Structure-activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine N-acetyltransferase 1

Article abstract of DOI:10.1016/j.bmc.2014.03.015

A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure-activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pK_a, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors.

Full text of DOI:10.1016/j.bmc.2014.03.015

Bioorganic & Medicinal Chemistry 22 (2014) 3030–3054
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships and colorimetric properties
of specific probes for the putative cancer biomarker human
arylamine N-acetyltransferase 1
James E. Egleton a,b, Cyrille C. Thinnes a,b, Peter T. Seden a, Nicola Laurieri b, Siu Po Lee b,
Kate S. Hadavizadeh a,b, Angelina R. Measures a, Alan M. Jones a, Sam Thompson a, Amy Varney b,
Graham M. Wynne a, Ali Ryan b,c, Edith Sim b,c, Angela J. Russell a,b,
⇑
a Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
b Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
c Faculty of Science, Engineering and Computing, University of Kingston, KT1 2EE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer
biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color
change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling
alongside structure–activity relationship studies to advance the hit compound towards a potential probe
to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a
two-fold greater absorption coefficient compared to the initial hit have been synthesized; these
compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2.
A relationship between pKa, inhibitor potency and colorimetric properties has also been uncovered.
The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the
way for the development of inhibitors with improved intrinsic sensitivity which could enable detection
of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in
ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors.
Received 22 November 2013
Revised 3 March 2014
Accepted 10 March 2014
Available online 28 March 2014
Keywords:
Arylamine N-acetyltransferase
Breast cancer
Biomarker
Colorimetric probe
Naphthoquinone
Ó 2014 Published by Elsevier Ltd.
1. Introduction
Proteomic6 and microarray7 studies have identified human
arylamine N-acetyltransferase 1 (hNAT1) as one of the ten most
The worldwide annual incidence of breast cancer exceeds one
million cases, with statistics from the UK showing that the lifetime
risk of a woman developing the disease is 1 in 8.1 Early detection of
tumors is known to be crucial for improving survival rates.2 The
estrogen receptor (ER) is overexpressed in a major subtype of
breast cancer, referred to as ‘ER-positive’ (ER+), and was the first
protein to be used as a diagnostic and prognostic biomarker for
breast cancer.3 ER+ tumors are often responsive to aromatase
inhibitors or to selective ER modulator (SERM) therapies.3
However, the clinical use of SERM therapies is limited by drug
resistance,4 and the use of immunohistochemical staining to detect
ERs for diagnostic purposes suffers from standardization
problems.5 Consequently, new therapeutic leads and diagnostic
biomarkers for ER+ tumors are desirable.
highly overexpressed genes in ER+ tumors; furthermore, this
overexpression inversely correlates to tumor grade.8 More recently
hNAT1 overexpression in male breast cancers has also been
reported.9 The corresponding enzyme hNAT1 is therefore of inter-
est as a surrogate diagnostic and prognostic biomarker for ER+
tumors. Furthermore, studies suggest that hNAT1 could also be a
novel therapeutic target against ER+ breast cancer.10 Key evidence
includes: the discovery of hallmarks of oncogenic potential in the
non-cancerous breast cell line HB4a induced to overexpress
hNAT1;6 and the reduction in cell proliferation and invasiveness
observed in the MDA-MB-231 breast cancer cell line (which
expresses high levels of hNAT1) when either hNAT1 was knocked
down by shRNA or the cells were treated with inhibitors of
hNAT1.11
NATs are a family of xenobiotic metabolizing enzymes found in
both eukaryotic and prokaryotic organisms, which catalyze the
transfer of an acetyl group from acetyl coenzyme A (AcCoA) to a
xenobiotic substrate, such as arylamines, arylhydroxylamines,
⇑
Corresponding author. Tel.: +44 1865275643.
E-mail address: angela.russell@chem.ox.ac.uk (A.J. Russell).
http://dx.doi.org/10.1016/j.bmc.2014.03.015
0968-0896/Ó 2014 Published by Elsevier Ltd.

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3031
arylhydrazines, and N-alkylarylamines.12 The human genome
codes for two functional NAT genes: hNAT1 and hNAT2. The gene
product hNAT2 is implicated in phase II drug metabolism and is
abundant in liver and intestinal cells,12a whilst hNAT1 has a wide-
spread tissue distribution13 and is reported to play a role in cofac-
tor homeostasis;14 studies suggest eukaryotic homologues of
hNAT1 are involved in growth and development.15 Estrogen levels
may also have an effect on hNAT1 expression.16
Aside from the discovery that the SERM tamoxifen 1 reduces
hNAT1 activity in vitro,17 the first compounds identified as hNAT1
inhibitors were found to be non-selective for hNAT1 and to act via
covalent modification of the catalytic Cys68 residue;18 these
include alkylating agents as well as the widely prescribed chemo-
therapeutic agent cisplatin.19
More recently, rhodanine 2 and naphthoquinone 3 have been
identified as inhibitors of hNAT1 (and its murine homologue
mNat2)22 and were found to be selective over a variety of other
NATs including hNAT2;20 additionally both 2 and 3 inhibited
hNAT1 activity in cell extracts from the ER+ breast cancer cell line
ZR-75-1 (Fig. 1 a).20,23
The reversible, competitive naphthoquinone inhibitor 3 proved
to be of particular interest, because it was found that 3 undergoes a
distinctive color change from red to blue in the presence of hNAT1
and mNat2, but not any other NAT isoform tested.23 Subsequently
spectroscopic, chemical, molecular modeling and biochemical
studies have been carried out on a close analogue of 3, the
dimethyl-substituted 4, which has a similar potency to 3 against
hNAT1 and mNat2 (Fig. 1b).21 These studies demonstrated that this
color change is driven by sequestration of the conjugate base of 4
(pKa ꢀ9.2), mediated by Arg127 in the active site of hNAT1 or
mNat2.21
here our preliminary structure–activity relationship and optimiza-
tion studies.
2. Results and discussion
2.1. Quantification of [hNAT1] in cell extracts from the breast
cancer cell line ZR-75-1
The approximate level of hNAT1 in a representative breast can-
cer cell line (ZR-75-1) was determined using immunoblotting, to
gain an understanding of the sensitivity required in a probe for
hNAT1 detection in cells.
The twelve C-terminal residues of hNAT1 and mNat2 are iden-
tical and distinct from those of hNAT2. This has allowed generation
of
a specific antibody which recognizes both hNAT1 and
mNat213a,15d,25 with equal levels of sensitivity.26
A Western blot was performed with both pure recombinant
mNat2 and ZR-75-1 cell extracts, and a single band at ꢀ34 kDa
(corresponding to the relevant NAT protein) was observed for all
samples (Fig. 2a). A comparison of lanes 3 and 6 suggested that
there is less than 62.5 ng/lL of hNAT1 (i.e., less than 62.5 ng hNAT1
per 5 ꢁ 104 cells).
Subsequently, dot-blotting studies were carried out, varying the
concentrations of pure recombinant mNat2 and ZR-75-1 cell
extracts, under both native and denaturing conditions (Fig. 2b).
Comparing lanes M2 and H2, there appeared to be greater than
19.5 ng of native hNAT1 per 5 ꢁ 104 cells. It can also be noted that
the antibody had a higher affinity for native hNAT1 than denatured
hNAT1, a result which is consistent with previous studies.26
These experiments therefore showed that the concentration of
hNAT1 in the ZR-75-1 cell extract is approximately 0.6–1.2
lM
(20–60 ng/ L); this informed us of the level of sensitivity required
l
This observed color change leads to the possibility that 4 could
act as a direct in vitro probe for the presence of native hNAT1,
without the conventional need for protein tagging or antibody
staining.24 Potentially, any specific small molecule inhibitors of
hNAT1 could additionally be valuable leads for a new drug therapy.
However, for the potential of these naphthoquinones as probes for
hNAT1 to be realized, it is believed that both the binding potency
and molar absorption coefficient need to be increased. We describe
of a colorimetric probe for detecting hNAT1 in such cell extracts.
Bradford assays show that the total cellular protein concentration
is 11.5 lg/lL, and hence the percentage of hNAT1 in the ZR-75-1
cell proteome is around 0.2–0.5%.
In separate experiments, the minimum hNAT1/inhibitor ratio to
enable the unambiguous determination of a colorimetric shift of 4
spectroscopically was determined. It was predicted that based on
the calculated Kd for 4 (4.9
l
M) and a 1:1 binding ratio,23 an excess
O
O
Me2N
(a)
H
N
I
Ph
S
NH
S
Et
O
NH
O
S
O
HO
Ph
O
I
2
3
1
O
(b)
X
N
S
Enzyme
O
NH
O
Compound
mNat2
WT
mNat2
R127G
mNat2
R127L
O
hNAT1
IC50 (μM)
5.3
1.9
51.7
102.5
4
5
4 X = H
λmax (nm)
585
625
498
498
λmax (pH 8) = 498 nm
IC50 (μM)
λmax (nm)
5.8
> 150
490
N.D.
N.D.
N.D.
N.D.
5 X = Me
476
λ
max (pH 8) = 496 nm
Figure 1. (a) Examples of hNAT1 inhibitors reported in the literature.6,20 (b) Compound 4 is deprotonated selectively in the presence of hNAT1 with a distinctive concomitant
color change; this process is driven by sequestration of the conjugate base of 4 by the Arg127 residue of hNAT1.21 (N.D. = not determined.).

