3 OC 2H 5 OC H + 2 5 OC 2H 5 Sodium acetate CH 3
(5) Spectral Data: EIMS m/z (rel. int. %): [M]+ 358(1), 281(1), 256(1), 254(1), 222(10), 221(100), 144(14), 115(4), 117(20),105(31), 91(27), 77(82), 51(46), 39(27).
1 H-NMR (CD3OD, 400 MHz) δ: 8.0(2H, d, J=7.9 Hz, CH-aromatic, H-2,6), 7.51 (2H, t, J=7.9, 7.1 Hz, CH-aromatic, H-3,5), 7.6(1H, t, J=7.12, Hz, CH-aromatic, H-4),
4.12 (4H, m, J=7.09 Hz, CH2, H-9, 11), 1.3 (6H, t, J=7.1, Hz, CH3, H-10, 12), 7.55 (2H, d, J=7.17 Hz, CH-aromatic, H-2’,6’), 7.4 (2H, t, J=7.1, 7.27 Hz, CH-aromatic, H-3’,5’), 7.47 (1H, t, J=7.1, Hz, CH-aromatic, H-4’)
3.2.6- Preparation of diethyl [(E)-1-benzoyl-2- (4-hydroxy-3-methoxyphenyl) vinyl] phosphonate (1a-5) (6) The compound (1) was derivatized to prepare compound (6) using Jiwane et. al. (2009) procedure [129]. The procedural details of the method and respective spectral data for the compound (6) are described as follows: 0.001 moles of 1a(0.256 g), 0.001 moles of vanillin/ 4-Hydroxy-3- methoxybenzaldehyde (0.152 g) and 0.002 moles of sodium acetate (0.164 g) were taken in 3 ml of acetic acid in a 25 ml round bottom flask. The mixture was refluxed for 3 hours and monitored with TLC (n-Hexane : ethylacetate 60 : 40). 30 ml of ice water was added to reaction mixture and extracted twice with ethyl acetate (2x50 mL). The ethyl acetate layer was separated, further washed with 50 mL water and dried to get (6).
38 Chapter-3: Experimental
O O O O O P P OC H H 2 5 OC 2H 5 OC H 2 5 + OC 2H 5 HO Sodium acetate O H 3C O
O H CH 3 (6) Spectral Data: EIMS m/z (rel. int. %): [M]+ 390(1), 313(1), 254(1), 176(10), 161(12), 147(25), 135(30), 120(6), 107(16), 105(80), 77(100), 51(15), 45(15), 39(15),
1 H-NMR (CD3OD, 400 MHz) δ: 8.07 (2H, d, J=7.9 Hz, CH-aromatic, H-2,6), 7.5 (2H, t, J=7.9, 7.1 Hz, CH-aromatic, H-3,5), 7.62(1H, t, J=7.12, Hz, CH-aromatic, H-
4), 4.1 (4H, m, J=7.1 Hz, CH2, H-9, 11 ), 1.3 (6H, t, J=7.1 Hz, CH3, H-10, 12), 7.75 (1H, s, CH, H-13), 7.4 (1H, s, CH-aromatic, H-2’), 7.03 (1H, d, J=8.34 Hz, CH- aromatic, H-5’), 7.36 (1H, d, J=8.34 Hz, CH-aromatic, H-6’)
3.2.7- Preparation of Diethyl benzylphosphonate (2a) (7) Lewis acid-mediated Michaelis-Arbuzov reaction has been reported for the preparation of arylmethyl and heteroarylmethyl phosphonate esters in good yields [130]. It has been used as template to prepare compound (7), which was further derivatized to afford five compounds 8-12, using Maloney et. al. (2009) procedure. It is a mild, high-yielding and general procedure for the preparation of β- ketophosphonates by condensation of esters and phosphonates. It provides products in high yields within minutes at 0°C. The reaction procedure is operationally simple and amenable to large-scale preparations [131]. The procedural details of the two methods and respective spectral data for the compounds 7-12 are described as follows: 0.1 mole (11.5 ml) of benzyl chloride was refluxed with 0.1 mole (17.14 ml) triethyl phosphite in a 50 ml round bottom flask for 5 hours along with 0.2 mole
ZnBr2 in DCM and monitored with TLC (n-Hexane : ethylacetate 3 : 2). Finally the reaction mixture was left overnight at room temperature, in fuminghood for the
removal of ethyl halide (C2H5X) formed in the reaction. The reaction mixture was put to column chromatography over silica gel. Solvent system swept from n-Hexane
39 Chapter-3: Experimental
(90%) towards Ethylacetate. Ten fractions were collected and the target compound was found present in fraction eight.
OC2H5 H5C2O OC2H5 Cl P + P OC2H5 O H5C2O
(7) Spectral Data: EIMS m/z (rel. int. %): [M]+ 228(29), 229(4), 185(4), 172(18), 124(25), 119(7), 118(30), 109(24), 105(11), 97(15), 96(10), 93(5), 92(28), 91(100), 89(5), 81(18), 65(21), 39(6), 29(10), 27(5).
1 H-NMR (CD3OD, 400 MHz) δ: 7.22(2H, d, J=7.7 Hz, CH-aromatic, H-2,6), 7.24 (2H, t, J=7.1,7.7 Hz, CH-aromatic, H3,5), 7.16 (1H, t, J= 7.1 Hz, CH-aromatic, H-4),
3.03 (2H, s, CH2, H-7), 4.0 (4H, m, J=7. 15 Hz, CH2, H-8,10), 1.21(6H, t, J=7.15 Hz,
CH3, H-9,11).
3.2.8- Preparation of diethyl [2-(2-hydroxyphenyl)-2-oxo-1-phenylethyl] phosphonate(2a-1) (8) The compound (7) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (8). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl salicylate (0.896 ml, 6.1 mmol), diethyl benzylphosphonate 2a (1.543 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (2a-1). 40 Chapter-3: Experimental
OH O OC 2H 5 OC 2H 5 O P OC 2H 5 LDA, THF OC H O + 2 5 0 °C OC 2H 5 OH P O (8) Spectral Data: EIMS m/z (rel. int. %): [M]+ 349(2), 271(1), 227(2), 211(7), 124(2), 122(15), 121(100), 118(3), 93(20), 92(3), 91(10), 65(16), 39(6).
1 H-NMR (CD3OD, 400 MHz) δ: 7.36 (2H, d, J=7.57 Hz, CH-aromatic, H-2,6), 7.23 ( 2H, t, J=7.57, 7.1 Hz, CH-aromatic, H-3,5), 7.0 (1H, t, J= 7.1 Hz, CH-aromatic, H-4),
5.68 (1H, s, CH, H-7), 4.15 (4H, m, J=7.1 Hz, CH2, H-8,10), 1.15 (6H, t, J=7.1Hz,
CH3, H-9, 11), 6.99 (1H, d, J=8.02Hz, CH-aromatic, H-3’), 7.64 (1H, t, J=8.0, 7.7Hz, CH-aromatic, H-4’), 6.9 (1H, t, J=8.1, 7.7Hz, CH-aromatic, H-5’), 7.8 (1H, d, J=8.1 Hz, CH-aromatic, H-6’).
3.2.9- Preparation of diethyl [(3E)-2-oxo-1,4-diphenylbut-3-en-1-yl]phosphonate (2a-2) (9) The compound (7) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (9). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl cinnamate (1.027 ml, 6.1 mmol), diethyl benzylphosphonate (7) (1.543 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (2a-2). 41 Chapter-3: Experimental
O OC 2H 5 OC 2 H 5 P OC 2 H 5 LDA, THF O O + 0 °C OC 2 H 5 OC 2H 5 P O (9) Spectral Data: EIMS m/z (rel. int. %): [M]+ 358(2), 281(2), 227(2), 132(15), 131(100), 103(15), 91(10), 77(25), 56(5), 51(4), 39(3).
1 H-NMR (CD3OD, 400 MHz) δ: 7.36 (2H, d, J=7.57Hz, CH-aromatic, H-2.6), 7.23 (2H, t, J=7.57,7.1Hz, CH-aromatic, H-3,5), 6.9 (1H, t, J=7.1 Hz, CH-aromatic, H-4),
4.31 (1H, s, CH, H-7), 4.11 (4H, m, J=7.1Hz, CH2, H-8,10), 1.15 (6H, t, J=7.1Hz, C
H3, H-9, 11), 6.86 (1H, d, J=16Hz, CH, H-13), 7.74 (1H, d, J=16Hz, CH, H-14), 7.48 (2H,d, J=7.7Hz, CH-aromatic, H-2’,6’), 7.4 (2H, t, J=7.1,7.7Hz, CH-aromatic, H- 3’,5’), 7. 36 (1H, t, J=7.1 Hz, CH-aromatic, H-4’).
3.2.10- Preparation of diethyl (2-oxo-1,2-diphenylethyl)phosphonate (2a-3) (10) The compound (7) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (10). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoate (0.872 ml, 6.1 mmol), diethyl benzylphosphonate (7) (1.543 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (2a-3). 42 Chapter-3: Experimental
O OC 2H 5 OC 2H 5 O P OC 2H 5 LDA, THF OC H O + 2 5 0 °C OC 2H 5 P O (10) Spectral Data: EIMS m/z (rel. int. %): [M]+ 332(1), 255(1), 227(1), 195(3), 106(7), 105(100), 90(7), 77(44), 65(7), 63(2), 51(12), 50(4), 39(4).
1 H-NMR (CD3OD, 400 MHz) δ: 7.34 (2H, d, J=7.57 Hz, CH-aromatic, H-2,6), 7.23 (2H, t, J=7.52, 7.1 Hz, CH-aromatic, H-3,5), 7.04 (1H, t, J=7.1 Hz, CH-aromatic, H-
4), 5.67 (1H, s, CH, H-7), 4.15 (4H, m, J=7.1 Hz, CH2, H-8,10), 1.15 (6H, t, J=7.1 Hz,
CH3, H-9,11), 8.08 (2H, d, J=7.6 Hz, CH-aromatic, H-2’,6’), 7.56 (2H, t, J=7.6, 7.1 Hz, CH-aromatic, H-3’,5’), 7.67 (1H, t, J=7.1 Hz, CH-aromatic, H-4’).
3.2.11- Preparation of diethyl (2,4-dioxo-1-phenylpentyl)phosphonate (2a-4) (11) The compound (7) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (11). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl acetoacetate (0.77 ml, 6.1 mmol), diethyl benzylphosphonate (7) (1.543 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled to -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (2a-4).
43 Chapter-3: Experimental
H 3C O OC 2H 5 OC 2H 5 O O P + LDA, THF O O H 3C OC 2H 5 0 °C OC 2H 5 OC 2H 5 P O
(11) Spectral Data: EIMS m/z (rel. int. %): [M]+ 312(4), 227(3).175(5), 118(12), 90(15), 85(100), 77(25), 43(29), 65(13), 63(5), 39(5),
1 H-NMR (CD3OD, 400 MHz) δ: 7.41(2H, d, J=7.37 Hz, CH-aromatic, H-2,6), 7.12 (2H, t, J=7.57, 7.1 Hz, CH-aromatic, H-3,5), 6.94 (1H, t, J=7.1 Hz, CH-aromatic, H-
4), 5.08 (1H, s, CH, H-7), 3.57 (2H, s, CH2, H-9), 1.97 (3H, s, CH3, H-11), 4.11 (4H,
m, J=7.1 Hz, CH2, H-12,14), 1.15 (6H, t, J=7.1 Hz, CH3, H-13,15).
3.2.12- Synthesis of diethyl (2,4-dioxo-1,4-diphenylbutyl)phosphonate (2a-5) (12) The compound (7) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (12). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoylacetate (1.056 ml, 6.1 mmol), diethyl benzylphosphonate 2a (1.543 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (2a-5).
44 Chapter-3: Experimental
OC 2 H 5 O O O OC 2 H 5 P LDA, THF O + OC 2 H 5 O 0 °C OC 2 H 5 OC 2 H 5 P O
(12) Spectral Data: EIMS m/z (rel. int. %): [M]+ 374(2), 297(2), 237(3), 227(3), 147(68), 106(7), 105(100), 78(12), 77(70), 65(13), 63(5), 51(20), 39(5),
1 H-NMR (CD3OD, 400 MHz) δ: 7.48 (2H, d, J=7.5 Hz, CH-aromatic, H-2,6), 7.1 (2H, t, J=7.5,7.1, Hz, CH-aromatic, H-3,5), 6.94 (1H, t, J=7.1 Hz, CH-aromatic, H-4),
5.54 (1-H, s, CH, H-7), 3.3 (2H, s, CH2, H-9), 4.1 (4H, m, J=7.1 Hz, CH2, H-11, 13),
1.15 (6H, t, J=7.1 Hz, CH3, H-12, 14), 8.06 (2H, d, J=7.6 Hz, CH-aromatic, H-2’,6’), 7.51 (2H, t, J=7.6, 7.1 Hz, CH-aromatic, H-3’,5’), 7.61 (1H, t, J=7.1 Hz, CH- aromatic, H-4’ ), 3.2.13-Preparation of Ethyl (diethoxyphosphoryl)acetate (3a) (13)
Simple keto- and ester-substituted phosphonates are prepared by the Arbusov reaction - alkylation of a trialkyl phosphite with an alkyl bromide or iodide [128]. The procedure has been used as template to prepare compound (13). The procedural details for preparation and spectral data for target compound (13) are described as follows: 0.1 mole (10.7 ml) of ethyl chloroacetate was refluxed with 0.1 mole (17.14 ml) triethyl phosphite (TEP) in a 50 ml round bottom flask for 2 hours while monitored with TLC (n-Hexane : ethylacetate 3 : 2) and finally left overnight at room temperature for the removal of ethyl chloride formed in the reaction and any traces of unreacted TEP.
OC2H5 H5C2O H5C2O OC2H5 OC2H5 P Cl + P OC2H5 O O O H5C2O (13) 45 Chapter-3: Experimental
Spectral Data: EIMS m/z (rel. int. %): [M]+ 224(4), 197(81), 180(2 ), 179(74), 169(31), 152(46), 151(69), 137(2 ), 125(2 ), 123(100), 122(1 ), 109(54), 105(2 ), 99(1 ), 97(2 ), 96(1 ), 88(53), 81(34), 65(2 ), 60( 1),45(2 ), 43(2 ), 42(3 ), 31(2 ), 29(52), 28(2 ), 27(3 ).
1 H-NMR (CD3OD, 400 MHz) δ: 1.29 (3H, t, J=6.88 Hz, CH3, H-1), 4.18 (2H, m,
J=6.88 Hz, CH2, H-2), 2.78 (2H, s, CH2, H-3), 4.15 (4H, m, J=7.1 Hz, CH2, H-4,6),
1.33 (6H, t, J=7.1 Hz, CH3, H-5,7).
3.2.14-Preparation of Diethyl(oxiran-2-ylmethyl)phosphonate(4a)(14)
Simple keto- and ester-substituted phosphonates are prepared by the Arbusov reaction - alkylation of a trialkyl phosphite with an alkyl bromide or iodide [128]. The procedure has been used as template to prepare compound (14) which was further derivatized to afford five compounds 15-19, using Maloney et. al. (2009) procedure. It is a mild, high-yielding and general procedure for the preparation of β- ketophosphonates by condensation of esters and phosphonates. It provides products in high yields within minutes at 0°C. The reaction procedure is operationally simple and amenable to large-scale preparations [131]. The procedural details of the two methods and respective spectral data for the compounds 14-19 are described as follows: 0.1 mole (7.84 ml) of epichlorohydrin was refluxed with 0.1 mole (17.14 ml) triethyl phosphite in a 50 ml round bottom flask for 2 hours while monitored with TLC (n-Hexane : ethylacetate 3 : 2) and finally left overnight at room temperature for the removal of ethyl chloride formed in the reaction and any traces of unreacted TEP. OCH H C O 2 5 5 2 OC2H5 P Cl + P OC2H5 O O O H5C2O (14) Spectral Data: EIMS m/z (rel. int. %): [M]+ 194(10), 151(5), 124(5), 118(5), 81(4), 57(100), 43(36), 31(16), 29(29), 28(10), 27(29), 26(6).
46 Chapter-3: Experimental
1 H-NMR (CD3OD, 400 MHz) δ: 2.75 (2H, d, J=4.2 Hz, CH2, H-1), 3.09 (1H, m,
J=4.2, 6.8 Hz, CH, H-2), 2.63 (2H, d, J=6.82 Hz, CH2, H-3 ), 4.13 (4H, m, J=7.15 Hz,
CH2, H-4,6), 1.14 (6H, t, J=7.15 Hz, CH3, H-5, 7).
3.2.15- Preparation of Diethyl [2-(2-hydroxyphenyl)-1-oxiran-2-yl-2-oxoethyl] phosphonate (4a-1) (15) The compound (14) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (15). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl salicylate (0.896 ml, 6.1 mmol), diethyl (oxiran-2-ylmethyl) phosphonate 4a (1.299 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester as determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (4a-1).
O OH
OC2H5 OC2H5 O P OC2H5 LDA, THF + OC H O O 2 5 OH 0 °C OC2H5 P O O (15) Spectral Data: EIMS m/z (rel. int. %): [M]+ 314(1), 271(2), 193(3), 181(4), 177(15), 124(5), 122(8), 121(100), 118(5), 31(12), 29(25), 28(8), 27(25), 26(5).
1 H-NMR (CD3OD, 400 MHz) δ: 3.24 (2H, d, J=4.5 Hz, CH2, H-1), 3.86 (1H, m , J=4.5,6.0 Hz, CH, H-2), 4.55 (1H, d, J=6.0 Hz, CH, H-3), 4.15 (4H, m, J=7.15 Hz, 47 Chapter-3: Experimental
CH2, H-4,6), 1.16 (6H, t, J=7.15 Hz, CH3, H-5,7), 6.93 (1H, d, J=8.02 Hz, CH- aromatic, H-2’), 7.63 (1H, t, J=7.76,8.02 Hz, CH-aromatic, H-3’), 6.87 (1H, t, J=8.12,7.76 Hz, CH-aromatic, H-4’), 7.76 (1H, d, J=8.12 Hz, CH-aromatic, H-5’).
3.2.16- Preparation of Diethyl [(3E)-1-oxiran-2-yl-2-oxo-4-phenylbut-3-en-1-yl] phosphonate (4a-2) (16) The compound (14) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (16). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl cinnamate (1.027 ml, 6.1 mmol), diethyl (oxiran-2-ylmethyl)phosphonate 4a (1.299 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester as determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (4a-2).
O OC 2 H 5 OC 2 H 5 P OC 2 H 5 LDA, THF + O O O 0 °C OC 2 H 5 OC 2 H 5 P O O (16) Spectral Data: EIMS m/z (rel. int. %): [M]+ 324(1), 131(90), 124(4),118(4),104(10), 103(100), 102(12), 81(3), 77(57), 51(40), 43(35), 31(18), 29(30), 28(12), 27(30), 26(8),
1 H-NMR (CD3OD, 400 MHz) δ: 3.24 (2H, d, J=4.49 Hz, CH2, H-1) 3.56 (1H, m,
J=4.49 Hz, CH, H-2), 3.05 (1H, d, J=6.0Hz, CH, H-3), 4.10 (4H, m, J=7.15 Hz, CH2,
48 Chapter-3: Experimental
H-4,6), 1.16 (6H, m, J=7.15 Hz, CH3,, H-5,7), 6.85 (1H, d, J=16 Hz, CH, H-9), 7.72 (1H, d, J=16 Hz, CH, H-10), 7.47 (2H, d, J=7.69 Hz, CH-aromatic, H-2’, 6’), 7.40 (2H, t, J=7.17,7.69 Hz, CH-aromatic, H-3’,5’), 7.36 (1H, t, J=7.17 Hz, CH-aromatic, H-4’).
3.2.17- Preparation of diethyl(1-oxiran-2-yl-2-oxo-2-phenylethyl)phosphonate (4a-3) (17) The compound (14) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (17). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoate (0.872 ml, 6.1 mmol), diethyl (oxiran-2-ylmethyl) phosphonate 4a (1.299 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester as determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (4a-3).
O OC 2 H 5 OC 2 H 5 O P OC 2 H 5 LDA, THF + OC H O O 2 5 0 °C OC 2 H 5 P O O (17) Spectral Data: EIMS m/z (rel. int. %): [M]+ 298(2), 255(2), 193(3), 181(3), 124(4), 118(4), 106(8), 105(100), 78(6),77(40), 51(11), 31(11), 29(26), 28(9), 27(25), 26(6).
1 H-NMR (CD3OD, 400 MHz) δ: 3.24 (2H, d, J=4.49 Hz, CH2, H-1), 3.86 (1H, t, J=4.49, 6.0 Hz, CH, H-2), 4.55 (1H, d, J=6.0 Hz, CH, H-3), 4.14 (4H, m, J=7.1 Hz,
CH2, H-4,6), 1.16 (6H, t, J=7.1 Hz, CH3, H-5,7), 8.04 (2H, d, J=7.6 Hz, CH-aromatic, 49 Chapter-3: Experimental
H-2’,6’), 7.5 (2H, t, J=7.6,7.12 Hz, CH-aromatic, H-3’,5’), 7.68 (1H, t, J= 7.12 Hz, CH-aromatic, H-4’).
