Synthesis of Novel Nitric Oxide Donors and Prodrugs of 5- Fluorouracil

Home , Indazole, Isobutyl nitrite

SYNTHESIS OF NOVEL NITRIC OXIDE DONORS AND PRODRUGS OF 5- FLUOROURACIL

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Tingwei Cai, M.S.

*****

The Ohio State University 2005

Dissertation Committee: Approved by Professor Peng George Wang, Advisor

Professor Robert S. Coleman ______Professor Matthew S. Platz Advisor Graduate Program in Chemistry

ABSTRACT

This dissertation describes my Ph.D. work that focused on the synthesis and evaluation of novel nitric oxide donors, and the synthesis of derivatives of 5-fluorouracil.

In chapter 1, a series of alkyl and aryl N-hydroxyguanidines were synthesized and demonstrated to act as substrates of nitric oxide synthases (NOS). The discovery of these non-amino acid hydroxyguanidines as novel substrates also led to the discovery of a novel-binding mode of NOS.

In chapter 2, in order to achieve site specific delivery of nitric oxide (NO), a new class of glycosidase activated NO donors has been developed. Glucose and galactose were covalently coupled to 3-morphorlinosydnonimine (SIN-1), a mesoionic heterocyclic NO donor, via a carbamate linkage at the anomeric position. The β-glycosides were successfully prepared for these conjugates, while the α glycosidic compounds were very unstable. The new stable sugar-NO conjugates could release NO in the presence of glycosidases. Such NO prodrugs may be used as enzyme activated NO donors in biomedical research.

In chapter 3, a new sialated diazeniumdiolate has been synthesized and the glycosylation product was exclusively an α anomer. This new nitric oxide donor exhibited significantly improved stability as compared to its parent diazeniumdiolate salts and it

ii could be efficiently hydrolyzed by neuraminidase to release nitric oxide with a Km of 0.14 mM. The sialic acid-NO conjugate would be a valuable prodrug that targets NO to influenza viruses.

In chapter 4, two classes of prodrugs of 5-fluorouracil (FU) were synthesized. The first type is FU-NO conjugate. The synthesized compounds showed greater cytotoxicities than 5-fluorouracil for DU 145 human prostate and HeLa cancer cells. The second type is carbohydrate-FU conjugate. A new reverse glycosadition was also developed to prepare sugar conjugates.

In chapter 5, 7-carboxylindazole, as well as 5- and 6-carboxylindazoles was synthesized.

These compounds were designed as isosteres of 7-nitroindazole, a well-known inhibitor of NOS. However, they did not show potent inhibition to NOS.

iii

This dissertation is dedicated to my wife, Zhenling, whose love, understanding, devotion and support will never be forgotten.

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my advisor, Professor Peng George

Wang, for his support, scientific guidance and allowing me the freedom to explore the wonderful world of chemistry in his lab during the past five years.

I would like to thank my Ph.D. committee: Professor Robert S. Coleman and

Professor Matthew Platz for their invaluable advice and time.

I am indebted to the following professors for their inspiring courses: Dr. Robert S.

Coleman, Dr. Martin Newcomb, Dr. Maarten Postema, Dr. John Montgomery, Dr. Mark

Spaller, Dr. Ashok Bhagwat, Dr. David Benson, Dr. David Rorabacher.

I am thankful to all my colleagues and members of the Wang’s research group. I would especially like to thank Dr. Xiaoping Tang, Dr. Peter Andreana, Dr. Adam

Janczuk, Dr. Ming Xian, Dr. Lizhi Zhu, Dr. Guisheng Zhang, Mr. Yun Zhang, Mr.

Yalong Zhang, Ms. Dongning Lu, Ms. Megan Landerholm, Mr. Xuejun Wu and other people helped me in the research.

I also thank Dr. Duxin Sun, Dr. Paul Braunschweiger and Dr. Richard Silverman for their assistance in biological assay.

v I would like to thank Dr. David Hart, Mrs. Judith Brown, Ms. Jennifer Bates and all the administrative staff for their very friendly and resourceful assistance.

Finally, I would like to thank my beloved parents and brothers. My whole Ph.D. work would not have been possible if not for the love, appreciation, patience and consideration from my wife Zhenling.

VITA

November, 1969...... Born – Jilin, China

July, 1992...... Bachelor of Science Beijing Medical University

July, 1995...... Master of Science Beijing Medical University

2000 – 2003...... Teaching Asst. Wayne State University.

2003 – 2004...... Teaching Asst. The Ohio State University.

2004 – 2005...... Research Asst. The Ohio State University.

PUBLICATIONS

Research Publications

15. Cai, T. B.; Lu, D.; Tang, X.; Zhang, Y.; Landerholm, M.; Wang, P. G. “New glycosidase activated nitric oxide donors: glycose and 3-morphorlinosydnonimine conjugates”, J. Org. Chem. 2005, 70, 3518-3524.

14. Cai, T. B.; Lu, D.; Wang, P. G. “N-Hydroxyguanidines as substrates of nitric oxide synthases”, Curr. Topics Med. Chem. 2005, in press.

13. Cheng, H; Cao, X.; Xian, M.; Fang, L.; Cai, T. B.; Ji, J. J.; Tunac, J. B.; Sun, D; Wang, P. G. “Synthesis and enzyme-specific activation of carbohydrate- geldanamycin conjugates with potent anticancer activity”, J. Med. Chem. 2005, 48, 645 -652.

12. Cai, T. B.; Lu, D.; Landerholm, M.; Wang, P. G. “ Sialated diazeniumdiolate: a new sialidase-activated nitric oxide donor”, Org. Let. 2004, 6, 4203-4205.

vii

11. Wang, P. G.; Cai, T. B.; Taniguchi, N. (book eds) “Nitric oxide donors: for pharmaceutical and biological applications” 2005, Wily-VCH, Weinheim, Germany.

10. Cai, .T. B.; Wang, P. G. “Recent development of anticancer NO donors” Exp. Opin. Ther. Patents 2004, 14, 849-857.

9. Cai, T. B.; Tang, X.; Nagorski, J.; Brauschweiger, P. G.; Wang, P. G. “Synthesis and cytotoxicity of 5-fluorouracil/diazeniumdiolate conjugates” Bioorg. Med. Chem. 2003, 11, 4971-4975.

8. Jia, Q.; Cai, T.; Huang, M.; Li, H.-Y.; Xian, M.; Poulos, T. L.; Wang, P. G. “Isoform-selective Substrates of nitric oxide synthase” J. Med. Chem. 2003, 46, 2271-2274.

7. Tang, X. ; Cai, T.; Wang, P. G. “Synthesis of beta-lactamase activated nitric oxide donors” Bioorg. Med. Chem. Lett. 2003, 13, 1687-1690.

6. Li, H.-Y.; Shimizu, H.; Flinspach, M.; Jamal, J.; Yang, W.-P.; Xian, M.; Cai, T.; Wen, E. Z.; Jia, Q.; Wang, P. G.; Poulos, T. L. “Novel binding mode of N-alkyl- N'-hydroxyguanidine to neuronal nitric oxide synthase reveals insights into NO biosynthesis” Biochemistry, 2002, 41, 13868-13875.

5. Xian, M.; Fujiwara, N.; Wen, Z.; Cai, T.; Kazuma, S.; Janczuk, A. J.; Tang, X.; Telyatnikov, V. V.; Zhang, Y.; Chen, X.; Miyamoto, Y.; Taniguchi, N.; Wang, P. G. “Novel substrates for nitric oxide synthases” Bioorg. Med. Chem. 2002, 10, 3049-3055.

4. Cai, T.; Xian, M.; Wang, P. G. “Electrochemical and peroxidase oxidation study of N'-Hydroxyguanidine derivatives as NO donors” Bioorg. Med. Chem. Lett. 2002, 12, 1507-1510.

3. Jia, Q.; Janczuk, A.; Cai, T.; Xian, M.; Wen, Z.; Wang, P. G. “Nitric oxide donors as anti-cancer agents” Exp. Opin. Ther. Patents 2002, 12, 819-826.

2. Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A. J. "Nitric oxide donors: chemical activities and biological applications" Chem. Rev. 2002, 102, 1091-1134.

1. Cai, T.; Min, J.; Zhang, L. “Synthesis of lactosyl phosphate diester derivatives of nucleosides” Carbohydr. Res. 1997, 303, 113-117.

viii FIELDS OF STUDY

Major Field: Chemistry

TABLE OF CONTENTS

P a g e

Abstract...... ii

Dedication...... iv

Acknowledgments ...... v

Vita ...... vii

List of Schemes...... xii

List of Tables...... xiv

List of Figures ...... xv

List of Abbreviations ...... xvii

Chapter:

1 N-Hydroxyguanidines as novel nitric oxide donors ...... 1

1.1 Introduction ...... 1 1.2 Synthesis and NO releasing ability of N-hydroxyguanidines as NOS substrates...... 5 1.3 Discovery of a novel binding mode for NOS...... 11 1.4 Electrochemical and peroxidase oxidation studies of N-hydroxyguanidines...... 16 1.5 Conclusion...... 23 1.6 Experimental...... 24

2 New carbohydrate and 3-morphorlinosyndnonime conjugates...... 33

2.1 Introduction...... 33 2.2 Synthesis of sugar-SIN1 conjugates...... 36 2.3 Enzymatic decomposition of sugar-SIN1 conjugates...... 40

x 2.4 Conclusion...... 46 2.5 Experimental...... 46

3 Design and synthesis of sialic acid-NO conjugates...... 58

3.1 Introduction...... 58 3.2 Design and synthesis of sialic acid-NO conjugates...... 62 3.3 Hydrolysis of sialic acid/NO conjugates by sialidase...... 68 3.4 Conclusion...... 71 3.5 Experimental...... 72

4 Synthesis of prodrugs of 5-fluorouracil...... 80

4.1 Introduction...... 80 4.2 Design and synthesis of FU and NO conjugates...... 84 4.3 NO releasing and cytotoxicity of FU-NO conjugates...... 87 4.4 Design and synthesis of carbohydrate-FU conjugates...... 91 4.5 Conclusion...... 98 4.6 Experimental...... 99

5 Synthesis of carboxyl indazoles as NOS inhibitors...... 112

5.1 Introduction...... 112 5.2 Synthesis of carboxylindazoles ...... 114 5.3 Conclusion...... 116 5.4 Experimental...... 117

References...... 120

Appendix: Selected NMR spectra...... 129

LIST OF SCHEMES

Scheme Page

1.1 Biological generation and functions of NO ...... 2

1.2 Synthetic methods of N-hydroxyguanidines ...... 6

1.3 Two modes of catalytic reactions of NOS ...... 14

1.4 Design and synthesis of N-isopropyl-N’-hydroxyarginine ...... 15

1.5 Proposed electrochemical oxidation of N-hydroxyguanidines ...... 19

2.1 NO releasing from SIN-1...... 35

2.2 Synthesis of SIN-1...... 36

2.3 Synthesis of glucose, galactose and SIN-1 conjugates ...... 38

2.4 Synthesis of galactose and GEA 3126 conjugate ...... 39

2.5 Synthesis of the phenol linkage on galactose ...... 40

2.6 Proposed NO releasing from 37b ...... 46

3.1 NO releasing from diazeniumdiolates ...... 59

3.2 Attempts to synthesize type I sialic acid-NO conjugates ...... 64

3.3 Attempts to synthesize type II sialic acid-NO conjugates...... 65

3.4 Synthesis of sialic acid and SIN-1 conjugates ...... 67

3.5 Synthesis of sialic acid-diazeniumdiolate conjugates...... 68

xii 3.6 NO releasing mechanism of 63 ...... 71

4.1 Anticancer mechanisms of FU ...... 81

4.2 Structures and mechanism of FU prodrugs ...... 83

4.3 Synthesis of 66...... 85

4.4 Synthesis of 72...... 86

4.5 Synthesis of 73-75...... 87

4.6 The hydrolysis pathway of conjugate 72...... 90

4.7 Synthesis of 79 via 1,6-anhydroglucose ...... 93

4.8 An alternative synthesis of 79 ...... 94

4.9 Synthesis of 83...... 94

4.10 Synthesis of 88...... 95

4.11 Synthesis of 89...... 96

4.12 Synthesis of 95...... 97

4.13 Synthesis of 98, 100 and 102...... 98

5.1 General methods of synthesis of indazoles...... 115

5.2 Synthesis of 7-carboxylindazole ...... 116

5.3 Synthesis of 5-carboxyindazole and 6-carboxyindazole ...... 116

xiii

LIST OF TABLES

Table Page

1.1 Initial rates of NO formation from oxidation of N-hydroxyguanidines by NOS ...... 8

1.2 NO formation from the oxidation of N-aryl-N’-hydroxyguanidines by NOS ...... 10

1.3 The oxidation potentials of N-substituted-N’-hydroxyguanidines...... 18

1.4 Nitrite concentration in the reaction of substrates with HRP for 20 min ...... 21

2.1 The kinetic parameters of 37a/b as substrates of glycosidases ...... 43

2.2 Nitrite concentration in the incubation of 37a/b with or without the glycosidases ...... 44

3.1 Nitrite formation from 61 and 63...... 71

4.1 Median effect dose for HeLa and Du145 cell lines...... 89

5.1 IC50 of indazole derivatives for nNOS...... 117

xiv

LIST OF FIGURES

Figure Page

1.1 Schematic structure of the active site of NOHA-bound iNOS ...... 3

1.2 Synthesized hydroxyguanidines and guanidines...... 7

1.3 Schematic structures of BHG bound to nNOS...... 12

1.4 Schematic structures of IHG bound to nNOS...... 13

1.5 Cyclic voltammogram of 13a in acetonitrile ...... 18

2.1 Structures of sugar-NO conjugates and SIN-1...... 34

2.2 The UV absorbance changing of 37b ...... 41

2.3 Lineweaver-Burk plot of 37a as the substrate of β-glucosidase ...... 42

2.4 Lineweaver-Burk plot of 37b as the substrate of β-galactosidase . . . . . 42

2.5 Measurement of NO profiles of 37a, 37b and SIN-1 ...... 45

3.1 Half-lives of diazeniumdiolates ...... 59

3.2 Prodrugs of diazeniumdiolates ...... 61

3.3 Influenza virus and NO prodrug design ...... 62

3.4 Design of sialic acid-NO donor conjugates ...... 64

3.5 Crystal structure of 51 ...... 65

3.6 The UV absorbance changing of 63...... 69

xv 3.7 Time course of NO release from 63...... 70

4.1 NO releasing from prodrug 72 and 75 in a pH = 8.0 buffer ...... 88

4.2 NO releasing from prodrug 72 and 75 in the presence of esterase . . . . . 88

4.3 Illustration of ADEPT ...... 92

5.1 Structure of 7-NI and 7-CI...... 114

xvi

LIST OF ABBREVIATIONS

α alpha

[α] specific rotation

Ac acetyl

ADEPT antibody-directed enzyme prodrug therapy br broad (IR and NMR)

β beta

BH4 5,6,7,8-tetrahydrobiopterin

BHG N-butyl-N'-hydroxyguanidine

BOC tert-butoxycarbonyl

BSA N, O-bistrimethylsilyl-acetamide n-Bu normal-butyl t-Bu tert-butyl

BTEAC benzyltriethylammonium chloride

Bz benzoyl

°C degrees Celsius calcd calculated

7-CI 7-carboxyl-1-H-indazole

xvii δ chemical shift in parts per million downfield from tetramethylsilane d doublet (spectra); day(s)

DCC dicyclohexylcarbodiimide

DCE 1,2-dichloroethane

5'dFUrd 5'-deoxy-5-fluorouridine

DIPEA diisopropylethylamine

DMAN 1,8-bis(N,N-dimethylamino)naphthalene

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

DPD dihydropyrimidine dehydrogenase dTMP thymidine-5'-monophosphate

DTT dithiothreitol dTTP thymidine-5'-triphosphate dUMP 2’-deoxyuridine-5’-monophosphate

EDCI 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride

EI electron impact

EMFU 1-ethoxymethyl-5-fluorouracil eNOS epithelial nitric oxide synthase eq. equivalent

ESI electrospray ionisation

Et ethyl

FAD flavin adenine dinucleotide

FdUMP 5-fluoro-2'-deoxyuridine-5'-monophosphate

xviii FMN flavin mononucleotide

FTO Ftorafur

FU 5-fluorouracil

FUTP 5-fluorouridine-5'-triphosphate g gram(s)

GEA Mesoionic oxatriazole-5-imine h hour(s)

HA Hemagglutinin

HRMS high resolution mass spectrometry

HRP horseradish peroxidase

IHG N-butyl-N'-hydroxyguanidine iNOS inducible nitric oxide synthase

IR infrared

J coupling constant in Hz (NMR) k kilo

L liter(s) m milli; multiplet (NMR)

μ micro

M moles per liter

Mc chloromethylsulfonyl

Me methyl

MHz megahertz min minute(s)

xix mol mole(s)

MeTHF 5,10-methylene tetrahydrofolate

MS mass spectrometry; molecular sieves m/z mass to charge ratio (MS)

NADPH nicotinamide adenine dinucleotide phosphate (reduced form)

NeuAc N-acetylneuraminic acid

7-NI 7-nitro-1-H-indazole

NMM 4-methylmorpholine

NMO 4-methylmorpholine N-oxide

NMR nuclear magnetic reasonance nNOS neuronal nitric oxide synthase

NO nitric oxide

NOHA N-hydroxy-L-arginine

NOS nitric oxide synthase p para

Ph phenyl ppm parts per million

PSA prostate specific antigen py pyridine q quartet (NMR)

RNS reactive nitrogen species rt room temperature s singlet (NMR); second(s)

xx SIN-1 3-Morphorlinosydnonimine

SOD superoxide dismutase t tertiary (tert) t triplet (NMR)

TBAF tetrabutylammonium fluoride

TBS t-butyldimethylsilyl

Tf trifluoromethanesulfonyl

THF tetrahydrofuran

TLC thin layer chromatography

TMS trimethylsilyl

TS thymidylate synthase

UTP uridine-5'-triphosphate

xxi

CHAPTER 1

N-HYDROXYGUANIDINES AS NOVEL NITRIC OXIDE DONORS

1.1 Introduction

Nitric oxide (NO), a messenger molecule, plays an important role in numerous physiological and pathophysiological processes, such as neuronal communication, blood vessel modulation and immune response. Endogenous NO is produced almost exclusively by L-arginine catabolism to L-citrulline in a reaction catalyzed by a family of three NO synthases (NOS) (Scheme 1.1).1 The three isoforms of NOSs share structural similarities and have nearly identical catalytic mechanisms. Each of the isoforms catalyzes the stepwise formation of NO and L-citrulline from L-arginine, O2 and

NADPH-derived electrons. During the 5 electron oxidation process, N-hydroxy-L- arginine (NOHA) serves as a key intermediate.

Three main isoforms of NOS exist in human cells, each associated with a particular physiological process (Scheme 1.1). Constitutive endothelial NOS (eNOS or

NOS III) regulates smooth muscle relaxation and blood pressure; constitutive neuronal

NOS (nNOS or NOS I) involves in neurotransmission and long-term potentiation; the NO produced from inducible NOS (iNOS or NOS II) in activated macrophage cells acts as a cytotoxic agent in normal immune defense against microorganisms and tumor cells. The

1 constitutive isoforms (nNOS and eNOS) require added Ca2+ and calmodulin for activity and produce relatively small amount of NO, while the inducible isoform (iNOS) has tightly bound Ca2+ and calmodulin, and produces relatively large amount of NO.

NH NOH O NOS NOS HO2C HO2C HO2C N NH2 N NH2 N NH2 NO H NADPH, O2 H NADPH, O2 H NH2 NH2 NH2 L-Arginine N-Hydroxyl-L-arginine L-Citruline

NOSs Locations Characteristics Major biological functions

nNOS (NOS-1) brain, spinal cord, peripheral constitutive, Ca2+ dependent Neuromediator iNOS (NOS-2) macrophages, other tissues inducible, Ca2+independent Host defender, cytotoxic

2+ eNOS (NOS-3) endotheliun constitutive, Ca dependent Vasodilator tone modulator

Scheme 1.1: Biological generation and functions of NO

Crystal structures of arginine and NOHA bound NOS show that the substrate binds close to the heme by a network of hydrogen bonds.1 L-Arginine is located immediately above the heme group. In the structure of murine iNOS oxygenase domain

(Fig. 1.1), the arginine guanidine group forms two hydrogen bonds to both carboxy oxygens of Glu371 localizing the substrate over the heme, and one hydrogen bond to the carbonyl of Trp366. Additional hydrogen bonds are formed between the arginine carboxy group and Tyr367 and the carboxy of Asp376. In addition, the amino group of

2 arginine is hydrogen-bonded with the propionate group of heme. The central guanidine- nitrogen atom of arginine is 3.8 Å away from the heme iron.

H Gly365 O Trp366 R N O N H R' N O H H H O O N N H H O NH Glu371 N H N O N Fe N O N O H O Asp376 O S + H2N Cys194 Tyr367 O H O O

Figure 1.1: Schematic structure of the active site of NOHA-bound iNOS

The guanidine fragment is distinct for the NO biosynthesis. It is the guanidine group that undergoes oxidation to the N-hydroxyguanidine and further to urea and NO by

NOS. The binding of the substrates with the heme in the catalytic pockets of NOS is largely due to the guanidine or hydroxyguanidine groups. So it is rational to expect that other N-hydroxyguanidine derivatives can be used as enzymatic NO donors. So far, only

3 very few compounds have been clearly shown to act as NOS substrates.2 Among them,

L-arginine and NOHA are natural NOS substrates, while homo-L-arginine and N- hydroxy-homo-L-arginine are arginine analogs with one more carbon on the aliphatic chain. Other L-arginine analogues, such as canavanine,3 ε-guanidino-carproic acid,3 agmatine, N-hydroxy-agmatine,4 and L-tyrosyl-L-arginine,5 have been proposed to be

NOS substrates because they caused endothelium-dependent vasorelaxation or accumulation of nitrite in cell cultures. However, only some of these results have been confirmed with purified NOSs and the precise nature of the transformations of these compounds is not yet known.6

The very limited number of substrates for NOS suggests that highly specific structural features are required for NO generation from NOS. Furthermore, because most substrates of NOS are arginine or NOHA analogs, it also suggests that the α-amino acid portions of those substrates play an important role in the catalysis.7 However, since some simple guanidines and isothioureas such as aminoguanidine, S-ethyl-isothiourea, and N- phenyl-S-methyl-isothiourea are strong NOS inhibitors through binding at the active site,8 it appears that the α-amino acid moiety of arginine can be removed without detrimental consequences, while the integrity of the guanidine function must be partially retained. It is logical then to assume that simple compounds bearing guanidine or hydroxyguanidine functions, not derived from L-Arginine or NOHA, can interact with NOS active site and possibly serve as substrates of NOS.

In an effort to better understand the structural factors that are important for the substrates of NOS and to find a new type of enzymatic NO donors, a series of compounds bearing an N-hydroxyguanidine functional group were synthesized and studied their 4 oxidation by recombinant NOS and horseradish peroxidase (HRP). It was found that several N-alkyl-N’-hydroxyguanidines can be efficiently oxidized by NOS and HRP to generate NO. Most interestingly, some N-aryl-N’-hydroxyguanidines have been found to be selective substrates for iNOS.

1.2 Synthesis and NO releasing ability of N-hydroxyguanidines as NOS substrates

Since the key step in the synthesis of hydroxyguanidine derivatives is the construction of the N-hydroxyguanidine functional group, the synthetic methods for hydroxyguanidines are summarized here. So far, three major methods have been documented for this class of compounds. The first one utilizes a cyanamide as the intermediate (Scheme 1.2(a)).9 It has been successfully employed in previous work and was shown to be a reliable pathway for the preparation of primary and secondary N- hydroxyguanidines including NOHA, homo-NOHA and nor-NOHA.10 The second method involves thioureas as a key intermediate (Scheme 1.2(b)).9,11 Then methylation, oxidation or elimination of sulfur gives intermediates which are ready to do the substitution or addition with hydroxylamine. This method was used in the first reported synthesis of NOHA.9 It could also be used in the synthesis of N, N’-disubstituted-N’’- hydroxyguanidines. A convenient reagent, 1-benzyloxy-3-benzyloxycarbonylthiourea, was developed for N-hydroxyguanylation.12 When treated with an amine, it will give N- and O-protected hydroxyguanidine derivatives. Catalytic hydrogenation will remove the protecting groups and give the final product (Scheme 1.2(c)). This reagent has been used to introduce hydroxyguanidine functional group to relatively complex molecules.13

However, this reagent is not commercially available and it has to be prepared first.

OH NH OH N BrCN H 2 (a) R NH2 RCNN R N NH2 H

S CH I R 3 N NH H OH S SO3H N 1. Thiophosgene H2O2 NH OH R NH R R 2 R 2 N NH N NH 2. NH 2 Na MoO N NH2 (b) 3 H 2 4 H H HgO RCNHN Et3N

S O Bn N OH CbzN NOBn H , Pd/C N H H 2 R NH2 R Cbz R (c) HgCl2 Et3N N N N NH2 H H H

Scheme 1.2: Synthetic methods of N-hydroxyguanidines

Starting from the corresponding amines, 24 N-substituted hydroxyguanidines (1-

16) (Fig. 1.2) were synthesized using a general procedure (Scheme 1.2(a)) previously reported14 with some modification (see the experimental section for detail). These compounds were fully characteristized by 1H, 13C NMR, and high-resolution mass spectroscopy. Activity of these N-hydroxyguanidine compounds as substrates of NOS was evaluated by hemoglobin assay.15 For our evaluation, NOHA was used as a control indicating 100% activity. The results were summarized in Table 1.1 and Table 1.2 (the compounds which are not in the tables were not substrates of NOS).

