mono and bicyclic 6-aminopyridinols and 2-aminopyrimidinols as novel antioxidants and prostaglandin h2 synthase inhibitors

79 Inhibition of Prostaglandin H; synthase PGHS-catalyzed lipid peroxidafion...-- chon 1 tk kg re 80 Inhibition of lipid peroxidation in LDL particle: free radical lipid peroxidation..

MONO AND BICYCLIC 6-AMINOPYRIDINOLS AND 2-AMINOPYRIMIDINOLS AS NOVEL ANTIOXIDANTS AND PROSTAGLANDIN H2 SYNTHASE INHIBITORS

By

Tae-gyu Nam

Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University

in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

In

Chemistry May, 2006 Nashville, Tennessee

UMI Number: 3229939

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ACKNOWLEDGEMENTS

I still remember the first moment I saw Dr Porter’s picture in the website; a smiling gentleman with gray beard in burgundy polo shirt The gentleman and I have happened to run across in the hallway for several years since that moment

I hope he found it enjoyable to nurture me as much as I enjoyed being a part of his research group Besides the chemistry and biology of lipids, how to appreciate, communicate and collaborate with the colleagues are what I have learned in Porter group

I should mention that I was lucky to be given the incredible and endless patience and attention All the Porter group members are to be acknowledged This dissertation could not be written without their valuable friendship and advices Ms Marianne Beebe can not

be forgotten for her exceptional assistance in administrative affairs

Collaboration with Dr John A Oates and Dr Olivier Boutaud was one of the most precious and exciting experiences during my Ph.D study All the advices, discussions and encouragements provided by Oates group members are unforgettable

I also thank my Ph.D committee members, Dr Carmelo J Rizzo, Dr Piotr Kaszynski, Dr David Wright and Dr Olivier Boutaud for their kind and priceless guidance for my study

My families have always been the biggest supporters Without their love, trust and

support, I would not be able to pursue this long journey with full of joy and hope

This dissertation is dedicated to my parents

ii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS QC nh HH nhe ng tàn hệt ii

LIST OF TABLES L2 2122122112112 111 HH Hàn HH ket vii

LIST OF FIGURES TQ He nnnn nh nh nn TT TEED th viii

LIST OF SCHEMES L 0 220222 HH HH HH ng tren xi

LIST OF ABBREVIATIONS cc cccccccctterereeeeeeteteeeee tere et ties trệt XIV

Part |

SYNTHESIS OF C(7)-MONO AND DIALKYL SUBSITUTED-1,8-TETRAHYDRO-

NAPHTHYRIDINOLS AS ANTIOXIDANTS Chapter

I._ INTRODUCTION 22t ng HT ng ng TH ng tk ke 2

Lipid peroxidafiOn - nàn kg 2

Mechanism of antioxidanfs - c - TQ nnnn TH nghành hen 7

Other naturally occurring phenolic antioxidanfs - +: 10

Il SYNTHESIS OF C(7)-MONO AND DIALKYL SUBSTITUTED-1,8-

TETRAHYDRONAPHTHYRIDINOLS : NAPHTHYRIDINE ROUTE 17 œ-Tocopherol analogs - previous antioxidanfs - : 17 Phenol ring modificafion - nghe Hy 17 Fused ring modificafion - cọ nho re 18 Side chain modification - ch HH raờ 20

Aminopyridinol s†rucfUr@ -. -cnnnnhhnhHhHHHHHHhret 22

Synthesis of Naphthyridinol antioxidants — Naphthyridine route 24

Alkyllithium addition to 1,8-naphthyridine :

Solvent and chelation effect ccceeee reece ee eee ee eeneneeneeseeeeeey 26 Regioselective alkyllithium addition to 1,8-naphthyridine 30 Modification of reagent cece rere reeeerreetnntisereeenenies 30 Modification of substrate 0 eerie 31

3-Bromo-1,8-naphthyridine ce eteeeeeeeeeeerenes 31

1H

1,8-Naphthyridin-4(1H)-one - he 32 Protected amino acid ánh hHHHre 33 Umpolung type reacfion cà eo 33 Cyano acfivafion che 35 Introduction of co-chelation Qroup ccceeceseseeeeeeeeeeees 36

Ye 100) 007-110) 8 EEE 40 2-Nitro-rn-xylene hydroxylafion . cào nnnnnseeehheHre 40 Formyl intermediate nhe ndờ 44 Amino intermediate che 45 Organothallium intermediate cnneenhhhhereo 47 Hydroxymethylafion - nh nghe Hườ 48 Synthesis of the short chain naphthyridinols - 50 Synthesis of Cig chain attached naphthyridinols 54 Synthesis of Naphthyridinol antioxidants: Vitamin Bạ route 60 Synthesis of N,O-ketals nh nh nhe 61

Six-membered bicyclic: no substitution at C(7) posifion 63

C(7)-substituted five- and six-membered bycyclic system 64 Five-membered bicyclic sys†em nhe 65 Perspective : six-membered bicyclic system 67 Synthesis of naphthyridinols moiefy 68 Asymmetric synthesis of C15 side chain 69 REPEFENCES 0.0 iitt 71

II PHYSICO-CHEMICAL AND BIOLOGICAL STUDIES 78

BDE, IP and kịạn measuremert: Antioxidant in the bulk solution 79

Inhibition of Prostaglandin H; synthase (PGHS)-catalyzed

lipid peroxidafion chon 1 tk kg re 80 Inhibition of lipid peroxidation in LDL particle:

free radical lipid peroxidation nha re 82 Perspective : inhibition of lipid peroxidation in LDL:

Macrophage lipoxygenase catalyzed lipid peroxidation 85 References LLcL Q.0 n TT Tnhh nh Khen k7 9 85

IV SUMMARYY 0Q Q Sn HT TH HH ke k1 K1 12711111110 88

V EXPERIMENTAL SECTION - 2c Tnhh nghe hg 90

0-1211 ằằằ a aHAee seep reas eeteaaeeeetaas 90 GONE AL iii ecec cece cece reer nnn E nành ng 90

Synthesis LH nh HH HH nhat Ha Hoà 91

Dyhedral angle prediction : MM2 molecular dynamics, MOPAC and Mechanics energy minimization .- 153 LDL oxidation experiment - sen eg 153 PGHS inhibition assay - che 154

IV

Part Il 6-AMINOPYRIDINOLS AND 2-AMINOPYRIMIDINOLS AS ACETAMINOPHEN ANALOGS : NOVEL PROSTAGLADIN H2 SYNTHASE INHIBITORS

Chapter

I INTRODUCTION Úc nành Hot khe và 156

Prostaglandin Hạ Synthases and Acetaminophen 156 Acetaminophen toxicify Tnhh HH hờ 160 Previously reported acetaminophen analogs ‹- - << 162 References 0 0n n HT TT TH HT ng ng TT ng TK ko 164

II AMINOPYRIDINOLS AND AMINOPYRIMIDINOLS AS

ACETAMINOPHEN ANALOCS TQ SH hai 168 Design of the analogs . - - 2 nh re, 168 Synthesis of the analogs 229222 2H nh nhen 170 Synthesis of acetamido analogs cà nnnnhhhnnee 170 Synthesis of dimethylamino analogs -.ccccŸằằ 175 Synthesis of ring dimethyl analogs - cớ 176

PPHP experiment chen nhu 191

Myoglobin redox experimenfS chen eg 194 Reduction of oxidized myoglobin heme by phenolic compounds 194 Inhibition of myoglobin-mediated arachidonic acid oxidation 202 Perspective : toxicify f©SÍ - nh nhe 204

IV SUMMARYY ng TH nn TT Tnhh KT g8 ki 207

V EXPERIMENTAL SECTION Hs Tnhh khe 209

SynthesiS nh nh HH khang Ha ren 209

pKaạ measuremenIs - - ‹ L Lành hen 11 Ta 220

PGHS experimenfS .-.- - Shin 221 Inhibition of PGHS-1 and PGHS-2 erie 221

pH dependent inhibition of PGHS-1 c che 222

PPHP reduction asSay nen nho hgườn

Reduction of oxidized myoglobin -. che

vi

Dihedral angle (9) and calculated BDEo.n and Kịnn 20

BDE and IP values of phenol, pyridinol and pyrimidinol 23 BDE, IP and kịn of the aminopyridinols 24 Effect of solvent polarity and reaction temperature on

005) 0I8N-ioieliiie8:ebobdiiiẳảả 28 Chelation directed MeLi addifion - - cc.ằ 39 AgNO; catalyzed hydroxymethylation of 1,8-naphthyridine 49 BDE, IP and Kịnn ValU©S -.-cQcnn SH HH nh khe khe 78 Dihedral angles œ and B from MM2 molecular dynamies 79

Comparison of dihedral angels of the compounds reported in literature 80

!Czo against PGHS-1 of representative compounds 82

Comparison of BDE, IP and pKạ values - 169

BDE, IP, Kinn and pK, values of acetamido and dimethylamino

ICzo values of the ApAP analogs against PGHS-1 183

Comparison of Kreg and PPHP index of phenolic compounds 200

vil

Structure of tocomonoenols cc nen he nhe nhe nhe he 10 Structure of flavonoid polyphenols cà ằì 11 Intra(and inter)molecular hydrogen bond(s) in catechols 12 Calculated BDEs of some catechin analogs 12 Examples of orfho f-butyl subsfitufion - se nnhnhinhhHhrree 18 Orbital overlapping between phenolic oxygen and heteroatom

lone pair in the fused ring -cc nen nnn nh nhe nh erent 19 Example of side chain modificafion chen 21 Synthetic antioxidants containing naphthofuran and benzofuran rings 22 Examples of a-TOH analogs with lOW IP cece reece erate 23 Chelation of 1,8-naphthyridine as a bidentate ligand 30

Cyclic chelation with co-chelafion group :: - cà sehhhhhhhenieo 37

Product distribution from 2-nitro-m-xylene hydroxylation - 41 TH-NMR and Mass spectroscopy of the N-adduct : 44

Vill

I-21,

I-22

I-23

I-24,

lI-1

lI-2

H-3

II-4

II-5

II-6

ll-7

II-8

II-9

I-10

H-11

I-12

H-13

H-14

I-15

I-16

H-17

I-18

Hybridization of naphthyridinol core and a-TOH side chain 55

Dihedral angles in naphthyridinol strucfure 79

Inhibition of lipid peroxidation by PyrOH in isolated LDL 84

Examples of LOX inhibitor : Nordihydroguaiaretic acid 85

Branched chain mechanism of PGHS nhe 157 Branched chain of PGHS and two hypothetical sites for inhibition by acetaminophen con bì kì nh nh bàn Hành He tr 158 Possible mechanisms for the oxidation of ApAP by PGHS and P-450 159

