Synthesis and biological evaluation of some D

Helwan University Faculty of Science

SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME D-GLUCURONIC ACID DERIVATIVES.

A dissertation submitted by

Mr. Frady Gamal Adly Gouany (B.Sc., Chemistry Department, 2006)

For partial fulfillment of the requirements for

The Master Degree of Science (Organic Chemistry)

To Chemistry Department, Faculty of Science, Helwan University (2011)

‫سما ِء يُ ْع ِطينا النَّجاح‪،‬‬ ‫إِلو ال َّ‬ ‫ون ْح ُن عبِي ُدهُ نقُو ُم ون ْبنِي‬ ‫(نحميا ‪)22 :2‬‬

Approval Sheet for Submission We hereby recommend that the thesis entitled "Synthesis and Biological Evaluation of some D-Glucuronic Acid Derivatives." prepared by Mr. Frady Gamal Adly Gouany under our supervision is accepted for fulfilling the requirements of The Master Degree of Science.

Approved by Prof. Dr. Yehya Mahmoud El-Kholy Prof. of Organic Chemistry Chemistry Department, Faculty of Science, Helwan University.

Signature:‫ــــــــــــــــــــــــــــــــــــــ‬

Prof. Dr. Atef Gobran Hanna Azaria Prof. of Organic Chemistry Chemistry of Natural Compounds Department, National Research Center (NRC).

Signature:‫ــــــــــــــــــــــــــــــــــــــ‬

Dr. Ahmed Omar Hussein Omar El-Nezhawy Associate Prof. of Organic Chemistry Chemistry of Natural and Microbial Products Department, National Research Center (NRC).

Signature:‫ـ ـــــــــــــــــــــــــــــــــــــ‬

Dr. Shahenaz Hassan El-Sayed Lecturer of Organic Chemistry Chemistry Department, Faculty of Science, Helwan University.

Signature:‫ــــــــــــــــــــــــــــــــــــــ‬

Abstract

Name: Frady Gamal Adly Gouany Degree: Master Degree of Science. Title of the thesis: Synthesis and Biological Evaluation of Some D-Glucuronic Acid Derivatives. Research Institute: National Research Center (NRC).

Twenty seven new D-glucuronic acid derivatives were chemically synthesized including acetylated (5-12) and deacetylated (14-21) uronamides, Δ4,5-uronamides (22-29) and N-glucuronides (33-34a,b) starting from the Dglucuronic acid itself by means of protection/deprotection, activation, basecatalyzed elimination and condensation protocols. Structure elucidation for all products along with optimization of the synthetic steps is described. The synthesized compounds were evaluated for their in vitro antitumor activity against MCF-7, TK-10 and UACC-62 cell lines. The compounds 6, 10-12, 14, 17, 21, 23, 24, 28 and 29 were the most active against TK-10 cell line. On the other hand, the most active compounds against the MCF-7 cell line were 20, 21, 24, 28 and 31. However, compounds 9-12, 16, 18-20, 23-25 and 27-29 were the most active against the UACC-62 cell line.

Keywords: D-glucuronic acid, D-glucuronamide, amide linkage, antitumor, TK-10, MCF-7, UACC-62.

Acknowledgments I would like to start expressing a sincere acknowledgment to my supervisors and to thank them for believing in me and giving me the opportunity to research under their guidance and supervision. I received motivation, encouragement and support form them during my study. I gratefully acknowledge Prof. Dr. Yehya Mahmoud El-Kholy for his crucial contribution and supervision, which made him a backbone of this research and so to this thesis. I thank him also for using his precious time to read this thesis and giving his suggestions and comments about it. I thank Prof. Dr. Atef Gobran Hanna for the so many discussions and advices. I admire very much his efficient way in making talks, his enthusiasm and endless energy. His truly scientist intuition has made him as a constant oasis of ideas and passions in science, which enriched my growth as a researcher. I thank Dr. Ahmed Omar Hussein Omar El-Nezhawy for his continuous scientific guidance which gave me an extraordinary experience through out the work which helped me to develop as an independent researcher. I am grateful for his guidance, constant support and advice. I thank him also for his careful reading of the thesis, his suggestions and comments for each version which helped me in improving it more and more. I would like to extend my thanks to Dr. Shahenaz Hassan El-Sayed for her effective contribution and supervision. Further, I am grateful in every possible way and I would like to express my special thanks to Prof. Dr. Sahar Awad-Allah Mahmoud, Nessrin Mahmoud Hegazy and Reham Tharwat El-Shaarawi from the NMR spectroscopy unit for their continuous help regarding the NMR measurements necessary for the research described in this thesis.

Also, I wish to thank all members of the Medicinal Chemistry & Drug Design Lab. for their help, assistance, valuable advices and for the overall laboratory atmosphere. I’m forever indebted to my family for all the feelings, support and encouragement they have given to me. Above all, my deepest thanks go to my fiancée, Mona, who supported me through all the years of this thesis with her continuous love, understanding and encouragement, however my mood was. Last but not least, I would like to thank everybody who was important to the successful realization of this thesis and I also apologize to anyone who may not be mentioned personally, here.

The Author Frady G. Adly

To My Family

i List of Schemes Scheme 1. .......................................................................................................... 2 Scheme 2. .......................................................................................................... 8 Scheme 3. .......................................................................................................... 9 Scheme 4. ......................................................................................................... 9 Scheme 5. ........................................................................................................ 10 Scheme 6. ....................................................................................................... 11 Scheme 7. ........................................................................................................ 12 Scheme 8. ........................................................................................................ 13 Scheme 9. ........................................................................................................ 13 Scheme 10. ...................................................................................................... 14 Scheme 11. ...................................................................................................... 15 Scheme 12. ...................................................................................................... 15 Scheme 13. ...................................................................................................... 16 Scheme 14. ...................................................................................................... 16 Scheme 15. ...................................................................................................... 17 Scheme 16. ...................................................................................................... 18 Scheme 17. ...................................................................................................... 20 Scheme 18. ...................................................................................................... 21 Scheme 19. ...................................................................................................... 22 Scheme 20. ...................................................................................................... 22 Scheme 21. ...................................................................................................... 23 Scheme 22. ...................................................................................................... 23 Scheme 23. ...................................................................................................... 24 Scheme 24. ...................................................................................................... 24 Scheme 25. ...................................................................................................... 26 Scheme 26. ...................................................................................................... 27 Scheme 27. ...................................................................................................... 27 Scheme 28. ...................................................................................................... 28 Scheme 29. ...................................................................................................... 30

ii Scheme 30. ...................................................................................................... 31 Scheme 31. ...................................................................................................... 33 Scheme 32. ...................................................................................................... 34 Scheme 33. ...................................................................................................... 39 Scheme 34. ...................................................................................................... 42 Scheme 35. ...................................................................................................... 43 Scheme 36. ...................................................................................................... 44 Scheme 37. ...................................................................................................... 44 Scheme 38. ...................................................................................................... 46 Scheme 39. ...................................................................................................... 47 Scheme 40. ...................................................................................................... 48 Scheme 41. ...................................................................................................... 49 Scheme 42. ...................................................................................................... 50 Scheme 43. ...................................................................................................... 52 Scheme 44. ...................................................................................................... 56 Scheme 45. ...................................................................................................... 63 Scheme 46. ...................................................................................................... 66 Scheme 47. ...................................................................................................... 67 Scheme 48. ...................................................................................................... 67 Scheme 49. ...................................................................................................... 68 Scheme 50. ...................................................................................................... 69 Scheme 51. ...................................................................................................... 69 Scheme 52. ...................................................................................................... 70 Scheme 53. ...................................................................................................... 71 Scheme 54. ...................................................................................................... 72 Scheme 55. ...................................................................................................... 75 Scheme 56. ...................................................................................................... 85 Scheme 57. ...................................................................................................... 86 Scheme 58. ...................................................................................................... 92 Scheme 59. .................................................................................................... 104 Scheme 60. .................................................................................................... 105

iii Scheme 61. .................................................................................................... 106 Scheme 62. .................................................................................................... 107 Scheme 63. .................................................................................................... 108 Scheme 64. .................................................................................................... 110 Scheme 65. .................................................................................................... 115

iv List of Figures Figure 1. ............................................................................................................ 4 Figure 2. ............................................................................................................ 5 Figure 3. ............................................................................................................ 6 Figure 4. .......................................................................................................... 25 Figure 5. .......................................................................................................... 29 Figure 6. .......................................................................................................... 31 Figure 7. .......................................................................................................... 32 Figure 8. .......................................................................................................... 32 Figure 9. .......................................................................................................... 37 Figure 10. ........................................................................................................ 45 Figure 11. ........................................................................................................ 51 Figure 12. ........................................................................................................ 53 Figure 13. ........................................................................................................ 54 Figure 14. ........................................................................................................ 55 Figure 15. ........................................................................................................ 57 Figure 16. ........................................................................................................ 59 Figure 17. ........................................................................................................ 61 Figure 18. ........................................................................................................ 62 Figure 19. ........................................................................................................ 65 Figure 20. ........................................................................................................ 73 Figure 21. ........................................................................................................ 74 Figure 22. ........................................................................................................ 77 Figure 23. ........................................................................................................ 79 Figure 24. ........................................................................................................ 91 Figure 25. ........................................................................................................ 93 Figure 26. ........................................................................................................ 95 Figure 27. ........................................................................................................ 96 Figure 28. ........................................................................................................ 98 Figure 29. ........................................................................................................ 99 Figure 30. ...................................................................................................... 109

v Figure 31. ...................................................................................................... 112 Figure 32. ...................................................................................................... 113 Figure 33. ...................................................................................................... 114

vi List of Tables Table 1. ........................................................................................................... 35 Table 2. ........................................................................................................... 38 Table 3. ........................................................................................................... 40 Table 4. ......................................................................................................... 184 Table 5. ......................................................................................................... 186 Table 6. ......................................................................................................... 187

vii Abbreviations and Acronyms Ac

Acetyl

AIBN

Azobisisobutyronitrile

All

Allyl

Alloc

Allyloxycarbonyl

aq.

Aqueous

Ar

Aryl

BAIB

[Bis(acetoxy)iodo]benzene

Bn

Benzyl

Bu

Butyl

t-Bu

tert-Butyl

Bz

Benzoyl

Cbz

Carboxybenzyl

DBU

1,8-Diazabicyclo[5.4.0]undec-7-ene

DCC

Dicyclohexylcarbodiimide

DCM

Dichloromethane

DEPT

Distortionless Enhancement by Polarization Transfer

DIAD

Diisopropyl azodicarboxylate

DIB

Iodobenzene diacetate

DIPEA

N,N-Diisopropylethylamine

DMAP

4-Dimethylaminopyridine

DMF

Dimethylformamide

DMSO

Dimethylsulfoxide

DMTST

Dimethyl(methylthio)sulfonium triflate

DPSO

Diphenylsulfoxide

EDCI

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Et

Ethyl

GlcNAc

N-Acetyl--D-glucosamine

GlcNAcA

N-Acetyl--D-glucosaminuronic acid

viii HATU

2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate Methanaminium

HBTU

O-Benzotriazole-N,N,N’,N’-tetramethyl-uroniumhexafluorophosphate

HOBt

1-Hydroxy-benzotriazole

HPLC

High Performance Liquid Chromatography

i-Pr

isoPropyl

IR

Infra Red

MBz

para-Methoxybenzoyl

Me

Methyl

MRP2

Multidrug Resistance Protein 2

MW

Microwave

NBS

N-Bromosuccinimide

NIS

N-Iodosuccinimide

NMM

N-Methylmorpholine

NMR

Nuclear Magnetic Resonance

NOE

Nuclear Overhouser Enhancement

P

Protecting group

PDC

Pyridinium dichromate

Ph

Phenyl

Phth

Phthaloyl

Piv

Pivaloyl

PMB

para-Methoxybenzyl

Py

Pyridine

Rf

Retention factor

ROE

Rotating-frame Overhauser Enhancement

rt

Room temperature

TBAHS

Tetrabutylammonium hydrogen sulfate

TBS

tert-Butyldimethylsilyl

ix TEA

Triethylamine

TEMPO

2,2,6,6-Tetramethylpiperidine-1-oxyl

Tf

Triflouromethanesulfonate (Triflate)

TfOH

Triflouromethanesulfonic acid

TFA

Triflouroacetic acid

THF

Tetrahydrofuran

TIPS

Tetraisopropyldisiloxyl

TMS

Trimethylsilyl

TPS

Triisopropylsilyl

Tr

Trityl

Ts

para-Toluenesulfonyl (Tosyl)

p-TsOH

para-Toluenesulfonic acid

UDP

Uridine diphosphate

x Contents Summary ....................................................................................................... xii Introduction ..................................................................................................... 1 1. Preface ..........................................................................................................1 2. D-Glucuronic Acid Mimetic: Occurrence and Biological Aspects .................. 2 2.1. O-, N- and S-glucuronides .......................................................................2 2.2. Nucleosides and Aza-Sugars ................................................................... 4 2.3. 4,5-Unsaturated Derivatives ....................................................................5 3. Chemical Preparation of D-glucuronic acid Derivatives ................................. 7 3.1. Oxidative Methods .................................................................................. 7 3.1.1. Platinum Group / O2 .........................................................................7 3.1.2. Chromium Reagents .........................................................................8 3.1.3. TEMPO-Catalyzed Oxidations ......................................................... 9 3.1.4. Two-Step Oxidation Protocols........................................................ 11 3.1.5. Fortes Protocol on 6-phenylthio-D-glucose .................................... 12 3.2. Large Scale Preparation ........................................................................ 13 4. Chemistry of D-Glucuronic Acid and Derivatives ........................................ 14 4.1. Estrification .......................................................................................... 14 4.1.1. Esterification of Carboxylic Acid Group ........................................ 14 4.1.2. Esterification of Hydroxyl Groups .................................................. 18 4.2. Etherification ........................................................................................ 19 4.3. Lactonization ........................................................................................ 22 4.4. Elimination ........................................................................................... 24 4.4.1. Δ1,2 Elimination .............................................................................. 24 4.4.2. Δ4,5 Elimination .............................................................................. 24 4.5. Decarboxylation.................................................................................... 25 4.6. Isomerization ........................................................................................ 28 4.7. Glucuronidation .................................................................................... 32 4.7.1. Alkyl and Aryl glucuronides .......................................................... 33 4.7.2. Acyl glucuronides .......................................................................... 45 4.7.3. 1,2-Cis-glucuronides ...................................................................... 48

xi 5. Glycosyl amides .......................................................................................... 51 5.1. N-Glycosyl amides ................................................................................ 51 5.2. C-Glycosyl amides ................................................................................ 56 5.3. Sugar Lactams ...................................................................................... 68 Results and Discussion .................................................................................. 71 Experimental Section .................................................................................. 116 Spectral Data ............................................................................................... 131 Antitumor Activity ...................................................................................... 181 Biological Activity Results and Disscusion ................................................ 188 Structure-Activity Relationship (SAR) ....................................................... 191 References .................................................................................................... 193 Arabic Summary

SUMMARY

SUMMARY

xii

Summary Studies were initiated by acetylation of the commercially available Dglucuronic acid with acetic anhydride and catalytic amounts of iodine as an acetyl transfer reagent. Compound 1 was subjected to reaction with water to afford the corresponding 1,2,3,4-tetra-O-acetyl--D-glucuronic acid 2 in 99% yield (Scheme I).

Scheme I. When compound 2 was left to crystallize, the 1H NMR of the crystals formed indicated an -anomeric configuration. Involvement of one of the two oxycarbenium ions (3 or 4) could give an explanation for such anomerization.

SUMMARY

xiii

Compound 1 was subjected to the reaction with an appropriate amine solely in

anhydrous

DCM

which

furnished,

after

extractive

work-up

and

chromatographic purification, the per-O-acetylated secondary amides 5-12 in moderate yields (Scheme II).

Scheme II. On the other hand, the reaction of the fully acetylated mixed anhydride 1 with 4-aminouracil or its N,N-dimethyl derivative under the previously established conditions revealed no formation of the amide products (13a,b) even after changing the reaction solvent to acetonitrile (Scheme III).

SUMMARY

xiv

Scheme III. Next, subsequent removal of acetyl protecting groups was found to be essential. Base catalyzed saponification of each of the glucuronamide derivatives 5-12 were achieved at pH 13 using 0.05 M LiOH to afford the products 14-21 in an equilibrium anomeric mixture and the  ratio was determined to be 1:1. Also, removal of acetyl groups using Zemplén’s conditions was tried on the N-allyl derivative and it revealed the same result with approximately equal yield (Scheme IV).

Scheme IV.

SUMMARY

xv

Next, the attention was shifted to the synthesis of analogues to the 2,3unsaturated sialic acid,

5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-

galacto-non-2-enonic acid (Neu5Ac2en), which is found in free form in nature. A straight forward one pot procedure was used starting from acid 2 in which, in addition to the amide bond formation, a base-promoted elimination of acetic acid was achieved. The coupling of 2 with an amine in the presence of ethyl chloroformate and TEA at -20 oC gave, after extractive work-up, the desired 4,5unsaturated amides (22-27) along with the non-elimination products which were also identified by comparing their TLC mobilities with the previously synthesized. Fortunately, these products could be separated by a slow silica gel column chromatography (Scheme V).

Scheme V. Interestingly, the attempt to use 2-aminopyrazine as the amine in this reaction gave a single reaction product as indicated by TLC, but spectral data inspection indicated that the reaction did not give the desired N-(pyrazin-2-yl)-

SUMMARY

xvi

1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (28), but instead it gave ethyl 1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronate (29) (Scheme VI).

Scheme VI. The focus was shifted to find an alternative route for the preparation of N(pyrazin-2-yl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide.

Alternatively,

a solution of the readily synthesized N-(pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl-βD-glucopyran-uronamide (9) in anhydrous DCM at 0 oC was treated with DBU (Scheme VII).

xvii

SUMMARY

Scheme VII. The DBU promoted elimination procedure was also tried for the preparation of 4,5-unsaturated compounds 22-27 from the corresponding 1,2,3,4-tetra-Oacetyl-β-D-glucopyranuronamides and it successfully yielded the desired products in moderate yields except for N-(1H-benzimidazol-2-yl)-1,2,3,4-tetraO-acetyl--D-glucuronamide (8) in which the reaction suffered from series of complications (Scheme VIII).

Scheme VIII. Next, the attention was shifted to the synthesis of a series of N-acyl-N-arylD-glucopyranuronosyl amine derivatives. The retrosynthetic analysis on which the synthetic strategy was based is outlined in the following scheme.

SUMMARY

xviii

Scheme IX. After refluxing the acid 2 in DCM with thionyl chloride for 2 h, TLC analysis of the reaction mixture identified the formation of a material that was more mobile than the starting material which was completely consumed. Direct treatment of the generated acid chloride with allyl alcohol in presence of pyridine for 1 h followed by extractive work-up furnished the allyl ester substrate 31 in 98% yield (Scheme X).

xix

SUMMARY

Scheme X. Next, a regioselective removal of the anomeric O-acetyl group was carefully performed with benzyl amine in THF which is mild enough to leave the base sensitive allyl ester unscathed. TLC analysis of the reaction mixture, after 19 h at ambient temperature, identified the formation of a material that was less mobile than the starting material 31. After silica gel purification, the C1hemiacetal 32 was obtained in a moderate yield (52%) (Scheme XI).

SUMMARY

xx

Scheme XI. In the reaction with para-flouroaniline, a pure -anomer was obtained after silica gel purification.

while, in the case of para-bromoaniline, the ring reforming proceeds on both the si- and re-faces of the imino group, consequently, a mixture of - and -Nglycosides was obtained in 70% overall yield. The anomeric ratio was 1:2 as indicated from the signal integration in the 1H NMR spectrum. Anomers were

SUMMARY

xxi

separated on a reversed phase silica gel column using H 2O-MeOH (20%) as an eluant and identified.

The formed N-glycoside was subjected to acylation either by reacting with acyl chlorides or by coupling with carboxylic acids using DCC as a carboxylic group activator, but all these attempts were unsuccessful. Further, methylation was also tried using MeI in presence of pyridine, TEA and NaH separately, but also all these attempts were unsuccessful (Scheme XII).

Scheme XII.

INTRODUCTION

INTRODUCTION

1

Introduction 1. Preface Carbohydrates or saccharides are the most abundant group of natural compounds found in nature, as well as their glycoconjugates. They are involved in such important function as cellcell recognition & communication, inflammation, immunological response, bacterial and viral infection, tumor genesis and metastasis (Bechthold & Fernández, 1999; Dwek, 1996; Feizi, 1993; Karlsson, 1991; Sharon & Lis, 1993; Sofia, 1998; Varki, 1993; WeymouthWilson, 1997; Zhang et al., 1999). Also, the saccharide portions of various classes of natural products function as a key molecular recognition elements important to the biological properties of the natural compound (Kahne, 1997; Sofia, 1998; Weymouth-Wilson, 1997). Our understanding to the relationship between carbohydrate structure and biological function is still far behind that of proteins and nucleic acids. Initially, carbohydrates were only recognized as structural and energy storage molecules (e.g. cellulose, chitin, starch and glycogen). The development of novel tools to study carbohydrate function has permitted additional biological functions to be elucidated. Their roles in protein folding, cell signaling, fertilization, pathogen binding to host tissue, leukocyte trafficking and associated inflammatory responses, tumor cell metastasis, and regulation of hormone and enzyme activities are just a few selected examples. These findings led to an immense interest in the preparation of oligosaccharides and glycoconjugates as well as the understanding of their interaction with their natural receptors (Ernst et al., 2000). The tremendous medicinal potential of glycol structures has already been acknowledged by the development of synthetic carbohydrates based vaccines and therapeutics. The elucidation of the mechanisms of carbohydrate involvement in disease progression would be further improved if we could rely

INTRODUCTION

2

on the detailed knowledge of the structure, conformation and properties of the carbohydrate molecule. Therefore, the development of effective methods for the synthesis of complex carbohydrates, including solid phase synthesis (Plante et al., 2001), and methods for enzymatic synthesis (Chen et al., 2002), has become critical for the field of glycoscience in both basic research and pharmacological interest. 2. D-Glucuronic Acid Mimetic: Occurrence and Biological Aspects Of the three naturally occurring hexuronic acids, the compound name known as D-glucuronic acid appears to be by far the most widely distributed. It has not been found free in nature, except possibly in small amounts in blood and urine, but it occurs conjugated to wide variety of molecules. The importance of Dglucuronic acid depends not only on its relationship to carbohydrates, but also on the fact that it plays an important part in the metabolism of many types of organic compounds (Bray, 1953). 2.1. O-, N- and S-glucuronides Conjugation with D-glucuronic acid (Glucuronidation) is considered a fundamental mechanism in nature for eliminating and detoxifying lipophilic drug molecules, toxic substances or other substances that cannot be used as energy source from the body (Smith, 1966). Glucuronides are any substance produced by linking D-glucuronic acid to another substance via glycosidic bond (Scheme 1).

Scheme 1. Natural glucuronidation.

INTRODUCTION

3

Natural glucuronidation takes place in presence of UDP-glucuronosyltransferases (UGT) enzymes, that are present in hepatic and extra-hepatic tissues in all animal species (Burchell et al., 1995; Dutton, 1980; Dutton & Burchell, 1977; Mulder et al., 1990) and catalyzes the conjugation of the compounds possessing a nucleophilic acceptor group (-OH, -COOH, -NH2, -NHR, -NR2 and -SH groups) with D-glucuronic acid, a relatively bulky hydrophilic moiety, whose carboxylic acid functional group is ionized at physiological pH, thus forming metabolites with significantly different chemical and biochemical properties to the parent aglycon and, in most of cases, with significantly decreased affinity for the receptor or enzymes responsible for the biological activity of the parent compound. In addition, due to the presence of the anionic glucuronic acid moiety, glucuronide conjugates are substrates for the human MRP2 (rodent Mrp2) efflux transporter localized on hepatic canalicular membranes and renal tubular brush border membranes, facilitating their transport into bile and urine, respectively. Due to their hydrophilicity, glucuronide conjugates are unlikely to be passively reabsorbed into the epithelial cells lining the biliary or urinary tracts, thus glucuronidation coupled with transporter-mediated efflux (and renal glomerular filtration) facilitates the excretion of endobiotics and xenobiotics from the body (Sallustio, 2008). Glucuronide prodrugs are widely used in Cancer Targeted Enzyme Prodrug Therapy. Glucronides of drug molecules having a -configuration are substrates for -glucuronidase enzyme. For better selectively and efficacy with reduction in systematic toxicity of cancer chemotherapy, -glucuronidase enzyme, that are selectively expressed at the tumor area, is used for conversion of relatively nontoxic glucuronide prodrugs into the corresponding parent cytotoxic agents (Graaf et al., 2002).

INTRODUCTION

4

2.2. Nucleosides and Aza-Sugars A variety of natural nucleosides were isolated with the carbohydrate moiety is a D-glucuronic acid derivative (Timoshchuk, 1995) (Figure 1). Gougerotin, Bagougeramines A & B, and Blasticidin C are nucleoside antibiotics which exhibit a wide spectrum of biological activities including antibacterial and antiviral activities by inhibition of peptide bond formation. They also exhibit an antitumor activities (Collins, 2006).

Name

R

R1

Gougerotin

Bagougeramine A

Bagougeramine B

Figure 1. Natural nucleoside antibiotics containing a D-glucuronic acid derivative.

INTRODUCTION

5

A series of nucleosides which contain a D-glucuronic acid group were synthesized including Purine (Kishikawa & Yuki, 1966b), Pyrimidine (Kishikawa et al., 1966a), Benzimidazole (Kishikawa, 1969), Uracil and Cytosine nucleosides (Kishikawa & Yuki, 1964). D-Glucuronic acid mimicking aza-sugars have been isolated as well as synthesized (Kim et al., 2000; Nishimura et al., 1996; Nishimura et al., 2000; Pandey & Kapur, 2002; Xie et al., 2004; Yoshimra et al., 2008; Zhao et al., 2001). 5-Aza-D-glucuronic acid (35) is a natural product isolated from the seeds of Baphia racemosa and found to be a specific inhibitor of human liver -Dglucuronidase (Manning et al., 1985) and, recently, Cipolla et al. (Cipolla et al., 2007) succeeded to prepare its bicyclic analogue (36). Examples for iminosugars derived from D-glucuronic acid as potential anticancer agents are illustrated in Figure 2 (Compain & Martin, 2007).

Figure 2. Iminosugars derived from D-glucuronic acid. 2.3. 4,5-Unsaturated Derivatives The 2,3-dehydro-silaic acids, widely distributed in nature (Schukla et al., 1983), are essentially a -C-glycoside of a ∆4,5-D-glucopyranosiduronic acid.

INTRODUCTION

6

The substituent at C-5 of the sialic acid corresponds to the C-2 substituent of the ∆4,5-D-glucuronic acid.