H H H H H H2N N H2N N H2N N H2N N H2N N N N N N N HO HO HO HO HO 5 1 2 34 H H H H H N N H H2N N 2 H2N N H2N N H2N N N N N N N HO HO HO HO HO 10 6 7 8 9 H H H H H H2N N H N N H2N N H2N N H2N N 2 NH2 N N N N HO X N HO HO HO HO 15 11 12 13 X = H (a), MeO (b), 14 Me (c), Cl (d), Br (e)

H H H2N N H H H2N N X H2N N H2N N N NH HO NH NH X 16 X = H (a), MeO (b), 17 18 19 X = H (a), MeO (b), Me (c) Me (c), Cl (d), NO2 (e)

Figure 1.2: Synthesized hydroxyguanidines and guanidines

NOH iNOS nNOS eNOS R H2N N H

R = H 0 0 0

R = ethyl 3 1 6

R = n-propyl 15 70 24

R = i-propyl 32 76 38

R = cyclopropyl 0.5 78 26

R = n-butyl 42 64 20

R = n-pentyl 32 31 0

R = n-hexyl 3 4 0

R = cyclohexyl 3 3 0

Table 1.1: Initial rates of NO formation from oxidation of N-hydroxyguanidines by NOS ( Percentage compared to NOHA)

As expected, most of the synthetic hydroxyguanidine derivatives were not as active as L-Arginine or NOHA. Without the α-amino acid moiety that allowed L-Arg or

NOHA to be specifically recognized by NOS, these synthetic compounds may fail to interact most favorably with the corresponding binding site of NOS. However, the results listed in Table 1.1 indicated that the activity of these compounds was related to the structure of substituents. Without any substituents, N-hydroxyguanidine itself was not a substrate. When a small alkyl group (such as ethyl, 1) was attached to hydroxyguanidine, only very low reactivity was observed. Surprisingly, one additional methylene group, n-

8 propyl substitution in 2, significantly enhanced the activity, especially for nNOS (up to

70% of NOHA). As the size of the alkyl chain increased to isopropyl or n-butyl group, 3 and 4 turned out to be good substrates. The Km values of 3 was 77 μM for iNOS and Km

= 56 μM for nNOS, whereas the Km values of 4 was 33 μM for iNOS and 67 μM for nNOS. N-Cyclopropyl-N’-hydroxyguanidine (12) was not only the best substrate for nNOS, but also a selective substrate for nNOS over iNOS. However, if the alkyl substituents became too long or too bulky (such as n-hexyl and cyclohexyl), the activity decreased significantly.

Since N-isopropyl-N’-hydroxyguanidine (3) and N-n-butyl-N’-hydroxyguanidine

(4) are excellent substrates for NOS, their corresponding derivatives, isopropylguanidine

(17) and butylguanidine (18) were also tested. These two compounds cannot generate

NO by NOS even at high concentration (10 mM). This suggests that the two steps in the

NO biosynthesis are distinct processes. The first oxidation step seems to require an exact

“natural” substrate such as L-arginine. Such substrate specificity can not be compromised by replacing L-arginine with simple N-substituted guanidine compounds.

In contrast, the second oxidation step of NOS reaction is much more compromising.

Some N-substituted hydroxyguanidines can be oxidized to produce NO in this step.

R NOH iNOS nNOS eNOS

H2NN H

R = H 32 <0.5 <0.5

R = OCH3 29 <0.5 <0.5

R = CH3 24 <0.5 <0.5

R = Br 15 <0.5 <0.5

R = Cl 13 <0.5 <0.5

Table 1.2: NO formation from the oxidation of N-aryl-N’-hydroxyguanidines by NOS (Percentage of NO formation (nmol/min/mg protein) to NOHA)

Next a series of N-aryl-N’-hydroxyguanidines (13a-e) were investigated (Table

1.2). Most interestingly, it was found that these compounds were selective substrates for iNOS. N-Phenyl-N’-hydroxyguanidine (13a) was the best substrate in this series (Km =

243 μM). The rate of NO formation, when using 500 μM of 13a, was 39 nmol/min/mg protein, as high as 70% of L-Argine and 32% of NOHA. The NO formation rates of other substrates were in the range of 16-35 nmol/min/mg protein. When used in the assay for nNOS and eNOS, none of them produced detectable NO even at high concentrations

(10 mM). This is in sharp contrast to that of their aliphatic counterparts (1-12), which exhibited good activity but hardly any isoform selectivity. These N-aryl-N’- hydroxyguanidine compounds are the first reported isoform selective NOS substrates so far. Given the high active-site conservation and high structural similarity among NOS isoforms, this observation deserves further mechanistic consideration. 10 Further study on the effects of incubation conditions on the formation of nitrite from these substrates indicated that the characteristics of this reaction were very similar to those of the NOS-dependent oxidation of the endogenous NOHA. The reaction required the presence of both the active enzyme and other cofactors (NADPH, FAD,

FMN, BH4, and CaCl2). This process could be strongly inhibited by classical NOS inhibitors such as N-nitro-L-arginine and N-methyl-L-arginine. It indicated that the oxidation of these substrates should occur in the active site of iNOS and the mechanism was similar to NOHA.

To further explore the structural effect on the activity, some structure-related compounds (14-16) were prepared and assayed. When the phenyl ring was replaced by a naphthalene ring (14) or by a benzyl group (16), the activity of substrate was destroyed completely. This increase in size might over-grow the “specific” phenyl-binding pocket in iNOS. As for N-alkyl substituted compounds, when the hydroxyguanidine group was changed to a guanidine group (19), none of them acted as a substrate of NOS.

1.3 Discovery of novel binding modes for NOS

To better understand how these unnatural substrates interact with NOS, we collaborated with Poulos’ laboratory in University of California at Irvine and obtained the crystal structures of rat nNOS complexed with N-butyl-N'-hydroxyguanidine (BHG, 4) and N-isopropyl-N'-hydroxyguanidine (IHG, 3), which are the most active substrates among N-substituted N'-hydroxyguanidines. The bound models of these compounds were illustrated in Fig. 1.3 and Fig. 1.4.

11 Gln478 Trp587 Pro565 Val567 O H CH3 OH N O H NH Phe584 H NN N Glu592 O Fe N N

S OH Cys194 OH O

Figure 1.3: Schematic structures of BHG bound to nNOS

The orientation of the guanidino plane of BHG found at the nNOS active site is similar to that of NOHA (Fig. 1.3). It is hydrogen-bonded to the Glu592 side chain and

Trp587 carbonyl oxygen similar to the NOHA complex. The BHG OH group points toward backbone amide of Gly586 with a tilt angle of 16 degrees from the guanidino plane, which is also similar to NOHA. The main difference between BHG and NOHA is the location of the butyl group in BHG compared to the methylene carbons of NOHA.

The end of the butyl group curls toward Gln478 and Val567 away from the binding site for the substrate or NOHA -amino and carboxylate groups. The side chain of Gln478 has to swing away slightly to avoid close van der Waals contact to the ligand while the butyl group is about 3.7Å from Val567 which probably provides favorable non-bonded interactions.

Trp587 Pro565 Val567 O HO H CH3 N O H N Phe584 NH N H N CH3 Glu592 O Fe N N

S OH Cys194 OH O

Figure 1.4: Schematic structures of IHG bound to nNOS

To our surprise, the binding mode of IHG was totally unexpected (Fig. 1.4). In this case, the isopropyl-attached nitrogen atom takes the position normally occupied by the OH-substituted terminal nitrogen atom in the other two complexes. Two methyl groups of isopropyl moiety make favorable van der Waals contacts with side chains of both Val567 and Phe584. This ligand orientation places the OH group into the corner between the Glu592 carboxylate and the Trp587 carbonyl. The OH oxygen atom is only

2.48Å from the Trp587 carbonyl oxygen while the two nitrogen atoms of guanidine are involved in the bifurcate H-bonds with the Glu592 carboxylate oxygen atoms. The OH group also bends downward toward the heme by 15 degrees from the guanidine plane.

13 This unexpected binding mode of the IHG in the active site provides new insights into the mechanism of hydroxyguanidine oxidation to generate NO.

E592 E592

OO OO

HHH O W587 HH H NNO R NNH O W587 + O + O N + NO + NO H R N R N NH2 O R N NH2 H . H H O H O . O O G586 O O G586 FeIII FeIII N-isopropyl-N'-hydroxyguanidine N-butyl-N'-hydroxyguanidine and NOHA

Scheme 1.3: Two modes of catalytic reactions of NOS

Since the OH group in IHG structure locates even farther away from the heme iron, it suggests that the Nω atom of NOHA is the source of hydrogen atom supplied to the ferric-superoxy species. For hydroxyguanidine compounds, all three nitrogen atoms are equivalent and hydrogen-bearing under physiological condition, being charged and electron delocalized. In the case of IHG, it is the alkyl-substituted nitrogen that donates hydrogen to ferric-superoxy species in the reaction. A hydrogen-bearing nitrogen atom in the vicinity of ferric-superoxy species appears to be the key feature for NOS substrates.

The orientation of hydroxy group, either pointing to the Gly586 backbone amide or to the

Trp587 carbonyl, appears not to be that critical (Scheme 1.3). However, the central carbon atom of the guanidine group still remains close enough to the heme iron. 14

Molecular design: Hydroxy-Guanidine binding

OH Hydrophobic N binding COO- NH N H + Amino acid binding NH3

Synthesis:

CO2H a CO2tBu b CbzHN CbzHN

NH2 NH2 20 21

c CO2tBu CO2tBu d CbzHN H2N NHBoc NHBoc 22 23

S OH N CO2tBu e NH N CO2tBu H NH N NHBoc H 24 NHBoc 25 OH N f CO2H NH N H NH2 26

Scheme 1.4: Design and synthesis of N-isopropyl-N’-hydroxyarginine Reagents and conditions: (a) t-BuOAc, HClO4, rt, 2 d, 84%; (b) (BOC)2O, CH2Cl2, rt, 3 h, 96%; (c) H2, Pd-C, rt, 5 h, 98%; (d) (i) CSCl2, CaCO3, rt, 16 h; (ii) iPr-NH2, rt, 3 h, 83%; (e) HgO, NH2OH, rt, 12 h, 51%; (f) TFA, rt, 2 h, 96%.

Suggested from the crystal structure of IHG with NOS, N-isopropyl-N’- hydroxyarginine was first proposed as an unnatural substrate of NOS, since it could have the combined interactions with NOS for both the hydrophobic and amino acid bindings.

It was then synthesized (Scheme 1.4) and evaluated. Enzymatic assay showed that it did

15 not have NO releasing ability. The reason was proposed that the hydrophobic and amino acid binding are not fit for this molecule. In another word, there is a strain for one molecule to bind the two directions.

1.4 Electrochemical and peroxidase oxidation studies of N-hydroxyguanidines

Since the mechanism of NO formation in vivo was disclosed, scientists have found that a series of heme proteins could catalyze the oxidation of physiological and non-physiological hydroxyguanidine-containing compounds to release NO.16 For example, N-hydroxydebrisoquine could be oxidized by liver microsomes to release NO.

Recently, N-hydroxyl derivatives of guanidine based cardiovascular drugs shown good

17 NO-releasing ability under the oxidation by HRP in the presence of H2O2. Studies indicated that non-physiological guanidines and N-hydroxyguanidines could be regarded as nitric oxide donors. These results may explain the cardiovascular activity of some therapeutic reagents.18 It has been shown that N-alkyl-N’-hydroxyguanidines can release

19 NO by oxidation with some chemical oxidants, such as Pb(OAc)4 and K3FeCN6/H2O2.

To study this new type of enzymatic NO donors, the electrochemical properties and NO- release abilities of substituted N-hydroxygunidines by HRP were studied, so as to find some relationship between oxidation potentials and NO releasing ability.

The oxidation potentials of N-hydroxyguanidines were measured by cyclic voltammetry (Table 1.3). A typical voltammogram obtained from compound 13a is shown in Fig. 1.5. At 100 mV/s, two irreversible oxidation peaks were observed at Eox1 =

+0.60 V and Eox2 = +1.61 V. The oxidation peak of lower intensity at 1.10 V may be due to a decomposition product of the first oxidation. There was absolutely no sign of reversibility in either step. This indicates that the oxidation of N-hydroxyguanidines is a

16 totally irreversible process. For phenyl substituted N-hydroxyguanidines (13a–e), the substituents on the phenyl ring may have an effect on the value of the peak potentials.

Electron-donating groups lower the oxidation potentials, while electron-withdrawing groups have the opposite effect. But the differences are not pronounced. There is no obvious trend in oxidation potentials of benzyl N-hydroxyguanidines (16a–e), because the aromatic rings do not conjugate with the guanidine group. The oxidation mechanism of C=N-OH group of amidoximes has been proposed,20 so N-hydroxyguanidines should have the same oxidation pattern (Scheme 1.5). It involves one electron oxidation with formation of an iminoxyl radical intermediate. The second step is the one electron oxidation of this radical leading to nitrosoimine. Elimination of HNO from the nitrosoimine would give the cyanamide (path A) whereas a further one-electron oxidation would generate the cyanamide and NO (path B). The appearance of two oxidation peaks supported the two-electron oxidation in agreement with previous report from Ingold,22 that is, the removal of an electron from N-hydroxyguanidine at Eox1 is followed by a rapid, irreversible deprotonation. The iminoxyl radical formed is further oxidized at the higher potential and the resulting cation is again capable of loosing a proton irreversibly. The oxidation potentials of unnatural N-hydroxyguanidines are also comparable with N-

22 hydroxyarginine (Eox1 = +0.47 V and Eox2 = +1.05 V).

Figure 1.5: Cyclic voltammogram of 13a in acetonitrile

Compounds Eox1 (V) Eox2 (V)

13a 0.60 1.61

13c 0.57 1.48

13b 0.54 1.24

13d 0.61 1.72

13e 0.62 1.70

16a 0.65 1.44

16c 0.63 1.24

16b Not obtained 1.27

16d 0.51 1.14

16e 0.60 1.45

Table 1.3: The oxidation potentials of N-substituted-N’-hydroxyguanidines

+. OH OH O . O N Eox1 N very fast N Eox2 N + -e - H -e, - H+ RHN NH2 RHN NH2 RHN NH2 RN NH2 iminoxyl radical

A B -e-, -H+

RNHC N + HNO RNHC N + NO

Scheme 1.5: Proposed electrochemical oxidation of N-hydroxyguanidines

As shown in Scheme 1.1, the second step involved in the NOS-catalyzed oxidation of L-arginine is an oxidative cleavage of the C=NOH bond of N- hydroxyarginine with the formation of citrulline and NO. Besides NOS, cytochromes

P450 have also been found to catalyze the oxidative cleavage of the C=NOH bond not only of N-hydroxyarginine but also of many other compounds such as ketoximes, amidoximes.16,22

Previous study showed that HRP catalyzed the oxidation of NOHA by H2O2 with formation of citrulline and NO.23 In this study, it was found N-hydoxyguanidine derivatives, compounds 13a-e and 16a-e, could be catalyzed by HRP with the formation of NO. HRP is one of the most studied peroxidases,24 which has been shown to be an effective oxidizing enzyme for arylamidoximes by hydrogen peroxide. The products

19 were O-(arylimidoyl)arylamidoximes and HNO/NO.20,25 This peroxidase effects a one- electron oxidation of the substrate and is regenerated by transferring two electrons to a molecule of H2O2. It is also a model for in vivo systems, which can oxidize these types of compounds to generate NO. Compounds 13a-e and 16a-e together with NOHA and L- arginine were incubated with HRP and H2O2 separately. Twenty minutes later, the

- 26 concentration of NO2 generated in the solution was determined by Griess method. The results are shown in Table 1.4.

- Substrate [NO2 ] μM

L-Arginine 0

NOHA 12

13a 28

13c 6

13b 24

13d 63

13e 54

16a 33

16c 113

16b 49

16d 151

16e 65

Table 1.4: Nitrite concentration in the reaction of substrate with HRP for 20 min (substrate: 600 μM, HRP: 80 μg/mL, H2O2: 600 μM)

21 Incubation the L-arginine or other substituted guanidines, such as phenylguanidine, with peroxidase did not result in the generation of nitrite, while incubation of all other N-hydroxyguanidines with the enzyme produced different amount of nitrite. 16d was the best NO donor among these tested compounds, and it produced

151 μM nitrite in 20 min. Except 13c, all other N-hydroxyguanidines were more potent

NO donors than NOHA under peroxidase oxidation. In control experiments, incubation of these compounds with H2O2 in the absence of HRP did not produce any nitrite. The differences of NO generation may be due to the difference of oxidation potentials or different binding affinity to the enzyme. Examination of the results in Table 1.3 and

Table 1.4, it seems that there is no direct correlation between oxidation potentials and NO release. Thus, oxidation potentials may not have a crucial effect on NO releasing. It has been found that classical peroxidases oxidize the substrate by electron transfer to heme center and bind the substrate near the heme edge.27 The binding affinity of the substrate to the active site is speculated to be the main factor affecting NO generation.

HRP, cytochromes P450 and NOSs are similar in the types of reactions that they catalyze.28 They have similar heme binding sites. Like P450s, HRP’s oxidation of N- hydroxyguanidine is a rather general reaction.29 P450’s process needs NADPH to form the high valent iron oxo heme intermediate, which contains the ferryl group [Fe=O]+3,

30 responsible for the oxidation of substrates. In this study, H2O2 could substitute NADPH to generate the iron oxo form directly from the resting state of the heme. The NO releasing mechanism of N-hydroxyguanidines by oxidation of HRP and H2O2 may be similar to NOSs’, in that they generate the similar products.

22 This study suggested a new possible way of endogenous NO formation different from the pathway, which only involves NOSs and NOHA. This new way of NO formation is the oxidation of exogenous N-hydroxyguanidines to NO by some hemeproteins in cells not containing NOSs. Using this strategy, it is possible to form NO in sites where it is needed but lacking NOSs. N-Hydroxyguanidines would act as a relatively stable and transportable precursor of NO.

The experimental data clearly show that N-hydroxyguanidine derivatives are potential NO donors under the catalysis of peroxidase. There was no clear relationship between the oxidation potentials and enzymatic NO releasing ability. Such enzymatic controlled NO generating compounds have potential for use in a variety of biomedical applications.

1.5 Conclusion

In summary, an extensive structural screening of N-hydroxyguanidine compounds revealed that relatively simple exogenous compounds, not bearing an amino acid function, could be oxidized by NOS in a manner similar to NOHA, with a significant rate of NO formation. The structure-activity relationship showed that a potent NOS substrate shared at least two characteristics: (1) an N-hydroxyguanidine functional group, capable of anchoring the substrate in the NOS active site and furnishing the second step of the NOS reaction; (2) a suitable hydrophobic chain that interacts favorably with the hydrophobic cavity in NOS active site. Moreover, to our best knowledge, N-aryl-N’- hydroxyguanidines (13a-e) are the first series of isoform selective substrates for NOS.

This finding is significant not only for understanding the NOS mechanism, but also in using such compound as isoform-specific probe in biomedical experiments.

1.6 Experimental

General procedure for synthesis of N-hydroxyguanidines. Step 1: Synthesis of corresponding cyanamide. A solution of corresponding amine (10 mmol) and

o anhydrous Et3N (1.51 g, 15.1 mmol) in dry CH2Cl2 (30 mL) was cooled to 0 C under argon atmosphere. To the solution was added BrCN (3.5 mL 3.0 M in CH2Cl2, 10.5 mmol) dropwise. After stirring 10 h at rt, the mixture was filtered through a short silica gel column. The filtrate was concentrated. The crude product was purified by flash chromatography in 55-85% yield.

Step 2: N-Substituted-N’-hydroxyguanidines. A mixture of cyanamide (0.6 mmol), hydroxyamine hydrochloride (43 mg, 0.61 mmol) and anhydrous K2CO3 (173 mg, 1.21 mmol) in anhydrous EtOH (5 mL) was stirred at rt for 5 hr. Then the mixture was filtrated through celite. After removing the solvent by rota-vap, the crude product was purified with flash chromatography to give N-hydroxyguanidine (40-78%).

1 N-Ethyl-N’-hydroxyguanidine (1): H NMR (500 MHz, CD3OD) δ 3.27 (q, 2H, J = 7.0

13 Hz,), 1.21 (t, 3H, J = 7.0 Hz,); C NMR (125 MHz, CD3OD) δ 159.0, 36.2, 13.4; HRMS calcd for C3H9N3O m/z 103.0746, found 103.0741.

1 N-n-Propyl-N’-hydroxyguanidine (2): H NMR (400 MHz, CD3OD) δ 3.16 (t, 2H, J =

13 7.6 Hz,), 1.57-1.65 (m, 2H), 0.97 (t, 3H, J = 7.6 Hz,); C NMR (100 MHz, CD3OD) δ

158.6, 42.7, 22.0, 10.1; HRMS calcd for C4H11N3O m/z 117.0902, found 117.0899.

1 N-i-Propyl-N’-hydroxyguanidine (3): H NMR (400 MHz, CD3OD) δ 3.70-3.78 (m,

13 1H), 1.23 (d, 6H, J = 6.4 Hz); C NMR (100 MHz, CD3OD) δ 158.4, 43.9, 21.4; HRMS calcd for C4H11N3O m/z 117.0902, found 117.0901. 24 1 N-n-Butyl-N’-hydroxyguanidine (4): H NMR (500 MHz, CD3OD) δ 3.00 (t, 2 H, J =

7.0 Hz), 1.45-1.53 (m, 2H), 1.34-1.38 (m, 2H), 0.993 (t, 3 H, J = 7.5 Hz); 13C NMR (125

MHz, CD3OD) δ 157.9, 40.7, 31.7, 19.9, 13.0; HRMS calcd for C5H13N3O m/z 131.1059, found 131.1060.

1 N-sec-Butyl-N’-hydroxyguanidine (5): H NMR (400 MHz, CD3OD) δ 3.52 (m, 1H),

1.56 (m, 2H), 1.20 (d, 2 H, J = 6.4 Hz), 0.95 (t, 3 H, J = 7.2 Hz); 13C NMR (100 MHz,

CD3OD): δ 160.0, 50.8, 30.4, 20.5, 10.7; HRMS calcd for C5H13N3O m/z 131.1059, found 131.1059.

1 N-iso-Butyl-N’-hydroxyguanidine (6): H NMR (500 MHz, CD3OD) δ 2.86 (t, 2 H, J =

13 6.0 Hz), 1.79 (m, 1H), 0.94 (d, 6 H, J = 6.9 Hz); C NMR (125 MHz, CD3OD) δ 159.2,

49.7, 29.5, 20.5; HRMS calcd for C5H13N3O m/z 131.1059, found 131.1060.

1 13 N-t-Butyl-N’-hydroxyguanidine (7): H NMR (500 MHz, CD3OD) δ 1.40 (s, 9H); C

NMR (125 MHz, CD3OD) δ 159.4, 53.3, 29.2; HRMS calcd for C5H13N3O m/z 131.1059, found 131.1062.

1 N-n-Pentyl-N’-hydroxyguanidine (8): H NMR (500 MHz, CD3OD) δ 3.17 (t, 2 H, J =

7.0 Hz), 1.56-1.61 (m, 2H), 1.32-1.39 (m, 4H), 0.931 (t, 3 H, J = 6.5 Hz); 13C NMR (125

MHz, CD3OD) δ 159.3, 41.1, 28.7, 28.5, 22.2, 13.1; HRMS calcd for C6H15N3O m/z

145.1215, found 145.1217.

1 N-Isoamyl-N’-hydroxyguanidine (9): H NMR (500 MHz, CD3OD) δ 3.02 (q, 2 H, J =

7.5 Hz), 1.65 (m, 1H), 1.40 (q, 2 H, J = 7.0 Hz), 0.92 (d, 6 H, J = 6.5 Hz); 13C NMR (125

MHz, CD3OD): δ 159.1, 40.4, 39.6, 26.9, 22.8; HRMS calcd for C6H15N3O m/z 145.1215, found 145.1216.

25 1 N-Hexyl-N’-hydroxyguanidine (10): H NMR (400 MHz, CD3OD) δ 3.01 (t, 3 H, J =

7.6 Hz), 1.50-1.54 (m, 2H), 1.30-1.37 (m, 6 H), 0.91 (t, 3 H, J = 6.4 Hz); 13C NMR (100

MHz, CD3OD) δ 159.2, 42.2, 32.8, 30.6, 27.7, 23.7, 14.4; HRMS calcd for C7H17N3O m/z 159.1372, found 159.1367.

1 N-Cyclohexyl-N’-hydroxyguanidine (11): H NMR (400 MHz, CD3OD) δ 3.34-3.40

13 (m, 1H), 1.63-1.92 (m, 5H), 1.16-1.40 (m, 5H); C NMR (100 MHz, CD3OD) δ 158.2,

50.8, 32.4, 25.0, 24.6; HRMS calcd for C7H15N3O m/z 157.1215, found 157.1209.

1 N-Cyclopropyl-N’-hydroxyguanidine (12): H NMR (400 MHz, CD3OD) δ 2.55 (m,

13 1H), 0.85-0.90 (m, 2H), 0.63-0.67 (m, 2H); C NMR (400 MHz. CD3OD) δ 160.3, 22.2,

6.5; HRMS calcd for C5H11N3O m/z 115.07456, found 115.07428.

1 N-Phenyl-N’-hydroxyguanidine (13a): H NMR (400 MHz, CD3OD) δ 7.30-7.20 (m, 4

13 H), 7.03-6.98 (m, 1 H); C NMR (100 MHz, CD3OD) δ 155.4, 139.4, 129.0, 122.9,

120.4; HRMS calcd for C7H9N3O 151.0746, found 151.0747.

1 N-(4-Methoxyl)-phenyl-N’-hydroxyguanidine (13b): H NMR (500 MHz, CD3OD) δ

7.12 (d, 2 H, J = 9.0 Hz), 6.80 (d, 2 H, J = 9.0 Hz), 3.73 (s, 3H); 13C NMR (125 MHz,

CD3OD) δ 159.5, 158.6, 127.5, 126.8, 115.0, 55.0; HRMS calcd for C8H11N3O2 m/z

181.0851, found 181.0851.

1 N-(4-Methyl)-phenyl-N’-hydroxyguanidine (13c): H NMR (400 MHz, CD3OD) δ 7.09

13 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 2.24 (s, 3H). C NMR (100 MHz, CD3OD)

δ 154.6, 138.5, 131.2, 129.2, 119.3, 19.5. HRMS calcd for C8H11N3O 165.0902, found

165.0903.

26 1 N-(4-Chloro)-phenyl-N’-hydroxyguanidine (13d): H NMR (500 MHz, CD3OD) δ

13 7.19 (d, 2 H, J = 8.5 Hz), 7.14 (d, 2 H, J = 8.5 Hz); C NMR (125 MHz, CD3OD) δ

153.7, 140.4, 128.4, 125.3, 119.4; HRMS calcd for C7H8ClN3O m/z 185.0356, found

185.0355.

1 N-(4-Bromo)-phenyl-N’-hydroxyguanidine (13e): H NMR (400 MHz, CD3OD) δ

13 7.28 (d, 2 H, J = 9.0 Hz), 7.14 (d, 2 H, J = 9.0 Hz); C NMR (100 MHz, CD3OD) δ

153.5, 141.0, 131.4, 119.6, 112.3; HRMS calcd for C7H8BrN3O m/z 228.9851, found

228.9854.

1 N-2-Naphthyl-N’-hydroxyguanidine (14): H NMR (400 MHz, CD3OD) δ 7.64-7.78 (m,

13 4H), 7.26-7.42 (m, 3H); C NMR (100 MHz, CD3OD) δ 153.3, 144.4, 134.6, 130.5,

128.6, 127.4, 126.8, 126.2, 123.9, 120.4, 117.0; HRMS calcd for C11H11N3O m/z

201.0902, found 201.0899.

1 N-(3-Aminomethyl-phenyl)- N’-hydroxyguanidine (15): H NMR (400 MHz, CD3OD)

13 δ 7.56-7.34 (m, 4 H), 4.21 (s, 2 H); C NMR (400 MHz, CD3OD) δ 157.8, 135.4, 135.2,

130.7, 128.0, 125.7, 124.0, 42.8; ESI-MS 181 (M+H)+.