3.2.18- Preparation of Diethyl(1-oxiran-2-yl-2,4-dioxopentyl)phosphonate(4a-4) The compound (14) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (18). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl acetoacetate (0.77 ml, 6.1 mmol), diethyl (oxiran-2-ylmethyl) phosphonate 4a (1.299 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester as determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (4a-4).
H 3C O
OC 2H 5 O O OC 2H 5 O P LDA, THF + H 3C OC 2H 5 OC H O O 2 5 0 °C OC 2H 5 P O O (18) Spectral Data: EIMS m/z (rel. int. %): [M]+ 278(1), 235(1), 193(3), 181(4), 124(4), 118(5), 85(28), 43(100), 42(30), 31(11), 29(35), 28(10), 27(44), 26(15), 15(19),
1 H-NMR (CD3OD, 400 MHz) δ: 3.24 (2H, d, J=4.49 Hz, CH2, H-1), 3.86 (1H, t,
J=4.49, 6.0 Hz, CH, H-2), 3.85 (1H, d, J=6.0 Hz, CH, H-3), 3.55 (2H, s, CH2, H-5),
1.97 (3H, s, CH3, H-7), 4.10 (4H, m, J=7.15 Hz, CH2, H-8,10), 1.16 (6H, t, J=7.15 Hz,
CH3, H-9,11).
50 Chapter-3: Experimental
3.2.19-Preparation of Diethyl (1-oxiran-2-yl-2, 4-dioxo-4-phenyl butyl) phosphonate (4a-5) (19) The compound (14) was derivatized using Maloney et. al.(2009) procedure [131] as template to prepare compound (19). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoylacetate (1.056 ml, 6.1 mmol), diethyl (oxiran-2-ylmethyl) phosphonate 4a (1.299 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester as determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (4a-5).
O O O OC 2 H 5 OC 2 H 5 P LDA, THF O + OC 2 H 5 O O 0 °C OC 2 H 5 OC 2 H 5 P O O (19) Spectral Data: EIMS m/z (rel. int. %): [M]+ 340(1), 297(1), 193(4), 181(3), 147(80), 124(2), 118(3), 105(100), 78(3), 77(50), 69(20), 51(15), 31(11), 29(20), 28(10), 27(20), 26(6),
1 H-NMR (CD3OD, 400 MHz) δ: 3.24 (2H, d, J=4.49 Hz, CH2, H-1), 3.83 (1H, t,
J=4.49, 6.0 Hz, CH, H-2), 4.31 (1H, d, J=6.0 Hz, CH, H-3), 3.27 (2H,s, CH2, H-5),
4.10 (4H, m, J-7.15 Hz, CH2, H-7,9), 1.16 (6H, t, J=7.15 Hz, CH3, H-8,10), 8.05 (2H, d, J=7.6 Hz, CH-aromatic, H-2’,6’), 7.51 (2H, t, J=7.6,7.12 Hz, CH-aromatic, H- 3’,5’), 7.6 (1H, t, J=7.12 Hz, CH-aromatic, H-4’).
51 Chapter-3: Experimental
3.2.20- Preparation of Diethyl (2-nitrobenzyl) phosphonate (5a) (20)
Simple keto- and ester-substituted phosphonates are prepared by the Arbusov reaction - alkylation of a trialkyl phosphite with an alkyl bromide or iodide [128]. The procedure has been used as template to prepare compound (20). The procedural details and respective spectral data for the compounds 20 are described as follows: 0.03 mole (5.178 g) of o-nitrobenzyl chloride was refluxed with 0.03 mole (5.144 ml) triethyl phosphite in a 50 ml round bottom flask for 5 hours while monitored with TLC (n-Hexane : ethylacetate 3 : 7) and finally left overnight at room temperature for the removal of ethyl chloride formed in the reaction and any traces of unreacted TEP.
OC 2H5 H C O OC 2H5 Cl 5 2 P + P OC 2H5 O NO H C O 2 5 2 NO 2 (20) Spectral Data: EIMS m/z (rel. int. %): [M]+ 273(4), 151(3), 136(55), 124(10), 107(15), 96(20), 92(80), 90(45),89(100), 79(25), 78(48), 77(50), 65(74), 63(49), 51(31),
1 H-NMR (CD3OD, 400 MHz) δ: 8.08 (1H,d,J=8.15Hz, CH-aromatic, H-2), 7.37(1h,t,J=7.34,8.15Hz, CH-aromatic, H-3), 7.7(1H,t,J=7.77, 7.34Hz, CH-aromatic,
H-4), 7.67 (1H,d,J=7.77Hz, CH-aromatic, H-5), 3.77(2H, s, CH2, H-7),
3.94(4H,m,J=7.1Hz, CH2, H-8,10), 1.16(6H,t,J=7.1Hz, CH2, H-9, 11).
3.2.21- Preparation of Tetraethyl ethane-1,2-diylbis(phosphonate) (6a) (21) Simple keto- and ester-substituted phosphonates are prepared by the Arbusov reaction - alkylation of a trialkyl phosphite with an alkyl bromide or iodide [128]. The procedure has been used as template to prepare compound (21) which was further derivatized to afford five compounds 22-26, using Maloney et. al. (2009) procedure [131]. The procedural details of the two methods and respective spectral data for the compounds 21-26 are described as follows: 52 Chapter-3: Experimental
0.1 mole (8.657 ml) of 1,2-dibromoethane was refluxed with 0.2 mole (34.295 ml) triethyl phosphite in a 50 ml round bottom flask for 5 hours while monitored with TLC (n-Hexane : ethylacetate 3 : 2) and finally left overnight at ~50 °C in an open beaker; placed in fuming hood to remove ethyl bromide and any traces of unreacted TEP. O OC H H5C2O 2 5 P OC2H5 Br Br + P OC2H5 H5C2O P OC2H5 H5C2O O (21) Spectral Data: EIMS m/z (rel. int. %): [M]+ 302(3), 257(19), 229(8), 201(7), 173(32), 166(30), 165(100), 155(5), 139(5), 138(15), 137(18), 111(7), 109(29), 91(8), 82(6), 81(14), 65(120). 1 H-NMR (CD3OD, 400 MHz) δ: 2.08 (4H,t, J=6.73Hz, CH, H-1,2), 3.45 (8H, m,
J=7.1Hz, CH2,H-3,5,7,9), 1.25 (12H,t,J=7.1, Hz, CH3, H-4,6,8,10).
3.2.22- Preparation of derivative A of tetraethyl ethane-1,2-diylbis(phosphonate) (6a-1) (22) The compound (21) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (22). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl salicylate (0.896 ml, 6.1 mmol), tetraethyl ethane-1,2-diylbis(phosphonate) 6a (2.09 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (6a-1).
53 Chapter-3: Experimental
OH
O O O OC2H5 O P OC2H5 H5C2O OC H OC2H5 LDA, THF 2 5 H5C2O P + P OC2H5 P O 0 °C H5C2O OC H OH O 2 5 (22) Spectral Data: EIMS m/z (rel. int. %): [M]+ 422(1), 301(1), 285(7), 271(2), 192(5), 164(15), 148(15), 122(8), 121(100), 93(15), 65(20), 63(4), 39(10), 53(2).
1 H-NMR (CD3OD), 400 MHz) δ: 2.73(2H,d, J=4.71Hz,CH2,H-1), 5.21(1H,t, J=4.71
Hz, CH,H-2), 4.14(4H,m,J=7.15Hz, CH2, H-4,6), 1.15 (6H,t,J=7.15 Hz, CH3, H-5,7),
3.98(4H,m,J=7.15 Hz, CH2, H-8,10), 1.23 (6H,t,J=7.15Hz, CH3, H-9, 11), 7.06 (1H,d, J=8.02 Hz, CH-aromatic, H-2’), 7.63(1H,t,J=7.76,8.02Hz, CH-aromatic, H-3’), 7.01 (1H,t, J=7.76, 8.12 Hz, CH-aromatic, H-4’), 7.81 (1H,d,J=8.12Hz, CH-aromatic,H- 5’).
3.2.23- Preparation of derivative B of tetraethyl ethane-1,2-diylbis(phosphonate) (6a-2) (23) The compound (21) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (23). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl cinnamate (1.027 ml, 6.1 mmol), tetraethyl ethane-1,2-diylbis(phosphonate) 6a (2.09 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled to -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (6a-2).
54 Chapter-3: Experimental
O O OC 2H5 P OC H O 2 5 OC 2H5 LDA, THF H5C2O P + O OC 2H5 H5C2O OC H O 0 °C P 2 5 P H5C2O OC H O 2 5 (23) Spectral Data: EIMS m/z (rel. int. %): [M]+ 434(1), 301(1), 295(2), 281(3), 192(5), 164(15),131(95), 104(10), 103(100), 102(12), 77(58), 51(30), 50(14),
1 H-NMR (CD3OD, 400 MHz) δ: 2.59 (2H,d,J=4.71Hz,CH2,H-1), 4.03 (1H,t, J=4.71Hz, C H, H-2), 6.78(1H,d,J=16Hz,CH,H-4), 7.72 (1H,d,J=16Hz,CH, H-5),
4.10(4H,m, J=7.15 Hz, CH2, H-6,8), 1.15(6H,t,J=7.15Hz,CH3,H-7,9), 3.98 (4H,m,
J=7.15 Hz, CH2, H10, 12), 1.23 (6H,t,J=7.15Hz,CH3, H-11,13), 7.47 (2H,d,J=7.69Hz, CH-aromatic, H-2’,6’), 7.40 (2H,t, J=7.69,7.17Hz, CH-aromatic, H-3’,5’), 7.36 (1H,d,J=7.17Hz, CH-aromatic, H-4’).
3.2.24-Preparation of derivative C of Tetraethyl ethane-1,2-diylbis(phosphonate) (6a-3) (24) The compound (21) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (24). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoate (0.872 ml, 6.1 mmol), tetraethyl ethane-1,2-diylbis(phosphonate) 6a (2.09 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The
55 Chapter-3: Experimental
combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (6a-3).
O O O OC 2H 5 O P OC 2H5 H5C 2O OC H OC 2H5 LDA, THF 2 5 H5C 2O P + P OC 2H5 P O 0 °C H5C2O OC H O 2 5 (24) Spectral Data: EIMS m/z (rel. int. %): [M]+ 406(1), 301(1). 269(3), 255(3),164(3), 145(9), 133(14), 119(10), 123(5), 107(1), 106(8), 105(100), 91(5), 78(5), 77(36), 51(10), 29(9).
1 H-NMR (CD3OD, 400 MHz) δ: 2.72 (2H,d,J=4.71Hz,CH2,H-1), 5.22(1H,t,J=4.71
Hz, CH, H-2), 4.14 (4H,m,J=7.15Hz, CH2, H-4,7), 1.15 (6H,t,J=7.15Hz, CH3,H-5,7),
3.98 (4H,m, J=7.15Hz, CH2, H-8,10), 1.23 (6H,t,J=7.15 Hz,CH3, H-9, 11), 8.09 (2H,d, J=7.6 Hz, CH-aromatic, H-2’,6’), 7.63 (2H,t, J=7.6,7.12Hz, CH-aromatic, H- 3’,5’), 7.66 (1H,t, J=7.12 Hz, CH-aromatic, H-4’).
3.2.25- Preparationof derivative D of Tetraethyl ethane-1,2-diylbis(phosphonate) (6a-4) (25) The compound (21) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (25). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl acetoacetate (0.77 ml, 6.1 mmol), tetraethyl ethane-1,2-diylbis(phosphonate) 6a (2.09 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (6a-4). 56 Chapter-3: Experimental
H3C O O O O OC2H5 P OC2H5 O H5C2O P LDA, THF O + H3C OC2H5 OC2H5 H5C2O OC H O 0 °C P 2 5 P H5C2O OC H O 2 5 (25) Spectral Data: EIMS m/z (rel. int. %): [M]+ 386(1), 257(2), 173(3), 165(3), 164(10), 137(2), 112(8), 109(3), 85(29), 43(100), 42(35), 27(48), 26(15), 15(15).
1 H-NMR (CD3OD, 400 MHz) δ: 2.56 (2H,d, J=4.71Hz, CH2,H-1), 4.77 (1H,t,J=4.71
Hz, CH, H-2), 3.65 (2H,s,CH2,H-4), 1.97 (3H,s, CH3,H-6), 4.10 (4H,m,J=7.15 Hz,
CH2, H-7, 9), 1.15 (6H,t,J=7.15 Hz, CH3, H-8,10), 3.98 (4H,m,J=7.15Hz, CH2,H-
11,13), 1.23 (6H,t, J=7.15 Hz, CH3,H-12, 14).
3.2.26- Preparation of derivative E of Tetraethyl ethane-1,2-diylbis(phosphonate) (6a-5) (26) The compound (21) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (26). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoylacetate (0.77 ml, 6.1 mmol), tetraethyl ethane-1,2-diylbis(phosphonate) 6a (2.09 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated to yield β-ketophosphonate (6a-5).
57 Chapter-3: Experimental
O O O O OC2H5 P OC2H5 H C O P LDA, THF O 5 2 + OC2H5 OC2H5 O O 0 °C H5C2O OC H P 2 5 P H5C2O OC H O 2 5 (26) Spectral Data: EIMS m/z (rel. int. %): [M]+ 448(1), 256(3), 174(90), 171(2), 165(3), 164(8), 161(60), 147(68), 136(3), 109(2), 105(100), 78(12), 77(51), 69(50), 51(19).
1 H-NMR (CD3OD, 400 MHz) δ: 2.62(2H,d,J=4.71Hz, CH2,H-1), 5.23 (1H,t,J=4.71
Hz, CH, H-2), 3.38 (2H, s, CH2,H-4), 4.10 (4H,m, J=7.15Hz, CH2, H-6,8), 1.15(6H,t,
J=7.15 Hz, CH3, H-7,9), 3.98 (4H,m,J=7.15Hz, CH2, H-10,12), 1.23 (6H,t, J=7.15
Hz, CH3, H-11,13), 8.05 (2H,d,J=7.6Hz, CH-aromatic, H-2’,6’), 7.51 (2H,t, J=7.6,7.12Hz, CH-aromatic, H-3’,5’), 7.60 (1H,t, J=7.12 Hz, CH-aromatic, H-4’).
3.2.27- Preparation of Diethyl methylphosphonate (7a) (27) Simple keto- and ester-substituted phosphonates are prepared by the Arbusov reaction - alkylation of a trialkyl phosphite with an alkyl bromide or iodide [128]. The procedure has been used as template to prepare compound (27) which was further derivatized to afford five compounds 28-32, using Maloney et. al. (2009) procedure [131]. The procedural details of the two methods and respective spectral data for the compounds 27-32 are described as follows: 0.1 mole (6.28 ml) of methyl iodide was refluxed with 0.1 mole (17.14 ml) triethyl phosphite in a 50 ml round bottom flask for 5 hours while monitored with TLC (n-Hexane : ethylacetate 3 : 2) and finally left overnight at ~70 °C in an open beaker; placed in fuming hood to remove ethyl iodide and any traces of unreacted TEP.
H5C2O OC2H5 H3C OC2H5 H3C I + P OC2H5 P H5C2O O (27) 58 Chapter-3: Experimental
Spectral Data: EIMS m/z (rel. int. %): [M]+ 152(4), 137(7), 125(72), 123(9), 109(8), 108(23), 107(21), 97(99), 93(5), 91(6), 81(19), 80(38), 79(100), 65(17), 47(18), 45(13), 43(7), 29(22), 28(8), 27(19), 15(8),
1 H-NMR (CD3OD, 400 MHz) δ: 1.40 (3H,s, CH3, H-1), 4.01 (4H,m,J=7.15Hz,
CH2,H-2,4), 1.19 (6H,t,J=7.15 Hz, CH3, H-3,5).
3.2.28- Preparation of Diethyl [2-(2-hydroxyphenyl)-2-oxoethyl] phosphonate (7a-1) (28) The compound (27) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (28). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl salicylate (0.896 ml, 6.1 mmol), diethyl methylphosphonate 7a (1.08 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (7a-1).
O OH OC H 2 5 OC H H3C OC2H5 OC H LDA, THF 2 5 P 2 5 OC2H5 + P O 0 °C OH O O (28) Spectral Data: EIMS m/z (rel. int. %): [M]+ 272(4), 151(4), 135(26), 125(8), 122(8), 121(100), 108(3), 97(10), 93(14), 81(2), 80(4), 79(10), 65(20), 63(4), 39(11).
59 Chapter-3: Experimental
1 H-NMR (CD3OD, 400 MHz) δ: 3.54 (2H,s, CH2, H-1), 3.55 (4H,m,J=7.15 Hz,
CH2,H-2,4), 1.30 (6H,t,J=7.15 Hz,CH3, H-3,5), 6.97 (1H,d,J=8.02 Hz, CH-aromatic, H-2), 7.63 (1H,t,J=8.0 Hz, CH-aromatic, H-3), 6.90 (1H,t,J=7.76,8.12 Hz, CH- aromatic, H-4), 7.83 (1H,d,J=8.12Hz, CH-aromatic, H-5). 3.2.29- Preparation of Diethyl [(3E)-2-oxo-4-phenylbut-3-en-1-yl] phosphonate (7a-2) (29) The compound (27) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (29). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl cinnamate (1.027 ml, 6.1 mmol), diethyl methylphosphonate 7a (1.08 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (7a-2).
O OC H 2 5 OC H H3C OC 2H5 OC H LDA, THF 2 5 P 2 5 OC 2H5 + P O 0 °C O O (29) Spectral Data: EIMS m/z (rel. int. %): [M]+ 282(2), 179(1), 145(45), 131(95), 123(7), 105(1), 104(12), 103(100), 102(13), 97(8), 79(8), 77(60), 51(35), 50(12). 1 H-NMR (CD3OD, 400 MHz) δ: 3.05 (2H,s,CH2,H-1), 6.86 (1H,d,J=15.54 Hz, CH,
H-3), 7.77 (1H,d,J=15.54 Hz, CH, H-4), 3.51 (4H,m, J=7.15 Hz, CH2, H-5,7), 1.27
(6H,t, J=7.15, Hz, CH3, H-6,8), 7.46 (2H,d,J=7.69 Hz, CH-aromatic, H-2’,6’), 7.41 (2H,t, J=7.69,7.17 Hz, CH-aromatic, H-3’,5’), 7.36 (1H,t,J=7.17Hz, CH-aromatic, H- 4’).
60 Chapter-3: Experimental
3.2.30- Preparation of diethyl (2-oxo-2-phenylethyl)phosphonate (7a-3) (30) The compound (27) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (30). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoate (0.872 ml, 6.1 mmol), diethyl methylphosphonate 7a (1.08 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (7a-3).
O OC H 2 5 OC H H3C OC 2H5 OC H LDA, THF 2 5 P 2 5 OC 2H5 + P O 0 °C O O (30) Spectral Data: EIMS m/z (rel. int. %): [M]+ 256(4), 257(1), 151(3), 147(3), 146(11), 123(7), 120(14), 106(8), 105(100), 103(5), 91(7), 81(6), 78(7), 77(36), 71(12), 65(6), 51(11), 29(11), 27(7).
1 H-NMR (CD3OD, 400 MHz) δ: 3.56 (2H,s,CH2,H-1), 3.55 (4H,m,J=7.15Hz, CH2,
H-3,5), 1.30 (6H,t,J=7.15Hz, CH3, H-4,6), 8.23 (2H,d, J=7.6 Hz,CH-aromatic, H-2,6), 7.49 (2H,t, J=7.6,7.12 Hz, CH-aromatic, H-3, 5), 8.05 (1H,t, J=7.12 Hz, CH- aromatic, H-4).
61 Chapter-3: Experimental
3.2.31- Preparation of diethyl (2,4-dioxopentyl)phosphonate (7a-4) (31) The compound (27) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (31). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl acetoacetate (0.77 ml, 6.1 mmol), diethyl methylphosphonate 7a (1.08 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (7a-4). O O OC2H5 H C OC H OC2H5 3 2 5 LDA, THF H C OC H P H3C OC2H5 3 2 5 + P O 0 °C O O O (31) Spectral Data: EIMS m/z (rel. int. %): [M]+ 236(2), 193(3), 179(3), 125(6), 106(3), 99(25), 97(10), 85(32), 80(4), 79(12), 43(100), 42(36), 27(50), 26(13), 15(30).
1 H-NMR (CD3OD, 400 MHz) δ: 3.24 (2H,s, CH2,H-1), 2.72 (2H,s, CH2,H-3), 2.03
(3H,s, CH3,H-5), 4.16 (4H,m, J=7.15 Hz, CH2,H-6,8), 1.62 (6H,t, J=7.15 Hz, CH3, H- 7,9).
