Rob Barker Part II Thesis

Synthesis of Fluorescent Inhibitors of the

Breast Cancer Biomarker hNAT1

Honour School of Chemistry 2014

Robert Barker

Keble College

Summary

i

Human arylamine N-acetyltransferase 1 (hNAT1) has been identified as a highly

overexpressed gene in estrogen-receptor-positive breast cancers,1 and hence hNAT1 is a

an attractive biomarker for tumour diagnosis. Naphthoquinone 1 is a selective inhibitor

of hNAT1 and its murine homologue, mNAT2, and exhibits a characteristic colour

change from red to blue in the presence of hNAT1/mNAT2 as the enzyme selectively

recognises the conjugate base [1]- (Fig. 1).

2 SAR studies on 1 resulted in more potent

and sensitive enzymatic inhibitors, but Δλmax in cell lysates remained unobservable.3 A

fluorescent mode of detection was therefore sought, and biaryl analogue 2 was

synthesised as the fluorescence properties of biaryls are widely reported.4

Figure 1: Left: Visible spectra of 1;2 Centre: Colour change mechanism; Right: Fluorescent biaryl 2.

Nine biaryl-substituted analogues based on 2 were initially synthesised, and seven were

potent mNAT2 inhibitors. All but one exhibited pH-dependent fluorescence (λex = 372,

408 nm; λem = 424 nm), which was observed at pH 8 but reversibly quenched in base

(Fig. 2A). Interestingly, 1 and other previously-synthesised inhibitors which lack a C3

anilino biaryl appendage displayed analogous fluorescence. This yields mechanistic

insight that deprotonation of the sulfonamide-NH quenches fluorescence attributable to

the naphthoquinone core, acting as a pH-dependent ‘on-off’ switch.

It was found that substitution at positions C5-C8 on the naphthoquinone core did not

increase fluorescence intensity significantly. Compounds with a methylene linker

between the C3 amine and the biaryl were thus synthesised, in an attempt to generate an

‘off-on’ probe based on photoelectron transfer (PET). para-Substituted 3 showed the

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Summary

ii

greatest potency against mNAT2 (Fig. 2B) and the characteristic pH-dependent

naphthoquinone fluorescence peak, but also a pH-insensitive peak (λex = 252 nm; λem =

344 nm) attributed to its biaryl moiety, yielding the first example of a ratiometric probe

in this study; however PET activity was not observed. Attention returned to a

conjugated scaffold and compounds 4 and 5 were synthesised. Fluorene 4 exhibited an

intense pH-dependent peak (λex = 279 nm; λem = 374 nm) which could be detected at

1 μM. This enabled a fluorescence titration against mNAT2, but unfortunately λex of 4

overlaps with that of tryptophan residues. Coumarin 5 possesses both high fluorescence

intensity and a λex orthogonal to tryptophan, but the coumarin peak is not sensitive to

sulfonamide deprotonation; however, modulation of its pKa might resolve this issue.

Figure 2A: Left: In silico modelling of 2 with hNAT1 (pdb: 2PQT); Right: Fluorescence spectra of 2.

Figure 2B: Left: Synthesised analogues 3, 4 and 5; Right: Fluorescence spectra of fluorene 4.

This study has therefore generated the first pH-sensitive fluorometric probes which are

potent mNAT2 inhibitors and elucidated the mechanism of fluorescence switching.

Optimisation lead to a five-fold increase in sensitivity, which has enabled detection of

these compounds at concentrations suitable for use in cell lysate studies.

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1. S. Tozlu et al., Endocrine-Related Cancer, 2006, 13, 1109-1120.

2. N. Laurieri J. E. Egleton, et al., PLoS ONE, 2013, 8, e70600.

3. J. E. Egleton et al. Bioorganic & Medicinal Chemistry, 2014, 22, 3030-3054.

4. J. W. Bridges et al., The Biochemical journal, 1965, 96, 872-878.

B

2

Acknowledgements

iii

Acknowledgements

Firstly, I’d like to thank Dr. Angie Russell for taking me on as a Part II this year: your

ability to give sound advice and encouragement to a soundtrack of ‘The Wheels on the

Bus go Round and Round’ and many other classics has been greatly appreciated.

My deepest and most sincere thanks to James Egleton (genuine appreciation managing

to break through the wall of Northern-ness there). Jimmy, Jeggles, my own personal

Pumba, time and again you have gone far beyond the call of duty to help in any way

you can. Your ridiculously large intellect, your radially extensive nose and your

willingness to listen to the (mostly daft) ideas I have has meant everything. You may be

second at everything else, but you’ll always be first to me.

Thanks of course go to Dr. Gu Lui Liu, my surrogate supervisor from day 1. Your

boundless depth of conversation topics (favourite colours, animals, ways to kill people

etc.) have kept me sane through the most arduous columns, as well as your sound

chemistry advice. Luthia, got radio? Get music. Thanks for putting up with me in the lab

all year, which has at times been an oasis of calm (although there always seems to be

some quacking sound…). I dearly hope that one day you will finally get to visit Spain,

and learn the language you seem to be so keen on getting to grips with. Thank you

Fernando for always being willing to help, and possessing an incredible gift for winding

Gu up (eh? eh? eh?).

Although I only ventured into G8 to annoy everyone with the sonicator, I thank you all

for making the group such a special place this year. Beth, I’m so grateful to have found

someone who loves cats even more than me, if only to prove that I’m not completely

mental. And thanks for actually going through with the baking thing you mentioned at

the start of the year – didn’t pan out that well for me. Joe, although your visit to G7 was

but brief, I’ll always remember your ability for conversations that could even rival Gu’s.

AVAR, thanks for bringing the winning attitude into the lab (we are STRONG) and Aini

for your questionable Finnish sweets (but far superior baking). Thanks to all the post-

docs, Carole, Noelia, Dave, Diana and Graham for all your wisdom and insights.

Thanks to Jason Sengel and the MIW group for putting up with my bumbling attempts

to clean cuvettes and questionable Spotify playlist.

Thanks to my parents for asking ‘how I’m getting on with my story’, and learning that

organic chemistry is ‘the one with the carbons and the pictures’. And Rachel, for not

only putting up with a year of stressing (and pretending to find 13

C assignments

interesting), but for making it so enjoyable. Thanks to all, for everything.

And InCl3. <3.

Abbreviations

iv

Abbreviations

Miscellaneous Terms

Ac Acetyl L Litre

app. Apparent lit. Literature

Aq. Aqueous LRMS Low Resolution Mass Spectrometry

Ar Aromatic LUMO Lowest Unoccupied Molecular Orbital

a.u. Arbitrary Units OD Optical Density

Boc tert-Butyl Carbamate M Molar

br. Broad m meta

C Celcius m Multiplet

cm-1

Wavenumber m/z Mass/Charge Ratio

conc. Concentrated Me Methyl

Chemical Shift min Minute

ΔECT Energy Difference in Charge

Transfer State mol Mole

ΔELE Energy Difference in Locally

Excited State mp Melting Point

Reagents, Solvents, Amino Acids, Biomolecules and NAT Enzymes

AcCoA Acetyl Coenzyme A hNAT1 Human NAT 1

7-AMC 7-Amino-4-Methylcoumarin hNAT2 Human NAT 2

Arg Arginine LB Luria-Bertani

Asp Aspartic Acid Leu Leucine

BlaC β-lactamase C MES 2-(N-Morpholino)ethanesulfonic Acid

Boc tert-Butyloxycarbonyl mNAT2 Mouse Nat 2

CAPS N-cyclohexyl-3-

Aminopropanesulfonic Acid NAT Arylamine N-acetyltransferase

Cys Cysteine Ni-NTA Nickel Nitrilotriacetic Acid

CHES N-cyclohexyl-2-

Aminoethanesulfonic Acid pABA para-Aminobenzoic Acid

CoA Coenzyme A Pet ether Petroleum Ether 30-40 ○C Fractions

3,5-

DMA 3,5-Dimethyaniline Phe Phenylalanine

DMF N,N-Dimethylformaldehyde RNA Ribonucleic Acid

DMSO Dimethylsulfoxide Ser Serine

DNA Deoxyribonucleic Acid shRNA Small Hairpin RNA

DTNB 5,5’-Dithio-bis(2-nitrobenzoic

acid) TFA Trifluoroacetic Acid

DTT Dithiothreitol THF Tetrahydrofuran

EDTA Ethylenediaminetetraacetic Acid TNB Thionitrobenzoate

Enz Enzyme Tris Tris(hydroxymethyl)aminomethane

Gly Glycine Trp Tryptophan

HEPES 4-(2-hydroxyethyl-1-

piperazineethanesuulfonic Acid Tyr Tyrosine

His Histidine XPhos 2-Dicyclohexylphosphino-2’,4’,6’-

triisopropylbiphenyl

IPTG Isopropyl β-D-1-

thiogalactopyranoside

Abbreviations

v

Δλmax Change in Wavelength of

Maximum Absorption MS Mass Spectroscopy

d Doublet ND Not Determined

dd Doublet of Doublets nm Nanometer

d.p. Decimal Point nM Nanomolar

dppf 1,1'-

Bis(diphenylphosphino)ferrocene NMR Nuclear Magnetic Resonance

dt Doublet of Triplets p para

ε Molar Absorption Coefficient pdb Protein Data Bank

εCB Molar Absorption Coefficient

(Conjugate Base) PET Photoinduced Electron Transfer

εN Molar Absorption Coefficient

(Neutral Species) pH -log10[H

+]

EDG Electron-Donating Group Ph Phenyl

Enz Enzyme Pin Pinacol

eq. Equivalents pKa -log10[Ka]

ER Estrogen Receptor pKaH pKa of the Conjugate Acid

ER+ Estrogen Receptor Positive ppm Parts per Million

ESI Electospray Ionisation q Quartet

Et Ethyl qRT-PCR Quantitative Real-Time Polymerase

Chain Reaction

EWG Electron-Withdrawing Group RCF Relative Centrifugal Force

FI Field Ionisation rpm Revolutions per Minute

[Fluor] Fluorophore Substituent RT Room Temperature

FRET Fluorescence Resonance Energy

Transfer s Singlet

FT Fourier Transform SAR Structure-Activity Relationship

g Gram sat. Saturated

GCT Gas Chromatography Time of

Flight

SDS-

PAGE

Sodium Dodecyl Sulfate-

Polyacrylamide Gel Electrophoresis

h Hours SERM Selective Estrogen Receptor

Modulator

HOMO Highest Occupied Molecular

Orbital t Triplet

HPLC High Performance Liquid

Chromatography tBu tert-Butyl

HRMS High Resolution Mass

Spectrometry td Triplet of Doublets

Hz Hertz TLC Thin Layer Chromatography

hν Photon Excitiation Energy TOF Time of Flight

IC50 Half Maximal Inhibitory

Concentration tt Triplet of Triplets

ICT Intramolecular Charge Transfer μM Micromolar

μw Microwave

IR

Infrared UK United Kingdom iPr iso-Propyl UV Ultraviolet

IR Infrared V Voltage; Volts

J Coupling Constant υmax Infrared Absorption

λem Wavelength of Emission vs. Versus

λex Wavelength of Excitation (v/v) Concentration by Volume

λmax Wavelength of Maximum

Absorption wrt With Respect To

Table of Contents

vi

Table of Contents

Summary ............................................................................................................................ i

Acknowledgements ........................................................................................................... iii

Abbreviations ................................................................................................................... iv

Table of Contents ............................................................................................................. vi

Chapter 1: Introduction ........................................................................................... 1

1.1. Project Overview ....................................................................................................1

1.2. Breast Cancer ..........................................................................................................1

1.3. Arylamine N-Acetyltransferases (NATs) ................................................................2

1.4. Development of an hNAT1-Specific Colorimetric Probe .......................................3

1.5. Fluorescence and its Use in the Detection of Analytes...........................................6

1.6. Aims of the project .................................................................................................8

Chapter 2: Synthesis of Fluorescent Probes ........................................................... 9

2.1. Introduction: Design of an Initial Fluorescent Inhibitor Library ............................9

2.2. Synthesis of the Initial Biaryl Library ..................................................................10

2.3. Pharmacological Evaluation of the Biaryl Library ...............................................14

2.4. Colorimetric Evaluation of the Biaryl Library .....................................................16

2.5. Fluorometric Evaluation of the Biaryl Library .....................................................16

2.6. Conclusions ..........................................................................................................20

Chapter 3: Optimisation of the Fluorophore ....................................................... 21

3.1. Fluorescence Studies on C5-C8 Substituted Species .............................................21

3.2. Synthesis of Compounds Containing a Methylene Linker ...................................22

3.3. Pharmacological and Optical Evaluation of the Linked Species 63-65 ...............24

Table of Contents

vii

3.4. Restoration of Conjugation: 2-Aminofluorene as the C3 Substituent ...................27

3.5. In Search of a New Fluorophore: Synthesis of a Coumarin Derivative ...............29

3.6. Pharmacological and Optical Evaluation of Coumarin 85 ...................................32

3.7. Conclusions and Further Work .............................................................................34

Chapter 4: Experimental ....................................................................................... 36

4.1. General Experimental ...........................................................................................36

4.2. General Synthetic Procedures ...............................................................................37

4.3. Preparation and Characterisation of Reported Compounds..................................38

Bibliography .................................................................................................................. 59

Appendix 1: Supplementary Experimental Data ...................................................... 61

Appendix 2: Protocols for Biological and Optical Evaluation .................................. 70

A 2.1. Attempted Expression and Purification of hNAT1 ...............................................70

A 2.2. Protocols for Compound Evaluation ....................................................................71

Appendix 3: Supplementary Pharmacological Data ................................................. 76

A3.1. pKa Curves of 12 ...................................................................................................76

A3.2. Representative IC50 Curves...................................................................................76

A3.3. Titration of 85 against mNAT2 .............................................................................77

Appendix 4: Summary of all Pharmacological and Spectroscopic Data ................. 78

Chapter 1: Introduction

1


1.1. Project Overview

The work presented in this thesis describes the synthesis, pharmacological and

spectroscopic evaluation of a family of molecules identified as inhibitors and potential

fluorescent probes for the putative estrogen-receptor-positive breast cancer biomarker

human arylamine N-acetyltransferase 1 (hNAT1).

1.2. Breast Cancer

Breast cancer is the second most prevalent form of cancer worldwide, with an estimated

1.67 million new cases diagnosed in 2012 corresponding to 25% of all cancers;1 early

diagnosis is known to be key to long-term survival.2 Approximately 70% of breast

tumours overexpress the estrogen receptor (ER),3 comprising the subtype of breast

cancers referred to as ER-positive (ER+). Oncogenesis in this subtype has been

hypothesised to take place via one of two mechanisms: either enhanced agonism of ERs

by estrogen results in a stimulation of mammary gland cell proliferation as well as an

increased risk of DNA replication errors; or genotoxic byproducts of estrogen

metabolism cause direct damage to DNA.4

Current therapies for ER+ breast cancers include aromatase inhibitors such as

Exemestane 2 which inhibits the formation of estradiol 3 thereby suppressing the

signalling pathway,5 and Selective Estrogen Receptor Modulators (SERMs) such as

Tamoxifen 4, whose downstream metabolites 5 and 6 act as estrogen receptor

antagonists (Figure 1.1).6 However, the use of these therapies is limited due to intrinsic

or acquired resistance.7, 8

Moreover, immunohistochemical staining for the detection of

ER+ tumours can be difficult to standardise and is often non-quantitative, limiting the

accuracy and rapidity of diagnoses.9 There is therefore a clear and present need for the


2

development of new therapeutic targets and diagnostic biomarkers for the diagnosis and

treatment of ER+ breast cancers, to improve long-term survival rates.

Figure 1.1: Current therapies for ER+ breast cancer – Aromatase inhibitor 2 arrests biosynthesis of

Estradiol 3, and Tamoxifen metabolites 5 and 6 antagonise ERs.

Human arylamine N-acetyltransferase 1 (hNAT1) has been identified as one of the 10

most highly overexpressed genes in ER+ breast cancers through proteomic and

microarray studies.10

Furthermore, an inverse correlation between overexpression and

tumour grade has been revealed,11

indicating hNAT1 could be an attractive surrogate

biomarker for the diagnosis and prognosis of ER+ tumours.

1.3. Arylamine N-Acetyltransferases (NATs)

NATs are a family of xenobiotic metabolising enzymes found in a variety of both

eukaryotic and prokaryotic organisms that catalyse the transfer of an acetyl group from

acetyl coenzyme A (AcCoA) to xenobiotic substrates, including arylamines,

alkylarylamines, hydrazines and arylhydroxylamines.12

In doing so, these enzymes

facilitate drug metabolism, detoxification, and, paradoxically, carcinogenesis (Figure

1.2). Crystallographic studies have implicated a catalytic triad comprising Cys68,

His107 and Asp122 in the mediation of acetyl transfer,13

and kinetic data is consistent

with a Ping-Pong Bi-Bi mechanism.14


3

Figure 1.2: The effects of hNATs on metabolism of arylamines in vivo – N-acetylation leads to

detoxification through excretion, whereas O-acetylation allows the formation of carcinogenic species.

There are two functional human NAT isoforms, hNAT1 and hNAT2. Despite possessing

a sequence homology of ~80%,15

these proteins have differing substrate specificity

profiles,11

endogenous roles, and tissue distribution.16

hNAT2 is abundant in the liver

and intestines, and has been implicated in phase II drug metabolism (having been shown

to N-acetylate drug-like substrates such as isoniazid),17

whereas the function of hNAT1

is less well defined. hNAT1 has a widespread tissue distribution,16

and may play a role

in growth, development and folate catabolism;18

recent studies also reveal it can act as

an AcCoA hydrolase in the presence of folate.19

Inhibition of hNAT1 has been

suggested as a therapeutic approach to targeting ER+ breast cancer, as an shRNA

knockdown of hNAT1 in the breast cancer cell line MDA-MB-231 resulted in a

significant reduction in the proliferative and invasive capacity of the cells.20

1.4. Development of an hNAT1-Specific Colorimetric Probe

Many of the first inhibitors of hNAT1 which were identified were either non-specific

(e.g. Tamoxifen 4, the anti-cancer properties of which have been attributed to

interactions with other proteins)21

or were irreversible modulators of enzymatic activity

(e.g. cisplatin,22

and small molecule nitrosoarene or N-arylhydroxylamine compounds,23

which form a covalent adduct with the catalytic Cys68 residue).22


4

A medium-throughput screen and subsequent optimisation studies identified rhodanine

7 and naphthoquinone 8 as competitive, reversible inhibitors of hNAT1 (and its murine

homologue, mNAT2, which shares 82% sequence identity, homology in substrate

specificity, and tissue distribution with hNAT1)24

(Figure 1.3); these were shown to be

selective inhibitors for hNAT1 and mNAT2 over a range of other eukaryotic and

prokaryotic NATs and additionally inhibited NAT activity in cell extracts from the

breast cancer cell line ZR-75-1, which overexpresses hNAT1.25

Rhodanine 7 was also

shown to reduce cell proliferation in the MDA-MB-231 cell line, but only at

significantly higher concentrations than its IC50 against recombinant hNAT1;20

this

result may therefore be also due in part to off-target effects as the promiscuity of

rhodanine substrates in biological systems is well documented.26, 27

Figure 1.3: Small molecules identified as specific hNAT1 inhibitors: rhodanine 7 and naphthoquinone 8.

The latter exhibited a characteristic colour change in the presence of hNAT1 and mNAT2, hypothesised to

occur via sequestration of the conjugate base [8]-.

Interestingly, in the presence of hNAT1 or mNAT2, naphthoquinone 8 was observed to

undergo a distinctive colour change from red (λmax = 498 nm) to blue (λmax = 625 nm for

hNAT1).28

A similar bathochromic shift was observed in 4 M NaOH solution

(λmax = 561 nm) but not in the presence of any other NAT tested, suggesting that the

colour change could be due to deprotonation of the acidic sulfonamide proton in 8.

Titration of 8 revealed a pKa of 9.2, which is greater than the assay pH of 8.0, and so it

was hypothesised that the colour change was mediated by selective recognition of the

conjugate base of naphthoquinone 8 by the enzyme (Figure 1.3).28


5

In silico modelling studies implicated the Arg127 residue of hNAT1 in this

phenomenon, and further chemical and biochemical experiments have shown that the

presence of both the acidic sulfonamide proton and the Arg127 residue are essential for

the colour change event to occur (Figure 1.4).29

This naphthoquinone could therefore

act as a pH-dependent colorimetric probe for hNAT1, which has a potential use in vitro

for the quantitative detection of native hNAT1 in biopsy cell lysates, thereby removing

the reliance upon current methods of detection which are either non-quantitative30

or

require the tagging of proteins:31

such probes could potentially be used for diagnosis

and prognosis of some ER+ breast cancers.

Figure 1.4: Left: Treatment of 8 with aq. NaOH or mNAT2 afforded a colour change, but use of mNAT2

mutants lacking Arg127 did not. Right: 9, a methylated analogue of 8, was found to inhibit hNAT1, but

without a concomitant spectral shift.29

Over 100 analogues of naphthoquinone 8 have been synthesised in

Structure-Activity-Relationship (SAR) studies in an effort to increase both the potency

and the absorption coefficient of the conjugate base, εCB, at λmax (Figure 1.5).32

These

studies have generated compounds such as 10, which has a forty-fold increase in

potency and 1.5-fold increase in εCB over 8, and 11, which has a ten-fold increase in

potency and two-fold increase in εCB over 8.32

However, upon testing compounds 10 and 11 against hNAT1 in cell extracts from the

ZR-75-1 breast cancer cell line, the characteristic colour change could not be observed

over background noise at the low concentrations required for the study ([hNAT1] in the

IC50 (hNAT1) = 5.8 µM λmax (pH 8) = 486 nm

λmax (hNAT1) = 486 nm

IC50 (hNAT1) = 4.1 µM

λmax (pH 8) = 498 nm

λmax (hNAT1) = 625 nm

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ZR-75-1 lysate is reported to be 0.6-1.2 μM).32

These colorimetric probes thus appear

not to exhibit the intrinsic level of sensitivity required for reliable detection of hNAT1

in vitro, and so a different approach to improving sensitivity must be undertaken.