1 N-Benzyl-N’-hydroxyguanidine (16a): H NMR (400 MHz, CD3OD) δ 7.38-7.24 (m,

13 5H), 4.45 (s, 2 H), C NMR (100 MHz, CD3OD) δ 159.2, 136.6, 128.7, 127.8, 127.1,

44.4; HRMS calcd for C8H11N3O m/z 165.0902, found 165.0908.

1 N-(4-Methoxyl)-benzyl-N’-hydroxyguanidine (16b): H NMR (400 MHz, CD3OD) δ

7.25 (d, 2 H, J = 9.0 Hz), 6.92 (d, 2 H, J = 9.0 Hz), 4.35 (s, 2H), 3.78 (s, 3H); 13C NMR

(100 MHz, CD3OD) δ 159.8, 159.2, 128.5, 128.3, 114.0, 54.3, 44.0; HRMS calcd for

C9H13O2N3 m/z 195.1008, found 195.1010.

27 1 N-(4-Methyl)-benzyl-N’-hydroxyguanidine (16c): H NMR (400 MHz, CD3OD) δ 7.19

(d, 2 H, J = 8.0 Hz), 7.11 (d, 2 H, J = 8.0 Hz), 4.13 (s, 2H), 2.29 (s, 3H); 13C NMR (100

+ MHz, CD3OD) δ 157.8, 136.6, 136.4, 128.9, 127.3, 44.7, 20.0; EIMS m/z 180.2 (M+H );

+ HRMS calcd for C9H13N3O (M -O) m/z 163.1100, found 163.1107.

1 N-(4-Chloro)-benzyl-N’-hydroxyguanidine (16d): H NMR (400 MHz, CD3OD) δ

13 7.37(d, 2 H, J = 8 Hz), 7.33 (d, 2H, J = 8Hz), 4.93(s, 2H); C NMR (100 MHz, CD3OD)

δ 154.5, 138.6, 128.8, 128.3, 118.6, 44.0; HRMS calcd for C8H10ON3Cl m/z 199.0512, found 199.0513.

1 N-(4-Nitro)-benzyl-N’-hydroxyguanidine (16e): H NMR (400 MHz, CD3OD) δ 8.20

13 (d, 2 H, J = 9.0 Hz), 7.59 (d, 2 H, J = 9.0 Hz), 4.99 (s, 2H); C NMR (100 MHz, CD3OD)

δ 158.6, 147.4, 145.8, 127.9, 123.5, 43.8; EIMS 211.0 (M+H+); HRMS calcd for

+ C8H9N3O3 (M -NH) m/z 195.0644, found 195.0643.

1 N-Phenylguanidine (19): H NMR (400 MHz, CD3OD) δ 7.45-7.52 (m, 2 H), 7.32-7.38

13 (m, 1 H), 7.25-7.32 (m, 2 H); C NMR (100 MHz, CD3OD) δ 152.1, 134.9, 129.9, 127.6,

125.6; HRMS calcd for m/z C7H9N3 135.0796, found 135.0801.

1 N-(4-Methoxyl)-phenylguanidine (19b): H NMR (500 MHz, CD3OD) δ 7.21 (d, 2 H, J

13 = 9.0 Hz), 7.01 (d, 2 H, J = 9.0 Hz), 3.83 (s, 3H); C NMR (125 MHz, CD3OD) δ 159.8,

157.6, 127.8, 127.0, 115.0, 55.1; HRMS calcd for C8H11N3O 165.0902, found 165.0904.

1 N-(4-Methyl)-phenylguanidine (19c): H NMR (500 MHz, CD3OD) δ 7.23 (d, 2 H, J =

13 8.0 Hz), 7.16 (d, 2 H, J = 8.0 Hz), 2.34 (s, 3H); C NMR (125 MHz, CD3OD) δ 156.9,

137.9, 132.0, 130.5, 125.6, 19.9; HRMS calcd for C8H11N3 m/z 149.0953, found

149.0951.

28 Nα-(Tert-butyloxycarbonyl)-L-ornithine tert-butyl ester (23): Step 1: Commercially available Nδ−(benzyloxy-carbonyl)-L-ornithine (1.78 g, 6.7 mmol), tert-butyl acetate

(100 mL) and perchloric acid (0.0073 mmol) were mixed and stirred at rt for 2 d. Water was then added to the reaction mixture followed by the addition of NaOH until the solution is basic and subsequent extraction with ethyl acetate. Rotary evaporation of the organic extracts afforded 21 as oil (1.80 g, 84%).

Step 2: The product (3.60 g, 11.2 mmol) from step 1 was dissolved in 20 mL of CH2Cl2 and cooled in ice bath. Then tert-butyl pyrocarbonate (2.80 g, 12.88 mmol) was added.

The mixture was stirred 1 h at 0 °C and 3 h at room temperature. The solvent was removed by rotary evaporation and the product isolated by silica gel chromatography using hexane/ethyl acetate (3:1). The product 22 was colorless oil (4.48 g, 95%). 1H

NMR (400 MHz, CDCl3) δ 7.31-7.34 (m, 5 H), 5.07 (s, 2 H), 4.11 (t, 1 H), 3.19 (t, 2 H),

1.9-1.5 ( m, 4 H ), 1.43 (s, 9 H), 1.42 (s, 9 H).

Step 3: 22 (4.22g, 10 mmol) in 40 mL of methanol was added 815 mg of 10% Pd/C.

The mixture was shaken under H2 (60 psi) for 5 h. Filtration and evaporation of the

1 solution afforded 3.43 g of 23 as an oil (100%). H NMR (400 MHz, CD3OD) δ 5.51 (br,

1H), 4.05 (br, 1H), 2.74 (br, 2 H), 1.5-1.8 (m, 4 H), 1.40 (s, 9 H), 1.37 (s, 9 H).

Nα-(Tert-butyloxycarbonyl)-δ-(N-isopropyl-thioureido)-L-norvaline tert-butyl ester

(24): 23 (5 mmol), thiophosgene (15 mmol) and Calcium Carbonate (515 mg, 5.15 mmol) in chloroform (40 mL) and water (5 mL) were stirred vigorously for 16 h. The suspension was filtered and the organic layer was separated. After concentration, the residue was taken up in methanol (20 mL) and cooled in ice bath. Isopropyl amine 50 mmol was added and then the mixture was stirred for 3 h at rt. Evaporation and 29 chromatography (EtOAc/hexane, 4:1) afforded a white solid (1.617 g, 83%). 1H NMR

(400 MHz, CDCl3) δ 6.51 (br, 1H), 6.09 (br, 1H), 5.21 (d, 1 H, J = 8 Hz), 4.22 (br, 1 H),

4.04-4.06 (m, 1 H), 3.44 (m, 2 H), 1.8-1.5 (m, 4 H), 1.41 (s, 9 H), 1.38, (s, 9 H), 1.16 (d,

13 6 H, J = 8 Hz); C NMR (400 MHz, CDCl3) δ 180.5, 171.8, 155.9, 82.6, 80.2, 53.5, 46.1,

43.9, 30.9, 28.7, 28.5, 28.2, 25.0, 22.8; ESI MS 390 (M+H)+.

Nα-(Tert-Butyloxycarbonyl)-Nω-isopropyl-L-arginine tert-butyl ester (25):

Triethylamine (3 mL) was added to a stirred mixture of 24 (214 mg, 0.55 mmol), HgO

(120 mg, 0.55 mmol), and hydroxylamine hydrochloride (38 mg, 0.55 mmol) under a nitrogen atmosphere. Diethyl ether (7 mL) was added to the mixture which after being vigorously stirred for over night. The mixture was subjected to column chromatography with chloroform-methanol (10:1) as eluent to give the title compound as an oil (83.6 mg,

1 39%). H NMR (500 MHz, CD3OD) δ 4.30 (br, 1 H), 3.96 (br, 1 H), 3.46 (br, 2 H), 1.80-

1.55 (m, 4 H), 1.46 (s, 9 H), 1.44, (s, 9 H), 1.17 (d, 6 H, J = 6.5 Hz); 13C NMR (500

MHz, CD3OD) δ 172.4, 156.9, 81.4, 79.3, 54.5, 43.3, 28.9, 27.6, 27.1, 25.7, 21.5.

Nω’-Hydroxy-Nω-isopropyl-L-arginine (26): 25 (83.6 mg, 0.29 mmol) was stirred in trifluoro acetic acid (10 mL) for 2 h. After evaporation in vacuo, 10 mL of water and 10 mL of ether were added. Water layer was separated and lyophilized. The title compound

1 was a white solid (50 mg, 100%). H NMR (300 MHz, CD3OD) δ 3.93 (t, 1 H), 3.77

(sept, 1 H), 3.15 (dt, 2 H), 2.0-1.5 (m, 4 H), 1.09 (d, 6 H, J = 8 Hz); 13C NMR (300 MHz,

+ D2O) δ 171.9, 52.7, 46.8, 43.2, 27.1, 24.3, 21.4, 8.4; ESI-MS 233 (M+H) ; HPLC, retention time 7.32 min, purity 96% (C-18 column, water:CH3CN (80:20) ).

30 Biochemical assay

Assay of the NOS catalyzed NO generation from N-hydroxygualidines. The initial rate of NO synthesis was determined at 37 ºC using the classical spectrophotometric oxyhemoglobin assay for NO. Briefly, 20-30 µL aliquots containing NOS, 5 µM BH4, and 2-5 mM dithiothreitol (DTT) were added to a prewarmed cuvette that contained 50 mM HEPES (pH 7.4), supplemented with 15 µM oxyhemoglobin, 100 units/mL SOD,

100 units/mL catalase, 200 µM NADPH, 4 µM FAD, 4 µM FMN, 5 µM BH4, and 0.5 mM substrate at the desired concentration, to give a final volume of 0.9 mL. In the case of nNOS and eNOS, 1 mM CaCl2 and 10 µg/mL Calmodulin were present. For iNOS, 1 mM magnesium acetate was added. The reference cuvette had the same composition except that 50 mM HEPES, 5 µM BH4, and 2-5 mM DTT were added instead of NOS- containing solutions. The NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored over time as an increase in absorbance at 401 nm and quantitated using an extinction coefficient of 38 mM-1 cm-1.

Assay of peroxidase catalyzed NO generation from N-hydroxygualidines. This enzymatic reaction was carried out in PBS buffer (pH 7.4) with 1 mM EDTA. Incubated mixtures containing 0.6 mM substrate, 0.6 mM H2O2, and 80 μg/mL horseradish peroxidase in a 500 μL final volume were shaken at 25 °C for 20 min. The concentration

- of NO2 generated in the system was determined by addition of the Griess reagent.

Absorbances were measured at 548 nm. Calibration curves were made from identical incubated mixtures without the enzyme and containing various concentration of NaNO2

- to properly determine the amounts of NO2 formed in the enzymatic reaction.

31 Electrochemical Measurement

Cyclic voltammetry was performed with a BAS-100B/W electrochemical analyzer

(Bioanalytical Systems, Inc) in 1.5 mM dry CH3CN solution under an argon atmosphere

(sweep rate, 100 mV/s). n-Bu4NPF6 (0.1 M) was employed as the supporting electrolyte.

A standard three-electrode cell consisted of a glassy carbon disk as working electrode, a platinum wire as counter electrode, and Ag/AgCl (3M NaCl) as a reference electrode.

CHAPTER 2

NEW CARBOHYDRATE AND 3-MORPHORLINOSYDNONIMINE CONJUGATES

2.1 Introduction

NO can be easily converted into a variety of reactive nitrogen species (RNS)31, such as dinitrogen trioxide (N2O3), nitrogen dioxide (NO2), and the peroxynitrite anion

(ONOO-).32 While these RNS are highly reactive, their reactivity can be utilized to achieve beneficial results. It has been reported that sufficient generation of RNS can lead to cellular dysfunction and cytotoxicity.33 By controlling the generation of a RNS to a localized area in which it is desirable to kill cells, such as in a solid tumor, the reactivity of the RNS can be used to achieve a desired result.

In order to achieve site-specific delivery of NO, spontaneous NO releasing agents can be converted into stable prodrugs by attaching them to carriers, which can be specially recognized by certain targets. Novel NO donors by taking advantage of the role of carbohydrates in cell recognition and internalization processes have been designed and synthesized in Wang’s group, since sugar derivatives can be delivered by hexose transporters on the cell membrane.34 Two types of carbohydrate-linked NO donors have been developed in our group. The first type is the sugar-S-nitrosothiol. For example,

Glyco-SNAP-1 (27) is a water soluble and stable NO donor, which showed superior

33 cytotoxicity in vitro and is commercially available for biological research.35 The second type is the sugar-NONOate. For example, glucose-NONOate (28) could release NO by the action of glycosidases.36 The previous studies also showed that carbohydrates could stabilize the NO releasing moieties. The sugar-linked approach could be a good solution for the instability that most NO donors suffer from. Here new glycosyl carbamate linked

NO donors (29) were developed. The carbohydrate moiety ensures that the NO donor compound is inactive until it encounters an appropriate activating enzyme, such as a glycosidase, and also increases the water solubility. The development of carbamate- linked sugars is more interesting, because this linkage is stable in aqueous solution and could be hydrolyzed by glycosidases37 and no other toxic groups are released from the linkage portion of the molecule.

OH OH NHAc O HO O H HO O N CH3 O N HO HO N N OH CH3 OH O S N O 27 28

O Cl- (OH)n H H N O N NO Donor 2 N N O O O N 29 30

Figure 2.1: Structures of sugar-NO conjugates and SIN-1

34 3-Morphorlinosydnonimine (SIN-1) (30) was first chosen as a model NO donor moiety.38 SIN-1 is a vasodilator and believed to release NO non-enzymatically. At physiological or alkaline pH, SIN-1 undergoes rapid hydrolysis to the ring-open form from which the NO radical is released (Scheme 2.1). The activation of SIN-1 also produces the superoxide anion, which combines with NO to produce the highly reactive peroxynitrite anion. This reactive species can damage biomolecules and induce cytotoxicity.39

O O O2 O2 OH- O N N N CH CN + N 2 N N - NH N CH2CN O N O N O SIN-1 SIN-1A

NO O H+ O N N CHCN N N SIN-1C CH2CN

Scheme 2.1: NO releasing from SIN-1

35 2.2 Synthesis of sugar-SIN1 conjugates

SIN-1 (30) as one of the starting materials was synthesized from 4- aminomorpholine by a two-step transformation following literature procedures.40 As shown in Scheme 2.2, reaction of 4-aminomorpholine with aldehyde sodium bisulfite followed by the addition of potassium cyanide provided the 1-

(cyanomethylamino)morpholine, which was directly nitrosated with sodium nitrite aqueous solution in the presence of HCl. To the resulting nitrosation compound was added an excess amount of methanolic hydrochloric acid. The SIN-1.HCl was collected as an off-white solid, which could be further purified by recrystallization from ethyl ether and methyl alcohol.

Cl- a b H2N O N NH2 O N NH CN N N O O N 31 32 30

Scheme 2.2: Synthesis of SIN-1 Reagents and conditions: (a) KCN, HOCH2SO3Na, 60 °C, 12 h, 50 %; (b) (i) HCl, NaNO2, rt, 15 min; (ii) HCl, MeOH, rt, 2 h, 69%.

Glucose and galactose derivatives (34a and 34b) with unprotected anomeric hydroxyl groups were obtained by treating peracetylated glycoses with benzylamine in

THF at an ambient temperature (Scheme 2.3).41 After purification by silica gel column chromatography, the glycoses were coupled with one equivalent of 4-nitrophenyl

36 chloroformate in the presence of three equivalents of triethylamine in methylene chloride at 0 oC, thereby producing 4-nitrophenyl (peracetyl-D-glycosyl) carbonate (35a and 35b) as α/β mixtures (molar ratio 1:3). The α/β mixtures could be separated in the following step. The coupling reaction of SIN-1 with protected glycosyl carbonate was carried out at room temperature in anhydrous pyridine.42 The crude product was purified by silica gel chromatography and the anomeric mixtures of the products were separated. For this step, since SIN-1 itself was unstable in the solution, the best yield obtained was 40%.

Then the acetyl protecting groups were removed in anhydrous methanol with sodium methoxide as a catalyst. This deprotection step required several hours at room temperature before the reaction mixture was neutralized with a strong acidic resin

(Amberlyst® 15 ion-exchange resin). Complete assignment of the chemical shifts of 37a and 37b was based on the 2D COSY and HMQC NMR spectra. The β configuration at the anomeric position was also confirmed by the coupling constant (7.5 Hz and 8.0 Hz) between H1 and H2. The deprotection of the α anomers of 36a and 36b failed. NMR showed that the heterocyclic ring decomposed. It seems that α conjugates were unstable after removing of acetyl protecting groups.

37 R1 OAc R1 OAc R1 OAc O R O O R2 b 2 R a O O NO2 2 OAc AcO OH AcO AcO OAc OAc OAc O 35a: R =H, R =OAc, 90% 33a: R1=H, R2=OAc 34a: R1=H, R2=OAc, 92% 1 2 35b: R =OAc, R =H, 88% 33b: R1=OAc, R2=H 34b: R1=OAc, R2=H, 90% 1 2

R1 OR R O c 2 RO O N N N O OR O O N

36a: R1=H, R2=OAc, R=Ac, 35% 36b: R =OAc, R =H, R=Ac, 40% 1 2 d 37a: R1=H, R2=OH, R=H, 100% 37b: R1=OH, R2=H, R=H, 100%

Scheme 2.3: Synthesis of glucose, galactose and SIN-1 conjugates Reagents and conditions: (a) BnNH2, THF, rt, 30 h; (b) p-NO2C6H4OCOCl, NEt3, CH2Cl2, rt, 4.5 h; (c) 30, pyridine, rt, 12 h; (d) NaOMe, MeOH, rt, 3 h.

Applying our strategy to other heterocyclic NO donors, more carbohydrate and

NO donor conjugates could be developed. Mesoionic oxatriazole-5-imines (GEAs) are structurally isosteric to sydnonimines. They are potent anti-platelet, fibrinolytic, thrombolytic, and bronchiectatic agents.43 A member of this type of NO donors, GEA

3162 (40), was synthesized according to a reported procedure.44 The reaction of 3,4- dichloroohenylhydrazine with potassium thiocyanate gave the corresponding thiosemicarbazide, which was nitrosated in acidic methanol with isobutylnitrite to give the GEA 3162. Coupling of the GEA 3162 with 4-nitrophenyl (2,3,4,6-tetra-O-acetyl-

α/β-D-galactopyranosyl) carbonate (35b) following a previously established method gave the desired Gal-GEA3162 conjugates (41) in fair yields (Scheme 2.4). However, deprotection of acetyl groups caused the decomposition of the oxatriazole ring. 38

Cl Cl Cl S a b Cl N N Cl NHNH2 Cl NHNH NH2 N NH HCl O 38 39 40

AcO OAc O Cl c AcO O N N N Cl OAc O O N

Scheme 2.4: Synthesis of galactose and GEA 3126 conjugate Reagents and conditions: (a) KSCN, EtOH, reflux, 7 h, 62%; (b) isobutyl nitrite, HCl, 0 °C, 20 min, 94%; (c) 35b, py, rt, 12 h 31%.

Galactose-NO conjugates with an aromatic linker were also designed to insure that they will be a good substrate for β-galactosidase. This self-immolative spacer has been used in prodrug design and the pharmacokinetics has been reported.45 Thus 2- amino-4-nitrophenol was methylated using methyl iodide, and the monomethyl derivative was isolated by chromatography. The amine function of 43 was protected as a BOC derivative 44 and the protected α-bromogalactose was introduced by phase-transfer- catalyzed glycosalation.46 The BOC protecting group was then removed to give 46, which was further converted into 47 upon the treatment of chloromethyl chloroformate.

However, the substitution of chloride by SIN-1 did not happen by a variety of conditions.

TLC showed that both compound 47 and SIN-1 could decompose in the reactions.

39 CH Boc H N 3 AcO OAc H C Boc 2 a HN b H C N c 3 N 3 O HO NO 2 HO NO HO NO AcO O 2 2 OAc 42 43 NO 44 45 2 AcO O d OAc MeHN e AcO OAc O MeN O Cl AcO O O OAc AcO O OAc NO2 NO 46 47 2

Scheme 2.5: Synthesis of the phenol linkage on galactose Reagents and conditions: (a) MeI, Et3N, 40 °C, 3 h, 55%; (b) (BOC)2O, K2CO3, THF, rt, 12 h, 64%; (c) BTEAC, CHCl3, H2O, NaOH, 60 °C, 3 h, 57%; (d) TFA, rr, 2 h, 97%; (e) chloromethyl chloroformate, DMAN, CHCl3, rt, 29 h, 98%.

2.3 Enzymatic decomposition of sugar-SIN1 conjugates

In order to test the molecular design, the decomposition of 37b by β- galactosidase was detected by the decay of the absorbance at 307 nm (Fig. 2.2), which is the characteristic of the NO donating heterocycle. The enzymatic hydrolysis was measured on a UV-VIS spectrophotometer fitted with an electrically thermostated cell block. The half-life of 37b with β-galactosidase was estimated to be 0.8 hour, while 37b was fairly stable in the buffer without the enzyme. It was noticed that 37b decomposed very slowly in the UV spectrophotometer. The reason may be that UV causes the decomposition of the SIN-1. To study the details of enzymatic kinetics, both 37a and

37b were incubated with β-glucosidase and β-galactosidase respectively, and then the solutions were analyzed by LC-MS (Shimadzu, 2010A). The kinetic data were obtained by the Lineweaver-Burk plots for 37a (Fig. 2.3) and 37b (Fig. 2.4). Km, Vmax and kcat are listed in Table 2.1. The Km for both substrates is within the ranges of known substrates 40 (from about 0.05 to 85 mM). The turnover numbers (kcat) are relatively large in the range of known substrates. It indicates that both 37a and 37b are good substrates of the correspondent glycosidases when enzymes are fully saturated with substrates. At low substrate concentrations, the hydrolysis is still efficient, since kcat/KM is also large. From the kinetic data, we can see that glycosidases showed the good catalytic efficiency for this type of carbohydrate conjugates with carbamate linkages.

0.8

0.7

0.6

0.5

0.4 Absorbance (AU) Absorbance 0.3

0.2 01234 Time (hour)

Figure 2.2: The UV absorbance changing of 37b ( In the presence (●) and in the absence (○) of β-galactosidase (0.05 mg/mL), 37b (0.04 mM) were incubated in 150 mM phosphate buffer, pH 7.4, 37 °C. UV was measured at 307 nm.)

41 14 12 10 8 (s/mM)

-1 6 4

Rate 2 0 01234 -1 -1 [S] (mM )

Figure 2.3: Lineweaver-Burk plot of 37a as the substrate of β-glucosidase (β-Glucosidase (0.5 mg/mL) and substrate ([S]) 37a (0.1, 0.2, 0.4, 0.8, 1.25 mg/mL) were incubated in 150 mM Tris-CF3CO2H buffer for 10 seconds at pH 7.30.)

15 s/mM -1 10

Rate 5

0 01234 -1 -1 [S] (mM )

Figure 2.4: Lineweaver-Burk plot of 37b as the substrate of β-galactosidase (β- Galactosidase (0.5 mg/mL) and substrate ([S]) 37b (0.1, 0.2, 0.4, 0.8, 1.25 mg/mL) were incubated in 150 mM Tris-CF3CO2H buffer for 10 seconds at pH 7.30)

KM Vmax kcat kcat/KM

-1 -1 -1 Substrate (mM) (mM/s) (s ) (mM s )

37a 0.311 0.181 2.75×105 8.87×105

37b 1.47 0.327 6.54×105 4.45×105

Table 2.1: The kinetic parameters of 37a/b as substrates of glycosidases

The NO releasing ability of 37a and 37b in the presence and absence of glycosidases was evaluated by measuring the formation of nitrite ion, since nitrite ion is

- generated from the oxidation of NO radical by oxygen. The nitrite (NO2 ) anion in solution was quantified by the Griess method. The results are summarized in Table 2.2.

Both 37a and 37b were relatively stable under physiological conditions without enzymes.

The trace amount of nitrite formed might come from the photo-decomposition of the heterocycle.47 In contrast, SIN-1 decomposed quite easily in the aqueous solution.

- Compound 37a and 37b generated comparable amounts of NO2 compared to SIN-1 only in the presence of glycosidases.

- Substrate (23 μM) [NO2 ]/μM

SIN-1 3.1

37a negligible

37a + β-glucosidase 2.4

37b 0.3

37b+ β-galactosidase 2.8

Table 2.2: Nitrite concentration in the incubation of 37a/b with or without the glycosidases (The amount of nitrite released was measured using the Griess method. Glycosidases (0.05 mg/mL) and 37a or 37b (23 μM) were incubated for 17 hours in 150 mM phosphate buffer, pH 7.4, 37 °C. UV absorbance was measured at 548 nm.)

The hydrolysis of the glycosides was also studied by monitoring of the NO release with an Electrochemical ISO-NO marker II isolated Nitric Oxide Meter manufactured by World Precision Instruments, Inc. NO was not detected, when 37a or

37b was incubated in aqueous solution at neutral pH, even in the presence of

- glycosidases. This is presumed to be, because NO/O2 reaction is so rapidly that NO is consumed. Yet, when 0.3 mg/ml superoxide dismutase (SOD) was present, NO formation was observed (Figure 2.5). This property is the same as SIN-1. A possible decomposition pathway of 37b is proposed in Scheme 2.6. β-Galactosidase first hydrolyzes the glycosidic linkage of 37b to generate SIN-1. At the physiological condition, SIN-1 undergoes rapid hydrolysis to form the ring opened product, SIN-1C. 44 NO radical is released in this process.48 Stoichiometric amounts of superoxide anions

• - • - ( O2 ) are formed as a result of oxygen reduction. Since NO is known to react with O2 at an almost diffusion-controlled rate,49 peroxynitrite (OONO-) production is inevitable.

Since the most NO released from both glycosides were converted to peroxynitrite, which

- could isomerize to nitrate (NO3 ), only small amount of nitrite was detected by Griess assay. Peroxynitrite is a well-known active species which can cause damage of biomolecules.

50 45 40 M) μ 35 30 25 20 15

NO Concentration ( 10 5 0 0 5 10 15 Time (minutes)

Figure 2.5: Measurement of NO profiles of 37a, 37b and SIN-1 (100 μM SIN-1 (♦) and SOD (0.3 mg/mL) were incubated; 100 μM 37a (■) and SOD (0.3 mg/mL) were incubated with β-glucosidase (0.005 mg/mL); 100 μM 37b (○) and SOD (0.3 mg/mL) with β-galacosidase (0.005 mg/mL) were incubated, in 150 mM phosphate buffer, pH 7.4, 37 oC.)