62 Chapter-3: Experimental
3.2.32- Preparation of diethyl (2,4-dioxo-4-phenylbutyl)phosphonate (7a-5) (32) The compound (27) was derivatized using Maloney et. al. (2009) procedure [131] as template to prepare compound (32). The procedural details and spectral data are described as follows: 50 ml three neck round-bottomed flask equipped with addition funnel and magnetic stirrer was charged with ethyl benzoylacetate (1.056 ml, 6.1 mmol), diethyl methylphosphonate 7a (1.08 g, 6.7 mmol) and 10 mL of THF. The reaction mixture was cooled at -5 °C while a 2.0 M solution of LDA (6.4 mL) was added dropwise via addition funnel keeping the internal temperature below 0 °C. After complete addition, the reaction mixture was stirred at 0 °C until complete consumption of ester was determined by TLC analysis. The reaction mixture was then carefully quenched with 5 M HCl to adjust the pH to ca. 4 and diluted with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated to yield β-ketophosphonate (7a-5).
O O OC H 2 5 OC H H3C OC2H5 OC H LDA, THF 2 5 P 2 5 OC2H5 + P O 0 °C O O O (32) Spectral Data: EIMS m/z (rel. int. %): [M]+ 298(1), 222(1), 193(2),179(3), 161(95), 147(70), 125(2), 105(100), 97(4), 80(3), 79(6),78(11), 77(45), 69(51), 51(16).
1 H-NMR (CD3OD, 400 MHz) δ: 3.31 (2H,s, CH2,H-1), 2.53 (2H,s,CH2,H-3), 4.16
(4H,m,J=7.15 Hz, CH2, H-5,7), 1.61 (6H,t,J=7.15 Hz, CH3, H-6,8), 8.05 (2H,d, J=7.6 Hz, CH-aromatic, H-2’,6’), 7.51 (2H,t,J=7.6,7.12 Hz, CH-aromatic,H-3’,5’), 7.60 (1H,t, J=7.12Hz, CH-aromatic, H-4’).
63 Chapter-3: Experimental
3.3:-Bioassay Guided Extraction & Isolation from Plants 3.3.1:- Compositae Family Twenty five plants were screened for BLase Inhibition activity by subjecting their methanolic/ ethanolic extracts to preliminary screening for BLase inhibition activity by Shahwar et. al. method [29]. Only Cichorium intyus, Holarrhena antidysenterica, Calotropis procera, Alstonia scholaris, Carissa opaca and Ageratum conyzoides were found active and processed further. 3.3.1.1:- Cichorium intybus [Kasni] The plant was collected in April 2008 from green-fodder fields at village Rajoke, near Daskah, District Sialkot, Islamic Republic of Pakistan. It was identified by Dr. Zaheer-ud-din Khan and Muhammad Ajaib, at the Department of Botany, GC University, Lahore, where a voucher specimen was submitted (GCU-Herb-Bot-895) at Dr. Sultan Ahmad Herbarium. The roots, including lower stem of Cichorium intybus (10 Kg) were dried in shade, grinded and soaked twice in Ethyl alcohol (~ 95%) for seven days each with intermittent shaking. The two filtrates were mixed. Ethanol was recovered from filtrate by rotary evaporator under reduced pressure and low temperature (40 °C). The crude viscous extract was subjected to GCMS analysis as well as β-Lactamase (BLase) inhibition assay using an efficient assay to screen β-Lactamase inhibitors from plant extracts developed at our Lab and later on published [29]. The Crude extract (123 g) was dissolved in 600 ml distilled water and was successively extracted with n-Hexane and Chloroform (each 4 x 250 ml). The remaining aqueous layer was acidified (pH 3.0) with acetic acid (AcOH) and was extracted with chloroform (4 x 250 ml). Then the remaining acidic aqueous layer was basified (pH 10) with ammonia and again extracted with chloroform (4 x 250 ml). The remaining aqueous layer was then neutralized using AcOH. This was then extracted successively with Eethyl acetate and n-Butanol (4 x 250 ml). Moisture was removed by filtering over the bed of anhydrous sodium sulphate. Solvents from dry filtrates were recovered by rotary evaporator. Dry isolated material was stored in vials. All the extracts were subjected to BLase inhibition assay and GCMS analysis. Neutral Chloroform (CIRCHNe), acidic Chloroform (CIRCHAc) and Ethyl acetate extracts (CIR-EA) showed significant activity. Which were subjected to Thin Layer Chromatography (TLC) as 64 Chapter-3: Experimental
shown in Scheme-3.1. TLC plates were visualized using one or more of: UV-light, Ceric sulphate and Draggondorff’s reagent. Bands containing plant material were scratched and washed repeatedly with methanol, chloroform and ethyl acetate. Which were further put to spectrophotometic BLase inhibition assay. The active components were put to GCMS and 1H-HMR analysis for identification.
Cichorium intybus (Dried Roots & Stem 3 Kg)
Crude Ethanolic Extract(123 g) dissolved in H2O
n-Hexane H2O Layer CIRHex
Chloroform H2O Layer CIRCHNe* Lupeol
Preparative TLC CHCl3 : Hexane (40 : 60) beta-Sitsterol 20% AcOH, pH = 3
Chloroform CIRCHAc* Benzene-4-hydroxy acetic acid
iso Vanillic acid Preparative TLC MeOH:CHCl3 (20 : 80 ) Syringic acid H2O Layer
Vanillic acid 20% NH4OH, pH = 9
Chloroform H2O Layer CIRCHBa
20% AcOH, pH = 7
H2O Layer Ethyl acetate CIREANe* Esculetin
Scopoletin Preparative TLC MeOH : CHCl3 Umbeliferone n-Butanol H2O Layer (30 : 70 ) CIRBUNe Kaempherol
* Showed BLase Inhibition activity Scheme-3.1:- Scheme for Bioassay guided Extraction, Fractionation and . Isolation from Cichorium intybus.
65 Chapter-3: Experimental
3.3.1.1.1- CIRCHNe-1 : Lupeol (33) Preparative TLC (chloroform : Hexane :: 40 : 60) of neutral Chloroform extract (CIRCHNe) yielded two compounds in handsome amount. The material recovered from top layer was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Lupeol. BLasé inhibition activity was tested.
CH2 29
H3C 28 30 20 21 13 18 19 14 12 CH3 17 24 CH3 3 4 11 27 16 2 5 10 15 CH3 1 CH326 25 8 9 6 HO 7 CH H3C 3 22 23 (33) EIMS m/z (rel. int. %): 43(100), 68(98), 55( 87), 67(78), 81(76), 69(73), 95(72), 93(68), 41(67), 109(61), 107(61), 121(57), 207(52), 135(52), 189(49), 79(46), 79(46), 57(41), 119(38), 218(39) ), 123(37), 91(36), 105(34). 147(27), 133(25), 71(25), 149(23), 136(22), 136(22), 94(21), 175(20), 134(17), 162(11),
1H-NMR (CD3OD, 400 MHz) δ: 3.19 (1H, t, J=6.39 & 9.17Hz, H-1), 1.55 & 1.88 (2H, dd, J=6.39 & 9.17 Hz, H-2a & 2b), 1.41 & 1.84 (2H, dd, J=3.67 Hz, H-3), 1.07 (1H, s, H-4), 0.86 & 1.33 (2H, dd, J=5.07 Hz, H-6a & 6b), 1.46 & 1.59 (2H, dd, J=5.07 Hz, H-7a & 7b), 0.62 (1H, s, H-9), 3.3.1.1.2- CIRCHNe-2 : β-Sitosterol (34) reparative TLC (chloroform : Hexane :: 40 : 60) of neutral Chloroform extract (CIRCHNe) yielded two compounds in handsome amount. The material recovered from bottom layer was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data
66 Chapter-3: Experimental
interpretation and comparison with literature confirmed it as β-Sitosterol. BLasé inhibition activity was tested.
H3C CH3 29 28 27 22 H3C 20 CH3 21 24 26 23 25 12 CH3 18 11 17 16 13 CH 1 93 19 8 2 14 15 10 3 7 HO 5 4 6 (34) EIMS m/z (rel. int. %): 43(100), 55(36), 41(33), 57(31), 107(30), 81(28), 414(27), 95(27), 105(25), 69(24), 414(27), 79(23), 145(19), 93(19), 67(19), 91(19), 161(18), 133(18), 159(16), 71(16), 119(15), 213(14), 131(13), 83(11), 109 (11), 143(10). H1-NMR (CD3OD, 400 MHz) δ: 3.53 (2H, m, J=7.5 & 9.0 Hz, H-1), 1.5 & 1.8 (2H, dd, J=6.84 & 9.0 Hz, H-2a & 2b), 1.05 & 1.85 (1H, t, J=6.84 Hz, H-3), 0.9 (2H, dd, J=12.0 Hz, H-4), 1.45 (1H, m, J=6.88 & 12.0 Hz, H-5), 1.87 & 1.97 (2H, m, J=6.88 & 5.25 Hz, H-6a & 6b), 5.35 (1H, t, J=5.25 Hz, H-7), 2.3 & 2.2 (2H, dd, J=7.5 Hz, H-8a & 8b), 1.1 (1H, t, J=9.0 Hz, H-11), 1.17 & 2.0 (2H, dd, J=5.07 Hz, H-13a & 13b), 1.45 (2H, m, J=5.07 & 3.62 Hz, H-14a & 14b), 1.07 & 1.58 (2H, m, J=9.0 &8.5 Hz, H-15a & 15b), 1.26 &1.87 (2H, m, J=8.5 & 8.6 Hz, H-16a & 16b), 1.13 (1H, t, J=8.6 Hz, H-17), 1.00 (3H, s, H-18), 0.68 (3H, s, H-19), 1.01 (1H, m, J=6.1 & 13.5 Hz, H- 20), 0.82 & 1.41 (2H, m, J=13.5 & 7.1 Hz, H-21a & 21b), 1.13 & 1.31 (2H, m, J=7.1 & 2.27 Hz, H-22a & 22b), 0.9 (1H, m, J=2.27 & 5.13 Hz, H-23), 0.84 & 0.99 (2H, m, J=2.27 & 7.67 Hz, H-24a & 24b), 0.85 (3H, t, J=7.67 Hz, H-25), 0.93 (3H, d, J=6.12 Hz, H-26), 1.55 (1H, m, J=6.67 Hz, H-27), 0.78 (6H, d, J=6.67 Hz, H-28 & 29).
3.3.1.1.3- CIRCHAc-1: p-hydroxy phenyl acetic acid (35) Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and 67 Chapter-3: Experimental
comparison with literature confirmed it as p-hydroxy phenyl acetic acid. BLasé inhibition activity was tested.
OH
7 O
2 1 6
3 4 5
OH (35) EIMS m/z (rel. int. %): 107( 100), 152( 29), 77( 13), 108 (08), 51(05), 78(04), 39(03), 53(03). 1H-NMR (CD3OD, 400 MHz) δ: 6.78 (2H, d, J=8.47 Hz, H-3 & 5), 7.12 (2H, d, J=8.47 Hz, H-2 & 6), 3.38 (2H, s, H-7).
3.3.1.1.4- CIRCHAc-2: Isovanillic acid / 3-hydroxy-4-methoxy benzoic acid (36) Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Isovanillic acid. BLasé inhibition activity was tested
O OH
6 1 2
3 5 4 OH
O H3C 7 (36) .EIMS m/z (rel. int. %): 168 (100), 153(78), 97( 32), 125( 23), 15( 18), 51(14), 53 (10), 52(10), 79(10), 169(10). 1H-NMR (CD3OD, 400 MHz) δ: 7.59 (1H, s, H-1), 6.98 (1H, d, J=8.14 Hz, H-5), 7.67 (1H, d, J=8.14 Hz, H-6), 3.84 (3H, s, H-7).
68 Chapter-3: Experimental
3.3.1.1.5- CIRCHAc-3: Syringic acid (37)
Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-3 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Syringic acid. BLasé inhibition activity was tested.
CH3 OH CH3 8 7 O O 4 5 3
6 1 2
O OH (37)
EIMS m/z (rel. int. %): 198 (100), 183 (28), 127(20 ), 109 ( 15 ), 199 (11 ), 65 (8 ), 39 (07 ), 93 (07 ), 81 (07 ), 155 (6 ). 1H-NMR (CD3OD, 400 MHz) δ: 7.39 (2H, s, H-2 & 6), 3.92 (6H, s, H-7 & 8).
3.3.1.1.6- CIRCHAc-4: Vanillic acid (38)
Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-4 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Syringic acid. BLasé inhibition activity was tested.
69 Chapter-3: Experimental
OH CH3 7 O 5 4 3
6 1 2
O OH (38)
EIMS m/z (rel. int. %): 168(100), 153( 55), 97 (28), 125 (23), 15( 14), 51 (11), 52 (11), 169(09). 1H-NMR (CD3OD, 400 MHz) δ: 7.74 (1H, s, H-2), 7.05 (1H, d, J=8.49 Hz, H-5), 7.83 (1H, d, J=8.49 Hz, H-6), 3.84 (3H, s, H-7).
3.3.1.1.7- CIREANe-1: Esculetin/ 6,7-dihydroxy coumarine (39)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Esculetin. BLasé inhibition activity was tested. 8 HO O O 9 1 7 2
6 10 3 HO 5 4 (39) EIMS m/z (rel. int. %): 178 9100), 150(60), 179(14), 69(13), 75(10), 51(10), 76(08), 94(08), 50(07), 53(06). 1H-NMR (CD3OD, 400 MHz) δ: 6.24 (1H, d, J=9.55 Hz, H-3), 7.70 (1H, d, J=9.55 Hz, H-4), 6.87 (2H, s, H-5 & 8).
70 Chapter-3: Experimental
3.3.1.1.8- CIREANe-2: Scopoletin (40)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Scopoletin. BLasé inhibition activity was tested. 8 HO O O 9 1 7 2
H C 6 3 10 3 11 O 5 4 (40) EIMS m/z (rel. int. %): 192 ( 100), 177 (59), 149( 43), 69( 40), 164( 25), 51( 24), 121 (18), 79 (18), 193 (09), 50( 08). 1H-NMR (CD3OD, 400 MHz) δ: 6.18 (1H, d, J=9.55 Hz, H-3), 7.82 (1H, d, J=9.55 Hz, H-4), 6.93 (1H, S, H-5), 6.97 (1H, s, H-8), 3.90 (3H, s, H-11).
3.3.1.1.9- CIREANe-3: Umbelliferone (41)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-3 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Umbelliferone. BLasé inhibition activity was tested. 8 HO O O 9 1 7 2
6 10 3 5 4 (41)
71 Chapter-3: Experimental
EIMS m/z (rel. int. %): 162(100), 134( 87), 78(25), 105(19), 51(17), 77(13), 67(11), 163(11), 39(10), 63(09). 1H-NMR (CD3OD, 400 MHz) δ: 6.10 (1H, d, J=9.55 Hz, H-3), 7.71 (1H, d, J=9.55 Hz, H-4), 7.37 (1H, d, J=8.58 Hz, H-5), 6.71 (1H, d, J= 8.58 Hz, H-6), 6.69 (1H, s, H- 8). 3.3.1.1.10- CIREANe-4: Kaempferol (42)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-4 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Scopoletin. BLasé inhibition activity was tested. 15 OH 16 14 8 HO O 13 9 1 2 11 7 12 3 6 5 10 4 OH
OH O (42)
EIMS m/z (rel. int. %): 286(100), 285(31), 287(17), 121(10), 258(07), 143(06), 257(06), 229(06). 1H-NMR (CD3OD, 400 MHz) δ: 6.28 (1H, s, H-6), 6.54 (1H, s, H-8), 8.1 (2H, d, J=8.5 Hz, H-12 & 16), 7.00 (2H, d, J=8.5 Hz, H-13 & 15).
72 Chapter-3: Experimental
3.3.1.2:- Ageratum conyzoides
The plant material was kindly provided, after identification by Dr. Zaheer-ud- din Khan and Muhammad Ajaib from the Department of Botany, GC University, Lahore, where a voucher specimen was submitted (GCU-Herb-Bot-789) at Dr. Sultan Ahmad Herbarium. The plant material was dried in shade, grinded and soaked thrice in methyl alcohol for five days each time with intermittent shaking. The three filtrates were mixed. Methanol was recovered from filtrate by rotary evaporator under reduced pressure and low temperature (40 °C). The crude viscous extract was subjected to GCMS analysis as well as β-Lactamase (BLase) inhibition assay using an efficient assay to screen β-Lactamase inhibitors from plant extracts developed at our Lab and later on published [29]. The Crude extract was dissolved in 400 ml distilled water, filtered. The filtrate was successively extracted with n-Hexane, Chloroform, Ethyl acetate and n-Butanol (each 4 x 250 ml). Moisture was removed by filtering over the bed of anhydrous sodium sulphate. Solvents from dry filtrates were recovered by rotary evaporator. Dry isolated material was stored in vials. All the extracts were subjected to BLase inhibition assay and GCMS analysis. n-Hexane extract (AC-Hex) showed significant activity which was subjected to column chromatography. All the five fractions from column were subjected to GCMS analysis and preliminary screening for BLasé inhibition potential. The active fraction-2 was further run over TLC as shown in Scheme-3.2. TLC plates were visualized using one or more of: UV- light, Ceric sulphate and Draggondorff’s reagent. Bands containing plant material were scratched and washed repeatedly with methanol, chloroform and ethyl acetate which were further put to spectrophotometic BLasé inhibition assay. The active components were put to GCMS and 1H-HMR analysis for identification.
73 Chapter-3: Experimental
Ageratum conyzoides
Methanolic Extract
Dissolved in H2O and filtered.
Filterate
Solvent Extraction
Aq. Layer Hexane AC-Hex* Column Chromatography
H
H H
H
H
e
e e
e
e
x
x x
x
x
:
: :
Aq. Layer : Chloroform : E
E E
E
E
t
t t
O t
t
O O
O . AC-CH O
A
A A
A
A
c
c c
c c
(
( (
8 (
(
0 4
1 6
0
0 0
0 0
:
0
: :
:
2
:
1 6
4
0
0 0
0
0
)
0 )
0
)
)
) Aq. Layer Ethyl acetate . AC-EA Fraction-2
Preparative T L C Aq. Layer n-Butanol Hex : EtOAc : CH2Cl2 . AC-BU 6 : 3 : 1 Stigmasterol
* Showed BLase Inhibition activity Scheme-3.2:- Scheme for Bioassay guided Extraction, Fractionation and . Isolation from Ageratum conyzoides.
74 Chapter-3: Experimental
3.3.1.2. 1- AC-Hex-Fr-2: Stigmasterol (43)
Fraction-2 (Hexan : Ethylacetate :: 60 : 40) was found BLasé inhibitor. Its
preparative TLC (Hex : EtOAc : CH2Cl2 :: 6 : 3 :1) yielded one pure compound. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Stigmasterol. BLasé inhibition activity was tested.
H3C 29 28 22 H3C 20 25 CH3 21 24 26 23 12 CH3 18 CH 11 17 16 3 13 27 CH 1 93 19 8 2 14 15 10 3 7 HO 5 4 6 (43) EIMS m/z (rel. int. %): 55 (100), 43 (65), 81 (48), 69(47), 83 (43), 41 (36), 79 (29), 95 (27), 67 (26), 97 (25), 107 (23), 145 ( 16), 121 (11), 271 (11), 109 (10), 123 (14), 147 (10), 91 (24), 57 (17), 71 (10), 93 (20), 133 (19), 255 (10), 119 (14), 159 (16), 412 (14). 1H-NMR (CD3OD, 400 MHz) δ: 0.76 (3H, d, J = 6.3 Hz, H-26), 0.77 (3H, s, H-19), 0.80 (3H, t, H-29), 0.81 (3H, s, H-18), 0.87 (3H, d, J = 6.3 Hz, H-27), 1.00 (3H, d, J = 5.9 Hz, H-21), 1.15 (2H, t, H-12), 1.23 (2H, m, H-28), 1.26 (2H, m, H-15), 1.27 (2H, t, H-16), 1.30 (1H, m, H-17), 1.31 (1H, m, H-24), 1.35 (2H, t, H-1), 1.44 (2H, m, H- 2), 1.46 (2H, m, H-11), 1.50 (2H, t, H-4), 1.57 (1H, m, H-14), 1.58 (1H, m, H-25), 1.80 (1H, m, H-9), 1.95 (1H, m, H-5), 2.03 (1H, m, H-20), 2.67 (1H, m, H-8), 3.28 (1H, m, H-3), 5.03 (1H, t, H-22), 5.11 (1H, t, H-23), 5.35 (1H, t, H-7), 5.57 (1H, t, H- 6)
75 Chapter-3: Experimental
3.3.2:- Apocynaceae Family Six plants were screened for BLase Inhibition activity by subjecting their methanolic/ ethanolic extracts to preliminary screening for BLasé inhibition activity by Shahwar et. al. method[29]. Only Hollarhena antidysenterica, Carissa opaca and Alstonia Scholaris were found active and processed further. 3.3.2.1:- Hollarrhena antidysenterica (Kurchi bark) The plant bark was kindly provided by Dr. Muhammad Akram Chaudhary; Chief Executive DMA Homoeo Laboratories Lahore. It was basically imported from Part Khor Sac. , India. It was identified by Dr. Zaheer-ud-din Khan and Muhammad Ajaib, at the Department of Botany, GC University, Lahore, where a voucher specimen was submitted (GCU-Herb-Bot-997) at Dr. Sultan Ahmad Herbarium. The dry bark of Hollarrhena antidysenterica (4 Kg) was grinded and soaked twice in 20 liters of Ethyl alcohol (~ 95%) for seven days each time with intermittent shaking. The two filtrates were mixed. Ethanol was recovered from filtrate by rotary evaporator under reduced pressure and low temperature (40 °C). The reddish viscous crude extract was subjected to GCMS analysis as well as β-Lactamase (BLase) inhibition assay using an efficient assay to screen β-Lactamase inhibitors from plant extracts developed at our Lab and later on published [29]. The Crude extract was dissolved in 600 ml 2M HCl (pH = 3), kept overnight and filtered. The filtrate was basified with ammonium hydroxide (pH = 9) and was successively extracted with n- Hexane, Chloroform, Eethyl acetate and n-Butanol (4 x 250 ml). Moisture was removed by filtering over the bed of anhydrous sodium sulphate. Solvents from dry filtrates were recovered by rotary evaporator. Dry isolated material was stored in vials. All the extracts were subjected to BLase inhibition assay and GCMS analysis. Chloroform (HA-CH), Ethyl acetate (HA-EA) and n-Butanol extracts (HA-BU) showed significant activity which were subjected to Thin Layer Chromatography (TLC) as shown in Scheme-3.3. TLC plates were visualized using one or more of: UV-light, Ceric sulphate and Draggondorff’s reagent. Bands containing plant material were scratched and washed repeatedly with methanol, chloroform and ethyl acetate. They were further put to spectrophotometic BLase inhibition assay. The active components were put to GCMS and 1H-HMR analysis for identification.