Figure 1.5: SAR studies to date have yielded 10 and 11, featuring improved potency and εCB over 8

1.5. Fluorescence and its Use in the Detection of Analytes

It has long been known that fluorescent detection techniques can be orders of magnitude

more sensitive than their colorimetric counterparts33

due to less background noise at key

wavelengths, which in some instances have even allowed single-molecule detection.34

As such, many colorimetric assay techniques have been superseded by fluorescent

systems35, 36

and are readily commercially available.37

Whilst it had been common in the

past to use fluorescent probes in vitro by utilising the accumulation of ‘always on’

fluorescent compounds at a site of interest,38

more modern approaches to fluorescent

sensing rely on activation rather than simply accumulation,39

allowing the potential for

an ‘off-on’ fluorescent switch to indicate the presence of an analyte. Today, there are

several design strategies for fluorescent probes, including fluorescence resonance

energy transfer (FRET),40

photoinduced electron transfer (PET),41

intramolecular charge

transfer (ICT)42

and spirocylization.43

An example of a pH-dependent PET fluorescent

probe has been recently reported:44

as the reported activity of the existing colorimetric

probes for hNAT1 is based upon a deprotonation event, this model of fluorescent probe

design could be a useful basis for this study. However, as one could argue that the


7

naphthoquinone core and its aromatic C3 substituent comprise a single conjugation

system, the ICT model may also prove applicable (Figure 1.6).

Figure 1.6: Top Left: Schematic of how an electronically excited state can decay, either radiatively or

non-radiatively. Top Right: Schematic of ICT fluorescence. Bottom Left: Schematic of PET

fluorescence. Bottom Right: Fluorescent hNAT1 inhibitor 12.

PET is the process by which an electron moves from one excited donor site to another

acceptor site within the molecule. These sites must be within a close proximity as the

wavefunctions of initially excited and product states must overlap. In these systems,

usually only one of the states is light emissive (reactant or product), and the electron

transfer leads either to a switch ‘on’ or switch ‘off’ of fluorescence, without any spectral

shift.45

ICT is, in principle, also an electron transfer, although as the process occurs

within the same electronic system, a charge-polarized state is yielded, rather than a

charge-separated one. In this case, some quenching may be observed but with a change

of intensities.33

There is also a concomitant spectral shift, as ∆𝐸𝐿𝐸 (energy difference in

locally excited state) differs from ∆𝐸𝐶𝑇 (energy difference in charge transfer state).33

This shift is also dependent on the environment: in polar solvents Stokes shifts may


8

become larger, due to an increased solute-solvent dipole interaction.46

Biosensors that

employ either of these techniques are often ratiometric in design: that is, there is more

than one fluorescence peak, and binding to the analyte provokes a different response

from each of these peaks.47

This orthogonal approach allows accurate analysis within

biological systems, removing issues with dilution and application.

Fluorescent studies on some of the biphenyl derivatives used as C3 appendages in the

ongoing SAR studies of 8 were reported nearly 50 years ago, and they were shown to be

mildly fluorescent.48

It was hypothesised that the inclusion of these biaryl moieties

might create a fluorescent analogue of 8, and indeed naphthoquinone 12 was found to

fluoresce under UV light. Having identified an example of an inhibitor that displays

fluorescent behaviour, there is a need to identify what features lend the compound its

fluorescence, and to observe its behaviour upon binding with the target protein.

1.6. Aims of the project

The present overall aims of the project are to synthesise further analogues of

naphthoquinone inhibitors based on the biaryl-substituted 12, which display both

increased potency against hNAT1/mNAT2, and exhibition of a pH-dependent change in

fluorescent output which is sensitive enough to enable detection of such a change in the

presence of recombinant hNAT1/mNAT2. In order to realise these aims, a variety of

biaryl groups and other small fluorophores will be appended to the naphthoquinone core

at the C3 position, and the fluorescent behaviour of these compounds upon

deprotonation will be analysed.

Figure 1.7: Evolution of hit

compound 8 to a potential

fluorometric probe

Chapter 2: Synthesis of Fluorescent Probes

9


2.1. Introduction: Design of an Initial Fluorescent Inhibitor Library

SAR studies have previously been carried out at the C2, C3 and C5-C8 positions of the

naphthoquinone core of hit compound 8, and whilst some amount of variation is

tolerated at positions C2 and C5-C8, it is at C3 where the greatest range of substituents

are permitted (Figure 1.5);32

docking studies suggest that radially extensive C3 groups

project out of the enzyme active site into the bulk solvent (Figure 2.1).49

Recently, in an

attempt to enhance εCB such that the characteristic colour change of compounds such as

8 could be observed at lower concentrations of inhibitor, a series of biaryl anilino

substituents were installed at the C3 position in order to create an extended

chromophore. Whilst this did have the desired effect of increasing absorption, the

colorimetric mode of detection was nonetheless found to be insufficiently sensitive for

in vitro detection at the required concentration of sub-1 μM.49

Figure 2.1: In silico modelling with hNAT1

(pdb: 2PQT).50

Top Left: First docking

studies of p-substitution of phenyl ring (13).49

Bottom Left, Centre: Docking simulations

of biaryl inhibitor 12 suggest a similar mode

of binding, showing extension from active

site and interaction with Arg127 and Tyr129;

Bottom Right: Biaryl precursor to 12.

The concept of applying fluorescent sensing to this probe was therefore considered, and

fortuitously an amino-substituted biaryl precursor 14 to inhibitor 12 was found to


10

fluoresce under long wave UV lamps (Figure 2.1). Fluorescence spectra of the adduct

12 showed that this species also exhibited weak fluorescence,51

and docking predicts 12

could be tolerated in the hNAT1 active site. This therefore presented a starting point for

investigating the potential of a fluorescent inhibitor to be a probe for hNAT1. However,

it is unknown as to how substitution on the biphenyl system affects the intensity of

fluorescence, or the wavelengths of excitation (λex) and emission (λem). Furthermore, it

is unknown how a change in pH or the presence of hNAT1 might affect the fluorescent

properties of such molecules. Therefore, a library was devised that might shed some

light on the relationship between substitution on these aromatic rings, molecular

fluorescence, and behaviour in the presence of hNAT1/mNAT2 (Table 2.1).

Table 2.1: Library of naphthoquinone derivatives to be synthesised, featuring the above C3 substituents.

Two main investigations were envisaged: firstly, 12 contains both an electron-donating

group (EDG) and an electron-withdrawing group (EWG) on its biaryl moiety.

Substitution of the methoxy group at differing positions on the biaryl should enable

elucidation of how the regiochemistry of nitro vs. methoxy affects molecular

fluorescence (compounds 15, 16 and 17). Secondly, compounds were devised which

bore either only one EWG or EDG on the biaryl system (compounds 18 and 19

respectively), or alternatively two of each such substituent (compounds 20 and 21).

2.2. Synthesis of the Initial Biaryl Library

A convergent synthetic procedure was devised in order to maximise efficiency from a

selection of appropriately-substituted aromatic starting materials (Scheme 2.1).

Compound

number 12 15 16 17 18 19 20 21

C3

Substituent


11

Scheme 2.1: Synthesis of inhibitors containing a C3 biaryl substituent. Reagents and conditions: (i)

NH2SO2Ph (1.0 eq.), Cs2CO3 (1.4 eq.), DMF, RT, 5 h; (ii) Naphthoquinone 23 (1.0 eq.), CeCl3∙7H2O

(1.0 eq.), MeOH, RT, 1.5 h, then add requisite aniline (3.0 eq.), 90 °C, 16 h; (iii) 70% HNO3:conc. H2SO4

(5:4), 50 °C, 16 h; (iv) Boc2O (1.2 eq.), NaOH (2.2 eq.), THF:H2O (1:1), 60 °C, 16 h; (v) Boc2O (1.0 eq.),

InCl3 (0.01 eq.), 35 °C, 45 min; (vi) B2Pin2 (1.5 eq.), KOAc (3.0 eq.), Pd(PPh3)2Cl2 (0.05 eq.), 1,4-

dioxane, 120 °C, 3 h; (vii) Arylbromide 28 (0.5 eq.), Pd(PPh3)2Cl2 (0.05 eq.), THF:sat. aq. NaHCO3

(22:5), 100 ºC, 16 h (yields shown wrt arylbromide 28); (viii) TFA:CH2Cl2 (1:5), RT, 2 h; (ix) 10% Pd/C

(0.1 eq.), H2 (1 atm), MeOH, RT, 16 h.

Authentic samples of naphthoquinones 8, 12, 15 and 20 were synthesised via the above

route (Scheme 2.1). Firstly, hit compound 8 was synthesised from dichlone 22 via two

sequential conjugate addition-elimination reactions: firstly with benzenesulfonamide,

and secondly with 3,5-dimethylaniline. The biaryl derivatives were synthesised from

aryl bromides 26 and 29. While 2-methoxy-4-nitrobromobenzene 29 was commercially

available, the regioisomer was not, so a sample of 26 was accessed via a nitration

reaction on 3-methoxybromobenzene 24,52

although the isolated yield was modest

(35%) due to the production of both regioisomers 25 and 26 (in a 1:1 ratio as

determined by 1H NMR analysis of the reaction mixture) which proved challenging to

separate. Aryl bromides 29 and 26 were converted to the corresponding boronic esters

30 and 31 via a palladium-catalysed cross-coupling reaction with B2Pin2.

Meanwhile, 4-bromoaniline 27 was N-protected with Boc2O to yield the required

carbamate 28. Under basic conditions (iii), this reaction was inefficient and gave a poor


12

isolated yield (34%). After trialling an alternative literature protocol utilising catalytic

InCl3 (conditions (iv)),53

a much more efficient protection method was identified which

did not require any organic solvent, only required heating to 35 °C, and gave near-

quantitative yields without the need for any further purification.

Boronic esters 30 and 31 then underwent Suzuki coupling54

with this carbamate 28 to

yield biphenyl products 32 and 33. These were subsequently subjected to N-

deprotection under mild conditions in TFA:CH2Cl2 (1:5) to afford anilines 14 and 34 in

good yield. The anilines were installed at the C3 position of the benzenesulfonamide

intermediate 23 via a conjugate addition-elimination reaction, yielding naphthoquinones

12 and 15. Finally, the 8″-OMe substituted analogue 12 underwent catalytic

hydrogenation to afford the reduced amino-naphthoquinone 20 in a quantitative yield.

Scheme 2.2: Comparison of strategies to synthesise protected biaryl amino 37. Reagents and conditions:

(i) B2Pin2 (1.5 eq.), KOAc (3.0 eq.), Pd(PPh3)2Cl2 (0.05 eq.), 1,4-dioxane, 120 °C, 3 h; (ii) Requisite

arylbromide (0.5 eq.) Pd(PPh3)2Cl2 (0.05 eq.), THF:sat. aq. NaHCO3 (22:5), 100 ºC, 16 h (yield shown

wrt arylbromide); (iii) B2Pin2 (1.05 eq.), KOAc (3.00 eq.), PdOAc (0.03 eq.), DMF, 80 °C, 3 h; then

Cs2CO3 (1.5 eq.), aryl bromide 28 (0.5 eq.), Pd(PPh3)4 (0.01 eq.), 80 °C, 16 h.

Attempts to furnish nitro-substituted biaryl 18 proved more troublesome. After

successful synthesis of the requisite boronic ester 36 (Scheme 2.2), the Suzuki coupling

with carbamate 28 gave poor conversion to 37 (10% as determined by 1H NMR of the

reaction mixture). It was hypothesised that this lack of reactivity was due to the

electron-deficient nature of the para-nitro-substituted phenyl boronic ester 36,

prohibiting effective transmetallation with the intermediate Pd(II) complex. A literature

protocol was therefore trialled in which the Suzuki coupling55

was carried out after the


13

formation of boronic ester in situ, to minimise loss of yield through work-up and

purification. However, the resultant reaction mixture could not be purified via column

chromatography. Therefore, it was decided to switch the bromo and boronic ester

coupling partners: the boronic ester moiety was installed at the para-bromo position of

tert-butyl carbamate 28, thereby generating a more electron-rich boronic ester to react

with the electron deficient para-nitro-bromobenzene 35 to give biaryl 37 in 56% yield.

This strategy exploits the inherent preference for electron-poor halides to undergo

efficient oxidative addition and electron-rich boronic esters to undergo efficient

transmetallation,54

and was used for the synthesis of all biaryls which contained a more

electron-poor nitro-substituted ring (Scheme 2.3; Table 2.1; steps (iv), (v) and (vi) are

analogous to those in the original Scheme 2.1).

Scheme 2.3: Revised general synthetic procedure of inhibitors containing a C3 biaryl substituent.

Reagents and conditions: (i) Boc2O (1.0 eq.), InCl3 (0.01 eq.), 35 °C, 45 min; (ii) B2Pin2 (1.5 eq.), KOAc

(3.0 eq.), Pd(PPh3)2Cl2 (0.05 eq.), 1,4-dioxane, 120 °C, 3 h; (iii) Requisite arylbromide (0.5 eq.),

Pd(PPh3)2Cl2 (0.01 eq.), THF:sat. aq. NaHCO3 (22:5), 100 ºC, 16 h; (yields shown wrt arylbromide) (iv)

TFA:CH2Cl2 (1:5), RT, 2 h; (v) Naphthoquinone 23, CeCl3∙7H2O (1.0 eq.), MeOH, RT, 1.5 h, then add

requisite aniline (3.0 eq.), 90 °C, 16 h; (vi) 10% Pd/C (0.1 eq.), H2 (1 atm), MeOH, RT, 16 h; (vii) 1 M

aq. NaOH (5 eq.), MeOH, 60 °C, 48 h. Yields for steps (i)-(v) are presented in Table 2.2.


14

Table 2.2: Yields for steps (i)-(v) shown in Scheme 2.3.

To synthesise a final product bearing two EWGs on the biaryl system, the para-nitro

and ortho-ester groups were selected (compound 21). Ester 21 also provided an

opportunity for facile synthesis of carboxylic acid 52 via an ester hydrolysis (conditions

(vii) in Scheme 2.3): carboxylic acid derivatives of hit compound 8 were shown to be

potent inhibitors in previous SAR investigations,49

so 52 was synthesised to investigate

whether this functionality confers the same effect on a biaryl analogue.

2.3. Pharmacological Evaluation of the Biaryl Library

Synthesised inhibitors were initially evaluated in the “DNTB” assay for their activity

against mNAT2 (expressed and purified by J. Egleton)56

at a final concentration of

30 μM. Attempts to express and purify recombinant hNAT1 from E. coli proved

challenging (protocol outlined in Appendix 2.1); however, this enzyme is notoriously

difficult to express and has lower stability than its murine homologue which has been

shown to be a reliable in vitro model of hNAT1. NAT activity was determined via an

end-point assay which measured the rate of AcCoA hydrolysis (for protocol see

Appendix 2.2.1). Probes which showed inhibition greater than 50% at 30 μM were

carried forward into dose-response assays in order to ascertain IC50 values (Table 2.3).

By comparison of the singly-substituted 18 and 19, it can be seen that conversion of an

EWG to an EDG leads to an increase in potency, whilst 20 indicates that multiple EDG

substitution does not give a cumulative increase in potency. Comparison of 12, 15, 16

and 17 highlights little variation through varying methoxy regioisomers with the

exception of 16 which is a poor inhibitor. The carboxylic acid functionality in 52

Compound

Number R1 R2

Yield for step

(i) (ii) (iii) (iv) (v)

18 All H All H 89% 78% 56% 86% 23%

17 2″-OMe All H 96% 72% 73% 73% 86%

16 3″-OMe All H 88% 25% 88% 88% 10%

21 All H 8″-CO2Me 89% 78% 77% 77% 88%


15

meanwhile confers a high inhibitory potency against mNAT2. It is hypothesised that the

acid, which will be ionised at the assay pH of 8, could lead to increased solvation – the

analogous ester 21, whilst structurally similar but incapable of carboxylate formation, is

a poor mNAT2 inhibitor. A possible electrostatic interaction between the carboxylate

and Ser214 has also been implicated (Figure 2.2). Other carboxylic acid derivatives of

hit compound 8 also demonstrate high potency, so this result is consistent with previous

SAR studies.49

Compound 8 12 15 16 17

C3 substituent

IC50 (mNAT2) (μM) 4.9 ± 2.9 2.8 ± 1.1 2.0 ± 1.2 27.9 ± 15.6 1.0 ± 1.5

Compound 18 19 20 21 52

C3 substituent

IC50 (mNAT2) (μM) 2.7 ± 1.3 0.64 ± 0.16 1.6 ± 0.7 >30 0.51 ± 0.12

Table 2.3: IC50 values against mNAT2, quoted ± one standard deviation.

Figure 2.2: Left: In silico modelling showing interactions between carboxylic acid 52 and Ser214 in

hNAT1 (pdb:2PQT);50

Centre: A surface representation of the interaction; Right: Potent inhibitor 52.


16

2.4. Colorimetric Evaluation of the Biaryl Library

In order to allow direct comparison of this library to previous SAR studies, the

following colorimetric properties of each inhibitor were measured: εN (absorbance

coefficient of neutral species), εCB (absorbance coefficient of its conjugate base), pKa,

λmax, Δλmax in basic conditions and Δλmax upon enzymatic binding. pKa values were

generated via a titration experiment using a spectrophotometer to detect a shift in λmax

(for protocol see Appendix 2.2.3), and are quoted ± one standard deviation.

Compound 8 12 15 16 17 18 19 20 21 52

pKa 9.2 ±

0.2

11.4 ±

0.5

10.5 ±

0.3

9.5 ±

0.2

10.7 ±

0.3

9.9 ±

0.3

8.9 ±

0.4

9.2 ±

0.4

7.7 ±

0.3

8.1 ±

0.3

λmax pH 8 (nm) 498 518 513 504 528 484 528 529 529 484

Δλmax pH 13 (nm) +79 +47 +53 -41 +38 +64 +44 +54 +13 +78

Δλmax mNAT2 (nm) +112 +45 +86 ND +40 +112 +106 +104 +93 +148

Table 2.4: A summary of colorimetric properties of the initial biaryl-substituted library.

The pKa values measured (Table 2.4) are consistent with previous C3 amino-substituted

species,32

and show that they each possess a suitable pKa for use in enzyme assays; that

is, higher than the assay pH, but lower than the pKaH of the Arg127 residue to allow

formation of the conjugate base in the presence of the enzyme. εN and εCB values (see

Appendix 4) are coherent with previous biaryls – they can be up to two-fold greater

than the values for hit compound 8 (εN = 9344 M-1

cm-1

, εCB = 4462 M-1

cm-1

), but still

not high enough to be useful as in vitro probes.49

Interestingly, the carboxylic acid 52

has a relatively low εN (4537 M-1

cm-1

) and εCB (2532 M-1

cm-1

) values, as observed

with other carboxylic acid derivatives of 8.49

Crucially, the observation of a shift in

Δλmax in the presence of mNAT2 indicates a similar binding mode of these compounds

with the enzyme to that of previous analogues tested.

2.5. Fluorometric Evaluation of the Biaryl Library

When the concept of a fluorescent probe for hNAT1 was first conceived, it was based

on the assumption that the inclusion of an electron-poor naphthoquinone and electron-

rich biaryl together would form the basis of an inhibitor species that could undergo


17

fluorescence switching via photoinduced electron transfer (PET), as described in Figure

2.3. This would result in an efficient ‘off-on’ fluorescent sensor for hNAT1, with a

potentially much higher degree of sensitivity than its colorimetric counterparts.

Figure 2.3: Proposed mechanism for PET quenching and fluorescence of hNAT1/mNAT2 inhibitors.

Hit compound 8 and the synthesised biaryl library presented here underwent

fluorescence evaluation as 5% DMSO solutions in pH8 Tris.HCl buffer – the same

conditions as those used for the present enzymatic and colorimetric assays. Initial scans

utilised relatively high final concentrations of 150 µM in each test run, in an attempt to

guarantee no fluorescent behaviour was overlooked due to an inability to resolve low

intensity fluorescence (see protocol outlined in Appendix 2.2.5).

Upon systematically exciting the sample of inhibitor 12 at discrete wavelengths

(measurements were taken at 5 nm increments, beginning at λem = 200 nm), an emission

peak under pH 8 conditions was identified at λem = 424 nm, which corresponds to

excitation peaks of λex = 372 nm or λex = 408 nm (Figure 2.4). Intriguingly, whilst this

result could be reproduced under acidic conditions, no such peak could be observed

under basic conditions. Upon reacidification of the basic solution by addition of 5 µL of

1 M aq. HCl to return the solution pH to ~8, the emission peak was once again detected.


18

This result gave the tantalising indication of a pH-dependent ‘on-off’ fluorescent

switch, in contrast to the ‘off-on’ sensor predicted by the initial hypothesis of PET

transfer within the molecule.