45 OH-, O H+ O 2 O HO OH N N O β-galactosidase N N HO O N N NH CHCN N N O OH O O O N NO SIN-1 O2 SIN-1C 37b

CO2 ONOO-

HO OH O HO OH OH Galactose

Scheme 2.6: Proposed NO releasing from 37b

2.4 Conclusion

In summary, a new class of glycosidase dependent NO donors, glycosyl carbamate of nitric oxide donors, were prepared. SIN-1 as an NO releasing moiety was coupled with glucose and galactose via well-defined chemical steps. The carbohydrate-

SIN-1 conjugates are much more stable than SIN-1 itself. It was also found that glycosyl carbamate of NO donors could be hydrolyzed by glycosidases and form NO. Such prodrugs may find applications in biological research and Antibody-Directed Prodrug

Therapy (ADEPT).

2.5 Experimental

1-(Cyanomethylamino) morpholine (32). 4-Aminomorpholine (2.04 g, 20.0 mmol) and aldehyde-sodium bisulfite (2.68 g, 20.0 mmol) was dissolved in water (15 mL) and stirred at room temperature for 3 h. To this reaction mixture was added KCN (1.3 g, 20

46 mmol) and stirring was kept overnight at 60 oC, then extracted with EtOAc (100 mL × 2) and washed with brine. The extract was dried over anhydrous Na2SO4 and then concentrated to give 1-(cyanomethylamino) morpholine (1.4 g, 49.5%) as a yellow oil. 1H

NMR (500 MHz, CDCl3) δ 3.68 (t, 4H, J = 5.0 Hz, H-6 & H-2), 3.65 (s, 2H, CH2 of

13 cyanomethylamino), 2.72 (s, 4H, H-5 & H-3); C NMR (CDCl3, 125 MHz) δ 118.23

(CN), 66.67 (C-2 & C-6), 56.38 (C-5 & C-3), 37.48 (CH2 of cyanomethylamino); ESI

+ + MS m/z 142 (M+H) ; HRMS calc. for C6H11N3O (M) m/z 141.0902 found, 141.0902.

3-Morpholinosydnonimine hydrochloride (30). To a solution of 1-

(cyanomethylamino) morpholine (1.4 g, 9.9 mmol) in water (20 mL) and conc. HCl (1.0 mL) was added dropwise a solution of NaNO2 (821 mg, 11.9 mmol) in H2O (5 mL). The reaction mixture was stirred for 15 min and then extracted with EtOAc (100 mL × 2).

The extract was dried over anhydrous Na2SO4 then concentrated to give yellow oil, to which was added an excess amount of methanolic hydrochloric acid. The solution was stirred for 2 h and concentrated to afford an off-white solid, which was collected by filtration and washed with ether. 3-Morpholinosydnonimine (1.40 g, 68.7%) was obtained as a white crystal after recrystalization from ethanol. 1H NMR (500 MHz,

DMSO-d6) δ 9.64 (s, 2H, NH2), 8.28 (s, 1H, H-4), 3.85 (t, 4H, H-6’ & H-2’, J = 5.0 Hz),

13 3.59 (t, 4 H, H-5’& H-3’, J = 5.0 Hz); C NMR (125 MHz, DMSO-d6) δ 168.54 (C-5),

96.48 (C-4), 64.65 (C-2’ & C-6’), 53.40 (C-5’& C-3’); ESI MS m/z 171 (M-HCl+H)+,

341 (2(M-HCl)+H)+.

2, 3, 4, 6-Tetra-O-acetyl-D-glucopyranose (34a). A solution of pentaacetate-D-glucose

(3.9 g, 10.0 mmol) and BnNH2 (1.2 mL, 11.0 mmol) in THF (45 mL) was stirred at ambient temperature for 30 hours. The solvent was removed and the residue was 47 dissolved in CH2Cl2 (100 mL), then washed with 1 N HCl (100 mL × 2), water (100 mL) successively. The organic layer was concentrated and chromatographed on silica gel with EtOAc/hexane (2:1-1:1), giving 34a (3.2 g, 92%) as a 3:1 (α/β) mixture of anomers

1 as judged by NMR. H NMR (400 MHz, CDCl3) δ 4.41 (d, 1H, J = 5.6 Hz, H-1 of β-

13 anomer), 3.89 (d, 1H, J = 3.2 Hz, H-1 of α-anomer); C NMR (100 MHz, CDCl3) δ 96.0,

90.3 (C-1).

4-Nitrophenyl (2, 3, 4, 6-tetra-O-acetyl-D-glucopyranosyl) carbonate (35a). To a

o cooled solution (0 C ) of 6a (2.48 g, 7.13 mmol) in anhydrous CH2Cl2 (50 mL) were successively added 4-nitrophenyl chloroformate (1.72 g, 8.53 mmol) and triethylamine

(2.96 mL, 21.3 mmol). After stirring for 4.5 h at rt, the crude mixture was diluted with another portion of CH2Cl2 (50 mL) and then washed with brine and water. The CH2Cl2 layer was dried over anhydrous Na2SO4 then concentrated. After purification by silica gel chromatography using EtOAc/hexane (1:2) as an eluent, 35a (3.28 g, 90%) was obtained as a 1:3 (α/β) mixture of anomers as judged by 1H-NMR. 1H NMR (500 MHz,

CDCl3) δ 6.28 (d, 1H, J = 3.0 Hz, H-1 of α-anomer), 5.66 (d, 1H, J = 8.0 Hz, H-1 of β-

13 + anomer); C NMR (125 MHz, CDCl3) δ 95.8, 94.1 (C-1); ESI MS m/z 536 (M+Na) ,

1049 (2M+Na)+.

N-(2, 3, 4, 6-Tetra-O-acetyl-β-D-glucopyranosyl)-carbonyl-3-morpholinosydnonimine (36a). To a solution of 35a (1.53 g, 2.98 mmol) in anhydrous pyridine (20 mL) was added 30 (678 mg, 3.28 mmol). The reaction mixture was stirred overnight and then the solvent was removed in vacuo to give sticky oil. The residue was purified by silica gel chromatography using EtOAc/CH2Cl2 (1:1) as an eluent. 36a (568 mg, yield

35%) and the α-anomer (164 mg, 10%) were obtained as foams. 36a: 1H NMR (500 48 MHz, CDCl3) δ 7.70 (s, 1 H, H-4’), 5.72 (d, 1H, J = 8.5 Hz, H-1), 5.26 (t, 1 H, J = 9.0 Hz,

H-3), 5.20~5.10 (m, 2 H, H-4 & H-2), 4.25 (dd, 1 H, J = 12.5, 4.0 Hz, H-6), 4.12 (dd, 1

H, J = 12.0, 2.0 Hz, H-6), 3.97 (t, 4 H, J = 5.0 Hz, H-2’’ & H-6’’), 3.85 (dd, 1 H, J = 4.0,

2.0 Hz, H-5), 3.54 (t, 4 H, J = 5.0 Hz, H-3’’& H-5’’), 2.06~1.98 (m, 12 H, COCH3 × 4);

13 C NMR (125 MHz, CDCl3) δ 174.3 (C-carbamate), 170.7 (C=O of Ac), 170.2 (C=O of

Ac), 169.4 (C=O of Ac × 2), 158.3 (C-5’), 99.8 (C-4’), 93.3 (C-1), 73.1 (C-3), 72.2 (C-5),

70.4 (C-2), 67.8 (C-4), 65.4 (C-2’’ & C-6’’), 61.5 (C-6), 54.5 (C-3’’& C-5’’), 20.7 (CH3

+ + of Ac × 2 ), 20.6 (CH3 of Ac × 2 ); ESI MS m/z 545 (M+H) , 567 (M+Na) , 1089

+ + + (2M+H) , 1111 (2M+Na) ; HRMS calcd for C21H28N4O13Na (M+Na) m/z 567.1545,

1 found m/z 567.1564. α-anomer: H NMR (500 MHz, CDCl3) δ 7.77 (s, 1 H, H-4’), 6.31

(d, 1 H, J = 3.5 Hz, H-1), 5.63 (t, 1 H, J = 10.0 Hz, H-3), 5.15 (t, 1 H, J = 10.0 Hz, H-4),

5.06 (dd, 1 H, J = 10.0, 4.0 Hz, H-2), 4.28~4.26 (m, 2 H, H-5 & H-6), 4.10~4.00 (m, 1 H,

H-6), 3.97 (t, 4 H, J = 5.0 Hz, H-2’’ & H-6’’), 3.54 (t, 4 H, J = 5.0 Hz, H-3’’& H-5’’),

13 2.10~1.97 (m, 12 H, COCH3 × 4); C NMR (125 MHz, CDCl3) δ 174.7 (C-carbamate),

170.7 (C=O of Ac), 170.0 (C=O of Ac), 169.8 (C=O of Ac), 169.6 (C=O of Ac), 159.2

(C-5’), 99.7 (C-4’), 90.1 (C-1), 70.1 (C-3), 69.6 (C-2), 69.0 (C-5), 68.0 (C-4), 65.4 (C-2’’

& C-6’’), 61.5 (C-6), 54.6 (C-3’’& C-5’’), 20.7 (CH3 of Ac), 20.6 (CH3 of Ac × 2 ), 20.6

+ + + + (CH3 of Ac);. ESI MS m/z 545 (M+H) , 567 (M+Na) , 1089 (2M+H) , 1111 (2M+Na) .

N-(β-D-Glucopyranosyl)-carbonyl-3-morpholinosydnonimine (37a). To a solution of

36a (15 mg, 27.5 mmol) in anhydrous methanol (5 mL) was added NaOCH3 to adjust the solution pH value to 8-9. The reaction mixture was stirred at room temperature for several hours and then strong acid ion-exchange resin was added to neutralize the reaction mixture. After removing the solvent in vacuo, the residue was purified by silica 49 gel chromatography using CH2Cl2 /CH3OH (4:1) as an eluent. 37a (10 mg) was obtained

1 as colorless syrup in quantitative yield. UV 307 nm; H NMR (500 MHz, CD3OD) δ

8.14 (s, 1 H, H-4’), 5.39 (d, 1 H, J = 7.5 Hz, H-1), 3.95 (t, 4 H, J = 5.0 Hz, H-6’’ & H-

2’’), 3.83 (dd, 1 H, J = 11.5, 2.0 Hz, H-6), 3.66 (dd, 1 H, J = 12.0, 5.0 Hz, H-6), 3.62 (t, 4

H, H-3’’& H-5’’), 3.40 (t, 1 H, J = 8.5 Hz, H-3), 3.38-3.32 (m, 2 H, H-5 & H-4),

13 3.32~3.29 (m, 1 H, H-2); C NMR (125 MHz, CD3OD) δ 169.0 (carbamate), 101.4 (C-

4’), 97.4 (C-1), 78.6, 78.0, 74.0, 71.2 (C-2,3,4,5), 65.6 (C-2’’ & C-6’’), 62.5 (C-6), 55.5

(C-3’’& C-5’’); ESI MS m/z 377 (M+H)+, 399 (M+Na)+, 753 (2M+H)+, 775 (2M+Na)+;

+ HRMS calcd for C13H20N4O9Na (M+Na) m/z 399.1122, found 399.1120.

2, 3, 4, 6-Tetra-O-acetyl-D-galactopyranose (34b). Prepared from 33b. Obtained as

1 foams (92%). H NMR (400 MHz, CDCl3) δ 5.50-5.30 (m, 3 H), 5.20-5.00 (m, 2 H),

4.69 (d, 0.25 H, J = 6.6 Hz), 4.43 (m, 1 H), 4.10-4.00 (m, 3 H), 4.00-3.90 (m, 0.25 H),

3.00-2.70 (m, 1 H), 2.13, 2.11, 2.06, 2.02, 2.01, 1.96 (s, 15 H); 13C NMR (100 MHz,

CDCl3) δ 95.8, 90.4 (C-1).

4-Nitrophenyl (2, 3, 4, 6-tetra-O-acetyl-D-galactopyranosyl) carbonate (35b).

1 Prepared from 34b. Obtained as foams (88%). H NMR (500 MHz, CDCl3) δ 6.33 (d, 1

H, J = 3.0 Hz, H-1 of α-anomer), 5.63 (d, 1 H, J = 8.0 Hz, H-1 of β-anomer); 13C NMR

(125 MHz, CDCl3) δ 96.3, 94.8 (C-1).

N-(2, 3, 4, 6-Tetra-O-acetyl-β-D-galactopyranosyl)-carbonyl-3-morpholinosydnonimine (36b). Prepared from 35b. Obtained as foams (40%). 36b: 1H NMR (500

MHz, CDCl3) δ 7.73 (s, 1 H, H-4’), 5.69 (d, 1 H, J = 8.5 Hz, H-1), 5.41 (d, 1 H, J = 3.5

Hz, H-4), 5.36 (dd, 1 H, J = 10.0, 8.5 Hz, H-2), 5.09 (dd, 1 H, J = 10.0, 3.5 Hz, H-3),

4.14~4.09 (m, 2 H, H-6 & H-6), 4.09~4.04 (m, 1 H, H-5), 3.97 (t, 4 H, J = 5.0 Hz, H-2’’ 50 13 & H-6’’), 3.55 (m, 4 H, H-3’’& H-5’’), 2.15~1.96 (m, 12 H, COCH3 × 4); C NMR (125

MHz, CDCl3) δ 174.2 (C-carbamate), 170.3 (C=O of Ac), 170.2 (C=O of Ac), 170.1

(C=O of Ac), 169.4 (C=O of Ac), 158.2 (C-5’), 100.0 (C-4’), 93.8 (C-1), 71.1 (C-3 & C-

5), 67.9 (C-2), 66.9 (C-4), 65.9 (C-2’’ & C-6’’), 60.9 (C-6), 54.5 (C-5’’& C-4’’), 20.8

+ (CH3 of Ac), 20.6 (CH3 of Ac × 2 ), 20.5 (CH3 of Ac); ESI MS m/z 545 (M+H) , 567

+ + + + (M+Na) , 1089 (2M+H) , 1111 (2M+Na) ; HRMS calcd for C21H28N4O13Na (M+Na)

1 m/z 567.1545, found 567.1518. α-anomer (12%): H NMR (500 MHz, CDCl3) δ 7.76 (s,

1 H, H-4’), 6.35 (d, 1 H, J = 3.5 Hz, H-1), 5.52~5.50 (m, 2 H, H-3 & H-4), 5.29 (dd, 1 H,

J = 11.0, 3.5 Hz, H-2), 4.44 (t, 1 H, J = 7.0 Hz, H-5), 4.15~4.00 (m, 2 H, H-6 & H-6),

4.10~4.00 (m, 1 H, H-6), 3.97 (t, 4 H, J = 5.0 Hz, H-2’’ & H-6’’), 3.53 (t, 4 H, J = 5.0 Hz,

13 H-3’’& H-5’’), 2.14~1.94 (m, 12 H, Ac × 4); C NMR (125 MHz, CDCl3) δ 174.7 (C- carbamate), 170.3 (C=O of Ac), 170.3 (C=O of Ac), 170.1 (C=O of Ac), 169.7 (C=O of

Ac), 159.4 (C-5’), 99.6 (C-4’), 90.7 (C-1), 68.1 (C-5), 67.7 (C-3), 67.5 (C-4), 66.9 (C-2),

65.3 (C-2’’ & C-6’’), 61.2 (C-6), 54.6 (C-3’’& C-5’’), 20.7 (CH3 of Ac), 20.6 (CH3 of

+ + + Ac), 20.6 (CH3 of Ac × 2); ESI MS m/z 545 (M+H) , 567 (M+Na) , 1089 (2M+H) , 1111

(2M+Na)+.

N-(β-D-Galactopyranosyl)-carbonyl-3-morpholinosydnonimine (37b). Prepared from

1 36b. Obtained as syrup (100%). UV 307 nm; H NMR (500 MHz, CD3OD) δ 8.16 (s, 1

H, H-4’), 5.35 (d, 1 H, J = 8.0 Hz, H-1), 3.96 (t, 4 H, J = 5.0 Hz, H-6’’ & H-2’’), 3.88 (d,

1 H, J = 3.0 Hz, H-4), 3.74~3.66 (m, 3 H, H-6, H-6 & H-2), 3.65~3.60 (m, 5 H, H-5, H-

13 3’’& H-5’’), 3.56-3.52 (m, 1 H, H-3); C-NMR (125 MHz, CD3OD) δ 175.3 (C- carbamate), 160.6 (C-5’), 101.5 (C-4’), 98.1 (C-1), 77.4 (C-5), 74.9 (C-3), 71.3 (C-2),

71.1 (C-4), 66.6 (C-2’’ & C-6’’), 62.3 (C-6), 55.4 (C-3’’& C-5’’); ESI MS m/z 377 51 + + + + (M+H) , 399 (M+Na) , 775 (2M+Na) ; HRMS calcd for C13H20N4O9Na (M+Na ) m/z

399.1122, found 399.1129.

3,4-Dichlorophenylthiocarbazide (39). A mixture of 3,4-dichlorophenylhydrazine hydrochloride (2.14 g, 10.0 mmol) and KSCN (1.17 g, 12.0 mmol) was dissolved in 30 mL of ethanol. The mixture was gently refluxed for 7 h. After cooled to room temperature, the solvent was evaporated, and the residue was recrystallized from a mixture of methanol (30 mL) and water (30 mL). About 1.47 g (62%) of crystalline

1 product was isolated after filtration. H NMR (DMSO-d6, 500 MHz): δ 9.37 (s, 1 H),

8.30 (s, 1 H), 7.89 (s, 1 H), 7.60 (s, 1 H), 7.39 (d, 2 H, J = 9.0 Hz), 6.73 (d, 1 H, J = 4.0

13 Hz), 6.60 (dd, 1 H, J = 9.0, 4.0 Hz); C NMR (DMSO-d6, 125 MHz): δ 182.47 (C=O),

148.32, 131.18, 130.70, 120.07, 113.39, 112.84.

3-(3,4-Dichlorophenyl)-1,2,3,4-oxatriazole-5-imine hydrochloride (40). 3,4-

Dichlorophenylthiocarbazide (1.4 g, 5.9 mmol) was dissolved in 20 mL of methanol and

0.5 mL of 37% hydrochloric acid while being stirred at room temperature. The mixture was cooled to 0 °C by an ice bath, and then 2.1 mL (17.7 mmol) of isobutyl nitrite was added via a syringe. The mixture was stirred for another 20 minutes, then the yellow precipitate was filtered off and the solvent was evaporated to give brown solid, which was further washed with ethyl ether to give 1.48 g (94%) of the product. 1H NMR

(DMSO-d6, 500 MHz): δ 11.34 (br, 1 H), 8.52 (d, 1 H, J = 2.5 Hz), 8.20 (dd, 1 H, J = 8.5,

13 2.5 Hz), 8. 11 (dd, 1 H, J = 8.5 Hz); C NMR (DMSO-d6, 125 MHz): δ 171.69, 138.58,

133.53, 132.93, 131.79, 124.07, 122.61.

N-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-carbonyl-3-(3,4-dichlorophenyl)-

1,2,3,4-oxatriazole-5-imine (41). Prepared from 40 and 35b. Obtained as a red powder 52 1 (31%). 41: H NMR (500 MHz, CDCl3) δ 8.35 (d, 1 H, J = 2.5 Hz), 8.10 (dd, 1 H, J =

8.5, 2.5 Hz), 7.80 (d, 1 H, J = 8.5 Hz), 5.73 (d, 1 H, J = 8.5 Hz, H-1), 5.41-5.38 (m, 2 H,

H-4 & H-2), 5.09 (dd, 1 H, J = 10.0, 3.5 Hz, H-3), 4.14~4.06 (m, 2 H, H-6, H-5), 2.13,

13 2.01, 1.99, 1.97 (4 OAc); C NMR (125 MHz, CDCl3) δ 209.8, 170.6, 170.4, 170.3,

169.5 (C=O of Ac), 157.0, 140.2, 135.6, 132.4, 123.7, 120.8, 94.3 (C-1), 71.7 (C-3), 71.4

(C-5), 68.0 (C-2), 67.1 (C-4), 61.2 (C-6), 21.0, 20.9, 20.8 (CH3 of Ac); ESI MS m/z 627

+ + + (M+Na) , 1231 (2M+Na) ; HRMS calcd for C22H22Cl2N4O12Na (M+Na) m/z 627.0503,

1 found 627.0488. α anomer (26%): H NMR (500 MHz, CDCl3) δ 8.35 (d, 1 H, J = 2.5

Hz), 8.10 (dd, 1 H, J = 9.0, 2.5 Hz), 7.80 (d, 1 H, J = 9.0 Hz), 6.41 (d, 1 H, J = 3.5 Hz, H-

1), 5.52-5.48 (m, 2 H, H-3 & H-4), 5.37 (dd, 1 H, J = 11.0, 3.5 Hz, H-2), 4.47 (t, 1 H, J =

6.5 Hz, H-5), 4.11 (m, 2 H, H-6), 2.16, 2.04, 2.01, 1.99 (4Ac); 13C NMR (125 MHz,

CDCl3) δ 209.6, 170.4, 170.2, 170.1, 170.0 (C=O of Ac), 156.7, 140.1, 135.4, 132.3,

132.1, 123.4, 120.5, 91.4 (C-1), 68.6 (C-5), 67.6 (C-3), 67.6 (C-4), 66.7 (C-2), 61.3 (C-6),

+ + 20.7, 20.7, 20.6 (CH3 of Ac); ESI MS m/z 627 (M+Na) , 1231 (2M+Na) .

(2-Hydroxy-5-nitro-phenyl)-methyl-carbamic acid tert-butyl ester (44). To a solution of 2-amino-4-nitrophenol (7.7 g, 50 mmol) and methyl iodide (5 mL, 80 mmol) in methanol (50 mL) and triethylamine (10 mL, 72 mmol) was added in an ice-bath. The bath was removed and the mixture was heated at 40 °C. After1 hour, methyl iodide (5 mL) and triethylamine (10 mL) were added again and the mixture was stirred for 2 h at

40°c. The reaction mixture was evaporated to dryness and extracted with ethyl acetate.

The organic layer was washed with 2 M sodium acetate (20 mL) and dried over Na2SO4.

The crude material was chromatographed on silica gel and eluted with CHCl3/CH3OH

53 (95:5) to give 2-(N-methylamino)-4-nitrophenol (43) (4.6 g, 55%) as a red solid (mp 148

°C).

2-(N-methylamino)-4-nitrophenol (2.09 g, 12.5 mmol), di-tert-butyl dicarbonate (7 g, 32 mol), and K2CO3 (2 g) were stirred in water (25 mL) and THF (25 mL) for overnight at room temperature. The mixture was neutralized with an aqueous 1 M HCl solution, and extracted with ethyl acetate, dried (Na2SO4) and concentrated. The residue was chromatographed on silica gel and eluted with CH2Cl2/CH3OH (39:1) to give 44 as the

1 yellow solid (2.15 g, 64%). H NMR (acetone-d6, 500 MHz) δ:8.06-8.10 (m, 2 H), 7.13

(d, 1 H, J = 9 Hz), 3.17 (s, 3 H), 3.04 (br, 1 H), 1.35 (s, 9 H).

2-[[(1,1-Dimethylethoxy)carbonyl]methylamino]-4-nitrophenyl 2,3,4-tri-O-acetyl-β-

D-galactopyranoside (45). A solution of the phenol 44 (2.44 g, 9 mmol) and 2,3,4,6- tetra-O-acetyl-α-D-galactopyranosyl bromide (3.7 g, 9 mmol) in chloroform (50 mL) was stirred at reflux with a solution of benzyltriethylammonium chloride (2.3 g, 10 mmol) and 1.25 M NaOH in 25 mL of water. After 3 h, the mixture was cooled and diluted with water (50 mL). The two phases were then separated and organic layer washed with 1.25

M NaOH (2×25 mL). The organic layer was dried with sodium sulfate, filtered and concentrated in vacuo. The crude product was purified with column chromatography on silica gel and eluted with hexane/ ethyl acetate (3:1) to afford 2.98 g of 45 (57%). 1H

NMR (CDCl3, 400 MHz) δ: 7.98 (m, 2 H), 7.15 (d, 1 H, J = 8.8 Hz), 5.39 (m, 2 H), 5.16

(d, 1 H, J = 7.6 Hz), 5.06 (dd, 1 H, J = 10.4, 4 Hz), 4.10 (m, 3 H), 2.99 (s, 3 H), 1.93,

13 1.92, 1.89, 1.88 (4s, 12 H), 1.24 (s, 9 H); C NMR (CDCl3, 100 MHz) δ: 171.1, 170.3,

170.2, 169.0, 157.2, 142.8, 134.1, 126.0, 125.1, 123.6, 115.8, 98.6, 80.8, 71.6, 70.7, 68.3,

54 67.0, 61.5, 60.4, 28.2, 21.0, 20.7, 20.6, 14.3; ESI MS, 599 (M+H)+, 621 (M+Na)+; HRMS

+ calc. for C26H34N2O14Na (M+Na) m/z 621.1902, found, 621.1904.

2-(Methylamino)-4-nitrophenyl 2,3,4-tri-acetyl-β-D-galactopyranoside (46).

Compound 45 (2.6 g, 4.5 mmol) was dissolved in 25 ml of trifluoroacetic acid. The solution was stirred for 2 hr at room temperature. After concentration, the mixture was poured into a excess of a saturated aqueous solution of NaHCO3, extracted with CH2Cl2, dried (Na2SO4) and concentrated again. The product was a sticky liquid (2.17 g, 97%).

1 H NMR (CDCl3, 400 MHz) δ: 7.52 (dd, 1H, J = 9.2, 2.4 Hz), 7.37 (d, 1H, J = 2.4 Hz),

6.90 (d, 1H, J = 9.2 Hz), 5.49 (m, 2H), 5.15 (dd, 1H, J = 10.8, 3.2 Hz), 5.06 (dd, 1H, J =

13 8.4 Hz), 4.20 (m, 3H), 2.90 (s, 3H), 2.19, 2.08, 2.07, 2.03 (4s, 12H); C NMR (CDCl3,

100 MHz) δ: 170.6, 170.5, 170.3, 170.2, 148.2, 140.5, 112.5, 112.0, 104.3, 99.8, 71.6,

70.4, 68.9, 66.9, 61.5, 30.2, 21.1, 20.9, 20.8, 20.7; ESI MS, 499 (M+H)+, 521 (M+Na)+;

+ HRMS calc. for C21H26N2O12Na (M+Na) m/z 521.1378, found, 521.1393.

2-[[(Chloromethoxy)carbonyl]methylamino]-4-nitrophenyl 2,3,4-tri-acetyl-β-D- galactopyranoside (47). The glycoside 46 (2.17 g, 4.4 mmol) and 1,8-bis(N,N- dimethylamino)naphthalene (1.9 g, 8.8 mmol) were dissolved in chloroform (75 mL).