76 Chapter-3: Experimental
* Showed BLase Inhibition activity Scheme-3.3:- Bioassay guided Extraction, Fractionation and Isolation from Holarrhena antidysenterica bark.
77 Chapter-3: Experimental
3.3.2.1.1. HA-CH-1 : Conessine (44)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 91.5 : 08 : 0.5) of Chloroform extract (HA-CH) yielded three compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Conessine. BLasé inhibition activity was tested.
H3C 25 CH3 N 22 19 18 1320 17 14 12 CH3 16 1 21 8 2 7 11 9 15
H C 3 3 10 6 23 N 4 5
CH3 24 (44) EIMS m/z (rel. int. %): 84 (100), 71 (15), 85(0 5), 80(04), 82(04), 341(03), 70(03).
1H-NMR (CD3OD, 400 MHz) δ: 2.07 & 2.38 (2H, dd, J=13.3 & 6.84 Hz, H-1a & 1b), 1.56 & 1.66 (2H, m, J=6.84, 13.3 &5.64 Hz, H-2a & 2b), 1.4 (1H, m, J=8.22, 5.64 & 4.67 Hz, H-3), 2.25 & 2.58 (2H, dd, J=4.67 Hz, H-4a & 4b), 5.33 (1H, t, J=5.25 Hz, H-5), 1.4 & 1.51 (2H, t, J=2.25 & 6.88 Hz, H-6a & 6b), 1.91 (1H, m, J=6.88, 12 & 5.8 Hz, H-7), 0.69 (1H, m, J=12 & 3.62 Hz, H-8), 1.01 (1H, m, J=5.8 & 9.0 Hz, H-11), 1.17 & 1.50 (2H, m, J=3.62 & 5.07 Hz, H-14a & 14b), 0.98 &1.46 (2H, m, J=9 & 8.5 Hz, H-15a & 15b), 1.63 & 1.35 (2H, m, J=8.5 & 6.17 Hz, H-16a & 16b), 2.17 (1H, m, J=6.17 & 5.9 Hz, H-17), 2.49 (1H, m, J=5.9 & 6.3 Hz, H-18), 2.34 & 2.42 (2H, d, J=12.0 Hz, H-20a & 20b), 0.99 (3H, s, H-21), 1.09 (3H, s, H-22), 2.25 (6H, s, H-23 & 24), 2.1 (3H, s, H-25).
78 Chapter-3: Experimental
3.3.2.1.2- HA-CH-2: Isoconessimine / Kurchinine (45)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 91.5 : 08 : 0.5) of Chloroform extract (HA-CH) yielded three compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Isoconessimine. BLasé inhibition activity was tested.
H3C 23 CH3 N 22 19 18 1320 17 14 12 CH3 16 1 21 8 2 7 11 9 15
H C 3 3 10 6 24 NH 4 5 (45)
EIMS m/z (rel. int. %): 71 (100), 327( 85), 70( 85 ), 342 (28), 273 (27), 328 (22), 341( 13), 56 (12), 72( 10), 343(08 ).
1H-NMR (CD3OD, 400 MHz) δ: 2.05 & 2.42 (2H, dd, J=13.3 & 6.84 Hz, H-1a & 1b), 1.51 & 1.60 (2H, m, J=6.84, 13.3 &5.64 Hz, H-2a & 2b), 1.41 (1H, m, J=8.22, 5.64 & 4.67 Hz, H-3), 2.25 & 2.51 (2H, dd, J=4.67 Hz, H-4a & 4b), 5.38 (1H, t, J=5.25 Hz, H-5), 1.4 & 1.59 (2H, t, J=2.25 & 6.88 Hz, H-6a & 6b), 1.85 (1H, m, J=6.88, 12 & 5.8 Hz, H-7), 0.71 (1H, m, J=12 & 3.62 Hz, H-8), 1.11 (1H, m, J=5.8 & 9.0 Hz, H-11), 1.22 & 1.50 (2H, m, J=3.62 & 5.07 Hz, H-14a & 14b), 0.98 &1.56 (2H, m, J=9 & 8.5 Hz, H-15a & 15b), 1.63 & 1.31 (2H, m, J=8.5 & 6.17 Hz, H-16a & 16b), 2.17 (1H, m, J=6.17 & 5.9 Hz, H-17), 2.45 (1H, m, J=5.9 & 6.3 Hz, H-18), 2.34 & 2.42 (2H, d, J=12.0 Hz, H-20a & 20b), 0.99 (3H, s, H-21), 1.09 (3H, s, H-22), 2.38 (6H, s, H-23), 2.1 (3H, s, H-24).
79 Chapter-3: Experimental
3.3.2.1.3- HA-CH-3: Conimine (46)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 91.5 : 08 : 0.5) of Chloroform extract (HA-CH) yielded three compounds in handsome amount. The material recovered from layer-3 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Conimine. BLasé inhibition activity was tested. H CH3 N 22 19 18 1320 17 14 12 CH3 16 1 21 8 2 7 11 9 15
H C 3 3 10 6 23 NH 4 5 (46) EIMS m/z (rel. int. %): 70(100), 259( 60), 57 (40), 260( 13), 56( 10), 71(0 8), 58(08), 55(08), 328(05), 91(05). 1H-NMR (CD3OD, 400 MHz) δ: 2.07 & 2.38 (2H, dd, J=13.3 & 6.84 Hz, H-1a & 1b), 1.56 & 1.66 (2H, m, J=6.84, 13.3 &5.64 Hz, H-2a & 2b), 1.4 (1H, m, J=8.22, 5.64 & 4.67 Hz, H-3), 2.25 & 2.58 (2H, dd, J=4.67 Hz, H-4a & 4b), 5.33 (1H, t, J=5.25 Hz, H-5), 1.4 & 1.51 (2H, t, J=2.25 & 6.88 Hz, H-6a & 6b), 1.91 (1H, m, J=6.88, 12 & 5.8 Hz, H-7), 0.69 (1H, m, J=12 & 3.62 Hz, H-8), 1.01 (1H, m, J=5.8 & 9.0 Hz, H-11), 1.17 & 1.50 (2H, m, J=3.62 & 5.07 Hz, H-14a & 14b), 0.98 &1.46 (2H, m, J=9 & 8.5 Hz, H-15a & 15b), 1.25 & 1.55 (2H, m, J=8.5 & 6.17 Hz, H-16a & 16b), 2.4 (1H, m, J=6.17 & 5.9 Hz, H-17), 2.84 (1H, m, J=5.9 & 6.28 Hz, H-18), 2.5 & 2.95 (2H, d, J=10.34 Hz, H-20a & 20b), 0.99 (3H, s, H-21), 0.98 (3H, s, H-22), 2.38 (3H, s, H-23).
80 Chapter-3: Experimental
3.3.2.1.4- HA-EA-1 : Kurchamine (47)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 88 : 11 : 01) of Ethyl acetate extract (HA-EA) yielded two compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Kurchamine. BLasé inhibition activity was tested.
H3C 21 20 NH2 13 CH3 19 17 14 12 CH3 16 1 18 8 2 7 11 9 15
H C 3 3 10 6 22 NH 4 5 (47) EIMS m/z (rel. int. %): 70(100), 44( 27), 261(20), 174(07), 71(06), 330(05), 262(05). 1H-NMR (CD3OD, 400 MHz) δ: 2.09 and 2.40 (2H,dd,J=6.84,13.3Hz,H1a and1b) ,1.38 and 1.55 (2H,m, J=6.84,13.4,5.64Hz, H-2a and 2b), 2.7 (1H,m, J=8.22, 5.64, 4.67 Hz, -3), 2.07 and 2.40 (2H, dd, J=4.67,17.33Hz, H-4a and 4b) ,5.31 (1H,t, J=5.25Hz, H-5), 1.49 and 1.60 (2H, t, J=5.25 and 6.88Hz, H-6a and 6b), 1.45 (Ht,m, J= 8.88, 12,5.8Hz, H-7) ,0.77(1H, m ,J=12,3.62Hz,H-8), 1.25 (1H, m, J=5.89Hz, H- 11), 0.98 and 1.74 (2H, dd, J=5.07,12.6Hz, H-13a and 13b), 1.45 and 1.55 (2H,m, J= 3.62, 5.07,13.2Hz, H-14a and14b), 1.35 and 1.83 (2H, m, J=9,8.5,10Hz, H-15a and 15b), 1.28 & 1.50 (2H, m, J=8.5, 10.86 Hz, H-16a & 16b), 1.99 (1H, m, J=8.6, 5.15 Hz, H-17), 0.99 (3H, s, H-18), 0.69 (3H, s, H-19), 3.73 (1H, m, J=5.15, 6.92 Hz, H- 20), 0.79 (3H, d, J=6.92 Hz, H-21), 2.38 (3H, s, H-22).
81 Chapter-3: Experimental
3.3.2.1.5-HA-EA-2 : Holaromine (48)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 88 : 11 : 01) of Ethyl acetate extract (HA-EA) yielded two compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Holaromine. BLasé inhibition activity was tested.
H3C HO 21 NH2 19 20 13 17 14 12 CH3 16 1 18 8 2 7 11 9 15
3 10 6 H N 2 4 5 (48) EIMS m/z (rel. int. %): 44(100), 56(75), 289( 22), 92 (10), 258( 08), 79(07), 57(07), 118(06), 55(06), 41(06).
1H-NMR (CD3OD, 400 MHz) δ: 2.11 and 2.42 (2H,dd,J=6.84,13.3 Hz,13.3Hz,H-1a and1b), 1.09 and1.29 (2H, m, J=6.84,13.43,8.22Hz, H-2a and 2b), 2.83 (1H, m, J=8.22,4.67Hz, H-3), 1.84 and2.16 (2H, dd, J=4.67 and 17.36Hz, H-4a and 4b), 5.35 (1H, t, J=5.25Hz, H-5), 1.52 and 1.64 (2H, t, J=5.25,6.88,18.35Hz, H-6a and 6b), 1.76 (1H, m, J=6.88,12,5.8Hz, H-7), 0.81(1H, m, J=12,3.62Hz, H-8), 1.16 (1H, m, J=5.8,9Hz, H-11), 0.99 and 1.89 (2H, dd, J= 5.07,12.6Hz, H-13a and 13b), 1.67 and 2.78 (2H, m, J= 3.62,5.61,13.2Hz, H-14a and 14b), 1.42 and 1.86 (2H, m, J=9,8.5,10Hz, H-15a and 15b), 1.33 and 1.61 (2H, m ,J =8.5,1OHz, H-16a and 16b), 2.21 (1H, m, J=8.6,5.15Hz, H-17), 0.99 (3H, s, H-18), 3.8 and 4.0 (2H, d, J=12.1Hz, H-19a and 19b), 3.68 (1H, m, J =5.15,6.92Hz, H-20), 0.82(3H, d, J= 6.92Hz, H-21)
82 Chapter-3: Experimental
3.3.2.1.6- HA-BU-1 : Kurchessine (49)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 88 : 11 : 01) of n-Butanol extract (HA-BU) yielded one compound in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Kurchessine. BLasé inhibition activity was tested.
CH3 25 H3C 21 N 20 13 CH3 19 CH3 17 24 14 12 CH3 16 1 18 2 8 11 9 7 15
H C 3 3 10 6 23 N 4 5
CH3 22 (49) EIMS m/z (rel. int. %): 72(100), 84( 30), 73(07), 301(03), 85(03), 286(03). 1H-NMR (CD3OD, 400 MHz) δ: 2.07 & 2.38 (2H, dd, J=13.3 & 6.84 Hz, H-1a & 1b), 1.56 & 1.66 (2H, m, J=6.84, 13.3 &5.64 Hz, H-2a & 2b), 1.4 (1H, m, J=8.22, 5.64 & 4.67 Hz, H-3), 2.25 & 2.58 (2H, dd, J=4.67 Hz, H-4a & 4b), 5.33 (1H, t, J=5.25 Hz, H-5), 1.4 & 1.51 (2H, t, J=2.25 & 6.88 Hz, H-6a & 6b), 1.91 (1H, m, J=6.88, 12 & 5.8 Hz, H-7), 0.69 (1H, m, J=12 & 3.62 Hz, H-8), 1.01 (1H, m, J=5.8 & 9.0 Hz, H-11), 1.17 & 1.50 (2H, m, J=3.62 & 5.07 Hz, H-14a & 14b), 0.98 &1.46 (2H, m, J=9 & 8.5 Hz, H-15a & 15b), 1.63 & 1.35 (2H, m, J=8.5 & 6.17 Hz, H-16a & 16b), 1.74 (1H, m, J=8.59 & 5.15 Hz, H-17), 0.99 (3H, s, H-18), 0.58 (3H, s, H-19), 3.17 (1H, m, J=5.15 & 6.84 Hz, H-20), 0.74 (3H, d, J=6.84 Hz, H-21), 2.25 (6H, s, H- 22 & 23), 2.08 (6H, s, H-24 & 25).
83 Chapter-3: Experimental
3.2.2.2:- Carissa opaca
The plant material (roots) was kindly provided, after identification by Dr. Zaheer-ud-din Khan and Muhammad Ajaib from the Department of Botany, GC University, Lahore, where a voucher specimen was submitted (GCU-Herb-Bot-735) at Dr. Sultan Ahmad Herbarium. The plant material was dried in shade, grinded and soaked thrice in ethyl alcohol (~ 95 %) for five days each time with intermittent shaking. The three filtrates were mixed. Ethanol was recovered from filtrate by rotary evaporator under reduced pressure and low temperature (40 °C). The crude viscous extract was subjected to GCMS analysis as well as β-Lactamase (BLase) inhibition assay using an efficient assay to screen β-Lactamase inhibitors from plant extracts developed at our Lab and later on published [29]. The Crude extract was dissolved in 500 ml distilled water, filtered and the filtrate was successively extracted with n-Hexane, Chloroform, Ethyl acetate and n-Butanol (each 4 x 250 ml). Moisture was removed by filtering over the bed of anhydrous sodium sulphate. Solvents from dry filtrates were recovered by rotary evaporator. Dry isolated material was stored in vials. All the extracts were subjected to BLase inhibition assay and GCMS analysis. Chloroform extract (CO- CH) showed significant activity. It was refluxed with methanolic KOH for two hours to remove fatty acids and filtered. The filtrate was extracted thrice with diethyl ether. Three extracts were mixed together, moisture removed by filtering over the bed of anhydrous sodium sulphate and solvent recovered by rotary evaporator equipped with chiller (- 5 Co) for condenser. Dry extract was subjected to column chromatography. All the three fractions from column were subjected to GCMS analysis and preliminary screening for BLase inhibition potential, as shown in Scheme-3.4. The active components were put to GCMS and 1H-NMR analysis for identification.
84 Chapter-3: Experimental
Carissa opaca
Ethanolic Extract
Dissolved in H2O and filtered.
Filterate
Solvent Extraction
Hexane Aq. Layer CO-Hex
Aq. Layer Chloroform . CO-CH**
Reflux with Methanolic KOH for 2 hrs. and Extract with Diethyl Ether Aq. Layer Ethyl acetate . CO-EA Column Chromatography of Dried Ether Extract
H
H
H
e
e
e
x
x
x
:
:
:
D
D
D
C
C
C
n-Butanol M
Aq. Layer M
M
. CO-BU
(
(
6
(
1
8
0
0
0
:
:
:
4
9
2
0
0
0
)
)
Frac-3 Campesterol Lupeol
*Showed BLase Inhibition activity Scheme-3.4:- Bioassay guided Extraction, Fractionation and Isolation from Carissa opaca.
85 Chapter-3: Experimental
3.3.2.2.1-CO-CH-Basic Ether-1: Lupeol (33)
Column chromatography with gradient elution (CH2Cl2 : Hex) of defatted chloroform extract (CO-CH-Basic Ether) yielded two compounds in handsome
amount. The material recovered from fraction-1 (CH2Cl2 : Hex :: 20 : 80) was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Lupeol (33), the same compound as from Cichorium intybus.
3.3.2.2.2- CO-CH-Basic Ether-2: Campesterol (50)
Column chromatography with gradient elution (CH2Cl2 : Hex) of defatted chloroform extract (CO-CH-Basic Ether) yielded two compounds in handsome
amount. The material recovered from fraction-2 (CH2Cl2 : Hex :: 20 : 80) was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Campesterol. BLasé inhibition activity was tested. 28
22 H3C 20 25 CH3 21 24 26 23 12 CH3 18 CH 11 17 16 3 13 27 CH 1 93 19 8 2 14 15 10 3 7 HO 5 4 6 (50)
EIMS m/z (rel. int. %): 43 (100), 55 (62), 41 (47), 57 (45), 81 (44), 95 (42), 107 (40), 71 (31), 105 (31), 145 (29), 69 (27), 79 (27), 109 (21), 119 (21), 91 (27), 121 (21), 161 (20), 67 (27), 93 (27), 400 (28), 213 (20), 289 (23).
1H-NMR (CD3OD, 400 MHz) δ: 1.05 & 1.85 (2H, d of t, J=6.84 & 13.3 Hz, H-1a & 1b), 1.5 & 1.8 (2H, d of m, J=6.84, 9.0 & 12.9 Hz, H-2a & 2b), 3.53 (1H, m, J=7.5 & 86 Chapter-3: Experimental
9.0 Hz, H-3), 2.2 & 2.3 (2H, dd, J=7.5 & 12.5 Hz, H-4a & 4b), 5.35 (1H, t, J=5.25 Hz, H-6), 1.87 & 1.97 (2H, d of t, J=5.25, 18.35 & 6.88 Hz, H-7a & 7b), 1.45 (1H, m, J=6.88, 12.0 & 5.8 Hz, H-8), 0.9 (1H, m, J=12.0 & 3.62 Hz, H-9), 1.45 & 1.55 (2H, d of m, J=3.62, 13.2 & 5.07 Hz, H-11a & 11b), 1.17 & 2.02 (2H, d of t, J=5.07 & 12.6 Hz, H-12a & 12b), 1.1 (1H, m, J=5.8 & 9.0 Hz, H-14), 1.07 & 1.57 (2H, d of m, J=9.0, 10.0 & 8.5 Hz, H-15a & 15b), 1.26 & 1.87 (2H, d of m, J=8.5, 10.0 & 8.6 Hz, H-16a & 16b), 1.13 (1H, m, J=8.6 & 10.9 Hz, H-17), 0.68 (3H, s, H-18), 1.01 (3H, s, H-19), 0.92 (1H, m, J=10.9, 6.1, 13.5 & 18.5 Hz, H-20), 0.92 (3H, d, J=6.12 Hz, H- 21), 0.8 & 1.4 (2H, d of m, J=13.5, 13.6 & 7.1 Hz, H-22a & 22b), 0.76 & 1.35 (2H, d of m, J=7.0, 13.6 & 18.5 Hz, H-23a & 23b), 1.15 (1H, m, J=18.5, 5.13 & 6.57 Hz, H- 24a & 24b), 1.98 (1H, m, J=5.13 & 6.76 Hz, H-25), 0.87 (6H, d, J=6.76 Hz, H-26 & 27), 0.97 (3H, d, J=6.57 Hz, H-28).