Figure 2.4: Left: Blank-corrected excitation and emission fluorescence spectrum of 12 measured at

λex = 408 nm and λem = 424 nm, at pH 8, under basic conditions, and reacidification to pH ~8, showing

return to near-original fluorescence intensity. Right: Structure of 12.

Table 2.5: A comparison of fluorescence intensities observed at pH 8 and λex = 408 nm (sensitivity

voltage, V = 800 V, a.u. = arbitrary units).

The whole library was screened in a similar fashion, taking readings at pH 8, and with

both acid and base. In each case, the peak at 424 nm was identified at varying levels of

intensity at acidic and neutral pHs, but not under basic conditions. Table 2.5 displays

the relative intensities observed with each compound at pH 8.

Compound 8 12 15 16 17

C3 substituent

Fluorescence intensity at pH 8

(λem = 424 nm) (a.u.) 61 126 67 8 75

Compound 18 19 20 21 52

C3 substituent

Fluorescence intensity at pH 8

(λem = 424 nm) (a.u.) 27 26 61 16 0

0

20

40

60

80

100

120

140

350 370 390 410 430 450 470

inte

nsi

ty (

arb

itrary u

nit

s)

Wavelength (nm)

pH 8

Basified

Reacidified

Excitation Emission


19

It appears that the most intense fluorescence is observed when both an EDG and EWG

are present on the biaryl system – the initially synthesised compound 12 fluoresces the

most strongly, followed by 15 and 17, each containing one nitro and one methoxy

substituent. Conversely, carboxylic acid 52 does not display any fluorescence at all,

potentially linked to its apparent intrinsically low εN and εCB values (vide supra).

However, perhaps the most curious result is that of 8: an analogue lacking the biaryl

moiety appears to fluoresce in the same manner.

Compound 23 53 54 55

Structure

Fluorescence

Intensity

(a.u.)

acid 50 108 17 68

pH 8 0 0 15 32

base 0 0 0 0

pKa 3.0 5.0 9.0 8.3

Table 2.6: Fluorescent data of representative compounds lacking biaryls previously synthesised in SAR

studies (sensitivity voltage V = 800 V, a.u. = arbitrary units).

Further compounds from previous SAR libraries32

which do not contain a biaryl system

were therefore tested under identical conditions, and each was found to feature this

distinctive fluorescent peak (Table 2.6), casting doubt on whether the C3 substituent is

responsible for this fluorescent activity. Naphthoquinones have been reported in the

literature to fluoresce,57-59

and benzenesulfonamide intermediate 23 was also found to

fluoresce under acidic conditions, but not at pH 8 or in base (pKa of 23 ~3.0). These

data are consistent with the fluorescence activity arising due to excitation/emission of

the naphthoquinone core, which is common to all species tested. It appears that

increasing electron density on the naphthoquinone core results in a quenching of

fluorescence, which could explain why a variety of fluorescent intensities are observed

all at the same λex and λem.


20

To provide further evidence that the absolute fluorescence quenching was due to a

deprotonation of the acidic sulfonamide, a titration experiment of 12 was run against

fluorescence intensity, in an analogous experiment to the colorimetric protocol using

Δλmax. The pKa value generated (10.5 ± 0.8) was roughly consistent with the pKa

obtained via the colorimetric method (11.4 ± 0.4) (titration curves in Appendix 3.1).

2.6. Conclusions

Synthesis of nine naphthoquinones containing a substituted biphenyl group at the C3

position demonstrated that seven of these are relatively potent inhibitors of mNAT2.

Upon investigating their fluorescence properties, all but one displayed a low intensity

fluorescence at λex = 372, 408 nm and λem = 424 nm which was quenched in base,

including hit compound 8. It was hypothesised that this fluorescence quenching was

due to a deprotonation event, analogous to the distinctive colour change phenomenon

previously reported for this family of compounds. Further studies suggested that the

core naphthoquinone feature lent each compound its characteristic fluorescence, rather

than the C3 substituent.

Despite identification of pH-dependent fluorescent probes which are potent inhibitors of

mNAT2, the sensitivity of the probe is still not nearly great enough to allow quantitative

detection of mNAT2 at the concentrations required for an in vitro assay.49

Optimisation

of the fluorescence activity is therefore required, and this could be achieved in one of

several ways. Firstly, by introducing a methylene linker between the biaryl moiety and

the naphthoquinone core, a separation of the two electronic systems could lead to a

more classical PET fluorescent probe, which may yield a more sensitive biosensor.

Alongside this, the effects of direct substitution of the naphthoquinone at positions

C5-C8 should be investigated, in order to establish whether altering the electron density

on the naphthoquinone itself could give an enhancement of fluorescence.

Chapter 3: Optimisation of the Fluorophore

21


3.1. Fluorescence Studies on C5-C8 Substituted Species

Evidence thus far suggests that the pH-dependent peak observed in the fluorescence

spectra of the synthesised inhibitor library (Figure 2.4) can be attributed to the

naphthoquinone core, and the quenching of this fluorescence was postulated to be due

to an increase in electron density upon deprotonation of the sulfonamide-NH. A library

of naphthoquinones bearing -NO2 and -NH2 groups at the C5-C8 positions had been

previously synthesised,32

enabling the study of whether substitution at these positions

had an impact on fluorescence intensity, and if certain groups could enhance fluorescent

activity sufficiently for in vitro studies (< 1 μM detection)49

(Figure 3.1).

Figure 3.1: Top Left: General structure of 11, 56-62; Top Right: Fluorescence intensities of C5-C8

substituted naphthoquinones (V = 800 V); Bottom: Representative emission spectrum of 62, 11 and 8

under acidic conditions. aPreviously determined by colorimetry;

32 bND = not determined.

Fluorescence spectra were recorded at λex = 408 nm and λem = 424 nm in acidic, neutral

and basic conditions. As expected, fluorescence quenching was observed in base for all

Compound

number R= pKa

a

Fluorescence Intensity (a.u.)

Acidic

conditions pH 8

Basic

conditions

56 5-NO2 8.2 99 77 0

57 5-NH2 NDb

47 47 0

58 6-NO2 7.5 43 35 0

59 6-NH2 NDb

21 32 0

60 7-NO2 8.0 115 106 0

61 7-NH2 NDb

44 49 0

62 8-NO2 8.6 94 85 0

11 8-NH2 8.4 78 86 0

0

20

40

60

80

100

120

418 423 428 433 438 443 448

Inte

nsi

ty (

arb

itra

ry

un

its)

Wavelength (nm)

62

11

8


22

compounds. For substitution at each position, there was a positive correlation between

how electron-withdrawing the substituent (H, NH2 or NO2) is, and the fluorescence

intensity of the species. However, as the greatest intensity observed was only 115 a.u. at

800 V, C5-C8 substitution does not appear to yield a sufficiently sensitive inhibitor for

use in enzymatic studies. Focus therefore turned towards the synthesis of a classical

PET probe, by introduction of a methylene linker between the naphthoquinone core and

a biaryl C3 substituent.

3.2. Synthesis of Compounds Containing a Methylene Linker

PET fluorescence has been successfully used in biological systems for the detection of

various analytes.41

In order to create a PET probe, two separate electronic systems must

be in close spatial proximity to allow transfer of electrons from an excited state to a

quencher state (Figure 1.6). In the first library of inhibitors synthesised in this study

(vide supra), the biaryl and naphthoquinone conjugate systems are connected via the

lone pair on the C3 nitrogen. An amino substituent is required at the C3 position in order

for the inhibitor to have an appropriate pKa; therefore, it was hypothesised that the

introduction of a methylene linker between the C3 nitrogen and the biaryl should

conserve a desirable pKa but prohibit inter-system conjugation between the biaryl and

naphthoquinone moieties. Ortho- (63), meta- (64) and para-substituted (65) biaryls

were all conceived as desirable targets to test this hypothesis, as the regiochemistry for

optimal enzymatic activity is unknown (Figure 3.2).

Figure 3.2: Proposed targets featuring a methylene linker between the naphthoquinone and biaryl.


23

Scheme 3.1: Different synthetic approaches for accessing the ortho-biaryl benzylic amine 69. Reagents

and conditions: (i) NH2SO2tBu (1.5 eq.), triethylsilane (1.1 eq.), 1 M triflic acid solution (0.05 eq.),

nitromethane, RT, 8 h; (ii) Pd(PPh3)2Cl2 (0.1 eq.), THF:sat. aq. NaHCO3 (22:5), 100 ºC, 16 h; (iii)

NaCNBH3 (3.0 eq.), 30% aq. NH3, sat. NH4OAc solution, 90 °C, 18 h; (iv) Boc2O (1.0 eq.), InCl3

(0.01 eq.), 35 °C, 45 min; (v) TFA:CH2Cl2 (1:5), 0 °C, 30 min.

Scheme 3.2: General synthetic route to final compounds 63-65. Reagents and conditions: (i) Boc2O

(1.0 eq.), InCl3 (0.01 eq.), 35 °C, 45 min; (ii) 2-methoxyphenylboronic acid (2.0 eq.), Pd(PPh3)2Cl2

(0.1 eq.), THF:sat. aq. NaHCO3 (22:5), 100 ºC, 16 h; (iii) TFA:CH2Cl2 (1:5), 0 °C, 30 min; (iv)

Naphthoquinone 23 (1.0 eq.), anhydrous CeCl3 (1.0 eq.), anhydrous toluene, RT, 1.5 h, then add requisite

aniline (3.0 eq.), 110 °C, 16 h.

Ortho-substituted biaryl 69 was synthesised in five steps from ortho-bromo

benzaldehyde 66 (Scheme 3.1 and 3.2). Initially, use of tert-butyl sulfonamide in the

reductive amination step (step (i) in Scheme 3.1) was trialled following a literature

protocol for aminations with analogous sulfonamides,60

to yield a protected amine in

one step. However, a complex mixture of products was obtained, perhaps attributable to

the acidic reaction conditions. Subsequently, a two-step procedure in which a Suzuki

coupling between benzaldehyde 66 and 2-methoxybenzeneboronic acid, followed by

reductive amination with ammonia to yield the benzylic amine 69 was attempted (steps

(ii) and (iii) in Scheme 3.1);61

however, the purification of this amine proved


24

challenging. Therefore, a lengthier four-step procedure to amine 69 was devised and

successfully executed. The initial reductive amination step (conditions (iii) in Scheme

3.1) produced the desired mono-substituted amine 72 and the di-substituted amine 71 in

a 3:2 ratio (as determined by 1H-NMR of the reaction mixture); nonetheless, 72 could

be isolated in 38% yield. Subsequent tert-butyl carbamate protection with catalytic

InCl3 yielded 73 in quantitative yield which was subjected to a Suzuki coupling54

to

give biaryl 74 in 68% yield. N-deprotection of this species in TFA:CH2Cl2 (1:5) for 2 h

at RT resulted in a complex mixture; thus milder reaction conditions (TFA:CH2Cl2 (1:5),

0 °C, 30 mins) were employed to yield amine 69 in quantitative yield without the need

for further purification. Finally, a conjugate addition-elimination with naphthoquinone

23 (step (iv) in Scheme 3.2) gave the desired final product 63 in 37% yield. In this step,

anhydrous toluene and CeCl3 were used, as 1H-NMR and LRMS data of crude reaction

mixtures in the presence of MeOH or H2O were consistent with cleavage between the

C3 amino group and the biaryl at the benzylic position to yield 2-benzenesulfonamido-

3-amino-1,4-naphthoquinone. meta- and para-Substituted 64 and 65 were synthesised

analogously (Scheme 3.2) from the commercially available benzylamine hydrochloride

salts 75 and 76.

3.3. Pharmacological and Optical Evaluation of the Linked Species 63-65

Initial potency tests on compounds 63, 64 and 65 at 30 μM against mNAT2 showed that

only the para-substituted derivative 65 displayed > 50% inhibition; dose-response

analysis indicated the IC50 value of 65 was 2.1 μM. The colorimetric analyses of these

compounds revealed relatively high pKa values compared to the first biaryl library: this

is possibly due to the increased electron donation from the C3 nitrogen into the

naphthoquinone core, as the linker prohibits delocalisation of the nitrogen lone pair into


25

the biaryl system. A shift in λmax upon treatment with mNAT2 for 63 and 65 indicates

that they bind in the same manner as previously synthesised inhibitors.

Table 3.1: Pharmacological, colorimetric and fluorometric data for compounds 63, 64 and 65 at

V = 800 V. IC50 and pKa values quoted ± one standard deviation (ND = not determined).

Compounds 63, 64, and 65 were also subjected to the same fluorescence analysis as the

preceding library, and each was again found to display the characteristic

naphthoquinone fluorescence at λex = 372 nm, λex = 408 nm and λem = 424 nm, which

was quenched upon the addition of 5 µL 1 M aq. NaOH solution. However, in the meta-

and para-substituted analogues 64 and 65, another peak was identified at λex = 252 nm

and λem = 344 nm which was not quenched in base (the same intensity was observed at

pH 8, and under either acidic or basic conditions) (Figure 3.3). The ortho-substituted

product 63 displayed no such fluorescence and only featured the naphthoquinone peak.

Further analysis showed that the meta- and para-biaryl benzylic amine precursors 81

and 82 to their respective final compounds displayed similar peaks at λex = 247 nm and

λem = 350 nm, whereas no fluorescence activity was detected for the ortho-substituted

benzylic amine 69. It is therefore likely that the fluorescence observed in inhibitors 64

Compound 63 64 65

Structure

IC50 (mNAT2) >30 >30 2.1 ± 1.1

pKa 11.6 ± 0.3 11.6 ± 0.4 11.7 ± 0.8

λmax pH 8 (nm) 483 474 471

Δλmax pH 13 (nm) +77 +86 +84

Δλmax mNAT2 (nm) +20 ND +12

Fluorescence

Intensity (λex =

408 nm) (a.u.)

pH 8 204 69 152

Basic 0 0 0

Fluorescence

Intensity (λex =

252 nm) (a.u.)

pH 8 0 102 55

Basic 0 107 55


26

and 65 at these wavelengths was due solely to the biaryl appendages. Meanwhile, it is

possible that steric repulsions between substituents in ortho-substituted species 63 and

its precursor 69 could prohibit formation of a planar electronically excited state, from

which radiative decay can occur to emit fluorescence.62

Figure 3.3: Emission spectra of 65. Left: Two λem peaks generated by 65 at pH 8 Right: Only one peak

observed under basic conditions (V = 800 V).

Although the methylene linker was introduced in this series in order to separate the

electronic systems to yield a more classical fluorescent probe based on PET, it appears

that the electronic separation results in a lack of sensitivity in the biaryl moiety, such

that deprotonation of the sulfonamide-NH does not affect the electronic state of the

biaryl fluorescent output. Whilst this does provide us with a probe that is somewhat

ratiometric in design (the intensity of the naphthoquinone peak could be directly

compared to that of the biaryl peak as an internal reference), as the biaryl peak does not

change in intensity upon deprotonation it has limited diagnostic use. This, compounded

by low absolute intensity of fluorescence displayed by the naphthoquinone moiety in

these compounds, indicates that further investigation into methylene-linked biaryls

would be of limited utility. Instead, optimisation of a directly-conjugated biaryl could

provide a greater scope for generating a pH-dependent probe with enhanced sensitivity

over the examples reported thus far.

0

20

40

60

80

100

120

140

160

270 320 370 420 470

Inte

nsi

ty (

arb

itra

ry u

nit

s)

Wavelength (nm)

0

20

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60

80

100

120

140

160

270 320 370 420 470

Inte

nsi

ty (

arb

itra

ry u

nit

s)

Wavelength (nm)

Excitation at λex = 252 nm

Excitation at λex = 408 nm


27

3.4. Restoration of Conjugation: 2-Aminofluorene as the C3 Substituent

As alluded to above, the ability to achieve planarity in a biaryl system is a key requisite

for fluorescence.62

Thus, the use of a directly conjugated biaryl moiety at C3 as before,

but one which is held rigidly in a planar conformation, could yield a compound which

has both a high fluorescence intensity and also remains sensitive to deprotonation of the

sulfonamide-NH: 2-aminofluorene was selected as an appropriate test C3 substituent. A

two-step synthesis (Scheme 3.3) yielded the aminofluorene-substituted naphthoquinone

via two sequential conjugate addition-elimination reactions in an overall yield of 95%.

Left: Scheme 3.3: Synthesis of aminofluorene-substituted naphthoquinone 83. Reagents and conditions:

(i) NH2SO2Ph (1.0 eq.), Cs2CO3 (1.4 eq.), DMF, RT, 5 h; (ii) CeCl3∙7H2O (1.0 eq.), MeOH, RT, 1.5 h,

then add 2-aminofluorene (3.0 eq.), 90 °C, 16 h. Right: Table 3.2: Pharmacological and colorimetric data

for 83. IC50 and pKa values quoted ± one standard deviation.

Naphthoquinone 83 is a relatively potent mNAT2 inhibitor (IC50 = 1.2 μM), and

displays a similar shift in λmax in the presence of base and enzyme to the biaryl

analogues in the initial library (Table 3.2). Interestingly, deprotonation of 83 does not

result in as great a decrease in the absorption coefficient as for its less rigid counterparts

– whilst εN(83)/εN(8) = 0.73, εCB(83)/εCB(8) = 1.16.

In fluorescence experiments, the characteristic naphthoquinone peak was again

observed for compound 83 at λem = 424 nm (54 a.u. at 800 V) at pH 8, but additionally,

as predicted, a much more intense peak (518 a.u. at 800 V) was also detected at

λex = 279 nm, λem = 374 nm (Figure 3.4). Upon treatment with base, a partial quenching

of the fluorescence at this lower wavelength was observed in addition to the total

IC50 (mNAT2) (μM) 1.2 ± 0.2

pKa 10.2 ± 0.4

λmax pH 8 (nm) 530

Δλmax pH 13 (nm) +43

Δλmax mNAT2 (nm) +32

εN (M-1

cm-1

) 6779

εN/εN(8) 0.73

εCB (M-1

cm-1

) 5178

εCB/εCB(8) 1.16


28

quenching of the λem = 424 nm naphthoquinone peak. Upon reacidification to pH ~8,

both peaks returned to near-original intensity (Figure 3.4).

The emission peak at 374 nm was sufficiently intense to enable detection in a 1 µM

solution of compound 83. Since 83 is a potent mNAT2 inhibitor, it was therefore finally

possible to attempt to observe the effect enzymatic binding would have on the

fluorescence output of an mNAT2 inhibitor.

Figure 3.4: Left: Excitation and emission spectra at pH 8, after basification and reacidification of 83 at

λex = 279 nm and λem = 374 nm (sensitivity voltage = 800 V); Right: excitation spectrum (λex = 279 nm at

800 V) observed when 83 is titrated with mNAT2.

Inhibitor 83 was titrated against mNAT2, with readings taken at λex = 279 nm and

λem = 374 nm. However, after addition of 2 µL of mNAT2 solution (5 mg/mL), a new

peak was detected at λex = 280 nm and λem = 333 nm. Upon further addition of mNAT2,

the intensity of this peak grew further, and began to obscure the fluorescence output of

83 (Figure 3.4). This peak has been attributed to the well-reported fluorescence of

tryptophan residues, which fluoresce at λex = 280 nm and λem = 300-350 nm.63

Despite

the synthesis of a truly ratiometric fluorescent probe 83 which is sensitive to

sulfonamide-NH deprotonation down to a concentration of 1 µM, the wavelengths of

excitation and emission of this probe are sufficiently close to those of tryptophan to

preclude compound 83 from being a useful enzymatic probe. This directly conjugated

0

100

200

300

400

500

600

300 350 400 450

Inte

nsi

ty (

arb

itrary u

nit

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Wavelength (nm)

0 μL mNAT2

2 μL mNAT2

4 μL mNAT2

0

100

200

300

400

500

600

220 270 320 370 420 470

Inte

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arb

itrary u

nit

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Wavelength (nm)

pH 8BasifiedReacidified

Excitation Emission


29

model may still prove fruitful though, if 2-aminofluorene is replaced with a fluorophore

which has an orthogonal λex to tryptophan.

3.5. In Search of a New Fluorophore: Synthesis of a Coumarin Derivative

Fluorescent substituents used as reporters vary greatly in both size and fluorescence

profile.64

It was proposed that attaching the known fluorophore 7-amino-4-

methylcoumarin 84 to the C3 position of the naphthoquinone core via the amino group

might give a species 85 which would be predicted to have a high fluorescent

brightness:size ratio,64

an appropriate pKa value, and bind well to the hNAT1 active site,

as suggested by previous SAR studies32

and in silico modelling (Figure 3.5).

Figure 3.5: Left: Well-reported fluorophore coumarin 84 and target 85; Centre, Right: In silico docking

of 85 with hNAT1 (pdb: 2PQT),50

showing predicted position in active site and interactions.

A three-step route to 85 was initially proposed from dichlone 22, in which m-

aminophenol is installed at the C3 position via a conjugate addition-elimination on

naphthoquinone intermediate 23, and subsequently undergoes a Pechmann

condensation65

to afford the coumarin-substituted product 85 (Scheme 3.4). Although

phenol 86 was obtained in good yield, the subsequent Pechmann condensation failed

when 86 was treated with conc. H2SO4 at either RT or 80 °C: at RT starting materials

were simply returned, whilst heating led to degradation of the starting material with no

evidence of product formation. A milder literature protocol was trialled, utilising ethyl

acetoacetate as the reaction solvent, and 0.1 eq. BiCl3 as a Lewis acid catalyst;66


30

however, no reaction was observed under these conditions. It was hypothesised that

BiCl3 might preferentially complex with the naphthoquinone carbonyl instead of ethyl

acetoacetate, and so the number of equivalents of BiCl3 were increased to 2.0: under

these conditions, HRMS data was consistent with formation of 85 (theoretical [M-H]-

m/z = 485.0829, observed m/z = 485.0821), but not in sufficient quantities to facilitate

isolation. 0.1 eq. of InCl3 was trialled as an alternative catalyst for the reaction, but

again despite HRMS consistent with product formation, isolation proved unsuccessful.