Figure 3. 4,5-unsaturated derivatives of D-glucuronic acid. 2,3-Dehydro-2-deoxy-sialic acids Neu5Ac2en, Neu5Gc2en, Kdn2en and their acetates, were found to inhibit both influenza virus and cholera microbe sialidases. Neu5Ac2en has been widely used as a base template in studies aimed for developing more potent neuraminidase inhibitors for the treatment of influenza viruses A and B. Zanamivir, designed and synthesized in von Itzstein laboratories, developed by GalxoSmithKline and Biota, and sold under the tradename Relenza®, is administrated as a nasal spray. The structure is based on Neu5Ac2en with the natural C-4 hydroxyl group replaced by a guanidino substituent (von Itzstein et al., 1994; von Itzstein et al., 1993). Oseltamivir is marketed by Roche and sold under the tradename of Tamiflu®. The amine substituent on the cyclohexene ring of oseltamivir is in the same position as the natural C-4 hydroxyl group of Neu5Ac2en and the glycerol

INTRODUCTION

7

side chain of Neu5Ac2en has been replaced with hydrophobic 3-pentyl ether. The result is a more lipophilic and orally bioavaliable inhibitor. Laninamivir is a neuraminidase inhibitor which is currently in Phase III clinical trials (Hayden, 2009; Yamashita et al., 2009). 3. Chemical Preparation of D-Glucuronic Acid Derivatives 3.1. Oxidative Methods The current most effective chemical method for the preparation of Dglucuronic acid or its derivatives depends upon oxidation of the primary hydroxyl group at C-6 of the corresponding aldose by catalytic or non-catalytic means. 3.1.1. Platinum Group / O2 The use of oxygen in the presence of metals of the platinum group (Pt/C (Mehltretter, 1951; Mehltretter, 1961; Mehltretter et al., 1951), Pd/C, Palladium boride on carbon (Csuros et al., 1974a; Csuros et al., 1974b) were used for selective oxidation of carbohydrates (Edye et al., 1991; Edye et al., 1994) and particularly in the synthesis of uronic acids. Li & Bugg (Li & Bugg, 2004) have achieved the oxidation of UDP-GlcNAc to the corresponding uronic acid using platinum and molecular oxygen, while Rejzek et al. (Rejzek et al., 2007) succeeded to oxidize UDP-GlcNAc by the reduced Adam's catalyst and molecular oxygen in water containing NaHCO3 (Scheme 2).

INTRODUCTION

8

Scheme 2. Example for Pt, O2 oxidation to corresponding uronic acid. 3.1.2. Chromium Reagents When acid labile protecting groups are not present in the substrate, the Jones oxidation (chromium(VI) oxide, sulfuric acid) can be applied for oxidation. For example, the Jones oxidation of methyl and allyl 2,3,4-tri-O-benzyl-α-Dglucopyranoside occurs with 2 equiv. of reagent to give the uronic acids in good yields (Jarosz, 1988; van Boeckel et al., 1985). Cleavage of acid labile protecting groups during the reaction can, in some cases, be an advantage. Worthy of note is the direct oxidation of trityl ether 37 to the corresponding uronic acid derivative 38 (Scheme 3) (Schell et al., 2001).

INTRODUCTION

9

Scheme 3. Direct oxidation of trityl ether. Another chromium(VI) oxidant is pyridinium dichromate (PDC) (Luzzio, 1998). Acetals and sulfides are stable under these conditions (Halkes et al., 1998; Westman & Nilsson, 1995). If tert-butanol is added to the reaction, the tert-butyl ester can be obtained directly as shown by the conversion of 39 into 40 (Nilsson et al., 1993) (Scheme 4).

Scheme 4. Formation of tert-butyl ester. 3.1.3. TEMPO-Catalyzed Oxidations Recently, the important TEMPO reagent has been introduced for selective oxidation of the primary hydroxyl group (De Souza, 2006). As an example, Mann et al.(Mann et al., 2006b) reported the synthesis of pivaloylated glucosaminuronate from glucosamine in 4 steps followed by oxidation of pivaloylated glucosamine 41 using the TEMPO oxidation approach (Scheme 5).

INTRODUCTION

10

Scheme 5. Example for oxidation using the TEMPO oxidation approach. A variety of partially protected saccharide derivatives have been successfully converted into the corresponding uronate derivatives using the TEMPO-KBr-NaOCl or TEMPO-KBr-Ca(OCl)2 oxidation systems (Lin et al., 2004; Rejzek et al., 2007; Ying & Gervay-Hague, 2003). A draw back with the TEMPO procedure is the need for several inorganic salts in the reaction mixture that can be difficult to remove in the work-up. This can be circumvented by using BAIB as the stoichiometric oxidant (Fraser-Reid et al., 2008). Selected literature examples are illustrated in Scheme 6 (Codée et al., 2005; Dinkelaar et al., 2009; Migawa et al., 2005; van den Bos et al., 2004; van den Bos et al., 2005)

INTRODUCTION

11

Scheme 6. Selected literature examples representing TEMPO/BAIB oxidations. 3.1.4. Two-Step Oxidation Protocols In some cases, a two-step protocol is a milder procedure for oxidation of a primary alcohol to a carboxylic acid. The first step is then oxidation of the alcohol to the aldehyde which can be achieved through Swern oxidation (Oscarson & Svahnberg, 2001), Pfitzner–Moffatt oxidation (Garegg et al., 1995; Oscarson et al., 2001) or Dess–Martin periodinane oxidation (Chambers et al., 2005) (Scheme 7).

12

INTRODUCTION

Scheme 7. Selected literature examples representing two step oxidations. 3.1.5. Fortes Protocol on 6-phenylthio-D-glucose A novel and effective procedure for the preparation of glucuronides has been developed (Yu et al., 2000; Yu et al., 2001) which employs 6-S-phenylhexopyranosides as precursors (Scheme 8).

INTRODUCTION

13

Scheme 8. Fortes protocol. 3.2. Large Scale Preparation The most recent method claimed for industrial preparation, by which Dglucuronic acid and/or D-glucuronolactone can be produced in high yield, is by oxidation of sucrose to give sucrose carboxylic acid by conventional methods, thereof, yeast is added so as to hydrolyze fructose residue and to assimilate the resulting product, and thus D-glucuronic acid and/or D-glucuronolactone is/are collected (Hamayasu et al., 2009) (Scheme 9).

Scheme 9. Large scale preparation.

INTRODUCTION

14

4. Chemistry of D-Glucuronic Acid and Derivatives 4.1. Estrification When applied to D-glucuronic acid, esterification may imply either combination of its carboxylic acid group with an alcohol, or esterification of the hydroxyl groups of the molecule. Esters of the former are usually referred to glucuronates. 4.1.1. Esterification of Carboxylic Acid Group A variety of methods were reported for esterification of the carboxyl group of D-glucuronic acid derivatives. These include treatment with methanolic hydrogen chloride (Moppett et al., 1971), methyl iodide and Na2CO3 in DMF (Lin et al., 2004), esterification with diazomethane (Kondo et al., 1972; Schmidt & Rucker, 1980) and its less-explosive replacement trimethylsilyldiazomethane. A common method for preparation of methyl D-glucuronate is by the esterification of D-glucuronolactone at room temperature in the presence of basic catalysts such as sodium hydroxide, sodium methoxide, triethylamine or anion exchange resins (Bollenback et al., 1955; Touster & Reynolds, 1952) (Scheme 10).

Scheme 10. Esterification from D-glucuronolactone. The 6,3-lactone reacts readily with methyl and allyl alcohols to give the methyl and allyl esters, respectively (Tosin et al., 2005a; Tosin & Murphy, 2005b) (Scheme 11).

INTRODUCTION

15

Scheme 11. Rat et al. (Rat et al., 2007) reported a method for obtaining methyl glucopyranuronate through 6,1-lactone opening under microwave irradiation in methanol and a catalytic amount of p-TsOH. On the other hand, the use of FeCl3 as promoter led to methyl ester 42 in 98% yields in short time (2 minutes). They prepared the butyl, octyl and dodecyl esters by the same method (Scheme 12).

Scheme 12. Methyl (Tosin & Murphy, 2002) and benzyl (Tietze et al., 2008) esters with fully acetylated hydroxyl groups glucuronate were obtained by stirring of the mixed anhydride 1 with methyl or benzyl alcohol, respectively (Scheme 13).

INTRODUCTION

16

Scheme 13. Trans-esterification procedure was used by Magnet et al. (Mignet et al., 1997) for the preparation of 1-chloro ethyl ester using Ti(OEt)4 in toluene. Bouvier et al. (Bouvier et al., 2003) also succeeded to prepare the benzyl ester either by the coupling between the carboxylic functional group and benzyl alcohol using DCC (Scheme 14a) or by trans-esterification of the methyl glucuronate 43 (Scheme 14b).

Scheme 14. Preparation of benzyl ester.

INTRODUCTION

17

Starting from D-glucuronic acid, the allyl ester can be directly obtained by reaction with allyl bromide under basic conditions (DBU) (El-Alaoui et al., 2006) (Scheme 15a). On the other hand, the fully acetylated allyl ester can be prepared via the reaction of its acid chloride with allyl alcohol in the presence of pyridine (Scheme 15b) (Tosin et al., 2004; Tosin et al., 2005c).

Scheme 15. Preparation of the allyl ester.

INTRODUCTION

18

4.1.2. Esterification of Hydroxyl Groups

Scheme 16. The acetylation of methyl D-glucopyranuronate with acetic anhydride in the presence of perchloric acid, zinc chloride or pyridine gave mixtures of the methyl 1,2,3,4-tetra-O-acetyl-- and -D-glucopyranuronates (Bishop, 1953; Bollenback et al., 1955; Goebel & Babers, 1934) from which the β-anomer 44

INTRODUCTION

19

can be easily separated by crystallization. Similarly, treatment with pivaloyl chloride (Gärtner et al., 2003) or isobutyryl chloride (Brown et al., 2000; Stanford & Stachulski, 2007) in presence of pyridine and after recrystalization, the -tetra-O-esters were obtained in an excellent anomeric purity (Scheme 16a). Tosin & Murphy (Tosin et al., 2002) reported that the acetylation of Dglucuronic acid by acetic anhydride in the presence of iodine followed by treatment with methanol in one pot gives the methyl ester 44 rather than the carboxylic acid as reported (Lellam et al., 1998; Malkinson et al., 2000). They found that isolation of the pure -anhydride was possible after crystallization of the residue obtained from acetylation of glucuronic acid (Scheme 16b); this could be converted into the desired acid by reaction with water. On the same pattern, the D-glucuronamide was converted to its -tetra-Oacetyl derivative by treatment with acetic anhydride and pyridine (McMillan et al., 2006) (Scheme 16c), as well, methyl D-glucuronate was readily benzoylated by reaction with benzoyl chloride and pyridine which was assigned to the anomeric configuration (Zorbach & Valliaveedan, 1964) (Scheme 16d). Also, all the hydroxyl groups of allyl D-glucuronate were esterified by treatment with an excess of allyloxy carbonyl chloride and pyridine (El-Alaoui et al., 2006) (Scheme 16e). 4.2. Etherification Methylation of the anomeric position and esterification of the carboxylate in anhydrous methanol containing acetyl chloride was reported (Jansen & Jang, 1946; Owen et al., 1941) (Scheme 17). During this reaction, an equilibrium between methyl (methyl -D-glucopyranosid)uronate and methyl -Dglucofuranosidurono-6,3-lactone (Owen et al., 1941) was observed, that was shifted towards the ester by heating of the reaction mixture.

INTRODUCTION

20

Scheme 17. On the other hand, the most efficient way for obtaining the benzylated Dglucuronic acid derivative is by oxidation of the appropriate glucose derivative (El-Alaoui et al., 2006; Kovac et al., 1974; Schmidt et al., 1980). Falkowski et al. (Falkowski et al., 1980) reported the preparation of the glucuronate 45 by treatment with trimethylsilyl chloride and pyridene in formamide (Scheme 18a). Also, treatment of 46 with either TBS-Cl or TIPS-Cl and imidazole in DMF gave 47 and 48, respectively (Krog-Jensen & Oscarson, 1998) (Scheme 18b,c). Mignet et al. (Mignet et al., 1997) prepared the TBS ether 49 using the same procedure (Scheme 18d), while, Bouvier et al. prepared the TBS ethers 50 and 51 using TBSOTf and DMAP (Bouvier et al., 2003) (Scheme 18e,f).

INTRODUCTION

Scheme 18. Preparation of silyl ethers.

21

INTRODUCTION

22 4.3. Lactonization

Formation of lactones or intramolecular esters is one of the most recognized features exhibited by D-glucuronic acid. When isolated from aqueous solution, it crystallizes as monoclinic plates in the form of its -lactone (Dglucuronolactone) (Dutton, 1966). Tosin et al. (Tosin et al., 2002) isolated 1,6-lactone 52 while exploring the introduction of stable azide at the anomeric center. Later, Poláková et al. succeeded to obtain lactone 52 by an improved and shorter procedure (Poláková et al., 2004) (Scheme 19).

Scheme 19. The 1,6-lactone 52 was also prepared by Rat et al. through microwave irradiation of D-glucuronic acid in the presence of acetic anhydride and iodine (Rat et al., 2007) (Scheme 20).

Scheme 20.

INTRODUCTION

23

Reaction of 53 with NIS gives the lactone 54 which was subsequently reduced using Bu3SnH/AIBN to give the 2-deoxylactone 55 (Poláková et al., 2004) (Scheme 21).

Scheme 21. 6,3-Lactone 57 can be obtained by the saponification of 56 followed by reacetylation (Tosin et al., 2005a; Tosin et al., 2005c) (Scheme 22).

Scheme 22. D-Glucfuranosid-urono-6,3-lactone 58 was obtained directly from Dglucuronic acid by microwave activation and best results were obtained while using p-TsOH (Rat et al., 2007) (Scheme 23).

INTRODUCTION

24

Scheme 23. 4.4. Elimination 4.4.1. Δ1,2 Elimination Reductive elimination of the protected 1-bromo sugar using zinc-vitamin B12 mixture yielded the Δ1,2 elimination product. (Forbes & Franck, 1999; Schell et al., 2001; Stanford et al., 2007). Also, the treatment with zinc dust, copper(II) sulfate and sodium acetate in aqueous acetic acid was successful (Durham & Miller, 2002; Poláková et al., 2004) (Scheme 24).

Scheme 24. Reductive elimination of the protected 1-bromo sugar. 4.4.2. Δ4,5 Elimination Adamczyk et al. (Adamczyk et al., 1992) noted that HPLC analysis of both commercial and synthetic estriol 16-glucuronide revealed two components. The minor component was tentatively assigned as a Δ4,5-glucuronide, and indeed when the precursor ester was treated with DBU in THF the -unsaturated ester 59 resulted in 79% yield (Figure 4); hydrolysis afforded the fully characterized Δ4,5-glucuronide, identical with the HPLC impurity.

INTRODUCTION

25

Figure 4. 4,5-Unsaturated estriol 16-glucuronide. Numerous bases could affect the Δ4,5 elimination but The use of DBU in DCM became the most popular method among the literature (Mann et al., 2006a; Murphy et al., 2002; Oscarson et al., 2001; Pitt et al., 2004; Stanford et al., 2007). 4.5. Decarboxylation Lefèvre & Tollens (Lefèvre & Tollens, 1907) observed that with concentrated hydrochloric acid, rapid and stoichiometric decarboxylation of Dglucuronic acid occurs. It is widely used for quantitative estimation of uronic acids. The use of hydriodic acid as an alternative was later described (Anderson et al., 1963). Perlin (Perlin, 1951) and Anderson et al. (Anderson et al., 1962) proposed that, thermal decarboxylation of D-glucuronic acid may be achieved by heating under reflux at the desired reaction temperature (diphenyl ether, 255 o

C; methyl phthalate, 280 oC). The uronoside structure also facilitates the reactions of the Hofer-Moest type

(also known as anodic decarboxylation or oxidative decarboxylation) (Stapley & BeMiller, 2006). Scheme 25 presents the mechanism as applied to Dglucuronoside (I) to produce (III) (xylo-pentodialose), where h+ symbolizes an anode.

INTRODUCTION

26

Scheme 25. Hofer-Moest decarboxylation of a D-glucuronopyranoside. Kitagawa & Yoshikawa (Kitagawa & Yoshikawa, 1977) discussed the anodic

decarboxylation

of

2,3,4-tri-O-methyl-D-glucuronopyranosides

in

methanol. Their reaction products and proposed mechanism are consistent with scheme 26 (R1 = Me).

INTRODUCTION

27

Scheme 26. Francisco et al. (Francisco et al., 1997) chemically decarboxylated Dglucuronic acid derivative 60 to give the D-xylo-pentodialdose 61 by treating with DIB and iodine (Scheme 27).

Scheme 27. Kitagawa et al. (Kitagawa et al., 1980; Kitagawa et al., 1981a; Kitagawa et al., 1981b) oxidized D-glucuronic acid in methanol anodically and obtained V, which has used as a synthetic intermediate (Scheme 28).

INTRODUCTION

28

Scheme 28. Methanolic Hofer-Moest decarboxylation of D-glucuronic acid. 4.6. Isomerization Siddiqui & Purves (Siddiqui & Purves, 1963) found that glucuronic acid was isomerized to a keto acid when treated with aqueous sodium hydroxide at room temperature. Fisher & Schmidt (Fischer & Schmidt, 1959) found that uronic acids were readily isomerized even at pH 7 (at 100oC) and reported that Dglucuronic acid formed mainly L-iduronic acid (by C-5 epimerization). In connection with studies of uronic acids (Nelson & Samuelson, 1968), it was observed that the isomerization of D-glucuronic acid at pH 7 resulted in a complex mixture of acids with D-lyxo-5-hexulosonic acid (5-keto-D-mannonic acid or 5-keto-L-gulonic acid) as the main reaction product. Later, the isomerization products from D-glucuronic acid in aqueous solution of pH 7 at 100 oC and 110 oC have been separated by anion exchange chromatography and identified (Carlsson & Samuelson, 1969). The predominant product was D-lyxo-5-hexulosonic acid and the others were L-ribo-5hexulosonic, D-mannuronic, D-altruronic and D-alluronic acids (the amounts of which decreased in the order given, Figure 5). L-Iduronic acid, previously stated as the main product by these conditions (Fischer et al., 1959), was not detected.

INTRODUCTION

29

Figure 5. Isomerization products from D-glucuronic acid in aqueous solution of pH 7 at 100 oC and 110 oC. On the other hand, inversion of the D-gluco- to the L-ido- series is of value, since L-iduronic acid is a constituent of some important natural products, but commercially very expensive. An ingenious approach by Sinaÿ, leading directly to a protected form of L-iduronic acid useful in synthesis, again takes advantage of the distinctive C-5 reactivity of glucuronic acid derivatives. In addition to the stabilization of C-5 anion, C-5 radicals are stabilized through capodative effect. Thus photobromination of ester 44, using NBS (Ferrier & Furneaux, 1977) or (better) bromine and a heat lamp (Blattner et al., 1980), affords the fairly stable 5-bromo derivative 62 in up to 89% yield. Radical reduction of 62 using tri-nbutyl tin hydride (Chiba & Sinaÿ, 1986; 2004) affords a mixture of 44 and the desired ido-product 63; though yield of 63 is modest, it may readily be separated on a multigram scale (Scheme 29).

INTRODUCTION

30

Scheme 29. The observation that base-catalyzed epimerization of D-glucuronic acid glycal 64 results mainly in the formation of L-iduronic acid glycal 67 reported by Thiem & Ossowski (Thiem & Ossowski, 1984), prompted Schell & coworkers (Schell et al., 2001) to investigate if D-glucuronic acid glycals could serve as key intermediates for the preparation of L-iduronic acid building blocks. Exploitation of such an epimerization strategy would allow ready access to these otherwise cumbersome to prepare differentially protected synthons. Treatment of protected glycols 65 and 66 with low concentration of sodium methoxide for short reaction times and separation of the mixture provided pure L-iduronic acid glycals 68 and 69, respectively (Scheme 30).

INTRODUCTION

31

Scheme 30. Recently, O'Brien et al. (O'Brien et al., 2007) described a conceivable pathway for the formation of 1,2-Cis-glycosides from D-glucuronic acid through anomerization using SnCl4 as a promoter (Figure 6).

Figure 6. Later, synthesis of closely related compounds to bacterial glycolipids containing -glucuronic acid residues was described where anomerization reaction is a key step. Very high stereoselectivites (>97:3 in favor of ) were observed from O-glycoside precursors (Pilgrim & Murphy, 2009) (Figure 7).

INTRODUCTION

32

Figure 7. 4.7. Glucuronidation Increased understanding of metabolic processes has led to a growing appreciation of the role and significance of phase II metabolites, glucuronides in particular. Glucuronides may also have significant biological activity in their own right. Morphine-6-glucuronide (70) is a very significant human metabolite of morphine. Not only, its analgesic activity is superior to that of morphine, but also it shows markedly reduced side effects (toxicity, nausea, respiratory depression and addiction) compared with the parent (Hidetoshi, 1969).

Figure 8. Morphine-6-glucuronide. Efficient methods of glucuronide synthesis are of considerable importance. O-Glucuronides may conveniently be divided into three classes, aryl, alkyl and

INTRODUCTION

33

acyl. In general, it may be said that the same synthetic methods are likely to be viable for both aryl and alkyl glucuronides. 4.7.1. Alkyl and Aryl glucuronides A clear majority of all glucuronides have been synthesized via acyl protected intermediates: the preparations of those most often used for coupling are summarized in the following schemes.

Scheme 31. Intermediates in glucuronidation, ester series. The protected precursor 44 may be converted to -bromo sugar 71. Both 44 and 71 are suitable precursors of the 1-hydroxy sugar 42. This hemiacetal is itself a useful glycosyl donor or it may be converted to the trichloroacetimidate 72 (Scheme 31).

INTRODUCTION

34

Scheme 32. Intermediates in glucuronidation, ether series. Hydrolysis of the glycosidic linkage of the fully protected glucuronic acid derivative 73 afforded the key 1-hydroxy sugar 74 which could be transformed into halo sugars 75 (Schmidt et al., 1980) or the imidate 76 (Schmidt & Grundler, 1981) (Scheme 32). 4.7.1.1. Bromo-Sugar Coupling (Koenigs-Knorr and other methods) The 1-bromo-sugar 38 (Bollenback et al., 1955; Bowering & Timell, 1960) is probably still the most popular glucuronidation intermediate and is commercially available. Its ‘highly unstable’ reputation is not entirely justified, but it must be kept dry: it may be stored for extended periods under desiccation at -20 oC. The Koenigs-Knorr method remains the most popular one for the synthesis of a wide range of alkyl and aryl glucuronides. Catalysts used are typically. AgI salts, especially Ag2CO3, AgClO4, or Ag2O. Of other used catalysts, AgOTf, Ag-Zeolite, Hg(CN)2, ZnBr2, and CdCO3. For easy reference, the following table lists examples for glucuronides prepared by this method.

INTRODUCTION

35

Table 1. Examples for glucuronides prepared by the Koenigs-Knorr method illustrating the promoter used.

Entry

aglycon

Promoter

Ref.

1

Ag2CO3

(Alonen et al., 2009)

2

Ag2CO3, Ag-Zeolite

(del Ruiz Ruiz et al., 2009)

3

Ag2CO3

(Marwah et al., 2001)

4

Ag2O

(Kamal et al., 2008)

5

AgOTf

(Arewång et al., 2007)

6

AgOTf

(Arewång et al., 2007)

7

Ag2O

(Engstrom et al., 2007)

36

INTRODUCTION

8

Ag2O

(Needs & Kroon, 2006)

9

Ag2O

(Rivault et al., 2004)

10

AgOTf, Ag2CO3

(Wang et al., 2003)

11

Hg(CN)2

(Gärtner et al., 2003)

12

ZnBr2

(Rukhman et al., 2001)

13

Ag2CO3, Quinoline

(Jones et al., 2009)

Murphy et al. (Murphy et al., 2002; Pitt et al., 2004) prepared a diverse of O-glucuronides (Figure 9) by reacting either the -bromo sugar with the hydroxyl group of the aglycons in presence of both AgCO3 and AgClO4 or by reacting the trichloroacetimidate (Section 3.7.1.4) with the hydroxyl group of the aglycons in the presence of BF3.OEt2.

INTRODUCTION

37

Figure 9. An alternative technique recently emerged employs glucuronidation using the phase transfer procedure described earlier for phenols (Hongu et al., 1999). The following table contains some glucuronidation examples using this method.

INTRODUCTION

38

Table 2. Glucuronidation examples using the phase transfer procedure.

Entry

aglycon

Conditions

Ref.

1

K2CO3, Bu4NBr, DCM, H2O, reflux

(Li et al., 2009)

2

Bu4NBr , K2CO3, CHCl3, H2O, rt

(Soidinsalo & Wähälä, 2007)

3

TBAHS, NaHCO3, DCM, H2O

(Mitchell & Whitcombe, 2000)

4.7.1.2. Perester Coupling An obvious saving result if the tetraacetate methyl ester 11 may be satisfactory coupled to the aglycon. Many aryl glucuronides have been made in this way. The reaction is stereochemically reliable giving only -anomers of the conjugates. The use of 1-ester (Bollenback et al., 1955; Bowering et al., 1960) is essential, the 1-anomer gives very little or no product. With the more powerful Lewis acid SnCl4, heating is unnecessary and reaction may proceed satisfactory at 0 oC (Sugihara et al., 1976). The glucuronide 77, a tentative metabolite of the anti-inflammatory substance naproxen, was synthesized by reaction of 44 with the aglycon methyl ester in presence of BF3.OEt2 as a Lewis acid followed by acetate removal (Arewång et al., 2007) (Scheme 33).

INTRODUCTION

39

Scheme 33. 4.7.1.3. 1-Hydroxy Sugar Coupling. The use of intermediate 42 is becoming more popular. Regioselective 1-Odeacylation can be carried out by several reagents such as bis(tributyltin)oxide, tributyltin methoxide (Nudelman et al., 1987) and nitrogenous nucleophiles as hydrazine acetate (Soliman et al., 2003) or benzyl amine (O'Brien et al., 2007). Intermediate 42 can also prepared by hydrolysis of the glycosyl bromide 71 with a silver salt (Pravdić et al., 1964). More often, however, Lewis acid catalyzed coupling of 42 has been used, particularly employing TMSOTf (Fischer et al., 1984). Both alcohols and phenols have been glucuronidated in this way, and the first intermediate is believed to be the silyl ether or its O-1 protonated form. The final stereochemical outcome then depends on delicate balance of factors. 4.7.1.4. Trichloroacetimidate Coupling Schmidt pioneering studies on glycosidation using trichloroacetimidates have led to an increasing number of applications to glucuronidation. The relatively mild catalysis required and very high -selectivity makes 72 an attractive intermediate. Reaction of CCl3CN with 42 was earlier performed using NaH (Fischer et al., 1984) or DBU (Jacquinet, 1990). Crystalline 72 may

INTRODUCTION

40 be stored at -20

o

C under desiccation for several weeks with little

decomposition. Table 3. Examples for glucuronides prepared by the trichloroacetimidate method illustrating the promoter used.

Entry

1

2

aglycon

Promoter TMSOTf BF3.OEt2

TMSOTf BF3.OEt2

Ref.

(Lucas et al., 2009)

(Lucas et al., 2009)

3

TMSOTf

(Casati et al., 2009)

4

BF3.OEt2

(Tietze et al., 2008)

5

BF3.OEt2

(Engstrom et al., 2007)

INTRODUCTION

41

6

BF3.OEt2

(Needs et al., 2006)

7

BF3.OEt2

(Pearson et al., 2005)

8

BF3.OEt2

(Brown et al., 2000)

TMSOTf

9

10

TMSOTf

(Iyer et al., 2003)

(Soliman et al., 2003)

The trichloroacetimidate of either tetra-O-acetyl (Tosin et al., 2005c) or terta-O-benzoyl (Pilgrim et al., 2009) allyl glucuronate are also useful glycosyl donors with alcohols in the presence of TMSOTf as a promoter. 4.7.1.5. Trifluroacetimidate Coupling Recently, Al-Maharik & Botting (Al-Maharik & Botting, 2006) reported an efficient and facile synthesis of isoflavone 7-O-glucuronide 80 employing the glycosyl trifluoroacetimidate donor. The method was based on the Lewis acid activated coupling of glycosyl N-4-methoxyphenyltrifluoroacetimidate 78 with the 7-hydroxyl group of isoflavone hexanoyl ester 79 (Scheme 34).