Oxidative metabolism of ApAP to NAPQI and GSH adduct formation 16

Previously reported ApAP analogs - c.c sec ằ 162 PGHS-1 inhibition by ApAP(8B), 6B and 7B 181

Correlation of BDE and IP versus lCzo against PGHS-1 182

ET and HAT mechanisms depending on deprotonation status of 3-pyridinol, a model compound ‹- cà cà cà nhe khe 183 Oxidation of arachidonic acid by PGHS-1 at various pH and buffers 184

Inhibition of PGHS-1 at various pH and buffers 185

Manually bound ApAP analog in PGHS-1 peroxidase active site 187

COX-1 and COX-2 inhibition by pyridinol 21 and pyrimidinol 22 188

Structure of SC-560 and Celecoxib e eter eee ees 189 Differences in oxidizable residues near peroxidase active site 190

Effect of ApAP and salicylate on the reduction of PPHP by PGHS-1 191

Effect of 21 and 22 on the reduction of PPHP by PGHS-1 193 Structure of resverafrol cọ HS SH nhe nh nhe kh he 194 UV-vis absorption of Mb c Sen nh nh nh hế nh kh kh ke nh 195

ix

Reduction of ferry Mb to ferric Mb by pyrimidinol 22 - 198

Parallel experiment on Azosnm increase with ApAP and 22 199 Inhibition of hemoprotein mediated AA oxidation by ApAP and

LIST OF SCHEMES

l-1 Mechanism of lipid peroxidation con nnn nh khe sen 3

|-2 Mechanism of inhibition of lipid peroxidation by œ-TOH - 9

I-3 Inverse electron demand Diels-Alder reaction for five-membered bicyclic system 3 c nen nen nhe 25 |-4 Retrosynthesis of naphthyridinols with quaternary center at C(7) pOSIẨÏON c Qn nnnnn TH nh n TH nh BE on BE ki th trẻ 26 I-5 | Examples of nucleophilic addition to 1,8-naphthyridine 27

I-6 Lithiation in 2-methyl substituted pyridine and 1,8-naphthyridine 29

|-7 Addition of alkylcerium reagent to 1,8-naphthyridine 31

|-8 Alkyllithium addition to 3-bromo-1,8-naphthyridine 32

|-9 1,8-Naphthyridin-4-(1Hj)-one HH nh nh nh nh ben kh khen 33 |-10 Protection of NH group of 9 uc uc con nen nhe nh nhe nhe 33 I-11 Construction of quaternary center by enolate alkylation 34

I-12 Dithiane as a carbonyl anion equivalent cà 34 |-13 Cyano group as a carbonyl anion equivalent 35

I-14 Attempted methylation on alanine methyl ester moiety 36

I-15 Introduction of co-chelation group các nà nen nhe He ne 38 I-16 Proposed mechanism of 2-nitro-rn-xylene hydroxylation 41

I-17 Formation of 2-nitroso-m-xylene adduct 42

I-18 Reactivity of O-adduct and N-adduct - 43

I-19 Formylation route for hydroxy product c cà ằ 45 |-20 Example of formylation route for pyridinol synthesis 45

XI

I-21,

I-22

|-23

I-24

I-25,

I-26

I-27

I-28,

I-29

I-30

I-31

I-32

I-33

1-34

1-35

1-36

|-37

1-38

I-39

I-40

I-41

I-42

I-43

Sandmeyer approach for hydroxy product c si 46

Proposed mechanism of copper-mediated hydroxylation/// ¬— 46 Example of indazole ring formation -ccccnnnnnnne nhe sớ 47

Organothallium route for hydroxy product 47

Hydroxymethylation of 1,8-naphthyridine 48

Synthesis of 7,7-dialkyl naphthyridinols 50

2-Nitro-m-xyleme hydroxylation for 7,7-dimethyl naphthyridinols 51

Synthesis of C(7)-monoalkyl naphthyridinols - 52

Nucleophilic aromatic subsfitufion : - cà chen hen nh khe 53 Cul-catalyzed alkoxylation - LH nh Hư han 53 Synthesis of di- and monoalkyl naphthyridinols using Cul-catalyzed benzyloxylafiOn ‹ EEE ne een EEE EEE EERE EES 54 Synthesis of Cig chain (early attemptS) c.c che nho neen 55 C+e—Li route for œ-TOH isosfere -.-.- cà cà nhe nee 56 Synthesis of Ca chain (revised) nen nh nhhhhnhneeese 57 Synthesis of a-TOH isostere using Wittig coupling - 58

Unexpected formation of tricyclic compound cc cà: 58 Attempted synthesis of œ-TOH isosftere ằằ 59 Synthesis of deoxypyridoxine (DOP) c cành eee eerie 61 Synthesis of N,O-kefals - ch nh nhì nh nh nho nen 62 Synthesis of œ-TOH isostere N,O-ketal ào 63 Synthesis of six-membered bicyclic sysfem à 64 Retrosynthetic scheme for the C(7)-subsfituted bicyclics 65

Synthesis of five-membered bicyclic system with C(6)-dialkyl

xii

SUDSTILUTION 00 ccc c ccc ccc cccccececceceeeceevesueeteeeeecenvunvaeneeenereteneeeen yas 67