87 Chapter-3: Experimental
3.3.2.3:- Alstonia scholaris The plant material (bark) was collected from Botanical Garden of GCU Lahore, identified by Dr. Zaheer-ud-din Khan and Muhammad Ajaib at the Department of Botany, GC University, Lahore, where a voucher specimen was submitted (GCU-Herb-Bot-743) at Dr. Sultan Ahmad Herbarium. The plant material was dried in shade, grinded and soaked thrice in methyl alcohol for five days each time with intermittent shaking. The three filtrates were mixed. Methanol was recovered from filtrate by rotary evaporator under reduced pressure and low temperature (40 °C). The crude viscous extract was subjected to GCMS analysis as well as β-Lactamase (BLase) inhibition assay using an efficient assay to screen β-Lactamase inhibitors from plant extracts developed at our Lab and later on published [29]. The Crude extract was dissolved in 400 ml distilled water, filtered and the filtrate was successively extracted with n-Hexane, Chloroform, Ethyl acetate and n-Butanol (each 4 x 250 ml). Moisture was removed by filtering over the bed of anhydrous sodium sulphate. Solvents from dry filtrates were recovered by rotary evaporator. Dry isolated material was stored in vials. All the extracts were subjected to BLase inhibition assay and GCMS analysis. Ethyl acetate extract (As- EA) showed significant activity which was subjected to column chromatography. All the five fractions from column were subjected to GCMS analysis and preliminary screening for BLasé inhibition potential. The active fractions-2 & 4 were further run over TLC as shown in Scheme-3.5. TLC plates were visualized using one or more of: UV-light, Ceric sulphate and Draggondorff’s reagent. Bands containing plant material were scratched and washed repeatedly with methanol, chloroform and ethyl acetate which were further put to spectrophotometic BLase inhibition assay. The active components were put to GCMS and 1H-HMR analysis for identification.
88 Chapter-3: Experimental
Alstonia scholaris
Methanolic Extract
Dissolved in H2O, filtered
Filterate
Aq. Layer Hexane AS-Hex
Aq. Layer Chloroform AS-CH
Aq. Layer EtOAc AS-EA Column Chromatograpy
M
H
H H
H
e
e e e
e
x
x x O
x
H
:
: :
:
n-BuOH
:
E Aq. Layer E E
E
E
t
t t
AS-BU t
O
O O
t
O
O
A
A A
A
A
c
c c
c
c
(
( (
(
0
1 3
(
7
0
0
0 0
0
2
0
:
:
:
:
:
1
7
3
9
0
0 0
0
8
0
0
)
)
)
)
)
Fraction-2 Fraction-4
Preparative TLC Preparative TLC Hex : EtOAc : CH2Cl2 Hex : EtOAc : MeOH 6 : 3 : 1 6 : 3 : 1
Quercetine Kaempherol
*Showed BLase Inhibition activity Scheme-3.5:- Bioassay guided Extraction, Fractionation and Isolation from Alstonia scholaris.
89 Chapter-3: Experimental
3.3.2.3.1-AS-EA-1: Quercetin (51)
Preparative Thin Layer Chromatography (Hex : EtOAc : MeOH :: 6:3:1) of (AS-EA-Fr-2) yielded one compound in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Quercetin. BLasé inhibition activity was tested. OH OH 2' 3' 4' 8 HO 9 O 2 5' 1 1' 7 6' 3 6 5 4 10 OH
OH O (51) EIMS m/z (rel. int. %): 302 (100), 137 (23), 69(19), 301 (17), 153 (17), 303 (16), 28 (16), 274 (11), 229 (10), 39 (10). 1H-NMR (CD3OD, 400 MHz) δ: 6.28 (1H,s,H-6), 6.54 (1H,s,H-8), 7.97 (1H, s, H- 2’), 7.40 (1H, d, J=8.48 Hz, H-5’), 7.70 (1H, d, J=8.48 Hz, H-6’)
3.3.2.3.2- AS-EA-2: Kaempferol (42)
Preparative Thin Layer Chromatography (Hex : EtOAc : MeOH :: 6:3:1) of (AS-EA-Fr-4) yielded one compound in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Kaempferol (42), the same compound as from Cichorium
90 Chapter-3: Experimental
3.3.3:- Asclepiadaceae Family Two plants were screened for BLase Inhibition activity by subjecting their methanolic/ ethanolic extracts to preliminary screening for BLasé inhibition activity by Shahwar et. al. method. Only Calotropis procera was found active and processed further. 3.3.3.1:- Calotropis procera: The plant was collected from Johar Town Lahore. It was identified by Dr. Zaheer-ud-din Khan and Muhammad Ajaib, at the Department of Botany, GC University, Lahore, where a voucher specimen was submitted (GCU-Herb-Bot-754) at Dr. Sultan Ahmad Herbarium. The dry herb was grinded and successively extracted with n-Hexane, Dichloromethane, Ethyl acetate and Metahnol. Solvents were recovered by rotary evaporator under reduced pressure and low temperature (40 °C). The crude extracts were subjected to GCMS analysis as well as β-Lactamase (BLase) inhibition assay using an efficient assay to screen β-Lactamase inhibitors from plant extracts developed at our Lab and later on published (Shahwar et. al. 2011). All the extracts were subjected to BLase inhibition assay and GCMS analysis. Dichloromethane (CP-DCM) extract showed significant activity which was subjected to column chromatography. All the five fractions were subjected to GCMS analysis and preliminary Screening for β-Lactamase (BLase) inhibition activity. The active fraction-3 was further column chromatographed (Scheme-3.6) to yield four fractions which were again preliminary screened for BLasé inhibition activity and run on GCMS. Fraction-3-3 was subjected to preparative Thin Layer Chromatography (TLC) as shown in Scheme-3.6. TLC plates were visualized using one or more of: UV-light, Ceric sulphate and Draggondorff’s reagent. Bands containing plant material were scratched and washed repeatedly with methanol, chloroform and ethyl acetate. They were further put to spectrophotometic BLase inhibition assay. The active components were put to GCMS and 1H-HMR analysis for identification.
91 Chapter-3: Experimental
Calotropis procera
Plant material successively soaked thrice and filtered.
Hexane Dichloromethane Ethylacetate Methanol CP_Hex CP-DCM ** CP-EA CP-ME
Column Chromatography
H
H
H
M
H
e
e
e
x
x
e
e
x
x
O
:
:
:
H
D
:
D
D
D
C
C
:
C
C
D M
M
M
M
C
M
(
(
1
(
5
(
2
(
0
0
0
0
0
0
:
0
1
:
5
:
:
:
0
7
0
1
9
)
0
0
0
9
)
)
0
) )
Fraction-1 Fraction-2 Fraction-3** Fraction-4 Fraction-5
Column Chromatography
H
M
H
H
e
e
e
e
x
O
x
x
H
:
:
:
D
:
D
D
C
D
C
C
M
C
M
M
M
(
( 5
(
(
0
2
0
0
1
0
0
:
: 5
:
:
0
9
7
1
)
9
0
0
)
)
0 )
Fraction-1 Fraction-3 Fraction-4 beta-Sitostrol
Preparative T L C Hex : EtOAc : CH2Cl2 60 : 25 : 15 alpha-Amyrin Ursolic acid
*Showed BLase Inhibition activity Scheme-3.6:- Scheme for Bioassay guided Extraction, Fractionation and Isolation from Calotropis procera.
92 Chapter-3: Experimental
3.3.3.1.1-CP-DCM-Fr-3-2: β-Sitosterol (34) CP-DCM-Fr-3 was further fractionated through column chromatography with gradient elution (Hex : DCM : MeOH) to yield four fractions. All the fractions were put to GCMS analysis. Fraction-2 (Hex : DCM :: 20:70) was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as β-Sitosterol (34), the same compound as from C.intybus.
3.3.3.1.2- CP-DCM-Fr-3-3A: α-Amyrin (52) CP-DCM-Fr-3 was further fractionated through column chromatography with gradient elution (Hex : DCM : MeOH) to yield four fractions. All the fractions were put to GCMS analysis. Fraction-3 (Hex : DCM :: 00:100) was further fractionated through Preparative Thin Layer Chromatography (Hex : EtOAc : DCM :: 60:25:15). It yielded two compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as alpha-Amyrin. BLasé inhibition activity was tested.
2 OH 1 3 H C 11 3 25 4 CH3 23 12 9 10 5 CH3 29 CH3 13 8 24 H3C 6 30 21 22 14 20 7 CH3 26 17 CH3 19 15 27
18 16 CH3 28 (52) EIMS m/z (rel. int. %): 218 (100), 219 (18), 203 (16), 189 (11), 135 (10), 122 (10), 95 (10), 426 (09), 207 (09), 109 (08). 1H-NMR (CD3OD, 400 MHz) δ: 1.41 & 1.84 (2H, d of t, J=3.67 & 12.6 Hz, H-1a & 1b), 1.55 & 1.9 (2H, d of m, J=3.67 & 12.4 Hz, H-2a & 2b), 3.41 (1H, t, J=6.39 & 9.17 Hz, H-3), 0.62 (1H, t, J=9.0 Hz, H-5), 1.39 & 1.61 (2H, d of m, J=9 & 5.07 Hz,
93 Chapter-3: Experimental
H-6a & 6b), 1.25 & 1.65 (2H, d of t, J=5.07 Hz, H-7a & 7b), 1.69 (1H, t, J=5.5 Hz, H- 9), 2.28 & 2.47 (2H, d of t, J=3.58, 5.55 & 17.9 Hz, H-11a & 11b), 5.14 (1H, t, J=3.58 Hz, H-12), 1.77 & 1.87 (2H, d of t, J=7.73 & 7.03 Hz, H-15a & 15b), 2.02 & 2.14 (2H, d of t, J=7.73 & 13.65 Hz, H-16a & 16b), 1.1 & 1.35 (2H, d of t, J=4.65 & 13.43 Hz, H-18a & 18b), 1.27 & 1.76 (2H, d of m, J=4.65, 12.0 & 3.45 Hz, H-19a & 19b), 1.54 (1H, m, J=3.45 & 8.07 Hz, H-20), 1.72 (1H, t, J=8.07 & 7.88 Hz, H-21), 1.15 (1H, d, J=7.88 Hz, H-22), 1.27 (3H, s, H-23), 1.16 (3H, s, H-24), 0.98 (3H, s, H-25), 0.94 (3H, s, H-26), 1.08 (3H, s, H-27), 0.81 (3H, s, H-28), 0.89 (3H, d, J=6.63 Hz, H- 29), 0.78 (3H, d, J=6.7 Hz, H-30).
3.3.3.1.3- CP-DCM-Fr-3-3B: Ursolic acid (53) CP-DCM-Fr-3 was further fractionated through column chromatography with gradient elution (Hex : DCM : MeOH) to yield four fractions. All the fractions were put to GCMS analysis. Fraction-3 (Hex : DCM :: 00:100) was further fractionated through Preparative Thin Layer Chromatography (Hex : EtOAc : DCM :: 60:25:15). It yielded two compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Ursolic acid. BLasé inhibition activity was tested. 2 OH 1 3 11 H3C 25 4 CH3 23 12 9 10 5 CH3 29 CH3 13 8 24 H3C 6 30 21 22 14 20 7 CH3 26 17 CH3 19 15 27
18 28 16 HO O (53) EIMS m/z (rel. int. %): 248 (100), 203 (47), 133 (35), 249 (31), 190 (27), 189 (23), 119 (19), 204 (13), 121 (11), 219 (10), 123 (05), 456 (06), 120 (05).
94 Chapter-3: Experimental
1H-NMR (CD3OD, 400 MHz) δ: 1.42 & 1.84 (2H, d of t, J=3.67 & 12.6 Hz, H-1a & 1b), 1.50 & 1.93 (2H, d of m, J=3.67 & 12.4 Hz, H-2a & 2b), 3.41 (1H, t, J=6.39 & 9.17 Hz, H-3), 0.65 (1H, t, J=9.0 Hz, H-5), 1.39 & 1.65 (2H, d of m, J=9 & 5.07 Hz, H-6a & 6b), 1.25 & 1.65 (2H, d of t, J=5.07 Hz, H-7a & 7b), 1.69 (1H, t, J=5.5 Hz, H- 9), 2.28 & 2.47 (2H, d of t, J=3.58, 5.55 & 17.9 Hz, H-11a & 11b), 5.42 (1H, t, J=3.58 Hz, H-12), 1.81 & 2.01 (2H, d of t, J=8.85 & 13.03 Hz, H-15a & 15b), 2.43 & 2.54 (2H, d of t, J=8.85 & 13.05 Hz, H-16a & 16b), 1.76 & 1.95 (2H, d of t, J=4.4 & 13.03 Hz, H-18a & 18b), 1.27 & 1.76 (2H, d of m, J=4.65, 12.0 & 3.45 Hz, H-19a & 19b), 1.60 (1H, m, J=3.45 & 8.07 Hz, H-20), 1.72 (1H, t, J=8.07 & 7.88 Hz, H-21), 2.15 (1H, d, J=7.88 Hz, H-22), 1.27 (3H, s, H-23), 1.16 (3H, s, H-24), 0.98 (3H, s, H-25), 0.94 (3H, s, H-26), 1.08 (3H, s, H-27), 0.99 (3H, d, J=6.63 Hz, H-29), 0.79 (3H, d, J=6.7 Hz, H-30).
95 Chapter-3: Experimental
3.4:- Β-Lactamase (BLase) Inhibition Assays
3.4.1:- Development of an Efficient Assay to Screen β-Lactamase Inhibitors from Plant Extracts [29]: A new, easy, efficient, economical and versatile growth based method was developed. The method has been used successfully in our laboratory and published [29]. The relevant experimental details of this method are described in section 3.4.2 and illustrated in Table-3.1 & Fig.-3.2.
3.4.2:- Preliminary Screening for BLase Inhibition Potential Assay was performed in test tubes (TTs) according to our developed method [29] as follows: Benzyl Penicillin, Clavulanic acid and sample (both isolated and synthesized) solutions were sterilized by micro-filtration with syringe-filters (20 μm, PTFE, Star Lab Scientific Co. Ltd.) into sterilized/autoclaved vials while working in the Laminar Flow Cabinet. 3.8 mL LB solution (25 g/dm3) was added to wider (38 mm × 200 mm) culture/test tubes (TTs), cotton plugged and autoclaved (15-20 min, 121 ºC, 0.11 MPa). After cooling to room temperature, according to scheme given in Table-3.1, 80 μL of each of sterilized plant extract solution (100 mg/mL) and Benzyl Penicillin solution (100 mg/mL) were added to make total volume 4 mL and hence get final concentration of 2 mg/mL each. For positive/negative controls water and corresponding solvents were added to make-up the volume. Plugged TTs were put on rotary shaker (ca. 50-100 rpm) to homogenize for 15-30 min. 15-18 h old inocculum was adjusted/ diluted to 10 6-8 CFU with sterile saline solution by spectrophotometry (530 nm) corresponding to 0.5 McFarland standard. Then 20 μL of inocculum were added to each TT, while working in the laminar flow cabinet. Put on rotary shaker (100-150 rpm) to assure homogeneity and to maintain aerobic conditions as well. Clavualnic acid and Augmentin (injection) were used as standard inhibitors. Observed during 10- 96 h any changes in turbidity/colour and reported according to Table-3.1
96 Chapter-3: Experimental
Table-3.1: Assay Scheme and Observations for BLase Inhibitory Potential TT# Contents of TT Observation (10-96 hrs) Inference/Result
It served as a colour L.Broth reference for comparison 1 Plant extract No Change as expected. (i.e -ve control) of Benzyl Penicillin experimental samples
Bacterial strain is Benzyl L.Broth Penicillin / Ampicillin TT contents turned turbid 2 Benzyl Penicillin resistant through BLase due to growth of bacteria E.coli(DH5α)Inocculum production ( i.e +ve control).
TT contents turned turbid Plant extract is not as compared with TT#-1 antibacterial in its own (i.e L.Broth due to growth of bacteria +ve control). Plant extract 3 Plant extract is itself E.coli(DH5a)inocculum TT contents show no effective against BL turbidity as compared with resistant mutant strain TT#-1 i.e. no growth of through some other bacteria mechanism TT contents turned turbid L.Broth Plant extract is not BLase as compared with TT#-1 Plant extract inhibitor. due to growth of bacteria 4 Benzyl Penicillin TT contents: no turbidity E.coli(DH5a)inocculum Plant extract is BLase- as compared with TT# 1 inhibitor i.e. no growth of bacteria
97 Chapter-3: Experimental
3.4.2:- Spectrophotometric Assay for Determining β-Lactamase Inhibition Activity using Nitrocefin Selected; isolated and synthetic samples were then finally spectrophotometrically screened for their BLase inhibitory potential using Nitrocefin.
Fig3.1- Nitrocefin ( a cephalosporinic β -lactam compound ): {3-(2,4-Dinitrostyryl)-(6R, 7R)-7-(2-thienylacetamido)-ceph-3-em-4-carboxylic Acid, E-isomer}
Nitrocefin (C21H16N4O8S2, Mr. 516.5) is a chromogenic (orange-yellow solid) β-lactamase substrate that undergoes distinctive color change from yellow (λmax = 390 nm at pH 7.0) to red (λmax = 486 nm at pH 7.0) as the amide bond in the β- lactam ring is hydrolyzed by BLase. Nitrocefin is sensitive to hydrolysis by all known lactamases produced by Gram-positive and Gram-negative bacteria. It has wide uses in competitive inhibition studies in developmental work on β-Lactamase-resistant antibiotics. (Ref.: Calbiochem data sheet). Phosphate Buffer-A: 100 mM phosphate; 1 mM EDTA, pH 7.0 Buffer solution-B: 0.1 M Tris-HCl, pH 7.0 with 1% gelatin. Nitrocefin solution-D: 01 mg Nitrocefin was dissolved in 100 μL dimethylsulfoxide (DMSO) and was vortexed. Then added 1.9 mL phosphate buffer-A to make total volume 2 mL. This yielded a 500μg/mL (approx. 1 mM) stock solution-C. Special care was taken to protect from light as Nitrocefin, particularly in solution, is very sensitive to light. Stock solution could have been stored at -20°C for maximum up to 2 weeks. The stock solution-C was diluted ten-fold in buffer-A to get working solution-D. (Reference: Calbiochem broucher).
98 Chapter-3: Experimental
Enzyme solution-E:
One unit of beta-lactamase II (mass of vial contents ÷ 50 = g: as each vial contained approximately 500 units beta-lactamase I and 50 units of beta-lactamase II) was reconstituted in 100mL of solution-B. (Reference MP Bio).
2+ Zn solution-F: . 0.1 M ZnSO4 Solution in water.
Enzyme Unit Definition: One unit is defined as the amount of enzyme which will hydrolyze 1.0 micromole of benzyl penicillin and 1.0 micromole of cephalosporin C per minute at 25°C in the presence of EDTA, pH 7.0 and Zn2+. (Reference MP Bio).
Typical Assay Method: 10μL sample solution (10 mg/mL; to carry in 100μg sample)
was mixed with mL ZnSO4 solution-F in two TTs each. 2mL of each of solution-B and enzyme solution-E were added to TT-1 (reference/control) and TT-2 (sample). After 5-10 minutes equilibration through gentle shaking, 1.0 mL of Nitrocefin working solution-D was added to each/TT-2(sample) test tube to carry in 0.1 μM (50 μg ) of substrate (Nitrocefin). The TTs were briskly shaken and contents transferred to cuvettes. Having autozeroed, spectrophotometric assay for β-Lactamase inhibition potential was carried out by monitoring changes in absorbance at 486 nm for 30 minutes. (NB: the molar extinction coefficient of hydrolyzed Nitrocefin at 486 nm is 20,500 M-1 cm-1). No increase in absorbance means the sample is BLase inhibitor as the inhibitors bind to the enzyme more effectively than Nitrocefin (and other β-lactam antibiotics) hence rendering it inactive. β -Lactamase I catalyzes the following reaction + Benzyl Penicillin + H2O D-Benzyl Penicillate + H β -lactamase II catalyzes the following reaction + Cephalosporin C + H2O (inactive) Cephalosporin + Acetate + 2H
99 Chapter-3: Experimental
Figure-3.2:- Pictorial Demonstration of Table 3.1
100 Chapter-4: Results & Discussion
4. RESULTS & DISCUSSION 4.1 Ketophosphonates
4.1.1- Diethyl (2-oxo-2-phenylethyl)phosphonate (1-a) (1)
CH3 9 10 O O 2 O 7 P 3 1 8 O
4 6 11 5 CH3 12
The pure compound (1-a) was obtained from column chromatography.
HREIMS confirmed molecular formula as C12H17O4P. The EIMS of compound indicated molecular ion [M+] at m/z 256, base peak at m/z 105 along with other characteristic peaks at 146, 123, 120, 106, 91, 78 and 77. In 1H-NMR (400MHz,
CD3OD) the five aromatic protons (H-2, H-3, H-4, H-5, H-6) appeared between δ 8.23- 7.49, two methylenic protons (H-8) showed a singlet at δ 3.56, four phospho- esteric methylenic protons (H-9,11) appeared as a multiplet between δ 4.11-3.55 and the six phospho-esteric methylic protons (H-10, 12) appeared as triplet at δ 1.3. β- Lactamase inhibition activity of the compound proved it to be more potent (Table 4.1 & 4.2) than Clavulanic acid; a widely used BLase inhibitor in modern chemotherapy.