INTRODUCTION

42

Scheme 34. Synthesis of isoflavone 7-O-glucuronide. 4.7.1.6. Thio-glucuronosides Coupling Thio-glucuronic acids proved to be useful as both donor and acceptor in sulfonium-mediated condensations (Codée et al., 2005; van den Bos et al., 2004).

INTRODUCTION

43

Scheme 35. Thioglucuronides were tested as donors in coupling reactions with unreactive carbohydrate alcohols using DMTST as promoter. The glucuronides were obtained with the desired -configuration because of the participating group at O-2 (Garegg et al., 1995). Later, glucuronate thioglycoside donors with a 2-O-nonparticipating group were used by Oscarson & Svahnberg (Oscarson et al., 2001) in synthesis of uronic acid containing xylans, with which the stereoselectivity in glycosylations can be controlled by the conditions employed. DMTST in diethyl ether gave glycosides, whereas NIS in DCM yielded -glycosides (Scheme 36).

INTRODUCTION

44

Scheme 36. The locked 1-thioglucuronides can directly be used as donors in glycosidations reactions using the DPSO/Tf2O reagent system (van den Bos et al., 2005) (Scheme 37).

Scheme 37. The stereodirecting effect of the glycosyl C-5 substituent has been investigated in a series of D-pyranosyl thioglycoside donors and related to their preferred positions in the intermediate 3H4 and 4H3 half-chair oxacarbenium ions (Dinkelaar et al., 2009). Computational studies showed that an axially

INTRODUCTION

45

positioned C-5 carboxylate ester can stabilize the 3H4 half-chair oxacarbenium ion conformer by donating electron density from its carbonyl function into the electron-poor oxacarbenium ion functionality (Figure 10).

Figure 10. The stereodirecting effect of the glycosyl C-5 substituent. 4.7.2. Acyl glucuronides Methods for the synthesis of acyl glucuronides have been reviewed (Stachulski et al., 2006; Stachulski & Jenkins, 1998). The preference now is for syntheses using minimal rather than global sugar protection (Bugianesi & Shen, 1971; DeMesmaeker et al., 1989), one of the most useful intermediates being allyl glucuronate 81. Mitsunobu coupling of 81 with a range of carboxylic acids followed by Pd deprotection was shown to be a viable method for the preparation of a range of acyl glucuronides. Yields were modest, however, and

 mixtures were invariably generated in the coupling step, requiring careful separation by preparative HPLC (Juteau et al., 1997) (Scheme 38).

46

INTRODUCTION

Scheme 38. Mitsunobu coupling. Later, some reports (Perrie et al., 2005; Plusquellec et al., 1986; Schmidt, 1986) had showed that selective acylation of 81 was an excellent alternative to the Mitsunobu procedure, affording higher yields and essentially pure products (Scheme 39). The advantage of the selective acylation method using 81 was explained as the kinetic anomeric effect was exploited to greatly favor 1acylation through the stereoelectronic enhancement of the nucleophilicity of the 1-alkoxide. After considerable experimentation with different carboxyl activators and bases, the group found that the combination of HATU for carboxyl activation and NMM as base gave best results for the acylation of 81. Bases weaker or stronger than NMM were less effective.

INTRODUCTION

47

Scheme 39. One drawback, when 81 was used, was the persistence of Pd traces in the final products after deprotection of the allyl ester using Pd reagents. This is not a major concern for the preparation of analytical standards, but would prohibit biological evaluation of material prepared in this way. While the resin-bounded form of the reagent greatly reduced the Pd amounts, the use of a different ester seemed to them a promising solution to the problem. Recently, Bowkett et al. (Bowkett et al., 2007) reported the use of benzyl glucuronate 82 as an alternative to allyl ester 81. Same conditions used for acylation of 81 were successfully applied to the selective acylation of 82 with a range of carboxylic acids (Scheme 40).

INTRODUCTION

48

Scheme 40. Deprotection is effected under mild conditions, using Pd for allyl esters and catalytic transfer (or conventional) hydrogenation using cyclohexa-1,4-diene (10% Pd–C, THF/i-PrOH, 60 oC, 1.5 h) for benzyl esters; these conditions are compatible with a range of functional groups. 4.7.3. 1,2-Cis-glucuronides While treatment of the 1-hydroxy sugar 74 with PBr3 afforded exclusively the 1-bromo sugar, either anomer of the chloro sugar could be isolated after SOCl2 treatment. Schmidt & Ruecker (Schmidt et al., 1980) found that reaction of any of these halo intermediates with various aglycones using silver perchlorate in acetonitrile led very largely to the -glucuronides at -15 oC. The common intermediate is believed to be the nitrilium conjugate (associated with further solvent molecules); other solvents gave different results (Scheme 41).

INTRODUCTION

49

Scheme 41. The synthesis of 1,2-Cis-glucronides can be achieved even in the presence of 2-acyl participating groups and the stereoselectivity can be explained by participation of remote acid or amide groups. Preliminary investigations were carried out by Tosin et al., (Tosin et al., 2002) to access the potential for using the 1,6-lactone 52 in the synthesis of O-glucuronosides in only the configuration. This methodology was used for the synthesis of a Kdn2en mimetic (Figure 3) with the -configuration.

INTRODUCTION

50

Scheme 42. Further investigations were done by Poláková et al. and they reported that the SnCl4-catalyzed coupling of silyl ethers, with lactone 52, provides a -Oglucuronides in significantly improved yields without loss of stereoselectivity. The methodology has been extended to the related 2-deoxylactones which give

- and -glycosides depending on the structure of the donor (Poláková et al., 2004). A proposed mechanistic pathway accounting for the results of these reactions is shown in Figure 11. It was suggested that the inversion of configuration at C-1 of 83 occurs for donors in which X=H or OAc without participation of the C-2 group when X=OAc, anchimeric assistance by the carboxylic group contributing to the stereochemical outcome.

INTRODUCTION

51

Figure 11. Possible mechanistic pathways to the - and -glycosidation products. The results observed for 85 were explained by iodine residue being better participator than the 2-acyl group and the carboxylate group. 5. Glycosyl amides 5.1. N-Glycosyl amides N-Glycosyl amides may be prepared by coupling a glycosyl amine with a suitable carboxylic acid derivative; however, problems associated with this method include anomeric interconversion and amine decomposition (Knauer et al., 2004). The use of Staudinger-type chemistry avoids many of these issues by treating a diastereomerically pure azide precursor with a phosphine and allowing the thus formed phosphinimine ylide to react with a carboxylic acid derivative to give the amide product, usually without loss of the integrity of the anomeric stereochemistry (Temelkoff et al., 2006a) (Scheme 43).

INTRODUCTION

52

Scheme 43. Amide linked glycosyl heterocycles have been reported (Murphy et al., 2002; Pitt et al., 2004) to posses interesting anti-angiogenesis activity therefore the creation of libraries of these compounds would be useful for further biological evaluation (Figure 12 & 13).

INTRODUCTION

Figure 12.

53

INTRODUCTION

54

Figure 13. The structural preference displayed by bivalent glycosyl amide 53 was investigated (Murphy et al., 2003). NMR spectroscopic investigations suggested that the major isomers adopted by 86 are 86a and 86b. For 86a the carbohydrates can be considered to be cis, whereas for 86b they are trans.

INTRODUCTION

55

Figure 14. Structural preference displayed by bivalent glycosyl amide 86. Later, grafting the -D-mannopyranoside headgroup onto hydroxyl groups of the core 86 to generate 87 with potential to crosslink mannose binding receptors (Tosin et al., 2005a) (Scheme 44).

INTRODUCTION

56

Scheme 44. Grafting the -D-mannopyranoside headgroup. 5.2. C-Glycosyl amides Tosin & coworkers (Tosin et al., 2005c) have reported, as an extension of earlier studies carried by other researchers (Avalos et al., 1998; Avalos et al., 1992), the structural preferences for glycosyl aromatic amides, which are shown to diverge depending on which are shown to diverge depending on the nature of the amide.

INTRODUCTION

57

Figure 15. Amide structure and nomenclature. The results given by Tosin & coworkers (Tosin et al., 2005c) provide evidence that Z amides are preferred for secondary anilides derived from glucuronic acid, whereas E-configured amides are preferred by the tertiary anilides (Figure 16). Molecular mechanics calculations suggested that the Z-anti structure is preferred for secondary anilides with Z-syn being a higher-energy structure and supported the proposal that the E-anti structure is preferred for tertiary anilides. The explanations for this are as follows: (i)

The greater steric interaction of the pyranose groups with the alkyl group destabilizes the Z-anti structure.

(ii)

The steric interaction of the alkyl group with H-4 destabilizes the Zsyn structure.

(iii)

The steric interaction of the aromatic group with H-4 destabilizes the E-syn structure.

INTRODUCTION

58

Thus, alkylation of secondary anilides to produce tertiary anilides induces a configurational switch that alters the orientation of the aromatic group with respect to the pyranose ring. The anomeric group is projected in a different orientation from the pyranose scaffold in a manner dependent on the structure of the amide. Secondary anilides had a Z configuration in the solid state and showed intraand intermolecular hydrogen bonding. However, on the basis of NMR and IR studies, there was generally no evidence for the same hydrogen bonding in solution.

INTRODUCTION

Figure 16. Summary of NOEs observed for secondary and tertiary amides.

59

60

INTRODUCTION Later, studies were extended to the structural analysis of bivalent

carbohydrates based on anilides of glucuronic acid (Tosin et al., 2005b; Velasco-Torrijos & Murphy, 2005). As for monomers, bivalent secondary anilides predominantly adopted the Zanti structure; there is also evidence for population of the Z-syn isomer. Bivalent tertiary anilides displayed two signal sets in their 1H NMR spectra, consistent with the presence of two slowly interconverting configurational isomers. The first of these, the major isomer, is C2-symmetric U-shaped and/or S-shaped where both amides have E configurations (EE) whereas the second isomer, the minor isomer, is explained by an isomer with the general features of the asymmetric L-shaped, where one amide is E and the other is Z (EZ)a. Qualitative NOE/ROE spectroscopic studies in solution support the proposal that the anti conformation is preferred for E amides (Tosin et al., 2005b) (Figure 17).

a

The U-shaped conformation has the carbohydrate groups on the same side of the plane defined by the aromatic ring; the S-shaped conformation has the carbohydrate on opposite sides of the ring.

INTRODUCTION

61

Figure 17. The x-ray crystal structure of 88 was discussed. 88 adopted the folded structure 88a (Figure 18), where each amide has different conformation: one is E-anti whereas the other is E-syn. The stacking was mediated by non-covalent interactions. Inter-atomic distances that support intramolecular Van der Waals interactions have been provided. There were clear interactions between (i) the oxygen atom of both pyranoses and the aromatic carbon and hydrogen atoms, (ii) the E-syn 2-acetate carbonyl oxygen atom and the E-anti pyranose ring protons, and (iii) the two azide groups (Tosin et al., 2004).

62

INTRODUCTION

Figure 18. The folded structure of compound 88. Later, a series of water soluble macrocyclic structures containing two saccharide units has been synthesized by ring closing metathesis (VelascoTorrijos et al., 2005) (Scheme 45).

INTRODUCTION

Scheme 45. Synthesis of macrocycles.

63

64

INTRODUCTION The 3D structure of these compounds has been studied. The results

suggested that the carbohydrate presentation within the macrocycle may diverge depending on macrocycle size. Furthermore, the larger macrocycle 89 derived from pentenyl glycosides showed switching phenomena similar to cyclodextrin (Tosin et al., 2005b; Velasco-Torrijos et al., 2005).

INTRODUCTION

65

Figure 19. Structural isomers of carbohydrate containing macrocycles. The synthesis of the N-substituted glucuronamides can be achieved by reaction of the mixed anhydride 1 with an amino group. The reaction is only

INTRODUCTION

66

successful for primary amines, as nucleophilic attack takes place at the carbonyl group nearest the pyranose in these cases, but it is not for secondary amines as the nucleophilic attack takes place at the less congested carbonyl group (Tosin et al., 2002) (Scheme 46).

Scheme 46. Preparation of D-glucuronamide derivatives can be carried out, also, from acid 57 via a coupling reaction of its acid chloride with an amine in DCM in the presence of pyridine (Temelkoff et al., 2006b; Tosin et al., 2002; Tosin et al., 2005b; Tosin et al., 2005c) or DIPEA (Velasco-Torrijos et al., 2005). Another route is direct coupling of the acid 2 with an amine promoted by HBTU/HOBt/DIPEA (Tosin et al., 2005a; Tosin et al., 2005b) or DCC/Et3N (Loukou et al., 2007; Tosin et al., 2005c) (Scheme 47).

INTRODUCTION

67

Scheme 47. Grafting of the -D-mannopyranoside headgroup on hydroxyl groups to produce potential to crosslink mannose binding receptors was also tried (Tosin et al., 2005a) (Scheme 48).

Scheme 48. Grafting of the -D-mannopyranoside headgroup.

INTRODUCTION

68 5.3. Sugar Lactams

The synthesis of -D-glucopyranosidurono-6,1-lactams of D-glucuronic acid derivatives were described by Loukou & coworkers (Loukou et al., 2007). The lactam derivative 91 was obtained from SnCl4-catalyzed reaction of methoxylamide derivative 90, which had been prepared by coupling of methoxylamine to the acid 2 (Scheme 49). The lactam 91 proved to be stable and could not be activated as a glycosyl donor in the presence of azidotrimethylsilane and SnCl4. This contrasted with the behavior of cyclicimidates such as 92 which gave glycosyl azides under similar conditions (Tosin et al., 2005c).

Scheme 49. A route to lactams was next explored from glycosyl azide 93. The catalytic hydrogenation of 94 provided an amine that spontaneously cyclised to give the 6,1-lactam 95 possessing a free hydroxyl group at C-3 (Scheme 50).

INTRODUCTION

69

Scheme 50. Attempts to obtain 67 by selective acetylation of 95 is not possible under the described conditions even at low temperature (Loukou et al., 2007) (Scheme 51).

Scheme 51. The synthesis of 99 was achieved from the glycosyl azide 98 (Scheme 52). The conversion of the -azide 98 to the desired 6,1-lactam 99 was achieved by the reaction of the acid 98 with DCC and HOBt in acetonitrile followed by addition of tributylphosphine which gave 99 in 38% yield. A similar yield (38%)

70

INTRODUCTION

but more straight forward purification of 99 was achieved by using EDCI and HOBt in acetonitrile and subsequent addition of tributylphosphine or trimethylphosphine in THF to effect the conversion to 99.

Scheme 52.

RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

71

Results and Discussion Scheme 53 shows the retrosynthetic analysis of the D-glucuronamide derivatives on which the synthetic strategy was based. Thus, appropriate disconnection led, retrosynthetically, to D-glucuronic acid as a starting material.

Scheme 53. Retrosynthetic analysis of N-substituted D-glucuronamides. The ideal intermediate for this synthesis should fulfill two important requirements: (1) It should possess a blocked hydroxyl groups with a suitable protecting groups. (2) It should possess an activated carboxylic acid group. Studies were initiated by acetylation of the commercially available Dglucuronic acid with acetic anhydride and catalytic amounts of iodine as an acetyl transfer reagent according to the previously described literature (Tosin et

RESULTS AND DISCUSSION

72

al., 2002) to give the 1,2,3,4-tetra-O-acetyl protected mixed anhydride 1 in 98% yield (Scheme 54). The formation of 1 was confirmed by 1H NMR spectroscopy. In particular, the acetoxy methyl signal that appeared at relatively low field (2.28 ppm) indicated the existence of the COOCOCH3 group, in addition to the higher field signals observed at 2.13 (3H), 2.05 (6H) and 2.04 ppm (3H) corresponding to the four O-acetyl groups.

Scheme 54. Acetylation of D-glucuronic acid. Compound 1 was subjected to reaction with water to afford the corresponding 1,2,3,4-tetra-O-acetyl--D-glucuronic acid 2 in 99% yield (Scheme 54). The formation of 2 was proven using 1H NMR which revealed the absence of the acetoxy methyl signal at 2.28 ppm and the appearance of a broad signal at 3.44 ppm corresponding to the carboxyl group proton (Tosin et al., 2002).

RESULTS AND DISCUSSION

73

Compound 2 is characterized by its stability at room temperature for long period of time without detection of any decomposition but when it was dissolved in diethyl ether and left to crystallize, the 1H NMR of the crystals formed was indicative to D-glucuronic acid tetra-acetate having an -anomeric configuration (Figure 20). In particular, 1H NMR signals for the anomeric proton of the -anomer was observed at 6.00 ppm (d, J1-2 = 8.1 Hz), while the corresponding signal for the -anomer appears at 6.38 ppm (d, J1-2 = 3.5 Hz).

Figure 20. 1,2,3,4-Tetra-O-acetyl--D-glucuronic acid. On the basis of the previously reported findings, mechanisms for such inversion could subsequently be offered (Figure 21).

RESULTS AND DISCUSSION

74

Figure 21. Suggested mechanisms for anomerization of 1,2,3,4-tetra-O-acetyl-

-D-glucuronic acid. The glucopyranuronic acid 2 may hydrolyze by a different mechanisms and this could involve an intermediate that adopts a 2SO conformation, which could give an oxycarbenium ion with a

2,5

B conformation (3; Figure 21, pathway a), or

RESULTS AND DISCUSSION

75

involve an intermediate that adopts a 1C4 conformation giving an oxycarbenium ion with a 3H4 half chair conformation (4; Figure 21, pathway b). In either case the carboxylic acid group is sufficiently proximate to the acetyl oxygen atom to facilitate intramolecular proton transfer (Figure 21, pathway a & b). Related intramolecular acid catalysis processes could be used to explain anomerization (Saunders & Timell, 1967; Saunders & Timell, 1968; Timell et al., 1965). Alternatively, protonation of the pyranose oxygen and subsequent cleavage of the C1-O5 bond (endocyclic cleavage) giving an open chair intermediate may also be considered (Figure 21, pathway c) (O'Brien et al., 2007). Fortunatly, the mixed anhydride 1 "perfectly fits" the proposed synthetic criteria as: (1) All the hydroxyl groups are protected with acetyl groups. (2) The carboxylic acid group is activated through mixed anhydride activation. On this basis, compound 1 was subjected to the reaction with an appropriate amine solely in anhydrous methylene chloride which furnished, after extractive work-up and chromatographic purification, the per-O-acetylated secondary amides 5-12 in moderate yields.

Scheme 55. Synthesis of glucuronamide derivatives. N-(Benzyl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide

(5)

was

obtained by reaction between benzyl amine and 1 in anhydrous DCM at room temperature overnight. The crude reaction product was purified by extractive

76

RESULTS AND DISCUSSION

work-up, followed by chromatographic purification.

The structural assignments based on NMR spectral data were in good agreement and confirmed the proposed structure. In particular, the 1H NMR spectrum showed the anomeric proton as doublet at 5.94 ppm with spin-spin coupling constant of 8.4 Hz corresponding to a diaxial orientation of H-1 and H2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.404.33 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.03, 1.97, 1.91 and 1.83 ppm. The five phenyl ring protons appeared as a multiplet in the 7.267.17 ppm region, while the methylene protons resonated at 4.20 ppm The splitting pattern of the methylene group was ddd (double of doublets for each methylene proton) which corresponds to an ABX system and it was reduced to a double of doublets after proton exchange experiment (Figure 22). This was accounted by the presence of chiral centers in the vicinity of the methylene group which makes a ‘non-equivalence’ between the two methylene protons and in turn a diastereotopic effect will be reflected in the 1H NMR spectrum and the CH2 group will appear as a double of doublets (doublet for each methylene proton). Further coupling with the neighboring amide NH proton will give the observed ddd pattern.

RESULTS AND DISCUSSION

77

Figure 22. Splitting pattern of the methylene protons before and after proton exchange.

78

RESULTS AND DISCUSSION On the other hand, the carbon skeleton was fully assigned with the aid of 13C

NMR spectra and DEPT experiment (Figure 23).

13

C NMR spectra were

characterized by a signal at 91.2 ppm corresponding to C-1 of the -Dglucopyranose ring. Another four signals at 72.9, 71.9, 70.1 and 68.9 ppm were assigned to C-2, C-3, C-4 and C-5. The four signals appearing at 169.8, 169.6, 169.2 and 168.7 ppm are due to the four acetoxy carbonyl carbons, while the signal at 165.8 ppm is due to the amide carbonyl carbon. The signals at 20.6 and 20.5 ppm are attributed to the acetate methyl carbons. The aglycon methylene carbon resonated at 42.9 ppm, while signals at 137.4, 128.7, 127.8 and 127.6 are due to the aglycon phenyl carbons.

Figure 23. DEPT Experiment for compound 5.

RESULTS AND DISCUSSION 79

80

RESULTS AND DISCUSSION

N-(Allyl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (6) was obtained by reaction between allyl amine and 1 in anhydrous DCM at room temperature overnight. The crude reaction product was purified by extractive work-up, followed by chromatographic purification.

The structural assignments of the product were based upon its NMR spectral data. In particular, the 1H NMR spectrum showed a doublet at 5.75 ppm which was assigned to H-1, and the magnitude of coupling between H-1 and H-2 (7.1 Hz) was indicative to their diaxial orientation (-anomeric configuration). The other four protons of the glucopyranosyl ring resonated in the 5.354.10 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.15, 2.08, 2.05 and 2.03 ppm. The allyl characteristic signals were also observed. It appeared as three multiplets at ~5.83 (=CH), ~5.35 (=CH2) and 3.86 ppm (CH2). N-(Thiazol-2-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (7) was obtained by reaction between 2-aminothiazole and 1 in anhydrous DCM at room temperature overnight. The crude reaction product was purified by extractive work-up, followed by chromatographic purification.

RESULTS AND DISCUSSION

81

The structural assignments based on NMR spectral data were in good agreement and confirmed the proposed structure. In particular, the 1H NMR spectrum showed the anomeric proton as doublet at 6.08 ppm with spin-spin coupling constant of 8.1 Hz corresponding to a diaxial orientation of H-1 and H2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.554.61 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.08, 2.03, 1.97 and 1.93 ppm. The thiazole ring protons appeared as two doublets at 7.50 and 7.31 ppm N-(1H-Benzimidazol-2-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (8) was obtained by reaction between 2-amino-1H-benzimidazole and 1 in anhydrous DCM at room temperature overnight. The crude reaction product was purified by extractive work-up, followed by chromatographic purification.

The structural assignments based on NMR spectral data were in good agreement and confirmed the proposed structure. In particular, the 1H NMR spectrum showed the anomeric proton as doublet at 6.09 ppm with spin-spin

RESULTS AND DISCUSSION

82

coupling constant of 9.7 Hz corresponding to a diaxial orientation of H-1 and H2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.564.65 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.10, 2.04, 1.98 and 1.96 ppm. The benzimidazole protons appeared as two doublets at 7.50 and 7.23 ppm N-(Pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (9) was obtained by reaction between 2-aminopyrazine and 1 in anhydrous DCM at room temperature overnight. The crude reaction product was purified by extractive work-up, followed by chromatographic purification.

The structural assignments of the obtained product were based on its NMR spectral data. In particular, the 1H NMR spectrum showed the anomeric proton as doublet at 5.82 ppm with spin-spin coupling constant of 8.1 Hz corresponding to a diaxial orientation of H-1 and H-2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.324.24 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.15, 2.10, 2.05 and 2.02 ppm. The pyrazine ring protons appeared as a singlet resonated at 9.40 ppm and two doublets resonated at 8.38 and 8.27 ppm N-(2-Chloro-4-nitrophenyl)-1,2,3,4-tetra-O-acetyl-β-Dglucopyranuronamide (10) was obtained by reaction between 2-chloro-4nitroaniline and 1 in anhydrous DCM at room temperature overnight. The crude

RESULTS AND DISCUSSION reaction product

was

purified

by

extractive

work-up,

83 followed

by

chromatographic purification.

The structural assignments based on NMR spectral data were in good agreement and confirmed the proposed structure. In particular, the 1H NMR spectrum showed the anomeric proton as doublet at 5.84 ppm with spin-spin coupling constant of 7.5 Hz corresponding to a diaxial orientation of H-1 and H2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.354.28 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.15, 2.10, 2.06 and 2.02 ppm. The three phenyl protons appeared as a doublet at 8.52 ppm, a singlet at 8.29 ppm and a multiplet at 8.15 ppm N-(4-Bromophenyl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide

(11)

was obtained by reaction between 4-bromoaniline and 1 in anhydrous DCM at room temperature overnight. The crude reaction product was purified by extractive work-up, followed by chromatographic purification.

84

RESULTS AND DISCUSSION

The structural assignments based on NMR spectral data were in good agreement and confirmed the proposed structure. In particular, the 1H NMR spectrum showed the anomeric proton as doublet at 6.06 ppm with spin-spin coupling constant of 8.4 Hz corresponding to a diaxial orientation of H-1 and H2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.534.46 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.09, 2.03, 1.97 and 1.92 ppm. The four phenyl protons appeared as a broad singlet at 7.52 ppm N-(Pyridin-4-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (12) was obtained by reaction between 4-aminopyridine and 1 in anhydrous DCM at room temperature overnight. The crude reaction product was purified by extractive work-up, followed by chromatographic purification to give the desired compound but with a dropped product yield (> 5 %). In an attempt to improve the yield, the reaction was repeated, and this time the work-up procedure was modified to direct subjection of the crude reaction to column chromatography. This modification to the work-up procedure highly improved the yield and gave the product in 50% yield.

RESULTS AND DISCUSSION

85

The structural assignments based on NMR spectral data were in good agreement and confirmed the proposed structure. In particular, the 1H NMR spectrum showed the anomeric proton as doublet at 6.02 ppm with spin-spin coupling constant of 8.0 Hz corresponding to a diaxial orientation of H-1 and H2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.244.55 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.04, 1.99, 1.93 and 1.88 ppm. The four pyridine protons appeared as two doublets at 8.41 and 7.54 ppm On the other hand, the reaction of the fully acetylated mixed anhydride 1 with 4-aminouracil or its N,N-dimethyl derivative under the previously established conditions revealed no formation of the amide products (13a,b) even after changing the reaction solvent to acetonitrile (Scheme 56).

Scheme 56. Reaction of anhydride 1 with aminouracil and its N,N-dimethyl derivative.

86

RESULTS AND DISCUSSION At this point, subsequent removal of acetyl protecting groups was found to

be essential. Base catalyzed saponification of each of the prepared glucuronamide derivatives 5-12 were achieved at pH 13 using 0.05 M LiOH to afford the products 14-21 in an equilibrium anomeric mixture (Scheme 57). The

 ratio for all products was found to be 1:1 ratio as determined by the signal integration in 1H NMR spectra. Also, removal of acetyl groups using Zemplén’s conditions (catalytic amount of sodium methoxide in methanol) was tried on the N-allyl derivative and it revealed the same result with approximately equal yield (Scheme 57).

Scheme 57. Deprotection of the synthesized glucuronamide derivatives. The complete removal of all acetyl groups was confirmed by NMR spectroscopy. For all products, both 1H NMR and 13C NMR spectra revealed the absence of signals corresponding to acetyl groups, in addition to an upfield shift for the glucopyranosyl ring protons was observed in the 1H NMR spectrum.

