SYNTHESIS OF FOSCARNET-PEPTIDE CONJUGATES AND DETECTION OF PYROPHOSPHATE ANALOGUES, BISPHOSPHONATES AND PHOSPHONOCARBOXYLATES

by

Kanokkarn Saejueng

Copyright 2008

PR EV

A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY)

IE
August 2008

W
Kanokkarn Saejueng

3329415

PR EV
2008

IE
3329415

DEDICATION

PR EV

To Luang Phor Jarun, my parents, teachers, sisters, and friends.

IE
ii

ACKNOWLEDGEMENT

I would like to thank my research adviser Professor Charles E. McKenna for his support and guidance during my graduate studies, Dr. Boris A. Kashemirov who trained me in organic synthesis. His expert advice was always very useful and valuable for my graduate works.

Talents Project (DPST, Thailand) and the Department of Chemistry, University of Southern California for the financial support before and during my graduate studies.

Residronate was provided as a gift from Proctor & Gamble Pharmaceuticals. Zoledronate and -hydroxy methylenebisphosphonate was prepared by Mr. James Hogan in our laboratory.

I would like to thank my graduate committee: Professor Robert Bau, Professor John A. Petruska, Assistant Professor Amy M. Barrios, Assistant Professor Peter Qin and Professor Surya Prakash. I would like to thank Heather Connor, graduate student adviser, Michele Dea, Marie de la Torre, Jaime Avila, Bruno Herreros, Ross Lewis and Allan Kershaw for all their help during my years in graduate school. I would like to thank Dr. Debbie C. Crans and Dr. Ernestas Gaidamauskas, Colorado State

PR EV

IE

I would like to thank The Development and Promotion of Science and Technology

iii

University, for their collaboration on Yb3+/ PV project (chapter 2). Without them, this work would not have been completed; working with them was a pleasure.

I would like to thank Dr. Elizabeth Erickson and Dr. Michael Quinlan for all their support and their kind heart during the time I worked with them as TA in Chem 105 class for 4 years and also Thuc Do who always helped me in Chem 105 experiements during that time. I would like to thank the McKenna group members: Greg, Mong,

for all the help. Special thanks for those who helped me get this thesis into writing, helped me in the lab and outside the lab, and for being such wonderful friends; Kasia, Jorge, Michaela, Paradise, Yue and Gian. I also would like to thanks those who are now so faraway but always listen and help: Chris and Edina. I would like to thank friends from Wat Phadhammachart, where I always go when I need to get peace.

Finally, I would like to thank my family and friends in Thailand who always listen, understand, care, encourage me to keep going and support me in all situations.

The people who I have mentioned above have contributed to this thesis and/ or supported me in some ways during my graduate studies, without them this thesis would not have been completed and I greatly appreciate their contributions.

PR EV

IE

JJ, Ulrika, Chris, James, Larryn, Joy, Tom, Chi, Dan, Shuting, Brian, Kirill and Ivan

iv

Table of Content

Dedication Acknowledgments List of Tables List of Figures Abstract Chapter 1: Introduction to Foscarnet 1.1 General information 1.2 In vitro activity and clinical activity 1.3 Mechanism of action 1.4 Dosage, administration and toxicities 1.5 Prodrugs of PFA 1.6 Detection and Quantitation of PFA 1.7 Conclusion 1.8 Chapter 1 References

ii iii xiii xiv xxvii 1 1 2 2 3 4 15 18 20 24

Chapter 2: Detection of Pyrophosphate Analogues, Phosphonoacetates, Bisphosphonates and ATP by Yb3+/ Pyrocatechol Violet Complex and LC/MS/MS 2.1 Introduction 2.2 Results and Discussion 2.2.1 Detection of phosphonates in buffer solution by Yb3+: PV complex. 2.2.1.1 Detection of foscarnet, phosphono acetate, bisphosphonate and ATP by Yb3+ and Pyrocatechol violet (2:1, Yin et al method). 2.2.1.2 An alternative method to obtain a linear foscarnet standard curve by using Yb3+/PV (2:1 molar ratio) 2.2.1.3 Reinvestigation of stoichiometry of putative Yb3+/PV complex 2.2.1.4 Comparison between 1:1 and 2:1 complex toward the detection and quantification of ATP and phosphate 2.2.1.5 Standard curve of foscarnet when 1:1 Yb3+: PV was used