101 Chapter-4: Results & Discussion
4.1.2- Diethyl [(E)-1-benzoyl-2-phenylvinyl]phosphonate (1a-1) (2)
CH3 9 10 O O O 2 7 P 11 3 8 1 O CH3 4 6 13 1' 12 5 2' 6'
3' 5' 4'
For the compound (1-a-1) HREIMS confirmed molecular formula as + C19H21O4P. The EIMS of compound indicated molecular ion [M ] at m/z 344, base peak at m/z 77 along with other characteristic peaks at 207, 130, 119, 105, 102 and 90. 1 In H-NMR (400MHz, CD3OD) the ten aromatic protons (H-2, H-3, H-4, H-5, H-6 & H-2/, H-3/, H-4/, H-5/, H-6/, ) from both rings (A & B) appeared between δ 8.07- 7.5, one methylynic proton (H-13) showed a singlet at δ 7.79, four phospho-esteric methylenic protons (H-9,11) appeared as a multiplet δ 4.10 and the six phospho- esteric methylic protons (H-10, 12) appeared as triplet at δ 1.3. The compound was found inactive for β-Lactamase inhibition activity.
102 Chapter-4: Results & Discussion
4.1.3- Diethyl [(E)-1-benzoyl-2-(2-hydroxyphenyl)vinyl]phosphonate(1a-2) (3)
CH3 9 10 O O O 2 7 P 11 3 8 1 O CH3 4 6 13 1' 12 HO 5 6' 2'
3' 5' 4'
For the compound (1-a-2), HREIMS confirmed molecular formula as + C19H21O5P. The EIMS of compound indicated molecular ion [M ] at m/z 360, base peak at m/z 77 along with other characteristic peaks at 118,117, 105 and 51. In 1H-
NMR (400MHz, CD3OD) the five aromatic protons from ring-A (H-2, H-3, H-4, H-5, H-6) appeared between δ 8.07- 7.5, the four aromatic protons from ring-B (H-3/, H-4/, H-5/, H-6/) appeared between δ 7.78- 6.7, one methylynic proton (H-13) showed a singlet at δ 7.91, four phospho-esteric methylenic protons (H-9,11) appeared as a multiplet at δ 4.1 and the six phospho-esteric methylic protons (H-10, 12) appeared as triplet at δ 1.3. The compound was found inactive for β-Lactamase inhibition activity.
103 Chapter-4: Results & Discussion
4.1.4- Diethyl [(1E,3E)-1-benzoyl-4-phenylbuta-1,3-dien-1-yl]phosphonate (1a-3) (4)
CH3 9 10 O O O 2 7 P 11 3 8 1 O CH3 4 6 13 14 12 5 1' 15 6' 2'
5' 3' 4'
For the compound (1-a-3), HREIMS showed molecular formula as C21H23O4P. The EIMS of compound indicated molecular ion [M+] at m/z 370, base peak at m/z 234 along with other characteristic peaks at 235, 233, 156, 128, 127, 105, 77 and 50. 1 In H-NMR spectrum (400MHz, CD3OD) the five aromatic protons from ring-A (H-2, H-3, H-4, H-5, H-6) appeared between δ 8.05- 7.51, the five aromatic protons from ring-B (H-2/, H-3/, H-4/, H-5/, H-6/) appeared between δ 7.7- 7.25, the two methylynic protons (H-13, 15) appeared (at δ 7.3, 6.9) as doublets while one methylynic proton (14-H) showed a triplet at δ 7.01, four phospho-esteric methylenic protons (H-9,11) appeared as a multiplet at δ 4.1 and the six phospho-esteric methylic protons (H-10, 12) appeared as triplet at δ 1.3. β- Lactamase inhibition activity of the compound proved it to be sufficiently active (Table 4.1 & 4.2).
104 Chapter-4: Results & Discussion
4.1.5- Diethyl [(1E)-1-benzoyl-2-phenylprop-1-en-1-yl]phosphonate (1a-4) (5)
CH3 9 10 O O O 2 7 P 11 3 8 1 O 13 CH3 4 6 1' 12 H3C 5 14 2' 6'
3' 5' 4'
For the compound (1-a-4), HREIMS showed molecular formula as C20H23O4P. The EIMS of compound indicated molecular ion [M+] at m/z 358, base peak at m/z 221 along with other characteristic peaks at 222, 144, 115, 117, 105, 77, 51 and 39. 1 In H-NMR spectrum (400MHz, CD3OD) the five aromatic protons from ring- A (H-2, H-3, H-4, H-5, H-6) appeared between δ 8.0- 7.51, the five aromatic protons from ring-B (H-2/, H-3/, H-4/, H-5/, H-6/) appeared between δ 7.55- 7.4, the three methylic protons (H-14) produced a singlet, four phospho-esteric methylenic protons (H-9,11) appeared as a multiplet at δ 4.12 and the six phospho-esteric methylic protons (H-10, 12) appeared as triplet at δ 1.3. The compound was found inactive for β-Lactamase inhibition activity.
105 Chapter-4: Results & Discussion
4.1.6- Diethyl[(E)-1-benzoyl-2-(4-hydroxy-3-methoxyphenyl)vinyl] phosphonate (1-a-5) (6)
CH3 9 10 O O O 2 7 P 11 3 8 1 O CH3 4 6 13 1' 12 5 2' 6'
3' H3C 4' 5' O OH
For the compound (1-a-5), HREIMS showed molecular formula as C20H23O6P. The EIMS of compound indicated molecular ion [M+] at m/z 390, base peak at m/z 77, along with other characteristic peaks at 176, 161, 147, 135, 120, 107, 105, 51, 45 and 1 39. In H-NMR spectrum (400MHz, CD3OD) the five aromatic protons from ring-A (H-2, H-3, H-4, H-5, H-6) appeared between δ 8.07- 7.5, the three aromatic protons from ring-B (H-2/, H-5/, H-6/) appeared at δ 7.4, 7.03 and 7.36 respectively, the one methylynic proton (H-13) produced a singlet at δ 7.75, four phospho-esteric methylenic protons (H-9,11) appeared as a multiplet at δ 4.1 and the six phospho- esteric methylic protons (H-10, 12) appeared as triplet at δ 1.3, . β- Lactamase inhibition activity of the compound proved it to be sufficiently active (Table 4.1 & 4.2).
106 Chapter-4: Results & Discussion
4.1.7- Diethyl benzylphosphonate (2-a) (7)
8 O CH3 9 7 P O 2 1 O 10 3 6
CH3 4 5 11
For the compound (2-a), HREIMS confirmed molecular formula as + C11H17O3P. The EIMS of compound indicated molecular ion [M ] at m/z 228, base peak at m/z 91 along with other characteristic peaks at 172, 124, 118, 109, 105, 97 and 1 92. In H-NMR (400MHz, CD3OD) spectrum the five aromatic protons (H-2, H-3, H- 4, H-5, H-6) appeared between δ 7.24- 7.16, two methylenic protons (H-7) showed a singlet at δ 3.03, four phospho-esteric methylenic protons (H-8,10) appeared as a multiplet at δ 4.0 and the six phospho-esteric methylic protons (H-9, 11) appeared as triplet at δ 1.21. β-Lactamase inhibition activity of the compound proved it to be more potent (Table 4.1 & 4.2) than Clavulanic acid; a widely used BLase inhibitor in modern chemotherapy.
107 Chapter-4: Results & Discussion
4.1.8- Diethyl [2-(2-hydroxyphenyl)-2-oxo-1-phenylethyl] phosphonate (2a-1) (8)
OH 3' 2' O 8 CH 1' 3 4' 12 O 9 7 P O 5' 6' 2 1 O 10 3 6
CH3 4 5 11
For the compound (2-a-1), HREIMS confirmed molecular formula as + C18H22O5P. The EIMS of compound indicated molecular ion [M ] at m/z 349, base peak at m/z 121 along with other characteristic peaks at 211, 122, 118, 93, 92, 91, 65 1 and 39. In H-NMR (400MHz, CD3OD) spectrum the five aromatic (ring-A) protons (H-2, H-3, H-4, H-5, H-6) appeared between δ 7.36- 70, the four aromatic (ring-B) protons (H-3/, H-4/, H-5/, H-6/) appeared between δ 6.9- 7.8, one methylynic proton (H-7) showed a singlet at δ 5.68, four phospho-esteric methylenic protons (H-8,10) appeared as a multiplet at δ 4.15 and the six phospho-esteric methylic protons (H-9, 11) appeared as triplet at δ 1.15. β- Lactamase inhibition activity was not shown by the compound during preliminary screening.
108 Chapter-4: Results & Discussion
4.1.9- Diethyl [(3E)-2-oxo-1,4-diphenylbut-3-en-1-yl] phosphonate (2a-2) (9) 3' 4' 2'
14 5' O 8 1' CH3 6' 12 O 9 13 7 P O 2 1 O 10 3 6
CH3 4 5 11
For the compound (2-a-2), HREIMS confirmed molecular formula as + C20H23O4P. The EIMS of compound indicated molecular ion [M ] at m/z 358, base peak at m/z 131, along with other characteristic peaks at 281, 227, 132, 103, 91, 77 1 and 56. In H-NMR (400MHz, CD3OD) the five aromatic (ring-A) protons (H-2, H- 3, H-4, H-5, H-6) appeared between δ 7.36- 6.9, the five aromatic (ring-B) protons (H-2/, H-3/, H-4/, H-5/, H-6/) appeared between δ 7.48- 7.36, one methylynic proton (H-7) showed a singlet at δ 4.31, four phospho-esteric methylenic protons (H-8,10) appeared as a multiplet at δ 4.11 and the six phospho-esteric methylic protons (H-9, 11) appeared as triplet at δ 1.15, the two methylynic protons ( H-13 & H-14) appeared as doublets at δ 6.86 & 7.74 respectively. The compound showed fairly good β- Lactamase inhibition activity (Table 4.1 & 4.2).
109 Chapter-4: Results & Discussion
4.1.10- Diethyl (2-oxo-1,2-diphenylethyl)phosphonate (2a-3) (10)
3' 2' O 8 CH 1' 3 4' 12 O 9 7 P O 5' 6' 2 1 O 10 3 6
CH3 4 5 11
For the compound (2-a-3), HREIMS confirmed molecular formula as + C18H21O4P. The EIMS of compound indicated molecular ion [M ] at m/z 332, base peak at m/z 105, along with other characteristic peaks at 195, 106, 90, 77, 65,63, 51 1 50 and 39. In H-NMR (400MHz, CD3OD) the five aromatic (ring-A) protons (H-2, H-3, H-4, H-5, H-6) appeared between δ 7.34- 7.04, the five aromatic (ring-B) protons (H-2/, H-3/, H-4/, H-5/, H-6/) appeared between δ 8.08- 7.56, one methylynic proton (H-7) showed a singlet at δ 5.67, four phospho-esteric methylenic protons (H- 8,10) appeared as a multiplet at δ 4.15 and the six phospho-esteric methylic protons (H-9, 11) appeared as triplet at δ 1.15. The compound was inactive towards β- Lactamase inhibition activity (Table 4.1).
110 Chapter-4: Results & Discussion
4.1.11- Diethyl (2,4-dioxo-1-phenylpentyl)phosphonate (2a-4) (11)
O
H3C O 12 11 10 CH3 O 13 9 8 7 P O 2 1 O 14 3 6
CH3 4 5 15
For the compound (2-a-4), HREIMS confirmed molecular formula as + C15H21O5P. The EIMS of compound indicated molecular ion [M ] at m/z 312, base peak at m/z 85, along with other characteristic peaks at 90, 118, 175, 77, 43 and 227. 1 In H-NMR (400MHz, CD3OD) the five aromatic (ring-A) protons (H-2, H-3, H-4, H-5, H-6) appeared between δ 7.34- 7.04, the five aromatic (ring-B) protons (H-2/, H-3/, H-4/, H-5/, H-6/) appeared between δ 8.08- 7.56, one methylynic proton (H-7) showed a singlet at δ 5.67, four phospho-esteric methylenic protons (H-8,10) appeared as a multiplet at δ 4.15 and the six phospho-esteric methylic protons (H-9, 11) appeared as triplet at δ 1.15. The compound was inactive towards β-Lactamase inhibition activity (Table 4.1).
111 Chapter-4: Results & Discussion
4.1.12- Diethyl (2,4-dioxo-1,4-diphenylbutyl)phosphonate (2a-5) (12)
3' 4' 2' O
5' O 11 1' 10 CH3 6' O 12 9 8 7 P O 2 1 O 13 3 6
CH3 4 5 14
The compound (2-a-5) was oily liquid. HREIMS confirmed molecular formula + as C20H23O5P. The EIMS of compound indicated molecular ion [M ] at m/z 374, base peak at m/z 105, along with other characteristic peaks at 147, 106, , 78, 77, 65, 63, 51 1 and 39. In H-NMR (400MHz, CD3OD) the five aromatic (ring-A) protons (H-2, H- 3, H-4, H-5, H-6) appeared between δ 7.48- 6.94, the five aromatic (ring-B) protons (H-2/, H-3/, H-4/, H-5/, H-6/) appeared between δ 8.06- 7.51, one methylynic proton (H-7) showed a singlet at δ 5.54, the diketonic methylene proton (H-9) produced a singlet at δ 3.3, four phospho-esteric methylenic protons (H-11, 13) appeared as a multiplet at δ 4.1 and the six phospho-esteric methylic protons (H-12, 14) appeared as triplet at δ 1.15. β-Lactamase inhibition activity of the compound proved it to be more potent (Table 4.1 & 4.2) than Clavulanic acid; a widely used BLase inhibitor in modern chemotherapy.
112 Chapter-4: Results & Discussion
4.1.13- Ethyl (diethoxyphosphoryl)acetate (3a) (13)
2 H3C O 5 1 CH3 O O 6 3 P O 4 O 7
CH3 8
For the compound (3-a) HREIMS confirmed molecular formula as C8H17O5P. The EIMS of compound indicated molecular ion [M+] at m/z 224, base peak at m/z 123, along with other characteristic peaks at 197, 179, 169, 152, 151 109, 88, 81 and 1 29. In H-NMR (400MHz, CD3OD) three methylic protons (H-1) appeared as a triplet at δ 1.29, two methylenic protons (H-3) showed a singlet at δ 2.78, four phospho- esteric methylenic protons (H-4, 6) appeared as a multiplet at δ 4.15 and the six phospho-esteric methylic protons (H-5, 7) appeared as triplet at δ 1.33. The compound failed to show any β-Lactamase inhibition activity (Table 4.1).
113 Chapter-4: Results & Discussion
4.1.14- Diethyl (oxiran-2-ylmethyl)phosphonate (4a) (14)
O 4 CH3 2 O 5 1 P O 3 O 6
CH3 7
For the compound (4a) HREIMS confirmed molecular formula as C7H15O4P. The EIMS of compound indicated molecular ion [M+] at m/z 194, base peak at m/z 57, along with other characteristic peaks at 151, 124, 118, 81, 43, 31, 29, 28, 27 and26. 1 In H-NMR (400MHz, CD3OD) two oxiranial protons (H-1) appeared as a doublet at δ 2.75, one oxiranial proton (H-2) appeared as a multiplet at δ 3.09, two methylenic protons (H-3) showed a doublet at δ 2.63, four phospho-esteric methylenic protons (H-4, 6) appeared as a multiplet at δ 4.13 and the six phospho-esteric methylic protons (H-5, 7) appeared as triplet at δ 1.14. The compound failed to show any β- Lactamase inhibition activity (Table 4.1).
114 Chapter-4: Results & Discussion
4.1.15-Diethyl[2-(2-hydroxyphenyl)-1-oxiran-2-yl-2-oxoethyl] phosphonate (4a-1) (15)
O 4 CH3 4' 2 O 5 1 3' 5' P O 3 2' 1' 8 O 6' 6 O OH CH3 7
For the compound (4-a-1), HREIMS confirmed molecular formula as + C14H19O6P. The EIMS of compound confirmed molecular ion [M ] at m/z 314, base peak at m/z 121, along with other characteristic peaks at 181, 177, 124, 122, 118, 81, 1 43, 31, 29, 28, 27 and26. In H-NMR (400MHz, CD3OD) two oxiranial protons (H-1) appeared as a doublet at δ 3.24, one oxiranial proton (H-2) appeared as a multiplet at δ 3.86, one methylenic proton (H-3) showed a doublet at δ 4.55, four phospho-esteric methylenic protons (H-4, 6) appeared as a multiplet at δ 4.15 and the six phospho- esteric methylic protons (H-5, 7) appeared as triplet at δ 1.16, the four aromatic protons (H-2/ H-3/, H-4/, H-5/) appeared between δ 6.87- 7.63 . The compound showed very good β-Lactamase inhibition activity (Table 4.1 & 4.2).
115 Chapter-4: Results & Discussion
4.1.16- Diethyl [(3E)-1-oxiran-2-yl-2-oxo-4-phenylbut-3-en-1-yl] phosphonate (4a-2) (16)
3' O 4 CH3 4' 2' 2 O 5 1 10 P O 5' 1' 3 6' 8 O 9 6 O
CH3 7
For the compound (4-a-2), HREIMS confirmed molecular formula as + C16H21O5P. The EIMS of compound confirmed molecular ion [M ] at m/z 324, base peak at m/z 103, along with other characteristic peaks at 131, 124, 122, 118, 104, 102, 1 81, 77, 51, 43, 31, 29, 28, 27 and26. In H-NMR (400MHz, CD3OD) two oxiranial protons (H-1) appeared as a doublet at δ 3.24, one oxiranial proton (H-2) appeared as a multiplet at δ 3.56, one methylenic proton (H-3) showed a doublet at δ 3.05, four phospho-esteric methylenic protons (H-4, 6) appeared as a multiplet at δ 4.10 and the six phospho-esteric methylic protons (H-5, 7) appeared as triplet at δ 1.16, the five aromatic protons (H-2/ H-3/, H-4/, H-5/, H-6/) appeared between δ 7.69- 7.36, the two methylynic protons (H-9, 10) gave doublets at δ 6.85 & 7.72 respectively . The compound showed no β-Lactamase inhibition activity (Table 4.1).
116 Chapter-4: Results & Discussion
4.1.17 -Diethyl (1-oxiran-2-yl-2-oxo-2-phenylethyl)phosphonate (4a-3) (17)
O 4 CH3 4' 2 O 5 1 3' 5' P O 3 2' 8 O 6' 6 1' O
CH3 7
For the compound (4-a-3), HREIMS confirmed molecular formula as + C14H19O5P. The EIMS of compound confirmed molecular ion [M ] at m/z 298, base peak at m/z 105, along with other characteristic peaks at 255, 193, 181, 124, 118, 77, 1 51, 31, 29, 28, 27 and26. In H-NMR (400MHz, CD3OD) two oxiranial protons (H-1) appeared as a doublet at δ 3.24, one oxiranial proton (H-2) appeared as a multiplet at δ 3.86, one methylenic proton (H-3) showed a doublet at δ 4.55, four phospho-esteric methylenic protons (H-4, 6) appeared as a multiplet at δ 4.14 and the six phospho- esteric methylic protons (H-5, 7) appeared as triplet at δ 1.16, the five aromatic protons (H-2/ H-3/, H-4/, H-5/, H-6/) appeared between δ 8.04- 7.50. The compound showed considerable β-Lactamase inhibition activity (Table 4.1& 4.2).
117 Chapter-4: Results & Discussion
4.1.18- Diethyl (1-oxiran-2-yl-2,4-dioxopentyl)phosphonate (4a-4) (18)
O 8 CH3 2 O 9 1 P O 3 5 4 O 10 H3C 6 O 7 O CH3 11
For the compound (4-a-4), HREIMS confirmed molecular formula as + C11H19O6P. The EIMS of compound confirmed molecular ion [M ] at m/z 278, base peak at m/z 43, along with other characteristic peaks at 235, 193, 181, 124, 118, 85, 1 42, 31, 29, 28, 27, 26 and 15. In H-NMR (400MHz, CD3OD) two oxiranial protons (H-1) appeared as a doublet at δ 3.24, one oxiranial proton (H-2) appeared as a triplet at δ 3.86, one methylenic proton (H-3) showed a doublet at δ 3.85, four phospho- esteric methylenic protons (H-8, 10) appeared as a multiplet at δ 4.10 and the six phospho-esteric methylic protons (H-9,11) appeared as triplet at δ 1.16, the two diketonic methylenic protons (H-5) appeared as a singlet at δ 3.55 and three methyl protons gave a singlet at δ 1.97. The compound showed no β-Lactamase inhibition activity (Table 4.1).
118 Chapter-4: Results & Discussion
4.1.19- Diethyl (1-oxiran-2-yl-2,4-dioxo-4-phenylbutyl) phosphonate (4a-5) (19)
O 7 CH3 2 O 8 1 3' P O 3 4' 2' 5 4 O 9 6 O 5' 1' 6' O CH3 10
For the compound (4-a-5), HREIMS confirmed molecular formula as + C16H21O6P. The EIMS of compound confirmed molecular ion [M ] at m/z 340, base peak at m/z 105, along with other characteristic peaks at 193, 147, 77, 69, 51, 31, 29, 1 28, 27 and 26. In H-NMR (400MHz, CD3OD) two oxiranial protons (H-1) appeared as a doublet at δ 3.24, one oxiranial proton (H-2) appeared as a multiplet at δ 3.83, one methylenic proton (H-3) showed a doublet at δ 4.31, two diketonic methylene protons (H-5) appeared as a singlet at δ 3.27, four phospho-esteric methylenic protons (H-7, 9) appeared as a multiplet at δ 4.10 and the six phospho-esteric methylic protons (H-8, 10) appeared as triplet at δ 1.16, the five aromatic protons (H-2/ H-3/, H-4/, H-5/, H-6/) appeared between δ 8.05- 7.51. The compound showed considerable β-Lactamase inhibition activity (Table 4.1& 4.2).