Scheme 3.4: Attempts to synthesise 85. Reagents and conditions: (i) CeCl3∙7H2O (1.0 eq.), MeOH, RT,

1.5 h, m-aminophenol (3.0 eq.), 90 °C, 16 h; (ii) BiCl3 (0.1 eq.) or BiCl3 (2.0 eq.) or InCl3 (0.1 eq.), ethyl

acetoacetate (excess), 110 °C, 16 h.

Hence an alternative synthetic route to 85 was devised in which 7-amino-4-methyl-

coumarin 85 was first synthesised and then appended to the naphthoquinone at C3 via

the standard conjugate addition-elimination methodology (Scheme 3.5). Coumarin 84

was initially synthesised in a 32% overall yield in 3 steps from m-aminophenol: firstly,

the aniline group was protected as the ethyl carbamate, then a Pechmann condensation

was successfully effected under acidic conditions, before the carbamate protecting

group was removed under heating in harsh acidic conditions.65

However, subsequently a

more efficient synthesis of 84 was also effected, in which m-aminophenol underwent a

Pechmann condensation with ethyl acetoacetate and a Lewis acid catalyst to give 84 in a

single step, albeit in moderate yield after purification (21%).

Whilst hydroxy-substituted analogues (umbelliferones) are reported in the literature to

act as nucleophiles,67

when 7-amino-4-methyl coumarin 84 was subjected to reaction


31

with naphthoquinone 23 and CeCl3∙7H2O in MeOH at 90 °C, only starting materials

were returned (Table 3.3). Increasing the reaction temperature to 110 °C or changing

the solvent to toluene also proved ineffective. At this stage, alternative literature

protocols for functionalising the naphthoquinone C3 position were trialled utilising the

commercially available and more nucleophilic 3,5-dimethylaniline as a test substrate.

Scheme 3.5: Attempts to install 7-amino-4-methyl coumarin 84 at C3. Reagents and conditions: (i) Ethyl

chloroformate (1.0 eq.), anhydrous Et2O, RT, 2 h; (ii) Ethyl acetoacetate (1.2 eq.), conc. H2SO4:EtOH

(7:3), RT, 6 h; (iii) Glacial acetic acid:conc. H2SO4 (1:1), 120 °C, 4 h; (iv) BiCl3 (0.1 eq.), ethyl

acetoacetate (excess), 75 °C, 48 h; (v) InCl3 (0.1 eq.), ethyl acetoacetate (excess), 75 °C, 48 h; (vi) 3,5-

dimethylaniline (1.5 eq.), tBuOK (1.5 eq.), PdCl2(dppf) (0.1 eq.), dppf (0.1 eq.), toluene, 80 °C, 16 h; (vii)

3,5-dimethylaniline (0.5 eq.) I2 (0.1 eq.), EtOH, ultrasonic radiation, 25 °C, 2 h; (viii-xii) Naphthoquinone

23 (1.0 eq.), CeCl3∙7H2O or CeCl3 (1.0 eq.), MeOH or toluene or anhydrous toluene, RT, 1.5 h, then add

coumarin 84 (3.0 eq.), for reaction temperature and time see Table 3.3.

Table 3.3: Comparison of conditions trialled in synthesis of 85. 3,5-DMA = 3,5-dimethylaniline;

7-AMC = 7-amino-4-methylcoumarin 84

However, neither a Buchwald-Hartwig coupling68

(Scheme 3.5 step (vi)) nor an iodine-

catalysed conjugate addition-elimination reaction facilitated by ultrasonic radiation

(Scheme 3.5 step (vii)) could install the aniline at C3 of intermediate 23. Returning to a

cerium-facilitated procedure, conjugate addition-elimination with the aminocoumarin 84

Reaction

Scheme Step Reagent Conditions Isolated Yield

(vi) 3,5-DMA tBuOK, cat. PdCl2(dppf) & dppf, toluene, 80 °C, 16 h 0%

(vii) 3,5-DMA cat. I2, ultrasonic radiation, RT, 2 h 0%

(viii) 7-AMC CeCl3∙7H2O, MeOH, 90 °C, 16 h 0%

(ix) 7-AMC CeCl3∙7H2O, MeOH, 110 °C, 16 h 0%

(x) 7-AMC CeCl3∙7H2O, toluene,110 °C, 16 h 0%

(xi) 7-AMC Anhydrous CeCl3, anhydrous toluene, 110 °C, 16h 0%

(xii) 7-AMC Anhydrous CeCl3, MeOH, 110 °C, 72 h 16%


32

was attempted with anhydrous CeCl3 in either toluene or MeOH, since use of anhydrous

Lewis acid had previously been demonstrated to result in a small increase in yield from

such reactions.32

This procedure proved effective, and heating at 110 °C in MeOH with

anhydrous CeCl3 for 3 days resulted in the total consumption of the starting material 23

as determined by TLC and 1H-NMR of the reaction mixture. Purification of final

compound 85 proved challenging, however: after failed attempts at column

chromatography and recrystallisation, 85 was finally isolated via preparative thin-layer

chromatography in 16% yield.

3.6. Pharmacological and Optical Evaluation of Coumarin 85

Docking studies showed that compound 85 could feasibly bind to hNAT1 in a similar

fashion to the biaryl-substitued analogues (Figure 3.5). Analysis revealed that 85 had an

IC50 value against mNAT2 of 250 nM, making 85 one of the most potent known

mNAT2 inhibitors, and displayed the characteristic shift in λmax (Figure 3.6). The

relatively low pKa of 7.34 which was observed could perhaps be rationalised by the

electron-withdrawing lactone: the lone pair of the C3 nitrogen will have a lower

propensity for mesomeric donation into the naphthoquinone core than was the case for

the analogous biaryls.

Fluorescence measurements on 85 again identified the distinctive naphthoquinone peak

at λem = 424 nm, as well as a more intense peak at λex = 344 nm and λem = 440 nm which

was attributed to the coumarin substituent. This peak was sufficiently intense to be

detected at 1 μM. Upon addition of 5 μL 1 M aq. NaOH, both fluorescent peaks were

quenched, but upon reacidification only the naphthoquinone peak returned to its original

intensity. This was presumed to be due to a ring opening of the lactone moiety by NaOH

as this reactivity of coumarin is reported in the literature,69

so a fluorescence


33

measurement was instead taken in a buffered 35 mM Na2HPO4.NaOH solution at

pH = 12.8. Here, although the naphthoquinone peak was once again quenched, the

intensity of the coumarin peak was unchanged. It would appear that the coumarin

installed on the naphthoquinone core is not sensitive to electronic changes of the

sulfonamide-NH deprotonation, and indeed in a titration experiment of 85 against

mNAT2, although the coumarin peak could be resolved throughout, no change in the

intensity of this peak was observed upon increasing enzyme concentration (Appendix

3.3). This could be attributed to the same factors contributing to the low pKa of 85.

Figure 3.6: Top Left: λex = 408 nm emission spectrum of 85; Bottom Left: λex = 344 nm emission

spectrum of 85; Top Right: Final compound 85; Bottom Right: Table of pharmacological, colorimetric

and fluorometric data for 85. IC50 and pKa values quoted ± one standard deviation. Fluorescence

sensitivity V = 800V.

However, in synthesising fluorene 83 and coumarin 85, two ratiometric, fluorometric

probes that are good inhibitors of mNAT2 have been developed which respectively

comprise two pH-sensitive fluorophores or exhibit strong fluorescence at a wavelength

orthogonal to that of intrinsic protein fluorescence.

IC50 (mNAT2) (μM) 0.25 ± 0.16

pKa 7.3 ± 0.3

λmax pH 8 (nm) 549

Δλmax pH 13 (nm) +6

Δλmax mNAT2 (nm) +55

Fluorescence

intensity λex

= 408 nm

pH 3.2 103

pH 8.0 10

pH 12.8 0

Fluorescence

intensity λex

= 344 nm

pH 3.2 202

pH 8.0 211

pH 12.8 177

0

20

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416 426 436 446 456 466

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pH 3.2

pH 8

pH 12.8

0

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370 420 470 520

Inte

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Wavelength (nm)


34

3.7. Conclusions and Further Work

A library of naphthoquinones bearing C3 anilino-biaryl substituents was synthesised in

an effort to create inhibitors of hNAT1 which could be used as fluorescent probes for

detection of the enzyme. Nine compounds were tested against mNAT2, and seven were

found to be potent inhibitors. Fluorescence analysis of this library revealed that

although reversible fluorescence quenching was displayed by all but one of the

inhibitors upon deprotonation of the sulfonamide-NH, the naphthoquinone core was

responsible for this fluorescent behaviour, rather than the appended biaryls (Table 2.6).

These inhibitors did not display an appropriate level of sensitivity, so attempts at

optimisation of the fluorophores were carried out. Introduction of a methylene linker

between the C3 amine and the biphenyl moiety generated the first example of a

ratiometric fluorescent probe 65 which is a potent inhibitor of mNAT2, but in which

only one of the two fluorophores was sensitive to sulfonamide deprotonation

(Figure 3.7). The original directly conjugated scaffold was thus optimised by

introducing 2-aminofluorene and 7-amino-4-methylcoumarin as C3 substituents. Both

are potent mNAT2 inhibitors, notably coumarin 85 (IC50 = 250 nM), and show

distinctive fluorescence peaks corresponding to the C3 moieties which can be detected

down to 1 μM. In the case of coumarin 85, the intensity of this fluorescent peak was not

sensitive to sulfonamide-NH deprotonation; however the fluorene 83 represents the first

example of a truly ratiometric, sensitive pH probe which is an mNAT2 inhibitor.

Unfortunately enzymatic detection is not possible due to the overlap of λex and λem

between aminofluorene and tryptophan residues. However, compounds 83 and 85

demonstrate there is a large scope for potential optimisation of such probes which could

allow this novel system to outperform any of the current methods of hNAT1 detection.


35

Figure 3.7: Optimisation from first fluorescent inhibitor identified to sensitive ratiometric probes.

Examples of biological probes featuring coumarins often utilise the fluorophore by

linking the core of the molecule to the coumarin moiety via the coumarin C3 position,

rather than the 7-amino, enabling greater conjugation.42

Future work towards

constructing a sensitive and responsive probe should aim to attach the coumarin in this

manner, via the proposed synthetic procedure outlined in Scheme 3.6. Alternatively,

work should be carried out to optimise the sensitivity of the current coumarin 85 to

deprotonation – installation of an EDG (such as a methoxy group) on the coumarin

system would greatly increase electron density on the C3 appendage, thereby increasing

propensity of the C3 nitrogen for lone pair donation into the naphthoquinone core. This

could result in the sulfonamide-NH deprotonation having a greater impact on the

electron density in the coumarin appendage, thereby affecting its fluorescent output in

the desired fashion.

Left: Scheme 3.6: Proposed synthesis of C3-C3″ linked naphthoquinone-coumarin 93. Reagents and

conditions: (i) 3-Chloroethyl acetoacetate (excess), InCl3 (0.1 eq.); (ii) B2Pin2 (1.5 eq.), KOAc (3.0 eq.),

Pd(PPh3)2Cl2 (0.05 eq.), 1,4-dioxane, 120 °C, 3 h; (iii) Naphthoquinone 23 (0.5 eq.) Pd(PPh3)2Cl2

(0.05 eq.), THF:sat. aq. NaHCO3 (22:5), 100 ºC, 16 h.

Right: Figure 3.8: Proposed coumarin derivative 94 predicted to be more sensitive to sulfonamide

deprotonation.

Chapter 4: Experimental

36


4.1. General Experimental

All reactions involving moisture-sensitive reagents were carried out under a nitrogen

atmosphere using standard vacuum line techniques and glassware that was flame-dried

before use. Solvents were dried following the procedure outlined by Grubbs et al.70

Water was purified by an Elix® UV-10 system. All other solvents and reagents were

used as supplied (analytical or HPLC grade) without prior purification. Organic layers

were dried over anhydrous MgSO4 or Na2SO4. Brine refers to a sat. aq. solution of

NaCl. In vacuo refers to the use of a rotary evaporator attached to a diaphragm pump.

Pet ether refers to the fraction of petroleum spirit boiling between 30 and 40 °C.

Thin layer chromatography was performed on Merck aluminium plates coated with 60

F254 silica. Plates were visualised using UV light (254 nm) or 1% aq. KMnO4. Flash

column chromatography was performed on Kieselgel 60 silica in a glass column.

Melting points were recorded on an EZ-Melt (SRS) apparatus and are uncorrected.

Infrared spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer, as neat

samples. Selected characteristic peaks are reported in wavenumbers (cm-1

). NMR

spectra were recorded on Bruker Avance spectrometers (AVII 400, AVII 500 or AV700)

in the deuterated solvent stated. The field was locked by external referencing to the

relevant deuteron resonance. Chemical shifts () are reported in parts per million (ppm)

and coupling constants (J), determined to 1 d.p. by analysis using ACD labs software,

are quoted in Hz. Low-resolution mass spectra were recorded an Agilent 6120 mass

spectrometer from solutions of MeOH. Accurate mass measurements were run on either

a Bruker MicroTOF internally calibrated with polyalanine, or a Micromass GCT

instrument fitted with a Scientific Glass Instruments BPX5 column (15 m 0.25 mm)


37

using amyl acetate as a lock mass, by the mass spectrometry department of the

Chemistry Research Laboratory, University of Oxford, UK. m/z values are reported in

Daltons and followed by their percentage abundance in parentheses.

4.2. General Synthetic Procedures

General Procedure 1: Substitution of N-(3-chloro-1,4-dioxo-1,4-

dihydronaphthalen-2-yl)benzenesulfonamide with anilines or amines

N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzenesulfonamide 23 (1 eq.) was

stirred with CeCl3∙7H2O or anhydrous CeCl3 (1.0 eq.) in MeOH or toluene, as stated, at

RT for 1.5 h in a microwave vial. The requisite aniline or amine (3.0 eq.) was added, the

vial was sealed and the mixture stirred at 90 ºC for 16 h, unless otherwise stated. The

solution was cooled to RT, sat. aq. NH4Cl (20 mL) was added and the organic product

was extracted with EtOAc (3 x 20 mL). The organic layer was collected, washed with

brine (2 x 20 mL), dried, filtered and concentrated in vacuo to give the crude product.

General Procedure 2: Formation of boron pinacol esters from arylbromides

The requisite arylbromide (1.0 eq.) was added to 1,4-dioxane in a microwave vial and

degassed whilst stirring. Bis(pinacolato)diboron (1.5 eq.), potassium acetate (3.0 eq.)

and bis(triphenylphosphine)palladium(II) dichloride (0.05 eq.) were added, the vial

sealed and the mixture stirred under N2 at 120 °C for 3 h. After cooling to RT, the

mixture was filtered through a Celite® pad, diluted with H2O (75 mL) and then extracted

with EtOAc (2 x 75 mL). The combined organic layers were washed with brine

(3 x 30 mL), dried, filtered and concentrated in vacuo to give the crude product.

General Procedure 3: Suzuki coupling of arylbromides with arylboronic esters

THF (22 parts) and sat. aq. NaHCO3 (5 parts) were added to a microwave vial

containing the requisite arylbromide (1.0 eq.) and degassed whilst stirring. The requisite


38

arylboronic ester (2.0 eq.) and bis(triphenylphosphine)palladium(II) dichloride (0.1 eq.)

were added, the vial sealed and heated to 100 ºC for 16 h. The solution was cooled to

RT and filtered through a Celite® pad, sat. aq. NH4Cl (50 mL) was added and the

organic product was extracted with EtOAc (3 x 50 mL). The combined organic layers

were washed with brine (2 x 30 mL), dried, filtered and concentrated in vacuo to give

the crude product.

General Procedure 4: tert-Butyl carbamate protection of anilines

The requisite aniline or amine (1.0 eq.), Boc2O (1.0 eq.) and InCl3 (0.01 eq.) were

stirred at 35 °C for 45 min, unless otherwise stated. The mixture was cooled to RT,

diluted with EtOAc (250 mL) and then washed with H2O (2 x 100 mL). The organic

layer was collected, dried, filtered and concentrated in vacuo to give the crude product.

General Procedure 5: tert-Butyl carbamate deprotection of protected anilines

Trifluoroacetic acid (1 part) was added slowly to the requisite tert-butyl carbamate

(1.0 eq.) in CH2Cl2 (5 parts) at 0 ºC. The solution was stirred at RT for 2 h, unless

otherwise stated. The reaction was poured onto H2O (50 mL) and extracted into CH2Cl2

(3 x 30 mL). The combined organic layers were washed with sat. aq. NaHCO3

(3 x 30 mL) and brine (2 x 30 mL), then dried, filtered and concentrated in vacuo to

give the crude product.

4.3. Preparation and Characterisation of Reported Compounds

N-(3-((3",5"-Dimethylphenyl)amino)-1,4-dioxo-1,4-dihydronaphthalen-2-

yl)benzenesulfonamide 825

Following General Procedure 1, using naphthoquinone 23 (0.300 g,

0.863 mmol), CeCl3.7H2O (0.322 g, 0.863 mmol), 3,5-

dimethylaniline (0.338 mL, 2.590 mmol) in MeOH (7 mL).


39

Purification via column chromatography (eluent pet ether:acetone 95:5 to 80:20) and

subsequent recrystallisation from boiling toluene gave 3-anilinonapthoquinone 8 as a

purple solid (0.127 g, 34%). mp 193-195 °C {lit. 178-182 °C}25

; δH (400 MHz, CDCl3)

2.32 (6H, s, 2 x Ar-Me), 6.68 (2H, s, H2″ and H6″), 6.83 (2H, s, H4″ and

sulfonamide-NH), 7.34 (2H, app. t, J 7.6, H3′ and H5′), 7.47 (1H, t, J 7.6, H4′), 7.62-7.71

(4H, m, H6, H7, H2′ and H6′), 7.89-7.91 (1H, m, H5 or H8), 8.02 (1H, br. s, aniline-NH),

8.05-8.09 (1H, m, H5 or H8); m/z (ESI-) 431 ([M-H]

-, 100%).

N-(3-((8″-Methoxy-10″-nitro-[4″,7″-biphenyl]-1″-yl)amino)-1,4-dioxo-1,4-

dihydronaphthalen-2-yl)benzenesulfonamide 1251

Following General Procedure 1, using naphthoquinone 23 (103 mg,

0.301 mmol), aniline 14 (220 mg, 0.902 mmol) and CeCl3∙7H2O

(112 mg, 0.301 mmol) in MeOH (7 mL). Upon workup a purple

precipitate was formed; this was collected by suction filtration and

washed with cold ethanol to yield 3-anilinonapthoquinone 23 as a

purple solid (131 mg, 78%). mp 253-257 °C; δH (500 MHz, DMSO-d6) 3.95 (3H, s,

OMe), 7.08 (2H, d, J 8.5, H2’’ and H6’’), 7.34-7.38 (2H, m, H3’ and H5’), 7.44 (2H, d, J

8.5, H3’’ and H5’’), 7.47-7.51 (1H, m, H4’), 7.56-7.61 (3H, m, H2’, H6’ and H12’’), 7.75-

7.83 (3H, m, H6, H7 and H8), 7.88 (1H, d, J 2.2, H9’’), 7.94 (1H, dd, J 8.4, 2.2, H11’’),

8.01-8.04 (1H, m, H5), 9.08 (1H, br. s, aniline-NH), 9.16 (1H, s, sulfonamide-NH); m/z

(ESI-) 554 ([M-H]

-, 100%).



Following General Procedure 1, using naphthoquinone 23 (47 mg, 0.14 mmol), aniline

34 (100 mg, 0.41 mmol) and CeCl3∙7H2O (51 mg, 0.14 mmol) in MeOH (6 mL).


40

Purification via column chromatography (eluent pet ether:acetone

80:20 to 0:100) gave 3-anilinonaphthoquinone 15 as a purple solid

(22 mg, 28%). mp 234-236 °C; νmax: (neat) 3314 (N-H), 3217 (N-

H), 1674 (C=O); δH (500 MHz, DMSO-d6) 4.06 (3H, s, OMe), 7.14

(2H, d, J 8.1, H2″ and H6″), 7.35 (2H, app. t, J 7.7, H3′ and H5′),

7.43 (1H, dd, J 8.5, 1.7, H12″), 7.48 (1H, t, J 7.7, H4′), 7.54 (1H, d, J 1.7, H8″), 7.57-7.60

(2H, m, H2′ and H6′), 7.70 (2H, d, J 8.1, H3″ and H5″), 7.75-7.81 (3H, m, H6, H7 and H8),

7.99 (1H, d, J 8.5, H11″), 8.03 (1H, d, J 6.5, H5), 9.17 (2H, br. s, sulfonamide and

aniline-NH); δC (125 MHz, DMSO-d6) 56.7 (OMe), 111.5 (C8″), 118.1 (C12″), 123.4 (C2″

and C6″), 125.7 (C8), 126.0 (C11″), 126.2 (C5), 126.4 (C2′ and C6′), 126.4 (C3″ and C5″),

128.5 (C3′ and C5′), 130.5 (C9), 131.4 (C10), 132.1 (C4′), 131.2 (C4″) 133.2 (C6), 134.8

(C7), 137.5 (C10″), 139.8 (C1″) 141.4 (C1′) 146.3 (C7″), 152.9 (C9″), 178.8 (C1), 182.1

(C4), C2 and C3 not observed; m/z (ESI-) 554 ([M-H]

-, 100%); HRMS (ESI

-)

C29H20O7N3S- requires 554.1027, found 554.1040.