PR EV

IE

24 29 29 29

33 35 39 42

2.3

Preliminary studies of detection of foscarnet in plasma sample by LC/MS 2.2.2.1 Detection of foscarnet by MS/MS 2.2.2.2 Solvent System for detection of foscarnet by MS/MS 2.2.2.3 Attempts to detect and quantify PFA by LC/MS/MS from rat plasma sample Experimental 2.3.1 Detection of bisphosphonates or phosphono carboxylate compounds using Yb3+/PV (2:1), Yin et al method. 2.3.1.1 Preparation of HEPES buffer 10 mM, pH 7 2.3.1.2 Preparation of Yb3+ 2 mM in HEPES buffer solution (10 mM), pH 7 2.3.1.3 Preparation of pyrocatechol violet 1 mM in HEPES buffer (10 mM), pH 7 2.3.1.4 Preparation of Yb3+ and PV complex, 2:1 molar ratio, 50 M in HEPES buffer (10 mM) at pH 7 2.3.1.5 Preparation of ATP solutions for measurement of UV-Vis spectra 2.3.1.5.1 Preparation of ATP stock solution 5 mM in HEPES buffer (10 mM), pH 7 2.3.1.5.2 Preparation of mixture of Yb3+/PV complex and ATP solution 2.3.1.6 Preparation of phosphonoformic acid (PFA) solutions for measurement of UV-Vis spectra. 2.3.1.6.1 Preparation of PFA stock solution in HEPES buffer (10 mM) solution, pH 7 2.3.1.6.2 Preparation of mixture of Yb3+/PV complex and PFA solutions in HEPES buffer (10 mM), pH 7 2.3.1.7 Preparation of ,-dichlorophosphonoacetic acid pyridinium salt solutions for measurement of UV-Vis spectra 2.3.1.7.1 Preparation of ,-dichlorophosphono acetic acid pyridinium salt stock solution 10 mM in HEPES buffer (10 mM) solution, pH 7 2.3.1.7.2 Preparation of mixture of Yb3+/PV complex and ,-dichlorophosphono acetic acid pyridinium Salt solutions in HEPES buffer (10 mM), pH 7 2.3.1.8 Preparation of pamidronate solutions for measurement of UV-Vis spectra. 2.3.1.8.1 Preparation of pamidronate stock solution in HEPES (10 mM) buffer solution, pH 7

2.2.2

43 44 45 47 51 51

52 52 52 53 53 53 53 54 54 54 55

PR EV

IE

55

55

56 56

vi

2.3.2 2.3.3

2.3.1.8.2 Preparation of mixture of Yb3+/PV complex and Pamidronate solutions in HEPES buffer (10 mM), pH 7 2.3.1.9 Preparation of methylenebisphosphonate solutions for measurement of UV-Vis spectra. 2.3.1.9.1 Preparation of methylenebisphosphonate stock solution 14 mM in HEPES buffer (10 mM) solution, pH 7 2.3.1.9.2 Preparation of mixture of Yb3+/PV complex and methylenebisphosphonate solutions in HEPES buffer (10 mM), pH 7 2.3.1.10 Preparation of thiophosphonoformic acid (TPFA) solutions for measurement of UV-Vis spectra 2.3.1.10.1 Preparation of TPFA stock solution 14 mM in HEPES buffer (10 mM) solution, pH 7 2.3.1.10.2 Preparation of mixture of Yb3+/PV complex and TPFA solutions in 10 mM HEPES buffer, pH 7 2.3.1.11 Preparation of zoledronate solutions for measurement of UV-Vis spectra 2.3.1.11.1 Preparation of zoledronate stock solution 15 mM in HEPES buffer (10 mM) solution, pH 7 2.3.1.11.2 Preparation of mixture of Yb3+/PV complex and zoledronate in HEPES buffer (10 mM), pH 7 2.3.1.12 Preparation of -hydroxy methylenebisphonate solutions for measurement of UV-Vis spectra 2.3.1.12.1Preparation of -hydroxy methylenebisphonate stock solution 14 mM in HEPES buffer (10 mM) solution, pH 7 2.3.1.12.2 Preparation of mixture of Yb3+/PV complex and hydroxy methylenebisphonate in HEPES buffer (10 mM), pH 7 2.3.1.13 Measurement of UV-Vis spectra Quantitative analysis experiments of PFA using Yb/PV 2:1 molar ratio complex (as discussed in 2.2.1.2) Detection of ATP, phosphate and foscarnet using Yb3+/PV (1:1) 2.3.3.1 Preparation of Stock Solutions 2.3.3.2 General procedure for UV-Vis spectroscopic analysis sample preparation 2.3.3.2.1 Blue YbPV complex samples 2.3.3.2.2 Measuring analyte concentrations

56

57 57

57

58 58 58

IE

59 59 59 60 60

PR EV

60

61 78 86 86 87 87 87 vii

2.4 2.5

LC/MS/MS, MS/MS experiment 2.3.4.1 Preparation of PFA standard solutions for LC/MS Experiment 2.3.4.2 Preparation of 10 mM ammonium formate buffer pH 7 in 20% methanol in water 2.3.4.3 Preparation of rat plasma 2.3.4.4 MS/MS and LC/MS/MS experiments Conclusion Chapter 2 References