The structural assignment for 14 was based on its spectral data. In particular, the 1H NMR showed signals at 7.277.20 ( aromatic H), 5.15 (J1-2 = 3.80

RESULTS AND DISCUSSION

87

Hz,  H-1), 4.57 (J5-4 = 7.65 Hz,  H-5), 4.32 ( CH2), 4.31 ( CH2), 4.14 (J5-4 = 9.2 Hz,  H-5) and 3.643.16 ppm ( H-4,  H-3 and  H-2). For both - and -anomers, the difference between the chemical shifts of the methylene diastereotopic protons is so small that neither this difference nor any coupling between them was easily detectable and appeared as two singlets at 4.32 ( CH2) and 4.31 ppm ( CH2). Additionally, the

13

C NMR showed signals at 171.3, 170.5, 137.6, 128.9,

127.6, 127.3, 96.2, 92.5, 75.4, 75.3, 71.8, 71.5 and 42.8 ppm which was in agreement with the expected product.

The structural assignment for 15 was based on its spectral data. In particular, the 1H NMR showed signals at 5.70 ( =CH), 5.13 (J1-2 = 3.05 Hz,  H-1), 5.074.99 ( =CH2), 4.09 (J5-4 = 9.95 Hz,  H-5), 3.76 (J5-4 = 9.95 Hz,  H5), 3.70 ( CH2), 3.583.37 ( H-4,  H-3 and  H-2), 3.19 ( CH2) and 3.15 ppm ( H-2). Additionally, the

13

C NMR showed signals at 171.3, 170.5,

133.2, 115.8, 96.3, 92.6, 73.9, 72.5, 71.6, 71.2, 41.4 and 30.4 ppm which was in agreement with the expected product.

RESULTS AND DISCUSSION

88

The structural assignment for 16 was based on its spectral data. In particular, the 1H NMR showed signals at 7.36 ( aromatic H), 7.12 ( aromatic H), 5.23 (J1-2 = 3.85 Hz,  H-1), 4.39 (J5-4 = 9.90 Hz,  H-5), 4.06 (J5-4 = 9.15 Hz,  H-5) and 3.693.23 ppm ( H-4,  H-3 and  H-2). Additionally, the 13

C NMR showed signals at 169.8, 168.2, 158.2, 137.1, 115.1, 96.4, 92.6, 75.3,

75.1, 73.7, 72.4, 71.6, 71.3, 71.2 and 71.1 ppm which was in agreement with the expected product.

The structural assignment for 17 was based on its spectral data. In particular, the 1H NMR showed signals at 7.12 ( aromatic H), 5.10 (J1-2 = 3.85 Hz,  H-1), 4.49 (J5-4 = 7.65 Hz,  H-5), 3.94 (J5-4 = 9.9 Hz,  H-5) and 3.583.04 ppm ( H-4,  H-3 and  H-2). Additionally, the

13

C NMR showed

signals at 169.0, 168.0, 150.2, 129.1, 123.5, 110.9, 95.9, 92.2, 76.9, 76.3, 75.6, 74.0, 72.6, 72.2, 71.9 and 71.4 ppm which was in a good agreement with the expected product.

RESULTS AND DISCUSSION

89

The structural assignment for 18 was based on its spectral data. In particular, the 1H NMR showed signals at 8.97 ( aromatic H), 8.24 ( aromatic H), 8.22 ( aromatic H), 5.24 (J1-2 = 3.1 Hz,  H-1), 4.34 (J5-4 = 9.9 Hz,  H-5), 4.02 (J5-4 = 9.95 Hz,  H-5) and 3.693.23 ppm (H-4, H- and  H2). Additionally, the

13

C NMR showed signals at 170.0, 169.0, 147.2, 143.1,

140.5, 137.1, 96.3, 92.6, 75.3, 73.8, 72.5, 71.7, 71.5, 71.4 and 71.1 ppm which was in a good agreement with the expected product.

The structural assignment for 19 was based on its spectral data. In particular, the 1H NMR showed signals at 8.33 ( aromatic H), 8.11 ( aromatic H), 8.01 ( aromatic H), 5.25 (J1-2 = 3.8 Hz,  H-1), 4.40 (J5-4 = 9.95 Hz,  H-5), 4.07 (J5-4 = 9.90 Hz,  H-5) and 3.693.24 ppm ( H-4,  H-3 and  H2). Additionally, the

13

C NMR showed signals at 169.9, 143.8, 139.6, 124.5,

123.8, 123.0, 121.3, 121.1, 97 .3, 93.1, 76.3, 74.9, 74.3, 73.2, 72.5, 72.0, 71.8 and 70.7 ppm which was in a good agreement with the expected product.

RESULTS AND DISCUSSION

90

The structural assignment for 20 was based on its spectral data. In particular, the 1H NMR showed signals at 7.39 ( aromatic H), 7.22 ( aromatic H), 5.16 ( H-1), 4.59 (J1-2 = 7.7 Hz,  H-1), 4.20 (J5-4 = 9.15 Hz,  H-5), 3.86 (J5-4 = 9.20 Hz,  H-5) and 3.633.17 ppm ( H-4,  H-3 and  H-2). Additionally, the 13C NMR showed signals at 169.9, 169.0, 135.3, 132.1, 123.9, 118.3, 96.3, 92.6, 75.7, 75.3, 73.8, 72.4, 71.7, 71.5 and 71.1 ppm which was in a good agreement with the expected product.

The structural assignment for 21 was based on its spectral data. In particular, the 1H NMR showed signals at 8.33 ( aromatic H), 7.55 ( aromatic H), 5.23 (J1-2 = 3.85 Hz,  H-1), 4.29 (J5-4 = 9.95 Hz,  H-5), 3.96 (J5-4 = 9.90 Hz,  H-5) and 3.623.23 ppm ( H-4,  H-3 and  H-2). Additionally, the 13

C NMR showed signals at 170.6, 169.6, 148.8, 145.8, 115.3, 96.3, 92.6, 75.3,

73.7, 71.7 and 71.3 ppm which was in a good agreement with the expected product. Next, the attention was shifted to the synthesis of analogues to the 2,3unsaturated sialic acid, 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-Dgalacto-non-2-enonic acid (Neu5Ac2en), which is found in free form in nature (Furuhata, 2004). Glucuronates derivatives were reported to readily undergo -cis-elimination to give the corresponding 4,5-unsaturated species by treatment with a base (Bazin et al., 1998; BeMiller & Kumari, 1972; Florio et al., 1999; Glänzer et al.,

RESULTS AND DISCUSSION

91

1991; Mann et al., 2006a; Mann et al., 2006b; Pitt et al., 2004). The electronwithdrawing effect of the carbonyl group makes the -proton (H-5) sufficiently acidic to be removed by a base. It has been proposed that there are three steps involved in the elimination. Firstly, removal of H-5 forming a carbanion, followed by conformational inversion, and finally, elimination to give the 4,5unsaturated derivative (BeMiller et al., 1972) (Figure 24).

Figure 24. -Elimination in glucuronic acid derivatives. A straightforward one pot procedure was employed starting from acid 2 in which, in addition to the amide bond formation, a base-promoted elimination of acetic acid was achieved. The coupling of 2 with an amine in the presence of ethyl chloroformate and TEA at -20 oC produced, after extractive work-up, the desired 4,5-unsaturated amides as the major product (22-27) beside a minor amount of a non-elimination products as an impurity which was identified by comparing their TLC mobilities with the previously synthesized. Fortunately, these products could be separated by a slow silica gel column chromatography (Scheme 58).

RESULTS AND DISCUSSION

92

Scheme 58. Preparation of 4,5-unsaturated glucuronamide derivatives.

-Elimination was accompanied by a conformational change of the ring from 4C1 chair to a half-chair to accommodate the newly formed double bond. This was reflected in the 1H NMR spectrums for all products by the reduction in the magnitude of the coupling between ring protons that were no longer in an axial-axial relationship with their neighbors. Further indication for -elimination was provided by the observed downfield shift of H-4 and the disappearance of the H-5 signal. The conformational change was also reflected in

13

C NMR spectrum by the

upfield shifts of signals corresponding to C-1, C-2 and C-3. Furthermore, the appearance of two downfield signals which are consistent with a C-4C-5 double bond carbons. The ring conformation of each product was determined by comparing the coupling constants with known glycosides of hex-4-enopyranuronate. It was concluded that all the prepared unsaturated compounds adapts preferentially the

RESULTS AND DISCUSSION 1

93

H2 confromation (Figure 25) as the coupling between H-1 and H-2 ranges

from1.6 to 4.6 Hz and between H-2 and H-3 ranges from 1.3 to 2.5 Hz which are characteristic to the equatorial-quasi-equatorial orientation of protons. Also, the coupling between H-3 and H-4 ranges from 4.3 to 4.8 Hz which is characteristic to the quasi-equatorial orientation of H-3. (Alföldie et al., 1975; Bazin et al., 1998). Further, a long rang coupling between H-1 and H-3, as well as, between H-2 and H-4 is observed which suggests a planner "W" arrangement of protons which is also consistent with the 1H2 conformation (Bazin et al., 1998).

Figure 25. The concluded conformation for all the prepared 4,5-unsaturated glucuronic acid derivatives. By using benzyl amine, the corresponding N-(benzyl)-1,2,3-tri-O-acetyl4,5

Δ -β-D-glucopyranuronamide (22) was obtained.

The

structural

assignment

for

22

was

confirmed

on

the

basis

RESULTS AND DISCUSSION

94

of its NMR spectral data. In particular, the anomeric

proton

at

6.21

ppm.

protons

appeared

at

5.17

and

olefinic

proton

appeared

at

The 5.04

6.18

other

1

H NMR showed the H-3

and

ppm,

respectively,

ppm.

The

acetoxy groups appeared as three singlets at

H-2

ring

while

the

remaining

three

1.99, 1.98 and 1.97

ppm. The five phenyl ring protons appeared as a multiplet in the 7.247.17 ppm region, while the methylene protons resonated at 4.39 ppm which appeared as ddd signal for the same reason described for CH2 group in compound 5 Additionally, 13C NMR interpretation assisted with DEPT (Figures 26 & 27) confirmed the structure by the observation of a signal at 88.7 ppm corresponding to C-1 atom. The other two signals at 67.5 and 64.1 ppm corresponds to C-2 and C-3, respectively, while the two signals at 144.8 and 103.8 ppm corresponds to C-5 and C-4 olefinic carbons, respectively. The four signals appearing in the 170.0160.6 ppm region are due to the four carbonyl carbon atoms, while the signals at 21.2 and 21.1 are due to the acetate methyl carbons. The benzyl group was also observed in the

13

C NMR by the appearance of

four signals at 137.9, 129.2, 128.4 and 128.1 ppm corresponds to the phenyl ring carbons, in addition to, the methylene carbon which appeared at 43.9 ppm.

Figure 26. DEPT Experiment for compound 22.

RESULTS AND DISCUSSION 95

Figure 27. DEPT Experiment for compound 22 (cont.).

96 RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

97

By using allyl amine, the corresponding N-(allyl)-1,2,3-tri-O-acetyl-Δ4,5-βD-glucopyranuronamide (23) was obtained.

The structural assignment for 23 was confirmed on the basis of its NMR spectral data. In particular, the 1H NMR showed the anomeric proton at 6.30 ppm with as spin-spin coupling constant of 3.1 Hz which is typical axial-quasiaxial coupling between H-1 and H-2 protons. The olefinic proton appeared at 6.25 ppm, while the other H-2 and H-3 ring protons appeared at 5.14 and 5.26 ppm, respectively. The remaining three acetoxy groups appeared as three singlets at 2.13, 2.12 and 2.09 ppm. The allyl protons were also observed as three multiplets at 5.85 (=CH), ~5.24 (=CH2) and 3.96 ppm (CH2). Additionally, 13C NMR interpretation assisted with DEPT (Figures 28 & 29) confirmed the structure by the observation of a signal at 88.2 ppm corresponding to C-1 atom. The other two signals at 67.0 and 63.5 ppm corresponds to C-2 and C-3, respectively, while the two signals at 144.2 and 103.1 ppm corresponds to C-5 and C-4 olefinic carbons, respectively. The four signals appearing in the 169.6160.0 ppm region are due to the four carbonyl carbon atoms, while the signals at 20.7 and 20.6 are due to the acetate methyl carbons. The allyl carbons were also observed by the appearance of two signals at 133.2 and 116.9 ppm corresponding to the two olefinic carbons and a signal at 41.7 ppm for the methylene carbon.

Figure 28. DEPT Experiment for compound 23.

98 RESULTS AND DISCUSSION

Figure 29. DEPT Experiment for compound 23 (cont.).

RESULTS AND DISCUSSION 99

RESULTS AND DISCUSSION

100

By using 2-aminothiazole, the corresponding N-(thiazol-2-yl)-1,2,3-tri-Oacetyl-Δ4,5-β-D-glucopyranuronamide (24) was obtained.

The

structural

assignment

for

24

was

confirmed

of its NMR spectral data. In particular, the anomeric

proton

6.346.32

ppm

appeared

at

overlapped region.

5.12

The

and

5.22

with

the

other

H-2

ppm,

1

on

the

basis

H NMR showed the

olefinic and

proton H-3

respectively.

in

ring The

the

protons remaining

three acetoxy groups appeared as three singlets at 2.05, 2.04 and 2.01

ppm.

Also,

the two

thiazole ring protons

appeared as

two

doublets at 7.44 and 6.98 ppm. Additionally,

13

C NMR interpretation confirmed the structure by the

observation of a signal at 88.8 ppm corresponding to C-1 atom. The other two signals at 67.3 and 63.8 ppm corresponds to C-2 and C-3, respectively, while the two signals at 143.6 and 106.1 ppm corresponds to C-5 and C-4 olefinic carbons, respectively. The four signals appearing in the 170.0157.5 ppm region are due to the four carbonyl carbon atoms, while the signals at 21.2 and 21.1 are due to the acetate methyl carbons. The three thiazole ring carbons were also observed at 158.1, 138.4 and 115.0 ppm. By using 2-aminobenzimidazole, the corresponding N-(1H-benzimidazol-2yl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (25) was obtained.

RESULTS AND DISCUSSION

101

The structural assignment for 25 was confirmed on the basis of its 1H NMR spectral data. In particular, it showed the anomeric proton at 6.35 ppm with as spin-spin coupling constant of 4.6 Hz which is typical axial-quasi-axial coupling between H-1 and H-2 protons. The olefinic proton appeared at 6.27 ppm, while the other H-2 and H-3 ring protons appeared at 5.12 and 5.23 ppm, respectively. The remaining three acetoxy groups appeared as three singlets at 2.06, 2.02 and 2.00 ppm. Also, the four benzimidazole ring protons appeared as two multiplets at 7.42 and 7.15 ppm each integrating two protons. By

using

2-chloro-4-nitroaniline,

the

corresponding

nitrophenyl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide

N-(2-chloro-4(26)

was

obtained.

The structural assignment for 26 was confirmed on the basis of its 1H NMR

RESULTS AND DISCUSSION

102

spectral data. In particular, it showed the anomeric proton at 6.44 ppm with as spin-spin coupling constant of 4.6 Hz which is typical axial-quasi-axial coupling between H-1 and H-2 protons. The olefinic proton appeared at 6.41 ppm, while the other H-2 and H-3 ring protons appeared at 5.20 and 5.32 ppm, respectively. The remaining three acetoxy groups appeared as three singlets at 2.15, 2.14 and 2.12 ppm. Also, the three phenyl ring protons appeared as three signals at 8.74, 8.33 and 8.20 ppm. By using 4-bromoaniline, the corresponding N-(4-bromophenyl)-1,2,3-tri-Oacetyl-Δ4,5-β-D-glucopyranuronamide (27) was obtained.

The structural assignment for 27 was confirmed on the basis of its NMR spectral data. In particular, the 1H NMR showed the anomeric proton at 6.37 ppm overlapped with the olefinic proton H-4, while the other H-2 and H-3 ring protons appeared at 5.16 and 5.28 ppm, respectively. The remaining three acetoxy groups appeared as three singlets at 2.14, 2.12 and 2.10 ppm. Also, the four phenyl ring protons appeared as two signals at 7.51 and 7.46 ppm with splitting pattern characteristic for the AB system Additionally,

13

C NMR interpretation confirmed the structure by the

observation of a signal at 88.3 ppm corresponding to C-1 atom. The other two signals at 67.0 and 63.4 ppm corresponds to C-2 and C-3, respectively, while the two signals at 143.9 and 104.3 ppm corresponds to C-5 and C-4 olefinic

RESULTS AND DISCUSSION

103

carbons, respectively. The four signals appearing in the 169.6157.9 ppm region are due to the four carbonyl carbon atoms, while the signals at 20.7 and 20.6 are due to the acetate methyl carbons. The six phenyl ring carbons were also observed at 135.8, 132.0, 121.6 and 117.6 ppm. Interestingly, the attempt to use 2-aminopyrazine as the amine in this reaction gave a single reaction product as indicated by TLC, but after purification and spectral data inspection, it was indicated that the reaction did not

give

the

desired

N-(pyrazin-2-yl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-

glucopyranuronamide (28), but instead it gave ethyl 1,2,3-tri-O-acetyl-Δ4,5-β-Dglucopyranuronate (29) (Scheme 59). The formation of 29 was an unexpected result, and the mechanism for its formation requires further investigation.

RESULTS AND DISCUSSION

104

Scheme 59. Reaction of 2 with 2-aminopyrazine. The product structure was assigned on the basis of its NMR spectral data. In particular the 1H NMR spectrum revealed nine signals; a quartet at 4.31 ppm and a triplet at 1.34 ppm corresponding to the ethyl group, three singlets at 2.12, 2.11 and 2.10 ppm corresponding to three acetyl groups, three signals at 6.41, 5.23 and 5.15 ppm corresponding to H-1, H-3, H-2 ring protons and a signal at 6.27 ppm corresponding to the olefinic ring proton. Additionally, the

13

C NMR spectra confirmed the suggested structure. The

two signals at 62.0 and 14.13 ppm were assigned for the ethyl carbon atoms,

RESULTS AND DISCUSSION

105

while the signal at 161.2 ppm was assigned for the ethyl ester carbonyl carbon. The three signals at 88.5, 66.7 and 63.6 ppm were assigned to C-1, C-2 and C-3 atoms, respectively, while the two signals at 143.2 and 106.6 ppm were assigned to the olefinic C-5 and C-4 atoms, respectively. The three signals appearing at 169.7, 169.1, and 168.4 ppm corresponds to the three acetoxy carbonyl carbons, and the other signals at 20.8 and 20.7 ppm corresponds to the acetate methyl carbons. The focus was shifted to find an alternative route for the preparation of N(pyrazin-2-yl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide.

Alternatively,

a solution of the readily synthesized N-(pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl-βD-glucopyranuronamide (9) in anhydrous DCM at 0 oC was treated with DBU. After 30 min., TLC analysis of the reaction mixture indicated the complete consumption of the starting material and a clean conversion to a product with a higher Rf. To work-up the reaction, the solvent was removed in vacuo and the residue was subjected to column chromatography to give the expected product 28 (Scheme 60).

Scheme

60.

Synthesis

of

N-(pyrazin-2-yl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-

glucopyranuronamide. The NMR spectroscopic analysis of the product confirmed the elimination as the 1H NMR spectrum showed an olefinic H-4 resonates at ~6.3 ppm and the absence of resonances for H-5. The

13

C NMR spectroscopy showed signals for

RESULTS AND DISCUSSION

106

olefinic carbons at 105.4 ppm (C-4) and 143.6 ppm (C-5) that were located downfield and consistent with a C-4C-5 double bond. Additionally, both the 1H NMR and

13

C NMR showed signals which corresponds to only three acetyl

groups. The DBU promoted elimination procedure was also tried for the preparation of 4,5-unsaturated compounds 22-27 from the corresponding 1,2,3,4-tetra-Oacetyl-β-D-glucopyranuronamides and it successfully yielded the desired products in moderate yields except for N-(1H-benzimidazol-2-yl)-1,2,3,4-tetraO-acetyl--D-glucuronamide (8) in which the reaction suffered from series of complications (Scheme 61).

Scheme 61. Reaction of N-(1H-benzimidazol-2-yl)-1,2,3,4-tetra-O-acetyl--Dglucuronamide with DBU. Next, the attention was shifted to the synthesis of a series of N-acyl-N-arylD-glucopyranuronosyl amine derivatives. The retrosynthetic analysis on which the synthetic strategy was based is outlined in scheme 62. It was disconnected into carboxylic acid and N-glycoside (cleavage 1); then a primary amine is removed, leaving behind a glucuronic acid donor (cleavage 2). The key step in this route is the formation of a Schiff’s base.

RESULTS AND DISCUSSION

107

Scheme 62. Retrosynthesis analysis of N-acyl-N-aryl-D-glucopyranuronosyl amine derivatives. After refluxing the acid 2 in DCM with thionyl chloride for 2 h, TLC analysis of the reaction mixture identified the formation of a material that was more mobile than the starting material which was completely consumed. Direct treatment of the generated acid chloride with allyl alcohol in presence of pyridine for 1 h followed by extractive work-up furnished the allyl ester substrate 31 in 98% yield (Scheme 63).

RESULTS AND DISCUSSION

108

Scheme 63. Synthesis of allyl ester substrate 31. Obviously, this procedure was found to be much efficient and simpler than the previously published method (Tosin et al., 2005c) as (1) the yield was superior to that achieved and (2) the product was obtained without the need for chromatographic purification. The formation of allyl ester was confirmed by the observation of a multiplet in the 1H NMR spectrum integrating for one proton at 5.82 ppm (=CH), in addition to, a multiplet integrating at ~5.1 ppm (=CH2) and a double of doublets integrating for two protons at 4.51 ppm (CH2), which are characteristic to the allyl group. The structure was confirmed once again by

13

C NMR assisted with the

DEPT experiment (Figure 30) which reveled three new allyl signals; two for olefinic carbons at 130.8 (=CH), 119.4 (=CH2) and one for methylene carbon at 66.5 ppm (CH2) which are characteristic for the allyl group.

Figure 30. DEPT Experiment for allyl ester substrate 31.

RESULTS AND DISCUSSION 109

RESULTS AND DISCUSSION

110

Next, a regioselective removal of the anomeric O-acetyl group was carefully performed with benzyl amine in THF which is mild enough to leave the base sensitive allyl ester unscathed. TLC analysis of the reaction mixture, after 19 h at ambient temperature, identified the formation of a material that was less mobile than the starting material 31. After silica gel purification, the C1hemiacetal 32 was obtained in moderate yield (52%) (Scheme 64). The 1H NMR spectrum of the product confirmed its formation. In particular, it showed signals corresponding to only three acetyl protons at 2.09, 2.04 and 2.02 ppm, in addition to, the upfield shift of the H-1 signal. The 1H NMR also confirmed the retention of the previously introduced allyl group by the appearance of its characteristic signals at 5.90 (=CH), ~5.16 (=CH2) and 4.61 ppm (CH2).

Scheme 64. Synthesis of the N-glycosides.

RESULTS AND DISCUSSION

111

Next, the N-(substituted-phenyl)-D-glucopyranuronyl amines were achieved via the reaction of equivalent amounts of the hemiacetal 32 as a glycosyl donor with para-bromo or para-fluroaniline as glycosyl acceptors in refluxing methanol. It is evident that the mechanism of this reaction takes place in three main steps. Firstly, the hemiacetal ring opens to give the straight chain aldo tautomer, followed by condensation reaction between the eliminated aldhydic group and the primary amine to yield an imine passing with the hemiaminal intermediate. Finally, the ring reforms to give the N-glycoside (Figure 31).

RESULTS AND DISCUSSION

112

Figure 31. Mechanism of the N-glycoside formation. In the reaction with para-flouroaniline, a pure -anomer was obtained after silica gel purification.

RESULTS AND DISCUSSION

113

Figure 32. Structure of 33. The structural assignment for 33 was based on its spectral data. In particular, the 1H NMR showed the anomeric proton as triplet at 5.39 ppm with spin-spin coupling constant corresponding to the -configuration. The other four protons of the glucopyranosyl ring resonated at 5.04, 4.73, ~4.67 and 4.14 ppm. The remaining three acetoxy groups appear as three singlets at 2.03 and 2.00 ppm. The allyl characteristic signals were observed as three multiplets at ~5.84 (=CH), ~5.25 (=CH2) and ~4.67 ppm (CH2). The four aromatic protons of the aglycon resonated at 6.88 and 6.61 ppm and each signal integrates two protons. Additionally, the 13C NMR confirmed the product. It was characterized by a signal at 85.2 ppm corresponding to the C-1 atom of the glucopyranosyl ring. The four signals appearing at 171.0, 170.0, 169.5 and 166.6 ppm are due to the three acetoxy carbonyl carbons and the allyl ester carbonyl carbon, respectively, while the three signals at 20.8, 20.6 and 20.7 ppm are attributed to the acetate methyl carbons. The six carbons of the aglycon resonated at 157.2, 140.4, 116.0, 115.8 and 115.7 ppm. Another four signals at 73.5, 72.3, 70.8 and 69.9 ppm were assigned to C-2C-5 carbons. The allyl carbons signals were observed at 131.2 (=CH), 119.5 (=CH2) and 66.7 ppm (CH2). In the case of para-bromoaniline, the ring reforming proceeds on both the siand re-faces of the imino group, consequently, a mixture of - and -Nglycosides was obtained in 70% overall yield. The anomeric ratio was 1:2 as indicated from the signal integration in the 1H NMR spectrum. Anomers were

RESULTS AND DISCUSSION

114

separated on a reversed phase silica gel column using H 2O-MeOH (20%) as an eluant and identified.

Figure 33. Structures of 34a and 34b. The structural assignment for 34a was based on its spectral data. In particular, the 1H NMR showed the anomeric proton as triplet at 5.47 ppm with spin-spin coupling constant corresponding to the -configuration. The other four protons of the glucopyranosyl ring resonated at 5.43, ~5.20, 5.12 and 4.49 ppm. The remaining three acetoxy groups appear as three singlets at 2.14, 2.09 and 2.05 ppm. The allyl characteristic signals were observed as three multiplets at ~5.90 (=CH), ~5.20 (=CH2) and 4.61 ppm (CH2). The four aromatic protons of the aglycon resonated at 7.31 and 6.82 ppm and each signal integrates two protons. The structural assignment for 34b was based on its spectral data. In particular, the 1H NMR showed the anomeric proton as triplet at 5.40 ppm with spin-spin coupling constant corresponding to the -configuration. The other four protons of the glucopyranosyl ring resonated at 5.05, 4.814.74 and 4.14 ppm. The remaining three acetoxy groups appear as three singlets at 2.04, 2.03 and 2.01 ppm. The allyl characteristic signals were observed as three multiplets at ~5.87 (=CH), ~5.20 (=CH2) and 4.60 ppm (CH2). The four aromatic protons of the aglycon resonated at 7.28 and 6.54 ppm and each signal integrates two protons.

RESULTS AND DISCUSSION

115

Next, the formed N-glycoside was subjected to acylation either by reacting with acyl chlorides or by coupling with carboxylic acids using DCC as a carboxylic group activator, but all these attempts were unsuccessful. Further, methylation was also tried using MeI in presence of pyridine, TEA and NaH separately, but also all these attempts were unsuccessful (Scheme 65).

Scheme 65. Unsuccessful acylation or methylation of the prepared Nglycosides.