(81 mg, 0.22 mmol) in MeOH (7 mL). Purification via column

chromatography (eluent pet ether:acetone 90:10 to 70:30) gave

3-anilinonaphthoquinone 23 as a red solid (12 mg, 10%). mp 200-

203 °C; δH (400 MHz, DMSO-d6) 3.77 (3H, s, OMe), 6.70-6.83 (2H, m, H2″ and H6″),

7.25 (1H, d, J 8.4, H5″), 7.32-7.40 (2H, m, H3′ and H5′), 7.49 (1H, t, J 6.8, H4′), 7.59 (2H,

d, J 7.8, H2′ and H6′), 7.76 - 7.86 (5H, m, H6, H7, H8, H8″ and H12″), 7.99 (1H, d, J 8.5,


41

H5), 8.27 (2H, d, J 8.5, H9″ and H11″), 9.10 (1H, br. s, aniline-NH), 9.28 (1H, s,

sulfonamide-NH); δC (125 MHz, DMSO-d6) 55.5 (OMe), 107.5 (C2″), 115.7 (C6″), 123.3

(C9″ and C11″), 125.6 (C8), 125.8 (C5), 126.4 (C2′ and C6′), 128.5 (C5″), 129.0 (C3′ and

C5′), 130.1 (C8″ and C12″), 130.5 (C4″), 131.5 (C9 or C10), 131.6 (C9 or C10), 131.8 (C4′),

133.3 (C6 or C7), 135.0 (C6 or C7), 141.5 (C1′), 141.5 (C1″), 145.3 (C7″ or C10″), 145.7

(C7″ or C10″), 153.8 (C3″), C1-C4 not observed; m/z (ESI-) 554 ([M-H]

-, 100%); HRMS

(ESI-) C29H20O7N3S

- requires 554.1027, found 554.1032.



Following General Procedure 1, using naphthoquinone 23

(117 mg, 0.34 mmol), aniline 48 (250 mg, 1.02 mmol) and

CeCl3∙7H2O (127 mg, 0.14 mmol) in MeOH (7 mL). Purification

via column chromatography (eluent pet ether:acetone 80:20 to

0:100) gave 3-anilinonaphthoquinone 17 as a purple solid (163 mg,

86%). mp 239-241 °C; νmax 3305 (N-H), 3225 (N-H) 1639 (C=O); δH (700 MHz,

DMSO-d6) 3.89 (3H, s, OMe), 7.13 (1H, d, J 1.7, H3″), 7.33 (1H, dd, J 8.2, 1.7, H5″)

7.35-7.37 (3H, m, H6″, H3′ and H5′), 7.49 (1H, t, J 7.3, H4′), 7.61 (2H, d, J 7.3, H2′ and

H6′), 7.76-7.81 (3H, m, H6, H7 and H8), 8.00 (1H, d, J 7.1, H5), 8.04 (2H, d, J 8.8, H8″

and H12″), 8.31 (2H, d, J 8.8, H9″ and H11″), 8.52 (1H, br. s, NH), 9.22 (1H, s, NH); δC

(175 MHz, DMSO-d6), 55.9 (OMe) 109.4 (C3″), 114.7 (C2) 118.7 (C5″), 124.0 (C9″ and

C11″), 125.7 (C8″), 126.2 (C5), 126.5 (C2′ and C6′), 127.5 (C8″ and C12″), 128.5 (C3′ and

C5′), 128.7 (C6″) 130.4 (C3), 131.4 (C10), 132.2 (C4′), 133.2 (C9), 133.8 (C4″), 134.9 (C6

and C7), 140.5 (C1′) 141.9 (C1″), 146.3 (C10″), 146.4 (C7″), 151.4 (C2″), 178.5 (C1), 181.8


42

(C4); m/z (ESI-) 554 ([M-H]

-, 100%); HRMS (ESI

-) C29H20O7N3S

- requires 554.1027,

found 554.1036.

N-(3-((10''-Nitro-[4",7"-biphenyl]-1″-yl)amino)-1,4-dioxo-1,4-dihydronaphthalen-

2-yl)benzenesulfonamide 18



(107 mg, 0.288 mmol) in MeOH (7 mL). Purification via column

chromatography (eluent pet ether:acetone 85:15 to 0:100) gave

3-anilinonaphthoquinone 18 as a purple solid (35 mg, 23%).

mp 212-215 °C; νmax (neat) 3325 (N-H), 3219 (N-H), 1632 (C=O); δH (500 MHz,

DMSO-d6) 7.16 (2H, d, J 8.7, H2'' and H6''), 7.33-7.38 (2H, m, H3' and H5'), 7.46-7.50

(1H, m, H4'), 7.56 (2H, dd, J 8.4 and 1.2, H2' and H6'), 7.69 (2H, d, J 8.7, H3'' and H5''),

7.76-7.83 (3H, m, H6, H7 and H8), 7.97-8.00 (2H, m, H8'' and H12''), 8.03-8.05 (1H, m,

H5), 8.29-8.32 (2H, m, H9'' and H11''), 9.17 (1H, s, sulfonamide-NH), 9.21 (1H, s,

aniline-NH); δC (125 MHz, DMSO-d6) 115.3 (C2), 124.1 (C2'' and C6''), 124.6 (C9'' and

C11''), 126.2 (C8), 126.7 (C5), 126.8 (C3'' and C5''), 127.0 (C2' and C6'), 127.6 (C8'' and

C12''), 129.0 (C3' and C5'), 130.9 (C9), 131.9 (C10), 132.4 (C4″), 132.7 (C4′), 133.6 (C6),

134.9 (C7), 140.2 (C1″), 141.3 (C1′), 142.0 (C3), 146.5 (C7″ or C10″), 146.7 (C7″ or C10″),

178.7 (C1 or C4), 182.3 (C1 or C4); m/z (ESI-) 524 ([M-H]

-, 100%); HRMS (ESI

-)

C28H18N3O6S- ([M-H]

-) requires 524.0922, found 524.0928.

N-(3-((10''-Amino-[4'',7''-biphenyl]-1″-yl)amino)-1,4-dioxo-1,4-


Naphthoquinone 18 (50 mg, 0.95 mmol) was stirred at RT with 10% Pd/C (11 mg,

0.10 mmol) in MeOH (5 mL) in a microwave vial. The vial was purged with H2 and


43

then stirred at RT under H2 for 16 h. The reaction mixture was

filtered through a Celite® pad and concentrated in vacuo.

Purification via column chromatography (eluent pet ether:acetone

70:30 to 0:100) gave amino-substituted 19 as a purple solid

(28 mg, 60%). mp 237-240 °C; νmax (neat) 3318 (N-H), 2360, 1608

(C=O); δH (500 MHz, DMSO-d6) 5.20 (2H, br. s, NH2), 6.65 (2H, d, J 8.7, H9″ and

H11″), 7.03 (2H, d, J 8.5, H2″ and H6″), 7.33-7.39 (4H, m, H3′, H5′, H8″ and H12″), 7.41

(2H, d, J 8.5, H3″ and H5″), 7.48 (1H, t, J 7.6, H4′), 7.56 (2H, d, J 7.4, H2′ and H6′), 7.73-

7.81 (3H, m, H6, H7 and H8), 8.02 (1H, d, J 7.1, H5), 9.04 (2H, br. s, sulfonamide and

aniline-NH); δC (125 MHz, DMSO-d6) 112.0 (C2), 114.3 (C9″ and C11″), 123.9 (C2″ and

C6″), 124.2 (C3″ and C5″), 125.6 (C8), 126.2 (C5), 126.5 (C2′ and C6′), 126.8 (C8″ and

C12″), 127.1 (C7″), 128.4 (C3′ and C5′), 130.3 (C9 or C10), 131.6 (C9 or C10), 132.1 (C4′),

132.9 (C6 or C7), 134.9 (C6 or C7), 135.9 (C4″), 136.4 (C1″), 141.0 (C1′), 141.9 (C3) 148.1

(C10″), 178.3 (C1 or C4), 182.3 (C1 or C4); m/z (ESI+) 496 ([M+H]

+, 100%), 518

([M+Na]+, 30%); HRMS C28H21O4N3NaS

+ requires 518.1145, found 518.1133.

N-(3-((10″-Amino-8″-methoxy-[4″,7″-biphenyl]-1″-yl)amino)-1,4-dioxo-1,4-


Naphthoquinone 12 (30 mg, 0.54 mmol) was stirred at RT with 10%

Pd/C (1 mg, 0.05 mmol) in MeOH (3 mL) in a microwave vial. The

vial was purged with H2 and then stirred at RT under H2 (1 atm) for

16 h. The reaction mixture was filtered through a Celite® pad and

concentrated in vacuo which gave amino-substituted 20 as a purple

solid (28 mg, quant.). mp 226-229 °C; νmax (neat) 3305 (N-H), 2360, 1608 (C=O);

δH (500 MHz, DMSO-d6) 3.71 (3H, s, OMe), 5.23 (2H, br. s, -NH2), 6.25 (1H, dd, J 8.2,


44

2.0, H11″), 6.33 (1H, d, J 2.0, H9″), 6.94-7.01 (3H, H2″, H6″ and H12″), 7.26-7.30 (2H, m,

H3″ and H5″) 7.33-7.39 (2H, m H3′ and H5′), 7.48 (1H, t, J 7.4, H4′), 7.54-7.59 (2H, m,

H2′ and H6′), 7.73-7.83 (3H, m, H6, H7 and H8), 8.01 (1H, d, J 7.3, H5), 8.94 (1H, br. s,

NH), 9.05 (1 H, br. s, NH); C (125 MHz, DMSO-d6) 55.1 (OMe), 97.7 (C11″), 106.5

(C9″), 113.4 (C2), 117.1 (C7″), 122.7 (C2″ and C6″), 125.6 (C8), 126.2 (C5), 126.5 (C2′ and

C6′), 127.8 (C3″ and C5″), 128.5 (C3′ and C5′), 130.4 (C9), 130.6 (C12″), 131.6 (C10), 132.1

(C4′), 132.9 (C6 or C7), 134.3 (C4″), 134.8 (C6 or C7), 136.1 (C1″), 141.0 (C1′), 141.8 (C3),

149.5 (C10″), 157.1 (C8″), 178.4 (C1), 182.3 (C4); m/z (ESI+) 526 ([M+H]

+, 100%), 548

([M+Na]+, 25%); HRMS (ESI

+) C29H24O5N3S

+ requires 526.1431, found 526.1427.

Methyl 1″-((1,4-dioxo-2-(phenylsulfonamido)-1,4-dihydronaphthalen-3-yl)amino)-

10″-nitro-[4″,7″-biphenyl]-8″-carboxylate 21



(85 mg, 0.27 mmol) in MeOH (7 mL) gave

3-anilinonaphthoquinone 21 as a purple solid (118 mg, 88%).

mp 258-260 °C; νmax (neat) 3345 (N-H), 3249 (N-H), 1736 (C=O);

δH (700 MHz, DMSO-d6) 3.73 (3H, s, -CO2Me), 7.06 (2H, d, J 8.5, H2″ and H6″), 7.22

(2H, d, J 8.5, H3″ and H5″), 7.33-7.37 (2H, m, H3′ and H5′), 7.47 (1H, t, J 7.5, H4′), 7.56-

7.64 (2H, m, H2′ and H6′), 7.75 (1H, d, J 8.5, H12″), 7.77-7.85 (3H, m, H6, H7 and H8),

8.02 (1H, d, J 7.3, H5), 8.44 (1H, dd, J 8.5, 2.6, H11″), 8.49 (1H, d, J 2.6, H9″), 9.22 (1H,

s, aniline-NH), 9.28 (1H, s, sulfonamide-NH); C (175 MHz, DMSO-d6) 52.8 (CO2Me),

115.6 (C2), 122.7 (C2″ and C6″), 124.3 (C9″), 125.7 (C11″), 125.9 (C8), 126.2 (C5), 126.5

(C2′ and C6′), 127.4 (C3″ and C5″), 128.5 (C3′ and C5′), 130.5 (C9), 131.4 (C10), 131.9

(C12″ or C4″), 131.9 (C12″ or C4″), 132.2 (C8″), 132.6 (C4′), 133.2 (C6 or C7), 134.9 (C6 or


45

C7), 139.4 (C1″), 140.9 (C1′), 141.4 (C3), 146.0 (C10″), 146.9 (C7″), 167.3 (-CO2Me),

178.8 (C1), 182.1 (C4); m/z (ESI-) 582 ([M-H]

-, 100%); HRMS (ESI

-) C30H20N3O8S

-

([M-H]-) requires 582.0977, found 582.1002.

2′-(3-Methoxy-4-nitrophenyl)-4′,4′,5′,5′-tetramethyl-1′,3′,2′-dioxaborolane 31

Following General Procedure 2, using arylbromide 26 (1.33 g, 5.71 mmol),

B2Pin2 (2.18 g, 8.57 mmol), KOAc (1.68 g, 17.13 mmol) and Pd(PPh3)2Cl2

(0.20 g, 0.29 mmol) in 1,4-dioxane (8 mL). Purification via column

chromatography (eluent pet ether:EtOAc 90:10) gave boronic ester 31 as a white solid

(1.07 g, 67%). mp= 141-143 °C; νmax (neat) 2976 (C-H), 1605, 1586; (400 MHz,

CDCl3) 1.37 (12H, s, C4′Me2 and C5′Me2), 4.01 (3H, s, OMe), 7.45 (1H, d, J 7.9, H6),

7.49 (1H, s, H2), 7.79 (1H, d, J 7.9, H5); C (125 MHz, CDCl3) 56.5 (OMe), 84.6 (C4′

and C5′), 119.2 (C2), 124.6 (C5), 126.6 (C6), 141.5 (C4), 151.9 (C3), C1 not observed; m/z

(ESI+) 250 (100%), 302 ([M+Na]

+, 100%); HRMS (ESI

+) C13H18BNO5Na

+ requires

302.1170, found 302.1142.

tert-Butyl (9-methoxy-10-nitro-[4,7-biphenyl]-1-yl)carbamate 33

Following General Procedure 3, using arylbromide 28 (0.50 g,

1.83 mmol), boronic ester 31 (1.02 g, 3.66 mmol), Pd(PPh3)2Cl2 (0.13 g,

0.15 mmol) and sat. aq. NaHCO3 (2.5 mL) in THF (11 mL). Purification

via column chromatography (eluent pet ether:EtOAc 95:5 to 80:20) gave

biaryl 33 as a light yellow solid (0.24 g, 46%). mp 133-135 °C; νmax (neat) 3401 (N-H),

2992 (C-H), 1716 (C=O); (500 MHz, CDCl3) 1.55 (9H, s, CMe3), 4.04 (3H, s, OMe),

6.61 (1H, s, NH), 7.20 (2H, m, H8 and H12), 7.50 (2H, d, J 8.6, H2 and H6), 7.54 (2H, d,

J 8.6, H3 and H5), 7.96 (1H, d, J 8.4, H11); C (125 MHz, CDCl3) 28.3 (CMe3), 56.6

(OMe), 81.0 (CMe3), 111.6 (C8), 118.6 (C12), 118.8 (C2 and C6), 126.6 (C11), 127.9 (C3


46

and C5), 133.5 (C4), 138.0 (C10), 139.3 (C1), 147.3 (C7), 152.5 (CO2tBu), 153.6 (C9); m/z

(ESI+) 345 ([M+H]

+, 50%), 367 ([M+Na]

+, 100%); HRMS (ESI

+) C18H20N2NaO5

+

requires 367.1264, found 367.1275.

9-Methoxy-10-nitro-[4,7-biphenyl]-1-amine 34

Following General Procedure 5, using carbamate 33 (243 mg, 0.71 mmol)

and TFA (3.3 mL) in CH2Cl2 (18 mL) gave aniline 34 as an orange solid

(120 mg, 69%). mp 134-136 °C: νmax (neat) 3381 (N-H), 2923 (C-H), 1599;

(400 MHz, CDCl3) 4.03 (3H, s, OMe), 6.78 (2H, d, J 8.5, H2 and H6), 7.15-7.23 (2H,

m, H8 and H12), 7.44 (2H, d, J 8.5, H3 and H5), 7.96 (1H, d, J 8.4, H11); C (125 MHz,

CDCl3) 56.5 (OMe), 110.9 (C8 or C12), 115.3 (C2 and C6), 118.0 (C8 or C12), 126.6

(C11), 128.4 (C3 and C5), 128.9 (C4), 137.34 (C10), 147.4 (C1), 147.9 (C7), 153.8 (C9);

m/z (ESI+) 245 ([M+H]

+, 100%), 267 ([M+Na]

+, 20%); HRMS (ESI

+) C13H18N2NaO3

+

requires 267.0740, found 267.0742.

tert-Butyl (10-nitro-[4,7-biphenyl]-1-yl)carbamate 37

Following General Procedure 3, using boronic ester 38 (1.50 g,

4.70 mmol), Pd(PPh3)2Cl2 (0.17 g, 0.24 mmol), 4-bromonitrobenzene 35

(0.48 g, 2.35 mmol), and sat. aq. NaHCO3 (2.5 mL) in THF (11 mL).

Purification via column chromatography (eluent pet ether:acetone 95:5 to

80:20) gave the biaryl 37 as an orange solid (0.83 g, 56%). mp 164-167 °C; νmax (neat)

3349 (N-H), 2918 (C-H), 1698 (C=O); δH (500 MHz, CDCl3) 1.55 (9H, s, CMe3), 6.62

(1H, s, NH), 7.51 (2H, d, J 8.6, H2 and H6), 7.58 (2H, d, J 8.6, H3 and H5), 7.71 (2H, d,

J 9.0, H8 and H12), 8.28 (2H, d, J 9.0, H9 and H11); δC (125 MHz, CDCl3), 28.3 (CMe3),

81.0 (CMe3), 118.8 (C2 and C6), 124.1 (C9 and C11), 127.2 (C8 and C12), 128.0 (C3 and

C5), 133.1 (C4), 139.3 (C1), 146.7 (C10), 147.0 (C7), 152.5 (C=O); m/z (ESI+) 337


47

([M+Na]+, 100%); HRMS (ESI

+) C17H18N2NaO4

+ ([M+Na]

+) requires 337.1159, found

337.1170.

tert-Butyl (2-methoxy-4-(4′,4′,5′,5′-tetramethyl-1′,3′,2′-dioxaborolan-2′-

yl)phenyl)carbamate 43

Following General Procedure 2, using aryl bromide 41 (3.20 g,

10.60 mmol), B2Pin2 (4.04 g, 15.90 mmol), KOAc (3.12 g, 31.80 mmol)

and Pd(PPh3)2Cl2 (0.37 g, 0.05 mmol) in 1,4-dioxane (18 mL). Purification

via column chromatography (eluent pet ether:EtOAc 95:5) gave boronic

ester 43 as a pale yellow solid (2.66 g, 72%). mp 69-71 °C; νmax (neat) 2977 (C-H),

2926 (C-H), 1735 (C=O); δH (400 MHz, CDCl3) 1.34 (12H, s, C4′Me2 and C5′Me2), 1.53

(9H, s, CMe3), 3.91 (3H, s, OMe), 7.24 (1H, s, NH), 7.25 (1H, d, J 1.0, H3), 7.43 (1H,

dd, J 7.8, 1.0, H5), 8.10 (1H, d, J 7.8, H6); δC (100 MHz, CDCl3) 25.0 (C4′Me2 and

C5′Me2), 28.3 (CMe3), 55.7 (OMe), 80.4 (CMe3), 83.6 (C4′ and C5′), 115.3 (C3), 116.9

(C6), 128.5 (C5), 131.0 (C1), 134.7 (C4), 146.6 (C2), 152.5 (CO2tBu); m/z (ESI

+) 294

(100%), 372 ([M+Na]+, 50%); HRMS C18H28BNNaO5

+ requires 372.1953, found

372.1950.

tert-Butyl (3-methoxy-4-(4′,4′,5′,5′-tetramethyl-1′,3′,2′-dioxaborolan-2′-

yl)phenyl)carbamate 44


9.27 mmol), B2Pin2 (3.53 g, 13.90 mmol), KOAc (2.73 g, 27.81 mmol)

and Pd(PPh3)2Cl2 (0.33 g, 0.52 mmol) in 1,4-dioxane (18 mL). Purification

via column chromatography (eluent pet ether:EtOAc 95:5) gave boronic

ester 44 as a pink solid (0.90 g, 25%). mp 79-81 °C; νmax (neat) 3329 (N-H), 2977

(C-H), 1725 (C=O); δH (400 MHz, CDCl3), 1.34 (12H, s, C4′Me2 and C5′Me2), 1.51 (9H,


48

s, CMe3), 3.83 (3H, s, OMe), 6.65 (1H, d, J 1.7, H2), 6.73 (1H, dd, J 8.1, 1.7, H6), 7.20

(1H, s, NH), 7.60 (1H, d, J 8.1, H5); δC (125 MHz, CDCl3), 24.8 (C4′Me2 and C5′Me2),

28.3 (CMe3), 55.8 (OMe), 80.68 (CMe3), 83.2 (C4′ and C5′), 100.5 (C2), 109.5 (C6),

137.6 (C5), 142.6 (C1), 152.3 (CO2tBu), 165.4 (C3), C4 not observed; m/z (ESI

+) 294

(100%), 350 ([M-H]+, 100%); HRMS (ESI

+) C18H28BO5NNa


372.1947.