2.3.4

87 88 89 89 89 100 101

Chapter 3: Synthesis of Amino Acid/ Peptide Foscarnet Derivatives via C-amide Bond Formation 3.1 Introduction 3.2 Results and Discussion 3.3 Experimental 3.3.1 Synthesis of Phenoxycarbonyl dimethylphosphonate (8) 3.3.2 Synthesis of Phenoxycarbonyl methylphosphonate (9) 3.3.3 Synthesis of sodium methyl phosphonoformate-N-BOCL-Lysine-L-Phenyalanine methyl ester (11) 3.3.4 Synthesis of disodium PFA-L-Lysine-L-Phenyalanine methyl ester (1) 3.3.5 Synthesis of N-Boc-N-Fmoc-L-Lysine; 2-tertButoxycarbonylamino-6-(9H-fluoren-9-yloxycarbony amino)-hexanoic acid (12) 3.3.6 Synthesis of N-Boc-N-Fmoc-L-Lysine-L-Phenylalanine ethyl ester; 2-[2-tert-Butoxycarbonylamino-6-(9H-fluoren9-yloxycarbonylamino)-hexanoylamino]-3-phenylpropionic acid ethyl ester (13) 3.3.7 Synthesis of N-Boc-L-Lysine-L-Phenylalanine ethyl ester; 2-(6-Amino-2-tert-butoxycarbonylaminohexanoylamino-3-phenyl-propionic acid ethyl ester (14) 3.3.8 Synthesis of disodium PFA-L-Lysine-L-Phenylalanine ethyl ester; 2-[2-Amino-6-(2-phosphono-formylamino] -3-phenyl-propionic acid ethyl ester (2) 3.3.9 Synthesis of N-Boc-N-Fmoc-L-Lysine-LPhenylalanine benzyl ester; 2-[2-tert-Butoxycarbonyl amino-6-(9H-fluoren-9-yloxycarbonylamino)-hexanoyl amino]-3-phenyl-propionic acid benzyl ester (15a) 3.3.10 Synthesis of N-Boc-L-Lysine-L-Phenylalanine benzyl ester; 2-(6-Amino-2-tert-butoxycarbonylaminoexanoylamino-3-phenyl-propionic acid benzyl ester (16a)

103 103 107 115 116 116 117 118 119

PR EV

IE

119

120

121

122

123

viii

3.3.11 Synthesis of PFA-L-Lysine-L-Phenylalanine; 2[2-Amino-6-(2-phosphono-formylamino)-hexanoyl amino]-3-phenyl-propionic acid (3) and determination of the amount of compound by UV method 3.3.12 Synthesis of N-Boc-N-Fmoc-D-Lysine-DPhenylalanine benzyl ester; 2-[2-tert-Butoxycarbonyl amino-6-(9H-fluoren-9-yloxycarbonylamino)hexanoyl amino]-3-phenyl-propionic acid benzyl ester (15b): 3.3.13 Synthesis of N-Boc-D-Lysine-D-Phenylalanine benzyl ester; 2-(6-Amino-2-tert-butoxycarbonyl amino-hexanoylamino-3-phenyl-propionic acid benzyl ester (16b) 3.3.14 Synthesis of disodium PFA-D-Lysine-D-Phenylalanine; 2-[2-Amino-6-(2-phosphono-formylamino)-hexanoylamino]-3phenyl-propionic acid (4) 3.3.15 Synthesis of N-Boc-N-Fmoc-L-Lysine-L-Tyrosine benzyl ester; 2-[2-tert-Butoxycarbonylamino-6(9H-fluoren-9-ylmethoxycarbonylamino-hexanoylamino] -3-(4-hydroxy-phenyl)-propionic acid benzyl ester (15c) 3.3.16 Synthesis of N-Boc-L-Lysine-L-Tyrosine benzyl ester; 2-(6-Amino-2-tert-butoxycarbonylamino-hexanoylamino) -3-(4-hydroxy-phenyl)-propionic acid benzyl ester (16c) 3.3.17 Synthesis of N-Boc-L-Lysine-L-Tyrosine; 2-(6-Amino2-tert-butoxycarbonylamino-hexanoylamino)-3-(4hydroxy-phenyl)-propionic acid (17c) 3.3.18 Synthesis of disodium PFA-L-Lysine-L-Tyrosine; 2-[2Amino-6-(2-phosphono-formylamino)-hexanoylamino] -3-(4-hydroxy-phenyl)-propionic acid (5) 3.3.19 Synthesis of N-Boc-N-Fmoc-L-Lysine-L-Tryptophan benzyl ester; 2-[2-tert-Butoxycarbonylamino-6-(9Hfluoren-9-ylmethoxycarbonylamino)-hexanoylamino]-3(1H-indol-3-yl)-propionic acid benzyl ester (15d) 3.3.20 Synthesis of N-Boc-L-Lysine-L-Tryptophan benzyl ester; 2-(6-Amino-2-tert-butoxycarbonylaminohexanoyl amino)-3-(1H-indol-3-yl)-propionic acid benzyl ester (16d) 3.3.21 Synthesis of N-Boc-L-Lysine-L-Tryptophan; 2-(6-Amino-2-tert-butoxycarbonylamino)-3-(1Hindol-3-yl)-propionic acid (17d) 3.3.22 Synthesis of disodium phosphonoformate -L-LysineL-Tryptophan; 2-[2-Amino-6-(2-phosphono-formyl amino)-hexanoylamino]-3-(1H-indol-3-ylpropionic acid (6)