EXPERIMENTAL SECTION

EXPERIMENTAL SECTION

116

Experimental Section General All starting materials and reagents were purchased from Sigma-Aldrich, BDH and Fluka and used without further purification. All solvents were either of analytical grades or dried and distilled immediately prior to use: DCM from calcium hydride, methanol from magnesium turnings and THF from sodium/benzophenone. All of the reactions were performed using oven-dried glassware. Melting points were measured on a Stuart-SMP10 melting point apparatus and are uncorrected. TLC was performed using Merck precoated Silica gel 60 F254 aluminum sheets (20 x 20 cm, layer thickness 0.2 mm) and Merck precoated Silica gel RP-C18 F254 aluminum sheets (20 x 20 cm, layer thickness 0.2 mm) and spots were visualized by UV (254 nm), KMNO 4 solution and/or charring with H2SO4-EtOH (5% v/v). Column chromatography was carried out on Silica Gel 60 (particle size 0.063–0.200 mm, 70230 mesh ASTM, Merck) and LiChroprep® RP-18 (prepacked column size B (31 x 2.5 cm), 0.0400.063 mm, Merck) using the specified eluents. All reaction products were stored refrigerated under 4 oC. NMR spectra were recorded with JEOL ECA-500, JEOL EX-270, and Varian Mercury-200BB spectrometers at room temperature in solvents given. Chemical shifts were expressed in parts per million (ppm) and reported either relative to an internal tetramethylsilane standard (TMS or relative to solvent peaks (CDCl3 = 7.2, HOD = 4.8, DMSO-d6 = 2.5) for 1H and (CDCl3 = 77.0, MeOH = 49.0) for

13

C.

Multiplicities are denoted as follows: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, ddd = double double doublet, m = multiplet, br = broad, apt = apparently.

13

C signals were assigned with the aid of DEPT.

Coupling constants (J) were reported in Hertz (Hz).

EXPERIMENTAL SECTION

117

1,2,3,4-Tetra-O-acetyl-β-D-glucopyranuronic acetic anhydride (1) D-Glucuronic acid (2 gm, 10.30 mmol) was suspended in acetic anhydride (30 mL) and stirred at 0 oC. Iodine (140 mg, 0.55 mmol) was added and the red solution was left to stir for 30 min at 0 oC and a further 2 h at room temperature. Acetic anhydride was mostly removed in vacuo and the formed solid was taken up in methylene chloride (50 mL), washed with 1M Na2S2O3 (2 x 30 mL), dried over anhydrous Na2SO4, filtered and evaporated to afford the title compound as a white solid (4.08 gm, 98%). 1H NMR (270 MHz, CDCl3): δ 5.80 (d, 1H, J1-2 = 6.9 Hz, H-1), 5.32 (m, 2H, H-3 and H-4 overlapping), 5.12 (apt t, 1H, H-2), 4.31 (d, 1H, J5-4 = 8.7 Hz, H-5), 2.28 (s, 3H, COCOOCH3), 2.13 (s, 3H, COCH3), 2.05 (s, 6H, 2 x COCH3), 2.04 (s, 3H, COCH3).

1,2,3.4-Tetra-O-acetyl-β-D-glucopyranuronic acid (2) 1,2,3,4-Tetra-O-acetyl-β-D-glucuronic acetic anhydride (1, 4.08 gm, 10.09 mmol) was dissolved in water and THF (90 mL, 1:2) and stirred overnight. The solution was concentrated and the product was extracted into methylene chloride (3 x 30 mL), the combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated in vacuo to yield the title compound as white foam (3.61 gm, 99%). 1H NMR (270 MHz, DMSO-d6): δ 6.00 (d, 1H, J1-2 = 8.1 Hz, H-1), 5.48 (apt t, 1H, H-3), 5.05 (apt t, 1H, H-4), 4.95 (apt t, 1H, H-2), 4.52 (d, 1H, J54=

8.2 Hz), 3.44 (br s, 1H, COOH), 2.08 (s, 3H, COCH3), 2.01 (s, 3H, COCH3),

1.97 (s, 6H, 2 x COCH3).

EXPERIMENTAL SECTION

118

N-Substituted-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamides General procedure for preparation of per-O-acetylated-N-substituted1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamides To a solution of 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acetic anhydride (1) in dry methylene chloride (5 mL/0.1 gm) and under argon, primary amine (1 equiv.) was added and the reaction mixture was stirred overnight at room temperature. The mixture was then diluted with methylene chloride, transferred to a separating funnel and washed with 1M HCl, saturated NaHCO 3, deionized water, dried over anhydrous Na2SO4 and filtered. The solvent was then removed and the residue was purified by column chromatography (1:3 EtOAc-petroleum ether) to afford the title compounds.

N-Benzyl-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (5) The protected precursor was prepared as a white solid (69%); mp 115-117 o

C; Rf = 0.50 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, DMSO-d6): δ

8.67 (t, 1H, NH), 7.267.17 (m, 5H, aromatic H), 5.94 (d, 1H, J1-2 = 8.4 Hz, H1), 5.40 (apt t, 1H, H-3), 5.12 (apt t, 1H, H-4), 4.95 (apt t, 1H, H-2), 4.33 (d, 1H, J5-4 = 10.0 Hz, H-5), 4.20 (ddd, 2H, CH2) 2.03, 1.97, 1.91, 1.83 (4s, 12H, 4 x COCH3);

13

C NMR (50 MHz, CDCl3): δ 169.8, 169.6, 169.2, 168.7 (4 x

COCH3), 165.8 (CONH), 137.4 (aromatic C), 128.7 (2 x aromatic CH), 127.8 (2 x aromatic CH), 127.6 (aromatic CH), 91.2 (C-1), 72.9, 71.9, 70.1, 68.9 (C-2C5), 42.9 (CH2), 20.6, 20.5 (4 x COCH3).

N-Allyl-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (6) The protected precursor was prepared as a white solid (64%); mp 182-184 o

C; Rf = 0.46 (1:1 EtOAc-petroleum ether); 1H NMR (270 MHz, CDCl3): δ 6.40

(t, 1H, NH), 5.845.81 (m, 1H, CH=CH2), 5.75 (d, 1H, J1-2 = 7.1 Hz, H-1),

EXPERIMENTAL SECTION

119

5.355.09 (m, 5H, CH=CH2, H-2, H-3 and H-4), 4.10 (d, 1H, J5-4 = 8.1 Hz, H5), 3.86 (m, 2H, OCH2-CH=CH2), 2.15, 2.08, 2.05, 2.03 (4s, 12H, 4 x COCH3).

N-(Thiazol-2-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (7) The protected precursor was prepared as a white solid (55%); mp 178-180 o

C; Rf = 0.37 (1:1 EtOAc-petroleum ether); 1H NMR (270 MHz, DMSO-d6): δ

12.49 (s, 1H, NH), 7.50 (d, 1H, aromatic H), 7.31 (d, 1H, aromatic H), 6.08 (d, 1H, J1-2 = 8.1 Hz, H-1), 5.55 (apt t, 1H, H-4), 5.34 (apt t, 1H, H-3), 5.12 (apt t, 1H, H-2), 4.61 (d, 1H, J5-4 = 8.9 Hz, H-5), 2.08, 2.03, 1.97, 1.93 (4s, 12H, 4 x COCH3).

N-(1H-Benzimidazol-2-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (8) The protected precursor was prepared as a white solid (42%); mp 142-144 o

C; Rf = 0.29 (1:1 EtOAc-petroleum ether); 1H NMR (270 MHz, DMSO-d6): δ

7.50 (s, 2H, aromatic H), 7.23 (s, 2H, aromatic H), 6.09 (d, 1H, J1-2 = 9.7 Hz, H1), 5.56 (apt t, 1H, H-4), 5.35 (apt t, 1H, H-3), 5.10 (apt t, 1H, H-2), 4.65 (d, 1H, J5-4 = 11.5 Hz, H-5), 2.10, 2.04, 1.98, 1.96 (4s, 12H, 4 x COCH3).

N-(Pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (9) The protected precursor was prepared as a white solid (51%); mp 108-110 o

C; Rf = 0.37 (1:1 EtOAc-petroleum ether); 1H NMR (270 MHz, CDCl3): δ 9.40

(s, 1H, aromatic H), 8.61 (s, 1H, NH), 8.38 (d, 1H, aromatic H), 8.27 (d, 1H, aromatic H), 5.82 (d, 1H, J1-2 = 8.1 Hz, H-1), 5.32 (m, 2H, H-3 and H-4 overlapping), 5.15 (apt t, 1H, H-2), 4.24 (d, 1H, J5-4 = 8.8 Hz, H-5), 2.15, 2.10, 2.05, 2.02 (4s, 12H, 4 x COCH3).

EXPERIMENTAL SECTION

120

N-(2-Chloro-4-nitrophenyl)-1,2,3,4-tetra-O-acetyl-β-Dglucopyranuronamide (10) The protected precursor was prepared as a white solid (61%); mp 169-170 o

C; Rf = 0.25 (1:3 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 8.90

(s, 1H, NH), 8.52 (d, 1H, aromatic H), 8.29 (s, 1H, aromatic H), 8.15 (m, 1H, aromatic H), 5.84 (d, 1H, J1-2 = 7.5 Hz, H-1), 5.35 (m, 2H, H-3 and H-4 overlapping), 5.18 (apt t, 1H, H-2), 4.28 (d, 1H, J5-4 = 9.8 Hz, H-5), 2.15, 2.10, 2.06, 2.02 (4s, 12H, 4 x COCH3).

N-(4-Bromophenyl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (11) The protected precursor was prepared as a white solid (63%); mp 173 oC (dec.); Rf = 0.52 (1:1 EtOAc-petroleum ether); 1H NMR (270 MHz, DMSO-d6): δ 10.30 (s, 1H, NH), 7.52 (br s, 4H, aromatic H), 6.06 (d, 1H, J1-2 = 8.4 Hz, H1), 5.53 (apt t, 1H, H-4), 5.26 (apt t, 1H, H-3), 5.08 (apt t, 1H, H-2), 4.46 (d, 1H, J5-4 = 9.7 Hz, H-5), 2.09, 2.03, 1.97, 1.92 (4s, 12H, 4 x COCH3).

N-(Pyridin-4-yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (12) To a solution of 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acetic anhydride in dry methylene chloride (2 mL/0.1 gm) and under argon, 4-aminopyridine (1 equiv.) was added and the reaction mixture was stirred overnight at room temperature. The solvent was evaporated and the residue was directly purified by chromatography (3% MeOH-DCM) to give the titled compound as a white solid (50%); mp 195-197 oC; Rf = 0.45 (EtOAc); 1H NMR (500 MHz, DMSOd6): δ 8.43 (s, 1H, NH), 8.41 (d, 2H, aromatic H), 7.54 (d, 2H, aromatic H), 6.02 (d, 1H, J1-2 = 8.0 Hz, H-1), 5.24 (apt t, 1H, H-4), 5.20 (apt t, 1H, H-3), 5.05 (apt t, 1H, H-2), 4.55 (d, 1H, J5-4 = 9.7 Hz, H-5), 2.04, 1.99, 1.93, 1.88 (4s, 12H, 4 x COCH3).

EXPERIMENTAL SECTION

121

N-Substituted-/ -D-glucopyranuronamides General procedure for deprotection of N-substituted-1,2,3,4-tetra-O-acetyl-

-D-glucopyranuronamides A solution of 0.05M LiOH in 2.5:1.0:0.5 MeOH-H2O-THF (6 equiv.) was added to the glycosylamide. The solution was stirred at 0 oC and the progress of the reaction was monitored by TLC (3:1 EtOAc-MeOH). After complete deprotection, the pH of the solution was adjusted to 6.0 by the addition of Amberlite IRC-50 (H+). The beads were filtered off, the solvents were removed in vacuo and the residue was purified by column chromatography (MeOHEtOAc) to give the desired compounds as an equilibrium mixture of the α- and β-anomers.

N-Benzyl-α/β-D-glucopyranuronamide (14) A portion of the intermediate (5, 183 mg, 0.65 mmol) was deprotected as described above to give 14 as a white solid (90 mg, 78%); ( 1:1); Rf = 0.57 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  7.277.20 (m, 10H,  aromatic H), 5.15 (d, 1H, J1-2 = 3.8 Hz,  H-1), 4.57 (d, 1H, J5-4 = 7.7 Hz,  H5) , 4.32 (s, 2H, CH2), 4.31 (s, 2H, CH2), 4.14 (d, 1H, J5-4 = 9.2 Hz,  H-5), 3.643.16 (6H,  H-4,  H-3 and  H-2); 13C NMR (125 MHz, D2O):  171.3, 170.5, 137.6, 128.9, 127.6, 127.3, 96.2, 92.5, 75.4, 75.3, 71.8, 71.5, 42.8.

N-Allyl-α/β-D-glucopyranuronamide (15) A portion of the intermediate (6, 110 mg, 0.47 mmol) was deprotected as described above to give 15 as a white solid (59 mg, 92%); ( 1:1); Rf = 0.57 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  5.70 (m, 2H,  CH=CH2), 5.13 (d, 1H, J1-2 = 3.1 Hz,  H-1), 5.074.99 (m, 4H,  CH=CH2), 4.09 (d,

EXPERIMENTAL SECTION

122

1H, J5-4 = 9.9 Hz,  H-5), 3.76 (d, 1H, J5-4 = 9.9 Hz,  H-5), 3.70 (s, 2H,  OCH2-CH=CH2), 3.583.37 (5H,  H-4,  H-3 and  H-2), 3.19 (s, 2H,  OCH2-CH=CH2), 3.15 (apt t, 1H,  H-2); 13C NMR (125 MHz, D2O):  171.3, 170.5, 133.2, 115.8, 96.3, 92.6, 73.9, 72.5, 71.6, 71.2, 41.4, 30.4.

N-(Thiazol-2-yl)-α/β-D-glucopyranuronamide (16) A portion of the intermediate (7, 200 mg, 0.72 mmol) was deprotected as described above to give 16 as a white solid (95 mg, 76%); ( 1:1); Rf = 0.53 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  7.36 (d, 2H,  aromatic H), 7.12 (d, 2H,  aromatic H), 5.23 (d, 1H, J1-2 = 3.9 Hz,  H-1), 4.39 (d, 1H, J5-4 = 9.9 Hz,  H-5), 4.06 (d, 1H, J5-4 = 9.2 Hz,  H-5), 3.693.23 (6H,

 H-4,  H-3 and  H-2); 13C NMR (125 MHz, D2O):  169.8, 168.2, 158.2, 137.1, 115.1, 96.4, 92.6, 75.3, 75.1, 73.7, 72.4, 71.6, 71.3, 71.2, 71.1.

N-(1H-Benzimidazol-2-yl)-α/β-D-glucopyranuronamide (17) A portion of the intermediate (8, 190 mg, 0.61 mmol) was deprotected as described above to give 17 as a white solid (79 mg, 60%); ( 1:1); Rf = 0.26 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  7.12 (m, 8H,  aromatic H), 5.10 (d, 1H, J1-2 = 3.9 Hz,  H-1), 4.49 (d, 1H, J5-4 = 7.7 Hz,  H-5), 3.94 (d, 1H, J5-4 = 9.9 Hz,  H-5), 3.583.04 (6H,  H-4,  H-3 and  H-2); 13

C NMR (125 MHz, D2O):  169.0, 168.0, 150.2, 129.1, 123.5, 110.9, 95.9,

92.2, 76.9, 76.3, 75.6, 74.0, 72.6, 72.2, 71.9, 71.4.

N-(Pyrazin-2-yl)-α/β-D-glucopyranuronamide (18) A portion of the intermediate (9, 170 mg, 0.63 mmol) was deprotected as described above to give 18 as a white solid (88 mg, 84%); ( 1:1); Rf = 0.38

EXPERIMENTAL SECTION

123

(1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  8.97 (d, 2H,  aromatic H), 8.24 (s, 2H,  aromatic H), 8.22 (m, 2H,  aromatic H), 5.24 (d, 1H, J1-2 = 3.1 Hz,  H-1), 4.34 (d, 1H, J5-4 = 9.9 Hz,  H-5), 4.02 (d, 1H, J5-4 = 10.0 Hz,  H-5), 3.693.23 (6H, H-4, H- and  H-2);

13

C NMR (125

MHz, D2O):  170.0, 169.0, 147.2, 143.1, 140.5, 137.1, 96.3, 92.6, 75.3, 73.8, 72.5, 71.7, 71.5, 71.4, 71.1.

N-(2-Chloro-4-nitrophenyl)-α/β-D-glucopyranuronamide (19) A portion of the intermediate (10, 177 mg, 0.51 mmol) was deprotected as described above to give 19 as a white solid (82 mg, 69%); ( 1:1); Rf = 0.76 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  8.33 (s, 2H,  aromatic H), 8.11 (d, 2H,  aromatic H), 8.01 (t, 2H,  aromatic H), 5.25 (d, 1H, J12

= 3.8 Hz,  H-1), 4.40 (d, 1H, J5-4 = 10.0 Hz,  H-5), 4.07 (d, 1H, J5-4 = 9.9

Hz,  H-5), 3.693.24 (6H,  H-4,  H-3 and  H-2);

13

C NMR (125

MHz, MeOD):  169.9, 143.8, 139.6, 124.5, 123.8, 123.0, 121.3, 121.1, 97.3, 93.1, 76.3, 74.9, 74.3, 73.2, 72.5, 72.0, 71.8, 70.7.

N-(4-Bromophenyl)-α/β-D-glucopyranuronamide (20) A portion of the intermediate (11, 81 mg, 0.23 mmol) was deprotected as described above to give 20 as a white solid (49 mg, 90%); ( 1:1); Rf = 0.53 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  7.39 (d, 4H,  aromatic H), 7.22 (d, 4H,  aromatic H), 5.16 (s, 1H,  H-1), 4.59 (d, 1H, J1-2 = 7.7 Hz,  H-1), 4.20 (d, 1H, J5-4 = 9.2 Hz,  H-5), 3.86 (d, 1H, J5-4 = 9.2 Hz,  H-5), 3.633.17 (6H,  H-4,  H-3 and  H-2); 13C NMR (125 MHz, D2O):  169.9, 169.0, 135.3, 132.1, 123.9, 118.3, 96.3, 92.6, 75.7, 75.3, 73.8, 72.4, 71.7, 71.5, 71.1.

EXPERIMENTAL SECTION

124

N-(Pyridin-4-yl)-α/β-D-glucopyranuronamide (21) A portion of the intermediate (12, 149 mg, 0.55 mmol) was deprotected as described above to give 21 as a white solid (61 mg, 67%); ( 1:1); Rf = 0.32 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O):  8.33 (d, 4H,  aromatic H), 7.55 (d, 4H,  aromatic H), 5.23 (d, 1H, J1-2 = 3.9 Hz,  H-1), 4.29 (d, 1H, J5-4 = 10.0 Hz,  H-5), 3.96 (d, 1H, J5-4 = 9.9 Hz,  H-5), 3.623.23 (6H,

 H-4,  H-3 and  H-2); 13C NMR (125 MHz, D2O):  170.6, 169.6, 148.8, 145.8, 115.3, 96.3, 92.6, 75.3, 73.7, 71.7, 71.3.

N-Substituted-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide General procedure for preparation of N-substituted-1,2,3-tri-O-acetyl-Δ4,5β-D-glucopyranuronamide Triethylamine (1 equiv.) was added to a stirred solution of 1,2,3,4-O-tetraβ-D-glucuronic acid (2) and ethylchloroformate (1 equiv.) in methylene chloride (5 mL/0.1 gm) cooled to -20 oC. After 15 min, the amine (1 equiv.) was added, and the mixture was allowed to warm to -5 oC over a period of 1 h. The reaction was then diluted with methylene chloride, transferred to a separating funnel and washed with 1M HCl, deionized water, saturated NaHCO3, deionized water again, dried over anhydrous Na2SO4 and filtered. The solvent was removed and the residue was purified by slow column chromatography (EtOAc-petroleum ether) to afford the title compounds.

N-Benzyl-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (22) White solid; (46%); mp 97-100 oC; Rf = 0.5 (1:1 EtOAc-petroleum ether); 1

H NMR (500 MHz, CDCl3): δ 7.247.17 (m, 5H, aromatic H), 7.05 (t, 1H,

NH), 6.21 (s, 1H, H-1), 6.18 (d, 1H, H-4), 5.17 (m, 1H, H-3), 5.04 (br s, 1H, H-

EXPERIMENTAL SECTION

125

2), 4.39 (ddd, 2H, CH2), 1.99, 1.98, 1.97 (3s, 9H, 3 x COCH3);

13

C NMR (50

MHz, CDCl3): δ 170.0, 169.6, 169.1 (3 x COCH3), 160.6 (CONH), 144.8 (C-5), 137.9 (aromatic C), 129.2 (2 x aromatic CH), 128.4 (2 x aromatic CH), 128.1 (aromatic CH), 103.8 (C-4), 88.7 (C-1), 67.5 (C-2), 64.1 (C-3), 43.9 (CH2), 21.2, 21.1 (3 x COCH3).

N-Allyl-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (23) Colorless oil; (45%); Rf = 0.44 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 6.67 (t, 1H, NH), 6.30 (d, 1H, J1-2 = 3.1 Hz, H-1), 6.25 (d, 1H, H-4), 5.85 (m, 1H, CH=CH2), 5.26 (m, 1H, H-3), 5.225.15 (m, 2H, CH=CH2), 5.14 (br s, 1H, H-2), 3.96 (m, 2H, OCH2-CH=CH2), 2.13, 2.12, 2.09 (3s, 9H, 3 x COCH3);

13

C NMR (50 MHz, CDCl3): δ 169.6, 169.1, 168.7 (3 x COCH3),

160.0 (CONH), 144.2 (C-5), 133.2 (CH=CH2), 116.9 (CH=CH2), 103.1 (C-4), 88.2 (C-1), 67.0 (C-2), 63.5 (C-3), 41.7 (OCH2-CH=CH2), 20.7, 20.6 (3 x COCH3).

N-(Thiazol-2-yl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (24) White solid; (43%); mp 103-104 oC; Rf = 0.5 (1:1 EtOAc-petroleum ether); 1

H NMR (500 MHz, CDCl3): δ 7.44 (d, 1H, aromatic H), 6.98 (d, 1H, aromatic

H), 6.346.32 (m, 2H, H-1 and H-4 overlapping), 5.22 (m, 1H, H-3), 5.12 (br s, 1H, H-2), 2.05, 2.04, 2.01 (3s, 9H, 3 x COCH3); 13C NMR (50 MHz, CDCl3): δ 170.0, 169.5, 168.8 (3 x COCH3), 158.1 (aromatic C), 157.5 (CONH), 143.6 (C5), 138.4 (aromatic C), 115.0 (aromatic C), 106.1 (C-4), 88.8 (C-1), 67.3 (C-2), 63.8 (C-3), 21.2, 21.1 (3 x COCH3).

EXPERIMENTAL SECTION

126

N-(1H-Benzimidazol-2-yl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (25) Yellow oil; (39%); Rf = 0.35 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 7.42 (m, 2H, aromatic H), 7.15 (m, 2H, aromatic H), 6.35 (d, 1H, J1-2 = 4.6 Hz, H-1), 6.27 (d, 1H, H-4), 5.23 (m, 1H, H-3), 5.12 (br s, 1H, H2), 2.06, 2.02, 2.00 (3s, 9H, 3 x COCH3).

N-(2-Chloro-4-nitrophenyl)-1,2,3-tri-O-acetyl-Δ4,5-β-Dglucopyranuronamide (26) Yellow oil; (43%); Rf = 0.85 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 9.15 (s, 1H, NH), 8.74 (d, 1H, aromatic H), 8.33 (d, 1H, aromatic H), 8.20 (dd, 1H, aromatic H), 6.44 (d, 1H, J1-2 = 4.6 Hz, H-1), 6.41 (dd, 1H, H-4), 5.32 (m, 1H, H-3), 5.20 (br s, 1H, H-2), 2.15, 2.14, 2.12 (3s, 9H, 3 x COCH3).

N-(4-Bromophenyl)-1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (27) Yellow oil; (41%); Rf = 0.74 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 8.20 (s, 1H, NH), 7.51 (d, 2H, aromatic H), 7.46 (d, 2H, aromatic H), 6.37 (m, 2H, H-1 and H-4 overlapping), 5.28 (m, 1H, H-3), 5.16 (br s, 1H, H-2), 2.14, 2.12, 2.10 (3s, 9H, 3 x COCH3);

13

C NMR (50 MHz,

CDCl3): δ 169.6, 169.2, 168.8 (3 x COCH3), 157.9 (CONH), 143.9 (C-5), 135.8 (aromatic C-Br), 132.0 (2 x aromatic CH), 121.6 (2 x aromatic CH), 117.6 (aromatic C-NH), 104.3 (C-4), 88.3 (C-1), 67.0 (C-2), 63.4 (C-3), 20.7, 20.6 (3 x COCH3).

127

EXPERIMENTAL SECTION

Ethyl 1,2,3-tri-O-acetyl-Δ4,5-β-D-glucpyranuronate (29) Colorless oil; (49%); Rf = 0.29 (1:3 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 6.41 (d, 1H, J1-2 = 2.3 Hz, H-1), 6.27 (dd, 1H, H-4), 5.23 (m, 1H, H-3), 5.15 (br s, 1H, H-2), 4.31 (q, 2H, CH2), 2.12, 2.11, 2.10 (3s, 9H, 3 x COCH3), 1.34 (t, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 169.7, 169.1, 168.4 (3 x COCH3), 161.2 (COOEt), 143.2 (C-5), 106.6 (C-4), 88.5 (C-1), 66.7 (C-2), 63.6 (C-3), 62.0 (OCH2CH3), 20.8, 20.7 (3 x COCH3), 14.13 (OCH2CH3).

N-(Pyrazin-2-yl) -1,2,3-tri-O-acetyl-Δ4,5-β-D-glucopyranuronamide (28) DBU (0.05 mL, 0.33 mmol) was added to a stirred solution of N-(Pyrazin-2yl)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronamide (9, 0.1 gm, 0.23 mmol) in dry methylene chloride (5 mL) cooled to 0 oC. After stirring for 30 min at 0 oC, the solvent was removed in vacuo and the dark brown residue was purified by column chromatography (1:3 EtOAc-petroleum ether) to afford the title compound as a colorless oil (73 mg, 81%); Rf = 0.38 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 9.51 (s, 1H, aromatic H), 8.77 (s,1H, NH), 8.33 (d, 1H, aromatic H), 8.25 (s, 1H, aromatic H), 6.36 (m, 2H, H-1 and H-4 overlapping), 5.26 (m, 1H, H-3), 5.14 (br s, 1H, H-2), 2.09, 2.07, 2.05 (3s, 9H, 3 x COCH3); 13C NMR (50 MHz, CDCl3): δ 169.5, 169.1, 168.5 (3 x COCH3), 158.2 (CONH), 147.2 (aromatic C), 143.6 (C-5), 142.3 (aromatic CH), 140.8 (aromatic CH), 137.1 (aromatic CH), 105.2 (C-4), 88.3 (C-1), 66.8 (C-2), 63.3 (C-3), 20.7, 20.6 (3 x COCH3).