3032
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
O
O
O
H
N
H
N
Ph
Ph
Cl
(i)
(ii)
S
S
O
O
O
O
30-89%
94%
Cl
NHR2
Cl
O
O
O
3-4, 6, 9-11
8
7
Scheme 1. Reagents and conditions: (i) PhSO2NH2 (1.0 equiv), Cs2CO3 (1.4 equiv),
DMF, rt, 5 h; (ii) either CeCl3ꢂ7H2O (1.0 equiv), MeOH, rt, 90 min then requisite
aniline (3.0 equiv), 110 °C, 16 h; or CeCl3ꢂ7H2O (1.0 equiv), toluene, rt, 90 min then
requisite amine (3.0 equiv), toluene, 110 °C, 6 h.
2.3. SAR at C3 of the naphthoquinone core
2.3.1. C3 amino-substituted analogues
Initially, compounds 3, 4 and 6, which we have reported previ-
ously,23 were synthesized in a two-step procedure from 2,3-dichlo-
ronaphthalene-1,4-dione 7 via successive addition-elimination
reactions (Scheme 1; Table 1). The reaction conditions were
optimized from those previously reported23 leading to improved
overall yields (for optimization see Scheme S1 and Table S1 in
Supporting information).
A small series of aliphatic amino groups were also introduced at
the C3 position by substitution of chloride from intermediate 8
with aliphatic amines. In comparison to anilines, aliphatic amines
possess higher conformational freedom and are unable to form
Figure 2. Immunohistochemical quantification of hNAT1 in ZR-75-1 cell lysate. (a)
Detection of purified recombinant mNat2, and hNAT1 in cell lysate by Western
blotting. Molecular weights and positions of the protein bands in the SeeBlueÒ
Plus2 Pre-Stained Standard used are shown on the left. Lanes: 1–4 0.0125, 0.125,
0.625 and 1.25 ng/lL mNat2; 5–7: 10-, 100- and 1000-fold diluted ZR-75-1 cell
lysate of 5 ꢁ 107 cells/mL. (b) Detection of purified recombinant mNat2 (M) and
hNAT1 in cell lysate (H) by dot blotting as native (N) and denatured (D) proteins.
Native protein samples were dissolved in an equal volume of buffer (200 mM
TrisꢂHCl (pH 8)), while denatured proteins were prepared in an equal volume of
p–p interactions within the NAT active site upon binding. A series
of analogues was chosen to allow comparison of effects of chain
extension, inclusion of polar heteroatoms and aromatic moieties
(Scheme 1; Table 1). Their synthesis was analogous to that of 4
except it was found that step (ii) proceeded in excellent yield
within only 6 h in toluene.
buffer with 2% (w/v) SDS and heated to 95 °C for 5 min. Lanes: M1 0.031 ng/lL; M2
0.0078 ng/
diluted lysate.
l
L; H1 500-fold diluted cell lysate of 5 ꢁ 107 cells/mL; H2 2500-fold
of the protein would be required for every inhibitor molecule to
bind and change color. The required hNAT1/inhibitor ratio would
also be expected to decrease with increasing enzyme active site
occupancy. With compound 4 this minimum ratio was found to
be 1.2:1. Therefore, 4 was added to ZR-75-1 cell extracts at a final
2.3.2. C3 aryloxy-substituted analogues
Aryloxy analogues of 4 were also synthesized, since these were
predicted to have similar conformational preferences to anilino sub-
stituents whilst possessing H-bond acceptor but not donor ability.
Initially, intermediate 8 was pre-treated with CeCl3ꢂ7H2O before
addition to sodium phenoxide (generated by treatment of phenol
with NaH) in toluene; however, none of the desired product was
obtained (Scheme 2). A Buchwald-type cross-coupling reaction
involving the bulky electron-rich ligand ditBuXPhos 12 between 8
and phenol was subsequently attempted,27 but returned only start-
ing materials. The same Buchwald conditions applied to the dichloro
precursor 7 yielded the monophenoxy-substituted derivative 13 in
moderate yield. Subsequent treatment of 13 with CeCl3ꢂ7H2O and
benzenesulfonamide, however, resulted in the sulfonamide displac-
ing the phenoxy substituent rather than the desired chloride leading
to formation of 8. Attempted cross-coupling between 13 and
PhSO2NH2 with Pd(dppf)2Cl2 as a catalyst also afforded 8.
concentration ranging from 0.6 lM to 2.5 lM, but the signal-to-
noise ratio was poor at these low concentrations and the kmax peak
(at 585 nm) could not be detected. This confirmed the hypothesis
that the potency and sensitivity of 4 must be increased in order
to enable hNAT1 detection in such cell extracts.
2.2. Overview of SAR studies
In this study we systematically varied substituents at the C2, C3
and C5–C8 positions of naphthoquinone 4, to improve our under-
standing of inhibitor-hNAT1 interactions which could guide the
rational design of colorimetric probes for specific hNAT1 detection
and inhibition (Fig. 3). Synthesis of novel inhibitors, guided by in
silico studies, sought to increase both the potency and absorption
coefficients of the inhibitor, whilst retaining both selectivity for
hNAT1 over hNAT2 and the ability to observe a distinctive color
change in the presence of hNAT1.
Given the susceptibility of phenoxy substituents to displace-
ment by benzenesulfonamide, an alternative route to aryloxy ana-
logues of 4 via the corresponding diaryloxy naphthoquinones was
developed. Dichloride 7 was treated with the requisite aryl alcohol
and cesium carbonate in THF at reflux to give the corresponding
O
1
O
H
N
8
5
NHSO2Ph
NH
R1
2
3
7
6
X
R3
Table 1
Identities of R2 substituents and yields for the amino analogue series
R2
4
R2
Yield of step (ii) (%)
O
O
Compound
R1 = Ph, Ar, CH2Ph, Aliphatic
2 = Ar, OAr, NHAr, NHR
R3 = H, NO2, NH2
3
4
6
9
10
11
Ph
63
57
30
87
89
42
4
300,500-Me2-C6H3
400-Br-C6H4
200-Methoxyethyl
Cyclopentyl
Benzyl
R
X = SO2, CO
Figure 3. Structure–activity relationships investigated in this study.

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3033
Table 3
O
O
O
O
Identities of R2 substituents and yields for the aryl analogue series
H
N
H
N
Ph (i) or (ii)
Ph
S
S
P(tBu)2
iPr
Compound
R2
Isolated yield step (ii) (%)
O
O
O
O
Cl
O
iPr
20
21
22
23
24
25
26
200-Cl-C6H4
300-Cl-C6H4
400-Cl-C6H4
300-CHO–C6H4
400-CHO–C6H4
200-Furanyl
300-Furanyl
48
35
47
31
24
45
23
8
17
12
iPr
O
O
O
O
O
Cl
Cl
OR2
OR2
(v)
63-89%
(ii)
68%
Table 4
IC50 values for the C3 analogue library
Cl
O
O
13
7
14-16
O
O
H
N
(iii) 72%
or (iv) 53%
(vi)
Ph
S
43-67%
O
O
R2
O
O
O
H
N
H
Ph
N
Ph
S
Compound R2
IC50 (mNat2) (
1.923
l
M) IC50 (hNAT1) (lM)
S
O
O
O
O
1.723
4.1
Cl
OR2
3
4
6
9
NHPh
O
NH-300,500-Me2-C6H3 2.4
8
17-19
NH-400-Br-C6H4
NHCH2CH2OMe
NH-Cyclopentyl
NHCH2Ph
1.7
>30
>30
>30
1.8
5.2
1.1
3.7
5.1
8.2
9.2
6.9
4.3
1.4
0.9
>30
>30
>30
2.8
12.1
1.9
Scheme 2. Reagents and conditions: (i) CeCl3ꢂ7H2O (1.0 equiv), toluene, rt, 90 min,
then NaOPh (1.0 equiv), reflux, 16 h; (ii) PhOH (1.2 equiv), Pd(OAc)2 (0.02 equiv), 12
(0.03 equiv), K3PO4 (2.0 equiv), toluene, reflux, 16 h; (iii) CeCl3ꢂ7H2O (1.0 equiv),
toluene, rt, 90 min, then PhSO2NH2 (3.0 equiv), reflux, 18 h; (iv) PhSO2NH2
(1.2 equiv), Pd(dppf)2Cl2 (0.02 equiv), Cs2CO3 (2.0 equiv), dioxane, reflux, 1 h; (v)
ArOH (2.2 equiv), Cs2CO3 (2.2 equiv), THF, reflux, 16 h; (vi), PhSO2NH2 (1.2 equiv),
Cs2CO3 (1.2 equiv), THF, reflux, 1 h.
10
11
17
18
19
20
21
22
23
24
25
26
OPh
O-300,500-Me2-C6H3
O-400-Br-C6H4
200-Cl-C6H4
8.0
300-Cl-C6H4
400-Cl-C6H4
300-CHO–C6H4
400-CHO–C6H4
Furan-200-yl
Furan-300-yl
14.5
12.1
8.4
10.3
9.6
diaryloxy-substituted naphthoquinones 14–16, which were subse-
quently treated with benzenesulfonamide and cesium carbonate in
THF at 80 °C for 1 h. Recrystallisation from toluene gave the desired
C3 aryloxy-substituted analogues 17–19 (Scheme 2; Table 2).
10.0
2.3.3. C3 aryl-substituted analogues
2.3.4. Pharmacological evaluation of the C3-substituted
analogues
A library of C3 aryl-substituted analogues, which are predicted
to have different conformational preferences to the anilino deriva-
tives and lack both H-bond donor and acceptor properties, was
synthesized. Suzuki cross-coupling reactions of chloride 8 and
the requisite boronic acid with Pd(PPh3)2Cl2 in 4:1 THF/satd aq
NaHCO3 proceeded in 70–80% yield; partial purification by column
chromatography followed by recrystallisation from toluene led to
isolation of analogues 20–26 in moderate to low yield (Scheme 3;
Table 3).
All of the synthesized analogues of compound 4 were initially
tested for inhibitory potency against recombinant mNat2 and
hNAT1, which were expressed and purified as outlined in Sec-
tion 4.2.1.22,28 NAT activity in the presence of an inhibitor was
determined by measuring the rate of acetyl coenzyme A (AcCoA)
hydrolysis using a previously described method.29 The IC50 value
Table 2
Identities of R2 substituents and yields for the aryloxy analogue series
Compound
R2
Isolated yield
step (v) (%)
Isolated yield
step (vi) (%)
17
18
19
Ph
63
89
72
67
57
43
300,500-Me2-C6H3
400-Br-C6H4
O
O
O
H
N
H
N
Ph
Ph
(ii)
Cl
Cl
(i)
S
S
O
O
O
O
R2
Cl
O
O
O
7
8
20-26
Figure 4. (a) In silico modeling of representative compounds 4 (carbons in grey), 17
(carbons in cyan) and 26 (carbons in yellow) in the active site of hNAT1 (pdb:
2PQT).12e (b) Surface-filled model for interaction of compound 4 with hNAT1,
depicting electrostatic surfaces and illustrating the active site pocket. Analysis
performed using GOLDÒ software.30
Scheme 3. Reagents and conditions: (i) PhSO2NH2 (1.0 equiv), Cs2CO3 (1.4 equiv),
DMF, rt, 5 h; (ii) Requisite boronic acid (2.0 equiv), Pd(PPh3)2Cl2 (0.1 equiv), 4:1
THF:satd aq NaHCO3, reflux, 18 h.