4.70 mmol), 4-bromonitrobenzene 35 (0.48 g, 2.35 mmol), Pd(PPh3)2Cl2

(0.17 g, 0.24 mmol) and sat. aq. NaHCO3 (2.5 mL) in THF (11 mL).

Purification via column chromatography (eluent pet ether:acetone 95:5 to

80:20) gave biaryl 45 as an orange solid (0.54 g, 72%). mp 149-151 °C; νmax (neat) 3435

(N-H), 2978 (C-H), 1727 (C=O); δH (400 MHz, CDCl3) 1.56 (9H, s, CMe3), 3.98 (3H, s,

OMe), 7.09 (1H, d, J 1.8, H3), 7.19 (1H, s, NH), 7.24 (1H, dd, J 8.2, 1.8, H5), 7.72 (2H,

d, J 8.8, H8 and H12), 8.22 (1H, d, J 8.2, H6), 8.28 (2H, d, J 8.8, H9 and H11);

δC (125 MHz, CDCl3) 28.3 (CMe3), 55.8 (OMe), 80.8 (CMe3), 108.7 (C3), 118.2 (C6),

120.4 (C5), 124.1 (C9 and C11), 127.3 (C8 and C12), 129.2 (C1), 132.7 (C4), 146.7 (C10),

147.4 (C7), 147.9 (C2), 152.6 (COtBu); m/z (ESI

+) 294 (100%), 372 ([M+Na]

+, 60%);

C18H20N2NaO5+ requires 367.1264, found 367.1280.



2.29 mmol), 4-bromonitrobenzene 35 (0.23 g, 1.15 mmol), Pd(PPh3)2Cl2

(0.08 g, 0.12 mmol) and sat. aq. NaHCO3 (1.5 mL) in THF (6 mL).

Purification via column chromatography (eluent pet ether:EtOAc 95:5)


49

gave biaryl 46 as a yellow solid (0.29 g, 73%). mp 134-136 °C; νmax (neat) 3347 (N-H),

2932 (C-H), 1705 (C=O); δH (700 MHz, CDCl3) 1.55 (9H, s, CMe3), 3.86 (3H, s, OMe),

6.62 (1H, s, NH), 6.85 (1H, dd, J 8.2, 2.0, H6), 7.25 (1H, d, J 8.2, H5), 7.39 (1H, app. s,

H2), 7.68 (2H, d, J 8.8, H8 and H12), 8.24 (2H, d, J 8.8, H9 and H11); δc (175 MHz,

CDCl3) 28.3 (CMe3), 55.6 (OMe), 81.0 (CMe3), 101.8 (C2), 110.5 (C6), 122.7 (C4),

123.2 (C9 and C11), 130.0 (C8 and C12), 130.8 (C5), 140.5 (C1), 145.2 (C7), 146.3 (C10),

152.5 (CO2tBu), 157.1 (C3); m/z (ESI

+) 367 ([M+Na]

+, 100%); HRMS (ESI

+)

C18H20N2NaO5+ requires 367.1264, found 367.1272.

Methyl 1-((tert-butoxycarbonyl)amino)-10-nitro-[4,7-biphenyl]-8-carboxylate 47


5.38 mmol), methyl-2-bromo-5-nitrobenzoate (0.70 g, 2.69 mmol),

Pd(PPh3)2Cl2 (0.19 g, 0.27 mmol) and sat. aq. NaHCO3 (2.5 mL) in THF

(11 mL). Purification via column chromatography (eluent pet ether:acetone

95:5 to 80:20) gave carbamate 47 as a yellow solid (0.45 g, 45%). mp 163 °C;

νmax (neat) 3425 (N-H), 1718 (C=O); δH (400 MHz, CDCl3) 1.54 (9H, s, CMe3), 3.75

(3H, s, CO2Me), 6.66 (1H, s, NH), 7.27 (2H, d, J 8.5, H2 and H6), 7.46 (2H, d, J 8.5, H3

and H5), 7.55 (1H, d, J 8.5, H12), 8.35 (1H, dd, J 8.5, 2.4, H11), 8.65 (1H, d, J 2.4, H9);

δC (125 MHz, CDCl3) 28.3 (CMe3), 52.6 (OMe), 80.9 (CMe3), 118.1 (C2 and C6), 125.2

(C9), 125.6 (C11), 128.9 (C3 and C5), 131.8 (C12), 131.9 (C8), 133.3 (C4), 139.0 (C1),

146.5 (C10), 148.1 (C7), 152.5 (CO2tBu), 167.2 (CO2Me); m/z (ESI

+) 395 ([M+Na]

+,

100%); HRMS (ESI-) C19H19O6N2

- requires 371.1249, found 371.1252.


Following General Procedure 5, using carbamate 45 (500 mg, 1.45 mmol) and TFA

(6.7 mL) in CH2Cl2 (35 mL) gave aniline 48 as an orange solid (259 mg, 73%). mp 123-


50

126 °C; νmax (neat) 3482 (N-H), 3383 (N-H), 2923 (C-H); δH (500 MHz,

CDCl3) 3.96 (3H, s, OMe), 6.81 (1H, d, J 8.0, H6), 7.06 (1H, d, J 1.9, H3),

7.13 (1H, dd, J 8.0, 1.9, H5), 7.68 (2H, d, J 8.8, H8 and H12), 8.26 (2H, d,

J 8.8, H9 and H11); δC (125 MHz, CDCl3) 55.6 (OMe), 109.2 (C3), 114.9 (C6), 120.6

(C5), 124.1 (C9 and C11), 126.6 (C8 and C12), 128.7 (C4), 137.5 (C1), 146.1 (C10), 147.5

(C2), 147.9 (C7); m/z (ESI+) 245 ([M+H]

+), 100%; HRMS (ESI

+) C13H12N2NaO3

+

requires 267.0740, found 267.0746.



and TFA (3.7 mL) in CH2Cl2 (20 mL) gave aniline 48 as an orange solid

(170 mg, 88%). mp 108-110 °C; νmax (neat) 3482 (N-H), 3384 (N-H), 2923

(C-H); δH (400 MHz, CDCl3) 3.81 (3H, s, OMe), 6.34 (1H, d, J 2.0, H2), 6.39 (1H, dd,

J 8.2, 2.0, H6), 7.17 (1H, d, J 8.2, H5), 7.67 (2H, d, J 8.9, H8 and H12), 8.21 (2H, d,

J 8.9, H9 and H11); δC (125 MHz, CDCl3) 55.4 (OMe), 98.4 (C2), 107.5 (C6), 118.4

(C10), 123.2 (C9 and C11), 129.7 (C8 and C12), 131.7 (C5), 145.8 (C4), 145.8 (C7), 148.7

(C1), 157.7 (C3); m/z (ESI+) 245 ([M-H]

+), 100%; HRMS (ESI

+) C13H12N2O3Na

+

requires 267.0740, found 267.0742.

Methyl 1-amino-10-nitro-[4,7-biphenyl]-8-carboxylate 51


and TFA (5.4 mL) in CH2Cl2 (28 mL) gave the aniline 51 as an orange

solid (246 mg, 77%). mp 182 °C; νmax (neat) 3463 (N-H), 3370 (N-H),

1713 (C=O); δH (400 MHz, CDCl3) 3.77 (3H, s, CO2Me), 6.76 (2H, d, J 7.7, H2 and H6),

7.16 (2H, d, J 7.7, H3 and H5), 7.54 (1H, d, J 8.3, H12), 8.32 (1H, d, J 8.3, H11), 8.61

(1H, s, H9); δC (100 MHz, CDCl3) 52.6 (OMe), 115.0 (C2 and C6), 125.1 (C9), 125.5


51

(C11), 128.9 (C4), 129.5 (C3 and C5), 131.5 (C12), 131.7 (C8), 146.0 (C10), 146.8 (C1),

148.5 (C7), 167.7 (CO2Me); m/z (ESI+) 273 ([M-H]

+, 100%), 295 ([M+Na]

+, 20%);

HRMS (ESI+) C14H12N2NaO4

+ ([M+Na]

+) requires 295.0689, found 295.0686.

1″-((1,4-Dioxo-2-(phenylsulfonamido)-1,4-dihydronaphthalen-3-yl)amino)-10″-

nitro-[4″,7″-biphenyl]-8″-carboxylic acid 52

1 M aq. NaOH (0.52 mL, 0.515 mmol) was added to ester 21

(60 mg, 0.103 mmol) in MeOH (3 mL), and the reactions mixture

refluxed at 60 °C for 48 h. After cooling to RT, diethyl ether (5 mL)

was added and the pH adjusted to 2 with 1 M aq. HCl. The organic

product was then extracted with EtOAc (3 x 10 mL), washed with

brine (2 x 10 mL), dried, filtered and concentrated in vacuo to yield the carboxylic acid

52 as a purple solid (59 mg, quant.). mp 231-233 °C; νmax (neat) 3520 (O-H), 3338

(N-H), 1723 (C=O); δH (500 MHz, DMSO-d6) 7.03 (2H, d, J 6.9, H2″ and H6″), 7.25

(2H, d, J 8.2, H3″ and H5″), 7.32-7.39 (2H, m, H3′ and H5′), 7.47 (1H, t, J 7.4, H4′), 7.58

(2H, d, J 7.7, H2′ and H6′), 7.68 (1H, d, J 8.5, H12″), 7.76-7.86 (3H, m, H6, H7 and H8),

8.02 (1H, d, J 7.4, H5), 8.41 (1H, dd, J 8.5, 2.4, H11″), 8.46 (1H, d, J 2.4, H9″), 9.14 (1H,

br. s, aniline-NH), 9.21 (1 H, br. s, sulfonamide-NH); C (125 MHz, DMSO-d6) 116.0

(C2), 122.5 (C2″ and C6″) 124.0 (C9″), 125.3 (C11″), 125.7 (C8), 126.3 (C5), 126.5 (C2′ and

C6′), 127.6 (C3″ and C5″), 128.6 (C3′ and C5′), 130.5 (C9), 131.4 (C10), 132.0 (C12″), 132.2

(C4′), 133.0 (C6 or C7 or C4″ or C8″), 133.2 (C6 or C7 or C4″ or C8″), 133.3 (C6 or C7 or

C4″ or C8″), 134.9 (C6 or C7), 139.3 (C1″), 140.9 (C1′), 141.5 (C3), 145.9 (C10″), 146.8

(C7″), 168.1 (-CO2H), 178.9 (C1), 182.1 (C4); m/z (ESI-) 568 ([M-H]

-, 100%); HRMS

C29H18N3O8S- requires 568.0815, found 5680.826.


52

N-(3-(((9″-Methoxy-[3″,8″-biphenyl]-2″-yl)methyl)amino)-1,4-dioxo-1,4-



0.13 mmol), amine 69 (80 mg, 0.38 mmol), and anhydrous CeCl3

(32 mg, 0.13 mmol) in anhydrous toluene (4 mL). Purification via

column chromatography (eluent pet ether: acetone 90:10 to 0:100)

gave naphthoquinone 63 as an orange solid (25 mg, 37%). mp 137-139 °C; νmax (neat)

3326 (N-H), 1672 (C=O), 1612 (C=O); H (500 MHz, DMSO-d6) 3.73 (3H, s, OMe),

4.67-4.89 (2H, m, 2 x H1″), 6.88-6.96 (2H, m, H12″ and amine-NH), 7.00 (1H, d, J 8.2,

H10″), 7.12-7.17 (2H, m, H4″ and H13″), 7.24 (1H,. ddd, J 8.2, 7.4, 1.7, H11″), 7.31-7.37

(3H, m, H5″, H6″ and H7″), 7.38-7.42 (2H, m, H3′ and H5′), 7.52 (1H, t, J 7.4, H4′), 7.63

(1H, dd, J 7.5, 1.3, H8), 7.66-7.71 (3H, m, H6, H2′ and H6′), 7.71-7.76 (1H, m, H7), 7.92

(1H, dd, J 7.6, 0.9, H5), 8.94 (1H, br. s, sulfonamide-NH); C (125 MHz, DMSO-d6)

45.9 (C1″), 55.3 (OMe), 109.0 (C2), 111.0 (C10″), 120.5 (C12″), 125.4 (C8), 126.1 (C5),

127.1 (C2′ and C6′), 127.2 (C5″ or C6″ or C7″), 127.6 (C5″ or C6″ or C7″), 128.0 (C5″ or C6″

or C7″), 128.5 (C3′ and C5′), 128.7 (C8″), 129.0 (C11″), 129.7 (C9), 130.4 (C4″ or C13″),

130.6 (C4″ or C13″),, 131.8 (C10), 132.4 (C4′), 132.5 (C6), 135.1 (C7), 137.0 (C2″ or C3″),

137.9 (C2″ or C3″), 140.3 (C1′), 145.2 (C3), 156.0 (C9″), 177.6 (C1), 182.0 (C4); m/z (ESI-)

523 ([M-H]-, 100%); HRMS (ESI

-) C30H23O5N2S

- requires 523.1333, found 523.1336.



Following General Procedure 1, using naphthoquinone 23 (87 mg, 0.25 mmol), amine

81 (160 mg, 0.75 mmol) and anhydrous CeCl3 (61 mg, 0.25 mmol) in anhydrous

toluene (4 mL). Purification via column chromatography (eluent pet ether: acetone


53

90:10 to 0:100) gave naphthoquinone 64 as an orange solid

(33 mg, 25%). mp 157-159 °C νmax (neat) 3332 (N-H), 1610

(C=O), 1572; H (400 MHz, DMSO-d6) 3.64 (3H, s, OMe), 5.01

(2H, br. s, 2 x H1″), 6.97-7.03 (1H, m, H12″), 7.06 (1H, d, J 8.2,

H10″), 7.25-7.38 (6H, m, H3″. H5″, H6″, H7″, H11″ and H13″), 7.40-7.46 (2H, m, H3′ and

H5′), 7.51-7.57 (1H, m, H4′), 7.63 (1H, d, J 7.3, H8), 7.66-7.77 (4H, m, H6, H7, H2′ and

H6′), 7.86 (1H, br. s, amine-NH), 7.96 (1H, d, J 7.0, H5), 9.07 (1H, br. s,

sulfonamide-NH); C (125 MHz, DMSO-d6) 46.6 (C1″), 55.3 (OMe), 109.7 (C2) 111.7

(C10″), 120.7 (C12″), 125.5 (C8), 125.6 (C3″ or C5″ or C7″), 126.1 (C11″), 126.2 (C5), 127.1

(C2′ and C6′), 128.0 (C6″), 128.4 (C3″ or C5″ or C7″), 128.5 (C3′ and C5′), 128.9 (C3″ or C5″

or C7″), 129.7 (C8″), 129.8 (C9), 130.4 (C13″), 131.8 (C10), 132.4 (C6), 132.6 (C4′), 135.1

(C7), 138.3 (C2″ or C4″), 139.1 (C2″ or C4″), 140.6 (C1′), 144.1 (C3), 156.1 (C9″), 177.8

(C1), 182.3 (C4) m/z (ESI-) 523 ([M-H]

-, 100%); HRMS (ESI

-) C30H23O5N2S

- requires

523.1333, found 523.1338.



Following General Procedure 1, using naphthoquinone 23

(65 mg, 0.19 mmol), amine 82 (120 mg, 0.56 mmol), and

anhydrous CeCl3 (46 mg, 0.19 mmol) in anhydrous toluene

(4 mL). Purification via column chromatography (eluent pet

ether:acetone 90:10 to 0:100) gave naphthoquinone 65 as an orange solid (37 mg, 38%).

mp 148-150 °C; νmax (neat) 3353 (N-H), 3134 (C-H), 1632 (C=O); H (400 MHz,

DMSO-d6) 3.73 (3H, s, OMe), 5.01 (2H, br. s, 2 x H1″), 6.96-7.04 (1H, m, H12″), 7.09

(1H, d, J 8.0, H10″), 7.25 (1H, d, J 7.6, H13″), 7.32 (1H, app. t, J 8.0, H11″), 7.35-7.47


54

(6H, m, H3′, H5′, H3″, H4″, H6″ and H7″), 7.52-7.60 (1H, m, H4′), 7.63 (1H, d, J 7.3, H8),

7.67-7.74 (2H, m, H6 and H7), 7.76 (2H, d, J 7.8, H2′ and H6′), 7.87 (1H, br. s,

amine-NH), 7.97 (1H, d, J 7.2, H5), 9.06 (1H, br. s, sulfonamide-NH); C (125 MHz,

DMSO-d6) 46.3 (C1″), 55.4 (OMe), 109.6 (C2), 111.7 (C10″), 120.8 (C12″), 125.5 (C8),

126.2 (C5), 127.1 (C2′ and C6′), 127.2 (C3″ and C7″), 128.5 (C3′ and C5′), 128.9 (C11″),

129.3 (C4″ and C6″), 129.5 (C8″), 129.9 (C9), 130.3 (C13″), 131.8 (C10), 132.4 (C6), 132.6

(C4′), 135.1 (C7), 136.9 (C5″), 137.9 (C2″), 140.7 (C1′), 144.2 (C3), 156.1 (C9″), 177.8

(C1), 182.3 (C4); m/z (ESI-) 523 ([M-H]

-, 100%); HRMS (ESI

-) C30H23O5N2S1 requires

523.1333, found 523.1338.

(9-Methoxy-[3,8-biphenyl]-2-yl)methanamine 69


and TFA (8 mL) in CH2Cl2 (40 mL), stirring at 0 °C for 30 min, gave

amine 69 as a yellow oil (242 mg, 99%). νmax (neat) 3060 (N-H), 3021 (N-H), 1479;

H (400 MHz, CDCl3) 3.76 (3H, s, OMe), 4.21 (1H, d, J 15.4, 1 x H1), 4.36 (1H, d,

J 15.4, 1 x H1), 6.96 (1H, d, J 8.4, H10), 7.01 (1H, app. t, J 7.5, H12), 7.15-7.21 (2H, m,

H4 and H13), 7.28-7.40 (3H, m, H5, H6 and H11), 7.47 (1H, d, J 7.5, H7); C (125 MHz,

CDCl3) 44.3 (C1), 55.4 (OMe), 110.7 (C10), 120.7 (C12), 126.7 (C5), 127.5 (C7), 127.9

(C6), 128.9 (C11), 129.9 (C8), 130.4 (C4), 131.1 (C13), 137.6 (C3), 141.4 (C2), 156.4 (C9);

m/z (ESI+) 214 ([M+H]

+, 100%); HMRS (ESI

+) C14H16ON


214.1220.

tert-Butyl ((9-methoxy-[3,8-biphenyl]-2-yl)methyl)carbamate 74


2.50 mmol), 2-methoxyphenylboronic acid (0.76 g, 5.00 mmol),

Pd(PPh3)2Cl2 (0.18 g, 0.25 mmol) and sat. aq. NaHCO3 (2.5 mL) in


55

THF (11 mL). Purification via column chromatography (eluent pet ether:acetone 95:5)

gave biaryl 74 as a white solid (0.53 g, 68%). mp 97-101 °C; νmax (neat) 3419 (N-H),

2975 (C-H), 1715 (C=O); δH (400 MHz, CDCl3) 1.42 (9H, s, CMe3), 3.78 (3H, s, OMe),

4.05-4.22 (2H, m, 2 x H1), 4.79 (1H, br. s, NH), 6.97 (1H, d, J 8.2, H10), 7.03 (1H, app.

t, J 7.5, H12), 7.15 (1H, dd, J 7.5, 1.6, H13), 7.18-7.21 (1H, m, H4), 7.30-7.40 (3H, m,

H5, H6 and H11), 7.45 (1H, d, J 7.3, H7); δC (125 MHz, CDCl3) 28.4 (CMe3), 42.6 (C1),

55.4 (OMe), 79.1 (CMe3), 110.7 (C10), 120.7 (C12), 127.2 (C5), 127.8 (C6), 128.1 (C7),

129.0 (C11), 129.6 (C8), 130.3 (C4), 131.0 (C13), 137.4 (C2), 138.0 (C3), 155.8 (CO2tBu),

156.3 (C9); m/z (ESI+) 336 ([M+Na]

+, 100%); HRMS (ESI

+) C19H23NNaO3

+ requires

336.1570, found 336.1560.



2.62 mmol), 2-methoxybenzylboronic ester (0.80 g, 5.24 mmol),

Pd(PPh3)Cl2 (0.18 g, 0.26 mmol) and sat. aq. NaHCO3 (2.5 mL) in

THF (11 mL). Purification via column chromatography (eluent pet ether:EtOAc 95:5 to

90:10) gave biaryl 79 as a white solid (378 mg, 46%). mp 65-67 °C; νmax (neat) 3347

(N-H), 2976 (C-H), 1691 (C=O);H (500 MHz, CDCl3) 1.49 (9H, s, CMe3), 3.82 (3H, s,

OMe), 4.38 (2H, br. s, H1), 4.90 (1H, br. s, -NH), 7.00 (1H, d, J 8.2, H10), 7.05 (1H, app.

td, J 7.5, 0.9, H12), 7.27 (1H, d, J 7.7, H7), 7.30-7.37 (2H, m, H11 and H13), 7.39 (1H,

app. t, J 7.7, H6), 7.43-7.47 (2H, m, H3 and H5); C (125 MHz, CDCl3) 28.4 (CMe3),

44.8 (C1), 55.5 (OMe), 79.4 (CMe3), 111.1 (C10), 120.8 (C12), 126.1 (C7), 128.2 (C6),

128.6 (C3 or C5 or C11), 128.6 (C3 or C5 or C11), 128.7 (C3 or C5 or C11), 130.3 (C8),

130.8 (C13), 138.5 (C2), 138.8 (C4), 155.8 (CO2tBu), 156.4 (C9); m/z (ESI

+) 336

([M+Na]+, 100%); HRMS C14H16NO

+ requires 214.1226, found 214.1222.