123

125

126

127

128

IE

129

PR EV

130

130

131

132

133

133

ix

3.4 3.5

Conclusion Chapter 3 References

135 136

Chapter 4: Synthesis of Cyclic Serine Foscarnet Derivatives via P-ester Bond Formation Using Pivaloyl Chloride and Mitsunobu Reaction 4.1 Introduction 4.2 Results and Discussion 4.3 Experimental 4.3.1 Synthesis of Me-PFA-L-SerOEt; sodium ethyl N[(methoxyphosphinato)carbonyl]serinate (5) 4.3.2 PyBOP reaction of 5 4.3.3 Study of stability of free acid of 5 4.3.3.1 Preparation of DOWEX 50 WX8-200 4.3.3.2 Stability study of free acid of 5 4.3.4 Synthesis of cyclic PFA-L-SerOEt (1a): ethyl 2-methoxy3-oxo-1,4,2-oxazaphosphinane-5-carboxylate 2-oxide 4.3.5 Purification of dehydroalanine PFA derivative (6): ethyl 2({[hydroxy(methoxy)phosphoryl]carbonyl}amino)acrylate 4.3.6 Study of stability of cyclic PFA-L-SerOEt; ethyl 2-methoxy3-oxo-1,4,2-oxazaphosphinane-5-carboxylate 2-oxide (1a) in CD3OD/ silica gel 4.3.6.1 Stability of 1a in CD3OD 4.3.6.2 Stability of 1a in silica gel 4.3.7 Synthesis of diammonium-PFA-amino propanol (8) 4.3.8 Synthesis of ditriethyl ammonium PFA-amino propanol (9) 4.3.9 Attempt to synthesize ammonium 3-oxo-1,4,2oxazaphosphepan-2-olate 2-oxide (10) from reaction of pivaloyl chloride and 8 4.3.10 Synthesis of diammonium (methoxycarbonyl)phosphonate (12) 4.3.11 Synthesis of ammonium methyl (methoxycarbonyl) phosphonate (13) by reaction of pivaloyl chloride, 12 and methanol 4.3.12 Synthesis of Me-PFA-L-SerOBzl: (sodium methyl [({1-[(benzyloxy)carbonyl]propyl}amino)carbonyl] phosphonate, 14) 4.3.13 Synthesis of diammonium benzyl N-(phosphonato carbonyl)serinate (15) 4.3.14 Attempt to synthesize ammonium 5-[(benzyloxy)carbonyl] -3-oxo-1,4,2-oxazaphosphinan-2-olate 2-oxide (1b), reaction of pivaloyl chloride and 15

183 183 188 224 225 226 226 226 226 226 227 228

PR EV

IE

228 228 228 229 229

230 230

231

231 232

4.3.15 Attempt to synthesize benzyl 2-methoxy-3-oxo -1,4,2-oxazaphosphinane-5-carboxylate 2-oxide (1c) by Mitsunobu reaction of 14 4.3.16 Attempt to synthesize sodium 5-[(benzyloxy)carbonyl]-3oxo-1,4,2-oxazaphosphinan-2-olate 2-oxide (1d) by Mitsunobu reaction of 14 4.3.17 Attempt to synthesize benzyl 2-hydroxy-3oxo-1,4,2-oxazaphosphinane-5-carboxylate 2-oxide (1e) by Mitsunobu reaction of disodium salt of 14 4.3.18 Attempt to synthesize Boc-Ser-Bn-PFA-Ph: (16) by Mitsunobu reaction 4.3.19 Sodium iodide reaction of Mitsunobu reaction crude between Phenylcarbonyl methylphosphonate (4) and Boc-L-Ser-OBzl (4.3.18) 4.3.20 Separation of 17 from NaI reaction crude (4.3.19) on TLC plastic plate 4.3.21 Purification of Boc-Ser-Bn-PFA-Boc-Ser-Bn (18) from NaI reaction crude (4.3.19) 4.3.22 Synthesis of Ser-Bn-PFA-Ser-Bn (19); deprotection of Boc group from Boc-Ser-Bn-PFA-Boc-Ser-Bn (18) 4.3.23 Imitation of Mitsunobu conditions to understand mechanism of how Boc-Ser-Bn-PFA-Boc-Ser-Bn or Boc-Ser-Bn-PFA-Me were generated 4.3.23.1 Reaction of phenoxycarbonyl dimethylphosphonate with DIAD, TPP, Boc-Ser-Bn 4.3.23.2 Reaction of phenoxycarbonyl dimethylphosphonate with benzyl alcohol 4.3.23.3 Stability of phenoxycarbony methylphosphonate in free acid form in methanol 4.3.23.4 Stability of phenoxycarbony methylphosphonate in free acid form in the presence of Boc-Ser-Bn in dioxane 4.3.24 Transesterification of phenoxycarbonyl methyl phosphonate (4) in the presence of DBU 4.3.25 Basic hydrolysis of phenoxycarbonyl dimethylphosphonate 4.3.26 Basic hydrolysis of phenoxycarbonly methylphosphonate (4) 4.3.27 Attempts to cleave C-phenyl ester of phenoxycarbonly dimethylphosphonate by AlCl3 / KI 4.3.28 Attempts to cleave C-phenyl ester of phenoxycarbonly methylphosphonate (4) by AlCl3 / KI 4.3.29 Synthesis of benzyloxycarbonyl dimethylphosphonate (22) 4.3.30 Synthesis of benzyloxycarbonyl methylphosphonate (23)