Perspective: synthesis of naphthyridinols moiefy 68 Perspective: asymmetric synthesis of C;z side chain 70 PGHS-catalyzed peroxidation of arachidonic acid 81 Synthesis of 6B LH HH He nền nh nh nh nh thà nh 171 Synthesis of 7 -.- LH HH nh nh nh nh nh nh bon 172 Mono and diacetyl products of †4 {cà ee 173 Demethylation of 17 with NaCN nen neee 174 2-Nitro-m-xylene rOUÍ@ -.ccQQQ SH nn TH nh nh nhe Bá 174 Synthesis of dimethylamino analogs - - 176 Synthesis of ring dimethyl analogs: pyrimidinol 177 Synthesis of ring dimethyl analogs: pyridinol : 177 Schematic diagram for GSH adduct formation with

dimethylamino analogS cceceeeee teeter eee eee eee nent seen ee ner een ens 204

Xili

ASA Acetyl salicylic acid

BDE Bond dissociation enthalpy

BHA t-Butyl hydroxyanisole

BnOH Benzyl alcohol

BOC (-Butyloxycarbonyl

BzCl Benzoyl chloride

y-CEHC 2,7,8-Trimethyl-2-(B-carboxylethyl)-6-hydrochroman

Ch-Lin Cholesteryl linoleate

CoQH2 reduced Co enzyme QH2

COX Cyclooxygenase

m-CPBA m-Chloroperbenzoic acid

CSA Camphorsulfonic acid

Electron donating group

Electron spin resonance

Electron transfer Flavin adenine dinucleotide Glutathione peroxidase reduced Glutathione oxidized Glutathione Hydrogen atom transfer Hemoglobin

High density lipoprotein

Hydroxy eicosatetraenoic acid 4-Hydroxynonenal

Hydroperoxy eicosatetraenoic acid

Horseraddish peroxidase Inhibition concentration

lonization potential

lsoprostanes Liquid chromatorgraphy-mass spectroscopy Lithium diisopropylamide

XV

Non-radical product Nonsteroidal anti-inflammatory Drug Phosphate buffered saline

Prostaglandin Go Prostaglandin H2 Prostaglandin H2 synthase

Polyunsaturated fatty acid

Reactive nitrogen species

Reactive oxygen species Superoxide dismutase

Tetran-butylammonium fluoride Thiobarbituric acid reactive substances t-Butyihydroperoxide

Tocopherol binding protein t-Butyldimethylsilyl chloride

2,2,6,6-Tetramethyl-1-piperidyloxyl radical

Trifluoroacetic acid Tetrahydrofuran

Thin layer chromatography

2,3,5,6-Tetramethyl-4-methoxyphenol Tocopherol mediated peroxidation Trimethylsily! iodide

a-Tocopherol Tris(hydroxymethyl)aminomethane

Tocopherol transfer protein

Very low-density lipoprotein

XVii

Part |

SYNTHESIS OF C(7)-ALKYLATED TETRAHYDRO-1,8-NAPHTHYRIDINOLS

AS NOVEL ANTIOXIDANTS

CHAPTER |

INTRODUCTION

Lipid peroxidation From the historical perspective,’ lipid peroxidation has long been noticed as

‘rancidity’ from the observation that lipids and fats such as vegetable oils and lard turned bad in long term storage Around 1800, the Swiss chemist Nicolas-Theodore de Saussure first reported that a layer of walnut oil exposed to air was able to absorb about 150 times its own volume of oxygen during a year period Criegee,’ Farmer 3-4 and Bolland * found

in early 1940’s that hydroperoxides are the major product of autoxidation of the hydrocarbons In early 1970’s, with the help of new analytical instruments such as HPLC,GC and tandem Mass spectroscopy, a tremendous amount of new knowledge was

accumulated at a rate previously not observed, leading to a new era of lipid studies in

chemistry, biochemistry and medicine fields Porter reported the identification and the mechanism of formation of complex hydroperoxide products from polyunsaturated fatty acids (PUFAs) 68 and membrane phospholipids.’

Mechanistically, lipid peroxidation is a free radical oxidation of PUFAs such as linoleic acid and arachidonic acid It is a type of autoxidation,!° oxidation by molecular oxygen (O2) The special reactivity of O2 is an important feature of autoxidation Molecular oxygen is in a triplet in ground state In other words, it has two unpaired electrons which is a characteristic of a free radical species, making it an efficient trap for radicals The basic mechanism of lipid peroxidation is found in the radical chain reaction

observed in typical autoxidation: initiation-propagation-termination (Scheme I-1) The primary products of lipid peroxidation are lipid hydroperoxides (LOOH) There are also examples of enzyme-catalyzed oxidation that appears to mimic lipid peroxidation The examples include lipoxygenases (LOXs) and cyclooxygenase (COXs, or, prostaglandin H2 synthases (PGHSs)) When arachidonic acid (AA, (52,82,11Z,14Z)-eicosa-5,8,11,14-

tetraenoic acid) is a substrate, isomers of HpETEs (hydroperoxy eicosatetraenoic acids) and HETEs (hydroxy eicosatetraenoic acids) are formed as primary lipid peroxidation products from LOX activity Some P-450 enzymes can directly induce the formation of HETEs (monooxygenase activity) COXs catalyze the formation of a rather specific hydroperoxide (PGG2) and hydroxy product (PGH2) from AA: The mechanism of lipid peroxidation is shown in Scheme I-1

(Initiation) Initiator ———®~ init *

LH + init e — | *+ ni-H

(Propagation) Le + ©, —~> LOO* (kop = 109 M18")

LOOs + LH ——* LOOH + Le® (kp=10- 10? Ms")

(Termination) LOO*®+ L(OO)s —> Non-RadicalProduct (kp = 10- 10? M's‘)