56



2.50 mmol), 2-methoxybenzylboronic acid (0.76 g, 5.00 mmol),

Pd(PPh3)2Cl2 (0.18 g, 0.25 mmol) and sat. aq. NaHCO3

(2.5 mL) in THF (11 mL). Purification via column chromatography (eluent pet

ether:EtOAc 95:5 to 85:15) gave biaryl 80 as a white solid (0.59 g, 75%). mp 104-

107 °C; νmax (neat) 3385 (N-H), 2922 (C-H), 1677 (C=O); δH (400 MHz, CDCl3) 1.49

(9H, s, CMe3), 3.82 (3H, s, OMe), 4.37 (2H, d, J 4.6, 2 x H1), 4.86 (1H, br. s, NH), 7.00

(1H, d, J 8.3, H10), 7.04 (1H, td, J 7.5, 1.0, H12), 7.29-7.37 (4H, m, H3, H7, H11 and H13),

7.51 (2H, d, J 8.1, H4 and H6); δC (125 MHz, CDCl3) 30.9 (CMe3), 45.9 (C1), 55.5

(OMe), 77.0 (CMe3) 111.1 (C10), 120.8 (C12), 127.0 (C3 and C7), 128.6 (C11), 129.8 (C4

and C6), 130.3 (C8), 130.8 (C13), 137.4 (C5), 140.4 (C2), 156.4 (C9), 161.8 (CO2tBu); m/z

(ESI+) 336 ([M+Na]

+, 100%); HRMS (ESI

+) C19H23NNaO3


336.1565.



and TFA (4 mL) in CH2Cl2 (20 mL) gave amine 81 as a grey oil (170 mg,

quant.). νmax (neat) 3045 (N-H), 3022 (N-H), 1563;H (500 MHz, CDCl3)

3.80 (3H, s, OMe), 3.95 (2H, br. s, H1), 6.98 (1H, d, J 8.5, H10), 7.01-7.04 (1H, m, H12),

7.29-7.35 (3H, m, H7, H11 and H13), 7.38 (1H, t, J 7.5, H6), 7.44 (1H, d, J 7.5, H5), 7.48

(1H, s, H3); C (125 MHz, CDCl3) 45.9 (C1), 55.5 (OMe), 111.2 (C10), 120.8 (C12),

126.1 (C7), 128.3 (C6), 128.6 (C5), 128.7 (C11 or C13), 128.7 (C3), 130.4 (C8), 130.9 (C4),

130.9 (C11 or C13), 138.9 (C2), 156.4 (C9); m/z (ESI+) 214 ([M+H]

+, 100%); HMRS

(ESI+) C14H16NO

+ requires 214.1226, found 214.1228.


57


Following General Procedure 5, using carbamate 80 (250 mg,

0.80 mmol) and TFA (4 mL) in CH2Cl2 (20 mL) gave amine 82 as a

green oil (128 mg, 75%). νmax (neat) 3038 (N-H), 3010 (N-H), 1511; H (400 MHz,

CDCl3) 3.82 (3H, s, OMe), 3.94 (2H, br. s, H1), 6.99 (1H, d, J 8.2, H10), 7.04 (1H, t,

J 7.5, H12), 7.29-7.41 (4H, m, H3, H7, H11 and H13), 7.52 (2H, d, J 7.6, H4 and H6);

C (125 MHz, CDCl3) 45.9 (C1), 55.5 (OMe), 111.2 (C10), 120.8 (C12), 127.0 (C3 and

C7), 128.6 (C11), 129.8 (C4 and C6), 130.3 (C8), 130.8 (C13), 137.4 (C5), 140.5 (C2),

156.4 (C9); m/z (ESI+) 214 ([M+H]

+, 100%), 236 ([M+Na]

+, 30%); HMRS (ESI

+)

C14H16NO+ requires 214.1226, found 214.1222.

N-(3-((13″H-Fluoren-1″-yl)amino)-1,4-dioxo-1,4-dihydronaphthalen-2-



0.24 mmol), 2-aminofluorene (133 mg, 0.73 mmol) and

CeCl3∙7H2O (91 mg, 0.24 mmol) in MeOH (7 mL). Subsequent

recrystallization from boiling MeOH gave naphthoquinone 83 as a

purple solid (125 mg, quant.). mp 251-255 °C; νmax (neat) 3325 (N-H), 1627 (C=O),

1602 (C=O); (500 MHz, DMSO-d6) 3.87 (2H, s, 2 x H13″), 7.05 (1H, dd, J 8.2, 1.7,

H6″), 7.18 (1H, s, H2″), 7.26-7.33 (3H, m, H3′, H5′ and H10″), 7.35-7.40 (1H, m, H11″),

7.40-7.45 (1H, t, J 7.4, H4′), 7.52 (2H, d, J 8.4, H2′ and H6′), 7.57 (1H, d, J 7.4, H9″),

7.72 (1H, d, J 8.2, H5″), 7.75-7.83 (3H, m, H6, H7 and H8), 7.85 (1H, d, J 7.6, H12″), 8.03

(1H, d, J 7.3, H5), 9.10 (1H, br. s, sulfonamide-NH), 9.16 (1H, s, aniline-NH);

C (125 MHz, DMSO-d6) 36.4 (C13″), 113.5 (C2), 118.9 (C5″), 119.6 (C12″), 120.2 (C2″),

122.3 (C6″), 125.0 (C9″), 125.7 (C8), 126.1 (C5 or C10″), 126.2 (C5 or C10″), 126.4 (C2′


58

and C6′), 126.8 (C11″), 128.4 (C3′ and C5′), 130.3 (C9), 131.6 (C10), 132.0 (C4′), 133.0

(C6), 134.9 (C7), 136.8 (C4″), 137.4 (C1″ or C3″), 141.1 (C1′), 141.2 (C7″), 142.0 (C3),

142.3 (C1″ or C3″), 143.0 (C8″), 178.6 (C1), 182.4 (C4); m/z (ESI-) 491 ([M-H]

-, 100%);

HRMS (ESI-) C29H19O4N2S

- requires 491.1071, found 491.1074.

N-(3-(7″-amino-4″-methylcoumarin)-1,4-dioxo-1,4-dihydronaphthalen-2-



0.48 mmol), coumarin 84 (250 mg, 1.43 mmol) and anhydrous

CeCl3 (117 mg, 0.48 mmol) in MeOH (2 mL), at 110 ºC for 72 h.

Purification via preparative thin layer chromatography (eluent

CH2Cl2:MeOH 98:2) gave naphthoquinone 85 as a red solid (38 mg, 16%). mp 219-

222 °C; νmax (neat) 3352 (N-H), 1730 (lactone C=O), 1600 (C=O); δH (500 MHz,

DMSO-d6) 2.42 (3H, s, C4″Me), 6.26 (1H, s, H3″), 6.88 (1H, app. s, H8″), 7.04 (1H, dd,

J 8.2, 1.7, H6″), 7.32 (2H, app. t, J 8.2, H3′ and H5′), 7.45 (1 H, t, J 8.2, H4′), 7.56 (3H,

app. d, J 8.2, H2′, H6′ and H5″), 7.77-7.85 (3H, m, H6, H7 and H8), 8.02-8.06 (1H, m, H5),

9.30 (1H, br. s, NH), 9.36 (1H, s, NH); δC (125 MHz, DMSO-d6) 18.2 (C4″Me), 109.4

(C8″), 112.0 (C3″) 114.6 (C9″) 118.9 (C6″), 124.2 (C5″), 125.8 (C8), 126.3 (C5), 126.4 (C2′

and C6′), 128.5 (C3′ and C5′), 130.5 (C9), 131.3 (C10), 132.2 (C4′), 133.4 (C6 or C7), 134.9

(C7 or C7), 140.8 (C1′) 142.6 (C4″ or C7″), 152.9 (C4″ or C7″ or C10″), 153.4 (C4″ or C7″ or

C10″), 160.3 (C2″), 179.1 (C1), 182.1 (C4), C2 and C3 not observed; m/z (ESI-) 485

([M-H]-, 100%); HRMS (ESI

+) C26H19O6N2S

+ requires 487.0958, found 487.0755.

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Appendix 1: Supplementary Experimental Data

61



Following General Procedure 5, using carbamate 32 (0.55 g, 1.60 mmol)

and TFA (8 mL) in CH2Cl2 (40 mL). Purification via column

chromatography (eluent pet ether:acetone 80:20) gave the aniline 14 as an

orange solid (0.34 g, 86%). mp 133-135 °C; δH (400 MHz, CDCl3) 3.83 (3H, s, CMe3),

6.61 (2H, d, J 8.5, H2 and H6), 7.24 (2H, d, J 8.5, H3 and H5), 7.38 (1H, d, J 8.4, H12),

7.69 (1H, d, J 2.2, H9), 7.73 (1H, dd, J 8.4, 2.2, H11); m/z (ESI+) 245 ([M+H]

+, 10%),

267 ([M+Na]+, 100%).

N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzenesulfonamide 2325

2,3-Dichloronaphthalene-1,4-dione 22 (2.00 g, 8.80 mmol),

benzenesulfonamide (1.38 g, 8.80 mmol) and Cs2CO3 (4.00 g,

12.32 mmol) were stirred in DMF (10 mL) at RT for 5 h. 1 M aq. HCl (50 mL) was

added and the crude product was extracted with EtOAc (3 x 50 mL). The organic layers

were combined and washed with brine (2 x 50 mL), dried, filtered and concentrated in

vacuo. Purification via column chromatography (eluent pet ether:acetone 80:20 to

50:50) gave sulfonamide 23 as a yellow solid (2.95 g, 96%). mp 234-237 °C {lit. 218-

223 °C}25

; δH (400 MHz, DMSO-d6) 7.59-7.64 (2H, m, H3′ and H5′), 7.65-7.71 (1H, m,

H4′), 7.83-7.91 (2H, m, H6 and H7), 7.92-7.98 (3H, m, H2′, H6′ and H5 or H8), 8.03-8.07

(1H, m, H5 or H8); m/z (ESI-) 346 ([M(

35Cl)-H]

-, 100%), 348 ([M(

37Cl)-H]

-, 33%).

4-Nitro-3-methoxy-1-bromobenzene 2652

70% HNO3 (10 mL) was stirred at 0 °C, and conc. H2SO4 (8 mL) was added

dropwise over 15 mins. 3-Bromoanisole 24 (5.4 mL, 42.8 mmol) was then

added dropwise, and the mixture subsequently stirred at 50 °C for 16 h. The reaction


62

mixture was cooled to RT, poured over ice cold water (200 mL), and extracted with

EtOAc (3 x 60 mL). The organic layers were collected and washed with water

(2 x 100 mL) and brine (2 x 100 mL), dried, filtered and concentrated in vacuo.

Purification via column chromatography (eluent pet ether:diethyl ether 0:100 to 5:95)

gave nitrophenyl 26 as a yellow solid (1.55 g, 16%). mp 105-109 °C {lit. 91-92}71

;

δH (400 MHz, CDCl3) 3.98 (3H, s, OMe), 7.19 (1H, dd, J 8.6, 1.8, H6), 7.25 (1 H, d,

J 1.8, H2), 7.77 (1H, d, J 8.6, H5); m/z (ESI+) 254 ([M(

79Br)+Na]

+, 100%), 256

([M(81

Br)+Na]+, 100%).

tert-Butyl (4-bromophenyl)carbamate 2853

Method 1: 4-bromoaniline 27 (5.00 g, 29.0 mmol) was dissolved in THF

(120 mL) at RT and a solution of Boc2O (7.63 g, 35.0 mmol) and NaOH

(2.56 g, 64.0 mmol) in H2O (120 mL) was added portionwise over 30 min.

The reaction mixture was then stirred at 60 °C for 16 h. The resulting mixture was

cooled to RT and extracted with CH2Cl2 (3 x 100 mL). The organic layers were

combined, washed with H2O (2 x 50 mL), dried, filtered, and concentrated in vacuo.

Purification via column chromatography (eluent pet ether:EtOAc 95:5) gave carbamate

28 as a white solid (2.79 g, 35%).

Method 2: Following General Procedure 4, using 4-bromoaniline (5.00 g, 29.0 mmol),

Boc2O (7.61 g, 34.9 mmol) and InCl3 (105 mg, 0.29 mmol). Purification via column

chromatography (eluent pet ether:EtOAc 95:5) gave carbamate 28 as a white solid

(7.04 g, 89%). mp 102-104 °C {lit. 102 °C}53

; δH (400 MHz, CDCl3) 1.52 (9H, s,

CMe3), 6.46 (1H, br. s, NH), 7.26 (2H, d, J 8.5, H3 and H5), 7.38 (2H, d, J 8.5, H2 and

H6); m/z (ESI+) 394 ([M(

79Br)+Na]

+, 100%), 396 ([M(

81Br)+Na]

+, 100%).


63

2′-(2-Methoxy-4-nitrophenyl)-4′,4′,5′,5′-tetramethyl-1′,3′,2′-dioxaborolane 3051

Following General Procedure 2, using 2-bromo-5-nitroanisole 29 (2.00 g,

8.62 mmol), B2Pin2 (3.28 g, 12.93 mmol), potassium acetate (2.53 g,

25.86 mmol) and Pd(PPh3)2Cl2 (0.30 g, 0.43 mmol) in 1,4-dioxane (12 mL).

Purification via column chromatography (eluent pet ether:EtOAc 90:10 to 50:50) gave

boronic ester 30 as a white solid (1.38 g, 58%). mp 101-104 °C; δH (400 MHz, CDCl3)

1.38 (12H, s, C4′Me2 and C5′Me2), 3.93 (3H, s, OMe), 7.67 (1H, s, H3), 7.79 (2H, app. s,

H5 and H6); m/z (ESI+) 302 ([M+Na]

+, 100%).

tert-Butyl (10-nitro-8-methoxy-[4,7-biphenyl]-1-yl)carbamate 3251


4.31 mmol), aryl bromide 28 (0.59 g, 2.16 mmol), Pd(PPh3)2Cl2 (0.15 g,

0.22 mmol) and sat. aq. NaHCO3 (2.1 mL) in THF (10 mL). Purification

via column chromatography (eluent pet ether:acetone 95:5 to 80:20) gave

the biaryl 32 as a yellow solid (0.60 g, 81%). mp 151-155 °C; δH (400 MHz, CDCl3)

1.55 (9H, s, CMe3), 3.92 (3H, s, OMe), 6.57 (1H, br. s, NH), 7.43-7.47 (3H, m, H2, H6

and H12), 7.48-7.52 (2H, m, H3 and H5), 7.82 (1H, d, J 2.2, H9), 7.91 (1H, dd, J 8.5, 2.2,

H11); m/z (ESI+) 367 ([M+Na]

+, 100%).

tert-Butyl (4-(4′,4′,5′,5′-tetramethyl-1′,3′,2′-dioxaborolan-2′-yl)phenyl)carbamate

3872


11.0 mmol), B2Pin2 (4.20 g, 16.5 mmol), KOAc (3.24 g, 33.1 mmol) and

Pd(PPh3)2Cl2 (0.39 g, 0.6 mmol) in 1,4-dioxane (18 mL). Purification via

column chromatography (eluent pet ether:EtOAc 90:10 to 50:50) gave

boronic ester 38 as a pale yellow solid (2.75 g, 78%). mp 118-122 °C; δH (400 MHz,


64

CDCl3) 1.34 (12H, s, C4′Me2 and C5′Me2), 1.53 (9H, s, -CMe3), 6.55 (1H, br. s, NH),

7.37 (2H, d, J 8.4, H3 and H5), 7.74 (2H, d, J 8.4, H2 and H6); m/z (ESI+) 342 ([M+Na]

+,

100%).

tert-Butyl (4-bromo-2-methoxyphenyl)carbamate 4173

Following General Procedure 4, using 4-bromo-2-methoxyaniline 39

(2.50 g, 12.4 mmol), Boc2O (2.70 g, 12.4 mmol) and InCl3 (28 mg, 0.13

mmol) gave carbamate 41 as a black oil (3.56 g, 96%). δH (400 MHz,

CDCl3) 1.52 (9H, s, CMe3), 3.86 (3H, s, OMe), 6.96 (1H, d, J 2.0, H3), 7.01 (1H, s,

NH), 7.07 (1H, dd, J 8.5, 2.0, H5), 7.97 (1H, d, J 8.5, H6); m/z (ESI+) 246 (100%), 248

(100%), 324 ([M(79

Br)+Na]+, 50%), 326 ([M(

81Br)+Na]

+, 50%).

tert-Butyl (4-bromo-3-methoxyphenyl)carbamate 42

Following General Procedure 4, using 4-bromo-3-methoxyaniline 40

(2.00 g, 9.90 mmol), Boc2O (2.18 g, 10.00 mmol) and InCl3 (22 mg, 0.01

mmol) gave carbamate 42 as a white solid (2.63 g, 88%). mp 135-139 °C;

νmax (neat) 3327 (N-H), 2979 (C-H), 1697 (C=O); δH (400 MHz, CDCl3) 1.53 (9H, s,

CMe3), 3.91 (3H, s, OMe), 6.51 (1H, s, NH), 6.63 (1H, dd, J 8.5, 2.4, H6), 7.32 (1H,

app. s, H2), 7.39 (1H, d, J 8.5, H5); δC (125 MHz, CDCl3) 28.3 (CMe3), 56.2 (OMe),

80.9 (CMe3), 102.7 (C2), 104.5 (C4), 111.3 (C6), 133.0 (C5), 139.0 (C1), 152.4

(-CO2tBu), 156.2 (C3); m/z (ESI

+) 324 ([M(

79Br)+Na]

+, 100%), 326 ([M(

81Br)+Na]

+,

100%); HRMS (ESI+) C12H16BrNNaO3

+ requires 324.0206, found 324.0203.

10-Nitro-[4,7-biphenyl]-1-amine 5074

Following General Procedure 5, using carbamate 37 (570 mg, 1.82 mmol) and

TFA (8.4 mL) in CH2Cl2 (44 mL) gave amine 50 as an orange solid (336 mg,

86%). mp 206 °C {lit. 202-203 °C}74

; δH (400 MHz, CDCl3) 3.90 (2H, br. s,


65

NH2), 6.79 (2H, d, J 8.5, H2 and H6), 7.48 (2H, J 8.5, H3 and H5), 7.67 (2H, J 8.8, H8

and H12), 8.25 (2H, J 8.8, H9 and H11); m/z (ESI+) 215 ([M+H]

+, 50%), 237 ([M+Na]

+,

100%).

9-Methoxy-[3,8-biphenyl]-2-carbaldehyde 70

Following General Procedure 3, using 2-bromobenzaldehyde 66 (152 mg,

0.82 mmol), 2-methoxyphenylboronic acid (250mg, 1.65 mmol),

Pd(PPh3)2Cl2 (57 mg, 0.08 mmol) and sat. aq. NaHCO3 (2.5 mL) in THF

(11 mL).Purification via column chromatography (eluent pet ether:EtOAc 95:5to 90:10)

gave biaryl 70 as a white solid (108 mg, 62%). mp 92-98 °C; νmax (neat) 2838 (C-H),

1694 (C=O), 1596; δH (500 MHz, CDCl3) 3.75 (3H, s, OMe), 6.99 (1H, d, J 8.2, H10),

7.10 (1H, app. td, J 7.6, 0.9, H12), 7.30 (1H, dd, J 7.6, 1.7, H13), 7.37 (1H, dd, J 7.6, 0.8,

H4), 7.41-7.45 (1H, m, H11), 7.49 (1H, app. t, J 7.6, H6), 7.65 (1H, app. td, J 7.6, 1.4,

H5), 8.01 (1H, dd, J 7.6, 1.4, H7), 9.80 (1H, s, H1); δC (125 MHz, CDCl3) 55.4 (OMe),

110.6 (C10), 121.0 (C12), 126.6 (C7), 126.8 (C8), 127.7 (C6), 130.0 (C11), 131.2 (C4),

131.4 (C13), 133.7 (C5), 134.0 (C2), 141.8 (C3), 156.5 (C9), 192.7 (C1); m/z (ESI+) 213

([M+H]+, 100%), 235 ([M+Na]

+, 100%) HRMS (ESI

+) C14H13O2

+ requires 213.0910,

found 213.0910.