232

233

233

234 236

238 238 238 239

PR EV

IE

239 239 241 241 241 242 242 246 248 250 250

xi

4.4 4.5 5.0

4.3.31 Synthesis of Boc-Ser-Bn-PFA-Bn (24) by Mitsunobu reaction and NaI 4.3.32 Synthesis of Ser-Bn-PFA-Bn (25) from Boc-Ser-Bn-PFA-Bn (24) 4.3.33 Attempts to synthesize Ser-PFA (2b) from Ser-Bn-PFA-Bn (25) by catalytic hydrogenation: Ser-Bn-PFA-Bn, 25 4.3.34 Attempts to synthesize phosphinecarboxylic acid (20) from benzyloxycarbonyl methylphosphonate (23) by catalytic hydrogenation Conclusion Chapter 4 References Bibliography

251 257 257 258

258 259 290

PR EV

IE
xii

List of Tables

2.2.1

Comparison between sensitivity of each solvent system for detection of foscarnet by MS/MS Preparation of mixture of Yb3+/PV complex and ATP solutions for detection and standard curves experiment Preparation of mixture of Yb3+/PV complex and PFA solutions for detection and standard curves experiment Preparation of mixture of Yb3+/PV complex and , dichlorophosphonoacetic acid solutions for detection and standard curves experiment.

46

2.3.1

54

2.3.2

55

2.3.3

56

2.3.5

2.3.6

PR EV

Preparation of mixture of Yb3+/PV complex and methylenebisphosphonate solutions for detection and standard curves experiment. Preparation of mixture of Yb3+/PV complex and TPFA solutions for detection and standard curves experiment Preparation of mixture of Yb3+/PV complex and zoledronate solutions for detection and standard curves experiment

IE

2.3.4

Preparation of mixture of Yb3+/PV complex and pamidronate solutions for detection and standard curves experiment.

57

58

59

2.3.7

60

2.3.8

Preparation of mixture of Yb3+/PV complex and -hydroxy methylenebisphonate solutions for detection and standard curves experiment

61

2.3.9

Preparation of standard PFA stock solutions

88

xiii

List of Figures

1.1

Chemical structure of phosphonoformic acid (PFA) and pyrophosphate (PPi) Acyloxyalkyl ester analogue of foscarnet Strucutre of ODG-PFA Alkoxy propane foscarnet conjugates; n= 21, 17,18, 11, 7

1.2 1.3 1.4

6 7 7

1.6

1.7

1.8 1.9 1.10 1.11 1.12

PR EV

General structures of PFA derivative conjugated with polyalcohol Cyclic diester analogue of PFA General structures of SATP-PFA Decomposition process of SATP test model Structure of BUdR and its foscarnet prodrug derivative

IE

General structures of sulfur-containing phosphonoformate derivatives

1.5

General structure of 1-O-octadecyl-2-X-sn-glycero-3 -PFA

10

11 12 12 13 14

General structure of conjugates of dideoxynucleoside with thiophosphonoformate

1.13 1.14 2.1.1 2.1.2

General structure of AZT-PFA derivatives

Chemical structure of P-N foscarnet-amino acid conjugates

14
15

Chemical structure of pyrocatechol violet (PV) Titration of PV by Yb3+ and Yb3+/PV complex by ATP by Yin et al.