119 Chapter-4: Results & Discussion
4.1.20- Diethyl (2-nitrobenzyl) phosphonate (5a) (20)
O 2 + N 3 1 - O 8 CH3 6 O 9 4 5 P O 7 O 10
CH3 11
For the compound (5-a), HREIMS confirmed molecular formula as + C11H16O5NP. The EIMS of compound confirmed molecular ion [M ] at m/z 273, base peak at m/z 89, along with other characteristic peaks at 151, 136, 124, 107, 96, 92, 90, 1 79 and 78. In H-NMR (400MHz, CD3OD) four aromatic protons (H-2, 3, 4, 5) appeared between chemical shifts δ 8.08-7.37, one methylenic proton (H-7) showed a singlet at δ 3.77, four phospho-esteric methylenic protons (H-8, 10) appeared as a multiplet at δ 3.94 and the six phospho-esteric methylic protons (H-9, 11) appeared as triplet at δ 1.16. The compound showed no β-Lactamase inhibition activity (Table 4.1).
120 Chapter-4: Results & Discussion
4.1.21- Tetraethyl ethane-1,2-diylbis (phosphonate) (6a) (21)
7 H3C O 8 O P CH3 9 2 4 O O 1 3 H3C P O 10 O CH3 5 6
For the compound (6-a), HREIMS confirmed molecular formula as + C10H24O6P2. The EIMS of compound confirmed molecular ion [M ] at m/z 302, base peak at m/z 165, along with other characteristic peaks at 257, 229, 201, 173, 166, 155, 1 139, 138, 137, 111, 109, 91, 82, 81 and 65. In H-NMR (400MHz, CD3OD) four methylenic protons (H-1, 2) showed a triplet at δ 2.08, eight phospho-esteric methylenic protons (H-3, 5, 7, 9) appeared as multiplet at δ 3.45 and the twelve phospho-esteric methylic protons (H-4, 6, 8, 10) appeared as triplet at δ 1.25. The compound showed no β-Lactamase inhibition activity (Table 4.1).
121 Chapter-4: Results & Discussion
4.1.22- Derivative A of tetraethyl ethane-1,2-diylbis (phosphonate) (6a-1) (22) 2' HO 3' 1' 6' H3C 4' 11 10 3 5' CH3 O 7 O O P 2 6 8 P O 1 O O H3C 9 4 CH3 5
For the compound (6-a-1), HREIMS confirmed molecular formula as + C17H28O8P2. The EIMS of compound confirmed molecular ion [M ] at m/z 422, base peak at m/z 121, along with other characteristic peaks at 285, 192, 164, 148, 122, 93, 1 65, 63 and 39. In H-NMR (400MHz, CD3OD) two methylenic protons (H-1) showed a doublet at δ 2.73, the other one methylenic proton (H-2) shifted to δ 5.21 as a triplet, eight phospho-esteric methylenic protons (H-4, 6 & H-8,10) appeared as multiplets at δ 4.14 & δ 3.98 respectively while the twelve phospho-esteric methylic protons (H-5, 7 & H-9, 11) appeared as triplets at δ 1.15 δ 1.23 respectively, the four aromatic protons (H-2/, 3/, 4/, 5/,) appeared between δ 7.81-7.01. The compound showed moderate β-Lactamase inhibition activity (Table 4.1 & 4.2).
122 Chapter-4: Results & Discussion
4.1.23- Derivative B of Tetraethyl ethane-1,2-diylbis(phosphonate) (6a-2) (23) 3' 2' 4'
1' 5 5' 6' H3C 9 8 O 4 CH3 O 3 11 O O P 2 10 6 P O 1 O O H3C 7 12 CH3 13
For the compound (6-a-2), HREIMS confirmed molecular formula as + C19H30O7P2. The EIMS of compound confirmed molecular ion [M ] at m/z 434, base peak at m/z 103, along with other characteristic peaks at 301, 295, 281, 12, 164, 131, 1 104, 102 and 77. In H-NMR (400MHz, CD3OD) two methylenic protons (H-1) showed a doublet at δ 2.59, the other one methylenic proton (H-2) shifted to δ 4.03 as a triplet, eight phospho-esteric methylenic protons (H-6, 8 & H-10, 12) appeared as multiplets at δ 4.10 & δ 3.98 respectively while the twelve phospho-esteric methylic protons (H-7, 9 & H-11, 13) appeared as triplets at δ 1.15 δ 1.23 respectively, the five aromatic protons (H-2/, 3/, 4/, 5/, 6/,) appeared between δ 7.40-7.36, the two methylynic protons (H-4, 5) appeared as doublets at δ 6.78 & δ 7.72 respectively. The compound showed no β-Lactamase inhibition activity (Table 4.1).
123 Chapter-4: Results & Discussion
4.1.24- Derivative C of tetraethyl ethane-1,2-diylbis(phosphonate) (6a-3) (24) 3' 2' 4'
1' H3C 5' 11 10 O 6' CH3 O 3 7 O O P 2 6 8 P O 1 O O H3C 9 4 CH3 5
For the compound (6-a-3), HREIMS confirmed molecular formula as + C17H28O7P2. The EIMS of compound confirmed molecular ion [M ] at m/z 406, base peak at m/z 105, along with other characteristic peaks at 269, 255, 164, 145, 133, 119, 1 123, 106, 91, 78 and 77. In H-NMR (400MHz, CD3OD) two methylenic protons (H- 1) showed a doublet at δ 2.72, the other one methylenic proton (H-2) shifted to δ 5.22 as a triplet, eight phospho-esteric methylenic protons (H-4, 6 & H-8, 10) appeared as multiplets at δ 4.14 & δ 3.98 respectively while the twelve phospho-esteric methylic protons (H-5, 7 & H-9, 11) appeared as triplets at δ 1.15 & δ 1.23 respectively, the five aromatic protons (H-2/, 3/, 4/, 5/, 6/,) appeared between δ 8.09-7.66. The compound showed fairly good β-Lactamase inhibition activity (Table 4.1 & 4.2).
124 Chapter-4: Results & Discussion
4.1.25- Derivative D of Tetraethyl ethane-1,2-diylbis (phosphonate) (6a-4) (25)
O CH3 6 5 H3C 12 11 O 4 CH3 O 3 8 O O P 2 7 13 P O 1 O O H3C 14 9 CH3 10
For the compound (6-a-4), HREIMS confirmed molecular formula as + C14H28O8P2. The EIMS of compound confirmed molecular ion [M ] at m/z 386, base peak at m/z 43, along with other characteristic peaks at 257, 173, 165, 164, 137, 112, 1 109, 85, 42, 27, 26 and 15. In H-NMR (400MHz, CD3OD) two methylenic protons (H-1) showed a doublet at δ 2.56, the other one methylenic proton (H-2) shifted to 4.77 as a triplet, two diketonic methylene protons (H-4) gave a singlet at δ 3.65, three methylic protons (H-6) produced a singlet at δ 1.97, eight phospho-esteric methylenic protons (H-7, 9 & H-11, 13) appeared as multiplets at 4.10 & δ 3.98 respectively while the twelve phospho-esteric methylic protons (H-8, 10 & H-12, 14) appeared as triplets at δ 1.15 δ 1.23 respectively,. The compound showed no β-Lactamase inhibition activity (Table 4.1).
125 Chapter-4: Results & Discussion
4.1.26- Derivative E of Tetraethyl ethane-1,2-diylbis(phosphonate) (6a-5) (26) 3' 2' 4'
1' O 5' 5 6' H3C 11 10 O 4 CH3 O 3 7 O O P 2 6 12 P O 1 O O H3C 13 8 CH3 9
For the compound (6-a-5), HREIMS confirmed molecular formula as + C19H30O8P2. The EIMS of compound confirmed molecular ion [M ] at m/z 448, base peak at m/z 105, along with other characteristic peaks at 448, 256, 174, 164, 161, 147, 1 78, 77, 69 and 51. In H-NMR (400MHz, CD3OD) two methylenic protons (H-1) showed a doublet at δ 2.62, the other one methylenic proton (H-2) shifted to δ 5.23 as a triplet, the diketonic methylene (H-4) gave singlet at δ 3.38, eight phospho-esteric methylenic protons (H-6, 8 & H-10, 12) appeared as multiplets at δ 4.10 & δ 3.98 respectively while the twelve phospho-esteric methylic protons (H-7, 9 & H-11, 13) appeared as triplets at δ 1.15 δ 1.23 respectively, the five aromatic protons (H-2/, 3/, 4/, 5/, 6/) appeared between δ 8.05-7.51. The β-Lactamase inhibition activity of the compound (6-a-5) was found comparable with Clavulanic acid; a widely used β- Lactamase inhibitor in modern medicine (Table 4.1 & 4.2).
126 Chapter-4: Results & Discussion
4.1.27- Diethyl methylphosphonate (7a) (27)
CH3 3 O 2 H3C 1 P O O 4 CH3 5
For the compound (7-a), HREIMS confirmed molecular formula as C5H13O3P. The EIMS of compound confirmed molecular ion [M+] at m/z 152, base peak at m/z 79, along with other characteristic peaks at 125, 108, 107, 97, 81, 80, 65, 47, 45, 19, 1 27 and 15. In H-NMR (400MHz, CD3OD) three methylic protons (H-1) showed a singlet at δ 1.40 four phospho-esteric methylenic protons (H-2, 4) appeared as multiplet at δ 4.01 and the six phospho-esteric methylic protons (H-3, 5) appeared as triplet at δ 1.19. The compound was found inactive towards β-Lactamase inhibition activity (Table 4.1).
127 Chapter-4: Results & Discussion
4.1.28- Diethyl [2-(2-hydroxyphenyl)-2-oxoethyl] phosphonate (7a-1) (28) 2' OH 3' 1'
4' O 6' CH3 5' 6 3 O 2 1 P O O 4 CH3 5
For the compound (7-a-1), HREIMS confirmed molecular formula as + C12H17O5P. The EIMS of compound confirmed molecular ion [M ] at m/z 272, base peak at m/z 121, along with other characteristic peaks at 151, 135, 125, 122, 97, 93, 1 79, 65 and 39. In H-NMR (400MHz, CD3OD) two methylic protons (H-1) showed a singlet at δ 3.54, four phospho-esteric methylenic protons (H-2, 4) appeared as multiplet at δ 3.55 and the six phospho-esteric methylic protons (H-3, 5) appeared as triplet at δ 1.30, four aromatic protons (H-2, 3, 4, 5) appeared between δ 7.83-6.90. The β-Lactamase inhibition activity of the compound (7-a-1) was found fairly good (Table 4.1 & 4.2).
128 Chapter-4: Results & Discussion
4.1.29- Diethyl [(3E)-2-oxo-4-phenylbut-3-en-1-yl] phosphonate (7a-2) (29)
O O 2' 4 O CH3 2 P 6 3' 5 1' 3 1 O 4' 6' 7 5' CH3 8
For the compound (7-a-2), HREIMS confirmed molecular formula as + C14H19O4P. The EIMS of compound confirmed molecular ion [M ] at m/z 282, base peak at m/z 103, along with other characteristic peaks at 145, 131, 123, 104, 102, 97, 1 79, 77 and 51.. In H-NMR (400MHz, CD3OD) two methylic protons (H-1) showed a singlet at δ 3.05, the two methylynic protons (H-3 & H-4) produced doublets at δ 6.86 & δ 7.77 respectively, four phospho-esteric methylenic protons (H-5, 7) appeared as multiplet at δ 3.51 and the six phospho-esteric methylic protons (H-6, 8) appeared as triplet at δ 1.27. The five aromatic protons (H-2, 3, 4, 5, 6) appeared between δ 7.46- 7.36. The β-Lactamase inhibition activity of the compound (7-a-2) was found much better than Clavulanic acid; a widely used β-Lactamase inhibitor in modern medicine (Table 4.1 & 4.2).
129 Chapter-4: Results & Discussion
4.1.30- Diethyl (2-oxo-2-phenylethyl) phosphonate (7a-3) (30)
O O 2' O CH3 2 P 4 3' 3 1' 1 O 4' 6' 5 5' CH3 6
For the compound (7-a-3), HREIMS confirmed molecular formula as + C12H17O4P. The EIMS of compound confirmed molecular ion [M ] at m/z 256, base peak at m/z 105, along with other characteristic peaks at 146, 123, 120, 106, 91, 81, 1 77, 71, 51 and 29. In H-NMR (400MHz, CD3OD) two methylic protons (H-1) showed a singlet at δ 3.56 four phospho-esteric methylenic protons (H-3, 5) appeared as multiplet at δ 3.55 and the six phospho-esteric methylic protons (H-4, 6) appeared as triplet at δ 1.30, five aromatic protons (H-2, 3, 4, 5, 6) appeared between δ 8.23- 7.49. The β-Lactamase inhibition activity of the compound (7-a-3) was found good (Table 4.1 & 4.2).
130 Chapter-4: Results & Discussion
4.1.31- Diethyl (2,4-dioxopentyl) phosphonate (7a-4) (31)
O O O O CH3 2 P 7 4 6 H3C 5 3 1 O
8
CH3 9
For the compound (7-a-4), HREIMS confirmed molecular formula as + C9H17O5P. The EIMS of compound confirmed molecular ion [M ] at m/z 236, base peak at m/z 43, along with other characteristic peaks at 193, 179, 125, 106, 99, 97, 85, 1 79, 42, 27, 26 and 15. In H-NMR (400MHz, CD3OD) two methylenic protons (H-1) showed a singlet at δ 3.24, two diketo-methylenic protons (H-3) showed a singlet at δ 2.72, three methylic protons (H-5) showed a singlet at δ 2.03, four phospho-esteric methylenic protons (H-6, 8) appeared as multiplet at δ 4.16 and six phospho-esteric methylic protons (H-7, 9) appeared as triplet at δ 1.62. The compound (7-a-4) was found inactive towars β-Lactamase inhibition activity (Table 4.1 & 4.2).
131 Chapter-4: Results & Discussion
4.1.32- Diethyl (2,4-dioxo-4-phenylbutyl) phosphonate (7a-5) (32)
O O O 2' O CH3 2 6 1' P 3' 4 5 3 1 O
4' 6' 7 5' CH3 8
For the compound (7-a-5), HREIMS confirmed molecular formula as + C14H19O5P. The EIMS of compound confirmed molecular ion [M ] at m/z 298, base peak at m/z 105, along with other characteristic peaks at 179, 161, 147, 97, 79, 78, 77, 1 69 and 51. . In H-NMR (400MHz, CD3OD) two methylenic protons (H-1) showed a singlet at δ 3.31, two diketo-methylenic protons (H-3) showed a singlet at δ 2.53, four phospho-esteric methylenic protons (H-5, 7) appeared as multiplet at δ 4.16 and six phospho-esteric methylic protons (H-6, 8) appeared as triplet at δ 1..61, the five aromatic protons (H-2, 3, 4, 5, 6) appeared between δ 8.05-7.51. The β-Lactamase inhibition activity of the compound (6-a-5) was found much better than Clavulanic acid; a widely used β-Lactamase inhibitor in modern medicine (Table 4.1 & 4.2).
132 Chapter-4: Results & Discussion
4.1.33- β-Lactamase (BLase) Inhibition Assay – Results:
Table 4.1: Preliminary Screening of Ketophosph(on)ates for β-Lactamase Inhibition Activity
Preliminary Screening of Ketophosph(on)ates for S β-Lactamase Inhibition Activity : Observations till . Compound Chemical r Mr. . . Code formula # 12 24 36 48 60 72 84 96 Comment hrs hrs hrs hrs hrs hrs hrs hrs s 256 √ 1 1-a C12H17O4P √ √ √ √ √ √ x Active 344 x 2 1-a-1 C19H21O4P x x x x x x x Inactive 360 x 3 1-a-2 C19H21O5P x x x x x x x Inactive 370 x 4 1-a-3 C21H23O4P √ √ √ √ x x x Active 358 x 5 1-a-4 C20H23O4P x x x x x x x Inactive 390 √ 6 1-a-5 C20H23O6P √ √ √ √ x x x Active 228 √ 7 2-a C11H17O3P √ √ √ √ √ √ x Active 349 x 8 2-a-1 C18H22O5P x x x x x x x Inactive 358 x 9 2-a-2 C20H23O4P √ √ √ √ x x x Active 1 332 x 2-a-3 C H O P x x x x x x x Inactive 0 18 21 4 1 312 x 2-a-4 C H O P x x x x x x x Inactive 1 15 21 5 1 374 √ 2-a-5 C H O P √ √ √ √ √ √ x Active 2 20 23 5 1 224 x 3-a C H O P x x x x x x x Inactive 3 8 17 5 1 194 x 4-a C H O P x x x x x x x Inactive 4 7 15 4 1 314 √ 4-a-1 C H O P √ √ √ √ x x x Active 5 14 19 6 1 324 x 4-a-2 C H O P x x x x x x x Inactive 6 16 21 5
133 Chapter-4: Results & Discussion
Preliminary Screening of Ketophosph(on)ates for S β-Lactamase Inhibition Activity : Observations till . Compound Chemical r Mr. . . Code formula # 12 24 36 48 60 72 84 96 Comment hrs hrs hrs hrs hrs hrs hrs hrs s 1 298 x 4-a-3 C H O P √ √ √ x x x x Active 7 14 19 5 1 278 4-a-4 C H O P x x x x x x x x Inactive 8 11 19 6 1 340 4-a-5 C H O P √ √ √ √ x x x x Active 9 16 21 6 2 C H O 273 5-a 11 16 5- x x x x x x x x Inactive 0 NP 2 C H O P 302 6-a 10 24 6 x x x x x x x x Inactive 1 2 2 C H O P 422 6-a-1 17 28 8 √ √ √ x x x x x Active 2 2 2 C H O P 434 6-a-2 19 30 7 x x x x x x x x Inactive 3 2 2 C H O P 406 6-a-3 17 28 7 √ √ √ √ x x x x Active 4 2 2 C H O P 386 6-a-4 14 28 8 x x x x x x x x Inactive 5 2 2 C H O P 448 6-a-5 19 30 8 √ √ √ √ √ x x X Active 6 2 2 152 7-a C H O P x x x x x x x X Inactive 7 5 13 3 2 272 7-a-1 C H O P √ √ √ x x x x x Active 8 12 17 5 2 282 7-a-2 C H O P √ √ √ √ √ √ √ x Active 9 14 19 4 3 256 7-a-3 C H O P √ √ √ x x x x x Active 0 12 17 4 3 236 7-a-4 C H O P x x x x x x x x Inactive 1 9 17 5 3 298 7-a-5 C H O P √ √ √ √ √ √ √ x Active 2 14 19 5 3 Clavulanc C H NO 199 √ √ √ √ √ √ x x Active 3 acid 8 9 5
134 Chapter-4: Results & Discussion
Table 4.2: Spectrophotometric Screening of Ketophosph(on)ates for β-Lactamase Inhibition Activity
Spectrophotometric Screening of Ketophosph(on)ates for β-Lactamase Sr Compound Chemical Inhibition Activity Mr. # code formula Absorbance after % inhibition 15 minutes [(Ablank –Asample) x (λ max 486 nm) 100] /Ablank
01 1-a C12H17O4P 256 0.530 81 % ± 2
02 1-a-3 C21H23O4P 370 1.428 49 % ± 3
03 1-a-5 C20H23O6P 390 1.064 62 % ± 2
04 2-a C11H17O3P 228 0.784 78 % ± 1
05 2-a-2 C20H23O4P 358 1.316 53 % ± 2
06 2-a-5 C20H23O5P 374 0.448 84 % ± 2
07 4-a-1 C14H19O6P 314 1.204 57 % ± 3
08 4-a-3 C14H19O5P 298 1.764 37 % ± 1
09 4-a-5 C16H21O6P 340 1.344 52 % ± 1
10 6-a-1 C17H28O8P2 422 1.596 43 % ± 2
11 6-a-3 C17H28O7P2 406 1.484 47 % ± 2
12 6-a-5 C19H30O8P2 448 1.064 62 % ± 1
13 7-a-1 C12H17O5P 272 1.708 39 % ± 1
14 7-a-2 C14H19O4P 282 0.504 82 % ± 2
15 7-a-3 C12H17O4P 256 1.652 41 % ± 1
16 7-a-5 C14H19O5P 298 0.420 85 % ± 3 Clavulanic 17 C H NO 199 0.840 70 % ± 1 acid 8 9 5 18 Blank 2.800 00 % ± 0
135 Chapter-4: Results & Discussion
4.2 Bioassay Guided Extraction & Isolation Table 4.3:- Compounds Isolated from Plants
Sr Extraction Plant Extract Code Compound Name Formula Mr. # Solvent 01 CIRCHNe-1 Lupeol C30H50O 426
02 CIRCHNe-2 β-Sitosterol C29H50O 414 p-hydroxy phenyl 03 CIRCHAc-1 C H O 152 acetic acid 8 8 3 Chloroform 04 CIRCHAc-2 Isovanillic acid C8H8O4 168
05 Cichorium CIRCHAc-3 Syringic acid C9H10O5 198 intybus 06 CIRCHAc-4 Vanillic acid C8H8O4 168
07 CIREANe-1 Esculetin C9H6O4 178
08 CIREANe-2 Scopoletin C10H8O4 192 Ethyl acetate 09 CIREANe-3 Umbelliferone C9H6O3 162
10 CIREANe-4 Kaempferol C15H10O6 286 Ageratum 11 Hexane AC-Hex-Fr-2 Stigmasterol C H O 412 conyzoides 29 48 12 HA-CH-1 Conessine C24H40N2 356 Isoconessimine / 13 Chloroform HA-CH-2 C H N 342 Kurchinine 23 38 2 14 HA-CH-3 Conimine C22H36N2 328 Hollarhena antidysenterica 15 HA-EA-1 Kurchamine C22H38N2 330 Ethyl acetate 16 HA-EA-2 Holaromine C21H36N2O 332
17 n-Butanol HA-BU Kurchessine C25H44N2 372
18 CO-CH-1 Lupeol C30H50O 426 Chloroform- Carissa opaca Basic-Ether 19 CO-CH-2 Campesterol C28H48O 400
20 AS-EA-1 Quercetin C15H10O7 302 Alstonia scholaris Ethyl acetate 21 AS-EA-2 Kaempferol C15H10O6 286
22 CP-DCM-Fr-3-2 β -Sitosterol C29H50O 414 Calotropis Dichloro CP-DCM-Fr-3- 23 á-Amyrin C H O 426 procera methane 3A 30 50 CP-DCM-Fr-3- 24 Ursolic acid C H O 456 3B 30 48 3
136 Chapter-4: Results & Discussion
4.2.1 COMPOSITAE FAMILY Among twenty five screened plants for BLase Inhibition activity, Cichorium intyus, Holarrhena antidysenterica, Carissa opaca, Clotropis procera,Alstonia scholaris and Ageratum conyzoides were found active. Further bioassay guided extraction yielded twenty four pure compounds. 4.2.1.1 Cichorium intybus: Crude methanolic extract from Cichorium intyus was found active during preliminary screening for Blasé Inhibition. After solvent extraction the Chloroform (CI-CH) and Ethyl acetate (CI-EA) extracts showed satisfactory enzyme inhibition activity. All the extracts were run on GCMS and from GC chromatogram number and amounts of compounds in solvent extracts were assessed. Later on preparative TLC yielded six pure compounds from chloroform extract and four from ethyl acetate exract as described below. 4.2.1.1.1- CIRCHNe-1 : Lupeol (33) Preparative TLC (chloroform : Hexane :: 40 : 60) of neutral Chloroform extract (CIRCHNe) yielded two compounds in handsome amount. The material recovered from top layer was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Lupeol. It exhibited weak BLase inhibition activity (Table 4.4 & 4.5).