Chloromethyl chloroformate (0.49 mL, 5.5 mmol) was added in the solution. The mixture was kept under argon for 29 h. After removal of the solvent, the residue was dissolved in CH2Cl2 and extracted successively with water, 0.01 M HCl, and water. The organic phase was dried up over Na2SO4, filtered and evaporated to yield 2.54 g of

1 product 47 (98%). H NMR (CDCl3, 500 MHz) δ: 8.18 (m, 2 H), 7.14 (d, 1 H, J = 8.5

Hz), 6.10 (m, 1 H), 5.88 (m, 2 H), 5.46 (s, 2 H), 5.12 (m, 2 H), 4.17 (m, 3 H), 3.21 (s, 3

13 H), 2.19, 2.07, 2.06, 2.00 (4s, 12 H); C NMR (CDCl3, 100 MHz) δ: 170.5, 170.4, 170.2, 55 169.7, 157.3, 143.2, 143.0, 132.3, 125.6, 125.0, 114.8, 98.7, 85.2, 71.9, 70.6, 68.0, 66.7,

+ + 61.6, 37.9, 21.0; ESI MS, 613 (M+Na) , 629 (M+K) ; HRMS calc. for C23H27ClN2O14Na

(M+Na)+ m/z 613.1043, found, 613.1054.

Measurement of enzymatic kinetics. Six 37a or 37b solutions of concentrations from

0.2 to 5 μM (0.5 mL) were incubated for 10 seconds with 10 μL of β-glucosidase or β- galactosidase (50 mg /mL) respectively. Then the solutions were injected into HPLC for analysis. The concentrations of substrates were quantified by the areas of the corresponding peaks. The reaction rates and concentrations of substrates were treated with the classical Lineweaver-Burk plot to obtain the Km, Vmax and kcat. The final data were the average of three repetitive experiments.

Measurement of nitrite formation. The stock solution of 37b (10 mM) was diluted to

0.023 mM using phosphate buffer (pH 7.4). Next the solution of β-galactosidase (E.C.

3.2.1.23, from Aspergillus Oryzae) in phosphate buffer (pH = 7.4) was added, and the mixture was incubated at 37 °C for about 17 hours before the addition of Griess reagent

[150 μL of 1% sulfaniamide in 1 M HCl and 150 μL of 0.1% N-(1-naphthyl)- ethylenediamine in 1 M HCl]. The absorbance of the solution was then measured at 548 nm. Calibration curves were made from known concentrations of aqueous NaNO2 in the

- same buffer solution so as to determine the amount of NO2 formed after the reactions.

Measurement of nitric oxide. NO measurement was carried out with an Electro- chemical ISO-NO Mark II Isolated Nitric Oxide Meter (World Precision Instruments,

Inc.). Calibration plot was obtained by measurements of standard NO solutions with current changes. For the assay, 37a, superoxide dismutase (SOD) (0.3 mg/mL) and β-

56 glucosidase (0.005 mg/mL) (E.C. 3.2.1.21) were incubated in pH 7.4 phosphate buffer.

The current changes were recorded every minute.

CHAPTER 3

DESIGN AND SYNTHESIS OF SIALIC ACID-NO CONJUGATES

3.1 Introduction

Design and synthesis of site-specific NO donors are current tasks in our laboratory. NO releasing moieties have been attached to carriers, which can be recognized by certain targets. Wang’s group has reported several classes of enzyme activated NO donors, previously. For example, the peptide-diazeniumdiolate conjugates were synthesized by the attachment of a diazeniumdiolate to the substrates of prostate specific antigen, and they were demonstrated to release NO upon the activation by

Prostate Specific Antigen (PSA).50 In addition, the conjugates of cephalosporin with 3- morpholinosydnonimine (SIN-1) release NO in the presence of β-lactamase.51 It was also found that the diazeniumdiolate functional group could be attached to the anomeric position of monosaccharide, and those glycosylated diazeniumdiolates could be hydrolyzed by glycosidases.52 Here new sialated NO donors were developed, which are promising to be utilized as an NO donor that targets influenza viruses.

58 H+ NO NO O- O N R1 N R R1 R 1 1 + N N H N+ N H N+ N H N O- O- O- R R 2 R2 R2 2

Scheme 3.1: NO releasing from diazeniumdiolates

Diazeniumdiolates (formerly known as NONOates) are important NO donors in biomedical research.53 The generic structure and NO releasing mechanism are shown in

Scheme 3.1. They are blessed with three attributes that make them an especially attractive starting point for designing solutions to important clinical problems, namely structural diversity, dependable rates of NO release, and a rich derivatization chemistry that facilitates targeting of NO to specific sites of need--a critical goal for therapeutic uses of a molecule with natural bioeffector roles in virtually every organ.

O- O- O- N N -O + -O + N N+ N N +H N N NH N N ONa 3 2 +H N 3 NH2

t = 2.8 s 1/2 t1/2 = 3 h t1/2 = 20 h

Figure 3.1: Half-lives of diazeniumdiolates

59 The major feature of this class of NO donors is that they can spontaneously release NO under physiological conditions with a range of half-lives from a few seconds to several days at pH 7.4 and 37 °C, depending on the identity of the R1R2N group (Fig.

3.1). They are minimally affected by metabolism and are thus fundamentally different from currently available clinical nitrovasodilators that require redox activation before NO is released. The enormous diversity of diazeniumdiolates can be extended even further by covalently binding different groups to the terminal oxygen of the ionic diazeniumdiolate moiety. This can lead to a variety of well-defined outcomes. For

2 example, when a methoxymethyl (MOM) group is attached at O position and R1R2N is a piperazine ring (Fig. 3.2), the diazeniumdiolate (MOM-PIPERAZI/NO) is still a spontaneous NO releaser at physiological pH, but the rate-limiting step is hydrolytic cleavage of the MOM group; in this case, the MOM derivative's half-life for NO generation is 17 days.54 Most other diazeniumdiolates with covalently bound to O2 have proven effectively stable toward spontaneous hydrolysis, but by choosing different groups that are vulnerable to cleavage by specific enzymes, agents capable of cell- or organ-selective NO generation after system-wide administration can be prepared. A case in point is AcOM-PYRRO/NO, a cell-permeant neutral molecule that has proven stable in cell culture medium but undergoes hydrolysis upon catalysis by intracellular esterases.

This feature was exploited in an in vitro test of NO's antileukemic activity in which the ability of AcOM-PYRRO/NO to concentrate NO release inside the cell made it orders of magnitude more cytotoxic than the ion produced on esterase-induced AcOM-

PYRRO/NO hydrolysis (PYRRO/NO, which generates extracellular NO homogeneously throughout the medium with a half-life of 3 s).55 O2-Glycosylated diazeniumdiolates

60 were developed in Wang’s group, such as β-Glc-PYRRO/NO, shown to be activated for

NO release by β-D-glucosidase.52

- O- O + OH -O + N N HN NN N+ N N O O HO O N O O O N HO O OH MOM-PIPERAZI/NO AcOM-PYRRO/NO Glc-PYRRO/NO

Figure 3.2: Prodrugs of diazeniumdiolates

The cytotoxicity of NO to viruses is an exciting area which can potentially provide better understanding of human immunity to viral infections as well as provide more information on antiviral drug designs.56 The influenza virus is especially attractive to us, since the replication of influenza A and B viruses could be severely impaired by

NO donors.57 Hemagglutinin (HA) and neuraminidase are embedded in the lipid bilayer of influenza virus to bind and cut the terminal sialic acid, respectively. Neuraminidase (a type of sialidase) can catalyze the hydrolysis of the α-O-glycosidic linkage of N- acetylneuraminic acid (NeuAc) connected to a variety of aglycons on the cellular surface.58 Therefore, N-acetylneuraminic acid–NO donor conjugates as novel NO prodrugs targeting influenza viruses were synthesized in this project (Fig. 3.3). If these

61 NO donors could bind and be hydrolyzed by influenza neuraminidases, the NO will be released around viruses and obtain the targeting effect. Our proposed synthetic NeuAc-

NO conjugates can also be used as probes to selectively deliver NO to viruses. Such conjugates may also be used to test a neuraminidase activated prodrug design for antiviral therapy.

Neuraminidase OH HO COOH

HO O AcNH Sialic Acid + NO HO NO donor

Influenza virus

Figure 3.3: Influenza virus and NO prodrug design

3.2 Design and synthesis of sialic acid-NO conjugates

Initially the self-activated prodrugs with linkers between sialic acid and NO donors were designed (Fig. 3.4). Considering the efficiency and stability of the prodrugs, two classes of aromatic spacers were chosen as the linkers.59 The first type of prodrug

62 was supposed to be initiated by an unmasked hydroxyl group that may cyclize into a cyclic carbamate with elimination of the NO donor moieties.60 While the second type could take advantage of the “trimethyl lock” effect to accelerate lactonization and release

NO.61 The linker parts were prepared according to synthetic routs in Scheme 2.5 and

Scheme 3.3. The donor for the sialidation was synthesized by a two-step reaction in nearly quantitative yields. Then the sialidation conditions were investigated. There are several methods available for the synthesis of aryl α-sialisides. The traditional Koenigs-

Knorr method usually resulted in elimination rather than substitution.62 In the literature, there were two conditions which gave satisfactory yields. The phase-transfer-catalyzed method was first tried.63 N-Acetylneuraminyl chloride (49) and the phenol linker (44) were refluxed in chloroform-aqueous alkali with benzyltriethylammonium chloride as a catalyst. After two hours, only small amount of product formed. Then another method in which 49 and 44 reacted in diisopropylethylamine and acetonitrile solution was tried.64

After the solution was stirred at room temperature for three hours, the α-sialiside (50) was obtained in 71% yield (Scheme 3.2). However when deprotecting the BOC group, the aromatic amine attacked the carboxyl carbon and formed a spiral ketal (51), which was confirmed by the X-ray crystallography (Fig. 3.5). Next we used the same sialidation procedure as before to prepare the type II conjugates (Scheme 3.3). Very low yields were obtained for the compound 55 in a variety of conditions.

63 Sialic acid Linker NO donor

O OH HO HOOC MeN O NO donor Type I HO O O AcNH HO NO2

OH HO HOOC O NO donor Type II O HO O O AcNH HO

Figure 3.4: Design of sialic acid-NO donor conjugates

OH OAc OAc HO OH AcO Cl AcO COOCH3 ab O HO O COOH AcO O COOCH3 AcO O AcNH AcNH AcNH HO AcO AcO H3C N NO2 49 BOC 48 50

CH3 OAc O N NO c AcO 2

AcO O O AcNH AcO 51

Scheme 3.2: Attempts to synthesize type I sialic acid-NO conjugates Reagents and conditions: (a) (i) MeOH, TFA, rt, 3 d; (ii) AcCl, AcOH, rt, 24 h, 100%; (b) 44, DIPEA, CH3CN, rt, 3 h, 71%; (c) CH2Cl2, TFA, rt, 4 h, 98%.

Figure 3.5: Crystal structure of 51

O OH OH O a b OH

52 53 54

OAc OH c AcO H3COOC

AcO O O AcNH AcO 55

Scheme 3.3: Attempts to synthesize type II sialic acid-NO conjugates Reagent and conditions: (a) (CH3)2C=CHCOOCH3, CH3SO3H, 70 °C, 24 h, 80%; (b) LAH, THF, 0 °C, 12 h, 72%; (c) 49, DIPEA, CH3CN, rt, 3 h, 26%. 65

The sialic acid/SIN-1 conjugate with a carbamate linkage was then designed and prepared. The synthetic route was slightly different from other monosaccharides due to the one more carboxyl at the anomeric carbon (Scheme 3.4). After the protection of carboxyl and hydroxyl groups with methyl ester and acetyl groups, the free anomeric OH form of sialic acid (57)65 as coupled with 4-nitrophenyl chloroformate in the presence of triethylamine to give 58. The following reaction with SIN-1 failed in the previously described condition in Chapter 2. Optimization of the reaction conditions showed that this reaction worked better in aqueous THF solution, which could give the protected sialic acid/SIN-1 conjugate (59) in 40% yield. The anomeric configurations of 58 and 59 could not be determined by NMR until the deprotected compound was obtained. The following two steps of deprotection afforded the sialic acid/SIN-1 conjugate (60) successfully. The anomeric configuration of the final product was determined as a β- sialoside, since the chemical shift of H3e (2.28 ppm) filled in the range of 2.32 ± 0.08 ppm according to the general rule.66 There were no α anomers obtained after column chromatography. The reason may be the anomeric effect, which made the thermodynamically favored β anomer dominant in the products. It was shown that this β- sialoside was not the substrate of the neuraminidase, which hydrolyzes the α-sialosides.

66 O OH OAc HO OH AcO OH a b OAc AcO O O NO2 O O HO COOCH3 AcO COOCH3 O AcNH AcNH AcO COOCH HO AcO AcNH 3 AcO 56 57 58

O - O N N N c OR1 O R1O O N O R1O COOR2 AcNH 59 R1= Ac, R2= CH3 R1O d 60 R1= H, R2= H

Scheme 3.4: Synthesis of sialic acid and SIN-1 conjugates Reagents and conditions: (a) Ac2O, HClO4, 2 h, 75%; (b) p-NO2C6H4OCOCl, NEt3, CH2Cl2, 4.5 h, 64%; (c) 30, NaHCO3, THF-H2O, 10 h, 40%; (d) NaOCH3, CH3OH, 2 h then NaOH, H2O, 10 h, 68%.

Since preparation of the suitable sialic acid and spacer conjugates failed, I started to consider the sialic acid and NO conjugates without the linker portion. PYRRO/NO

(61), which had a half-life about 3 seconds, was chosen as a representative of NO donors.

Acetonitrile was still used as the solvent for the sialidation, as previous experiments proved that it is an excellent solvent to obtain the α-anomer. Compound 49 was stirred with 61 in anhydrous acetonitrile at room temperature. The reaction was monitored by

TLC. After 2 days, the starting material totally disappeared and there was one product shown on TLC. After column chromatography on silica gel, the α-anomer of sialyl diazeniumdiolate (62) was obtained in 70% yield (Scheme 3.5). Since the α-anomer is thermodynamically disfavored and there is no suitable neighboring participation at C-3, it would be difficult to control the anomeric selectivity. However, the result came out successfully. The reason might be the formation of β-acetonitrilium ion as an 67 intermediate which provides predominantly α-sialosides.67 Then deprotection of the acetyl and ester groups afforded the first sialated diazeniumdiolate (63). The anomeric

66 configuration was determined as α by the chemical shift of H3e( 2.70 ppm ).

OAc AcO COOCH OH O a 3 b HO COOH N N O O N O N N ONa AcO N N HO O AcNH AcNH N N AcO O HO O 61 62 63

Scheme 3.5: Synthesis of sialic acid-diazeniumdiolate conjugates Reagents and conditions: (a) 49, CH3CN, rt, 2 d, 70%; (b) (i) MeOH, MeONa, rt, 2 h; (ii) 0.1 M NaOH, rt, 1 h, 100%.

3.3 Hydrolysis of sialic acid/NO conjugates by sialidase

This novel NO donor (63) was water-soluble and very stable in the solid state. It could be stored at room temperature without any decomposition. The stability of this novel NO donor in solution was tested in buffers with different pH. Compound 63 was very stable in both neutral and basic (pH = 10) solution, and did not decompose within several days. While in acidic solution (pH = 1), it showed a half-life about 19.6 hours.

Additionally, this compound decomposed readily after addition of neuraminidase

(3.2.1.18, Clostridium perfringens, Sigma). The decomposition went steadily, as determined by the decay of the absorption at 255 nm (Fig. 3.6).

0.450

0.400

0.350

0.300

0.250

Absorbance Absorbance 0.200

0.150

0.100 0 50 100 150 200 250 300 Time (s)

Figure 3.6: The UV absorbance changing of 63. (in the presence ( ) and in the absence ( ) of neuraminidase (0.05 mg/ml), 63 (0.15 mM) was incubated at pH 7.4 phosphate buffer, 37 °C. UV was measured at 255 nm.)

The enzymatic activity of neuraminidase on 63 was determined on a by UV-VIS spectrophotometer (Hewlett-Packard 8453) fitted with an electrically thermostated cell block. Michaelis constant (Km) was determined from the Lineweaver-Burk plot. The Km

68 (0.14 mM) was smaller than Km values (0.6-1.6 mM) for oligosaccharides as substrates, which indicted the binding between 63 and neuraminidase was stronger than the natural sialosides.

The enzymatic decomposition of 63 was also examined by NO measurement.

The amount of NO generated could be quantitatively measured with an electrochemical

ISO-NO Mark-II isolated nitric oxide meter (World Precision Instruments, Inc. Sarasota, 69 Florida). As shown in Fig. 3.7, 63 was stable in the buffer solution (pH=5.0), whereas substantial amount of NO would be generated after the addition of neuraminidase. In addition, the Griess method also showed the formation of nitrite ion from the enzymatic decomposition of 63, because the NO was oxidized to nitrite by oxygen from exposure to air (Table 3.1). At pH 7, nitrite formation from 63 was comparable with that of 61 after

0.5 hour at 37 °C, while no nitrite was formed from 63 without the enzyme. In addition,

63 could also slowly release NO in acidic solution. The two assays confirmed the decomposing pathway of 63 (Scheme 3.6). Neuraminidase first hydrolyzes the glycosidic bond, and then the diazeniumdiolate automatically decomposes to give two equivalents of NO.

1100

900

700

500 [NO] (nM) 300

100

-100 0 100 200 300 400 500 600

Figure 3.7: Time course of NO release from 63. (in the presence (■) and in the absence (♦ ) of neuraminidase (0.05 mg/ml), 63 (0.2 mM) was incubated at pH 5.0 phosphate buffer, 25°C.)

70 - Substrate (23 μM) [NO2 ] (μM) 61 5.6 63 Negligible 63 + neuraminidase 4.1 63 at pH = 1 0.2

Table 3.1: Nitrite formation from 61 and 63

Neuraminidase OH HO N HO COOH 63 N N + O HO O OH AcNH HO H Sialic acid N 2 NO +

Scheme 3.6: NO releasing mechanism of 63

3.4 Conclusion

These results first present that the diazeniumdiolate group could be tethered to the anomeric position of N-acetylneuraminic acid with α-stereoselectivity. Moreover, the properties of the novel sialidase activated NO donor were studied. Since sialic acids and enzymes that metabolize them have been implicated in the pathogenicity of a number of infectious microorganisms,69 this class of NO donors would have great potential in biomedical applications.

71 3.5 Experimental

Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-didexoy-β-D-glycero-D-galacto-2- nonulopyronosyl chloride) onate (49). N-acetylneuraminic acid (800 mg, 2.6 mmol) was dissolved in dry methanol (100 mL), and CF3COOH (0.2 mL) was added. The reaction was stirred at room temperature for 3 d until the solution became clear. Then the solvent was removed under reduced pressure and the residue coevaporated with 5 ml of dry methanol. Glacial acetic acid (10 mL) and acetyl chloride (30 mL) were poured into the flask, and the mixture was stirred for 24 h at room temperature. Co-evaporation of the reaction mixture with toluene afforded 49 as a syrup in quantitative yield. 1H NMR

(CDCl3, 400 MHz) δ: 7.17 (d, 1 H, J = 7.2 Hz), 5.36-5.48 (m, 3 H), 5.17 (td, 1 H, J = 5.6

Hz, 2.4 Hz), 4.42 (dd, 1 H, J = 12.8, 2.4 Hz), 4.35 (dd, 1 H, J = 10.4, 2.4 Hz), 4.06 (dd, 1

H, J = 12, 5.6 Hz), 3.88 (s, 3 H), 2.78 (dd, 1 H, J = 14, 4.8 Hz), 2.28 (dd, 1 H, J = 14, 11

Hz), 2.12 (s, 3 H), 2.08 (s, 3 H), 2.06 (s, 3 H), 2.05 (s, 3 H), 1.92 (s, 3 H).

Methyl (2-[[(1,1-dimethylethoxy)carbonyl]methylamino]-4-nitrophenyl 5- acetamido-4,7,8,9-tetra-O-acetyl-3,5 dideoxy-α-D-glycero-D-galacto-2- nonulopyranosid)onate (50). 49 (2.6 mmol, 1.3 g) was added to a solution of 44 (5.2 mmol, 1.4 g) and diisopropylethylamine (4.16 mmol, 537 mg) in acetonitrile (15 mL).

The solution was stirred at room temperature for 3 h. Then the solution was concentrated and chromatographed on silica gel eluted with ethyl acetate. The product 50 was

1 obtained as a slightly yellow solid (1.37 g, 71%). H NMR (CD3COCD3, 500 MHz) δ:

8.07-8.14 (m, 2 H), 7.98-8.02 (m, 1 H), 7.44 (br, 1 H), 5.33-5.37 (m, 2 H), 4.97 (t, 1 H, J

= 5 Hz), 4.69-4.72 (m, 1H), 4.22-4.24 (m, 2 H), 4.07-4.08 (m, 1 H), 3.75 (s, 3 H), 3.18 (s,

3 H), 2.81 (dd, 1 H, J = 14, 5 Hz), 2.18-2.21 (m, 1 H), 2.03 (s, 3 H), 2.02 (s, 3 H), 2.01 (s, 72 13 6 H), 1.98 (s, 3 H), 1.38 (s, 9 H); C NMR (CD3COCD3, 125 MHz) δ: 170.1, 170.0,

169.8, 169.6, 155.4, 154.3, 125.0, 123.6, 118.0, 80.0, 78.6, 74.2, 68.5, 67.5, 62.3, 53.3,

48.6, 46.3, 38.8, 36.1, 27.7, 22.4, 20.5, 20.3, 20.2, 20.1; ESI-MS, 764 (M+Na)+.

Preparation of 51: A solution of 50 (1.0 g, 1.35 mmol) in CH2Cl2 (4 mL) and TFA (4 mL) was stirred for 4 h at rt. After concentration, the mixture was poured into an excess of a saturated aqueous solution of NaHCO3, extracted with CH2Cl2, dried (Na2SO4) and

1 concentrated to afford 51 (805 mg, 98%) as a white solid. H NMR (CDCl3, 400 MHz) δ:

7.99 (dd, 1 H, J = 8.8, 2.4 Hz), 7.92 (d, 1 H, J = 2.4 Hz), 7.23 (d, 1 H, J = 8.8 Hz), 5.84-

5.87 (m, 1H), 5.70 (td, 1 H, J = 10.4, 5.6 Hz), 5.17 (dd, 1 H, J = 6.4, 1.6 Hz), 4.84 (td, 1

H, J = 7.6, 3.2 Hz), 4.23 (q, 1 H, J = 10 Hz), 3.69-3.80 (m, 2 H), 3.45 (s, 3 H), 2.71 (dd, 1

H, J = 13, 5.6 Hz), 2.16 (t, 1 H, J = 13 Hz), 2.04 (s, 3H), 2.02 (s, 3 H), 2.00(s, 3 H),

13 1,85(s, 3 H), 1.84(s, 3 H); C NMR (CDCl3, 400 MHz) δ: 170.0, 169.5, 169.3, 169.0,

168.7, 159.9, 145.5, 142.6, 127.8, 119.0, 117.0, 95.8, 76.4, 76.1, 75.8, 72.5, 68.9, 68.4,

66.2, 61.1, 47.9, 35.7, 27.9, 22.1, 20.0 19.8, 19.7, 19.4; ESI MS, 610 (M+H)+, 632

+ + (M+Na) ; HRMS, calcd for C26H31N3O14 (M) m/z 609.1806, found 609.1809.

2-(3-Hydroxy-1,1-dimethyl-propyl)-3,5-dimethyl-phenol (54). 2,5-Dimethylphenol

(33 mmol, 3.94 g) was added to methanesulfonic acid (4.7 mL) followed by the addition of methyl 3,3-dimethylacrylate (4.1 g, 36 mmol). The mixture was heated to 70°C for 24 h. After cooling to room temperature, the solution was poured into water (100 mL). The mixture was extracted with ethyl acetate (150 mL). The organic layer was washed with

5% NaHCO3 and brine (100 mL) and dried over anhydrous MgSO4. The solvent was removed in vacuo, and hexane added to the residue to precipitate a pale white solid which was collected by filtration and washed with hexane to give 4,4,5,8-tetramethyl-3,4- 73 1 dihydrocoumarin (53) as a pale white solid (5.35 g, 80%). H NMR (CDCl3, 400 MHz) δ:

6.73-6.74 (m, 2 H), 2.58 (s, 2H), 2.45 (s, 3 H), 2.26 (s, 3 H), 1.42 (s, 3 H), 1.43 (s, 3 H);

13C NMR δ: 168.7, 151.7, 137.8, 136.3, 129.7, 126.8, 116.4, 46.0, 35.2, 28.0, 23.3, 20.8;

ESI MS, 205 (M+H)+, 243 (M+K)+.

A solution of 4,4,5,8-tetramethyl-3,4-dihydrocoumarin (53) (23 mmol, 4.6 g) in anhydrous THF (50 mL) was cooled in an ice bath and slowly added to a suspension of

LAH (47.4 mmol, 1.8 g) in diethyl ether (50 mL) under nitrogen. The mixture was stirred overnight at 0°C and then allowed to warm to room temperature. THF (500 mL) was added slowly with condenser attached, followed by 6 mL of saturated aqueous ammonium chloride solution to quench excess LAH, and the mixture was filtered. The solid was washed with THF (50 mL) and the solvent was removed in vacuo from the filtrate. The residue was purified by column chromatography (10-20% EtOAC in toluene)

1 to give 3.42 g (72%) of product 54. H NMR (CDCl3, 500 MHz) δ: 6.48 (s, 1 H), 6.33 (s,

1 H), 5.98 (br, 1 H), 3.62 (t, 2 H, J = 7 Hz), 2.47 (s, 3 H), 2.23 (t, 2 H, J = 7 Hz), 2.16 (s,

3 H), 1.63 (br, 1 H), 1.55 (s, 6 H); 13C NMR δ: 155.6, 138.1, 136.4, 128.8, 127.2, 116.5,

+ 61.7, 45.2, 39.8, 32.1, 25.8, 20.5; HRMS calcd for C13H20O2 (M) m/z 208.1462, found

208.1465.

Methyl (2-[2-(3-hydroxy-1,1-dimethyl-propyl)-3,5-dimethyl]-phenyl 5-acetamido-

4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosid)onate

(55). 49 (1.33 g, 2.6 mmol) was added to a solution of 54 (1.08 g, 5.2 mmol) and diisopropylethylamine (537 mg, 4.16 mmol) in acetonitrile (15 mL) at room temperature for 3 h. The reaction mixture was concentrated in vacuo and the residue was charged on a column of silica gel eluted with ethyl acetate. The product (55) was obtained as a white 74 1 solid (458 mg, 26%). H NMR (CD3OD, 300 MHz) δ: 6.40 (s, 1 H), 6.36 (s, 1 H), 5.15-

5.41 (m, 3 H), 4.71 (dd, 1 H, J = 4.2, 1.8 Hz), 4.03-4.12 (m, 2 H), 3.77-3.86 (m, 2 H),

3.69 (m, 1 H), 3.60 (s, 3 H), 2.97 (dd, 1 H, J = 13, 6 Hz), 2.46 (s, 3 H), 2.26 (t, 1 H, J =

13 Hz), 2.13, 2.10, 2.06, 1.99, 1.96 (5×s, 15 H), 1.84-1.86 (m, 2 H), 1.50 (s, 6 H); 13C

NMR δ: 172.6, 171.2, 170.6, 170.3, 170.2, 168.5, 156.7, 137.7, 135.3, 128.0, 125.7,

115.4, 98.9, 71.8, 69.3, 68.4, 67.4, 63.9, 62.2, 52.1, 41.4, 40.0, 38.0, 31.4, 25.0, 21.4, 20.0,

19.8, 19.6, 19.4; ESI MS, 682 (M+H)+, 704 (M+Na)+, 720 (M+K)+.