Scheme I-1 Mechanism of lipid peroxidation The second propagation step that has rate constant k, is the rate determining step where chain breaking antioxidants operate

The other important feature of lipid peroxidation is found in the bis-allylic hydrogen(s) in PUFAs (Figure I-1) Because of a low C-H bond dissociation enthalpy (BDE),'! the bis-allylic hydrogen can be easily abstracted by an initiator radical to form a carbon centered pentadieny] radical, L» (initiation step, Scheme I-1)

Figure I-1 Representative PUFAs and bis-allylic hydrogen C-H BDE varies depending

on R and R’ group: the more double bond in R and/or R’, the lower bis-allylic C-H BDE

The lipid radical (Le) then reacts with molecular oxygen to form lipid peroxyl radical (LOOs) at the diffusion-controlled rate, ko, In the first step, LOOs abstracts a bis- allylic hydrogen from another lipid molecule (LH) to afford lipid hydroperoxide (LOOH) and another lipid radical (L*), which continues the radical chain reaction This propagation step is quite slow (kp = 10' ~ 10’ M's"), and is the rate determining step of

the whole chain reaction

Lipid peroxidation has been proposed to be related to various diseases and pathological states Therefore, it has been of great interest not only for its mechanistic aspects but also for the biological and pharmacological aspects One of the most biologically important lipid peroxidations occurs in low-density lipoprotein (LDL) LDL

is the major carrier of cholesterol and cholesteryl esters of fatty acids and this lipoprotein

is commonly called ‘bad’ cholesterol, since it is associated with increased risk of cardiovascular disease

mm Unesterified cholesterol g-TOH, 6-12 molecules /LDL

Ml Phospholipid — — - y-TOH, 0.5 molecule / LDL

= cree CoQH, 0.8 molecule / LDL

Figure I-2 Structure and composition of LDL particle '?

As shown in Figure I-2, the most abundant lipid component in an LDL particle is the neutral cholestery! linoleate ester (Ch-Lin) residing in the inner lipophilic core When LDL particle is oxidized, Ch-Lin undergoes lipid peroxidation It is noticeable that o-

tocopherol (a-TOH), nature’s best lipophilic antioxidant, also resides in the inner core of

LDL and is the most abundant antioxidant found in the LDL particle LDL oxidation has been proposed to play a central role in the early development of atherosclerosis (Figure I- 3) LDL is oxidized by various initiation mechanisms, including those of macrophage-origin, the vascular 12/15 lypoxygenase activities, and free radical mechanism Macrophages in intima then scavenge the oxidized LDL Accumulation of

oxidized LDL in the macrophage turns it into foam cell, which is believed to be the initial

event in the formation of lipid streak on the artery wall If this condition progresses, the artery wall will be stiffer and narrower, a characteristic of atherosclerosis

Figure I-3 Proposed mechanism of early event in fatty streak formation 8

Various reactive aldehydes are generated from degradation of oxidized lipids

(Figure I-4) They include malondialdehyde (MDA), 4-hydroxy-2-alkanals (ie 4- hydroxy-2-nonenal (4-HNE)) and y-ketoaldehydes (i.e (iso)levuglandins)

These aldehydes react with cellular nucleophiles such as proteins, nucleic acids and other macromolecules to form a variety of adducts Formation of those adducts as

well as the oxidative modification of the lipids are related to various pathologies and

diseases, including cancer!®, diabetes'’, Alzheimer disease 18 etc DNA adducts (M\G, malondialdehyde-guanine (pyrimido-[1,2a]purin-10(3)-one)) with endogenous MDA detected in healthy subjects provides a new etiology of human genetic diseases and cancer.'” It was also suggested that MDA can be formed by PGHSs.” Figure I-5 demonstrated the mechanism of the formation of the representative aldehyde, MDA

21-23 »rostaglandin-like compounds formed in vivo Although isoprostanes (IsoPs),

by non-enzymatic free radical oxidation of arachidonic acid, are considered as the ‘gold standard’ to assess oxidative stress, adducts formed from those aldehydes such as thiobarbiturate-MDA adducts (for TBARS test *4) and ascorbate-HNE 25,26 adducts have also proposed to be good biomarkers for oxidative stress

Mechanism of inhibition: antioxidants

Proteins, nucleic acids and other cellular components are constantly damaged by various oxidants including reactive oxygen species (ROS) and reactive nitrogen species (RNS) Such oxidative damage of cellular components is called oxidative injury or oxidative stress ROS include various radical and non-radical species as shown in Figure [-6

radicals : HO*,O,* ,LOO®

non-radicals : LOOH, H;O¿, Os

Figure I-6 Radical and non-radical ROS

There are cellular defensive mechanisms against oxidative stress, including

various enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) etc Non-enzyme defense mechanisms are mostly antioxidant small molecules such as flavonoids (Figure I-9), vitamin E (Figure I-7), vitamin C, lycopene, carotenes, CoQH)

Vitamin E is the major lipophilic antioxidant and it is a mixture of eight isomers consisting of four tocopherols and four tocotrienols Figure I-7 shows the structures of the eight isomers of vitamin E Although several biological roles 8.29 for a-tocopherol (a- TOH) have been suggested, a specific metabolic function has not been found Its major function appears to be, as a lipid soluble chain-breaking antioxidant, to protect PUFAs in cell membranes and lipoproteins a-TOH is the most potent and abundant in vivo component of the vitamin E isomers Antioxidant activity decreases in the following order: a > B = y >> 6-tocopherol in solution.*° If the substitution pattern of electron donating methyl groups on the phenol ring is identical, difference in Cig side chain between tocopherol versus tocotrienol does not result in a significant change in

antioxidant activity Scheme I-2 depicts the mechanism of inhibition of lipid peroxidation

by a-TOH It efficiently inhibits lipid peroxidation by scavenging lipid peroxyl radical (LOO»), the chain propagating radical, to afford the lipid hydroperoxide (LOOH) and a- TOH radical (a-TOs)