N-(Substituted-phenyl)-α/β-D-glucpyranuronamines Allyl 1,2,3,4-tetra-O-acetyl-β-D-glucpyranuronate (31) Thionyl chloride (1.6 mL, 21.92 mmol) was added to a stirred solution of 1,2,3,4-O-tetra-β-D-glucuronic acid (2, 3.60 gm, 9.94 mmol) and DMF (0.4 mL,

EXPERIMENTAL SECTION

128

5.17 mmol) dissolved in dry methylene chloride (25 mL) cooled to 0 oC. The mixture was stirred for 5 min at 0 oC and then heated to reflux for 2 h. the solvents were then removed under reduced pressure and the resulting solid was dissolved in dry methylene chloride (25 mL) under argon again and cooled to 0 o

C. Allyl alcohol (0.8 mL, 11.76 mmol) and pyridine (1.92 mL, 23.74 mmol)

were added and the mixture was stirred for 30 min at 0 oC and for further 30 min at room temperature. The mixture was then transferred to a separating funnel and washed with saturated NaHCO3 (2 x 100 mL), 1M HCl (2 x 100 mL), dried over anhydrous Na2SO4, filtered and the solvent was removed to give the title compound as a slightly brown solid (3.93 gm, 98%) which was used in the next step without any further purification. An analytical sample was prepared by column chromatography purification. 1H NMR (200 MHz, CDCl3): δ 5.82 (m, 1H, CH=CH2), 5.70 (d, 1H, J1-2 = 7.4 Hz, H-1), 5.295.00 (m, 5H, H-2, H-3, H4 and CH=CH2 overlapping), 4.51 (dd, 2H, OCH2-CH=CH2), 4.15 (d, 1H, J4-5 = 8.8 Hz, H-5), 2.02 (s, 3H, COCH3), 1.93 (3s, 9H, 3 x COCH3);

13

C NMR (50

MHz, CDCl3): δ 169.7, 169.2, 169.0, 168.6 (4 x COCH3), 166.0 (COOAll), 130.8 (CH=CH2), 119.4 (CH=CH2), 91.0 (C-1), 72.6, 71.6, 69.9, 68.6 (C-2C5), 66.5 (OCH2-CH=CH2), 20.5, 20.3 (4 x COCH3).

Allyl 2,3,4-tri-O-acetyl-β-D-glucpyranuronate (32); Allyl 1,2,3,4-tetra-O-acetyl--D-glucuronate (31, 4 gm, 9.94 mmol) was stirred in dry THF (28 mL). Benzylamine (2.2 mL, 20.12 mmol) was added dropwise and the solution was left to stir overnight at room temperature. The solvent was then removed in vacuo and the residue was purified by column chromatography (1:3 EtOAc-petroleum ether) to afford the hemiacetal as red oil (1.84 gm, 52%). 1H NMR (270 MHz, CDCl3): δ 5.90 (m, 1H, CH=CH2), 5.56 (d, 1H, J1-2 = 4.4 Hz, H-1), 5.395.16 (m, 5H, H-2, H-3, H-4 and CH=CH2

EXPERIMENTAL SECTION

129

overlapping), 4.92 (d, 1H, J4-5 = 10.0 Hz, H-5), 4.61 (dd, 2H, OCH2-CH=CH2), 2.09, 2.04, 2.02 (3s, 9H, 3 x COCH3).

General procedure for preparation of N-(substituted-phenyl)-2,3,4-tri-Oacetyl-D-glucpyranuronamines A solution of allyl 2,3,4-tri-O-acetyl-D-glucuronate (32) and parasubstituted aniline (1.5 equiv.) dissolved in anhydrous methanol (5 mL/0.1 gm) was refluxed for 25 h. The solvent was then remved under diminished pressure and the residue was purified by column chromatography (1:10 EtOAc-petroleum ether) to give the title compounds. In the case of bromo-substituted phenyl, a mixture of  anomers in a ratio of about 1:2 was obtained which were separated by C-18 reversed phase chromatography (20% H2O-MeOH) and identified, while in the case of fluoro-substituted phenyl, a pure -anomer was obtained.

N-(4-Fluorophenyl)-2,3,4-tri-O-acetyl- -D-glucpyranuronamine (33) White solid (63 %); mp 127 oC; Rf = 0.23 (1:4 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 6.88 (t, 2H, aromatic H), 6.61 (m, 2H, aromatic H), 5.84 (m, 1H, CH=CH2), 5.39 (apt t, 1H, J1-2 = J1-NH = 9.2 Hz, H-1), 5.325.20 (m, 3H, NH and CH=CH2 overlapping), 5.04 (apt t, 1H, H-4), 4.73 (apt t, 1H, H3), 4.67 (m, 3H, H-2 and OCH2-CH=CH2 overlapping), 4.14 (d, 1H, J4-5 = 10.0 Hz, H-5), 2.03 (s, 6H, 2 x COCH3), 2.00 (s, 3H, COCH3); 13C NMR (125 MHz, CDCl3): δ 171.0, 170.0, 169.5 (3 x COCH3), 166.6 (COOAll), 157.2 (aromatic C-F, JC-F = 229.9 Hz), 140.4 (aromatic C-NH), 131.2 (CH, CH=CH2), 119.5 (CH=CH2), 116.0, 115.8, 115.7 (4 x aromatic CH), 85.2 (C-1), 73.5, 72.3, 70.8, 69.9 (C-2C-5), 66.7 (OCH2-CH=CH2), 20.8, 20.6, 20.7 (3 x COCH3).

EXPERIMENTAL SECTION

130

N-(4-Bromophenyl)-2,3,4-tri-O-acetyl--D-glucpyranuronamine (34a) White solid (21 %); Rf = 0.42 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 7.31 (d, 2H, aromatic H), 6.82 (d, 2H, aromatic H), 5.90 (m, 1H, CH=CH2), 5.47 (apt t, 1H, J1-2 = J1-NH = 3.0 Hz, H-1), 5.43 (apt t, 1H, H-4), 5.355.23 (m, 2H, H-3 and CH=CH2 overlapping), 5.12 (m, 1H, H-2), 4.61 (m, 2H, OCH2-CH=CH2), 4.49 (m, 2H, H-5 and NH overlapping), 2.14, 2.09, 2.05 (3s, 9H, 3 x COCH3).

N-(4-Bromophenyl)-2,3,4-tri-O-acetyl- -D-glucpyranuronamine (34b) White solid (49 %); Rf = 0.42 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3): δ 7.28 (d, 2H, aromatic H), 6.54 (d, 2H, aromatic H), 5.87 (m, 1H, CH=CH2), 5.40 (apt t, 1H, J1-2 = J1-NH = 9.8 Hz, H-1), 5.335.10 (m, 3H, NH and CH=CH2 overlapping), 5.05 (apt t, 1H, H-4), 4.814.74 (m, 2H, H-3 and H-2 overlapping), 4.60 (m, 2H, OCH2-CH=CH2), 4.14 (d, 1H, J4-5 = 9.8 Hz, H-5), 2.04, 2.03, 2.01 (3s, 9H, 3 x COCH3).

SPECTRAL DATA

SPECTRAL DATA Spectral Data

131

132

SPECTRAL DATA

SPECTRAL DATA

133

134

SPECTRAL DATA

SPECTRAL DATA

135

136

SPECTRAL DATA

SPECTRAL DATA

137

138

SPECTRAL DATA

SPECTRAL DATA

139

140

SPECTRAL DATA

SPECTRAL DATA

141

142

SPECTRAL DATA

SPECTRAL DATA

143

144

SPECTRAL DATA

SPECTRAL DATA

145

146

SPECTRAL DATA

SPECTRAL DATA

147

148

SPECTRAL DATA

SPECTRAL DATA

149

150

SPECTRAL DATA

SPECTRAL DATA

151

152

SPECTRAL DATA

SPECTRAL DATA

153

154

SPECTRAL DATA

SPECTRAL DATA

155

156

SPECTRAL DATA

SPECTRAL DATA

157

158

SPECTRAL DATA

SPECTRAL DATA

159

160

SPECTRAL DATA

SPECTRAL DATA

161

162

SPECTRAL DATA

SPECTRAL DATA

163

164

SPECTRAL DATA

SPECTRAL DATA

165

166

SPECTRAL DATA

SPECTRAL DATA

167

168

SPECTRAL DATA

SPECTRAL DATA

169

170

SPECTRAL DATA

SPECTRAL DATA

171

172

SPECTRAL DATA

SPECTRAL DATA

173

174

SPECTRAL DATA

SPECTRAL DATA

175

176

SPECTRAL DATA

SPECTRAL DATA

177

178

SPECTRAL DATA

SPECTRAL DATA

179

180

SPECTRAL DATA

ANTITUMOR ACTIVITY

ANTITUMOR ACTIVITY

181

Antitumor Activity The compounds were divided into three groups: the 1 st group represents acetylated derivaties, the 2nd group represents de-acetylated derivatives and the 3rd one represents 4,5-unsaturated derivatives and their activities were represented in the tables 4,5 and 6, respectively.

Assay for cytotoxic activity: human cell lines The following three human cancer cell lines were used in these experiments: the human renal adenocarcinoma (TK-10), the human breast adenocarcinoma (MCF-7) and the human melanoma (UACC-62) cell lines. The human tumour cytotoxicities were determined following protocols established by NCI (Monks et al., 1991). TK-10, MCF-7, UACC-62 cell lines were cultured in RPMI 1640 medium (BioWhittaker®) containing 20% foetal calf serum (FCS), 2 mmol/L Lglutamine, 100 U/mL penicillin and 100 g/mL streptomycin. All cell lines were maintained at 37 oC in a 5% CO2 atmosphere with 95% humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. According to their growth profiles, the optimal plating densities of each cell line was determined (15 x 103, 5 x 103 and 100 x 103 cells/well for TK-10, MCF-7 and UACC-62, respectively) to ensure exponential growth throughout the experimental period and to ensure a linear relationship between absorbance at 492 nm and cell number when analyzed by the sulphorhodamine B (SRB) assay.

Testing procedure and data processing The sulphorhodamine B (SRB) assay was used in this study to assess growth inhibition. This colorimetric assay estimates cell number indirectly by staining total cellular protein with the SRB dye. For the assay, cells were detached with

182

ANTITUMOR ACTIVITY

0.1% trypsin-EDTA (Sigma Chemical Co.) to make single-cell suspensions, and viable cells were counted using a Coulter counter and diluted with medium to give final densities of 15 x 104, 5 x 104 and 100 x 104 cells/mL for TK-10, MCF7 and UACC-62, respectively. One hundred microlitres per well of these cell suspensions was seeded in 96-well microtiter plates and incubated to allow for cell attachment. After 24 h, the cells were treated with the serial concentrations of the synthesized compounds. They were initially dissolved in an amount of 100% DMSO (40 mmol/L) and further diluted in medium to produce five concentrations. One hundred microlitres per well of each concentration was added to the plates to obtain final concentration of 10 −4, 10−5, 10−6, 10−7 and 10−8 M for the synthesized compounds. The DMSO concentration for the tested dilutions was not greater than 0.25% (v/v), the same as in solvent control wells. The final volume in each well was 200 l. The plates were incubated for 48 h.

Sulphorhodamine B method After incubating for 48 h, adherent cell cultures were fixed in situ by adding 50 l of cold 50% (w/v) trichloroacetic acid (TCA) and incubating for 60 min at 4 oC. The supernatant was then discarded and the plates washed five times with deionised water and dried. One hundred microlitres of SRB solution (0.4% w/v in 1% acetic acid) was added to each microtiter well and the culture was incubated for 30 min at room temperature. Unbound SRB was removed by washing five times with 1% acetic acid then the plates were air-dried. Bound stain is solubilised with Tris buffer and the optical densities (OD) were read on an automated spectrophotometric plate reader at a single wavelength of 492 nm. At the end, GI50 values (concentrations required to inhibit cell growth by 50%), TGI (concentration resulting in total growth inhibition) and LC 50 (concentration causing 50% of net cell killing) were calculated according to the previously described protocols (Monks et al., 1991). Two or three experiments were carried

ANTITUMOR ACTIVITY

183

out for each compound. The data are given as the mean of two or three different assays ± S.E.M.

ANTITUMOR ACTIVITY

184

Table 4. Group I concentrations (µM) required to cause different inhibitions. Cell Line

Compound number

Inhibition parameter

TK-10

MCF-7

UACC-62

5

GI50

18.06 ± 2.64

>100

32.10 ± 6.27

TGI

>100

>100

9.97 ± 2.73

LC50

>100

>100

>100

GI50

0.21 ± 0.13

>100

>100

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

89.38 ± 4.92

>100

>100

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

92.74 ± 27.10

>100

79.53

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

23.88 ± 6.49

98.17 ± 23.75 0.42 ± 0.50

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

3.82 ± 1.09

>100

3.40 ± 1.00

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

0.82 ± 0.03

18.55 ± 5.82

0.87 ± 0.41

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

0.67 ± 0.08

>100

0.21 ± 0.03

TGI

>100

>100

5.49 ± 2.07

LC50

>100

>100

39.35 ± 9.23

GI50

20.49 ± 2.89

4.50 ± 0.13

15.66 ± 4.13

TGI

51.45 ± 7.00

>100

56.74 ± 11.82

LC50

>100

>100

>100

6

7

8

9

10

11

12

31

ANTITUMOR ACTIVITY 34

185

GI50

42.75 ± 8.22

58.79 ± 0.63

21.19 ± 0.91

TGI

88.99 ± 20.06

>100

79.33 ± 8.01

LC50

>100

>100

>100

The range of doses assayed was 10−4, 10−5, 10−6, 10−7 and 10−8 M. Results are mean ± S.E.M. (n = 3).

ANTITUMOR ACTIVITY

186

Table 5. Group II concentrations (µM) required to cause different inhibitions. Cell Line

Compound number

Inhibition parameter

TK-10

MCF-7

UACC-62

14

GI50

0.05 ± 0.11

>100

>100

TGI

16.15 ± 3.75

>100

>100

LC50

>100

>100

>100

GI50

47.60 ± 6.92

>100

50.33 ± 8.27

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

24.88 ± 1.50

8.17

0.049 ± 0.02

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

5.51 ± 0.04

>100

9.42 ± 2.98

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

19.77 ± 2.91

36.01

0.08 ± 0.03

TGI

43.56 ± 5.16

>100

3.11 ± 1.07

LC50

95.95 ± 23.73

>100

35.17 ± 7.31

GI50

10.72 ± 0.90

90.34

2.71 ± 0.35

TGI

60.02 ± 11.49

>100

8.09 ± 3.01

LC50

90.34 ± 15.83

>100

24.13 ± 7.81

GI50

17.05 ± 4.01

0.56

0.76 ± 0.11

TGI

>100

>100

54.19 ± 10.00

LC50

>100

>100

>100

GI50

0.23 ± 0.12

0.19

15.20 ± 3.56

TGI

1.29 ± 0.08

>100

>100

LC50

7.27 ± 0.17

>100

>100

15

16

17

18

19

20

21

The range of doses assayed was 10−4, 10−5, 10−6, 10−7 and 10−8 M. Results are mean ± S.E.M. (n = 3).

ANTITUMOR ACTIVITY

187

Table 6. Group III concentrations (µM) required to cause different inhibitions. Cell Line

Compound number

Inhibition parameter

TK-10

MCF-7

UACC-62

22

GI50

84.96 ± 13.48

>100

24.38 ± 2.01

TGI

>100

>100

>100

LC50

>100

>100

>100

GI50

0.23 ± 0.01

>100

0.036 ± 0.01

TGI

>100

>100

0.71 ± 0.03

LC50

>100

>100

15.06 ± 1.97

GI50

1.51 ± 0.80

0.32 ± 0.31

0.803 ± 0.21

TGI

3.44 ± 0.10

1.06 ± 0.10

2.57 ± 0.18

LC50

7.85 ± 1.18

3.55 ± 0.18

8.22 ± 1.92

GI50

6.00 ± 0.09

>100

1.53 ± 0.03

TGI

>100

>100

11.46 ± 2.81

LC50

>100

>100

85.67 ± 11.50

GI50

32.83 ± 6.81

25.71 ± 2.77

16.09 ± 3.17

TGI

72.13 ± 12.07

>100

36.31 ± 7.68

LC50

>100

>100

81.91 ± 16.00

GI50

14.43 ± 3.31

99.73 ± 21.63 2.14 ± 0.12

TGI

34.63 ± 8.22

>100

8.55 ± 0.50

LC50

83.05 ± 6.19

>100

34.12 ± 6.18

GI50

2.38 ± 0.04

3.04 ± 0.09

0.62 ± 0.02

TGI

7.71 ± 1.50

80.87 ± 19.11 4.67 ± 0.99

LC50

25.00 ± 5.16

>100

24.75 ± 4.75

GI50

0.14 ± 0.05

9.03 ± 1.11

0.18 ± 0.02

TGI

>100

>100

>100

LC50

>100

>100

>100

23

24

25

26

27

28

29

The range of doses assayed was 10−4, 10−5, 10−6, 10−7 and 10−8 M. Results are mean ± S.E.M. (n = 3).

ANTITUMOR ACTIVITY

188

Biological Activity Results and Disscusion The newly synthesized compounds were examined for in vitro activity against several human cancer cell lines, including renal adenocarcinoma (TK10), human breast adenocarcinoma (MCF-7) and human melanoma (UACC-62). Three response parameters (GI50, TGI, and LC50) were calculated for each cell line. The GI50 value (growth inhibitory activity) corresponds to the concentration of the compounds causing 50% decrease in net cell growth, the TGI value (cytostatic activity) is the concentration of the compounds resulting in total growth inhibition and the LC50 value (cytotoxic activity) is the concentration of the compounds causing net 50% loss of initial cells at the end of the incubation period. Tables 4-6 lists the GI50, TGI and LC50 values obtained for acetylated (group I), deacetylated (group II) and 4,5-unsaturated (group III) compounds, respectively. Regarding the activities of the acetylated derivatives 5-12, 31 and 34 in table 4, compounds 9, 11, 31 and 34 showed a growth inhibitory activity on the three mentioned tumoural cell lines at the tested doses. However, compounds 5, 8, 10 and 12 showed only an inhibitory effect against the growth of the TK-10 and UACC-62 cells, but did not posses any activity on the MCF-7 cell lines. The compounds 6 and 7 only affected in the renal adenocarcinoma (TK-10) cell line. The compounds 6 and 10-12 were the most cytotoxic on TK-10 (GI50 = 0.21, 3.82, 0.82 and 0.67 µM, respectively). On the other hand, the most compounds which were affected on the MCF-7 cell line were 11 and 31 (GI50 =18.55 and 4.50 µM, respectively). However, compounds 9-12 were the most effective on the UACC-62 cell line (GI50 = 0.42, 3.40, 0.87 and 0.21 µM, respectively). The growth of TK-10 cells was totally inhibited by 31 and 34 (TGI = 51.45 and 88.99 µM, respectively). At the same time, compounds 5, 12, 31 and 34 were showed cytostatic activity (TGI) against the UACC-62 cells with TGI values of 9.97, 5.49, 56.74 and 79.33 µM, respectively. However, all the tested

ANTITUMOR ACTIVITY

189

compounds did not demonstrate any cytostatic activity against the MCF-7 cell line. Furthermore, only 12 produced a cytotoxic activity (LC50) against MCF-7 cell line at the dose 39.35 µM. Regarding the activities of the deacetylated derivatives 15-21 in table 5, compounds 16 and 18-21 showed a growth inhibitory activity (GI50) on the three mentioned tumoural cell lines at the tested doses. However, compounds 15 and 17 showed only an inhibitory effect against the growth of the TK-10 and UACC-62 cells, but did not posses any activity on the MCF-7 cell lines. The compound 14 only affected in the renal adenocarcinoma (TK-10) cell line. The compounds 14, 17 and 21 were the most cytotoxic on TK-10 (GI50 = 0.05, 5.51 and 0.23 µM, respectively). On the other hand, the most compounds which were affected on the MCF-7 cell line were 20 and 21 (GI50 = 0.56 and 0.19 µM, respectively). However, compounds 16 and 18-20 were the most effective on the UACC-62 cell line (GI50 = 0.049, 0.08, 2.71 and 0.76 µM, respectively). The growth of TK-10 cells was totally inhibited by 14, 18, 19 and 21 (TGI = 16.15, 43.56, 60.02 and 1.29 µM, respectively). At the same time, compounds 18-20 were showed cytostatic activity against the UACC-62 cells with TGI values of 3.11, 8.09 and 54.19 µM, respectively. However, none the tested deacetylated compounds showed neither cytostatic nor cytotoxic activities against the MCF-7 cells. Regarding the activities of the 4,5-unsaturated derivatives 22-29 in table 6, compounds 24 and 26-29 showed a growth inhibitory activity on the three mentioned tumoural cell lines at the tested doses. However, compounds 22, 23 and 25 showed only an inhibitory effect against the growth of the TK-10 and UACC-62 cells, but did not posses any activity on the MCF-7 cell lines. The compounds 23, 24, 28 and 29 were the most cytotoxic on TK-10 (GI50 = 0.23, 1.51, 2.38 and 0.14 µM, respectively). On the other hand, the most compounds which were affected on the MCF-7 cell line were 24 and 28 (GI50 = 0.32 and

190

ANTITUMOR ACTIVITY

3.04 µM, respectively). However, all the tested 4,5-unsaturated derivatives, with exception of compounds 22 and 26, showed a high growth inhibitory activity against the UACC-62 cell line. The growth of TK-10 cells was totally inhibited by 24 and 26-28 (TGI = 3.44, 72.13, 34.63 and 7.71 µM, respectively). At the same time, all the tested 4,5-unsaturated derivatives, with exception of compounds 22 and 29, showed a cytostatic and a cytotoxic activities against the UACC-62 cell line. Compounds 24 and 28 demonstrated a cytostatic activity against the MCF-7 cell line with TGI values of 1.06 and 80.87 µM, respectively. Furthermore, only 24 produced a cytotoxic activity (LC50) against MCF-7 cell line at the dose of 3.55 µM.

ANTITUMOR ACTIVITY

191

Structure-Activity Relationship (SAR) By observing the activity of the three tested groups of compounds, the relationship between the structure variation and the activity can be concluded. The effect of deacetylation on the activity was established by comparing the activity of group I compounds (Table 4) with their corresponding derivatives in group II (Table 5). It was obvious that acetyl groups removal resulted in enhancement in the activity for all compounds against the three tumoural cell lines with the exception of the allyl, benzyl and benzimidazolyl derivatives against MCF-7 cell line, the allyl, p-bromophenyl and 2-chloro-4-nitrophenyl derivatives against TK-10 cell line and the benzyl and pyridyl derivatives against UACC-62 cell line, in which removal of the acetyl groups is accompanied by reduction in potency. On the other hand, the effect of double bond introduction between C-4 and C-5 was concluded by comparing the results of group I compounds (Table 4) with their corresponding derivatives in group III (Table 6). Considrable enhancement in the activity for all compounds against the three tested cell lines was observed with the exception of the allyl, p-bromophenyl and 2-chloro-4nitrophenyl for TK-10 cell line, the p-bromophenyl and 2-chloro-4-nitrophenyl derivatives for UACC-62 cell line and the p-bromophenyl derivative for MCF-7 cell line, in which the activity was reduced. The effect of separation of the aromatic ring from the amide linkage by a methylene group can be obtained by comparing the activity of the benzyl derivative in each group (5, 14 or 22) with the other phenyl derivatives in the same group. It was obvious that the activity of the three groups against the MCF-7 and UACC-62 cell lines was sufficiently reduced, while, in the case of TK-10 cell line, the activity of group I and II compounds were the only to be reduced

ANTITUMOR ACTIVITY

192

The effect of replacement of allyl group with benzyl group can be obtained by comparing the activities of the allyl derivative (6, 15 or 23) in each group.with the benzyl derivative (5, 14 or 22) in the same group. Replacement of the allyl group with a benzyl group in the unsaturated series resulted in a sufficient decrease in the activity against both TK-10 and UACC-62 cell lines, while in the acetylated series the activity increased against UACC-62 cells and decreased against TK-10 cells, which is inverted in the non-acetylated series. All the allyl and benzyl derivatives were totally inactive against the MCF-7 cell line, By comparing the activities of the benzimidazolyl derivative (8, 17 or 25) in each group and the pyranzinyl derivative (9, 18 or 28) in the same group, the effect of expanding the five membered ring into a six membered ring was concluded in which; the activities were efficiently increased for all groups against the three tested tumoral cell lines except for the group II against TK-10 cell line. Further, by comparing the activity of the benzimidazolyl derivative (8, 17 or 25) in each group with the thiazolyl derivative (7, 16 or 24) in the same group, it was obvious that the replacement of the NH with S atom in the five membered ring also enhanced the activity in most cases against the three tumoral cell lines. Also, during the evaluation of the derivatives prepared, it was noted that, for the six membered aromatic derivatives, C-4 position seems to play an important role in activity in which all the synthesized phenyl derivatives having C-4 substituent (Br, NO2) exhibited high activity in most cases with the bromo substituent better than the nitro substituent. Also, the pyridyl derivatives, in which the C-4 was replaced by a nitrogen atom, exhibit a high activity in most cases and if compared with the activity of the pyrazinyl derivatives.

REFERENCES

REFERENCES

193

References

Adamczyk, M.; Chen, Y.-Y.; Fishpaugh, J. R. (1992). Synthesis of estriol 16-4',5'-dehydroglucuronide. Org. Prep. Proced. Int., 24, 546-548. Al-Maharik, N.; Botting, N. P. (2006). A facile synthesis of isoflavone 7-Oglucuronides. Tetrahedron Lett., 47, 8703-8706. Alföldie, J.; Palovkl, R.; Peciar, C.; Hirsch, J.; Kovác, P. (1975). Conformation of some derivatives of methyl 4-deoxy- and -L-threo-hex-4enopyranuronate. Carbohydr. Res., 44, 133-137. Alonen, A.; Gartman, M.; Aitio, O.; Finel, M.; Yli-Kauhaluoma, J.; Kostiainen, R. (2009). Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides. Eur. J. Pharm. Sci., 37, 581-587. Anderson, D. M. W.; Garbutt, S.; Smith, J. F. (1962). Studies on uronic acid materials V: The thermal decarboxylation method of analysis. Talanta, 9, 689697. Anderson, D. M. W.; Garbutt, S.; Zaidi, S. S. H. (1963). Studies on uronic acid materials: The simultaneous determination of uronic acid and alkoxyl groups in polysaccharides by reflux with hydriodic acid. Anal. Chim. Acta, 29, 39-45. Arewång, C. J.; Lahmann, M.; Oscarson, S.; Tidén, A.-K. (2007). Synthesis of urine drug metabolites: glucuronic acid glycosides of phenol intermediates. Carbohydr. Res., 342, 970-974. Avalos, M.; Babiano, R.; Carretero, M. J.; Cintas, P.; Higes, F. J.; Jimenez, J. L.; Palacios, J. C. (1998). The structure of glycosyl amides: A combined study by NMR spectroscopy, x-ray crytallography, and computational chemistry. Tetrahedron, 54, 615-628. Avalos, M.; Babiano, R.; Duran, C. J.; Jimenez, J. L.; Palacios, J. C. (1992). NMR studies of sugar amides and thioamides. J. Chem. Soc., Perkin Trans. 2, 2205-2215.