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J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
Table 5
pKa values for the C3 library of inhibitors
Compound
3
4
6
9
10
11
17
18
19
20
21
22
23
24
25
26
pKa
9.5
9.2
10.4
9.8
10.1
10.4
5.0
5.0
5.0
4.9
4.5
5.0
5.0
4.6
5.1
5.0
Table 6
Spectrophotometric properties of the anilino- and amino-substituted inhibitors 3, 4, 6 and 9–11
Compound
at pH 8
Compound
at pH 13
Δλmax pH 13
(nm)
Δλmax mNat2
(nm)
εCB (M-1cm-1)
εCB / εCB(4)
Compound
R2
3
4
6
NHPh
+ 71
+ 79
+ 121
+ 112
7900
6590
1.20
1.00
NH-3’’,5’’-Me2-C6H3
NH-4’’-Br-C6H4
+ 70
+ 89
+ 93
N.D.
7790
1010
1.18
0.15
9
NHCH2CH2OMe
NH-Cyclopentyl
NHCH2Ph
+ 82
+ 74
+ 3
+ 5
4470
5770
0.68
0.88
10
11
N.D. = not determined.
Cl
R1
H2N
R1
(i)
S
O
S
O
O
O
c
c
R1
(ii)
=
)
29
hex ( ) 91%
27
hex (
R1
=
30
28
2-furyl ( ) quant.
2-furyl (
)
O
O
O
O
O
H
H
N
Cl
Cl
N
R1
R1
(iii)
S
S
O
O
O
O
NH
Cl
O
7
8, 31-40
4, 41-50
Scheme 4. Reagents and conditions: (i) NH3 (aq, 33%), rt, 30-100 min; (ii) requisite
sulfonamide (1.0 equiv), Cs2CO3 (1.4 equiv), DMF, rt, 5-16 h; (iii) CeCl3ꢂ7H2O
(1.0 equiv), MeOH, rt, 90 min, then 3,5-dimethylaniline (3.0 equiv), 90 °C, 16 h.
Table 7
Identities of R1 and yields for the C2 sulfonamide library
Compound R1
Isolated yield step (ii) (%) Isolated yield step (iii) (%)
4
Ph
91
39
64
57
65
32
49
20
33
26
25
59
66
53
41
42
43
44
45
46
47
48
49
50
Me
cHex
20-Me-C6H4 70
20-NO2-C6H4 44
20,60-F2-C6H3 67
30-NO2-C6H4 Quant.
40-Me-C6H4 66
40-F-C6H4
Furan-20-yl 85
Benzyl 81
63
of each synthesized compound is given in Table 4; squared corre-
lation coefficients for these data are presented in Table S2 in Sup-
porting information.
A wide variety of substituents appears to be tolerated at the C3
position, with examples of anilino, aryloxy and aryl groups at this
position all exhibiting inhibition of hNAT1 and mNat2. This
suggests that the N–H hydrogen bond donor capability of
anilino-substituted species such as 4 is not essential for binding
to the enzyme, since ligands with hydrogen bond acceptor
Figure 5. In silico modeling of 48 and 49 in the active site of hNAT1 (pdb: 2PQT),12e
highlighting the potentially key interactions between the inhibitors and enzyme.
Analysis performed using GOLDÒ software.30

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3035
capable of forming
p
–p
interactions with the C3 moiety; however,
any of the conformational preferences, electronic properties or
hydrophobicity of aromatic substituents may be a requisite of
binding (Fig. 4b). Examples of both electron-rich and electron-poor
aromatic moieties at C3 have shown similar levels of potency to the
hit compound 4.
The ground state rotational freedom of amino-substituted spe-
cies 9 and 11 and the concomitant loss of entropy on binding to
hNAT1 could also contribute to the low inhibitory potency of these
compounds. However, the less conformationally flexible cyclopen-
tylamine 10 is also a poor inhibitor, suggesting that an N-, O-, or
directly linked aromatic substituent is indeed preferred at the C3
position.
2.3.5. Spectrophotometric evaluation of the C3-substituted
analogues
Figure 6. The% residual activity of mNat2 and hNAT1 when dosed with 30 lM of
compounds 4 and 41–50. Experiments were conducted in triplicate and results are
shown as averages one standard deviation. The stars and brackets above the bars
show the significance of the difference between the mNat2 and hNAT1 data when a
Student’s T-Test was performed: no stars = P >0.05; ⁄P <0.05; ⁄⁄P <0.01.
For a probe to be clinically useful for quantification of hNAT1 in
biological samples, it not only needs to be a potent and selective
binder of hNAT1, but it must also possess appropriate colorimetric
properties, namely: the probe must have a pKa value for the acidic
sulfonamide proton above the assay pH of 8 but below the pKaH of
the Arg127 residue; the color change should be distinct, so that it
can be unambiguously determined from visible spectra; and the
conjugate base of the probe must have a high absorption coeffi-
properties at C3 (aryloxy-substituted species such as 17) and
ligands with neither hydrogen-bond-acceptor nor-donor proper-
ties at C3 (aryl-substituted species such as 26) bind with similar
potencies to that of compound 4. In silico modeling was carried
out on compounds 3, 4, 6 and 17–26 and suggests that the binding
modes of all the anilino-, aryloxy- and aryl-substituted species are
similar, supporting the experimental observations; representative
examples of modeling solutions are shown in Figure 4a.
cient (eCB) to enable a high sensitivity of detection.
The colorimetric properties of species 3, 4, 6, 9–11 and 17–26
were evaluated and are outlined in Tables 5 and 6. pKa values were
determined via titration experiments (outlined in Section 4.2.5.2.).
e
Values for both the neutral (eN) and conjugate base (eCB) forms of
The other key observation from the pharmacological data is that
all derivatives bearing an aliphatic substituent at C3 (9–11) display
poor levels of inhibition versus hNAT1 and mNat2. Docking sug-
gests that no residues around the entrance to the active site are
each compound were calculated following the experimental proce-
dure described in Section 4.2.5.3.
Despite the promising inhibitory potency of both the C3 aryl-
oxy- and aryl-substituted species, the utility of both of these series
Table 8
Spectrophotometric properties of the C2 sulfonamide-substituted inhibitors 4 and 41–50
Compound
at pH 8
Compound
at pH 13
Δλmax pH 13
(nm)
Δλmax mNat2
(nm)
Compound
R1
Ph
pKa
9.2
ε
CB (M-1cm-1)
6590
εCB / εCB(4)
1.00
4
+ 79
+ 87
+ 112
N.D.
41
Me
9.5
6770
1.03
42
43
cHex
10.6
9.3
+ 53
+ 17
- 7
6790
6430
1.03
0.98
2’-Me-C6H4
N.D.
44
45
46
47
2’-NO2-C6H4
2’,6’-F2-C6H3
3’-NO2-C6H4
4’-Me-C6H4
7.9
8.4
8.6
9.7
+ 32
+ 39
+ 42
+ 73
N.D.
+ 79
N.D.
- 8
7610
7780
5670
7170
1.16
1.18
0.86
1.09
48
49
50
4’-F-C6H4
Furan-2’-yl
Benzyl
9.9
8.3
+ 59
+ 32
+ 61
+ 122
+ 99
+ 57
3490
7770
6660
0.53
1.18
1.01
10.0
N.D. = not determined.

3036
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
O
O
R1
NH
O
O
R1
NH
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
Cl
(iv)
(i)
(i)
9-59%
81%
42%
Cl
R2
59
60
52
53, 54
64-67
Scheme 6. Reagents and conditions: (i) Requisite boronic acid (2.0 equiv),
Pd(PPh3)2Cl2 (0.1 equiv), 4:1 THF/satd aq NaHCO3, reflux, 18 h. For identities of R1
and R2, refer to Table 9.
O
Ph
O
Ph
O
O
NH
NH2
Cl
NH
(v)
(ii)
82%
69%
NH
Cl
Table 9
51
53
Yields in the synthesis of amides 64–67
Compound
R1
R2
Isolated yield step (i) (%)
64
65
66
67
Ph
Ph
Bn
Bn
300-CHO–C6H4
Furan-300-yl
300-CHO–C6H4
Furan-300-yl
15
59
44
9
n
n
O
O
O
O
Ph
O
O
Ph
NH
NH
NH
Cl
(iv)
(iii)
54
61
n = 1
n = 1
(IC50 >30 lM). However, none of the conjugate bases display signif-
icantly enhanced absorption coefficient values over that of the con-
jugate base of 4.
(57%)
(49%)
55 n = 2
(50%)
62 n = 2
(54%)
Therefore, attention was next turned to modifying the substitu-
ent at the C2 position on the naphthoquinone core instead.
O
O
O
O
Ph
(iv)
58%
O
O
Ph
O
O
NH
Cl
NH
NH
(vii)
(vi)
Ph
Ph
Cl
OH
2.4. SAR at C2 of the naphthoquinone core
80%
57
58
56
63
2.4.1. C2 sulfonamido-substituted species
In silico docking of compounds 48 and 49 in hNAT1 as represen-
tative examples of new analogues suggested that at the C2 position
there are key interactions between the sulfonamide group and
Arg127 (a hypothesis supported by our previous work)21 and
between the R1 substituent and Tyr129 (Fig. 5). This potential
interaction was investigated by synthesis of a library of analogues
of 4 containing examples of both aliphatic and aromatic R1 substit-
uents (Scheme 4; Table 7). All sulfonamides were commercially
available except for 29 and 30, which were prepared from the cor-
responding sulfonyl chlorides using standard methods.31
Scheme 5. Reagents and conditions: (i) MeCOCl (excess), concd H2SO4 (cat.), 50 °C,
1 h; (ii) NaH (3.3 equiv), THF, rt, 30 min then PhCOCl (1.3 equiv), rt, 1 h; (iii)
requisite acyl chloride (4.0 equiv), BF3ꢂOEt2 (1.0 equiv), toluene, 90 °C, 4 h; (iv)
CeCl3ꢂ7H2O (1.0 equiv), MeOH, rt, 90 min then 3,5-dimethylaniline (3.0 equiv),
110 °C, 16 h; (v) 3,5-dimethylaniline (1.2 equiv), Pd(OAc)2 (0.01 equiv), XPhos
(0.03 equiv), K2CO3 (1.4 equiv), H2O (0.04 equiv), tBuOH, 100 °C, 16 h; (vi) (COCl)2
(1.2 equiv), DMF, CH2Cl2, rt, 3 h; (vii) naphthoquinone 51 (0.25 equiv), BF3ꢂOEt2
(0.25 equiv), toluene, 90 °C, 4 h.
of compounds as colorimetric probes are limited by their low pKa
values, which are below that of physiological pH.
Conversely, all the anilino-substituted and amino-substituted
species synthesised show clear changes in kmax between pH 8
and pH 13. The anilino-substituted species 3, 4 and 6 also show a
color change in the presence of mNat2, whereas the amino-substi-
tuted species 9–11 do not; this is likely to be attributable to the
weak binding of the amino-substituted species against the enzyme
An analysis of the potency of these compounds against hNAT1
and mNat2 (Fig. 6) shows that many of these compounds are poor
inhibitors of both enzymes. IC50 values were subsequently
Table 10
Pharmacological and spectrophotometric properties of amides 64–67
Compound
64
65
66
67
IC50 (mNat2)
(μM)
36.8
23.5
30.4
14.5
IC50 (hNAT1)
(μM)
22.1
11.4
19.3
12.5
10.0
10.9
19.0
12.1
pKa
Compound at
pH 8
Compound at
pH 13
λmax shift at
pH 13?
Yes
No
Yes
No
Yes
Yes
Yes
No
λmax shift with
mNat2?
Figure 7. The % residual activity of mNat2 and hNAT1 when dosed with 30 lM of
εCB (M-1cm-1)
8491
1.29
2449
0.37
compounds 59–63. Experiments were conducted in triplicate and results are shown
as averages one standard deviation. The stars and brackets above the bars show
the significance of the difference between the mNat2 and hNAT1 data when a
Student’s T-test was performed: no stars = P > 0.05; ⁄P <0.05; ⁄⁄P <0.01.
2672
0.41
4817
0.73
εCB / εCB(4)