(inhibition) LOO+s + aTOH ——> LOOH + ơ-TO * (knn= 3x10Ê8M 1s)

(termination) œTOs + LOOs (oro-TOe) —> Non Radical Product

œ-TOH — œ-TO*s + He (BDE = 78.3 kcal/mol)

Scheme I-2 Mechanism of inhibition of lipid peroxidation by œ-TOH

Unlike L* and LOOs, a-TOs is stable enough not to propagate the radical chain reaction Thus, the radical reaction no longer propagates once LOO+ is trapped by a-TOH

Alternatively, œ-TOH can scavenge LOO* by forming non-radical products (NRP) (termination step, Scheme I-2) Antioxidant activity of œ—TOH 1s attributed to ifs low Ô-

H bond dissociation enthalpy (BDE) It is one of the primary factors to affect k¡m, the rafe constant for the reaction with LOO» Indeed, inn is the best indication of antioxidant

activity of a specific antioxidant in bulk solution In general, the lower the BDE, the greater kinn; therefore, the better hydrogen atom donor or the better antioxidant

Other naturally occurring phenolic antioxidants

The ninth tocopherol homolog other than the eight isomers shown in Figure I-7 was discovered from plants in 1995; œ-tocomonoenol 3! (Figure I-8) Another tocomonoenol was isolated from chum salmon eggs in cold-water environment and was

found to have the identical antioxidant activity as a-TOH in MeOH or liposomal

suspension at 37 °C,*? Later, it was named Marine-derived tocopherol (MDT) and found

to inhibit peroxidation of cholesterol-containing phosphatidylcholine liposomes to a

greater extent than did «-TOH at 0 °C Furthermore, the ratios of the rate constants for MDT and a-TOH to scavenge peroxyl radicals increased with decreasing rates of radical

flux in liposomes and fish oil at 0 °C, indicating that the enhanced activity of MDT at low temperature is attributed to its greater rate of diffusion in viscous lipids 33

10

Figure I-8 Structure of tocomonoenols

Flavonoids ** *° are another series of phenolic antioxidants present in nature, especially in green tea They include a variety of polyphenol compounds such as,

flavanones, flavones and flavanols (Figure I-9)

Rs O Rs

5,7,4-OH Naringenin 5,7,4'-OH Apigenin 3,5,7,3',4'-OH (+)-Catechin 3,5,7,3',4'-OH Taxifolin 3,5,7,4-OH Kaempferol

5,7,3 ,4-OH Luteolin 3,5,7,3',4'-OH Quercetin 3,5,7,3',4',5'-OH Myricetin

Figure I-9 Structure of flavonoid polyphenols

Flavonoids mostly depend on their antioxidant activity on the catecholic -OH group in B rỉng”” just as phenolic —OH in tocopherol analogs However, the two hydroxy] groups in the catechol moiety are not equivalent in their O-H BDE because of the

lãi

intramolecular hydrogen bond (Figure I-10) It is known that hydrogen bonded O-H has higher BDE than mono hydroxyl analog while non hydrogen-bonded O-H has significantly low BDE 37 Interests in the effect of the intramolecular hydrogen bonding

on the antioxidant (and anti-artherogenic) properties of these polyphenols found in various vegetables and fruits have been increasing recently The same catecholic effect can be found in many endogenous catechol amine neurotransmitters such as L-DOPA and

dopamine which are reported to have both toxic and antioxidant effect

on the antioxidant activity However, semi-empirical calculation suggested that catechins

are likely to donate C(2)-hydrogen atom rather than catecholic hydrogen atom due to the lower BDE * *° (= 64 kcal/mol vs 68 ~ 72 kcal/mol) (Figure I-11)

12

Epicatechin (EC) Epigallocatechin (EGC)

Figure I-11 °° Calculated BDEs of some catechin analogs

References

(1) www.cyberlipid.org

(2) Criegee, Ber 1939, 72, 1799

(3) Farmer, E H., Sutton, D A., J Chem Soc 1943, 119-122

(4) Farmer, E H., Koch, H P., Sutton, D A., J Chem Soc 1943, 541-547

(5) Bolland, Q Rev 1949, 3, 1

(6) Porter, N A.; Byers, J D.; Holden, K M.; Menzel, D B., Synthesis of Prostaglandin H) J Am Chem Soc 1979, 101, 4319-4322

(7) Porter, N A.; Wolf, R A Yarbro, E M.; Weenen, H., Autoxidation of

Arachidonic-Acid - Formation of the Proposed Srs-a Intermediate Biochem

Biophys Res Comm 1979, 89(4), 1058-1064

(8) Porter, N A.; Weber, B A.; Weenen, H.; Khan, J A., Autoxidation of Poly- Unsaturated Lipids - Factors Controlling the Stereochemistry of Product Hydroperoxides J Am Chem Soc 1980, 102, 5597-5601

(9) Porter, N A.; Wolf, R A.; Weenen, H., Free-Radical Oxidation of Poly- Unsaturated Lecithins Lipids 1980, 15(3), 163-167