194

REFERENCES

Bazin, H. G.; Capila, I.; Linhardt, R. J. (1998). Conformational study of synthetic Δ4-uronate monosaccharides and glycosaminoglycan-derived disaccharides. Carbohydr. Res., 309, 135-144. Bechthold, A.; Fernández, J. A. S. (1999). Combinatorial Chemistry: Synthesis, Analysis, Screening.; Jung, G., ed.; Wiley-VCH: Weinheim, pp. 381439. BeMiller, J. N.; Kumari, G. V. (1972). beta-Elimination in uronic acids: Evidance for an E1cB mechanism. Carbohydr. Res., 25, 419-428. Bishop, C. T. (1953). Isolation of the aldobiuronic acid, 3-(xylopyranosyl)-D-glucuronopyranoside, from wheat straw holocellulose and synthesis of its -isomer. Can. J. Chem., 31, 134-144. Blattner, R.; Ferrier, R. J.; Tyler, P. C. (1980). Unsaturated carbohydrates. Part 22. Alkenes from 5-bromohexopyranose derivatives. J. Chem. Soc., Perkin Trans. 1, 1535-1540. Bollenback, G. N.; Long, J. W.; Benjamin, D. G.; Lindquist, J. A. (1955). The synthesis of aryl-D-glucopyranosiduronic acids. J. Am. Chem. Soc., 77, 3310-3315. Bouvier, E.; Thirot, S.; Schmidt, F.; Monneret, C. (2003). A new paclitaxel prodrug for use in ADEPT strategy. Org. Biomol. Chem., 1, 3343-3352. Bowering, W. D. S.; Timell, T. E. (1960). Synthesis and characterization of 2-O-(-D-glucopyranosyluronic acid)-D-xylopyranose. J. Am. Chem. Soc., 82, 2827-2830. Bowkett, E. R.; Harding, J. R.; Maggs, J. L.; Park, B. K.; Perriea, J. A.; Stachulskia, A. V. (2007). Efficient synthesis of 1-O-acyl glucuronides via selective acylation of allyl or benzyl D-glucuronate. Tetrahedron, 63, 75967605. Bray, H. G. (1953). D-Glucuronic acid in metabolism. In Advances in Carbohydrate Chemistry and Biochemistry.; Hudson, C. S.; Wolfrom, M. L., eds.; Academic Press, Inc.: New York, Vol. 8, pp. 251-275.

REFERENCES

195

Brown, R. T.; Carter, N. E.; Mayalarpa, S. P.; Scheinmannb, F. (2000). A simple synthesis of morphine-3,6-di--D-glucuronide. Tetrahedron, 56, 75917594. Bugianesi, R.; Shen, T. Y. (1971). A chemical synthesis of 1-Oindomethacin--d-glucosyluronic acid. Carbohydr. Res., 19, 179-187. Burchell, B.; Brierley, C. H.; Rance, D. (1995). Specifity of human UDPglucuronosyltransferases and xenobiotic glucuronidation. Life Sci., 57, 819-831. Carlsson, B.; Samuelson, O. (1969). Isomerization of D-glucuronic acid in neutral aqueous solution. Acta Chem. Scand., 23, 261-267. Casati, S.; Ottria, R.; Ciuffreda, P. (2009). 17- and 17-boldenone 17glucuronides: Synthesis and complete characterization by 1H and 13C NMR. Steroids, 74, 250-255. Chambers, D. J.; Evansb, G. R.; Fairbanks, A. J. (2005). An approach to the synthesis of -(1→6)-C-disaccharides by tandem Tebbe methylenation and Claisen rearrangement. Tetrahedron, 61, 7184-7192. Chen, X.; Liu, Z.-Y.; Zhang, J.-B.; Zheng, W.; Kowal, P.; Wang, P. G. (2002). Reassembled biosynthetic pathway for large-scale carbohydrate synthesis: -Gal epitope producing superbug. ChemBioChem, 3, 47-53. Chiba, T.; Sinaÿ, P. (1986). Application of a radical reaction to the synthesis of L-iduronic acid derivatives from D-glucuronic acid analogues. Carbohydr. Res., 151, 379-389. Cipolla, L.; Fernandes, M. R.; Gregori, M.; Airoldi, C.; Nicotra, F. (2007). Synthesis and biological evaluation of a small library of nojirimycin-derived bicyclic iminosugars. Carbohydr. Res., 342, 1813-1830. Codée, J. D. C.; Stubba, B.; Schiattarella, M.; Overkleeft, H. S.; van Boeckel, C. A. A.; van Boom, J. H.; van der Marel, G. A. (2005). A modular strategy toward the synthesis of heparin-like oligosaccharides using monomeric building blocks in a sequential glycosylation strategy. J. Am. Chem. Soc., 127, 3767-3773.

196

REFERENCES

Collins, P. M. (2006). Dictionary of Carbohydrates with CD-ROM.; Chapman & Hall/CRC, Taylor & Francis Group, Boca Raton: London-New York. Compain, P.; Martin, O. R. (2007). Iminosugars, from synthesis to therapeutic applications.; J. Wiley: Weinheim. Csuros, Z.; Petro, J.; Fogassy, E.; Lengyel, A. (1974a). Catalytic air oxidation of the primary hydroxyl groups of di-O-isopropylidene hexoses. Period. Polytech. Chem. Eng., 18, 155-165. Csuros, Z.; Soos, R.; Fogassy, E.; Szabo, G. T.; Molnar, V. (1974b). Catalytic oxidation with air of 1,2-O-isopropylidene--glucofuranose and Dglucose. Period. Polytech. Chem. Eng., 18, 229-247. De Souza, M. V. N. (2006). TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) - An important reagent in alcohol oxidation and its application in synthesis of natural products. Mini-Rev. Org. Chem., 3, 155-165. del Ruiz Ruiz, M. C.; Amer, H.; Stanetty, C.; Beseda, I.; Czollner, L.; Shah, P.; Jordis, U.; Kueenburg, B.; Claßen-Houben, D.; Hofinger, A.; Kosma, P. (2009). Efficient synthesis of glycyrrhetinic acid glycoside/glucuronide derivatives using silver zeolite as promoter. Carbohydr. Res., 344, 1063-1071. DeMesmaeker, A.; Hoffmann, P.; Ernst, B. (1989). A new protected form of glucuronic acid for the synthesis of labile 1-O-acyl-β-D-glucuronides. Tetrahedron Lett., 30, 3773-3776. Dinkelaar, J.; de Jong, A. R.; van Meer, R.; Somers, M.; Lodder, G.; Overkleeft, H. S.; Codée, J. D. C.; van der Marel, G. A. (2009). Stereodirecting effect of the pyranosyl C-5 substituent in glycosylation reactions. J. Org. Chem., 74, 4982-4991. Durham, T. B.; Miller, M. J. (2002). Conversion of glucuronic acid glycosides to novel bicyclic -lactams. Org. Lett., 4, 135-138. Dutton, G. J. (1966). Glucuronic Acid, Free and Combined; Academic Press: New York and London. Dutton, G. J. (1980). Glucuronidation of Drugs and Other Compounds.; CRC Press: Boca Raton, Florida.

REFERENCES

197

Dutton, G. T.; Burchell, B. (1977). New aspects of glucuronidation. In Progress Drug Metabolism and Disposition.; Bridges, J. W.; Chasseaud, L. F., eds.; J. Wiley: London, Vol. 2, pp. 1-70. Dwek, R. A. (1996). Glycobiology: Toward understanding the function of sugars. Chem. Rev., 96, 683-720. Edye, L. A.; Meehan, G. V.; Richards, G. N. (1991). Platinum catalysed oxidation of sucrose. J. Carbohydr. Chem., 10, 11-23. Edye, L. A.; Meehan, G. V.; Richards, G. N. (1994). Influence of temperature and pH on the platinum catalysed oxidation of sucrose. J. Carbohydr. Chem., 13, 273-283. El-Alaoui, A.; Schmidt, F.; Monneret, C.; Florent, J.-C. (2006). Protecting groups for glucuronic acid: Application to the synthesis of new Paclitaxel (Taxol®) derivatives. J. Org. Chem., 71, 9628-9636. Engstrom, K. M.; Henry, R. F.; Marsden, I. (2007). Synthesis of the glucuronide metabolite of ABT-751. Tetrahedron Lett., 48, 1359-1362. Ernst, B.; Hart, G. W.; Sinay, P. (2000). Carbohydrates in Chemistry and Biology.; Wiley-VCH: Weinheim. Falkowski, L.; Stefanska, B.; Bylec, E.; Kolodziejczyk, P. (1980). An improved method of synthesis of glucuronamides. Pol. J. Chem., 54, 559-603. Feizi, T. J. (1993). Oligosaccharides that mediate cell-cell adhesion. Curr. Opin. Struct. Biol., 3, 701-710. Ferrier, R. J.; Furneaux, R. H. (1977). C-5 Bromination of some glucopyranuronic acid derivatives. J. Chem. Soc., Perkin Trans. 1, 1996-2000. Fischer, B.; Nudelman, A.; Ruse, M.; Herzig, J.; Gottlieb, H. E. (1984). A novel method for stereoselective glucuronidation. J. Org. Chem., 49, 4988-4993. Fischer, F. G.; Schmidt, H. (1959). Die epimerisierung der uronsäuren. Chem. Ber., 92, 2184-2188.

198

REFERENCES

Florio, P.; Thomson, R. J.; Alafaci, A.; Abo, S.; von Itzstein, M. (1999). Synthesis of Δ4--D-glucopyranosiduronic acids as mimetics of 2,3-unsaturated sialic acids for sialidase inhibition. Bioorg. Med. Chem. Lett., 9, 2065-2068. Forbes, C. L.; Franck, R. W. (1999). Improved version of the Fischer-Zach synthesis of glycals: Vitamin B-12 catalyzed reductive elimination of glycosyl bromides. J. Org. Chem., 64, 1424-1425. Francisco, C. G.; Gonzalez, C. C.; Suárez, E. (1997). A mild oxidative decarboxylation of carbohydrate acids. Tetrahedron Lett., 38, 4141-4144. Fraser-Reid, B. O.; Tatsuta, K.; Thiem, J. (2008). Oxidation, reduction, and deoxygenation. In Glycoscience.; Madsen, R., ed.; Springer: Berlin, Heidelberg, pp. 179-225. Furuhata, K. (2004). Chemistry of N-acetylneuraminic acid (Neu5Ac). Trends Glycosci. Glycotechnol., 16, 143-169. Garegg, P. J.; Olsson, L.; Oscarson, S. (1995). Synthesis of methyl (ethyl 2O-acyl-3,4-di-O-benzyl-1-thio-beta-D-glucopyranosid)uronates and evaluation of their use as reactive beta-selective glucuronic acid donors. J. Org. Chem., 60, 2200-2204. Gärtner, P.; Novak, C.; Einzinger, C.; Felzmann, W.; Knollmüller, M.; Gmeiner, G.; Schänzer, W. (2003). A facile and high yielding synthesis of 2,2,3,4,4-d5-androsterone--D-glucuronide - An internal standard in dope analysis. Steroids, 68, 85-96. Glänzer, B. I.; Györgydeak, Z.; Bernet, B.; Vasella, A. (1991). Analogues of sialic acids as potential sialidase inhibitors. Synthesis of C6 and C7 analogues of N-acetyl-6-amino-2,6-dideoxyneuraminic acid. Helv. Chim. Acta, 74, 343369. Goebel, W. F.; Babers, F. H. (1934). Derivatives of glucuronic acid: IV. The synthesis of - and -tetraacetylglucuronic acid methyl ester and of 1chlorotriacetylglucuronic acid methyl ester. J. Biol. Chem., 106, 63-69. Graaf, M.; Boven, E.; Scheeren, H. W.; Haisma, H. J.; Pinedo, H. M. (2002). beta-Glucuronidase mediated drug release. Curr. Pharm. Des., 8, 13911403.

REFERENCES

199

Halkes, K. M.; Slaghek, T. M.; Hyppönen, T. K.; Kruiskamp, P. H.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J. F. G. (1998). Synthesis of hyaluronic acid related oligosaccharides and analogues, as their 4-methoxyphenyl glycosides, having N-acetyl--D-glucosamine at the reducing end. Carbohydr. Res., 309, 161-174. Hamayasu, K.; Tadokoro, H.; Kishino, E.; Ito, T.; Fujita, K.; Hara, K. (2009). Process for production of glucuronic acid and/or glucuronolactone. US Patents 20090030194. Hayden, F. (2009). Developing new antiviral agents for influenza treatment: what does the future hold? Clin. Infect. Dis., 48, S3-S13. Hidetoshi, Y. (1969). Metabolism of drugs. LXII. Isolation and identification of morphine glucuronides in urine and bile of rabbits. Biochem. Pharmacol., 18, 279-286. Hongu, M.; Saito, K.; Tsujihara, K. (1999). Solid–liquid phase transfer catalyzed novel glycosylation reaction of phenols. Synth. Commun., 29, 27752781. Iyer, S. S.; Rele, S. M.; Baskaran, S.; Chaikof, E. L. (2003). Design and synthesis of hyaluronan-mimetic gemini disaccharides. Tetrahedron, 59, 631638. Jacquinet, J.-C. (1990). Synthesis of the methyl glycosides of the repeating units of chondroitin 4- and 6-sulfate. Carbohydr. Res., 199, 153-181. Jansen, E. F.; Jang, R. (1946). Esterification of galacturonic acid and polyuronides with methanol-hydrogen chloride. J. Am. Chem. Soc., 68, 14751477. Jarosz, S. (1988). Stereoselective reduction of higher sugar enones with zinc borohydride. Carbohydr. Res., 183, 201-207. Jones, A. E.; Wilson, H. K.; Meath, P.; Meng, X.; Holt, D. W.; Johnston, A.; Oellerich, M.; Armstrong, V. W.; Stachulski, A. V. (2009). Convenient syntheses of the in vivo carbohydrate metabolites of mycophenolic acid: Reactivity of the acyl glucuronide. Tetrahedron Lett., 50, 4973-4977.

200

REFERENCES

Juteau, H.; Gareau, Y.; Labelle, M. (1997). A convenient synthesis of acyl glucuronides. Tetrahedron Lett., 38, 1481-1484. Kahne, D. (1997). Combinatorial approaches to carbohydrates. Curr. Opin. Chem. Biol., 1, 130-135. Kamal, A.; Tekumalla, V.; Raju, P.; Naidu, V. G. M.; Diwan, P. V.; Sistla, R. (2008). Pyrrolo[2,1-c][1,4]benzodiazepine--glucuronide prodrugs with a potential for selective therapy of solid tumors by PMT and ADEPT strategies. Bioorg. Med. Chem. Lett., 18, 3769-3773. Karlsson, K.-A. (1991). Glycobiology: a growing field for drug design. Trends Pharmacol. Sci., 12, 265-272. Kim, Y. J.; Ichikawa, M.; Ichikawa, Y. (2000). Highly selective synthesis of 1-N-iminosugars of the D-glucose and -glucuronic acid types. J. Org. Chem., 65, 2599-2602. Kishikawa, T. (1969). Studies on chemotherapeutic agents V. A synthesis of benimidazole nuleosides of D-glucuronamide. Chem. Pharm. Bull., 17, 24942501. Kishikawa, T.; Yamazaki, T.; Yuki, H. (1966a). Studies on chemotherapeutic agents I. A synthesis of pyrimidine nuleosides of Dglucuronic acid and its derivatives. Chem. Pharm. Bull., 14, 1354-1360. Kishikawa, T.; Yuki, H. (1964). A synthesis of uracil and cytosine nucleosides containing D-glucuronic acid and its derivatives as sugar component. Chem. Pharm. Bull., 12, 1259-1261. Kishikawa, T.; Yuki, H. (1966b). Studies on chemotherapeutic agents II. A synthesis of purine nuleosides of D-glucuronic acid. Chem. Pharm. Bull., 14, 1360-1364. Kitagawa, I.; Kamigauchi, T.; Ohmori, H.; Yoshikawa, M. (1980). Saponin and Sapogenol. XXIX. Selective cleavage of the glucuronide linkage in oligoglycosides by anodic oxidation. Chem. Pharm. Bull., 28, 3078-3086. Kitagawa, I.; Kamigauchi, T.; Shirakawa, K.; Ikeda, Y.; Ohmori, H.; Yoshikawa, M. (1981a). Syntheses of aminocyclitols and aminocyclitol-

REFERENCES

201

oligoglycosides from uronic acids and glucuronide-saponins by means of electrolytic decarboxylation reaction. Heterocycles, 15, 349-354. Kitagawa, I.; Yoshikawa, M. (1977). Selective cleavage of glucuronide linkage in oligoglycoside. Heterocycles, 8, 783-811. Kitagawa, I.; Yoshikawa, M.; Kamigauchi, T.; Shirakawa, K.; Ikeda, Y. (1981b). Chemical transformation of uronic acids leading to aminocyclitols. III. Syntheses of aminocyclitols and aminocyclitol-oligoglycosides from uronic acids and glucuronide-saponins by means of electrolytic decarboxylation. Chem. Pharm. Bull., 29, 2571-2581. Knauer, S.; Kranke, B.; Krause, L.; Kunz, H. (2004). Amino sugars and glycosylamines as tools in stereoselective synthesis. Curr. Org. Chem., 8, 17391761. Kondo, T.; Nakai, H.; Goto, T. (1972). Synthesis of cytosinine, the nucleoside component of antibiotic blasticidin S. Tetrahedron Lett., 13, 18811884. Kovac, P.; Alfoldi, J.; Kosik, M. (1974). Synthesis and reactions of uronic acid derivatives. IV. Unambiguous synthesis of methyl (methyl - and -Dglucopyranosid)uronate. Chem. Zvesti, 28, 820-832. Krog-Jensen, C.; Oscarson, S. (1998). Efficient synthesis of differently protected methyl (ethyl 1-thio--D-glucopyranosid)uronates and their evaluation as glucuronic acid donors and acceptors. Carbohydr. Res., 308, 287-296. Lefèvre, K. U.; Tollens, B. (1907). Untersuchungen uber die glucuronsaure, ihre quantitative bestimmung und ihre farbenreaktionen. Ber. Deut. Chem. Ges., 40, 4513-4523. Lellam, B.; Drouillat, B.; Dekany, G.; Starr, M. S.; Toth, I. (1998). Synthesis and in vitro evaluation of lipoamino acid and carbohydrate-modified enkephalins as potential antinociceptive agents. Int. J. Pharm., 161, 55-64. Li, J.-F.; Chen, S.-J.; Zhao, Y.; Li, J.-X. (2009). Glycoside modification of oleanolic acid derivatives as a novel class of anti-osteoclast formation agents. Carbohydr. Res., 344, 599-605.

202

REFERENCES

Li, J. J.; Bugg, T. D. H. (2004). A fluorescent analogue of UDP-Nacetylglucosamine: application for FRET assay of peptidoglycan translocase II (MurG). Chem. Commun., 21, 182-183. Lin, F.; Peng, W.; Xu, W.; Han, X.; Yu, B. (2004). A facile preparation of uronates via selective oxidation with TEMPO/KBr/Ca(OCl) 2 under aqueous conditions. Carbohydr. Res., 339, 1219-1223. Loukou, C.; Tosin, M.; Müller-Bunz, H.; Murphy, P. V. (2007). Synthesis of sugar-lactams from azides of glucuronic acid. Carbohydr. Res., 342, 19531959. Lucas, R.; Alcantara, D.; Morales, J. C. (2009). A concise synthesis of glucuronide metabolites of urolithin-B, resveratrol, and hydroxytyrosol. Carbohydr. Res., 344, 1340-1346. Luzzio, F. A. (1998). The oxidation of alcohols by modified oxochromium (VI)-amine reagents. In Organic Reactions.; Paquette, L. A., ed.; John Wiley & Sons: New York, Vol. 53, pp. 1-221. Malkinson, J. P.; Falconer, R. A.; Toth, I. (2000). Synthesis of C-terminal glycopeptides from resin-bound glycosyl azides via a modified Staudinger reaction. J. Org. Chem., 65, 5249-5252. Mann, M. C.; Islam, T.; Dyason, J. C.; Florio, P.; Trower, C. J.; Thomson, R. J.; von Itzstein, M. (2006a). Unsaturated N-acetyl-D-glucoaminuronic acid glycosides as inhibitors of influenza virus sialidase. Glycoconj. J., 23, 127-133. Mann, M. C.; Thomson, R. J.; Dyason, J. C.; McAtamney, S.; von Itzstein, M. (2006b). Modelling, synthesis and biological evaluation of novel glucuronide-based probes of vibrio cholerae sialidase. Bioorg. Med. Chem., 14, 1518-1537. Manning, K. S.; Lynn, D. G.; Shabanowitz, J.; Fellows, L. E.; Singh, M.; Schrire, B. D. (1985). A glucuronidase inhibitor from the seeds of Baphia racemosa: Application of fast atom bombardment coupled with collision activated dissociation in natural product structure assignment. J. Chem. Soc., Chem. Commun., 127-129.

REFERENCES

203

Marwah, P.; Marwah, A.; Kneer, N.; Lardy, H. (2001). Ergosteroids IV: Synthesis and biological activity of steroid glucuronosides, ethers, and alkylcarbonates. Steroids, 66, 581-595. McMillan, K. G.; Tackett, M. N.; Dawson, A.; Fordyce, E.; Paton, R. M. (2006). Synthesis, structure and reactivity of 5-pyranosyl-1,3,4-oxathiazol-2ones. Carbohydr. Res., 341, 41-48. Mehltretter, C. L. (1951). Process for making D-glucuronic acid and its salts. US Patents 2562200. Mehltretter, C. L. (1961). D-Glucuronic acid lactone. US Patents 3012041. Mehltretter, C. L.; Alexander, B. H.; Mellies, R. L.; Rist, C. E. (1951). A practical synthesis of D-glucuronic acid through the catalytic oxidation of 1,2isopropylidene-D-glucose. J. Am. Chem. Soc., 73, 2424-2427. Migawa, M. T.; Risen, L. M.; Griffey, R. H.; Swayze, E. E. (2005). An efficient synthesis of gougerotin and related analogues using solid- and solutionphase methodology. Org. Lett., 7, 3429-3432. Mignet, N.; Chaix, C.; Rayner, B.; Imbach, J.-L. (1997). Synthesis and evaluation of glucuronic acid derivatives as alkylating agents for the reversible masking of internucleoside groups of antisense oligonucleotides. Carbohydr. Res., 303, 17-24. Mitchell, M. B.; Whitcombe, I. W. A. (2000). The synthesis of the glucuronide adduct of Trocade™. Tetrahedron Lett., 41, 8829-8834. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gays-Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. (1991). Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumour cell lines. J. Natl. Cancer Inst., 83, 757-766. Moppett, C. E.; Dix, D. T.; Johnson, F.; Coronelli, C. (1971). Total synthesis of gougerotin. J. Am. Chem. Soc., 94, 3272-3274. Mulder, G. J.; Coughtrie, M. M. H.; Burchell, B. (1990). Glucuronidation. In Conjugation Reactions in Drug Metabolism.; Mulder, G. J., ed.; Taylor and Francis: London, pp. 51-105.

204

REFERENCES

Murphy, P. V.; Bradley, H.; Tosin, M.; Pitt, N.; Fitzpatrick, G. M.; Glass, W. K. (2003). Development of carbohydrate-based scaffolds for restricted presentation of recognition groups. Extension to divalent ligands and implications for the structure of dimerized receptors. J. Org. Chem., 68, 56925704. Murphy, P. V.; Pitt, N.; O’Brien, A.; Enright, P. M.; Dunne, A.; Wilson, S. J.; Duaneb, R. M.; O’Boyleb, K. M. (2002). Identification of novel inhibitors of fibroblast growth factor (FGF-2) binding to heparin and endothelial cell survival from a structurally diverse carbohybrid library. Bioorg. Med. Chem. Lett., 12, 3287–3290. Needs, P. W.; Kroon, P. A. (2006). Convenient syntheses of metabolically important quercetin glucuronides and sulfates. Tetrahedron, 62, 6862-6868. Nelson, P. F.; Samuelson, O. (1968). Identification of acidic groups in kraft pulps from Eucalypt and Pinus radiata. Svensk Papperstidn, 71, 325-330. Nilsson, M.; Svahn, C.-M.; Westman, L. (1993). Synthesis of the methyl glycosides of a tri- and a tetra-saccharide related to heparin and heparin sulphate. Carbohydr. Res., 246, 161-172. Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeuchi, T.; Azetaka, M.; Fukuyasu, H.; Iizuka, Y. (1996). The first L-iduronic acid-type 1-Niminosugars having inhibitory activity of experimental metastasis. J. Am. Chem. Soc., 118, 3051-3052. Nishimura, Y.; Shitara, E.; Adachi, H.; Toyoshima, M.; Nakajima, M.; Okami, Y.; Takeuchi, T. (2000). Flexible synthesis and biological activity of uronic acid-type gem-diamine 1-N-iminosugars: A new family of glycosidase inhibitors. J. Org. Chem., 65, 2-11. Nudelman, A.; Herzig, J.; Gottlieb, H. E. (1987). Selective deacetylation of anomeric sugar acetates with tin alkoxides. Carbohydr. Res., 162, 145-152. O'Brien, C.; Polakova, M.; Pitt, N.; Tosin, M.; Murphy, P. V. (2007). Glycosidation-Anomerization reactions of 6,1-anhydroglucopyranuronic acid and anomerization of -D-glucopyranosiduronic acids promoted by SnCl4. Chem. Eur. J., 13, 902-909.

REFERENCES

205

Oscarson, S.; Svahnberg, P. (2001). Synthesis of uronic acid-containing xylans found in wood and pulp. J. Chem. Soc., Perkin Trans. 1, 873-879. Owen, L. N.; Peat, S.; Jones, W. J. G. (1941). Furanose and pyranose derivatives of glucurone. J. Chem. Soc., 339-343. Pandey, G.; Kapur, M. (2002). Design and development of a common synthetic strategy for a variety of 1-N-iminosugars. Org. Lett., 4, 3883-3886. Pearson, A. G.; Kiefel, M. J.; Ferrob, V.; von Itzsteina, M. (2005). Towards the synthesis of aryl glucuronides as potential heparanase probes. An interesting outcome in the glycosidation of glucuronic acid with 4-hydroxycinnamic acid. Carbohydr. Res., 340, 2077-2085. Perlin, A. S. (1951). Thermal decarboxylation of uronic acids. Can. J. Chem., 30, 278-290. Perrie, J. A.; Harding, J. R.; Holt, D. W.; Johnston, A.; Meath, P.; Stachulski, A. V. (2005). Effective synthesis of 1-acyl glucuronides by selective acylation. Org. Lett., 7, 2591-2594. Pilgrim, W.; Murphy, P. V. (2009). α-Glycosphingolipids via chelationinduced anomerization of O- and S-glucuronic and galacturonic acid derivatives. Org. Lett., 11, 939–942. Pitt, N.; Duane, R. M.; O'Brein, A.; Bradley, H.; Wilson, S. J.; O'Boyle, K. M.; Murphy, P. V. (2004). Synthesis of a glucuronic acid and glucose conjugate library and evaluation of effects on endothelial cell growth. Carbohydr. Res., 339, 1873-1887. Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. (2001). Automated solidphase synthesis of oligosaccharides. Science, 291, 1523–1527. Plusquellec, D.; Roulleau, F.; Bertho, F.; Lefeuvre, M.; Brown, E. (1986). Chimie des sucres sans groupements protecteurs - I - esterification regioselective de l'hydroxyle anomere du lactose, du maltose et du glucose. Tetrahedron, 42, 2457-2467. Poláková, M.; Pitt, N.; Tosin, M.; Murphy, P. V. (2004). Glycosidation reactions of silyl ethers with conformationally inverted donners derived from

206

REFERENCES

glucuronic acid: Stereoselective synthesis of glycosides and 2-deoxyglycosides. Angew. Chem. Int. Ed., 43, 2518-2521. Pravdić, N.; Keglević, D.; Andrews, E. D.; Harvey, W. E.; Bolton, R.; Casy, A. F.; Myers, J. L.; Mathiwson, D. W.; Whalley, W. B.; Blatchly, J. M.; Taylor, R.; Jansen, A. B. A.; Stokes, P. J.; Cheeseman, G. W. H.; Davidson, J. S.; Crabtree, J. M.; Gates, P. N.; Mooney, E. F.; Sutherland, J. K.; Widdowson, D. A.; Mongkolsuk, S.; Dean, F. M.; Kirby, G. W.; Tiwari, H. P.; Machin, D. J.; Morris, D. F. C.; Short, E. L.; Bluck, R. S.; Odell, A. L.; Olliff, R. W.; Burdon, J.; Hollyhead, W. B.; Patrick, C. R.; Salthouse, J. A.; Waddingt, T. C. (1964). Notes. J. Chem. Soc., 4633-4665. Rat, S.; Mathiron, D.; Michaud, P.; Kovensky, J.; Wadouachi, A. (2007). Efficient glycosylation and/or estrification of D-glucuronic acid and its 6,1lactone under solvent-free microwave irradiation. Tetrahedron, 63, 1242412428. Rejzek, M.; Mukhopadhyay, B.; Wenzel, C. Q.; Lam, J. S.; Field, R. A. (2007). Direct oxidation of sugar nucleotides to the corresponding uronic acids: TEMPO and platinum-based procedures. Carbohydr. Res., 342, 460-466. Rivault, F.; Tranoy-Opalinski, I.; Gesson, J.-P. (2004). A new linker for glucuronylated anticancer prodrugs. Bioorg. Med. Chem., 12, 675-682. Rukhman, I.; Yudovich, L.; Nisnevich, G.; Gutman, A. L. (2001). Selective synthesis of both isomers of morphine 6--D-glucuronide and their analogs. Tetrahedron, 57, 1083-1092. Sallustio, B. C. (2008). Glucuronidation-dependant toxicity and bioactivation. In Advances in Molecular Toxicology.; Fishbein, J. C., ed.; Elsevier Science: Amsterdam, Vol. 2, pp. 57-77. Saunders, M. D.; Timell, T. E. (1967). The acid hydrolysis of glycosides: V. Effect of the nature of the aglycon on the hydrolysis of glycosiduronic acids (glycuronides). Carbohydr. Res., 5, 453-460. Saunders, M. D.; Timell, T. E. (1968). The acid hydrolysis of glycosides: VI. Effect of substitution at C-3 and C-5. Carbohydr. Res., 6, 12-17.