4.2.1.1.2- CIRCHNe-2 : β-Sitosterol (34) Preparative TLC (chloroform : Hexane :: 40 : 60) of neutral Chloroform extract (CIRCHNe) yielded two compounds in handsome amount. The material recovered from bottom layer was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as β-Sitosterol. It exhibited BLase inhibition activity close to Lupeol (Table 4.4 & 4.5).
4.2.1.1.3- CIRCHAc-1: p-hydroxy phenyl acetic acid (35) Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak
137 Chapter-4: Results & Discussion
in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as p-hydroxy phenyl acetic acid. It exhibited no BLase inhibition activity (Table 4.4 & 4.5).
4.2.1.1.4- CIRCHAc-2: Isovanillic acid (36) Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Isovanillic acid. It was found slightly BLase inhibitor (Table 4.4 & 4.5).
4.2.1.1.5- CIRCHAc-3: Syringic acid (37)
Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-3 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Syringic acid. It exhibited BLase inhibition activity close to Isovanillic acid (Table 4.4 & 4.5). 4.2.1.1.6- CIRCHAc-4: Vanillic acid (38)
Preparative TLC (chloroform : Methanol :: 80 : 20) of acidic Chloroform extract (CIRCHAc) yielded four compounds in handsome amount. The material recovered from layer-4 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Syringic acid. It exhibited BLase inhibition activity close to Vanillic acid (Table 4.4 & 4.5). 4.2.1.1.7- CIREANe-1: Esculetin (39)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single
138 Chapter-4: Results & Discussion
significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Esculetin. It exhibited BLase inhibition activity slightly higher than compounds from CIRCHAc extract (Table 4.4 & 4.5).
4.2.1.1.8- CIREANe-2: Scopoletin (40)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Scopoletin. It exhibited BLase inhibition activity higher than Esculetin (Table 4.4 & 4.5). 4.2.1.1.9- CIREANe-3: Umbelliferone (41)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-3 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Umbelliferone. It was found least BLase inhibitor among compounds isolated from ethyl acetate extract (Table 4.4 & 4.5).
4.2.1.1.10- CIREANe-4: Kaempferol (42)
Preparative TLC (Chloroform : Methanol :: 70 : 30) of neutral Ethylacetate extract (CIREANe) yielded four compounds in handsome amount. The material recovered from layer-4 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Scopoletin. It exhibited highest BLasé inhibition activity among all the ten compounds isolated from Cichorium intybus (Table 4.4 & 4.5).
139 Chapter-4: Results & Discussion
4.2.1.2 Ageratum conyzoides: Crude methanolic extract from Ageratum conyzoides was found active during preliminary screening for BLasé Inhibition. After solvent extraction only n-Hexane (AC-Hex) extract showed satisfactory enzyme inhibition activity. AC-Hex was column chromatographed over silica gel with gradient elution (Hexan : Ethylacetate) to yield six fractions. All the six fractions were preliminary screened for BLasé inhibition activity and also run on GCMS. From GC chromatogram number and amounts of compounds in these fractions were assessed. Fraction-2 was found BLasé inhibitor.Later on preparative TLC yielded six pure compounds from chloroform extract and four from ethyl acetate exract as described below.
4.2.1.2. 1- AC-Hex-Fr-2: Stigmasterol (43)
Fraction-2 (Hexan : Ethylacetate :: 60 : 40) was found BLasé inhibitor. Its
preparative TLC (Hex : EtOAc : CH2Cl2 :: 6 : 3 :1) yielded one pure compound. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1- NMR data interpretation and comparison with literature confirmed it as Stigmasterol. It exhibited BLasé inhibition activity equal to Eculetin and Lupeol from Cichorium intybus (Table 4.4 & 4.5).
4.2.2 APOCYNACEAE FAMILY Total six plants were screened for BLasé inhibition activity from Apocynaceae family. Three plants: Hollarhena antidysenterica, Carissa opaca and Alstonia scholaris were found active hence processed further.
4.2.2.1: Hollarhena antidysenterica:
Crude ethannolic extract from Hollarhena antidysenterica was found active during preliminary screening for BLasé Inhibition. After solvent extraction the Chloroform (HA-CH), Ethyl acetate (HA-EA) and n-Butanol (HA-BU) extracts
140 Chapter-4: Results & Discussion
showed satisfactory enzyme inhibition activity. All the extracts were run on GCMS and from GC chromatogram number and amounts of compounds in solvent extracts were assessed. Later on preparative TLC yielded total six pure compounds from active exracts as described below.
4.2.2.1.1. HA-CH-1 : Conessine (44)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 91.5 : 08 : 0.5) of Chloroform extract (HA-CH) yielded three compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Conessine. It exhibited good BLasé inhibition activity (Table 4.4 & 4.5). 4.2.2.1.2- HA-CH-2: Isoconessimine / Kurchinine (45)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 91.5 : 08 : 0.5) of Chloroform extract (HA-CH) yielded three compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Isoconessimine. It exhibited BLasé inhibition activity higher than Conessine (Table 4.4 & 4.5).
4.2.2.1.3- HA-CH-3: Conimine (46) Preparative TLC (CHCl3 : MeOH : CH3COOH :: 91.5 : 08 : 0.5) of Chloroform extract (HA-CH) yielded three compounds in handsome amount. The material recovered from layer-3 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Conimine. It exhibited BLasé inhibition activity highest of all the three compounds from HA-CH extract (Table 4.4 & 4.5).
141 Chapter-4: Results & Discussion
4.2.2.1.4- HA-EA-1 : Kurchamine (47)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 88 : 11 : 01) of Ethyl acetate extract (HA-EA) yielded two compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Kurchamine. It exhibited BLasé inhibition activity higher than compounds isolated from HA-CH and HA-BU (Table 4.4 & 4.5). 4.2.2.1.5-HA-EA-2 : Holaromine (48)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 88 : 11 : 01) of Ethyl acetate extract (HA-EA) yielded two compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Holaromine. It exhibited BLasé inhibition activity highest of all the twenty four compounds isolated from six plants belonging to three families of plant kingdom (Table 4.4 & 4.5).
4.2.2.1.6- HA-BU-1 : Kurchessine (49)
Preparative TLC (CHCl3 : MeOH : CH3COOH :: 88 : 11 : 01) of n-Butanol extract (HA-BU) yielded one compound in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Kurchessine. It exhibited reasonable BLasé inhibition activity (Table 4.4 & 4.5). 4.2.2.2: Carissa opaca:
Crude ethannolic extract from Carissa opaca was found active during preliminary screening for BLasé Inhibition. After solvent extraction the Chloroform (CO-CH), extract showed satisfactory enzyme inhibition activity. All the extracts were run on GCMS and from GC chromatogram number and amounts of compounds in solvent
142 Chapter-4: Results & Discussion
extracts were assessed. Later on column chromatography of CO-CH after removal of fatty acids yielded two pure compounds from the active extract as described below.
4.2.2.2.1-CO-CH-Basic Ether-1: Lupeol (33)
Column chromatography with gradient elution (CH2Cl2 : Hex) of defatted chloroform extract (CO-CH-Basic Ether) yielded two compounds in handsome
amount. The material recovered from fraction-1 (CH2Cl2 : Hex :: 20 : 80) was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Lupeol. It exhibited weak BLasé inhibition activity (Table 4.4 & 4.5).
4.2.2.2.2- CO-CH-Basic Ether-2: Campesterol (50)
Column chromatography with gradient elution (CH2Cl2 : Hex) of defatted chloroform extract (CO-CH-Basic Ether) yielded two compounds in handsome
amount. The material recovered from fraction-2 (CH2Cl2 : Hex :: 20 : 80) was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Campesterol. It exhibited reasonable BLasé inhibition activity (Table 4.4 & 4.5).
4.2.2.3:- Alstonia scholaris : Crude methanolic extract from Alstonia scholaris was found active during preliminary screening for BLasé Inhibition. After solvent extraction the Ethyl acetate (AS-EA) extract showed satisfactory enzyme inhibition activity. All the extracts were run on GCMS and from GC chromatogram, number and amounts of compounds in solvent extracts were assessed. AS-EA was further fractionated through column chromatography with gradient elution (Hex : Ethyl acetate : Methanol) to yield five fractions. Fractions -2 and -4 were found active during preliminary screening for Blasé inhibition activity. Later on preparative TLC yielded two pure compounds from active fractions as described below.
143 Chapter-4: Results & Discussion
4.2.2.3.1-AS-EA-1: Quercetin (51)
Preparative Thin Layer Chromatography (Hex : EtOAc : MeOH :: 6:3:1) of (AS-EA-Fr-2) yielded one compound in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Quercetin. It exhibited highest BLasé inhibition activity among extracts from Alstonia scholaris (Table 4.4 & 4.5). 4.2.2.3.2- AS-EA-2: Kaempferol (42)
Preparative Thin Layer Chromatography (Hex : EtOAc : MeOH :: 6:3:1) of (AS-EA-Fr-4) yielded one compound in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Kaempferol. It exhibited reasonable BLasé inhibition activity (Table 4.4 & 4.5). 4.2.3:- Asclepiadaceae Family Two plants were screened for BLasé inhibition activity from Asclepiadaceae family. Only Calotropis procera was found active, hence processed further. 4.2.3.1: Calotropis procera : Crude Dichloromethanoic extract from Calotropis procera was found active during preliminary screening for BLasé Inhibition. CP-DCM was further fractionated through column chromatography with gradient elution (Hex : Ethyl acetate : Methanol) to yield five fractions. Fraction-3 (Hex : DCM :: 20:80) was found active during preliminary screening for BLasé inhibition activity. CP-DCM-Fr-3 was further fractionated through column chromatography with gradient elution (Hex : Dichloromethane : Methanol) to yield four fractions. Fractions -2 and -3 were found active during preliminary screening for Blasé inhibition activity. Later on preparative TLC yielded three pure compounds from active fractions as described below.
144 Chapter-4: Results & Discussion
4.2.3.1.1-CP-DCM-Fr-3-2: β-Sitosterol (34)
CP-DCM-Fr-3 was further fractionated through column chromatography with gradient elution (Hex : DCM : MeOH) to yield four fractions. All the fractions were put to GCMS analysis. Fraction-2 (Hex : DCM :: 20:70) was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as β-Sitosterol. It exhibited weak BLase inhibition activity (Table 4.4 & 4.5).
4.2.3.1.2- CP-DCM-Fr-3-3A: α-Amyrin (52)
CP-DCM-Fr-3 was further fractionated through column chromatography with gradient elution (Hex : DCM : MeOH) to yield four fractions. All the fractions were put to GCMS analysis. Fraction-3 (Hex : DCM :: 00:100) was further fractionated through Preparative Thin Layer Chromatography (Hex : EtOAc : DCM :: 60:25:15). It yielded two compounds in handsome amount. The material recovered from layer-1 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as alpha-Amyrin. It exhibited good BLasé inhibition activity (Table 4.4 & 4.5). 4.2.3.1.3- CP-DCM-Fr-3-3B: Ursolic acid (53)
CP-DCM-Fr-3 was further fractionated through column chromatography with gradient elution (Hex : DCM : MeOH) to yield four fractions. All the fractions were put to GCMS analysis. Fraction-3 (Hex : DCM :: 00:100) was further fractionated through Preparative Thin Layer Chromatography (Hex : EtOAc : DCM :: 60:25:15). It yielded two compounds in handsome amount. The material recovered from layer-2 was put to GC-MS analysis. It was found pure i.e. single significant peak in GC chromatogram. Later on Mass-Spectrum and H1-NMR data interpretation and comparison with literature confirmed it as Ursolic acid. It exhibited highest BLasé inhibition activity among all the fractions from Calotropis procera (Table 4.4 & 4.5).
145 Chapter-4: Results & Discussion
4.2.4 β-Lactamase (BLase) Inhibition Assay – Results:
Table 4.4:- Preliminary Screening of Plant Extracts for β-Lactamase Inhibition Activity
Preliminary Screening of Plants for β-Lactamase inhibition: Observations till . . . .
Sr # Plant Name Extract Code 12 24 36 48 60 72 84 96 Comments hrs hrs hrs hrs hrs hrs hrs hrs
1 CIR-EtOH √ x x x x x x x Active
2 CIR-Hex x x x x x x x x Inactive
3 CIR-CH-NE √ √ x x x x x x Active
4 Cichorium intybus CIR-CH-Ac √ √ x x x x x x Active
5 CIR-CH-Ba x x x x x x x x Inactive
6 CIR-EA-NE √ √ √ √ x x x x Active
7 CIR-Bu-NE x x x x x x x x Inactive
8 AC-MeOH √ x x x x x x x Active
9 AC-Hex √ √ x x x x x x Active
10 Ageratum conyzoides AC-CH x x x x x x x x Inactive
AC-EA x x x Inactive 11 x x x x x
AC-BU x x x Inactive 12 x x x x x
13 HA-EtOH √ √ x x x x x x Active
14 HA-Hex x x x x x x x x Inactive
Hollarhena 15 HA-CH √ √ √ x x x x x Active antidysenterica
16 HA-EA √ √ √ √ x x x x Active
17 HA-BU √ √ x x x x x x Active
18 CO-EtOH √ x x x x x x x Active
19 CO-Hex x x x x x x x x Inactive Carissa opaca 20 CO-CH √ √ x x x x x x Active
21 CO-EA x x x x x x x x Inactive
146 Chapter-4: Results & Discussion
Preliminary Screening of Plants for β-Lactamase inhibition: Observations till . . . .
Sr # Plant Name Extract Code 12 24 36 48 60 72 84 96 Comments hrs hrs hrs hrs hrs hrs hrs hrs
22 CO-BU x x x x x x x x Inactive
23 AS-MeOH √ √ x x x x x x Active
24 AS-Hex x x x x x x x x Inactive
25 Alstonia scholaris AS-CH x x x x x x x x Inactive
26 AS-EA √ √ √ x x x x x Active
27 AS-Bu x x x x x x x x Inactive
28 CP-Hex x x x x x x x x Inactive
29 CP-DCM √ √ √ √ x x x x Active
30 Calotropis procera CP-EA x x x x x x x x Inactive
31 CP-Bu x x x x x x x x Inactive
32 Clavulanic Standard √ √ √ √ √ √ x x Active acid
147 Chapter-4: Results & Discussion
Table 4.5:- Spectrophotometric Screening of Isolated Compounds for β- Lactamase Inhibition Activity
Spectrophotometric monitoring for β- Lactamase Inhibition Activity Sr Plant Extract Code Name Formula Mr % inhibition = % inhibition = # [(Ablank –Asample) x [(Ablank –Asample) x
100] /Ablank 100] /Ablank
01 CIRCHNe-1 Lupeol C30H50O 426 2.40 17
02 CIRCHNe-2 b-Sitosterol C29H50O 414 2.34 19
p-hydroxy phenyl 03 CIRCHAc-1 C8H8O3 152 2.90 00 acetic acid
04 CIRCHAc-2 Isovanillic acid C8H8O4 168 2.52 13
05 Cichorium CIRCHAc-3 Syringic acid C9H10O5 198 2.58 11 intybus 06 CIRCHAc-4 Vanillic acid C8H8O4 168 2.52 13
07 CIREANe-1 Esculetin C9H6O4 178 2.40 17
08 CIREANe-2 Scopoletin C10H8O4 192 2.29 21
09 CIREANe-3 Umbelliferone C9H6O3 162 2.46 15
10 CIREANe-4 Kaempferol C15H10O6 286 2.26 22
Ageratum 11 AC-Hex-Fr-2 Stigmasterol C29H48O 412 2.41 17 conyzoides
12 HA-CH Conessine C24H40N2 356 2.29 21
Isoconessimine / 13 HA-CH C23H38N2 342 2.08 28 Kurchinine
14 Hollarhena HA-CH Conimine C22H36N2 328 1.97 32 antidysenterica 15 HA-EA Kurchamine C22H38N2 330 1.88 35
16 HA-EA Holaromine C21H36N2O 332 1.74 40
17 HA-BU Kurchessine C25H44N2 372 2.26 22
148 Chapter-4: Results & Discussion
Spectrophotometric monitoring for β- Lactamase Inhibition Activity Sr Plant Extract Code Name Formula Mr % inhibition = % inhibition = # [(Ablank –Asample) x [(Ablank –Asample) x
100] /Ablank 100] /Ablank CO-CH- 18 Basic Ether- Lupeol C30H50O 426 2.41 17 1 Carissa opaca CO-CH- Campesterol 19 Basic Ether- C28H48O 400 2.46 15 2
C H O 20 Alstonia AS-EA-1 Quercetin 15 7 10 302 1.91 34 scholaris 21 AS-EA-2 Kaempferol C15H10O6 286 2.26 22
CP-DCM- 22 β-Sitosterol C29H50O 414 2.34 19 Fr-3 Calotropis CP-DCM- 23 α-Amyrin C30H50O1 426 2.42 17 procera Fr-3-3A CP-DCM- 24 Ursolic acid C30H48O3 456 2.23 23 Fr-3-3B
26 Clavulanic Acid C8H9NO5 199 0.87 70
27 Blank 2.90 00
149 Chapter-4: Results & Discussion
4.3. ACHIVEMENTS AND CONCLUSIONS:
The present works consists of Study of beta-Lactamase Inhibitory Potential of Synthetic Ketophosph(on)ates and Phytochemicals from Apocynaceae and Compositae Families. In this regard an entirely new method for β-Lactamase Inhibition Activity Studies was developed and published. For this study thirty two compounds were prepared and characterized by EIMS & 1H-NMR. Seventeen synthetic compounds showed little to moderate BLase inhibition potential. The compounds 1, 12, 29 & 32 showed BLase inhibition activities (81, 84, 82 & 85 % respectively) which are higher than Clavulanic acid (70% only): the most widely used commercial product around the globe. It means Ketophosph(on)ates are the potential candidates to make place in the market as chemotherapeutical agents against resistant strains of microbes. Extraction and bioassay guided isolation of six active plant extracts yielded twenty one known phytocompounds. Although in general all phytocompounds proved less active than expectation yet Kurchamine (47), Holaromine (48) and Quercetine (51) showed highest BLase inhibition activity among the isolated natural products. However these compounds could be taken as basic skeleton to look for new, active, semi-synthetic derivatives.
150 Chapter-5: References
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