(10) Howard, J A., In Free Radicals, ed.; Kochi, J K., Wiley: New York, 1973; Vol II, p3

13

J Am Chem Soc 2003, 125, 5801-5810

Stryer, L., Biochemistry, 4h ed.; W H Freeman and Company: New York, 1995;

Chaudhary, A K.; Nokubo, M.; Reddy, G R.; Yeola, S N.; Morrow, J D.; Blair, I A.; Marnett, L J., Detection of Endogenous Malondialdehyde-Deoxyguanosine Adducts in Human Liver Science 1994, 265, (5178), 1580-1582

Hecker, M.; Ullrich, V., On the Mechanism of Prostacyclin and Thromboxane-A2 Biosynthesis J Biol Chem 1989, 264, (1), 141-150

(21) Morrow, J D.; Hill, K E.; Burk, R F.; Nammour, T M.; Badr, K F.; Roberts, L J.,

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A Series of Prostaglandin-F2-Like Compounds Are Produced Invivo in Humans by

a Noncyclooxygenase, Free Radical-Catalyzed Mechanism Proc Natl Acad Sci

U S A 1990, 87, (23), 9383-9387

Yin, H Y.; Porter, N A.; Morrow, J D., Separation and identification of F2- isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods J Chromatogr B 2005, 827, (1), 157-164

Montuschi, P.; Barnes, P J.; Roberts, L J., Isoprostanes: markers and mediators of oxidative stress FASEB J 2004, 78, (15), 1791-1800

14

Sowell, J.; Conway, H M.; Bruno, R S.; Traber, M G.; Frei, B.; Stevens, J F., Ascorbylated 4-hydroxy-2-nonenal as a potential biomarker of oxidative stress response J Chromatogr B 2005, 827, (1), 139-145

Sowell, J.; Frei, B.; Stevens, J F., Vitamin C conjugates of genotoxic lipid peroxidation products: Structural characterization and detection in human plasma Proc Natl Acad Sci U S A 2004, 101, (52), 17964-17969

Marnett, L J., Lipid peroxidation-DNA damage by malondialdehyde Mutat Res

1999, 424, 83-95

Devaraj, S.; Jialal, I., alpha-Tocopherol decreases tumor necrosis factor-alpha mRNA and protein from activated human monocytes by inhibition of 5-

lipoxygenase Free Rad Biol Med 2005, 38, (9), 1212-1220

Wu, D Y.; Liu, L P.; Meydani, M.; Meycani, S N., Vitamin E increases

production of vasodilator prostanoids in human aortic endothelial cells through

opposing effects on cyclooxygenase-2 and phospholipase A(2) J Nutrition 2005,

135, (8), 1847-1853

Burton, G W.; Ingold, K U., Autoxidation of Biological Molecules 1 The Antioxidant Activity of Vitamin-E and Related Chain-Breaking Phenolic Antioxidants Invitro J Am Chem Soc 1981, 103, 6472-6477

(31) Matsumoto, A., Takahashi, S., Nakano, K., Kijima, S., J Jpn Oil Chem Soc 1995,

Yamamoto, Y.; Fujisawa, A.; Hara, A.; Dunlap, W C., An unusual vitamin E constituent (alpha-tocomonoenol) provides enhanced antioxidant protection in marine organisms adapted to cold-water environments Proc Natl Acad Sci U.S

4 2001, 98, (23), 13144-13148

Jovanovic, S V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic, M G., Flavonoids

as Antioxidants J Am Chem Soc 1994, 116, 4846-4851

Kondo, K.; Kurihara, M.; Fukuhara, K., Mechanism of antioxidant effect of catechins Flavonoids and Other Polyphenols 2001, 335, 203-217

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Foti, M C.; Barclay, L R C.; Ingold, K U., The role of hydrogen bonding on the H-atom-donating abilities of catechols and naphthalene diols and on a previously

overlooked aspect of their infrared spectra J Am Chem Soc 2002, 124, 12881-

12888

Hastings, T G.; Lewis, D A.; Zigmond, M J., Role of oxidation in the neurotoxic

effects of intrastriatal dopamine injections Proc Natl Acad Sci U S A 1996, 93, (5), 1956-1961

Kondo, K.; Kurihara, M.; Miyata, N.; Suzuki, T.; Toyoda, M., Mechanistic studies

of catechins as antioxidants against radical oxidation Arch Biochem Biophys

CHAPTER II

SYNTHESIS OF C(7)-MONO AND DIALKYL SUBSTITUTED-1,8-

TETRAHYDRONAPHTHYRIDINOLS : NAPHTHYRIDINE ROUTE

a- Tocopherol analogs: Previous antioxidants

Phenols are the most abundant structure found in natural and synthetic

antioxidants The best and most famous phenolic antioxidant in nature is a-TOH Since

isolated in 1927 as a substance (vitamin E) required by animals for normal reproduction,’ o-TOH has been a target of synthetic, mechanistic and biological studies and a model of synthetic antioxidants as well The absolute configuration of three chiral centers in a- TOH was assigned as (R),(R),(R) * and it was later confirmed by the first total synthesis

of naturally occurring a-TOH in 1963 by Mayer 3 Along with tremendous amount of biological studies using a-TOH as an antioxidant, various synthetic analogs have been developed to improve antioxidant activity and phyiso-chemical properties, i.e solubility and stability