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3037
Figure 8. Visible spectra of compound 64 (left) and 65 (right), showing the compounds at a final concentration of 15
l
M in 20 mM TrisꢂHCl buffer solution, pH 8 (yellow line),
4 M NaOH solution, pH 13.75 (purple line) and in 20 mM TrisꢂHCl buffer solution, pH 8, with 30
lM mNat2 (blue line).
(IC50
substituted 49 (IC50, mNat2 = 1.9
ylphenyl-substituted 43 and 20-nitrophenyl-substituted 44 show
weak activity with IC50 values against hNAT1 of 23.1 M and
21.8 M respectively.
,
mNat2 = 10.0
lM; IC50
,
hNAT1 = 10.0
l
M) and furan-20-yl-
M). 20-meth-
lM; IC50, hNAT1 = 7.0
l
l
l
First, these results suggest that at the C2 position, an aryl or het-
eroaryl substituent appears to be required for binding to hNAT1;
compound 42, which incorporates a fully saturated analogue of
the phenyl group within 4, shows poor levels of hNAT1 and mNat2
inhibition. This is possibly because
p–p interactions are instru-
mental in achieving high inhibitor potency, a conclusion which
supports the binding mode predicted by in silico modeling
(Fig. 5) which suggests an interaction between an aromatic C2 sub-
stituent and the Tyr129 residue of hNAT1.
Furthermore, the results show that substitution on the aromatic
R1 moiety is poorly tolerated, with examples of both electron-
donating and electron-withdrawing ortho-, meta- and para- substi-
tuted species showing poor levels of inhibition. The only substitu-
tion tolerated on an aromatic R1 moiety was found to be fluoro
substitution (compounds 45 and 48), which suggests that the R1
aromatic group occupies a tight pocket within the enzyme active
site. This hypothesis was further supported by the synthesis of
the benzyl substituted analogue 50, which also shows much poorer
levels of inhibition than 4.
Figure 9. In silico modeling of 65 in the active site of hNAT1 (pdb: 2PQT),12e
highlighting potential alternative binding mode for this furanyl analogue.
Distances between atoms highlighted by black dashed lines are given in Angstroms.
Analysis performed using GOLDÒ software.30 Docking was repeated 10 times giving
consistent results.
a
determined for species showing >50% inhibition at 30
compounds in this series which display comparable potency to
phenyl-substituted against one or both proteins are the
bioisosteric 40-fluorophenyl-substituted 48 (IC50, mNat2 = 3.9
M;
IC50, > 30
M), 20,60-difluorophenyl-substituted 45
lM. The only
The importance of the Tyr129 residue of hNAT1/mNat2 in bind-
ing of these naphthoquinone ligands was further investigated in a
study on the homologous enzyme from the Syrian hamster,
shNat2. hNAT1 and shNat2 share an 81% sequence homology and
4
l
=
l
hNAT1
O
O
O
O
O
NHSO2Ph
Cl
NHSO2Ph
NH
NHSO2Ph
NH
(iii)
(ii)
26%
36%
quant.
70%
O
O
Cl
Cl
NO2
70
NH2
O
O
NO2
(i)
74
79
NO2
NO2
O
O
NO2
NH2
NHSO2Ph
Cl
NHSO2Ph
NH
NHSO2Ph
NH
68
(ii)
(iii)
O
O
R
O
R
73
R
R
82
77
R = Me, quant.
R = Me, 57%
83 R = H, quant.
78 R = H, 26%
Scheme 7. Reagents and conditions: (i) PhSO2NH2 (1.0 equiv), Cs2CO3 (1.4 equiv), DMF, rt, 5 h; (ii) CeCl3ꢂ7H2O (1.0 equiv), MeOH, rt, 90 min then requisite aniline (3.0 equiv),
110 °C, 16 h; (iii) Pd/C 10% (0.1 equiv), H2, MeOH, rt, 16 h.

3038
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
O
O
O
NHSO2Ph
NH
NHSO2Ph
(iii)
13%
quant.
H2N
O2N
NH
O
O
O
O
NHSO2Ph
Cl
Cl
Cl
(i)
(ii)
80
75
O2N
55%
O2N
O
O
O
H2N
NHSO2Ph
NH
O2N
NHSO2Ph
NH
69
71, 72
(iii)
77%
12%
O
O
76
81
Scheme 8. Reagents and conditions: (i) PhSO2NH2 (1.0 equiv), Cs2CO3 (1.4 equiv), DMF, rt, 5 h; (ii) CeCl3ꢂ7H2O (1.0 equiv), MeOH, rt, 90 min then requisite aniline (3.0 equiv),
110 °C, 16 h; (iii) Pd/C 10% (0.1 equiv), H2, MeOH, rt, 16 h.
different preferred conformation to a sulfonamide group,33 which
is likely to affect the binding to hNAT1. Replacement of the sulfon-
amide functionality might also be expected to alter the colorimet-
ric properties of the molecules, such as kmax and
e values. It was
also anticipated the sulfonamide to amide switch would increase
the pKa by ꢀ7–8 log units.34 Since amides have shorter bond
lengths than their sulfonamide counterparts,35 it was predicted
that a larger R1 substituent than phenyl might be required to retain
the important
p–p interactions in the binding site; in particular,
computational modeling studies support the proposal of a benzyl
amide substituent as an isostere for a phenyl sulfonamide substit-
utent (see Fig. S2 in Supporting information).
Initially, a library of amides bearing a 3,5-dimethylanilino
group at C3 were synthesized for direct comparison with hit com-
pound 4. Literature procedures to generate the known intermedi-
ates 52 and 53 were followed (Scheme 5).36 However, the
attempted synthesis of 54 proceeded in low yield and thus a gen-
eral coupling procedure to access these intermediates was devel-
oped, using the conversion of 51 to 54 as the model reaction (see
Scheme S2 and Table S2 in Supporting information). The optimum
conditions were found to involve Lewis acid catalysis with BF3ꢂOEt2
in toluene; these improved conditions were subsequently used to
synthesize intermediates 54, 55 and 58 in good yield. From inter-
mediates 52, 54, 55 and 58, the desired final products could be
obtained by substitution with 3,5-dimethylaniline (under our stan-
dard conditions); however intermediate 53 was inert under these
reaction conditions. Final product 60 was therefore obtained using
a Buchwald–Hartwig coupling between naphthoquinone interme-
diate 53 and 3,5-dimethylaniline.37
Figure 10. In silico modeling of 81 (carbons in grey) and 82 (carbons in cyan) in the
active site of hNAT1 (pdb: 2PQT),12e highlighting potential interactions between the
7- or 8-amino substituent and the backbone carbonyl oxygen of Gly124 or Phe125.
Distances between atoms highlighted by black dashed lines are given in Angstroms.
Analysis performed using GOLDÒ software.30
homology in substrate specificity,32 but in shNat2, Tyr129 is
replaced by Leu (see Fig. S1 in Supporting information for sequence
alignment). Other key active site residues, in particular Arg127,
Phe125 and the catalytic Cys-His-Asp triad, are conserved between
shNat2, hNAT1 and mNat2. Compound 4 was found to be a poor
inhibitor of shNat2 (IC50 = 89 lM) although some evidence of a
color change was observed upon binding; this suggests that
Tyr129 does indeed play a crucial role in inhibitor recognition.
The colorimetric properties of this series of inhibitors were also
evaluated (Table 8). Varying the R1 substituent appears to have lit-
tle effect on the kmax value of the neutral species; however, elec-
The SAR at C2 for the amide series is steep, with only the benzyl
amide 61 showing a moderate level of mNat2 inhibition (IC50,
mNat2 = 28.1 lM), and all of the series have IC50 values of >30 lM
against hNAT1 (Fig. 7). The inactivity of phenyl amide 60 is possi-
bly attributable to the restricted rotation imposed on the molecule
through the use of the amide linker, which might prevent the key
tron-donating or electron-withdrawing groups can have
a
significant effect on both the pKa and CB of the inhibitor. Inhibitors
e
with lower pKa values (close to 8) are unsurprisingly found to exhi-
bit smaller shifts in kmax in the presence of either base or enzyme
relative to kmax at pH 8. 20,60-Difluorophenyl- and 2-furyl-substi-
tuted species 45 and 49 have undesirably low pKa values (and
p–p interactions with Tyr129 between the aryl rings from forming.
Introduction of a flexible –CH2– linker in benzyl amide 61 while
improving inhibitory activity still however does not lead to levels
of potency which are equivalent to sulfonamide 4.
hence
D
kmax), despite showing improved eCB values and similar
One alternative explanation for the lack of activity of these
amides is that all of these species 59–63 possess pKa values higher
than 14, which is likely to preclude formation of their conjugate
bases in the presence of hNAT1/mNat2 in an assay buffer of pH
8. We have previously established that the color change of sulfon-
amide inhibitors such as 4 in the hNAT1 active site is due to selec-
tive recognition of the conjugate base species by the enzyme.21 If
hNAT1 and mNat2 were to have higher affinities for the conjugate
base species of any given naphthoquinone inhibitor than the
potencies to those of the hit compound 4.
2.4.2. C2 amido-substituted species
On the basis of the in silico studies carried out (Figs. 4 and 5),
which was supported by and consistent with our initial SAR data,
we hypothesised that an amide group might be a suitable alterna-
tive to the sulfonamide as a binding partner for the guanidinium
group of Arg127. An amide group would be expected to have a