26 26

xiv

2.1.3 2.2.1 2.2.2 2.2.3 2.2.4

Detection of ATP by Yb3+: PV, 2:1 molar ratio by Yin et al Structures of test compounds Solution of Yb3+ / PV complex when PFA was added UV-Vis spectra of titration of PFA and Yb3+/PV complex Differences of absorbance (A) plotted against concentration of each analyte Standard curve of PFA by an alternative method The absorbance difference in 100 M Yb3+ and 50 M PV solution upon phosphate and ATP addition Continuous variation plot at three representative wavelengths for the Yb3+ and PV system Absorbance difference at 623 and 443 nm as a function of the [Yb3+]/ [PV] molar ratio

27 30 30 32 33

2.2.5 2.2.6

35 36

2.2.7

38

2.2.8

IE

39

2.2.9

PR EV

Visual assessment of color changes in PV (50 M) solutions in HEPES (10 mM, pH 7.0) upon addition of [Yb3+] = 0 - 50 M and then [ATP] = 0 - 50 M

40

2.2.10

Standard curves of phosphate and ATP when Yb3+/PV (1:1) were used UV-Vis spectra when PFA was added to Yb3+/PV complex (1:1)

41

2.2.11 2.2.12 2.2.13 2.2.14

42 43 45 48

Standard curve of PFA when 1:1 complex was used

Detection of foscarnet by MS/MS Study of the effect of plasma on detection of PFA by MS/MS (no column), solvent system: 20% MeOH/ water Study of the effect of plasma on detection of PFA by LC/MS/MS, solvent system: 20% MeOH /water

2.2.15

50

xv

2.3.1

UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and ATP solutions at difference concentrations UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and PFA solutions at difference concentrations UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and ,-dichlorophosphonoacetic acid solutions at difference concentrations UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and pamidronate solutions at difference concentrations UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and methylenebisphosphonate solutions at difference concentrations

62

2.3.2

63

2.3.3

64

2.3.4

65

2.3.5

66

PR EV

2.3.7

UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and zoledronate solutions at difference concentrations

IE

2.3.6

UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and TPFA solutions at difference concentrations

67

68

2.3.8

UV-Vis spectra of Yb3+ / PV (100 M Yb3+ and 50 M PV) and -hydroxy methylenebisphosphonate at difference concentrations

69

2.3.9

Standard curve of ATP when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used Standard curve of PFA when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used Standard curve of ,-dichlorophosphonoacetic acid when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used Standard curve of pamidronate when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used

70

2.3.10

71

2.3.11

72

2.3.12

73

xvi

2.3.13

Standard curve of methylenebisphosphonate when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used Standard curve of TPFA when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used Standard curve of zoledronate when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used Standard curve of -hydroxy methylenebisphosphonate when Yb3+ / PV (2:1; 50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was used Picture demonstrates how to find absorbance at the end point of each titration

74

2.3.14

75

2.3.15

76

2.3.16

77

2.3.17

79

PR EV

2.3.19

Titration curve of 20 M PFA. Yb3+/PV (50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was added

IE

2.3.18

An example of a standard curve obtained by plotting between absorbance at the end point of each solution and concentrations of each solution

79

80

2.3.20

Titration curve of 40 M PFA. Yb3+/PV (50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was added Titration curve of 60 M PFA. Yb3+/PV (50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was added Titration curve of 80 M PFA. Yb3+/PV (50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was added Titration curve of 100 M PFA. Yb3+/PV (50 M PV and 100 M Yb3+) solution in 10 mM HEPES, pH 7 was added Standard curve of PFA by multiple titrations method

81

2.3.21

82

2.3.22

83

2.3.23

84

2.3.24 2.3.25

85 91

Detection of foscarnet by MS/MS (no column), solvent system: 20% methanol in 10 mM ammonium formate

xvii

2.3.26

Detection of foscarnet by MS/MS (no column), solvent system: 20% methanol/ water Detection of foscarnet by MS/MS (no column), solvent system: 50% methanol/ water Detection of foscarnet by MS/MS (no column), solvent system: 20% acetonitrile/ water etection of foscarnet by MS/MS (no column), solvent system: 50% acetonitrile/ water Detection of foscarnet by LC/MS/MS, solvent system: 20% MeOH /water Study of the effect of plasma on detection of PFA by LC/MS/MS, solvent system: 20% MeOH/ water, collision energy 35 Study of the effect of plasma on detection of PFA by LC/MS/MS, solvent system: 50% MeOH /water Study of the effect of plasma on detection of PFA by LC/MS/MS, solvent system: 1% MeOH /water

92

2.3.27

93

2.3.28

94

2.3.29

95

2.3.30

96

2.3.31

97

2.3.32

IE

98

2.3.33

PR EV

99

3.1.1

Chemical structures of PFA, pyrophosphate (PPi) and P-N Foscarnet-Amino acid conjugates

103

3.1.2 3.1.3

Example of C-amide derivatives of foscarnet Structure of PFA-Lys derivatives with C-amide bonds connected between foscarnet moiety and peptides
31