REFERENCES

207

Schell, P.; Orgueira, H. A.; Roehrig, S.; Seeberger, P. H. (2001). Synthesis and transformations of D-glucuronic and L-iduronic acid glycals. Tetrahedron Lett., 42, 3811-3814. Schmidt, R. R. (1986). New methods for the synthesis of glycosides and oligosaccharides - are there alternatives to the Koenigs-Knorr method? Angew. Chem. Int. Ed., 25, 212-235. Schmidt, R. R.; Grundler, G. (1981). Einfache synthese von β-Dglucopyranosyluronaten. Synthesis, 885-887. Schmidt, R. R.; Rucker, E. (1980). Stereoselective glycosidations of uronic acids. Tetrahedron Lett., 21, 1421-1424. Schukla, A. K.; Schroder, C.; Nohle, U.; Shauer, Glycoconjugates. Lund-Ronneby, Sweden, pp. 155-156.

R.

(1983).

Sharon, N.; Lis, H. (1993). Carbohydrates in cell recognition. Sci. Am., 268, 82-89. Siddiqui, I. R.; Purves, C. B. (1963). The behavior of D-glucuronic, Dgalacturonic, and D-mannuironic acid in dilute aqueous sodium hydroxide. Can. J. Chem., 41, 382-386. Smith, R. L. (1966). The biliary excretion and enterohepatic circulation of drugs and other organic compounds. Fortschr. Arzneimittel-Forsch, 9, 299-360. Sofia, M. J. (1998). Combinitorial Chemistry and Molecular Diversity in Drug Discovery.; Gordon, E. M., ed.; Wiley-Liss, Inc.: New York, pp. 243-269. Soidinsalo, O.; Wähälä, K. (2007). Synthesis of daidzein 7-O--Dglucuronide-4'-O-sulfate. Steroids, 72, 851-854. Soliman, S. E.; Bassily, R. W.; El-Sokkary, R. I.; Nashed, M. A. (2003). Acetylated methyl glucopyranuronate trichloroacetimidate as a glycosyl donor for efficient synthesis of disaccharides. Carbohydr. Res., 338, 2337-2340. Stachulski, A. V.; Harding, J. R.; Lindon, J. C.; Maggs, J. L.; Park, B. K.; Wilson, I. D. (2006). Acyl glucuronides: biological activity, chemical reactivity, and chemical synthesis. J. Med. Chem., 49, 6931–6945.

208

REFERENCES

Stachulski, A. V.; Jenkins, G. N. (1998). The synthesis of O-glucuronides. Nat. Prod. Rep., 15, 173-186. Stanford, D.; Stachulski, A. V. (2007). Convenient synthesis of deoxypyranose sugars from glucuronolactone. Tetrahedron Lett., 48, 23612364. Stapley, J. A.; BeMiller, J. N. (2006). The Hofer-Moest decarboxylation of D-glucuronic acid and D-glucuronosides. Carbohydr. Res., 342, 610-613. Sugihara, H.; Ukawa, K.; Miyake, A.; Itoh, K.; Sanno, Y. (1976). Syntheses of 1,2-N-alkylimino-1,2,3,4-tetrahydronaphthalene derivatives and preparation of ring closed analog of salbutamol as a new β-adrenoceptor agent. Chem. Pharm. Bull., 24, 394-404. Temelkoff, D. P.; Smith, C. R.; Kibler, D. A.; McKee, S.; Duncan, S. J.; Zeller, M.; Hunsen, M.; Norris, P. (2006a). Applications of bis(diphenylphosphino)ethane (DPPE) in Staudinger-type N-glycopyranosyl amide synthesis. Carbohydr. Res., 341, 1645-1656. Temelkoff, D. P.; Zeller, M.; Norris, P. (2006b). N-Glycoside neoglycotrimers from 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl azide. Carbohydr. Res., 341, 1081-1090. Thiem, J.; Ossowski, P. (1984). Studies of hexuronic acid ester glycals and the synthesis of 2-deoxy--glycoside precursors. J. Carbohydr. Chem., 3, 287313. Tietze, L. F.; Schuster, H. J.; Schmuck, K.; Schuberth, I.; Alves, F. (2008). Duocarmycin-based prodrugs for cancer prodrug monotherapy. Bioorg. Med. Chem., 16, 6312-6318. Timell, T. E.; Enterman, W.; Spencer, F.; Soltes, E. J. (1965). The acid hydrolysis of glycosides: II. Effect of substituents at C-5. Can. J. Chem., 43, 2296-2305. Timoshchuk, V. A. (1995). Nucleosides of uronic acids as a componant of natural antibiotics (Review). Pharm. Chem. J., 29, 51-59. Tosin, M.; Müller-Bunz, H.; Murphy, P. V. (2004). Synthesis and x-ray single crystal structure of a bivalent glycocluster. Chem. Commun., 494-495.

REFERENCES

209

Tosin, M.; Gouin, S. G.; Murphy, P. V. (2005a). Synthesis of structurally diverse and defined bivalent mannosides on saccharide scaffolding. Org. Lett., 7, 211-214. Tosin, M.; Murphy, P. V. (2002). Synthesis of α-glucuronic acid and amide derivatives in the presence of a participating 2-acyl protecting group. Org. Lett., 4, 3675-3678. Tosin, M.; Murphy, P. V. (2005b). Synthesis of structurally defined scaffolds for bivalent ligand display based on glucuronic acid anilides. The degree of tertiary amide isomerism and folding depends on the configuration of glycosyl azide. J. Org. Chem., 70, 4107-4117. Tosin, M.; O'Brien, C.; Fitzpatrick, G. M.; Müller-Bunz, H.; Glass, W. K.; Murphy, P. V. (2005c). Synthesis and structural analysis of the anilides of glucuronic acid and orientation of the groups on the carbohydrate scaffolding. J. Org. Chem., 70, 4096-4106. Touster, O.; Reynolds, V. H. (1952). The synthesis and properties of -Dglucuronic acid-1-phosphate. J. Biol. Chem., 197, 863-868. van Boeckel, C. A. A.; Delbressine, L. P. C.; Kaspersen, F. M. (1985). The synthesis of glucuronides derived from the antidepressant drugs mianserin and Org 3770. Recl. Trav. Chim. Pays-Bas., 104, 259-265. van den Bos, L. J.; Codée, J. D. C.; van der Toorn, J. C.; Boltje, T. J.; van Boom, J. H.; Overkleeft, H. S.; van der Marel, G. A. (2004). Thioglycuronides: Synthesis and application in the assembly of acidic oligosaccharides. Org. Lett., 6, 2165-2168. van den Bos, L. J.; Litjens, R. E. J. N.; van den Berg, R. J. B. H. N.; Overkleeft, H. S.; van der Marel, G. A. (2005). Preparation of 1-thio uronic acid lactones and their use in oligosaccharide synthesis. Org. Lett., 7, 2007-2010. Varki, A. (1993). Biological roles of oligosaccharides: All of the theories are correct. Glycobiology, 3, 97-130. Velasco-Torrijos, T.; Murphy, P. V. (2005). Synthesis and conformational analysis of novel water soluble macrocycles incorporating carbohydrates, including a -cyclodextrin mimic. Tetrahedron: Asymmetry, 16, 261-272.

210

REFERENCES

von Itzstein, M.; Wu, W.-Y.; Jin, B. (1994). The synthesis of 2,3didehydro-2,4-dideoxy-4-guanidinyl-N-acetylneuraminic acid: a potent influenza virus sialidase inhibitor. Carbohydr. Res., 259, 301-305. von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Phan, T. V.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman, P. M.; Varghese, J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotham, V. J.; Cameron, J. M.; Penn, C. R. (1993). Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature, 363, 418-423. Wang, Y.; Yuan, H.; Wright, S. C.; Wang, H.; Larrick, J. W. (2003). Synthesis and preliminary cytotoxicity study of glucuronide derivatives of CC1065® analogues. Bioorg. Med. Chem., 11, 1569-1575. Westman, J.; Nilsson, M. (1995). Synthesis of non-glucosamino glucan oligosaccharides related to heparin and heparan Sulphate. J. Carbohydr. Chem., 14, 949–960. Weymouth-Wilson, A. C. (1997). The role of carbohydrates in biologically active natural products. Nat. Prod. Rep., 14, 99-110. Xie, J.; Güveli, T.; Hebbe, S.; Dechoux, L. (2004). Synthesis of novel 1-Niminosugars from chiral nonracemic bicyclic lactams. Tetrahedron Lett., 45, 4903-4906. Yamashita, M.; Tomozawa, T.; Kakuta, M.; Tokumitsu, A.; Nasu, H.; Kubo, S. (2009). CS-8958, a prodrug of the new neuraminidase inhibitor R125489, shows long-acting anti-influenza virus activity. Antimicrob. Agents Chemother., 53, 186-192. Ying, L.; Gervay-Hague, J. (2003). General methods for the synthesis of glycopyranosyluronic acid azides. Carbohydr. Res., 338, 835-841. Yoshimra, Y.; Ohara, C.; Imahori, T.; Saito, Adachi, I.; Takahata, H. (2008). Synthesis hydroxypipecolic acid derivatives equivalent to evaluation of their inhibitory activities against Chem., 16, 8273-8286.

Y.; Kato, A.; Miyauchi, S.; of both enantiomers of 5-azapyranuronic acids and glycosidases. Bioorg. Med.

Yu, B.; Zhu, X.; Hui, Y. (2000). A novel and effective procedure for the preparation of glucuronides. Org. Lett., 2, 2539–2541.

REFERENCES

211

Yu, B.; Zhu, X.; Hui, Y. (2001). 6-S-Phenyl-glycopyransides as ready precursors to the synthesis of glycuronides. Tetrahedron, 57, 9403-9413. Yu, H. N.; Furukawa, J.-I.; Ikeda, T.; Wong, C.-H. (2004). Novel efficient routes to heparin monosaccharides and disaccharides achieved via regio- and stereoselective glycosidation. Org. Lett., 6, 723-726. Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.; Wong, C.H. (1999). Programmable one-pot oligosaccharide synthesis. J. Am. Chem. Soc., 121, 734-735. Zhao, G.; Deo, U. C.; Ganem, B. (2001). Selective fowler reductions: Asymmetric total syntheses of isofagomine and other 1-azasugars from methyl nicotinate. Org. Lett., 3, 201-203. Zorbach, W. W.; Valliaveedan, G. D. (1964). Methyl (cortison-21-yl-2,3,4tri-O-acetyl--D-glucosid)uronate. J. Org. Chem., 29, 2462-2463.

ARABIC SUMMARY

‫‪ARABIC SUMMARY‬‬

‫‪11‬‬

‫بعد ذلك‪ ،‬تم تعريض كل الجميكوسـيدات الناتجـة لايسـمة أمـا بتثاعميـا مـع أسـيل كموريـد أو عـن طريـق‬ ‫األقتـران مــع األحمــاض الكربوكســيمية بأســتخدام الـ ـ ‪ DCC‬ولكــن جميــع ىــذه المحــاوالت بــاءت بالثشــل‪ .‬مــن‬ ‫ناحية أخري‪ ،‬تم تعريضيا لمميسمة في وجود قواعد مختمثة ولكن جميع ىذه المحـاوالت أيضـا بـاءت بالثشـل‬ ‫(مخطط ‪.)XII‬‬

‫مخطط ‪XII‬‬

‫‪ARABIC SUMMARY‬‬

‫‪9‬‬

‫مخطط ‪XI‬‬ ‫بتثاعل الييمي أسيتال ‪ 32‬مع البا ار‪-‬فموروانيمين تم الحصول عمي البيتا‪-‬أنومر ‪ 33‬في صورة نظيثة‪.‬‬

‫بينمـا‪ ،‬فــي حالــة البــا ار‪-‬برومـوانيمين تــم الحصــول عمــي خمــيط مــن الجميكوســيدات األلثــا (‪ )34a‬و البيتــا‬ ‫(‪ ،)34b‬و كانت النسبة االنوميرية ‪ 4:3‬كما كان مبين من التكامل في أطيا‬ ‫لمييدروجين‪ .‬وقد تم فصل و تعري‬

‫كل منيم‪.‬‬

‫الرنين النـووي المغناطيسـي‬

‫‪ARABIC SUMMARY‬‬

‫‪8‬‬

‫مخطط ‪X‬‬ ‫بعــد ذلــك‪ ،‬تمــت األ ازلــة االنتقاةيــة لمجموعــة االســيتيل األنوميريــة بعنايــة باســتخدام البنزيــل أمــين وىــو‬ ‫خثي‬

‫بما يكثي لترك باقي المجموعات الحساسة لمقواعد كما ىـي‪ .‬أوضـحت كروماتوجيرفيـا الطبقـة الرقيقـة‬

‫لخمــيط التثاعــل وجــود مــادة أقــل تــنقال مــن مــادة االبتــداء ‪ 31‬و بعــد فصــميا و تنقيتيــا حصــمنا عمــي الييمــي‬ ‫أسيتال ‪( )٪ 53( 32‬مخطط ‪.)XI‬‬

‫‪ARABIC SUMMARY‬‬

‫‪7‬‬

‫مخطط ‪IX‬‬ ‫بعد غميـان الحمـض ‪ ،2‬المـذاب فـي ميثيمـين كموريـد المـاةي‪ ،‬مـع الثيونيـل كموريـد لمـدة سـاعتين‪ ،‬بينـت‬

‫كروماتوجيرفيــا الطبق ــة الرقيق ــة لخم ــيط التثاع ــل وج ــود م ــادة أكث ــر ت ــنقال م ــن م ــادة االبت ــداء الت ــي كان ــت ق ــد‬ ‫أســتيمكت تمامــا‪ .‬و بالتثاعــل المباش ـرة لالســيد كموريــد النــاتج مــع األليــل الكحــول فــي وجــود بيريــدين لمــدة‬ ‫ساعة‪ ،‬حصمنا عمي األليل استر ‪( )٪ 99( 31‬مخطط ‪.)X‬‬

‫‪ARABIC SUMMARY‬‬

‫‪6‬‬

‫مخطط ‪VII‬‬ ‫نجحت تمك الطريقو أيضا في تحضير المركبات الغير مشبعة ‪ 27-22‬من الـ ‪-4،3،2،4‬تيت ار‪-O-‬‬

‫اسيتيل‪-‬بيتا‪-D-‬جموكوبيرانيوروناميدات المقابمة ماعدا الـ (بينزاميدازول‪-3-‬ايل)‪-4،3،2،4-‬تيت ار‪-O-‬‬ ‫اسيتيل‪-‬بيتا‪-D-‬جموكوبيرانيوروناميد (‪ )8‬حيث إن التثاعل عاني من الكثير من التعقيدات (مخطط‬ ‫‪.)VIII‬‬

‫مخطط ‪VIII‬‬

‫بعد ذلك تحول االنتباه إلـ تشـييد سمسـمة مـن ال ـ ‪-N‬أسـيل‪-N-‬أريـل‪-D-‬جموكوبيرانيرونوسـيل أمـين و كـان‬

‫تحميل التشييد العكسي المقترح لتحضير تمك السمسمة كما ىو مبين في المخطط التالي‪:‬‬

‫‪ARABIC SUMMARY‬‬

‫‪5‬‬

‫مخطط ‪VI‬‬ ‫تحول التركيز أليجاد طريقو أخري لتحضير الـ ‪(-N‬بي ارزين‪-3-‬ايل)‪-4،3،2-‬تراي‪-O-‬اسيتيل‪-‬‬ ‫‪-5,4Δ‬بيتا‪-D-‬جموكوبيرانيوروناميد‪ .‬بمعاممو محمول من الـ ‪(-N‬بي ارزين‪-3-‬ايل)‪-4،3،2،4-‬تيت ار‪-O-‬‬ ‫اسيتيل‪-‬بيتا‪-D-‬جموكوبيرانيوروناميد (‪ )9‬في الميثيمين كموريد مع ‪ DBU‬عند درجة ح اررة صثر درجة‬ ‫مةوية أعطت الناتج المطموب (مخطط ‪.)VII‬‬

‫‪ARABIC SUMMARY‬‬

‫‪4‬‬

‫مخطط ‪V‬‬ ‫من المثير لالىتمام أن محاولة استخدام ‪-3‬امينوبي ارزين كأمين في التثاعل السابق أعطت ناتج وحيد‬ ‫كما تبين من قبل كروماتوجيرفيا الطبقة الرقيقة و لكن بعد تنقيتة و تحميمة بالطرق الطيثية وجد أنو لم يكن‬ ‫الـ ‪(-N‬بي ارزين‪-3-‬ايل)‪-4،3،2-‬تراي‪-O-‬اسيتيل‪-5,4Δ-‬بيتا‪-D-‬جموكوبيرانيوروناميد (‪ )28‬المطموب و‬ ‫المتوقع و لكن بدال من ذلك حصمنا عمي الـ ايثيل ‪-4،3،2‬تراي‪-O-‬اسيتيل‪-5,4Δ-‬بيتا‪-D-‬‬ ‫جموكوبيرانيورينات (‪( )29‬مخطط ‪.)VI‬‬

‫‪ARABIC SUMMARY‬‬

‫‪3‬‬

‫مخطط ‪IV‬‬ ‫بعد ذلك‪ ،‬تحول االىتمام الي تشييد بعض المركبات كمشتقات لـ اسيتاميدو‪-3،6-‬انييدرو‪-2،5-‬‬ ‫دايديوكسي‪-D-‬جميسرو‪-D-‬جالكتو‪-‬نون‪-3-‬اينويك أسيد الموجود في الطبيعة‪ .‬بتثاعل ‪ 2‬مع األمينات‬

‫في وجود ايثيل كموروفورمات و تراي ايثيل أمين عند ‪ 3.-‬درجة مئوية‪ ،‬أنتج األميدات الغير مشبعة‬ ‫‪ 27-22‬بجانب كمية بسيطة من األميدات المشبعة والتي تم فصميا باستخدام الطرق الكروماتوجرافية‬ ‫(مخطط ‪.)V‬‬

‫‪ARABIC SUMMARY‬‬

‫‪2‬‬

‫مخطط ‪II‬‬ ‫من الناحية األخري‪ ،‬تثاعل المركب ‪ 1‬مع الـ ‪-4‬أمينويوراسيل )‪ (13a‬أو مشتقة )‪ (13b‬تحت نثس‬

‫الظرو‬

‫السابق ذكرىا لم يعطي أي نتيجة حتي بعد تغيير المذيب (مخطط ‪.)III‬‬

‫مخطط ‪III‬‬ ‫ثم بعد ذلك‪ ،‬وجد أنة من الضروري إزالة مجموعات االسيتيل وقد تم ذلك بأستخدام تثاعل التصبن‬ ‫لكل من مشتقات الجموكيروناميد ‪ 12-5‬و ذلك بأستخدام ‪ .0.5‬موالر من محمول ىيدروكسيد الميثيوم‬ ‫لمحصول عم‬

‫المركبات المقابمة (‪ )21-14‬كخميط متوازن من األلثا و البيتا التي أمكن تحديد النسبة‬

‫بينيم والتي كانت ‪ 4:4‬لكل المشتقات‪ .‬كذلك‪ ،‬إزالة مجموعات االسيتيل بأستخدام طريقة زمبمن أعطت‬ ‫نثس النتيجة (مخطط ‪.)IV‬‬

‫‪ARABIC SUMMARY‬‬

‫‪1‬‬

‫الممخص العربي‬ ‫بدأت الدراسة بأستمو حمض الجموكيورونيك المتاح تجاريا بتثاعمة مع األستك أنييدريد في وجود‬ ‫كميات محثزة من اليود كناقل لمجموعات األسيتيل‪ .‬و بتعريض المركب ‪ 1‬لمتثاعل مع الماء‪ ،‬حصمنا‬

‫عمي ‪-4،3،2،4‬تيت ار‪-O-‬أسيتيل‪-‬بيتا‪-D-‬حمض جموكيورونيك ‪ 2‬كناتج (‪( )٪ 99‬مخطط ‪.)I‬‬

‫مخطط ‪I‬‬ ‫عندما ترك المركب ‪ 2‬لمتبمور‪ ،‬أ وضح طي‬

‫الرنين النووي المغناطيسي لمييدروجين أن البمورات‬

‫المتكونة ىي األلثا أنومر و ليست بعد البيتا و يمكن تثسير ذلك التحول بتكون إحدي أيونات‬ ‫األكسيكربنيوم األتية‪:‬‬

‫المركب ‪ 1‬تعرض لمتثاعل مع العديد من األمينات في الميثيمين كموريد الالماةي كمزيب‪ ،‬فأنتج‬ ‫مجموعة األميدات الثانوية المقابمة ‪( 12-5‬مخطط ‪.)II‬‬

‫الملخص العربي‬

‫المستخمص العربي‬

‫األسم ‪ :‬فريدي جمال عدلي جوني‬ ‫الدرجة ‪ :‬درجة الماجستير في العموم‬ ‫عنوان الرسالة ‪ :‬تخميق و تقييم النشاط البيولوجي لبعض مشتقات حمض الجموكيورونيك‪.‬‬ ‫جهة البحث ‪ :‬المركز القومي لمبحوث‬

‫تضمن ىذا العمل تشييد سبعة و عشرون مشتق جديد لحمض الجموكيورونيـك والتـ تـم تقسـيميم الـ‬ ‫ثالث مجموعات‪ :‬األولـ جموكيوروناميـدات تحتـوي عمـي مجموعـات أسـيتيل او منزوعـة األسـيتيل و الثانيـة‬ ‫‪5,4‬‬

‫‪Δ‬‬

‫جموكيوروناميــدات و الثالثــة ‪-N‬جميكوســيدات و ذلــك ابتــداءا مــن حمــض الجموكيورونيــك عــن طريــق‬

‫اســتخدام الطــرق الكيمياةيــة المختمثــة مثــل طــرق الحمايــة ‪ /‬ال ـال حمايــة‪ ،‬التنشــيط‪ ،‬النــزع و التكثيـ ‪ .‬لقــد تــم‬ ‫اثبات التركيب الكيمياة لكل المركبات باستخدام الطرق الطيثية المختمثـة وكـذلك تـم وصـ‬

‫جميـع ظـرو‬

‫التثاعالت‪ .‬بعد ذلـك‪ ،‬تمـت د ارسـة النشـاط البيولـوجي لكـل المركبـات كمضـادات لـاورام ضـد الخاليـا ‪TK-‬‬ ‫‪ 10‬و ‪ MCF-7‬و ‪ .UACC-62‬كانـ ـ ـ ــت المركبـ ـ ـ ــات ‪ 28 ،24 ،23 ،21 ،17 ،14 ،10-12 ،6‬و ‪29‬‬ ‫ىـي األكثــر نشــاطا ضــد خاليــا ال ـ ‪ ،TK-10‬بينمــا كانــت المركبــات ‪ 28 ،24 ،21 ،20‬و ‪ 31‬ىــي األكثــر‬ ‫نشاطا ضد خاليا الـ ‪ ،MCF-7‬و كانـت المركبـات ‪ 23-25 ،18-20 ،16 ،9-12‬و ‪ 27-29‬ىـي االكثـر‬ ‫نشاطا ضد خاليا الـ ‪.UACC-62‬‬

‫الكممــات الماتاحيــة ‪ :‬حمــض الجموكيورونيــك‪ ،‬الجموكيوروناميــد‪ ،‬رابطــة األميــد‪ ،‬مضــادات األورام‪TK- ،‬‬

‫‪.UACC-62 ،MCF-7 ،10‬‬

‫أقـ ـ ـرار صالحيـــة الرسال ـــة‬ ‫نشيد بأن الرسالة المقدمة من السيد ‪ /‬فريدي جمال عدلي جوني تحت عنوان " تخميق و تقييم النشاط‬

‫البيولوجي لبعض مشتقات حمض الجموكيورونيك " مقبولة و توفي متطمبات الحصول عمي درجة‬

‫الماجستير في العموم‪.‬‬ ‫المشرفـ ـ ــــ ـ ـون‬

‫أ‪.‬د‪ .‬يحيي محمود الخولي‬ ‫أستاذ الكيمياء العضوية‬

‫قسم الكيمياء‪ ،‬كمية العموم‪ ،‬جامعة حموان‬

‫التوقيع ‪ :‬ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ‬

‫أ‪.‬د‪ .‬عاطف جبران حنا‬

‫أستاذ الكيمياء العضوية‬ ‫قسم كيمياء المركبات الطبيعية‪،‬‬

‫التوقيع ‪ :‬ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ‬

‫المركز القومي لمبحوث‬

‫أ‪.‬م‪.‬د‪ .‬أحمد عمر حسين عمر النزهاوي‬ ‫أستاذ مساعد الكيمياء العضوية‬

‫قسم كيمياء المنتجات الطبيعية و الميكروبية‪،‬‬

‫التوقيع ‪ :‬ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ‬

‫المركز القومي لمبحوث‬

‫د‪ .‬شاهيناز حسن السيد‬ ‫مدرس الكيمياء العضوية‬

‫قسم الكيمياء‪ ،‬كمية العموم‪ ،‬جامعة حموان‬

‫التوقيع ‪ :‬ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ‬

‫جـــــامعة حلــــوان‬ ‫كلـــــية العلـــــــوم‬

‫تخليق و تقيين النشاط البيولوجي لبعض هشتقات حوض‬ ‫الجلوكيورونيك‪.‬‬ ‫رسالـــة مقدمــــة من‬

‫فريذي جوال عذلي جوني‬ ‫)بكاليوريوس علوم‪ ،‬كيوياء‪(6002 ،‬‬

‫ألستيفاء متطلبات الحصول علي‬

‫درجة الواجستير في العلوم‬ ‫)الكيوياء العضوية(‬ ‫الي‬ ‫قسن الكيوياء‬ ‫كلية العلوم‬ ‫جاهعة حلواى‬ ‫)‪(6022‬‬