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3039
respective neutral species, then one might expect naphthoqui-
2.5. SAR at C5–C8 of the naphthoquinone core
nones with very high pKa values to be poor inhibitors.
Therefore, a small number of species with an amide substituent
at C2 but with an aryl substituent replacing the aniline substituent
at C3 were synthesized, to test the inhibitory potency and colori-
metric properties of amides with pKa values predicted to be within
a more appropriate range. These species were synthesized via a
Suzuki coupling reaction (Scheme 6; Table 9).
This small series of amide compounds were indeed found to
have lower pKa values than their C3 anilino-substituted counter-
parts (Table 10). Furthermore, all examples displayed improved
IC50 values against hNAT1 relative to the analogues with an anilino
substituent at C3. This supports a potential link between pKa and
potency which is currently under further investigation. The com-
pound in this series with the lowest pKa value, 64, is shown to
undergo a shift of kmax in the presence of enzyme (Fig. 8). Com-
Since the sulfonamide inhibitors generally appear to have more
favorable properties than their amide analogues, a series of nitro-
and amino-substituted species at the C5–C8 positions was synthe-
sized via a similar preparation to 4 but starting from 5- or 6-nitro-
2,3-dichloronaphtho-1,4-quinone 68 or 69, and utilizing 10% Pd on
carbon as a catalyst for the reduction in the final step (Schemes 7
and 8). It was also thought that the introduction of substituents at
the 5-, 6-, 7- or 8-positions might allow exploitation of new inhib-
itor-enzyme interactions, which could lead to an increase in inhib-
itory potency against hNAT1. In silico modeling studies suggest
that amino substituents at the 7- or 8-positions may be able to
interact with the backbone carbonyls of residues Gly124 or
Phe125 (Fig. 10); conversely it was hypothesized that nitro substit-
uents at these positions might not be tolerated. Docking did not
reveal any obvious additional polar contacts which might be
gained by substitution at the 5- or 6-positions.
In addition, it was hypothesized that amino substitution could
increase the absorption coefficient, e, of the ligands relative to that
of the unsubstituted species 4, since the amino group is auxochro-
mic and could donate electron density into the naphthoquinone
core.
When substituents were introduced directly onto the naphtho-
quinone core at positions 5–8, some clear SAR patterns emerged. A
5-nitro substituent (74) displayed similar IC50 values to those of
compound 4 against hNAT1 and mNat2, whilst those with 6-nitro,
7-nitro or 8-nitro substituents (75–77) were significantly less
active (Table 11; for squared correlation coefficients for IC50 data
see Table S2 in Supporting information). For the amino-substituted
series, 5-amino derivative 79 and 7-amino derivative 81 had
potencies comparable to that of 4, whilst the 6-amino derivative
pounds 65, 66 and 67 do not show corresponding
Dkmax shifts; this
could be due to their higher pKa values or because they have a dif-
ferent binding mode in the enzyme active site; in silico modeling
does propose a feasible alternative mode of binding for the fura-
nyl-substituted species 65 in which the furanyl oxygen is capable
of binding to Arg127 in place of the amide carbonyl oxygen
(Fig. 9), which would preclude a color change driven by recognition
between the conjugate base and Arg127.
Whilst studies on these compounds give an interesting mecha-
nistic insight into molecular interactions with the enzyme active
site, colorimetric studies show that they suffer from lower eCB val-
ues than the corresponding sulfonamides (Table 10). Furthermore,
when tested against hNAT2, it was found that amide species 64
and 65 were far less selective for hNAT1 over hNAT2 than their sul-
fonamide analogues, with 64 and 65 showing >50% inhibition of
hNAT2 at 30 lM.
Table 11
Pharmacological and spectrophotometric properties of nitro- and amino-substituted naphthoquinones 74–83
IC50
(mNat2)
(μM)
IC50
(hNAT1)
(μM)
Compound
at pH 8
Compound
at pH 13
Δλmax pH 13
(nm)
Δλmax mNat2
(nm)
Compound
pKa
εCB
εCB/ εCB(4)
74
75
76
+ 23
+ 73
+ 87
+ 95
+ 74
N.D.
8.2
7.5
8.0
4517
2241
3879
0.69
0.34
0.59
2.7
6.6
> 30
> 30
> 30
N.D.
77
78
79
80
+ 58
+ 74
+ 16
- 9
N.D.
+ 107
+ 16
8.6
8.4
*
2737
3012
0.42
0.46
2.40
1.90
> 30
> 30
5.7
> 30
> 30
4.2
15796
12542
N.D.
*
> 30
N.D.
81
82
83
+10
- 63
- 28
0
*
21892
12384
15842
3.32
1.88
2.40
2.5
2.6
- 62
- 12
8.4
8.2
0.43
0.27
0.54
0.12
N.D. = not determined.
⁄
Not possible to determine pKa via this method.

3040
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
2.6. Evaluation in cell extracts from the breast cancer cell line
ZR-75-1
Representative compounds from this study possessing a range
of functionality were tested for inhibitory activity of hNAT1 in
ZR-75-1 lysate at concentrations of 30 M, 10 M and 1 lM
l
l
(Fig. 12). All the representative sulfonamides which showed high
levels of potency against recombinant hNAT1 also showed high
levels of inhibition against hNAT1 in the lysate, with compounds
4, 48, 81 and 82 exhibiting >50% inhibition of hNAT1 at an inhibitor
concentration of 1 lM.
8-Amino substituted species 82 and 83 possessing a tenfold and
fiftyfold improved potency against hNAT1 over 4 respectively, and
with twofold improved eCB, were subsequently examined in spec-
trophotometric experiments with the ZR-75-1 cell extracts in an
attempt to detect the hNAT1 present in the cells. The probes were
tested at final concentrations ranging from 0.6 lM to 2.5 lM, but
again due to poor signal-to-noise ratios no kmax peak could be
detected at these low concentrations. A negative control concen-
Figure 11. The% residual activity of mNat2, hNAT1 and hNAT2 when dosed with
compounds 74, 77, 79 and 82 at 30 lM. Hit compound 4 is also included as a
control. Experiments were conducted in triplicate and results are shown as
averages one standard deviation. The stars and brackets above the bars show
the significance of the difference between pairs of data when a Student’s T-Test was
performed: no stars = P >0.05; ⁄P <0.05; ⁄⁄P <0.01; ⁄⁄⁄P <0.001.
tration of 20 lM was also used, clearly showing kmax corresponding
to the neutral (protonated) species in each case. This demonstrates
that further improvements to the sensitivity of the probes are
required for their clinical use; however, this study has provided
key underpinning insights to demonstrate that there is a scope
for variation around the naphthoquinone core of the original hit
compound 5 in order to achieve this.
80 showed low potency and the 8-amino derivative 82 possessed
IC50 values one order of magnitude more potent than 4. These
experimental results correlate well with the predictions from in
silico modeling.
Given the high potency of compound 82 against both hNAT1
and mNat2, with IC50 values of 540 nM and 430 nM respectively
against each enzyme, the analogous species 83 was also synthe-
sized. With IC50 values against hNAT1 and mNat2 of 120 and 270
nM respectively, 83 represents the most potent inhibitor of hNAT1
and mNat2 synthesized in this study. Its nitro-substituted precur-
3. Conclusions
In this study, we have designed and synthesized a family of
naphthoquinones which are capable of rapidly and unambiguously
detecting pure recombinant hNAT1 as they change color in the
presence of the protein through a mechanism which we have pre-
viously elucidated. As no similar colorimetric probes have been
reported to date in the literature, it proved necessary to determine
the key requirements for such probes to be clinically useful. This
study verifies that the potency, pKa and absorption coefficient of
sor 78 had low activity (>30 lM) against both enzymes.
Crucially, both 8-amino substituted species 82 and 83 possess
significantly improved absorption coefficients compared to com-
pound 4, whilst still leading to a discernible color change in the
presence of mNat2; compound 82 was also found to be highly
selective for hNAT1 and mNat2 over hNAT2 (Table 11; Fig. 11).
Compounds 82 and 83 were therefore subjected to further studies
in lysates from ZR-75-1 cells.
the conjugate base (eCB) are crucial parameters of probe design.
We have developed extensive SAR around the naphthoquinone
core of our hit compound 4, and have built up an in silico docking
model which is consistent with our experimental observations;
this should prove valuable for any future attempts to develop
hNAT1 inhibitors and is summarized in Figure 13. Compounds 82
and 83 show ten- and fifty-fold increases respectively in potency
and a two-fold increase in eCB over initial hit compound 4; 82 is
also shown to retain selectivity for hNAT1 over its isoenzyme
hNAT2. Although the conjugate bases of 82 and 83 could not be
clearly detected in absorption spectra with ZR-75-1 extracts, they
offer promising insights which aid ongoing work focused on
increasing the eCB of these probes whilst retaining high potency
and selectivity for hNAT1. The high potency of representative
examples of the sulfonamide probes against hNAT1 in ZR-75-1 cell
-NH2 favoured: ten-fold increase in
potency; two-fold increasein absorption
R1 = phenyl, 4-fluorophenyl, 2,6-
difluorophenyl, furyl tolerated
-NH2 tolerated
O
H
N
R1
R3
X
X = SO2 gives higher potency
than X = CO when R2 substituent
is anilino; possibly due to
sulfonamides having a lower pKa
R2
O
Figure 12. The % residual activity of hNAT1 in ZR-75-1 cell lysate when dosed with
representative compounds 3, 4, 45, 48, 49, 77, 81 and 82 at 30 lM, 10 lM and 1 lM,
Wide variety of aromatic species
tolerated; crucial substituent for
tuning of pKa
-NH2 and -NO2 tolerated
relative to a control experiment (vehicle only). Experiments were conducted in
triplicate and results are shown as averages one standard deviation. The stars
above the bars show the significance of the difference between the test data and its
respective control when a Student’s T-test was performed: no stars = P > 0.05; ⁄P
<0.05; ⁄⁄P <0.01; ⁄⁄⁄P <0.001.
Figure 13. Summary of SAR elucidated around the naphthoquinone core of hit
compound 4 in this study.