105 106

3.2.1

P NMR of PFA-L-Lys-L-PheOMe after removal of TEAC buffer

110

3.2.2

Chromatogram of analytical HPLC of PFA-L-lys-L-PheOEt (2) 113 after Preparative HPLC and removal of TEAA buffer P NMR spectrum of Phenoxycarbonyl dimethyl phosphonate (8)
31

3.3.1a

139

xviii

3.3.1b

H NMR spectrum of Phenoxycarbonyl dimethylphosphonate (8)

140

3.3.2a

31

P NMR spectrum of Phenoxycarbonyl methylphosphonate (9) H NMR spectrum of Phenoxycarbonyl methylphosphonate (9)
31

141

3.3.2b

142

3.3.3a

P NMR spectrum of sodium methyl phosphonoformateN-BOC-L-Lysine-L-Phenyalanine methyl ester (11)

1

143

3.3.3b

H NMR spectrum of sodium methyl phosphonoformateN-BOC-L-Lysine-L-Phenyalanine methyl ester (11) P NMR spectrum of disodium PFA-L-Lysine-LPhenyalanine methyl ester (1)

144

3.3.4a

31

145

3.3.4b

H NMR spectrum of disodium PFA-L-Lysine-LPhenyalanine methyl ester (1) HRMS of disodium PFA-L-Lysine-L-Phenyalanine methyl ester (1)

IE

146

3.3.4d

PR EV

3.3.4c

147

Chromatogram of HPLC separation of disodium PFAL-Lysine-L-Phenyalanine methyl ester (1), TEAC buffer Chromatogram of HPLC separation of disodium PFAL-Lysine-L-Phenyalanine methyl ester (1), TEAA buffer
1

148

3.3.4e

149

3.3.5 3.3.6

H NMR spectrum of N-Boc-N-Fmoc-L-Lysine (12)

150 151

H NMR spectrum of N-Boc-N-Fmoc-L-Lysine-LPhenylalanine ethyl ester (13)

1

3.3.7

H NMR spectrum of N-Boc-L-Lysine-L-Phenylalanine ethyl ester (14) P NMR spectrum of disodium PFA-L-Lysine-LPhenylalanine ethyl ester (2)

152

3.3.8a

31

153

xix

3.3.8b

H NMR spectrum of disodium PFA-L-Lysine-LPhenylalanineethyl ester (2)

154

3.3.8c

Chromatogram of preparative HPLC of disodium PFA-LLysine-L-Phenylalanine ethyl ester (2) Analytical HPLC chromatogram to test purity of 2 after disodium PFA-L-Lysine-L-Phenylalanine ethyl ester (2) H NMR spectrum of N-Boc-N-Fmoc-L-Lysine-LPhenylalanine benzyl ester (15a) H NMR spectrum of N-Boc-L-Lysine-L-Phenylalanine benzyl ester (16a)
31 1 1

155

3.3.8d

156

3.3.9

157

3.3.10

158

3.3.11a 3.3.11b 3.3.11c 3.3.11d

P NMR spectrum of PFA-L-Lysine-L-Phenylalanine (3)

159 160 161 162

HRMS of PFA-L-Lys-L-Phe (3)

PR EV
1

Chromatogram of preparative HPLC of PFA-L-LysineL-Phenylalanine (3) H NMR spectrum of N-Boc-N-Fmoc-D-Lysine-DPhenylalanine benzyl ester (15b)
1

IE

H NMR spectrum of PFA-L-Lysine-L-Phenylalanine (3)

3.3.12

163

3.3.13

H NMR spectrum of N-Boc-D-Lysine-D-Phenylalanine benzyl ester (16b)

31

164

3.3.14a

P NMR spectrum of disodium PFA-D-Lysine-DPhenylalanine (4)

165

3.3.14b

H NMR spectrum of disodium PFA-D-Lysine-DPhenylalanine (4) HRMS of disodium PFA-D-Lysine-DPhenylalanine (4) Chromatogram of preparative HPLC of disodium PFA-DLysine-D-Phenylalanine (4)

166

3.3.14c

167

3.3.14d

168

xx

3.3.15

H NMR spectrum of N-Boc-N-Fmoc-L-Lysine-LTyrosine benzyl ester (15c)

1

169

3.3.16

H NMR spectrum of N-Boc-L-Lysine-LTyrosine benzyl ester (16c) H NMR spectrum of N-Boc-L-Lysine-L-Tyrosine (17c) P NMR spectrum of disodium PFA-L-Lysine-L-Tyrosine (5)

170

3.3.17 3.3.18a 3.3.18b 3.3.18c 3.3.18d

171 172 173 174 175

31

H NMR spectrum of disodium PFA-L-Lysine-L-Tyrosine (5)

HRMS of disodium PFA-L-Lysine-L-Tyrosine (5)

3.3.20

PR EV

H NMR spectrum of N-Boc-L-Lysine-L-Tryptophan benzyl ester (16d)