Methyl 5-acetamido-3,5-dideoxy-β-D-galacto-2-nonulopyranosonate (56). N-

Actylneuraminic acid (800 mg, 2.6 mmol) was dissolved in dry methanol (100 mL), and

CF3COOH (0.2 mL) was added. The reaction mixture was stirred at room temperature for 3 d until the solution became clear. Then the solvent was removed under reduced pressure and the residue coevaporated twice with dry methanol (5 mL). The product was used without purification.

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-β-D-glycero-D-galacto-2- nonulopyranosonate (57). To a stirred, warmed (40 °C) solution of acetic anhydride

(2.88 g, 2.7 mL) and aqueous 60% perchlorice acid (20 μL) was added 56 (2.6 mmol) during 30 min in small portions. The mixtures was stirred for 2 h at 40 °C, then cooled to room temperature, diluted with cold water (40 mL), saturated with ammonium chloride, and extracted with dichloromethane (3 × 100 mL). The combined extracts were washed with saturated aqueous sodium hydrogencarbonate (40 mL) and water (40 mL), dried

(MgSO4), and concentrated to give a white solid. Crystallization from ethyl acetate- hexane gave 57 (1.00 g, 75%) as white needles, mp 147-148 °C; 1H NMR (400 MHz,

75 CDCl3), δ 5.79 (m, 1H, NH), 5.36 (m, 1H), 5.26-5.16 (m, 2H), 4.65 (br, 1H), 4.52 (dd, 1

H, J 12 Hz), 4.21-4.12 (m, 2 H), 4.02 (dd, 1 H, J =12, 8 Hz), 3.85 (s, 3 H), 2.28-2.16 (m,

13 2 H), 2.14, 2.10, 2.02, 2.01, and 1.89 (5 s, 15 H); C-NMR (100 MHz, CDCl3), 171.2

(2×), 170.5 (2×), 170.4, 169.3, 95.1, 71.6, 71.3, 69.5, 68.3, 62.8, 53.7, 49.6, 36.3, 23.4,

21.3, 21.1 (2×), 21.0 (2×); ESI MS, 514.2 (M+Na)+.

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-(4-nitrophenoxycarbonyloxy)-3,5- dideoxy-β-D-glycero-D-galacto-2-nonulopyranosonate (58). To a cooled solution (0

°C) of 57 (400 mg, 0.815 mmol) in anhydrous CH2Cl2 (8 mL) was successively added 4- nitrophenyl chloroformate (197 mg, 0.978 mmol) and triethylamine (247 mg, 2.45 mmol).

After stirring for 4.5 h at room temperature, the crude mixture was diluted with CH2Cl2

(50 mL). Column chromatography with ethyl acetate afforded 58 (342 mg, 64%) as a

1 white syrup. H-NMR (500 MHz, CD3OD), δ 8.34 (d, 2 H, J = 9 Hz), 7.52 (d, 2 H, J = 9

Hz), 5.41 (dd, 1 H, J = 6.5, 2 Hz), 5.24 (td, 1 H, J =11, 5 Hz), 5.17 (td, 1H, J =6, 2.5 Hz),

4.44 (dd, 1 H, J = 13, 2.5 Hz), 4.34 (dd, 1 H, J = 10.5, 2 Hz), 4.11 (t, 1 H, J = 10.5 Hz),

4.09 (dd, 1 H, J = 12, 7 Hz), 3.83 (s, 3 H), 2.68 (dd, 1 H, J =13.5, 5 Hz), 2.12 (s, 3 H),

2.03-1.99 (m, 1 H), 2.01 (s, 3 H), 2.00 (s, 3 H), 1.97 (s, 3 H), 1.88 (s, 3 H); 13C-NMR

(125 MHz, CD3OD), δ 172.3, 171.1, 170.6, 170.4, 170.3, 165.8, 155.2, 149.8, 146.1,

125.3, 122.0, 100.8, 73.1, 70.4, 68.4, 67.7, 62.0, 53.0, 48.5, 35.6, 21.6, 19.7, 19.6, 19.5,

+ + + 19.4; ESI MS, 679 (M+Na) , 695 (M+K) ; HRMS calcd for C27H32N2O17Na (M+Na) m/z 679.1593, found 679.1589.

N-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-β-D-glycero-D-galacto-2- nonulopyranosyl)onate]-carbonyl-3-morpholinosydnonimine (59). To a suspension

76 of 30 (350 mg, 1.68 mmol) in water (3 mL) was added NaHCO3 (155 mg, 1.85 mmol) with ice-cooling and stirring. After 10 min a solution of 58 (900 mg, 1.4 mmol) in THF

(5 mL) was added dropwise with stirring. The mixture was further stirred for overnight.

Then THF was removed in vacuo, and subsequent extraction with ethyl acetate (10 mL) for 3 times. The extract was applied on column chromatography (CHCl3/CH3OH, 20:1).

The product 59 was collected as white foams (400 mg, 40%). 1H NMR (300 MHz,

CD3OD), δ 8.12 (s, 1 H), 5.38 (dd, 1 H, J = 6.5, 2 Hz), 5.22 (td, 1 H, J = 11, 5 Hz), 5.10

(td, 1 H, J = 6, 2.5 Hz), 4.43 (dd, 1 H, J = 13, 2.5 Hz), 4.14 (dd, 1 H, J = 10.5, 2 Hz),

4.11-4.02 (m, 2 H), 3.95 (t, 4 H, J = 5 Hz), 3.74 (s, 3 H), 3.63 (t, 4 H, J = 5 Hz), 2.56 (dd,

1 H, J = 13.5, 5 Hz), 2.09 (s, 3 H), 2.06-2.01 (m, 1 H), 1.98 (s, 6 H), 1.90 (s, 3 H), 1.88 (s,

13 3 H); C-NMR (125 MHz, CD3OD), δ 174.4, 172.2, 171.2, 170.6, 170.3, 168.2, 157.7,

100.8, 96.9, 71.6, 70.1, 69.4, 67.4, 65.3, 61.6, 54.2, 52.,1, 48.7, 36.4, 21.5, 19.6 (2×), 19.5

+ + + (2×); ESI MS, 688 (M+H) , 710 (M+Na) ; HRMS calcd for C27H37N5O16Na (M+Na) m/z 710.2128, found 710.2160.

N-(5-acetamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosyl)-carbonyl-3- morpholinosydnonimine (60). Compound 59 (200 mg, 0.29 mmol) was dissolved in methanol (30 mL) and 0.1 M NaOCH3 in methanol (0.68 mL) was added. The mixture was stirred for 2 h at room temperature. Strong acid ion-exchange resin was added to neutralize the base. The resin was filtered off and twice washed with methanol. The solvent was concentrated in vacuo and the residue was dissolved in 0.1 M NaOH (70 mL).

The solution was stirred for overnight. After neutralization with acidic resin, the solution was concentrated and loaded on C-18 silica gel. Washing with water gave the product as

1 an off-white powder (100 mg, 68%). H NMR (500 MHz, D2O), δ 7.91 (s, 1H), 3.95 (td, 77 1H, J = 10, 5 Hz), 3.83 (t, 4H, J= 5 Hz), 3.79-3.76 (m, 1 H), 3.69 (d, 1H, J = 10.5 Hz),

3.63 (m, 1 H), 3.55 (dd, 1 H, J = 12.5, 3 Hz), 3.47 (t, 4 H, J = 5 Hz), 3.44 (d, 1H, J = 8

Hz), 3.36 (dd, 1 H, J = 12, 6 Hz), 2.28 (dd, 1 H, J = 13.5, 5 Hz), 1.87 (s, 3 H), 1.60 (t, 1 H,

13 J = 13 Hz); C NMR (125 MHz, D2O), δ 174.9, 174.6, 173.8, 158.7, 101.2, 98.9, 72.2,

71.3, 68.0, 66.9, 65.4, 63.0, 53.9, 51.7, 39.3, 22.2; ESI MS, 506 (M+H)+, 528 (M+Na)+,

+ + 544 (M+K) ; HRMS, calcd for C18H27N5O12Na (M+Na) m/z 528.1548, found 528.1551.

Sodium 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (61). A solution of pyrrolidine

(16.5 mL) in a mixture of ether (40 mL) and acetonitrile (12 mL) was bubbled with nitrogen for 20 min, then cooled with dry ice bath for 30 min. NO gas was slowly added into the solution for 1 h. Nitrogen was flushed to remove the extra NO. The crystalline product was collected by filtration with a fritted glass funnel and then washed with cold ether under an atmosphere of nitrogen. 10 M sodium hydroxide (10 ml) was added to the crystalline in a flask. After flooding the resulting mixture with ether (50 mL), the solid

1 sodium salt (5.6 g, 37%) was collected by filtration. H NMR (CD3OD, 400 MHz) δ:

3.22 (m, 4 H), 1.91 (m, 4 H).

O2-(N-acetyl-4,7,8,9-tetra-O-acetyl-1-methyl-α-neuraminosyl)-1-(pyrrolidin-1- yl)diazen-1-ium-1,2-diolate (62). 49 (1.33 g, 2.6 mmol) was dissolved in acetonitrile

(10 mL), to which was added 61 (600 mg, 3.9 mmol). The mixture was stirred for 2 d at room temperature. The solution was concentrated and charged on a column of silica gel eluted with ethyl acetate. Compound 62 was an off-white solid (1.09 g, 70%). 1H NMR

(CDCl3, 500 MHz) δ: 5.30-5.36 (m, 2 H), 5.14 (d, 1 H, J = 10.5 Hz), 4.89-4.94 (m, 1 H),

4.39 (dd, 1 H, J = 11, 2 Hz), 4.31 (dd, 1 H, J = 11, 2 Hz), 4.10-4.14 (m, 2 H), 3.78 (s, 3

H), 3.59 (dt, 2 H, J = 10.5, 6.5 Hz), 3.50 (dt, 2 H, J = 10.5, 6.5 Hz), 2.70 (dd, 1 H, J = 78 12.5, 4.5 Hz), 2.22 (t, 1 H, J = 12.5 Hz), 2.12 (s, 6 H), 2.04 (s, 3 H), 2.03 (s, 3 H), 1.93-

1.96 (m, 4 H), 1.89 (s, 3 H); 13C NMR δ: 171.2, 170.9, 170.4, 170.0, 167.5, 100.4, 73.5,

69.2, 68.8, 67.4, 62.3, 53.2, 50.7, 49.5, 36.1, 23.4, 23.2, 21.2, 21.1, 21.0; ESI MS, 627

+ + + (M+Na) , 643 (M+K) ; HRMS, calcd for C24H36N4NaO14 (M+Na) m/z 627.2126, found

627.2124.

O2-(N-acetyl-α-neuraminosyl) 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (63). 62

(100 mg, 0.17 mmol) was dissolved in anhydrous methanol (17 mL) and 0.1 M methanolic sodium methoxide solution (0.34 mL) was added. The mixture was stirred at room temperature for 2 h. The solvent was evaporated and the residue was dissolved in aqueous 0.1 M NaOH solution (35 mL). After being stirred for 1 hour, the mixture was neutralized with Dowex 50X4-400 ion-exchange resin and the solution was lyophilized to

1 yield 63 (71.7 mg, 100%) as an off-white powder. UV, 255 nm; H NMR (D2O, 400

MHz) δ: 4.02 (dd, 1 H, J = 11, 2 Hz), 3.91 (t, 1 H, J = 10 Hz), 3.76-3.88 (m, 3 H), 3.60-

3.66 (m, 2 H), 3.55 (t, 4 H, J = 7 Hz), 2.82 (dd, 1 H, J = 12, 5 Hz), 1.96 (t, 4 H, J = 3 Hz),

1.91 (t, 1 H, J = 12 Hz); 13C NMR δ: 175.3, 172.3, 103.1, 73.8, 71.9, 68.4, 67.9, 62.9,

51.9, 51.3, 38.4, 22.7, 22.5; ESI MS, 423 (M+H)+, 445 (M+Na)+; HRMS, calcd for

+ C15H26N4NaO10 (M+Na) m/z 445.1547, found 445.1544.

CHAPTER 4

SYNTHESIS OF PRODRUGS OF 5-FLUOROURACIL

4.1 Introduction

5-Fluorouracil (FU) is an example of a rationally designed anticancer agent. The observation that rat hepatomas utilize radiolabelled uracil more avidly than nonmalignant tissues implied that the enzymatic pathways for utilization of uracil differ between malignant and normal cells.70 Thus FU was synthesized in 1957 as an antimetabolite agent.71 In this molecule, the hydrogen atom in position 5 of uracil is replaced by the similar sized atom of fluorine, and was designed to occupy the active sites of enzyme targets thereby blocking metabolism in malignant cells. Although this antimetabolite is toxic, its efficacy makes it one of the most widely used agents against solid tumors, such as breast, colorrectal, and gastric cancers.

There are two mechanisms by which FU can generate antitumor activity (Scheme

4.1).72 One of the routes of metabolism of FU gives rise to 5-fluorouridine-5'- triphosphate (FUTP). During transcription, this fluoronucleotide thus mimics uridine-5'- triphosphate (UTP) and is recognized by RNA-polymerases. This leads to the incorporation of FU in all classes of RNA. It is thought that the full cytotoxicity stems

80 from a combination of the numerous modifications of RNA due to incorporation of FU rather than alteration of a single function.

O F NH

N O H

O O FU F F NH NH O O O O N O N O HO P O HO P O P O P O O O OH OH OH OH OH OH OH FUTP FdUMP

RNA THYMIDYLATE SYNTHASE INCORPORATION INHIBITION

Scheme 4.1: Anticancer mechanisms of FU

The second way of activation of FU by the formation of 5-fluoro-2'-deoxyuridine-

5'-monophosphate (FdUMP) could be the most significant. Indeed, this fluoronucleotide is an inhibitor of thymidylate synthase (TS), which is involved in the synthesis of DNA.

TS is an obvious target for cytotoxic agents since thymidine is the only nucleotide precursor specific to DNA. In the presence of the cofactor 5,10-methylene tetrahydrofolate (MeTHF) serving as the methyl donor, TS and 2’-deoxyuridine-5’-

81 monophosphate (dUMP) form a ternary complex, that enables transfer of a methyl group on carbon 5 of dUMP to form thymidine-5'-monophosphate (dTMP). Following FU exposure and adequate FdUMP formation, the methyl transfer does not take place because the fluorine atom in the C5 position of FdUMP is much more tightly bound than hydrogen. The enzyme is then trapped in a slowly reversible ternary complex. The presence of MeTHF (or its polyglutamates) is essential for tight binding of FdUMP to TS.

The formation of dTMP is therefore blocked, thereby decreasing the availability of thymidine-5'-triphosphate (dTTP) for DNA replication and repair. The “genotoxic stress” resulting from TS inhibition may activate programmed cell death pathways.

FU is relatively efficient, but has numerous drawbacks.73 First of all, it is relatively toxic, causing myelosuppression and gastrointestinal disorders, due mainly to phosphorylation of FU in the digestive tract. Toxicity also derives from the lack of selectivity of the drug towards tumors. Its efficiency is limited by the short half-life of

FU in plasma, by its low bioavailability due to the reduction by dihydropyrimidine dehydrogenase (DPD) and by the resistance to FU of some tumors with strong expression of TS, or low reserves of reduced folates. Its mode of administration is also a problem.

Any i.v. administration, whether by bolus or continuous infusion, requires the presence of the patient in hospital, whereas oral treatment can be taken at home with a corresponding reduction in stress. Furthermore, i.v. administration is not without risk of complications

(venous thrombosis or infection round the catheter). For these reasons, one of the challenges of cancer research is the development of prodrugs of FU that diminish or circumvent some of these disadvantages: reduction in toxicity by avoiding certain routes of degradation (prodrug not being a substrate for the enzymes of degradation) or by

82 targeting the tumor site (prodrugs that liberate the active principle selectively in tumor cells); enhancement of activity by reducing catabolism (use of DPD inhibitors) or by increasing anabolism; improvement in quality of life of the patient by developing oral prodrugs.

O O O F F F NH NH NH

N O N O H C N O 3 O O

O OH OH FTO EMFU 5’dFUrd

O O F F NH Hepatic microsomal NH O P450 enzymes F N O N O NH O O N O OH H FTO FU

Scheme 4.2: Structures and mechanism of FU prodrugs

Ftorafur (FTO) or 1-(2-tetrahydrofuryl)-5-fluorouracil (Scheme 4.2), the first prodrug of FU, was developed in the end of the 1960s.74 It was tested in the USA as a drug for i.v. administration, but it was soon found to be effective via the oral route.75

After oral administration, FTO is rapidly absorbed intact by the organism and then 83 hydroxylated and converted into FU mainly by microsomal P450 enzymes in the liver.6

The level of FU obtained from metabolism of FTO is lower than that of the parent compound (1/50 to 1/400 of the initial concentration of FTO).77 However, FU plasma level decays slowly indicating that the pharmacokinetics of FTO is comparable to that of

FU by continuous i.v. infusion.78 Other two examples of the prodrugs of FU are 1- ethoxymethyl-5-fluorouracil (EMFU)79 and 5'-deoxy-5-fluorouridine (5'dFUrd).80 They could have the same metabolism as FTO.

Novel prodrugs of 5-FU possessing a broader spectrum of antitumor activity and fewer toxicity have been sought diligently by many researchers.81 The common feature of these prodrugs is that they are all N1-modified derivatives through different biodegradable linkages.82 Here two classes of prodrugs of FU were synthesized. They are FU-NO and carbohydrate-FU conjugates.

4.2 Design and synthesis of FU and NO conjugates

The synergistic effects between NO and anticancer agents have been shown in several researches. In a study, the cytotoxicity of cisplatin was enhanced about 60-fold after NONOate pretreatment for 30 min.83 Another research demonstrated that cytotoxicity of Taxol was enhanced by S-nitrosocaptopril.84 This effect is primarily mediated via the increased influx of Taxol by NO into intracellar compartments, while

NO-induced cytotoxicity can not be excluded. In another study, Russian researchers found organic nitrates could also increase the efficiency of cytostatic therapy and retard the development of drug resistance.85 The combined therapy resulted in significant increase in life span and number of survivors among mice bearing leukemias P388 and L-

84 1210. Comparative studies of organic nitrates and a similar compound in which ONO2 moieties were replaced by OH groups demonstrated that the presence of NO2 is required for adjuvant activity of compounds and confirmed that NO modifies the antitumor effects of cytostatics. It was also shown that the NO donor retards the development of drug resistance to cyclophosphamide.

Since all previous studies used the combination of NO donors and anticancer drugs, FU/diazeniumdiolate conjugates were first designed basing on the mutual prodrug concept. Methylene or acyloxymethylene as the spacers were utilized with the aim to improve tumor selectivity, efficiency and safety.

The synthetic scheme toward the conjugate 66 is shown in Scheme 4.3. The chloromethyl NONOate 65 was obtained from the O-alkylation of sodium PYRRO/NO

(61) with chloromethyl methyl sulfide and subsequent treatment with sulfuryl chloride.86

Attachment of FU to 65 gave the desired N1 alkylation product 66 as the major product.

The N1, N3 bis-alkylation product 66a was also obtained from the same reaction.

O O O a NN b NN NN N O N ONa N O SCH3 Cl 61 64 65

O O F N F c HN N N O N O O N + O N N N O N N O N N O O 66 66a

Scheme 4.3: Synthesis of 66 Reagents and conditions: (a) ClCH2SCH3, KI, DMF/THF, rt, 12 h, 45%; (b) SO2Cl2, CH2Cl2, rt; 40 min; (c) FU, NEt3, DMF, rt, 30 min (66, 21%; 66a, 12%). 85

O O O S O b Cl O HO a OBn OBn OBn O O O 67 68 69

O O F F HN HN c d OON OON OH OBn O O O O 70 71

N N O O N O N F O N F N HN N N N O N e + O O O OON OON O O O O O O 72 72a

Scheme 4.4: Synthesis of 72 Reagents and conditions: (a) ClCH2SCH3, Cs2CO3, DMF, rt, 12 h, 88%; (b) SO2Cl2, CH2Cl2, rt, 40 min, 70%; (c) FU, NEt3, DMF, rt, 30 min, 54%; (d), 10%Pd-C, AcOH, 1,4-cyclohexadiene, rt, 3 h, 100%; (e) 65, Cs2CO3, DMF, rt, 30 min (72, 18%; 72a, 12%).

The stepwise synthesis of conjugate 72, an esterase sensitive prodrug, is illustrated in Scheme 4.4. The intermediate 71 could be obtained following a similar procedure reported by F. M. Menger and M. J. Rourk.87 Alkylation of succinic acid monobenzyl ester (67) with chloromethyl methyl sulfide and subsequent treatment with sulfuryl chloride generated the desired chloromethyl ester 69. Attachment of 5-FU to 69 provided the compound 70 as the only product, and there was no sign of the N3 alkylation product. Catalytic transfer hydrogenation debenzylation of 70 in glacial acetic acid, 86 using 1,4-cyclohexadiene as the hydrogen source, gave 71 in quantitative yield.

Coupling of 71 with chloromethyl PYRRO/NO (61) gave the desired N1 alkylation product 72, along with the N1, N3 bis-alkylation product 72a.

To compare the bioactivity of FU-NO conjugates with the NO donor alone.

Other two relative derivatives, acetal linked dimers of diazeniumdiolates were also synthesized by the reactions between 65 and 61 or cupferron.86 Another previously reported prodrug AcOM-PYRRO (75) was synthesized from 65 in high yield (Scheme

4.5).

O O NN NN N O ab N O 65 N O N O N N N c O O 74 73 O O O N N N O 75

Scheme 4.5: Synthesis of 73-75 Reagents and conditions: (a) 61, DMF, rt, 67%; (b) Cupferron, DMF, rt, 28%; (c) NaAc, DMF, rt, 51%.

4.3 NO releasing and cytotoxicity of FU-NO conjugates

The hydrolysis of the prodrug was studied by monitoring of the NO release with an Electrochemical ISO-NO marker II isolated Nitric Oxide Meter manufactured by

World Precision Instruments, Inc. (Sarasota, Florida). All of the prodrug 66, 72, and 75 87 were stable in aqueous solution at neutral pH. Both 72 and 75 slowly release NO at pH =

8 (Fig. 4.1), and in the presence of porcine liver esterase (Fig. 4.2). The NO release rate for compound 72 is much slower than 75 at the same condition. On the contrary, the prodrug 66 was very stable in aqueous solution. No substantial NO was detected even at either acidic or basic conditions.

m) μ NO (

Time (sec)

Figure 4.1: NO releasing from prodrug 72 () and 75 () in a pH = 8.0 buffer. m) μ NO (

Time (sec)

Figure 4.2: NO releasing from prodrug 72 () and 75 () in the presence of esterase.

Compound HeLa DU145

FU 278 204

66 112 98

72 50 129

72a 257 65

Table 4.1: Median effect dose (µM) for HeLa and DU145 cell lines

The cytotoxicities of FU/diazeniumdiolate conjugates were assessed by in vitro clonogenic cell survival assays as described in literature.88 The median effect dose

(ED50) for compound 66 in DU145 (98 µM) and HeLa (112 µM) cell lines indicated that this prodrug was about twice as potent as FU (204 µM in DU145 and 278 µM in HeLa).

Compound 72a (257 µM) and FU (278 µM) exhibited similar cytotoxic effects in HeLa cells, however compound 72 (50 µM) was twice as potent as compound 66 and 5 fold more toxic than FU and compound 72a. In DU145 cells, compound 72a (65 µM) exhibited the greatest cytotoxicity. It was about twice as potent as compound 72, and about three fold more potent than FU. Compound 73-75 had little or no effect in the tested cell lines. 89 Since both of the conjugate 66 and 72 have greater activity than FU, the antitumor nucleoside and NO could have synergetic effects in biological systems. The apparent difference between the cytotoxic activities of the two conjugates, 66 and 72, could be explained by the appreciation of the individual design. 72 was easily been hydrolyzed by esterases (Scheme 4.6), while the N-O ketal linker in compound 66 could not be hydrolyzed as easy as the spacer in compound 72.

O F HN 5-FU O N -H2CO H

O O F HN F esterase HN N N O N O O - + O O N -C2H4(COO )2 O N O O N N O O N O O -H2CO 72

N N NO O N O

Scheme 4.6: The hydrolysis pathway of conjugate 72

90 4.4 Design and synthesis of carbohydrate-FU conjugates

Passive diffusion is by far the major route for compounds crossing epithelial barriers and so is the most important target for improving bioavailability. However, monosaccharides are actively transported through the cell membrane. The use of carbohydrates as tools in drug delivery has three major benefits: firstly, the polyhydroxylated nature of sugars provides an efficient and biocompatible way of altering the physicochemical properties of a drug, particularly increasing water solubility; secondly, sugar units provide the opportunity of utilizing specific, active or facilitated transport pathways across some biological barriers, particularly the blood brain barrier;89 thirdly, the recognition of some carbohydrate structures by some receptors triggers receptor-mediated endocytosis, which allows for the targeting of drugs to particular cell lines or organs. The conjugation of saccharide units to bioactive compounds is being recognized as an effective method of manipulating their physicochemical characteristics, enzyme susceptibility and absorption. For example, glycosylation of neuroactive small peptides such as enkephalins and endorphins has been particularly successful in improving their potency and their ability to cross the blood brain barrier.90 The ability to target drugs to particular cells or organs using selective carbohydrate receptors is the most significant contribution that carbohydrates make to drug delivery science.

Another reason for the development of carbohydrate-FU conjugates is that those target compounds have the potential in antibody-directed enzyme prodrug therapy

(ADEPT) (Fig. 4.3).91 ADEPT is an approach to target cytotoxic drugs to neoplastic tissue. It has long been known that certain types of tumor cells present characteristic tumor-associated antigens on their surface. In the first step of this method, an enzyme- 91 monoclonal antibody is administered and allowed sufficient time to localize to the tumor and clear from circulation. Following this, the tethered enzyme catalytically metabolizes a separately-given prodrug, releasing an effector able to diffuse to and enter surrounding tumor cells.

Figure 4.3: Illustration of ADEPT

Carbohydrate-anticancer drug conjugates are frequently utilized in ADEPT. For example, glucuronic acid conjugates could be prodrugs activated by glucuronidase. β-

Galatosidase is very low in the serum (almost undetectable), which makes this enzyme a very attractive enzyme for activation of these conjugates if it is delivered to tumors. The

92 low levels of this enzyme would minimize the premature release of activate drug in blood in ADEPT study.