(ortho-Bromophenyl)methanamine 7275

2-bromobenzaldehyde 66 (1.45 mL, 12.5 mmol) was stirred in sat. ethanolic

NH4OAc (250 mL), before NaCNBH3 (2.35 g, 37.5 mmol) and 30% aq. NH3

(100 mL) were added, and the resulting mixture was refluxed for 18 h. The reaction

mixture was cooled to RT and basified with 1 M aq. NaOH (50 mL), extracted with

CH2Cl2 (3 x 100 mL), washed with brine (3 x 100 mL), dried, filtered and concentrated

in vacuo. Purification via column chromatography (eluent pet


66

ether:EtOAc:triethylamine 95:4.8:0.2 to 70:29.8:0.2), gave amine 72 as a colourless oil

(0.84 g, 36%). H (400 MHz, CDCl3) 4.48 (1H, app. s, 1 x H1), 4.55 (1H, app. s, 1 x H1),

7.09 (1H, app. t, J 7.6, H5), 7.24-7.30 (1H, m, H6), 7.37-7.56 (2H, m, H4 and H7); m/z

(ESI+) 186 ([M(

79Br)+H]

+, 100%), 188 ([M(

81Br)+H]

+, 100%).

tert-Butyl ortho-bromobenzylcarbamate 7376

Following General Procedure 4, using benzyl amine 72 (3.00 g,

13.5 mmol), Boc2O (2.94 g, 13.5 mmol) and InCl3 (30 mg,

0.14 mmol) gave carbamate 73 as a white solid (3.80 g, 98%). mp 59-61 °C {lit. 51-

53 °C}76

; (400 MHz, CDCl3) 1.46 (9H, s, CMe3), 4.40 (2H, d, J 6.0, 2 x H1), 5.03

(1H, br. s, NH), 7.15 (1H, app. td, J 7.7, 1.4, H5), 7.30 (1H, app. t, J 7.7 H6), 7.39 (1H,

d, J 7.5, H7), 7.55 (1H, d, J 7.7, H4); m/z (ESI+) 308 ([M(

79Br)+Na]

+, 100%), 310

([M(81

Br)+Na]+, 100%).

tert-Butyl meta-bromobenzylcarbamate 7777

Following General Procedure 4, using 3-bromobenzylamine

hydrochloride 75 (3.00 g, 16.1 mmol), Boc2O (3.52 g, 16.1 mmol),

InCl3 (36 mg, 0.2 mmol) and triethylamine (3.8 mL, 27.0 mmol) gave carbamate 77 as a

white solid (4.25 g, 92%). mp 81-84 °C {lit. 59-60 °C}77

; H (400 MHz, CDCl3) 1.47

(9H, s, CMe3), 4.29 (2H, d, J 5.5, 2 x H1), 4.91 (1H, br. s, NH), 7.19-7.22 (2H, m, H6

and H5 or H7), 7.38-7.41 (1H, m, H5 or H7), 7.43 (1H, s, H3); m/z (ESI+) 308

([M(79

Br)+Na]+, 100%), 310 ([M(

81Br)+Na]

+, 100%).

tert-Butyl para-bromobenzylcarbamate 7878

Following General Procedure 4, using 4-bromobenzylamine

hydrochloride 76 (3.00 g, 13.50 mmol), Boc2O (2.94 g,

13.50 mmol), InCl3 (30 mg, 0.14 mmol) and 1 M aq. NaOH (13.5 mL, 13.50 mmol)


67

gave carbamate 78 as a white solid (2.94 g, 76%). mp= 59-65 °C {lit. 92-93 °C}78

;

δH (400 MHz, CDCl3) 1.46 (9H, s, CMe3), 4.27 (2H, d, J 5.0, 2 x H1), 4.87 (1H, br. s,

NH), 7.17 (2H, d, J 8.2, H3 and H7), 7.45 (2H, d, J 8.2, H4 and H6); m/z (ESI+) 230

(50%), 232 (50%), 308 ([M(79

Br)+Na]+, 100%), 310 ([M(

81Br)+Na]

+, 100%).

7-Amino-4-methylcoumarin 8465

Method 1: Carbethoxyamine 89 (280 mg, 1.13 mmol) was added to a

mixture of H2SO4 (0.5 mL) and glacial acetic acid (0.5 mL) and stirred

at 120 °C for 4 h. The resulting mixture was poured onto ice-cold water (15 mL) and

left to stand for 16 h. The resulting suspension was basified with cold aq. 1 M NaOH,

and the precipitate collected via suction filtration, which was further washed with cold

water. Subsequent recrystallization from boiling ethanol afforded coumarin 84 as a light

brown solid (132 mg, 67%).

Method 2: A mixture of m-aminophenol 87 (0.55 g, 5.00 mmol), ethyl acetoacetate

(0.64 mL, 5.00 mmol) and InCl3 (55 mg, 0.25 mmol) were stirred at 75 °C for 48 h. The

reaction mixture was poured onto ice-cold water, and the precipitate filtered and washed

with cold water. Subsequent recrystallization from boiling ethanol afforded coumarin 84

as a light brown solid (182 mg, 21%). mp 198-200 °C {lit. 220-222 °C}65

; δH (400

MHz, DMSO-d6) 2.29 (3H, s, C4Me), 5.90 (1H, s, H3), 6.11 (2H, s, NH2), 6.40 (1H, s,

H8), 6.55 (1H, d, J 8.0, H6), 7.40 (1H, d, J 8.0, H5); m/z (ESI+) 176 ([M+H]

+, 100%),

198 ([M+Na]+, 40%).

N-(3-((3″-hydroxyphenyl)amino)-1,4-dioxo-1,4-dihydronaphthalen-2-


Following General Procedure 1, using naphthoquinone 23 (300 mg, 0.86 mmol),

m-aminophenol 87 (282 mg, 2.59 mmol), CeCl3∙7H2O (321 mg, 0.86 mmol) in MeOH


68

(7 mL). Subsequent recrystallization from boiling MeOH gave

naphthoquinone 86 as a purple solid (298 mg, 82%). mp 179-

183 °C; νmax (neat) 3300 (O-H), 2925 (C-H), 1674 (C=O);

H (400 MHz, DMSO-d6) 6.41-6.51 (3H, m, H2″, H4″ and H6″), 7.02 (1H, app. t, J 8.3,

H5″), 7.37 (2H, app. t, J 7.6, H3′ and H5′), 7.49 (1H, t, J 7.6, H4′), 7.60 (2H, d, J 7.6, H2′

and H6′), 7.69-7.82 (3H, m, H6, H7 and H8), 7.95-8.01 (1H, m, H5), 8.84 (1H, br. s,

aniline-NH), 8.98 (1H, br. s, sulfonamide-NH), 9.26 (1H, s, OH); C (125 MHz,

DMSO-d6) 99.4 (C2), 103.3 (C3), 110.9 (C2″ or C4″ or C6″), 111.4 (C2″ or C4″ or C6″),

114.9 (C2″ or C4″ or C6″), 126.0 (C8), 126.6 (C5), 127.0 (C2′ and C6′), 128.6 (C5″), 128.9

(C3′ and C5′), 131.9 (C10), 132.3 (C4′), 132.5 (C9), 133.5 (C6 or C7), 135.2 (C6 or C7),

140.5 (C1′), 157.2 (C3″), 158.3 (C1″), 179.0 (C1), 182.5 (C4); m/z (ESI-) 419 ([M-H]

-,

100%); HRMS (ESI-) C22H15O5N2S

- requires 419.0707, found 419.0713.

Ethyl (3-hydroxyphenyl)carbamate 8865

Ethyl chloroformate (1.00 g, 9.20 mmol) was added to a stirred

suspension of m-aminophenol 87 (1.0 g, 9.20 mmol) in anhydrous

diethyl ether (40 mL), immediately forming a white precipitate. The reaction mixture

was stirred for 2 h at RT. The precipitate was removed via suction filtration, and the

filtrate was concentrated in vacuo. Subsequent recrystallization from boiling pet ether

afforded carbamate 88 as a white solid (1.30g, 78%). mp 92-94 °C {lit. 91-92 °C}65

;

δH (400 MHz, CDCl3) 1.32 (3H, t, J 7.1, 3 x H2′), 4.24 (2H, q, J 7.2, 2 x H1′), 6.58 (1H,

ddd, J 8.2, 2.4, 0.7, H6), 6.62-6.72 (2H, m, H2 and H4) 7.14 (1H, app. t, J 8.2, H5), 7.34

(1H, br. s, NH); m/z (ESI+) 181 ([M+H], 25%), 204 ([M+Na], 100%).


69

7-Carbethoxyamino-4-methylcoumarin 8965

Phenol 88 (0.60 g, 3.31 mmol) and ethyl acetoacetate (0.51 mL,

4.00 mmol) were stirred in a 70% ethanolic H2SO4 solution for

5 h at RT. The reaction mixture was poured onto ice-cold water, and the precipitate

collected via suction filtration, which was further washed with cold water. Subsequent

recrystallization from boiling ethanol afforded carbethoxycoumarin 89 as a light brown

solid (0.50 g, 61 %). mp 170 °C {lit. 185-186 °C}65

; δH (400 MHz, DMSO-d6) 1.26 (3H,

t, J 7.1, 3 x H2′), 2.38 (3H, d, J 1.0, C4Me), 4.17 (2H, q, J 7.1, 2 x H1′), 6.23 (1H, d,

J 1.0, H3), 7.40 (1H, dd, J 8.8, 2.1, H6), 7.55 (1H, d, J 2.1, H8), 7.68 (1H, d, J 8.8, H5),

10.15 (1H, s, NH); m/z (ESI+) 248 ([M+H]

+, 100 %), 270 ([M+Na]

+, 30 %).

Appendix 2: Protocols for Biological and Optical Evaluation

70

Appendix 2: Protocols for Biological and Optical

Evaluation

A 2.1. Attempted Expression and Purification of hNAT1

E. coli Rosetta®(-DE3)pLysS cells containing pET28b(+), into which the hNAT1 open-

reading frame1 had been sub-cloned so as to allow the production of recombinant

hNAT1 with an N-terminal hexa-His tag, had been previously prepared by Kawamura et

al. and stored at -80 °C in LB medium with 10% (v/v) glycerol.2 10 μL of thawed stock

was used to inoculate 10 mL of LB medium, into which kanamycin (30 μg/mL) was

also added; this culture was incubated at 37 °C for 16 h with shaking (180 rpm). A

larger-scale culture was then prepared by the addition of 5 mL of starter culture to 1000

mL LB medium; kanamycin (30 μg/mL) was added and the culture incubated at 37 °C

with shaking.

The optical density of the solution at 600 nm (OD600) was monitored at hourly intervals

and when the absorbance reached 0.4, IPTG was added to a final concentration of 0.1

mM. After a further incubation period of 22 h at 37 °C with shaking (180 rpm), the

E. coli cells were harvested by centrifugation (5000 RCF, 4 °C, 30 min). These cells

were then re-suspended in 20 mL of lysis buffer (300 mM NaCl, 20 mM Tris.HCl (pH

8.0) containing one EDTA-free Complete Protease Inhibitor per 20 mL) and sonicated

on ice (30 cycles of 30 sec. on, 30 sec. off at 10 μm). The soluble lysate was then

separated from the cell debris by centrifugation (15000 RCF, 4 °C, 20 min).

In attempted purification of hNAT1 from the soluble lysate via Ni-NTA affinity

chromatography, 5 mL of Ni-NTA solution was used as the stationary phase and 20 mM

Tris.HCl (pH 8.0) containing 100 mM NaCl and increasing concentrations of imidazole

(0 mM, 10 mM, 25 mM, 50 mM, 250 mM and 500 mM) was used as the mobile phase.


71

1 mM DTT was added to the 250 mM imidazole solution to prevent enzyme

precipitation.

SDS-PAGE gels were run to determine which of the column fractions contained pure

hNAT1 using a 12% acrylamide separating gel and a 4% acrylamide stacking gel;

however, neither when stained with Coomassie blue nor with silver could any desired

protein bands be detected. hNAT1 is well-documented to express poorly and to possess

poor stability. In the interests of time, it was therefore decided to use recombinant

mNAT2 for the enzymatic experiments in this study, since mNAT2 is a robust model for

hNAT1.

A 2.2. Protocols for Compound Evaluation

A 2.2.1. Acetyl CoA Hydrolysis Assay

The acetyl CoA hydrolysis assay, or “DTNB assay”, monitors the rate of AcCoA

hydrolysis by NATs and is based upon the reaction outlined in Scheme A2.1.1 This

assay is an end-point assay used to test the inhibitory potency of synthesised compounds

against mNAT2. Assays were carried out in flat-bottomed 96-well plates (Greiner)

which were set up as outlined in Table A2.1, giving a final assay well volume of 100 μL

containing 5% (v/v) DMSO.

Scheme A2.1: Reaction utilised in the DTNB assay to monitor the rate of AcCoA hydrolysis by NATs.


72

Table A2.1: DTNB assay protocol. amNAT2 was used as a

1/400 dilution of stock mNat2 (5 mg/mL); 20

mM Tris.HCl (pH 8) was used as the dilution buffer. bReaction in different wells on the same plate may

be initiated with AcCoA at different time points as desired. For an initial activity test of an inhibitor at a

concentration of 30 μM, 4 wells were used in each assay, initiated with AcCoA after 0, 5, 10 and 15 min

respectively; quenching with DTNB occurred after 15 min. For an IC50 test, a single well was used in

each assay, initiated with AcCoA after 0 min. and quenched with DTNB after 10 min.

For an initial screen of inhibitory potency of a compound at a final concentration of

30 μM, assays were conducted in triplicate and the % specific NAT activity was

calculated from a graph of OD405 against time. The % specific activity was calculated

against a control assay without inhibitor.

In IC50 tests, a range of ten concentrations was selected for each compound, most often

beginning at 10, 30 or 50 μM and diluting by a factor of two in each subsequent well.

Assays were conducted in duplicate and % specific activity for each assay was

determined relative to a control. KyPlot® software was then used to plot a curve of %

specific activity against concentration using the IC50%FUN model. This software

determines the IC50 value, quoted ± one standard deviation, via the method of least

squares. R2 values are quoted in Appendix 4.

Stage Assay Well

Component

Stock Concentration Volume Final

Conditions

Preparation DMSO (Control)

or Inhibitor in

DMSO

>99% purity or

Various in >99% purity

5 μL 5% (v/v) or

Various in 5%

(v/v)

mNAT2a

0.0125 mg/mL 10 μL 125 ng

pABA

1.11 mM 45 μL 500 μM

Incubation Incubate at 370C for 5 min

Initiation AcCoA 1 mM 40 μL 400 μM

Reaction Incubate at 370C for desired reaction time (various

b)

Quench DTNB 5mM in 6.4 M Guanidine.HCl,

20mM Tris.HCl (pH 7.5)

25 μL 1mM

Observation Record absorbance at 405 nm on a FLUORSTAR Omega® plate reader


73

A2.2.2. Spectrophotometry

Visible spectra of each compound was recorded at pH 8, pH 13.75 and, for selected

compounds, in the presence of mNat2. Unless otherwise stated, the concentrations and

volumes of solutions used were as outlined in Table A2.2. All spectra were blank-

corrected and normalised.

Table A2.2: Protocol for spectrophotometry. All samples were made up in wells of 96-well flat-bottomed

plates (Corning). The final inhibitor concentration used was 15 μM and the final mNat2 concentration

used was 30 μM unless otherwise stated.

A2.2.3. Titration Experiments to Determine pKa

To determine the pKa of an inhibitor colorimetrically, a full absorbance spectrum was

recorded on a plate reader (Omega) for each compound in 12 differently buffered

solutions in 5% (v/v) DMSO (Table A2.3). 5 μL of the inhibitor in DMSO (stock

concentration 2 mM) was added to 95 μL of buffer solution in a flat-bottomed 96-well

plate (Corning), giving a final compound concentration of 100 μM. Recorded spectra

were blank-corrected and normalised before λmax was determined.

Table 2.3: Buffer solutions for different pH.

Spectral

Conditions

Volume and Type of

Buffer Solution

Added

Inhibitor

(Stock 300 μM in DMSO)

mNat2 (5 mg/mL in 20 mM

Tris.HCl (pH 8))

Volume

Added

Final

Concentration

Volume

Added

Final

Concentration

pH 8 95 μL 20mM Tris.HCl 5 μL 15 μM 0 μl 30 μM

pH 12.8 95 μL 35 mM

Na2HPO4.NaOH

5 μL 15 μM 0 μl 30 μM

With mNat2 70 μL 20 mM Tris.HCl 5 μL 15 μM 25 μl 30 μM

pH Buffer

4.0 20 mM Acetate.Acetic Acid, 20 mM NaCl

6.0 20 mM MES.Acetic Acid, 20 mM NaCl

7.0 20 mM HEPES.Acetic Acid, 20 mM NaCl

7.5 20 mM Tris.HCl

8.0 20 mM Tris.HCl

8.5 20 mM Tris.HCl, 20 mM NaCl

9.0 20 mM Tris.HCl, 20 mM NaCl

9.5 20 mM CHES.NaOH, 20 mM NaCl

10.0 20 mM CHES.NaOH, 20mM NaCl

11.0 20 mM CAPS.NaOH, 20 mM NaCl

12.0 70 mM Na2HPO4.NaOH

12.8 35 mM Na2HPO4.NaOH


74

For each inhibitor, a graph of λmax against pH was then plotted using GraphPad®

software. A sigmoidal dose-response (variable slope) model was used to determine the

pKa by the method of least squares. R2 values are quoted in Appendix 4.

A2.2.4. Determination of Absorption Coefficients

To determine molar absorption coefficients at pH 8 and 12.8, 5 μL of the inhibitor in

DMSO (stock concentrations 4 mM, 2 mM, 1 mM, 0.5 mM) was added to each of four

wells in a 96-well flat-bottomed plate (Corning) respectively. To this was added 95 μL

Tris.HCl (pH 8.0) and full visible spectra were recorded on a plate reader (Omega).

Experiments were performed in duplicate. Spectra were blank-corrected and normalised

and the absorbance at λmax was recorded for each final concentration (200 μM, 100 μM,

50 μM, 25 μM). A plot of absorbance against concentration then yielded εN as the

gradient divided by the path length of 0.315 cm.

To all of the wells, 10 μL 4 M NaOH was then added and the procedure repeated to

yield εCB, ensuring that a correction factor of 11

/10 was applied to the final inhibitor

concentration in each well and to the path length.

A2.2.5. Fluorescence Studies

Fluorescence spectra were recorded on a Varian Cary Eclipse Fluorescence

Spectrophotometer. 5 μL of a stock solution of 3 mM compound in DMSO was added

to 95 μL Tris.HCl buffer (pH 8.0) to yield a final concentration of 150 µM, unless

otherwise stated. Each scan consisted of an initial solvent blank measurement to remove

noise or solvent scattering from peak analysis, and was subsequently subtracted from

resultant spectra to yield blank-corrected spectra; a negative 5% DMSO:95% Tris.HCl

control was also run. Where possible, λex and λem were identified by ‘zero order’ scans

(illumination of the sample with the full available range of electromagnetic radiation


75

simultaneously), otherwise measurements were recorded at 5 nm increments, starting at

λex = 200 nm. Spectra were recorded at a sensitivity voltage of V = 800 V unless

otherwise stated. Acidic and basic conditions were achieved by addition of 5 µL of 1 M

aq. HCl or NaOH respectively.

A2.2.6. Docking Studies

All images showing protein structures were generated using the software PyMOL (W. L.

DeLano (2002) PyMOL, DeLano Scientific, San Carlos, CA). Prior to docking a ligand

into the hNAT1 active site (pdb: 2PQT),3 the ground state conformation of the ligand

was predicted using the molecular editor Avogadro,4 and the protein was protonated to

be consistent with the assay conditions of pH 8. The docking simulations and the

analysis of the possible interactions between the protein and the ligand were performed

using GOLD®

v. 5.2.5 The docking site was defined as a region of 10 Å within the

active pocket of the enzyme and the generated solutions were ranked using the GOLD®

Score Fitness function.5 Each docking simulation was repeated ten times to ensure the

observed solutions were consistent.

References

1.

Brooke, E. W.; Davies, S. G.; Mulvaney, A. W.; Pompeo, F.; Sim, E.; Vickers, R. J.; Bioorg Med

Chem, 2003, 11 (7), 1227-1234

2. Kawamura, A.; Westwood, I. M.; Wakefield, L.; Long, H.; Zhang, N.; Walters, K.; Redfield, C.; Sim,

E., Biochem Pharmacol, 2008, 75 (7), 1550-1560

3. Wu, H.; Dombrovsky, L.; Tempel, W.; Martin, F.; Loppnau, P.; Goodfellow, G. H.; Grant, D. M.;

Plotnikov, A. N.; J Biol Chem, 2007, 282 (41), 30189-30197

4. Hanwell, M. D.; Curtis, D. E.; Lonie, D. C.; Vandermeersch, T.; Zurek, E.; Hutchinson, G. R.,

Journal of Cheminformatics, 2012, 4 (1), 17

5. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D., Proteins: Structure,

Function and Bioinformatics, 2003, 52 (4), 609-623

Appendix 3: Supplementary Pharmacological Data

76

Appendix 3: Supplementary Pharmacological

Data

A3.1. pKa Curves of 12

A3.2. Representative IC50 Curves

0 5 1 0 1 5

4 5 0

5 0 0

5 5 0

6 0 0

p K a o f 1 2 - C o lo rim e tr ic T itra tio n

p H

m

ax

0 5 1 0 1 5

0

5 0

1 0 0

1 5 0

p K a o f 1 2 - F lu o re s c e n c e T itra tio n

p H

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

.)

Appendix 3: Supplementary Pharmacological Data

77

A3.3. Titration of 85 against mNAT2

0

5

10

15

20

25

30

370 420 470 520 570

Flu

ore

scen

ce I

nte

nsi

ty (

arb

itra

ry u

nit

s)

Wavelength (nm)

0 μL mNAT2

4 μL mNAT2

6 μL mNAT2

8 μL mNAT2

10 μL mNAT2

Appendix 4: Summary of all Pharmacological and Spectroscopic Data

78

Appendix 4: Summary of all Pharmacological and Spectroscopic Data

Rob Barker Part II Thesis

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