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3041
5'
2'
extracts also pave the way for the development of inhibitors with
6'
1'
4'
3'
O
O
improved physical properties such as solubility and cell permeabil-
ity which could potentially act as tools for elucidating the currently
unknown role which hNAT1 plays in ER+ breast cancer progres-
sion; these studies are also ongoing. Such selective inhibitors of
hNAT1 with appropriate properties for application in vivo might
also possess a valuable therapeutic use.
H
N
R'
8
5
1
4
10
9
2
3
7
6
X
R
Y
1''
6''
2''
R''
3''
5''
4''
4. Experimental
4.1. Chemistry
Figure 14. Numbering system employed throughout based on a generic represen-
tative naphthoquinone species.
4.1.1. General experimental
in MeCN. The flow rate was 1 mL/min and detection was at a wave-
length of 215 nm. Method B: RP-HPLC was performed on a Gilson
instrument equipped with Gilson 306 pumps, a Gilson 811C
dynamic mixer, a Gilson 806 manometric module with automated
sample injection on a Gilson 215 Liquid Handler, configured with a
Gilson 819 valve actuator. Separations were performed on a Varian
Chemicals were purchased from Sigma–Aldrich UK, TCI UK,
Apollo Scientific UK, Alfa Aesar UK, Fluorochem UK or Fisher Scien-
tific UK and used without further purification. Where appropriate,
all reactions involving moisture-sensitive reagents were carried
out under a nitrogen or argon atmosphere using standard vacuum
line techniques and glassware that was flame-dried before use.
Anhydrous DMF, anhydrous MeOH and anhydrous dioxane were
purchased from Sigma-Aldrich UK in SureSeal™ bottles and used
without further purification; other anhydrous solvents were dried
following the procedure outlined by Grubbs and co-workers.38
Water was purified by an ElixÒ UV-10 system. Organic layers were
dried over anhydrous MgSO4. Brine refers to a saturated aqueous
solution of sodium chloride. 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 silica gel 60 F254
aluminium-supported thin layer chromatography sheets. Plates
were visualised using UV light (254 nm), or thermal development
after dipping in 1% aq KMnO4. Flash column chromatography
was performed on Kieselgel 60 silica in a glass column, or on a Bio-
tage SP4 flash column chromatography platform.
Omnisphere
5 C18 (analytical) column (5 lm particle size,
150.0 mm ꢁ 4.6 mm). Experiments were performed under gradient
elution (eluent H2O containing 0.1% (v/v) TFA/MeCN 95:5 to 5:95
over 8 min then isocratic for 4 min). Sample injections consisted
of 20 lL of 1 mg/mL sample solution in DMSO. The flow rate was
1 mL/min Detection was at wavelengths of 220 and 254 nm using
a Gilson 170 Diode Array Detector. For each compound, retention
times (tR) are quoted to the nearest minute and are followed by
the% purity.
Compounds are characterized throughout this experimental
section by numbering aromatic carbons and any attached hydro-
gen nuclei as shown in Figure 14.
4.1.2. Representative Procedures
4.1.2.1. Representative Procedure 1: substitution of 2,3-dichlo-
ronaphthalene-1,4-diones with sulfonamides.
2,3-Dichlo-
Melting points were recorded on a Gallenkamp Hot Stage appa-
ratus and are uncorrected. Where relevant, the recrystallisation
solvent is reported in parentheses. Infrared spectra were recorded
on a Bruker Tensor 27 FT-IR spectrometer, neat or as KBr discs.
Selected characteristic peaks are reported in wavenumbers
(cmꢃ1). NMR spectra were recorded on Bruker Avance spectrome-
ters (DPX400, DQX400, AVII 500 or DRX500) in the deuterated sol-
vent stated. The field was locked by external referencing to the
relevant deuteron resonance. Chemical shifts (d) are reported in
parts per million (ppm) and coupling constants (J) are quoted in
Hz; both are reported to one decimal place. The coupling constants
were determined by analysis using ACD Labs software. Low-resolu-
tion mass spectra were recorded on either a VG MassLab 20–250 or
a Micromass Platform 1 spectrometer, operating in positive or neg-
ative mode, 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) using amyl ace-
tate 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.
ronaphthalene-1,4-dione (1.0 equiv), the requisite sulfonamide
(1.0 equiv) and Cs2CO3 (1.4 equiv) were stirred in DMF in a sealed
microwave vial at rt for 5 h. 1 M aq HCl (50 mL) was added and the
organic product extracted with EtOAc (3 ꢁ 20 mL). The organic
washings were combined, washed with brine (3 ꢁ 20 mL), dried
over magnesium sulfate, filtered and concentrated in vacuo to give
the crude product.
4.1.2.2. Representative Procedure 2: Substitution of N-(3-chloro-
1,4-dioxo-1,4-dihydronaphthalen-2-yl)sulfonamides
with
anilines. The requisite sulfonamide (1.0 equiv) was stirred
with cerium trichloride heptahydrate (1.0 equiv) in MeOH at rt in
a microwave vial for 1.5 h. The requisite aniline (3.0 equiv) was
added, the vial sealed and the reaction mixture heated to 90 °C for
16 h, unless otherwise stated. The solution was cooled to rt, satd
aq NH4Cl (30 mL) was added and the organic product was extracted
with EtOAc (3 ꢁ 20 mL). The organic washings were combined,
washed with brine (3 ꢁ 20 mL), dried over magnesium sulfate, fil-
tered and concentrated in vacuo to give the crude product.
Reverse-phase high-performance liquid chromatography
(RP-HPLC) was performed by one of two methods, as stated.
Method A: RP-HPLC was performed using a 1525 pump, 2707
autosampler and 2849 detector, all from Waters. Separations were
4.1.2.3. Representative Procedure 3: Substitution of N-(3-chloro-
1,4-dioxo-1,4-dihydronaphthalen-2-yl)sulfonamide with aliphatic
amines.
The requisite sulfonamide (1.0 equiv) was stirred
with cerium trichloride heptahydrate (0.4 or 1.0 equiv, as stated)
in toluene at rt in a microwave vial for 1.5 h. The requisite aniline
(3.0 equiv) was added, the vial sealed and the reaction mixture
heated to 110 °C for 6 h. The solution was cooled to rt, satd aq
NH4Cl (30 mL) was added and the organic product was extracted
with EtOAc (3 ꢁ 20 mL). The organic washings were combined,
performed on
(5
a Phenomonex Luma C18 (analytical) column
m particle size, 250.0 mm ꢁ 4.6 mm). Experiments were per-
l
formed under gradient elution (eluent H2O containing 0.1% (v/v)
TFA/MeCN 95:5 to 5:95 over 20 min then isocratic for 15 min).
Sample injections consisted of 40
lL of 2 mg/mL sample solution

3042
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
washed with brine (3 ꢁ 20 mL), dried over magnesium sulfate, fil-
188–192 °C (toluene); HPLC (method A) tR 17 min, 97%; dH
00
tered and concentrated in vacuo to give the crude product.
(400 MHz, DMSO-d6) 2.22 (6H, s, 2 ꢁ Ar-Me), 6.59 (2H, s, H2 and
00
00
0
0
H6 ), 6.67 (1H, s, H4 ), 7.34–7.40 (2H, m, H3 and H5 ), 7.47–7.51
0
0
0
4.1.2.4. Representative Procedure 4: Disubstitution of 2,3-
(1H, m, H4 ), 7.51–7.56 (2H, m, H2 and H6 ), 7.74–7.83 (3H, m, H6,
H7 and H5 or H8), 8.00–8.04 (1H, m, H5 or H8), 8.79 (1H, s, NH),
9.06 (1H, s, NH); m/z (ESIꢃ) 431 ([MꢃH]ꢃ, 100%); kmax (pH 8)
dichloronaphthalene-1,4-dione with phenols.
2,3-Dichlo-
ronaphthalene-1,4-dione (1.0 equiv), the requisite phenol
(2.2 equiv) and Cs2CO3 (2.2 equiv) were refluxed in THF for 16 h.
The solution was cooled to rt and partitioned between EtOAc
(50 mL) and 0.1 M aq NaOH (30 mL). The organic layer was washed
with satd aq NH4Cl (2 ꢁ 20 mL) and brine (3 ꢁ 20 mL), before being
dried over magnesium sulfate, filtered and concentrated in vacuo
to give the crude product.
498 nm
6590 Mꢃ1 cmꢃ1); pKa 9.2.
( (eCB
eN 11960 Mꢃ1 cmꢃ1), kmax (pH 13.75) 577 nm
4.1.5. N-(3-(400-Bromophenylamino)-1,4-dioxo-1,4-dihydronap-
hthalen-2-yl)benzenesulfonamide (6)23
Following Representative Procedure 2, using naphthoquinone 8
(300 mg, 0.86 mmol), 4-bromoaniline (446 mg, 2.59 mmol) and
CeCl3ꢂ7H2O (322 mg, 0.86 mmol) in MeOH (7 mL). Purification via
column chromatography on silica gel (eluent pet ether/EtOAc
90:10 to 50:50) gave 3-anilinonaphthoquinone 6 as a red solid
(133 mg, 30%). Mp 246–247 °C; HPLC (method A) tR 17 min, 96%;
dH (500 MHz, DMSO-d6) 6.98 (2H, d, J 8.4, H2’’ and H6’’), 7.36–7.41
4.1.2.5. Representative Procedure 5: Substitution of 2,3-diaryl-
oxynaphthalene-1,4-diones with benzenesulfonamide.
The
requisite diphenol (1.0 equiv), benzenesulfonamide (1.0 equiv) and
Cs2CO3 (1.2 equiv) were refluxed in THF for 1 h, unless otherwise
stated. The solution was cooled to rt and partitioned between
EtOAc (50 mL) and 1 M aq HCl (30 mL). The organic layer was
washed with brine (3 ꢁ 20 mL), before being dried over magne-
sium sulfate, filtered and concentrated in vacuo to give the crude
product.
0
0
0
(4H, m, H3 , H5 , H3’’ and H5’’), 7.51 (1H, t, J 7.0, H4 ), 7.57 (2H, d, J
0
0
7.0, H2 and H6 ), 7.75–7.83 (3H, m, H6, H7 and H8), 8.02 (1H, d, J
8.4, H5), 9.06 (1H, s, sulfonamide-NH), 9.13 (1H, s, aniline-NH);
m/z (ESIꢃ) 481 ([M(79Br)ꢃH]ꢃ, 83%), 483 ([M(81Br)ꢃH]ꢃ, 100%);
kmax (pH 8) 500 nm
(
eN 13330 Mꢃ1 cmꢃ1), kmax (pH 13.75)
4.1.2.6. Representative Procedure 6: Suzuki coupling of N-(3-
570 nm (eCB 7790 Mꢃ1 cmꢃ1); pKa 10.4.
chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-arylsulfonamide
with
arylboronic
acids.
The
requisite
sulfonamide
4.1.6. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzene-
sulfonamide (8)23
(1.0 equiv), the requisite boronic acid (2.0 equiv), Pd(PPh3)2Cl2
(0.1 equiv) and satd aq NaHCO3 were stirred in THF in a sealed
microwave vial under N2 and heated to 100 °C for 16 h. The solu-
tion was cooled to rt and partitioned between EtOAc (50 mL) and
satd aq NH4Cl (50 mL). The organic layer was collected, washed
with brine (3 ꢁ 20 mL), dried over magnesium sulfate, filtered
and concentrated in vacuo to give the crude product.
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione
7 (1.00 g, 4.40 mmol), benzenesulfonamide
(0.69 g, 4.40 mmol) and Cs2CO3 (2.00 g, 6.16 mmol) in DMF
(10 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 75:25) gave sulfonamide 8 as a yellow solid
(1.39 g, 91%). Mp 219–223 °C; dH (400 MHz, DMSO-d6) 7.56–7.64
0
0
0
(2H, m, H3 and H5 ), 7.66 (1H, app. t, J 7.1, H4 ), 7.81–7.90 (2H, m,
0
0
4.1.2.7. Representative Procedure 7: Reduction of nitronaph-
H6 and H7), 7.91–7.98 (H2 , H6 and H5 or H8), 8.01–8.06 (1H, m,
thalene-1,4-diones to aminonapthahlene-1,4-diones.
The
H5 or H8); m/z (ESIꢃ) 346 ([M(35Cl)ꢃH]ꢃ, 100%).
aromatic nitro species (1.0 equiv) and 10% Pd/C catalyst (0.2 equiv)
were stirred in MeOH in a sealed microwave vial under H2 (1 atm.)
at rt for 16 h. The mixture was filtered through CeliteÒ to remove
the Pd/C catalyst, and the filtrate concentrated in vacuo to give
the crude product.
4.1.7. N-(3-((200-Methoxyethyl)amino)-1,4-dioxo-1,4-dihydrona-
phthalen-2-yl)benzenesulfonamide (9)
Following Representative Procedure 3, using naphthoquinone 8
(200 mg, 0.58 mmol), 3-methoxyethylamine (150
lL, 1.74 mmol)
and CeCl3ꢂ7H2O (86 mg, 0.23 mmol) in toluene (10 mL). Purifica-
4.1.3. N-(3-Phenylamino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
benzenesulfonamide (3)23
tion via column chromatography (eluent pet ether/acetone
70:30) gave 3-aminonaphthoquinone
9 as an orange solid
Following Representative Procedure 2, using naphthoquinone 8
(195 mg, 87%). Mp 167–169 °C; HPLC (method B) tR 7 min, 97%;
mmax (KBr) 3312 (N–H), 3235 (N–H), 1676 (C@O), 1610 (C@O); dH
(100 mg, 0.29 mmol), aniline (79
l
L, 0.84 mmol) and CeCl3ꢂ7H2O
00
(108 mg, 0.29 mmol) in MeOH (5 mL). Purification via column
chromatography on silica gel (eluent pet ether/acetone 85:15) gave
3-anilinonaphthoquinone 3 as a red solid (80 mg, 63%). Mp 215–
221 °C; HPLC (method A) tR 18 min, >99%; dH (400 MHz, DMSO-
(400 MHz, CDCl3) 3.43 (3H, s, OMe), 3.67 (2H, t, J 5.2, 2 ꢁ H2 ),
00
4.16 (2H, q, J 5.2, 2 ꢁ H1 ), 6.64 (1H, br s, sulfonamide-NH), 6.72
0
0
(1H, br t, J 5.2, amine-NH), 7.32–7.39 (2H, m, H3 and H5 ), 7.45–
0
7.52 (1H, m, H4 ), 7.55–7.64 (2H, m, H6 and H7), 7.66–7.71 (1H,
00
00
00
00
0
0
d6) 6.99–7.09 (3H, m, H2 , H4 and H6 ), 7.23 (2H, app. t, J 7.9, H3
m, H5 or H8), 7.79 (2H, d, J 7.3, H2 and H6 ), 8.00–8.06 (1H, m, H5
or H8); dC (100 MHz, CDCl3) 44.2, 58.8, 70.6, 109.3, 125.9, 126.8,
127.8, 128.6, 130.1, 131.8, 132.4, 133.1, 134.9, 138.2, 143.9,
178.6, 181.7; m/z (ESIꢃ) 385 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C19H18-
N2NaO5S+ ([M+Na]+) requires 409.0829, found 409.0829; kmax (pH
8) 469 nm (eN 1330 Mꢃ1 cmꢃ1), kmax (pH 13.75) 558 nm (eCB
1010 Mꢃ1 cmꢃ1); pKa 9.8.
00
0
0
0
and H5 ), 7.33–7.41 (2H, m, H3 and H5 ), 7.45–7.52 (1H, m, H4 ),
0
0
7.52–7.58 (2H, m, H2 and H6 ), 7.73–7.84 (3H, m, H6, H7 and H5
or H8), 7.99–8.04 (1H, m, H5 or H8), 9.02 (2H, s, aniline-NH and sul-
fonamide-NH); m/z (ESIꢃ) 403 ([MꢃH]ꢃ, 100%); kmax (pH 8) 489 nm
(e e
N 8700 Mꢃ1 cmꢃ1), kmax (pH 13.75) 561 nm ( CB 7900 Mꢃ1 cmꢃ1);
pKa 9.5.
4.1.4. N-(3-(300,500-Dimethylphenylamino)-1,4-dioxo-1,4-dihydro-
naphthalen-2-yl)benzenesulfonamide (4)21,23
4.1.8. N-(3-(Cyclopentylamino)-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)benzenesulfonamide (10)
Following Representative Procedure 2, using naphthoquinone 8
(1.30 g, 3.75 mmol), 3,5-dimethylaniline (1.40 mL, 11.25 mmol)
and CeCl3ꢂ7H2O (1.39 g, 3.75 mmol) in MeOH (10 mL). Purification
via column chromatography on silica gel (eluent pet ether/acetone
90:10 to 50:50) and subsequent recrystallisation from toluene gave
3-anilinonaphthoquinone 4 as a red solid (919 mg, 57%). Mp
Following Representative Procedure 3, using naphthoquinone 8
(200 mg, 0.58 mmol), cyclopentylamine (172 lL, 1.74 mmol) and
CeCl3ꢂ7H2O (86 mg, 0.23 mmol) in toluene (10 mL). Purification
via column chromatography (eluent pet ether/acetone 70:30) gave
3-aminonaphthoquinone 10 as a red solid (204 mg, 89%). Mp 199–
201 °C; HPLC (method B) tR 9 min, 99%; mmax (KBr) 3318 (N–H),