O OH OH F OAc OH F a b O c HO O O AcO O O OH HO O HO AcO N NH OH OAc HO N NH OAc OAc OH O 76 O 77 78 79

Scheme 4.7: Synthesis of 79 via 1,6-anhydroglucose Reagents and conditions: (a) (i) TsOH, pyridine, 1.5 h, then at pH 9, 1.5 h; (ii) Ac2O, pyridine, rt, 24 h, 10%; (b) 2,4-bis-trimethylsilyoxy-5-fluoropyrimidine, SnCl4, CH3CN, reflux, 10 h, 40%; (c) MeOH, MeONa, rt, 2 h, 100%.

The first type of synthesized carbohydrate-FU conjugates in this projects belongs to pyranosyl nucleosides. The furanosides of FU, such as 5-fluorouridine and 5- fluorodeoxyuridine, already showed significant antitumor activities and have successfully been put to clinical use.92 Therefore, β-glucose pyranosides of FU (79 )was synthesized to evaluate the enzymatic stability and bioactivity. The synthetic strategy initially attempted to successfully prepare (79 ) was based on the stero-controlled glycosylation of trimethylsilyuracil with anhydro sugars.93 But due to the arduous process for the preparation of 1,6-anhydroglucose,94 an alternative method based on the stannic chloride catalyzed glycosylation. In this procedure, FU was first transferred to 2,4-bis- trimethylsilyoxy-5-fluoropyrimidine in situ. Then stannic chloride catalyzed the glycosylation with penta-O-acetyl-glucose. This method even worked for the synthesis

93 of disaccharide nucleosides. The nucleoside of β-D-gentiobiose and FU (83) were prepared by this method.

F F OAc OAc a O OH b O O O AcO AcO HO O OAc N NH N NH AcO AcO HO OAc OAc OH O O 33a 80 79

Scheme 4.8: An alternative synthesis of 79 Reagents and conditions: (a) FU, TMSOTf, BSA, DCE, reflux, 2 h, 52%; (b) MeOH, MeONa, 2 h, 100%.

OAc OAc OH O AcO AcO O F HO O AcO O a AcO O F OAc O b HO O OAc O O O OH AcO AcO O OAc N NH HO AcO AcO N NH OAc OAc HO O OH 81 82 83 O

Scheme 4.9: Synthesis of 83 Reagents and conditions: (a) FU, TMSOTf, BSA, DCE, reflux, 2 h, 51%; (b) MeOH, MeONa, 2 h, 100%.

The cytotoxicity of the two nucleosides of glucose was tested in the School of

Pharmacy of the Ohio State University. The results showed that they were totally inactive to a number of cancer cell lines. Then, a novel series of FU/sugar conjugates, which could be hydrolyzed by glycosidases, were synthesized. A methylene spacer is

94 inserted between N1 of FU and anomeric O of the sugar. In the routine glycosylation, the sugar moiety is the donor that has the potential to be modified on the anomeric carbon, while the alcohol is the accepter. Since the proposed 1-hydroxymethyl-5-FU is unstable and releases formaldehyde and FU, other methodology has to be utilized. A new reverse glycosylation catalyzed by NIS and TfOH was successfully applied in the synthesis of both glucose and galactose conjugates with FU. Both α and β glycosides could be obtained, but the β isomer was always the dominant product. The preliminary results showed that compound was inactive and it only was cytotoxic when the glycosidase added.

O OTMS F F a NH N N O F F N OTMS OAc OH O H C S b O c 84 3 85 O HO O AcO N NH O N NH AcO O HO OH OAc OAc O O AcO O 86 87 OH AcO OAc 34a

Scheme 4.10: Synthesis of 87 Reagents and conditions: (a) CH3SCH2Cl, SnCl4, CHCl3, 60 °C, 2 h, 75%; (b) NIS, TfOH, DCE, diethylether, 0 °C, 1 min, 73%; (c) MeOH, MeONa, 2 h, 100%.

95 O F F AcO OAc F NH AcOOAc O OH O O a b HO OH + N O O O AcO O N NH N NH OAc AcO HO O OAc H3C S O OH O 34b 85 88 89

Scheme 4.11: Synthesis of 89 Reagents and conditions: (a) NIS, TfOH, DCE, diethylether, 0 °C, 1 min, 74%; (c) MeOH, MeONa, 2 h, 100%.

The third type of carbohydrate-FU conjugates prepared in this project are the derivatives of aminosugars. It is known that hexose transporters can deliver the monosaccharide with a modification at the 2 position of pyranoses.95 So the over- expressed hexose transporters on the surface of tumor cells could assist the uptake of FU.

A number of aminosugars, such as glucosamine, galactosamine, and manosamine, are commercially available and not expensive. Another advantage is that the amine group is easy to be modified and tethered to FU. The initial strategy was chloroacetylation on the amino group. Then FU substituted the chloride to afford the conjugate. 1,3,4,6-Tetra-O- acetyl-β-D-glucosamine was obtained from D-glucosamine hydrochloride in a procedure that used p-anisaldehyde for protection of NH2, per-acetylation and removal of the p-

96 methoxybenzylidene group with 5 M HCl in actone. After the chloroacetylation of NH2, the intermediate reacted with FU in solution of TBAF and THF. It was found that the alkylation happened on both N1 and N3 of FU. The selective alkylation on N1 was difficult to achieve in this condition.

96 OH OAc OH OAc a O HO O b AcO O c HO OH OAc AcO O OH HO AcO OAc HO AcO NH +Cl- N N 3 OMe OMe NH2 90 91 92 93

OAc F d O e AcO O OAc O O AcO OAc OAc NH AcO N N OAc HN AcO N AcO Cl H O O AcO O O OAc 94 95

Scheme 4.12: Synthesis of 95 Reagents and conditions: (a) aq NaOH, MeO-C6H4-CHO, rt, 73%; (b) Ac2O, py, rt, 12 h, 88%; (c) acetone, 5 M HCl, rt, 2 h, 94%; (d) ClCH2COCl, Et3N, CH2Cl2, rt, 1 h, 88%; (e) FU, TBAF, THF, rt, 3 h, 69%.

In literature, 5-fluorouracil-1-acetic acid, which is easy to be prepared from FU, is frequently used as a building block for FU conjugates.97 Simple conjugates can be prepared by the formation of an amide bond between the carboxyl and amino groups.

The acetyl protected glucosamine was easily coupled with 5-fluorouracil-1-acetic acid.

The deprotection of acetyl groups afforded the glucosamine-FU conjugate (98). Another convenient synthetic pathway has also developed. Since the free hydroxyls do not affect the coupling reagents, such as DCC and EDCI, the commercially available aminosugars were coupled with 5-fluorouracil-1-acetic acid directly. Thus, the conjugates of galactosamine/FU and mannosamine/FU were synthesized (Scheme 4.13).

97 O OAc OH O F O F a NH b AcO F HO O NH OAc c OH F AcO HO N O HN N O N O HN N O H NH HOOC O O NH O O FU 96 97 98

HO OH HO OH d O O OH F OH HO HO HN N O NH2 O NH O 99 100

F O NH N O OH 2 e HN OH NH HO O O OH O HO HO OH HO 101 102

Scheme 4.13: Synthesis of 98, 100 and 102 Reagents and conditions: (a) BrCH2COOH, aq KOH, 40 °C, 2 h, 46%; (b) 93, EDCI, NMM, DMF, rt, 12 h, 70%; (c) MeOH, MeONa, rt, 2 h, 72%; (d) 96, EDCI, NMM, DMF, rt, 12 h, 56%; (e) 96, EDCI, NMM, DMF, rt, 12 h, 32%.

4.5 Conclusion

Two classed of conjugates of FU were synthesized. The FU-NO conjugates showed greater cytotoxicity than both FU and the NO donor. It first indicated that anticancer agents and NO could have synergistic effects when they formed conjugates. A new series of carbohydrate-FU conjugates were also prepared. During the development of synthetic methods, a novel reverse glycosylation was utilized. Those sugar-FU conjugates have the potential to be the prodrug in ADEPT. The anitumor activity of those compounds is being evaluated.

98 4.6 Experimental

O2-methylthiomethyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (64). A slurry of compound 3 (3.20 g, 20.9 mmol) and anhydrous sodium carbonate (2.22 g, 20.9 mmol) in

40 mL of dry THF was cooled with an ice-water bath, to which 2.28 mL (27.2 mmol) chloromethyl methylsulfide was slowly added via a syringe, followed by 15 mL of DMF.

After 5 min, the bath was removed, and catalytic amount of KI (200 mg) was added.

The reaction mixture was stirred under nitrogen overnight. Then it was diluted with 200 mL of ether, washed with cold water. The separated aqueous layer was extracted with ether again. The combined ether solution was washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatograph with 1:4 ethyl acetate/hexane, resulting in 1.80 g (45 %) of 64 as colorless

1 oil, which solidified when kept inside refrigerator. H NMR (CDCl3, 300 MHz): δ 5.09

13 (s, 2H), 3.44 (t, 4H), 2.15 (s, 3H), 1.83 (m, 4H); C NMR (CDCl3, 75 MHz): δ 78.06,

50.94, 23.02, 15.61; ESI MS m/z 214 (M+Na)+, 405 (2M+Na)+; HRMS calcd for

C6H13N3O2S m/z 191.0728, found 191.0727.

O2-chloromethyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (65). A solution of compound 64 (91 mg, 0.48 mmol) in 10 mL of dry CH2Cl2 was cooled with an ice-water bath, to which 0.48 mL of sulfuric chloride (1.0 M in CH2Cl2) was added dropwise.

After 10 min, the ice-water bath was removed, and the mixture was stirred for another 30 min. Evaporation under vacuum gave 65 as yellow oil, which was immediately used in

1 the following reactions without further purification. H NMR (CDCl3, 400 MHz): δ 5.80

13 (s, 2 H), 3.60 (t, 4 H), 1.95 (m, 4 H); C NMR (CDCl3, 100 MHz): δ 79.8, 50.8, 23.3.

99 5-FU/diazeniumdiolate conjugate (66). To a solution of 5-fluorouracil (133 mg, 1.02 mmol) in 5 mL of DMF was added triethyl amine (328 μL, 2.36 mmol), the mixture was stirred at room temperature for 30 min. Then a solution of compound 65 in 5 mL DMF, which was freshly prepared from 64 (150 mg, 0.78 mmol) and sulfuric chloride (0.78 mL,

1.0 M in CH2Cl2), was added dropwise, and the mixture was stirred overnight. The mixture was diluted with 100 mL of water, extracted with CH2Cl2 twice (50 mL × 2).

The separated organic phase was combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was loaded on the silica gel chromatograph column. Elution with 1:1 ethyl acetate/methylene chloride provided 45 mg (21%) 66 and 50 mg (12%) 66a. 66: colorless solid, mp 172-178 °C; 1H NMR

(acetone-d6, 400 MHz): δ 7.98 (d, 1H, J = 6.4 Hz), 5.67 (s, 2 H), 3.53 (m, 4 H), 1.95 (m,

4 H); 13C NMR (acetone-d6, 100 MHz): δ 129.53, 129.20, 78.13, 51.26, 23.39. ESI MS

+ + + m/z 296 (M+Na) , 569 (2M+Na) ; HRMS m/z calcd for C9H12FN5O4 274.0952 (M+H) ,

1 found 274.0962. 6a: H NMR (CDCl3, 500 MHz): δ 7.43 (d, 1 H, J = 5.0 Hz), 5.99 (s, 2

13 H), 5.67 (s, 2 H), 3.55 (m, 4 H), 1.95 (m, 4 H); C NMR (CDCl3, 125 MHz): δ 126.36,

126.09, 77.78, 71.17, 71.17, 50.65, 50.56, 23.04, 22.89; ESI MS 439 (M+Na)+, 855

(2M+Na)+.

Succinic acid benzyl ester (methylsulfanyl)methyl ester (67). To the stirred solution of succinic acid monobenzyl ester (4.0 g, 19.0 mmol) in 25 mL of DMF was added cesium carbonate (6.2 g, 19.0 mmol), the resulting suspension was stirred for 30 min at room temperature. Then chloromethyl sulfide (1.59 mL, 19.0 mmol) was added, and the mixture was stirred at room temperature overnight and at 70 °C for 10 min. The mixture

100 was cooled to room temperature, diluted with water, and extracted with ethyl acetate. The separated organic layer was washed with brine, dried over sodium sulfate, and

1 concentrated to give 4.5 g (88 %) yellow oil. H NMR (CDCl3, 400 MHz): δ 7.34 (m, 5

13 H), 5.12 (m, 4 H), 2.69 (m, 4 H), 2.21 (s, 3H); C NMR (CDCl3, 100 MHz): δ 171.88,

135.62, 128.50, 128.24, 128.18, 68.43, 66.54, 29.12, 28.96, 15.31.

Succinic acid benzyl ester chloromethyl ester (69). Following similar procedure for

1 the synthesis of 65. 70 % yield after flash column chromatography. H NMR (CDCl3,

13 500 MHz): δ 7.35 (m, 5 H), 5.68 (s, 2 H), 5.14 (s, 2 H), 2.72 (m, 4 H); C NMR (CDCl3,

125 MHz): δ 171.88, 170.70, 135.83, 128.83, 128.59, 128.52, 69.00, 66.96, 66.91, 64.22,

29.19, 28.98. ESI MS 279 (M+Na)+.

Succinic acid benzyl ester (5-fluro-2,4-dioxo-di-hydro-2H-pyrimidin-1-yl)methyl ester (70). Following similar procedure for the synthesis of 66. 54 % yield after flash

1 column chromatography; white solid, mp 125-127 °C; H NMR (CDCl3, 500 MHz): δ

9.04 (br, 1 H), 7.56 (d, 1 H, J = 4.5 Hz), 7.35 (m, 5 H), 5.62 (s, 2 H), 5.12 (s, 2 H), 2.70 (s,

13 4 H); C NMR (CDCl3, 125 MHz): δ 172.54, 171.73, 156.83, 149.09, 141.09, 139.18,

135.50, 128.59, 128.39, 128.29, 128.23, 69.75, 66.78, 28.80; ESI MS m/z 373 (M+Na)+,

723 (2M+Na)+.

Succinic acid mono[(5-fluro-2,4-dioxo-di-hydro-2H-pyrimidin-1-yl)methyl] ester

(71). To the stirred solution of compound 70 (470 mg, 1.34 mmol) in 10 mL of acetic acid was added 10 % Pd/C (0.5 g) and 1,4-cyclohexadiene (0.76 mL, 8.0 mmol). The mixture was stirred for 3 h at room temperature, then it was filtered and evaporated to

101 1 give 350 mg product 71 (100%). H NMR (DMSO-d6, 400 MHz): δ 8.08 (d, 1 H, J = 6.4

Hz, 1 H), 5.55 (s, 2 H), 2.48 (m, 4 H); ESI MS m/z 283 (M+Na)+, 543 (2M+Na)+.

5-FU/diazeniumdiolate conjugate (72) and (72a). To a solution of 5-fluorouracil (145 mg, 0.56 mmol) in 5 mL of DMF was added cesium carbonate (182 mg, 0.56 mmol).

The mixture was stirred at room temperature for 30 min. Then a solution of compound

65 in 5 mL DMF, which was freshly prepared from 64 (128 mg, 0.67 mmol) and sulfuric chloride (0.67 mL, 1.0 M in CH2Cl2), was added dropwise, and the mixture was stirred overnight. After the evaporation of solvent under vacuum, the residue was loaded onto a silica gel chromatograph column. Elution with 1:1 ethyl acetate/methylene chloride

1 provided 40 mg (18%) 72 and 36 mg (12%) 72a. 72: H NMR (CDCl3, 500 MHz): δ

9.38 (br, 1 H), 7.60 (d, 1 H, J = 6.4Hz), 5.72 (s, 2 H), 5.62 (2 H), 3.59 (m, 4 H), 2.66-2.73

13 (m, 4 H), 1.96 (m, 4 H); C NMR (CDCl3, 125 MHz): δ 171.2, 170.93, 149.17, 141.03,

139.14, 128.69, 128.42, 87.44, 87.30, 69.93, 50.69, 50.56, 28.78, 28.55, 23.02, 22.94;

+ + + ESI MS m/z 426 (M+Na) , 829 (2M+Na) ; HRMS m/z calcd for C14H19FN5O8 (M+H)

1 404.1218, found 404.1225. 72a: H NMR (CDCl3, 500 MHz): δ 7.63 (d, 1 H, J = 5.0

Hz), 5.96 (s, 2 H), 5.74 (s, 2 H), 5.66 (s, 2 H), 3.56 (m, 4 H), 3.52 (m, 4 H), 1.95 (m, 4 H),

13 1.91 (m, 4 H); C NMR (CDCl3, 125 MHz): δ 172.19, 170.55, 128.17, 127.77, 127.51,

87.40, 71.17, 70.78, 50.63, 28.74, 28.65, 28.52, 23.00, 22.92, 22.85; ESI MS m/z 569

+ + + (M+Na) , 1115 (2M+Na) ; HRMS calcd for C19H27FN8O10Na (M+Na) 569.1732, found

569.1752.

Diazeniumdiolate dimer (73). Compound 65 was freshly prepared from 64 (100 mg,

0.52 mmol) and sulfuric chloride (0.52 mL, 1.0 M in CH2Cl2), and it was dissolved in 5 102 mL of dry DMF. Then compound 3 (95 mg, 0.62 mmol) was added, the mixture was stirred at room temperature, and the reaction progress was monitored by TLC. After the disappearance of the starting material, the mixture was diluted with 100 mL of water, extracted with ethyl acetate twice (50 mL × 2). The separated organic phase was combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was recrystallized from acetate/hexane to provide 96 mg (67

1 % from 65) of 66 as colorless needles. H NMR (CDCl3, 500 MHz): δ 5.67 (s, 2 H), 3.56

13 (m, 8 H), 1.92 (m, 8 H); C NMR (CDCl3, 125 MHz): δ 95.65, 50.86, 23.19; ESI MS m/z 297 (M+Na)+, 571 (2M+Na)+.

Diazeniumdiolate/cupferon heterodimer (74). Prepared from compound 65 (28%) after flash chromatography, following a similar procedure for compound 73. 1H NMR

(CDCl3, 500 MHz): δ 7.97 (d, 2 H, J = 8.0 Hz), 7.51-7.43 (m, 3 H), 5.92 (s, 2 H), 3.57 (m,

13 4 H), 1.91 (m, 4 H); C NMR (CDCl3, 125 MHz): δ 131.64, 128.91, 121.27, 95.90,

50.49, 22.92; ESI MS 304 (M+Na)+, 585 (2M+Na)+.

O2-(Acetoxymethyl) 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (75). To a DMF (5 mL) solution of freshly prepared compound 65, which was obtained from 64 (110 mg,

0.58 mmol) and sulfuric chloride (0.58 mL, 1.0 M in CH2Cl2), was added sodium acetate

(158 mg, 1.16 mmol) in one portion. The mixture was stirred at room temperature for 7 h, then it was diluted with 100 mL of water, extracted with CH2Cl2 twice (50 mL × 2). The separated organic phase was combined, washed with brine, dried over anhydrous sodium

1 sulfate, and concentrated under vacuum to give 60 mg (51%) solid. H NMR (CDCl3,

103 13 400 MHz): δ 5.75 (s, 2H), 3.59 (m, 4 H), 2.11 (s, 3 H), 1.96 (m, 4 H); C NMR (CDCl3,

100 MHz): δ 129.99, 87.36, 50.71, 22.98, 20.91.

2,3,4-Tri-O-acetyl-1,6-anhydro-β-D-glucopyranose (77). D-Glucose (20 g, 0.11 mol) was put into pyridine (80 mL). To this suspension, a solution of p-toluenesulfonyl chloride (31.7 g, 0.17 mol, 1.5 equiv) in pyridine (80 mL) was added, keeping the internal temperature of the reaction between 20 and 25 °C with the aid of an ice-water bath. After stirring for 90 min, the reaction mixture was brought to pH 9 and carefully maintained at this value by addition of 3 N aqueous NaOH. After 90 min, the pH was carefully raised to 7 by addition of 3 N HCl, and the solvent were eliminated under reduced pressure. Azeotropic removal of pyridine with toluene gave a semisolid that was triturated with absolute ethanol and filtered through florisil (200 mesh). Impurities, which show up as a brown residue, were not allowed to penetrate through the pad.

Washing with ethanol was continued until the filtrate was free of sugar derivatives).

Rotary evaporation of the solvent afforded the desired crude anhydrosugar, which were dried under high vacuum. To the crude 1,6-anhydroglucopyranose were added acetic anhydride (10.4 mL, 0.11 mol), pyridine (15 mL). The solution was stirred for 24 h. The reaction mixture was dissolved in chloroform, and treated with solid sodium bicarbonate until CO2 evolution ceased. Evaporation of the solvents under reduced pressure gave the triacetate (3.34 g, 10%). Mp 106-108 °C.

5-Fluoro-1-(2,3,4-tri-O-acetyl-β-D-glucosyl)uracil (78). To a cooled solution of (2.32 g, 8.05 mmol) and 5-fluoro-2,4-bis(trimethylsilyoxy)pyrimidine in acetonitrile (100 mL) was added SnCl4 (2.0 g), and the mixture was boiled for 10 h. After evaporation of

104 acetonitrile, the residue was made neutral with NaHCO3 in water and extracted with hot

CHCl3 (100 mL × 3). Concentration of the solvent gave a crude product. Crystallization

1 from ethanol afforded 1.3 g of product (40%). mp 196-198 °C. H NMR (CDCl3, 400

MHz): δ 8.88 (s, 1 H), 7.66 (d, 1 H, J = 7.5 Hz), 5.95 (br, 1 H), 5.20 (m, 2 H), 4.72 (br, 1

H), 4.54 (dd, 1 H, J = 12.5, 2 Hz), 4.07 (dd, 1 H, J = 10, 2.5 Hz), 3.94 (m, 1 H), 1.91-2.02

(s, 9 H).

1-β-D-Glucopyranosyl-5-fluorouracil (79). FU (1.08 g, 8.32 mmol) and α-D- glucopenta-O-acetate (3.24 g, 8.32 mmol) in DCE (40 mL) was added BSA (4.8 mL, 20 mmol). The mixture was stirred under nitrogen at rt for 20 min. After addition of

TMSOTf (3.6 mL, 20 mmol), the mixture was heated under reflux for 2 h. After cooling to rt, the resulting brown mixture was evaporated in vacuo. The resulting oil was diluted in ethylacetate (200 mL) and washed with NaHCO3 and brine (100 mL). After drying over Na2SO4 and solvent evaporated, the resulted oil was purified by column chromatography (hexane/EtOAc 3:2) to give 80. Then the deprotection of acetyl groups

1 following the previous standard procedure afforded 1.2 g of 79 (52%). H NMR (D2O,

400 MHz): δ 7.78 (d, 1 H, J = 6.4 Hz), 5.69 (s, 1 H), 4.21 (m, 2 H), 4.16 (dd, 1 H, J = 8, 4

Hz), 4.10 (m, 1 H), 3.82 (dd, 1 H, J = 12, 4 Hz), 3.70 (dd, 1 H, J = 12, 4 Hz); 13C NMR

(D2O, 100 MHz): δ 125.0, 92.4, 82.0, 80.5, 74.4, 68.5, 63.3.

Hepta-O-acetyl-1-β-D-Gentiobiosyl-5-fluorouracil (82). To a suspension of FU (1.08 g, 8.32 mmol) and β-D-Gentiobiose octaacetate (5.65 g, 8.32 mmol) in DCE (40 mL) was added BSA (4.8 mL, 20 mmol). The mixture was stirred under nitrogen at rt for 20 min.

After addition of TMSOTf (3.6 mL, 20 mmol), the mixture was heated under reflux for 2

105 h. After cooling to rt, the resulting brown mixture was evaporated in vacuo. The resulting oil was diluted in ethylacetate (200 mL) and washed with NaHCO3 and brine

(100 mL). After drying over Na2SO4 and solvent evaporated, the resulted oil was purified by column chromatography (hexane/EtOAc 2:1) to give 82 (51%). 1H NMR

(CDCl3, 250 MHz): δ 9.78 (br, 1 H), 7.53 (d, 1 H, J = 5.9 Hz), 5.85 (d, 1 H, J = 10 Hz),

5.32 (t, 1 H, J = 10 Hz), 4.90-5.10 (m, 6 H), 4.47 (d, 1 H, J = 7.5 Hz), 4.10 (dd, 1 H, J =

7.5, 2.5 Hz), 3.85-3.95 (m, 1 H), 3.60-3.70 (m, 1 H), 3.50-3.55 (m, 1 H), 1.93-2.05 (7s,

+ + 21 H); ESI MS 771 (M+Na) ; HRMS calcd for C30H37FN2O19Na (M+Na ) m/z 771.1867, found 771.1894.

1-β-D-Gentiobiosyl-5-fluorouracil (83). The deprotection of acetyl groups following

1 the previous standard procedure to afford 1.2 g of 83 (100%). H NMR (D2O, 250 MHz):

δ 7.58 (d, 1 H, J = 5 Hz), 5.42 (d, 1 H, J = 7.5 Hz), 4.47 (d, 1 H, J = 7.5 Hz), 4.20 (d, 1 H,

13 J = 10 Hz), 3.3-3.9 (m, 11 H); C NMR (D2O, 63 MHz): δ 157.3, 144.5, 125.0, 124.4,

103.0, 83.9, 77.9, 76.5, 76.3, 76.0, 73.5, 71.4, 70.0, 69.5, 69.2, 61.1; HRMS calcd for

+ C16H23FN2O12Na (M+Na) m/z 477.1127, found 477.1144.

1-Methylsulfanylmethyl-5-fluoro-uracil (85). TMSOTf (2.15 mL, 11.9 mmol) was added to the solution of 84 and CH3SCH2Cl (1.14 g, 11.9 mmol) in CH2Cl2 (20 mL). The mixture was refluxed for 1 h. Then ethanol was added and the whole was evaporated.

The residue was taken up by CH2Cl2 (100 mL) and this solution was washed by water, dried and evaporated. Ether was added to the residue to solidify the product (2.3 g, 75%).

1 H NMR (CDCl3, 250 MHz): δ 9.20 (br, 1 H), 7.42 (d, 1 H, J = 5 Hz), 4.75 (s, 2 H), 2.12

(s, 3 H).

106 Preparation of 86. 34a (174 mg, 0.5 mmol) and 85 (57 mg, 0.3 mmol) were dissolved in

5 mL of DCE. Then a solution of NIS (68 mg, 0.3 mmol) and TfOH (4 μL, 45 μmol) in

DCE and ether (1:1, 3 mL) was added at 0 °C. The reaction was run for 1 min. After filtration, the filtrate was washed with 2 M Na2S2O3 and NaHCO3, dried and concentrated.