1

IE

3.3.19

H NMR spectrum of N-Boc-N-Fmoc-L-Lysine-LTryptophan benzyl ester (15d)

Chromatogram of preparative HPLC of disodium PFA-LLysine-L-Tyrosine (5)

176

177

3.3.21

H NMR spectrum of N-Boc-L-Lysine-L-Tryptophan (17d)

178 179

3.3.22a

31

P NMR spectrum of disodium PFA-L-Lysine-LTryptophan (6)

3.3.22b

H NMR NMR spectrum of disodium PFA-L-Lysine-LTryptophan (6) HRMS of disodium PFA-L-Lysine-L-Tryptophan (6)

180

3.3.22c 3.3.22d

181 182

Chromatogram of preparative HPLC of disodium PFA-LLysine-L-Tryptophan (6) Four types of foscarnet prodrug reported by Noren et al Examples of type III prodrugs of PFA that release parent PFA by enzymatic cleavage Two types of target Serine-PFA compounds (1, 2)

4.1.1 4.1.2

184 185

4.1.3

186 xxi

4.1.4

Structure of ethyl (ethoxycarbonyl) phosphonate (a) and its cyclic counter part (b) Stability study of PFA-Serine free acid form of 5 in CD3OD Stability of 2a on TLC silica gel
31

187

4.2.1 4.2.2 4.2.3

191 193 196

P NMR spectrum of reaction mixture of pivaloyl chloride reaction of PFA derivative 8 or 9

4.2.4

P NMR spectrum of reaction mixture of pivaloyl chloride reaction between di ammonium (methoxycarbonyl) phosphonate (12) and methanol
31

31

197

4.2.6

MS spectrum in negative mode of reaction mixture of pivaloyl chloride and diammonium benzyl N-(phosphonatocarbonyl)serinate (15)

IE

4.2.5

P NMR spectrum of reaction mixture of pivaloyl chloride reaction of diammonium benzyl N-(phosphonatocarbonyl) serinate (15)

199

200

4.2.7

PR EV

MS spectrum in negative mode of Mitsunobu reaction of L-Ser-OBzl-PFA (14)

202

4.2.8

MS spectrum in negative mode of reaction mixture Mitsunobu reaction of disodium benzyl N-(phosphonatocarbonyl)serinate
1

204

4.2.9

H NMR spectrum of crude reaction mixture of Mitsunobu reaction of disodium benzyl N-(phosphonatocarbonyl)serinate

205

4.2.10

MS spectrum in negative mode of Mitsunobu reaction crude of 4 and Boc-L-Ser-OBn

207

4.2.11

MS spectrum in negative mode of spot 2 extracted from a 1 X 3 TLC plate after separation of NaI reaction crude MS spectrum in negative mode of spot 3 extracted from a 1 X 3 TLC plate after separation of NaI reaction crude MS spectrum in negative mode of spot 1 extracted from a 1 X 3 TLC plate after separation of NaI reaction crude

208

4.2.12

209

4.2.13

210

xxii

4.2.14

MS spectrum in negative mode of TFA reaction crude performed directly from Mitsunobu and NaI reaction crude without purification MS spectrum in negative mode of a solution of phenoxycarbonyl methylphosphonate in free acid form in methanol MS spectrum in positive mode of a solution of phenoxycarbonyl methylphosphonate in free acid form in a presence of Boc-Ser-Bn/ dioxane MS spectrum in negative mode of mixture of 4, DBU and Boc-Ser-OBn
31

212

4.2.15

216

4.2.16

217

4.2.17

219

4.3.1a

P NMR spectra of Mitsunobu reaction of 4 and Boc-L-Ser-OBn, in an attempt to make 16

235

IE

4.3.1b

31

P couple with 1H NMR spectrum of crude Mitsunobu reaction after washing with NaHCO3

236

4.3.3

PR EV

4.3.2

P couple with 1H NMR spectra monitoring progress of NaI reaction. MS spectrum of reaction mixture of TFA and Boc- Ser-Bn-PFA-Boc-Ser-Bn (18) P NMR coupled with 1H spectrum of basic hydrolysis reaction mixture of phenoxylcarbonyl dimethylphosphonate P NMR coupled with 1H spectrum of basic hydrolysis reaction mixture of phenoxylcarbonyl methylphosphonate (4)

31

237

240

4.3.4

31

242

4.3.5a

31

243

4.3.5b

H NMR spectrum of basic hydrolysis reaction mixture of phenoxylcarbonyl methylphosphonate (4) MS spectrum in negative mode of basic hydrolysis reaction mixture of phenoxylcarbonyl methylphosphonate (4)
31

244

4.3.5c

245

4.3.6a

P NMR spectrum of reaction mixture of phenoxycarbonly dimethylphosphonate and AlCl3 / KI

246

xxiii