Phenol ring modification

Since the phenolic OH is the core structure of the antioxidant activity of «-TOH, substitution pattern of the phenol ring can vary antioxidant activity Lower antioxidant activities, reflected in inh, of B, y and 5-tocopherol than that of a-TOH demonstrated the importance of methyl groups on the phenol ring (a > B = y >> 5-tocopherol).’ It is consistent with the general trend that electron donating groups at the ortho and para positions to phenolic OH decrease BDEo.n, therefore, increasing finn The other role of

17

the ortho methyl(s) on the phenol ring is to decrease the reactivity oftocopheroxyl radical

(a-TO*) by generating steric hindrance so that «-TO* does not propagate the radical chain reaction or promote the other side reactions Representative examples of the steric effect include ortho t-butyl substituted BO-653 >-6 and Probucol ’ (Figure I-12) While BO-653 has comparable antioxidant activity against peroxyl radical to that of a-TOH, probucol, 8 a hypocholestrolemic and antioxidant drug, showed much less reactivity toward lipid peroxyl radical than a-TOH, demonstrating the importance of the para- substitution and the fused ring structure

Figure I-12 Examples of ortho t-butyl substitution

Fused ring modification

Extensive studies on the structure—activity relationships by Ingold *? showed that

antioxidant activities of tocopherol analogs are affected by the degree of stabilization of the phenoxy] radical This stabilization depend on two factors: (1) the extent of orbital overlap between the 2p type lone pair on the para oxygen (or hetero)atom and the aromatic x electron system and (ii) the electron-donating or withdrawing character of the para substituted group Orbital overlap is determined by the angle 6 (Figure I-13) and can

be estimated by dihedral angle (a-b) between the O-substuent and the aromatic ring plane As summarized in Figure I-13 and Table I-1, @ (=fab) of PMC (2,2,5,7,8-

18

pentamethy]-6-chromanol, Figure I-13(A) and Table I-1(Entry 1)) is 17 ° while Ø= 88.6°

in TMMP (2,3,5,6-tetramethyl-4-methoxyphenol, Figure I-13(B) and Table I-1(Entry 3)) and @= 6° in benzofuran (Table I-1, Entry 2) It turns out that kinn is inversely correlated with the dihedral angles The large dihedral angle of TMMP due to the steric interaction between the para -OMe group and the ring methyl groups leads to low rate constant In bicyclic systems, puckering of the second ring reduces steric interaction between the phenol ring methyls and the O-substituents generating better orbital overlap (PMC vs TMMP) In the furan analog, ring puckering in the benzofuran creates the best overlapping reflected in the smallest 0, consistent with the observed Kin, and calculated

The orbital overlap also accounts for the lower rate constant of the aza analog (Figure I- 13C, Table I-l(Entry 4)) than expected from the electron donating effect of a dialkylamino substituent, compared with oxy analog (Entry 5) Because of the steric interaction between phenol ring methyl! and amino substituent, nitrogen 2p-type orbital is

not effectively overlapped with phenoxyl radical orbital (Figure I-13C)

Table I-1.° Dihedral angle (@ and calculated BDEo.y and Kinn

* measured by x-ray crystallography

>» measured by inhibition of autoxidation of styrene at 30 °C

° caculated data from ref 1°

Side chain Modification

Side chain modification typically affects the solubility of the antioxidants A Cie isoprenoid chain of a-TOH makes it very lipid soluble and truncated side chain to methyl renders more water solubility to PMC Better water solubility is found in Trolox ® where

20

one of the methyl side chains of PMC is a carboxylic acid The same type of carboxylic acid function is found in the metabolite of y-TOH, y-CEHC (2,7,8-trimethyl-2-(p- carboxylethyl)-6-hydroxychroman) (Figure I-14(A)) Lipophilicity can be increased by incorporating the side chain of a-TOH based on the reports that the side chain plays an important role in its biological activity The a-TOH side chain has been coupled to a

polar antioxidant nucleus, such as a flavonoids, to make hybrid compounds A

representative example is a myricetin-tocopherol hybrid '! (Figure I-14B)

Figure 1-14 Example of side chain modification: modulation of solubility

Notable examples based on the structure-activity relationships described above are shown in Figure I-15 Naphthofuran analog (A)? showed almost 10 times better kinh (2.87 x 107 M Ìs}) than œ-TOH It was proposed that the naphthol moiety increases the stability of the aryloxyl radical than the core structure in tocopherols BO-653"? has a benzofuran skeleton coupled with di ¢-butyl substitution on the phenol ring 4 while IRFI

21

(Raxofelast®) features the dimethyl substituted benzofuran skeleton and acetic acid side chain It showed protective effect in oxidant stress mediated tissue injury in vivo 'S and is under clinical development (Figure I-15)

Figure 1-15 Synthetic antioxidants containing naphthofuran and benzofuran rings

Aminopyridinol structure

As mentioned, electron donating groups (EDGs) at the ortho and para position to the phenolic OH group decrease O-H BDE of the phenolic compounds ” Therefore, the primary strategy to increase antioxidant activity of phenolic compounds has been to incorporate EDGs However, this strategy has a drawback in that EDGs also decrease the ionization potential (IP) of the phenolic compound For example, compounds in Figure I-

16 (5,7,8-trimethyl-1,2,3,4-tetrahydroquinolin-6-ol (left) ? and 9-hydroxy-julolidine (right) '°) are better antioxidants than œ-TOH due to their low BDE from ør/ho and para EDGs, but they react directly with oxygen in the air * 18 because of a low IP The IP indicates the degree of ease with which a phenolic compound donates an electron from the aromatic ring

22