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3043
3243 (N–H), 1673 (C@O), 1612 (C@O); dH (400 MHz, CDCl3) 1.49–
4.1.11. N-(3-(300,500-Dimethylphenoxy)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide (18)
Following Representative Procedure 4, using 2,3-dichloronaph-
thalene-1,4-dione (454 mg, 2.00 mmol), 3,5-dimethylphenol
00
00
00
1.61 (2H, m, 1 ꢁ H2 and 1 ꢁ H5 ), 1.66–1.82 (4H, m, 2 ꢁ H3 and
00
00
00
2 ꢁ H4 ), 2.08–2.21 (2H, m, 1 ꢁ H2 and 1 ꢁ H5 ), 5.02 (1H, app.
00
sext, J 7.1, H1 ), 6.41 (1H, d, J 7.1, amine-NH), 6.68 (1H, s, sulfon-
7
0
0
amide-NH), 7.32–7.39 (2H, m, H3 and H5 ), 7.45–7.52 (1H, m,
0
(537 mg, 4.40 mmol) and Cs2CO3 (1434 mg, 4.40 mmol) in THF
(30 mL) gave intermediate diaryloxy 15 as an orange solid
(709 mg, 89%). Mp 175–177 °C; mmax (neat) 1665 (C@O), 1590
(C@O); dH (500 MHz, CDCl3) 2.18 (12H, s, 4 ꢁ Ar-Me), 6.65 (2H, s,
H4 ), 7.55–7.64 (2H, m, H6 and H7), 7.66–7.72 (1H, m, H5 or H8),
0
0
7.80 (2H, d, J 7.8, H2 and H6 ), 7.99–8.06 (1H, m, H5 or H8); dC
(100 MHz, CDCl3) 24.1, 34.5, 55.0, 109.1, 125.9, 126.7, 127.8,
128.6, 130.1, 131.9, 132.3, 133.1, 134.9, 138.3, 143.5, 178.5,
182.0; m/z (ESIꢃ) 395 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C21H20N2NaO4-
S+ ([M+Na]+) requires 419.1036, found 419.1036; kmax (pH 8)
0
00
0
0
00
00
H4 and H4 ), 6.73 (4H, s, H2 , H6 , H2 and H6 ), 7.88–7.93 (2H, m,
H6 and H7), 8.01–8.06 (2H, m, H5 and H8); dC (125 MHz, CDCl3)
20.7, 113.6, 124.3, 126.0, 131.0, 134.3, 138.8, 146.2, 156.7, 180.2;
m/z (ESI+) 399 ([M+H]+, 100%), 421 ([M+Na]+, 20%); HRMS (ESI+)
471 nm
( ) kmax (pH 13.75) 553 nm (eCB
eN 10080 Mꢃ1 cmꢃ1
4470 Mꢃ1 cmꢃ1); pKa 10.1.
C
26H22NaO+4 ([M+Na]+) requires 421.1410, found 421.1391. Then,
following Representative Procedure 5, using naphthoquinone 15
without further purification (400 mg, 1.01 mmol), benzenesulfon-
amide (158 mg, 1.01 mmol) and Cs2CO3 (393 mg, 1.21 mmol) in
THF (30 mL) and subsequent recrystallisation from toluene gave
2-sulfonamidonaphthoquinone 18 as an orange–brown solid
(248 mg, 57%). Mp 218–223 °C (toluene); HPLC (method A) tR
18 min, 96%; mmax (neat) 3242 (N–H), 1662 (C@O), 1641 (C@O);
4.1.9. N-(3-(Benzylamino)-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)benzenesulfonamide (11)
Following Representative Procedure 3, using naphthoquinone 8
(100 mg, 0.29 mmol), benzylamine (100 lL, 0.86 mmol) and CeCl3-
ꢂ7H2O (107 mg, 0.29 mmol) in toluene (4 mL). Purification via col-
umn chromatography (eluent pet ether/acetone 85:15) and
subsequent recrystallisation from toluene gave 3-aminonaphtho-
quinone 11 as an orange solid (50 mg, 42%). Mp 206–211 °C (tolu-
ene); HPLC (method A) tR 16 min, 99%; mmax (neat) 3342 (N–H),
1675 (C@O), 1612 (C@O); dH (500 MHz, CDCl3) 5.12 (2H, d, J 5.8,
benzyl-CH2), 6.56 (1H, s, sulfonamide-NH), 6.59 (1H, br s, amine-
00
dH (500 MHz, CDCl3) 2.22 (6H, s, 2 ꢁ Ar-Me), 6.28 (2H, s, H2 and
00
00
0
0
H6 ), 6.66 (1H, s, H4 ), 7.32–7.37 (2H, m, H3 and H5 ), 7.43–7.49
0
(1H, m, H4 ), 7.72–7.77 (3H, m, H6, H7 and NH), 7.80–7.85 (2H, m,
0
0
H2 and H6 ), 7.94–7.99 (1H, m, H5), 8.12–8.18 (1H, m, H8); dC
(125 MHz, CDCl3) 21.2, 114.5, 125.3, 126.8, 126.9, 127.1, 128.5,
129.8, 131.1, 131.1, 132.7, 133.9, 134.9, 138.8, 140.1, 141.2,
156.0, 178.8, 180.5; m/z (ESI+) 434 ([M+H]+, 100%), 456 ([M+Na]+,
60%); HRMS (ESI+) C24H19NNaO5S+ ([M+Na]+) requires 456.0876,
found 456.0857; kmax (pH 8) <400 nm, kmax (pH 13.75) 474 nm
0
0
00
NH), 7.30–7.35 (1H, m, H4’’), 7.35–7.40 (6H, m, H3 , H5 , H2’’, H3
,
0
H5’’ and H6’’), 7.50 (1H, t, J 7.7, H4 ), 7.57–7.64 (2H, m, H6 and H7),
0
0
7.67–7.71 (1H, m, H8), 7.82 (2H, d, J 7.6, H2 and H6 ), 8.02–8.05
(1H, m, H5); dC (125 MHz, CDCl3) 48.9, 109.8, 126.0, 126.8, 127.8,
127.9, 128.1, 128.7, 128.9, 130.0, 131.3, 132.5, 133.1, 134.9,
137.8, 138.3, 143.6, 178.8, 181.8; m/z (ESIꢃ) 417 ([MꢃH]ꢃ, 100%);
HRMS (ESI+) C23H18N2NaO4S+ ([M+Na]+) requires 441.0879, found
441.0865; kmax (pH 8) 486 nm (eN 10480 Mꢃ1 cmꢃ1), kmax (pH
13.75) 560 nm (eCB 5770 Mꢃ1 cmꢃ1); pKa 10.4.
(
eCB 7200 Mꢃ1 cmꢃ1); pKa 5.0.
4.1.12. N-(3-(400-Bromophenoxy)-1,4-dioxo-1,4-dihydronaph-
thalen-2-yl)benzenesulfonamide (19)
Following Representative Procedure 4, using 2,3-dichloronaph-
thalene-1,4-dione
(761 mg, 4.40 mmol) and Cs2CO3 (1434 mg, 4.40 mmol) in THF
(30 mL) gave intermediate diaryloxy 16 as yellow solid
(715 mg, 72%). Mp 182–185 °C; mmax (neat) 1677 (C@O), 1659
7
(454 mg, 2.00 mmol), 4-bromophenol
4.1.10. N-(3-Phenoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
benzenesulfonamide (17)
a
Following Representative Procedure 4, using 2,3-dichloronaph-
0
0
00
(C@O); dH (500 MHz, CDCl3) 6.78–6.82 (4H, m, H2 , H6 , H2 and
thalene-1,4-dione
7
(454 mg, 2.00 mmol), phenol (414 mg,
00
0
0
00
00
H6 ), 7.34–7.39 (4H, m, H3 , H5 , H3 and H5 ), 7.78–7.82 (2H, m,
H6 and H7), 8.10–8.15 (2H, m, H5 and H8); dC (125 MHz, CDCl3)
116.4, 118.3, 126.9, 130.6, 132.4, 134.5, 145.7, 155.4, 180.0; m/z
(FI) 498 ([M(79Br79Br)]+, 50%), 500 ([M(79Br)(81Br)]+, 100%), 502
([M(81Br)(81Br)]+, 50%); HRMS (FI) C22H12O4Br2 ([M(79Br79Br)]+)
requires 497.9092, found 497.9102. Then, following Representative
Procedure 5, using naphthoquinone 16 without further purification
(400 mg, 0.80 mmol), benzenesulfonamide (126 mg, 0.80 mmol)
and Cs2CO3 (313 mg, 0.96 mmol) in THF (30 mL) and subsequent
recrystallisation from toluene gave 2-sulfonamidonaphthoquinone
19 as a yellow solid (210 mg, 43%). Mp 215–218 °C (toluene); HPLC
(method A) tR 19 min, 97%; mmax (neat) 3251 (N–H), 1675 (C@O),
4.40 mmol) and Cs2CO3 (1434 mg, 4.40 mmol) in THF (30 mL) gave
intermediate diphenoxy 14 as an orange solid (430 mg, 63%). Mp
188–193 °C; mmax (neat) 1662 (C@O), 1591 (C@O); dH (400 MHz,
0
0
00
00
CDCl3) 6.88–6.94 (4H, m, H2 , H6 , H2 and H6 ), 7.02–7.09 (2H, m,
0
00
0
0
00
00
H4 and H4 ), 7.21–7.28 (4H, m, H3 , H5 , H3 and H5 ), 7.75–7.81
(2H, m, H6 and H7), 8.10–8.16 (2H, m, H5 and H8); dC (100 MHz,
CDCl3) 116.6, 123.6, 126.8, 129.4, 130.8, 134.3, 146.1, 156.5,
180.4; m/z (ESI+) 343 ([M+H]+, 80%), 365 ([M+Na]+, 100%); HRMS
(ESI+) C22H14NaO4+ ([M+Na]+) requires 365.0784, found 365.0768.
Then, following Representative Procedure 5, using naphthoquinone
14 without further purification (400 mg, 1.17 mmol), benzenesul-
fonamide (184 mg, 1.17 mmol) and Cs2CO3 (458 mg, 1.40 mmol)
in THF (30 mL) for 16 h and subsequent recrystallisation from tol-
uene gave 2-sulfonamidonaphthoquinone 17 as a yellow solid
(175 mg, 67%). Mp 216–219 °C (toluene); HPLC (method A) tR
18 min, 95%; mmax (neat) 3235 (N–H), 1668 (C@O), 1655 (C@O);
00
00
1649 (C@O); dH (500 MHz, CDCl3) 6.57–6.62 (2H, m, H2 and H6 ),
00
00
0
0
7.29–7.34 (2H, m, H3 and H5 ), 7.36–7.42 (2H, m, H3 and H5 ),
0
0
0
7.48–7.53 (1H, m, H4 ), 7.72–7.84 (5H, m, H6, H7, H2 , H6 and NH),
7.93–7.98 (1H, m, H5), 8.13–8.18 (1H, m, H8); dC (125 MHz, CDCl3)
116.1, 118.7, 126.9, 126.9, 127.0, 128.8, 129.7, 130.9, 131.0, 132.1,
133.0, 134.1, 135.1, 140.2, 140.4, 155.0, 178.5, 180.3; m/z (ESIꢃ)
482 ([M(79Br)ꢃH]ꢃ, 100%), 484 ([M(81Br)ꢃH]ꢃ, 100%); HRMS
(ESI+) C22H14BrNNaO5S+ ([M(81Br)+Na]+) requires 507.9649, found
507.9666; kmax (pH 8) <400 nm, kmax (pH 13.75) 473 nm (eCB
8550 Mꢃ1 cmꢃ1); pKa 5.0.
00
00
dH (500 MHz, DMSO-d6) 6.82–6.86 (2H, m, H2 and H6 ), 6.99–
00
00
00
7.04 (1H, m, H4 ), 7.18–7.24 (2H, m, H3 and H5 ), 7.46–7.51 (2H,
0
0
0
m, H3 and H5 ), 7.53–7.57 (1H, m, H4 ), 7.81–7.92 (5H, m, H5, H6,
0
0
H7, H2 and H6 ), 8.03–8.06 (1H, m, H8), 10.30 (1H, br s, NH); dC
(125 MHz, DMSO-d6) 116.4, 122.7, 126.1, 126.1, 126.3, 128.7,
129.1, 130.6, 130.6, 132.3, 132.4, 134.4, 134.5, 141.7, 145.1,
156.4, 179.1, 180.6; m/z (ESIꢃ) 405 ([MꢃH]ꢃ, 100%); HRMS (ESI+)
4.1.13. N-(3-(200-Chlorophenyl)-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)benzenesulfonamide (20)
Following Representative Procedure 6, using naphthoquinone 8
(200 mg, 0.58 mmol), 2-chlorophenylboronic acid (181 mg,
C
22H15NNaO5S+ ([M+Na]+) requires 428.0563, found 428.0565;
kmax (pH 8) <400 nm, kmax (pH 13.75) 473 nm (
pKa 5.0.
e
CB 7080 Mꢃ1 cmꢃ1);