The product (107 mg, 73%) was purified by chromatography (hexane/ EtOAc, 1:1). 1H

NMR (CDCl3, 500 MHz): δ 9.75 (br, 1 H), 7.35 (d, 1 H, J = 5 Hz), 5.35 (d, 1 H, J = 11

Hz), 5.21 (d, 1 H, J = 11 Hz), 5.17 (t, 1 H, J = 10 Hz), 5.05 (t, 1 H, J = 10 Hz), 4.93 (t, 1

H, J = 9.3 Hz), 4.76 (d, 1 H, J = 8 Hz), 4.11 (m, 2 H), 3.68 (dt, 1 H, J = 10 Hz, 3 Hz),

13 1.98-2.10 (4s, 12 H); C NMR (CDCl3, 125 MHz): δ 170.7, 170.2, 169.4, 169.3, 157.3,

149.9, 139.6, 127.2, 100.0, 75.4, 72.5, 70.9, 67.9, 61.4, 21.1, 21.0, 20.9, 20.8; HRMS

+ calcd for C19H23FN2O12Na (M+Na) m/z 513.1133, found 513.1121.

Preparation of 87. From the deprotection of 86. Obtained as foam (100%). 1H NMR

(D2O, 400 MHz): δ 7.75 (d, 1 H, J = 5 Hz), 5.48 (d, 1 H, J = 11 Hz), 5.34 (d, 1 H, J = 11

Hz), 4.66 (dd, 1 H, J = 8, 2 Hz), 3.87 (d, 1 H, J = 12 Hz), 3.75 (dd, 1 H, J = 12, 4.4 Hz),

13 3.40-3.55 (m, 3 H), 3.33 (td, 1 H, J = 8, 2 Hz); C NMR (D2O, 100 MHz): δ 171.1, 157.0,

140.4, 128.6, 101.4, 77.4, 76.0, 75.6, 72.8, 69.3, 60.4; HRMS calcd for C11H15FN2O8Na

(M+Na)+ m/z 345.0705, found 345.0706.

1 Preparation of 88. From 34b and 85. Obtained as foam (74%). H NMR (CDCl3, 400

MHz): δ 9.78 (br, 1 H), 7.43 (br, 1 H), 5.40-5.44 (m, 2 H), 5.17-5.26 (m, 2 H), 5.05 (d, 1

H, J = 10 Hz), 4.78 (d, 1 H, J = 7.6 Hz), 4.12 (m, 2 H), 3.68 (m, 1 H), 1.99-2.17 (4s, 12

13 H); C NMR (CDCl3, 100 MHz): δ 170.9, 170.6, 170.4, 169.9, 157.4, 150.2, 139.2,

107 128.0, 101.0, 76.0, 71.8, 70.9, 68.8, 67.4, 61.7, 21.1, 21.0, 21.0, 20.9; HRMS calcd for

+ C19H23FN2O12Na (M+Na) m/z 513.1127, found 513.1108.

Preparation of 89. From the deprotection of 88. Obtained as foam (100%). 1H NMR

(D2O, 400 MHz): δ 7.62 (d, 1 H, J = 5 Hz), 5.41 (d, 1 H, J = 11 Hz), 5.23 (d, 1 H, J = 11

Hz), 4.49 (d, 1 H, J = 8 Hz), 3.87 (d, 1 H, J = 3.2 Hz), 3.60-3.75 (m, 4 H), 3.47 (t, 1 H, J

13 = 8 Hz); C NMR (D2O, 100 MHz): δ 171.1, 158.4, 128.5, 128.2, 101.4, 77.1, 75.2, 72.6,

70.5, 68.4, 60.7.

1,3,4,6-Tetra-O-acetyl-D-glucosamine hydrochloride (93). To a solution of D- glucosamine hydrochloride (25 g, 0.12 mol) in 1 M NaOH (120 mL) under stirring was added p-anisaldehyde (17 mL, 0.14 mol). After a short time, crystallization began. The precipitated product was filtered off and washed with cold water, and followed by a mixture of 1:1 EtOH/Et2O to give 91 as white crystals (26.1 g, 73%, mp 164-165 °C).

This intermediate product (10 g, 0.034 mol) was added successively to a cooled (ice- water) mixture of pyridine (54 mL) and Ac2O (30 mL). The mixture was stirred in ice- bath for 1 h and then at room temperature overnight. The yellow solution was poured into 200 mL of ice-water. The precipitated white product was filtered, washed with cold water and dried. This product was identified as 1,3,4,6-tetra- O-acetyl-2-deoxy-2-[ p- methoxybenzylidene(amino)]-β-D-glucopyranose (92) (13.7 g, 88%): mp 181–183 °C.

Subsequently this compound was dissolved in warm acetone (120 mL), and then HCl (5

M, 6 mL) was added with immediate formation of a precipitate. The mixture was cooled, and after addition of Et2O (160 mL), it was stirred for 2 h and refrigerated overnight. The precipitated product was filtered, washed with Et2O and dried to give 93 (8.3 g, 94%): mp

1 at 235 °C decomposed; H NMR (D2O, 250 MHz): δ 5.92 (d, 1 H, J = 9 Hz), 5.45 (t, 1 H, 108 J = 10.5 Hz), 5.10 (t, 1 H, J = 9 Hz), 4.33(dd, 1 H, J = 8.3, 4.3 Hz), 4.16 (m, 2 H), 3.73 (t,

1 H, J = 9 Hz), 2.06-2.18 (4s, 12 H).

1,3,4,6-Tetra-O-acetyl-N-chloroacetyl-D-glucosamine (94). 93 (1.03 g, 2.68 mmol) and Et3N (10 mL) in CH2Cl2 (20 mL). Chloroacetyl chloride (363 mg, 3.2 mmol) was added at 0 °C. The mixture was stirred for 1 h at rt. After washing with 1 N HCl and sat.

1 NaHCO3. The residue crystallized from Et2O to give white solids (1.0 g, 88%). H NMR

(CDCl3, 250 MHz): δ 6.48 (d, 1 H, J = 9 Hz), 5.68 (d, 1 H, J = 9 Hz), 5.15 (t, 1 H, J = 9

Hz), 5.05 (t, 1 H, 9 Hz), 4.0-4.2 (m, 2 H), 3.88 (s, 2 H), 3.57(m, 1 H), 1.95-2.14 (4s, 12

13 H); C NMR (CDCl3, 63 MHz): δ 171.0, 170.6, 169.4, 169.3, 166.7, 92.1, 72.8, 72.1,

68.2, 61.8, 53.2, 42.3, 20.8, 20.7, 20.6, 20.5.

Preparation of 95. 94 (100 mg, 0.24 mmol) was dissolved in THF and TBAF (0.24 mL),

FU (39 mg, 0.3 mmol) stirred at rt for 2 h. Product 95 (150 mg, 69%) was separated by

1 column chromatography (hexane/ EtOAc, 1:1). H NMR (CDCl3, 250 MHz): δ 7.43 (d, 1

H, J = 5 Hz), 6.98 (d, 1 H, J = 9 Hz), 6.84 (d, 1 H, J = 9 Hz), 5.91 (d, 1 H, J = 9 Hz), 5.48

(d, 1 H, J = 9 Hz), 5.31 (t, 1 H, J = 10 Hz), 5.11-5.22 (m, 3 H), 4.84 (m, 2 H), 4.19-4.38

(m, 4 H), 4.10 (m. 1 H), 4.05 (m, 1 H), 3.73-3.84 (m, 2 H), 1.98-2.16 (8s, 24 H); 13C

NMR (CDCl3, 63 MHz): δ 173.1, 172.7, 171.1, 170.4, 169.4, 169.2, 167.0, 168.9, 166.5,

166.2, 156.4, 156.2, 149.8, 91.6, 91.3, 73.1, 72.5, 68.2, 67.6, 61.9, 61.7, 60.3, 53.1, 51.6,

+ 45.1, 21.0, 20.8, 20.7, 20.6, 20.5, 20.3, 20.2; HR MS calcd for C36H45FN2O22Na (M+Na) m/z 927.2402, found 927.2408.

5-Fluorouracil-1-acetic acid (96). FU (3.9 g, 30 mmol) dissolved in 20 mL of KOH aqueous solution (6.41 g, 11.5 mmol). At 40 °C, bromoacetic acid (6.25 g, 11.5 mmol) was added in 30 min. The mixture was stirred for 2 h, then cooled to rt. Concentrated 109 HCl was used to adjust the pH to 5.5. Then the solution was put in the fridge for 2 h.

Precipitate was filtered off. The solution was adjusted to pH 2 and stored in the fridge for

1 6 h. The product was filtered off as white crystals: mp 276-277 °C; H NMR (CDCl3,

250 MHz): δ 11.18 (br, 1 H), 8.09 (d, 1 H, J = 7 Hz), 4.40 (s, 2 H).

Preparation of 97. 93 (960 mg, 2.5 mmol) and 4-methylmorpholine (303 mg, 3 mmol) in DMF (10 mL) was treated with 96 (334 mg, 1.8 mmol). The mixture was cooled in an ice bath and subsequently treated with EDCI (480 mg, 2.5 mmol), stirred at rt for overnight. EtOAc(50 mL) was added to the mixture and washed with water for 10 times.

1 Concentration of the EtOAc layer gave the product as a syrup. H NMR (CDCl3, 400

MHz): δ 7.76 (d, 1 H, J = 6.4 Hz), 5.81 (d, 1 H, J = 9 Hz), 5.32 ( t, 1 H, J = 9.6 Hz), 5.05

( t, 1 H, J = 9.6 Hz), 4.32 (s, 2 H), 4.29 (m, 1 H), 4.12 (m, 2 H), 3.95 (m, 1 H), 2.01-2.11

13 (4s, 12 H); C NMR (CDCl3, 100 MHz): δ 171.0, 170.7, 169.9, 169.5, 168.3, 158.6,

149.9, 139.2, 130.3, 91.8, 72.4, 72.1, 68.3, 61.6, 52.9, 50.4, 19.4, 19.3, 19.2; ESI MS m/z

+ + 540 (M+Na) ; HR-MS calcd for C20H24FN3O12Na (M+Na) m/z 540.1236, found

540.1236.

Preparation of 98. From the deprotection of 97. Obtained as a syrup (72%). 1H NMR

(D2O, 400 MHz): δ 7.74 (d, 0.5 H, J = 5 Hz), 7.73 (d, 0.5 H, J = 5 Hz), 5.16 (d, 0.5 H, J =

13 3.6 Hz), 4.50, 4.51 (2s, 2 H), 3.60-3.85 (m, 5 H), 3.40-3.48 (m, 1.5 H); C NMR (D2O,

100 MHz): δ 169.5, 169.3, 161.5, 152.1, 141.8, 131.2, 94.7, 90.8, 75.9, 73.7, 71.6, 70.7,

70.0, 69.9, 60.7, 60.6, 57.1, 54.3, 50.9; ESI MS m/z 372 (M+Na)+; HRMS calcd for

+ C12H16FN3O8Na (M+Na) m/z 372.0814, found 372.0803.

Preparation of 100. D-Galactosamine (550 mg, 1.8 mmol) and 4-methylmorpholine

(303 mg, 3 mmol) in DMF (10 mL) was treated with 96 (334 mg, 1.8 mmol). The 110 mixture was cooled in an ice bath and subsequently treated with EDCI (480 mg, 2.5 mmol), stirred at rt for overnight. After concentration of the mixture, the residue was purified by column chromatography on C-18 silica gel. The product was a syrup (320 mg,

1 56%). H NMR (D2O, 400 MHz): δ 7.77(d, 1 H, J = 5 Hz), 5.21 (d, 0.5 H, J = 3.5 Hz),

13 4.48, 4.47 (2s, 2 H), 3.80-4.20 (m, 3.5 H), 3.60-3.70 (m, 3 H); C NMR (D2O, 100 MHz):

δ 169.5, 169.2, 160.0, 159.8, 150.8, 139.3, 131.6, 131.2, 95.2, 90.9, 75.2, 70.9, 70.6, 68.6,

67.9, 67.4, 61.2, 61.0, 54.2, 50.7, 50.6; ESI MS m/z 372 (M+Na)+; HRMS calcd for

+ C12H16FN3O8Na (M+Na) m/z 372.0814, found 372.0826.

Preparation of 102. From D-mannosamine and 96. Obtained as a syrup (32%). 1H

NMR (D2O, 400 MHz): δ 7.77 (d, 1 H, J = 5 Hz), 5.11 (d, 0.5 H, J = 0.8 Hz), 5.10 (d, 0.5

H, J = 0.8 Hz), 4.51-4.58(s, 2 H), 4.45 (m, 0.5 H), 4.32 (dd, 0.5 H, J = 4.5, 1 Hz), 4.03

(dd, 0.5 H, J = 10, 4.5 Hz), 3.75-3.86 (m, 3 H), 3.59 (t, 0.5 H, J = 10 Hz), 3.49 (t, 0.5 H, J

13 = 10 Hz), 3.39 (m, 0.5 H); C NMR (D2O, 100 MHz): δ 170.2, 169.3, 160.0, 159.8,

150.9, 150.8, 141.5, 139.2, 131.6, 131.2, 93.0, 92.9, 76.4, 72.0, 68.8, 66.8, 66.5, 60.4,

+ 54.6, 53.6, 50.6, 50.4; ESI MS m/z 372 (M+Na) ; HR MS calcd for C12H16FN3O8Na

(M+Na)+ m/z 372.0814, found 372.0813.

111

CHAPTER 5

SYNTHESIS OF CARBOXYLY INDAZOLES AS NOS INHIBITORS

5.1 Introduction

Increased generation of NO, either alone or in the presence of other free radicals, such as superoxide, has been implicated in pathophysiological changes in virtually every organ system. Evidence comes from studies of NO generation, NOS isoform expression and effects of NOS inhibitors. For example, induction of iNOS is seen in models of septic shock, inflammatory and non-inflammatory pain, arthritis and ischemia or trauma, as well as in various models of neurodegeneration or cerebral inflammation.98 iNOS is not the only isoform that can cause a pathophysiological increase in NO. In particular, increased nNOS activity is associated with certain types of neurotoxicity. NOSs can also generate superoxide through NADPH oxidation that is uncoupled from NO generation.

A glance at the NO literature reveals many apparently contradictory papers showing beneficial or harmful effects of endogenous NO.1 NO can promote or inhibit apoptosis, kill tumors or increase the potential for metastasis or vascularization, increase or protect against damage after stroke and trigger ischemic preconditioning in the heart and mediate late effects of preconditioning. Some of these dual effects of NO relate to different isoforms of NOS regulating different processes. For example, protective effects 112 in stroke seem to be mediated by eNOS, whereas harmful effects are due to nNOS activity (neuronal toxicity). Other dual effects might relate simply to the amount of NO generated or the background redox state of the cell; at low concentrations, NO seems to be anti-apoptotic, in part through inhibition of caspase activity by means of nitrosation, whereas at higher concentrations, it can indirectly activate caspases. The dual effects of

NO present one of the biggest challenges for the development of potential inhibitors.

Beneficial effects can be offset by harmful effects or turn into harmful effects, depending on the underlying rates of NO synthesis.

It is clear that blocking NO has the potential to produce therapeutic benefit. The difficulties lie in achieving isoform specificity, in targeting to specific cells or tissues and in ensuring that the correct degree of inhibition is achieved. As expected, most interest has focused on iNOS inhibitors, but with substantial interest also in nNOS inhibitors. A key issue for any inhibitor is the degree of selectivity over other NOS isoforms, and specificity for NOS over other potential targets. Some compounds are apparently selective for one or other isoform in vivo, but show no real selectivity at the level of the enzyme. For example, 7-nitroindazole (7-NI) is widely quoted as a selective inhibitor of nNOS, but in reality it is about equipotent as an inhibitor of all three NOS isozymes.99 Its apparent preferential effect on NO synthesis in the brain in vivo presumably relates to a kinetic property of the molecule that allows selective access or metabolism in certain tissues. However, the selectivity of 7-NI over the other isoforms remains low. The aim of this study was to pharmacomodulate the indazole nucleus in order to develop novel potent and specific inhibitors of nNOS. Since the carboxylic acid is the isostere the nitro group, the first target is to synthesize the 7-carboxyl-indazole (7-CI) (Fig. 5.1). 113

N N N N H NO H 2 COOH

7-Nitroindazole 7-Carboxylindazole

Figure 5.1: Structure of 7-NI and 7-CI

5.2 Synthesis of Carboxylindazoles

Most of the syntheses of the indazole derivatives reported in the literature proceed from the benzene precursors in which the pyrazole moiety was generated by ring closing reactions. The two most common methods are phase transfer catalyzed reaction from o- methylbenediazonium tetrafluoroborates (Method A)100 and cyclization of N-nitroso derivatives (Method B) (Scheme 5.1).101

114 CH3 Method A R + - 1. HBF4 N2 BF4 AcOK, 18-Crown-6

2. NaNO2 aq CH3 R R N NH2 N AcOH, HCl conc, H NaNO2

CH Method B 3 benzene, reflux R NHNO

Scheme 5.1: General methods of synthesis of indazoles

7-CI has the similar structure and electron distribution to 7-NI. It may have the same biological activity as 7-nitroindazole, but it has not been synthesized before. Starting from 2-Amino-3-methyl-benzoic acid, the two methods in Scheme 5.1 were tried, but failed. The reason was proposed that the acid and amino group have strong interaction, including hydrogen bonding, which interfere the cyclization. The carboxyl group must be generated after the formation of indazole ring. Thus, 2,6-dimethylanaline was used as the starting material. After the cyclization, the methyl was oxidized. To prevent the oxidation of the pyrazole, BOC was used to protect it. It was found that the BOC was deprotected while the formation of the carboxylic acid (Scheme 5.2). 5 and 6- carboxyindazoles were also synthesized by the nitrosation and cyclization (Scheme 5.3).

115 + NH2 N2

a b N N H

103 104 105

c d N N N N H BOC COOH 106 107

Scheme 5.2: Synthesis of 7-carboxylindazole Reagents and conditions: (a) HNO2, H2O, 0 °C, 30 min, 65%; (b) KOAc, 18-Crown-6, rt, 1 h, 70%; (c) (BOC)2O, Et3N, DMAP, rt, 12 h, 78%; (d) KMnO4, t-BuOH, H2O, reflux, 6 h, 35%.

CH3 a N HOOC N HOOC NH2 H

108 109

HOOC HOOC CH3 a N N NH2 H 110 111

Scheme 5.3: Synthesis of 5-carboxyindazole and 6-carboxyindazole Reagents and conditions: (a) (i) t-BuNO2, 0°C, 2 h; (ii) KOAc, 18-Crown-6, rt, 72 h (45% for 109, 70% for 111).

5.3 Conclusion Three new indazole carboxylic acids were successfully synthesized. They did not show inhibition for nNOS (Table 5.1) by hemoglobin assay. All indazole carboxylic 116 acids had very large IC50 (>1 mM). From the enzymatic results, it indicated that the electronic withdrawing group strongly enhanced the binding of indazole ring with heme. It also gave a hint for further structural modification of indazole to develop new NOS inhibitors.

Compounds IC50 (μM)

7-Nitroindazole 8.51

7-Carboxylindazole 5100

7-Methylindazole 2200

6-Carboxylindazole 3200

5-Carboxylindazole 3900

3-Carboxylindazole 11800

Table 5.1: IC50 of indazole derivatives for nNOS

5.4 Experimental 2,6-Dimethyl-phenyl-diazonium tetrafluoroborate (104). HCl (51 mL), water (100 mL), NaBF4 (35 g) were added 0.25 mol of 2,6-dimethyl-analine. The solution was cooled in ice bath. A solution of 17.3 g of NaNO2 (0.25 mol) in 35 mL of water was added slowly, and then stirred for an half hour. The mixture was filtered, washed with cold water, 50 ml ice cold methanol and 50 ml ether. The white solid was dried in the air overnight (35.6 g, 65%). 117 7-Methyl-1H-indazole (105). Diazonium tetrafluoroborate (2.2 g, 10 mmol) was added in one portion to a stirred of dried and powdered potassium acetate (2.0 g, 20 mmol) and

18-crown-6 (0.14 g, 0.5 mmol) in 100 mL ethanol-free chloroform. After stirring for 1 hour at room temperature, the mixture was filtered and the filtered solid was washed with

CHCl3. The filtrate and washings were combined, washed with water (3×50 mL), dried with sodium sulphate and evaporated in vacuo. The crude product was recrystallized from petroleum ether. The title compound was obtained as a yellow solid (1.41 g, 70%).

1 H NMR (400 MHz, CD3OD) δ 7.95 (s, 1H), 7.46 (d, J = 8Hz, 1H), 7.0-6.9 (m, 2H), 2.44

13 (s, 3H); C NMR (400 MHz, CD3OD) δ 140.5, 133.9, 126.6, 122.8, 121.1, 120.3, 118.0,

16.0.

Preparation of 106. 7-Methyl-indazole (10 mmol) in 20 mL of CH3CN, was treated with of (BOC)2O (2.84 g, 13 mmol), triethylamine (1.21 g), DMAP (244 mg). The mixture was stirred overnight at room temperature. Flash chromatograph of the mixture

1 afforded a liquid 1.806 g (78%). H NMR (400 MHz, CD3OD) δ 8.63 (s, 1 H), 7.43 (d, J

= 7.2 Hz, 1 H), 7.04-6.92 (m, 2 H), 2.52 (s, 3 H), 1.68 (s, 9 H); 13C NMR (400 MHz,

CD3OD) δ 171.7, 151.2, 148.1, 128.0, 125.2, 124.2, 122.0, 118.8, 86.7, 26.9, 16.0.

1H-Indazole-7-carboxylic acid (107). 106 (1 g, 4.3 mmol) in 200 mL tert-butanol was added 100 mL of water. During refluxing, 3.5 g of KMnO4 was added by portion in 6 h.

The hot reaction was filtered off, the solvent was removed by rotvap and the basic solution was extracted with ethyl acetate. The water layer was concentrated to a small amount, adjusted with 1M HCl to pH = 4-5. The solid product was filtered off (243 mg,

1 35%). H NMR (400 MHz, CD3OD) δ 8.15 (s, 1 H), 8.08 (d, 1 H, J = 8 Hz), 8.04 (d, 1 H,

13 J = 8 Hz), 7.25 (t, 1 H), 2.15 (s, 1 H), 1.29 (s, 1 H); C NMR (400 MHz, CD3OD) δ 118 167.7, 138.6, 134.3, 129.7, 126.5, 124.8, 120,2, 113.7; ESI MS 163 (M+H)+, 185

+ + (M+Na) ; HRMS calcd for C8H6N2O2 (M) m/z 162.0429, found 162.0426.

1H-Indazole-6-carboxylic acid (109). A solution of 3-amino-4-methyl benzoic acid

(1.51 g, 10 mmol) in 15 mL anhydrous THF was added dropwise to a –15°C solution of boron trifluoride etherate (14.6 mmol, 1.8 mL) in 45 mL of ethanol free chloroform over

15 min. The resulting mixture was then stirred for 5 min. To this mixture was added t- butyl nitrite (1.4 mL, 10.6 mmol) dropwise, and the reaction stirred at 0°C for 2 h. 4.9 g

(50 mmol) of potassium acetate were added portionwise, followed by 265 mg of 18- crown-6 in one portion. The reaction was allowed to stir at room temperature for 72 h, then was concentrated on a rotvap. Acetone: ethyl acetate (7:3) 50 mL and 1N HCl (15 mL) were added. After stirring for 2 h, brine 15 mL was added to the mixture and the mixture was filtered. The aqueous filtrate was extracted with acetone: ethyl acetate (3:7)

20 mL. The combined organic extracts were dried (MgSO4) and evaporated. The resulting residue was dissolved in hot acetic acid 25 mL, 25 mL 1 M ethereal HCl and 25 mL of ether. After cooling to room temperature, the precipitate was filtered and treated with acetone: ethyl acetate (3:7) 50 mL and brine 10 mL for 1 h. After the phases were separated, the aqueous layer was extracted with ethyl acetate 10 mL. The combined organic extracts were washed with brine, dried (MgSO4) and evaporated to afford the title

1 compound as a brown solid (729 mg, 45%). H NMR (400 MHz, CDCl3) δ 8.16 (s, 1 H),

8.15 (s, 1 H), 7.82 (d, 1 H, J = 8.8 Hz), 7.66 (d, 1 H, J = 8.8 Hz), 3.46 (broad, 1 H); 13C

NMR (400 MHz, CDCl3) δ 168.4, 140.1, 134.2, 129.0, 125.9, 121.2, 112,9; EI MS 162

(M)+.

119 1H-Indazole-5-carboxylic acid (111). Following the same procedure of 1H-indazole-6- carboxylic acid, starting from 4-amino-3-methyl benzoic acid, afforded 1H-indazole-5-

1 carboxylic acid as a slightly yellow solid (563 mg, 70%). H NMR (400 MHz, CDCl3) δ

8.54 (s, 1 H), 8.18 (s, 1 H), 8.02 (d, 1 H, J = 8.8 Hz), 7.56 (d, 1 H, J = 8.8 Hz), 4.93

13 (broad, 2 H); C NMR (400 MHz, CDCl3) δ 169.0, 135.3, 127.4, 123.6, 122.9, 109.7,

94.7; EI MS 163 (M+H)+.

120

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APPENDIX

SELECTED NMR SPECTRA

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OH F O AcO O AcO N NH OAc O 78 198

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8

OH F O HO O HO N NH OH O 79 199

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4

OH O HO F HO O OH O HO O N NH HO OH O 83 200

PPM 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

F OAc O O AcO N NH AcO O OAc O 86 201

PPM 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

F OAc O O AcO N NH AcO O OAc O 86

202

PPM 185.0 175.0 165.0 155.0 145.0 135.0 125.0 115.0 105.0 95.0 85.0 75.0 65.0 55.0 45.0 35.0 25.0 15.0 5.0

F OH O O HO N NH HO O OH O 87 203

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4

F OH O O HO N NH HO O OH O 87

204

PPM 200.0 180.0 160.0 140.0 120.0 100.0 80.0 60.0 40.0 20.0 0.0

F AcOOAc O O N NH AcO O OAc O 88

205

PPM 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

F HO OH O O N NH HO O OH O 89 206

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8

OAc O AcO OAc AcO NH2 93 207

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4

F AcO O OAc O O OAc NH N N OAc AcO N AcO H O O AcO O OAc 95 208

PPM 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

F AcO O OAc O O OAc NH N N OAc AcO N AcO H O O AcO O OAc 95 209

PPM 200.0 180.0 160.0 140.0 120.0 100.0 80.0 60.0 40.0 20.0 0.0

O F NH

N O

HOOC 96 210

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4

OH O HO OH F HO HN N O O NH O 98 211

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4

HO OH O OH F HO HN N O O NH O 100 212

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4

HO OH O OH F HO HN N O O NH O 100

213

PPM 200.0 180.0 160.0 140.0 120.0 100.0 80.0 60.0 40.0 20.0 0.0

F O N O HN OH NH O O HO OH HO 102 214

PPM 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4

F O N O HN OH NH O O HO OH HO 102

215

PPM 200.0 180.0 160.0 140.0 120.0 100.0 80.0 60.0 40.0 20.0 0.0

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