Post on 16-Apr-2017
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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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Chapter 1: Introduction
6
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 2: Synthesis of Fluorescent Probes
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
Chapter 2: Synthesis of Fluorescent Probes
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
Chapter 2: Synthesis of Fluorescent Probes
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
Chapter 2: Synthesis of Fluorescent Probes
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
Chapter 2: Synthesis of Fluorescent Probes
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.
Chapter 2: Synthesis of Fluorescent Probes
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%
Chapter 2: Synthesis of Fluorescent Probes
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.
Chapter 2: Synthesis of Fluorescent Probes
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
Chapter 2: Synthesis of Fluorescent Probes
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.
Chapter 2: Synthesis of Fluorescent Probes
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
Chapter 2: Synthesis of Fluorescent Probes
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.
Chapter 2: Synthesis of Fluorescent Probes
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
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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.
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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.
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Excitation at λex = 252 nm
Excitation at λex = 408 nm
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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
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4 μL mNAT2
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Excitation Emission
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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%
Chapter 3: Optimisation of the Fluorophore
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
Chapter 3: Optimisation of the Fluorophore
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
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Chapter 3: Optimisation of the Fluorophore
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.
Chapter 3: Optimisation of the Fluorophore
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
Chapter 4: Experimental
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)
Chapter 4: Experimental
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
Chapter 4: Experimental
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).
Chapter 4: Experimental
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%).
N-(3-((9″-Methoxy-10″-nitro-[4″,7″-biphenyl]-1″-yl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide 15
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).
Chapter 4: Experimental
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.
N-(3-((3″-Methoxy-10″-nitro-[4″,7″-biphenyl]-1″-yl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide 16
Following General Procedure 1, using naphthoquinone 23 (74 mg,
0.22 mmol), aniline 49 (160 mg, 0.66 mmol) and CeCl3∙7H2O
(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,
Chapter 4: Experimental
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.
N-(3-((2″-Methoxy-10″-nitro-[4″,7″-biphenyl]-1″-yl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide 17
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
Chapter 4: Experimental
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
Following General Procedure 1, using naphthoquinone 23 (100 mg,
0.288 mmol), aniline 50 (185 mg, 0.864 mmol) and CeCl3∙7H2O
(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-
dihydronaphthalen-2-yl)benzenesulfonamide 19
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
Chapter 4: Experimental
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-
dihydronaphthalen-2-yl)benzenesulfonamide 20
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,
Chapter 4: Experimental
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
Following General Procedure 1, using naphthoquinone 23 (77 mg,
0.27 mmol), aniline 51 (185 mg, 0.68 mmol) and CeCl3∙7H2O
(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
Chapter 4: Experimental
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
Chapter 4: Experimental
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
Chapter 4: Experimental
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
Following General Procedure 2, using aryl bromide 42 (2.80 g,
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,
Chapter 4: Experimental
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
+ requires 371.1989, found
372.1947.
tert-Butyl (2-methoxy-10-nitro-[4,7-biphenyl]-1-yl)carbamate 45
Following General Procedure 3, using boronic ester 43 (1.50 g,
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.
tert-Butyl (3-methoxy-10-nitro-[4,7-biphenyl]-1-yl)carbamate 46
Following General Procedure 3, using boronic ester 44 (0.80 g,
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)
Chapter 4: Experimental
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
Following General Procedure 3, using boronic ester 38 (1.72 g,
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.
2-Methoxy-10-nitro-[4,7-biphenyl]-1-amine 48
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-
Chapter 4: Experimental
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.
3-Methoxy-10-nitro-[4,7-biphenyl]-1-amine 49
Following General Procedure 5, using carbamate 46 (270 mg, 0.79 mmol)
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
Following General Procedure 5, using carbamate 47 (435 mg, 1.17 mmol)
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
Chapter 4: Experimental
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.
Chapter 4: Experimental
52
N-(3-(((9″-Methoxy-[3″,8″-biphenyl]-2″-yl)methyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide 63
Following General Procedure 1, using naphthoquinone 23 (47 mg,
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.
N-(3-(((9″-Methoxy-[4″,8″-biphenyl]-2″-yl)methyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide 64
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
Chapter 4: Experimental
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.
N-(3-(((9″-Methoxy-[5″,8″-biphenyl]-2″-yl)methyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide 65
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
Chapter 4: Experimental
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
Following General Procedure 5, using carbamate 74 (360 mg, 1.15 mmol)
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
+ requires 214.1226, found
214.1220.
tert-Butyl ((9-methoxy-[3,8-biphenyl]-2-yl)methyl)carbamate 74
Following General Procedure 3, using aryl bromide 73 (0.72 g,
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
Chapter 4: Experimental
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.
tert-Butyl ((9-methoxy-[4,8-biphenyl]-2-yl)methyl)carbamate 79
Following General Procedure 3, using aryl bromide 77 (0.75 g,
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.
Chapter 4: Experimental
56
tert-Butyl ((9-methoxy-[5,8-biphenyl]-2-yl)methyl)carbamate 80
Following General Procedure 3, using aryl bromide 78 (0.72 g,
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
+ requires 336.1570, found
336.1565.
(9-Methoxy-[4,8-biphenyl]-2-yl)methanamine 81
Following General Procedure 5, using carbamate 79 (250 mg, 0.80 mmol)
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.
Chapter 4: Experimental
57
(9-Methoxy-[5,8-biphenyl]-2-yl)methanamine 82
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-
yl)benzenesulfonamide 83
Following General Procedure 1, using naphthoquinone 23 (85 mg,
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′
Chapter 4: Experimental
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-
yl)benzenesulfonamide 85
Following General Procedure 1, using naphthoquinone 23 (165 mg,
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
Appendix 1: Supplementary Experimental Data
8-Methoxy-10-nitro-[4,7-biphenyl]-1-amine 1451
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
Appendix 1: Supplementary Experimental Data
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%).
Appendix 1: Supplementary Experimental Data
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
Following General Procedure 3, using boronic ester 30 (1.20 g,
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
Following General Procedure 2, using aryl bromide 28 (3.00 g,
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,
Appendix 1: Supplementary Experimental Data
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,
Appendix 1: Supplementary Experimental Data
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
Appendix 1: Supplementary Experimental Data
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)
Appendix 1: Supplementary Experimental Data
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-
yl)benzenesulfonamide 86
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
Appendix 1: Supplementary Experimental Data
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%).
Appendix 1: Supplementary Experimental Data
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.
Appendix 2: Protocols for Biological and Optical Evaluation
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.
Appendix 2: Protocols for Biological and Optical Evaluation
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
Appendix 2: Protocols for Biological and Optical Evaluation
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
Appendix 2: Protocols for Biological and Optical Evaluation
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
Appendix 2: Protocols for Biological and Optical Evaluation
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