3044
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
1.14 mmol), Pd(PPh3)2Cl2 (41 mg, 0.06 mmol) and satd aq NaHCO3
(5 mL) in THF (22 mL). Purification via column chromatography on
silica gel (eluent pet ether/acetone 75:25) and subsequent recrys-
tallisation from toluene gave 3-arylnaphthoquinone 20 as a yellow
solid (119 mg, 48%). Mp 194-196 °C (toluene); HPLC (method B) tR
10 min, 96%; mmax (KBr) 3206 (N–H), 1673 (C@O), 1656 (C@O); dH
yellow solid (74 mg, 31%). Mp 200–201 °C (toluene); HPLC
(method B) tR 9 min, >99%; mmax (KBr) 3229 (N–H), 1698 (C@O),
1671 (C@O), 1651 (C@O); dH (400 MHz, Acetone-d6) 7.37–7.45
00
00
00
00
0
0
(2H, m, two of H2 , H4 , H5 and H6 ), 7.49–7.55 (4H, m, H3 , H5
00
00
00
00
0
and two of H2 , H4 , H5 and H6 ), 7.63–7.70 (1H, m, H4 ), 7.75–
0
0
7.85 (2H, m, H2 and H6 ), 7.87–7.97 (2H, m, H6 and H7), 8.06–
8.16 (2H, m, H5 and H8), 8.69 (1H, br s, NH), 9.89 (1H, s, CHO); dC
(100 MHz, Acetone-d6) 126.5, 126.7, 126.9, 128.7, 129.2, 129.8,
131.2, 132.1, 132.5, 133.0, 133.4, 134.4, 135.2, 136.2, 136.5,
137.2, 139.6, 141.7, 181.7, 183.6, 192.0; m/z (ESIꢃ) 416 ([MꢃH]ꢃ,
100%); HRMS (ESIꢃ) C23H14NO5Sꢃ ([MꢃH]ꢃ) requires 416.0598,
found 416.0596; kmax (pH 8) <400 nm, kmax (pH 13.75) 474 nm
00
00
00
00
(400 MHz, Acetone-d6) 7.21–7.36 (4H, m, H3 , H4 , H5 and H6
)
0
0
0
7.44–7.53 (2H, m, H3 and H5 ), 7.59 (1H, s, H4 ), 7.67–7.76 (2H,
0
0
m, H2 and H6 ), 7.85–7.97 (2H, m, H6 and H7), 8.05–8.14 (2H, m,
H5 and H8), 8.61 (1H, br s, NH); dC (100 MHz, Acetone-d6) 126.7,
126.8, 126.8, 126.9, 129.1, 129.4, 130.8, 131.2, 131.6, 132.4,
132.6, 132.9, 134.3, 134.4, 134.8, 135.2, 140.7, 141.8, 181.2,
182.5; m/z (ESIꢃ) 422 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C22H14ClN-
NaO4S+ ([M+Na]+) requires 446.0224, found 446.0225; kmax (pH
(
eCB 5720 Mꢃ1 cmꢃ1); pKa 5.0.
8) <400 nm, kmax (pH 13.75) 474 nm (
e
CB 8860 Mꢃ1 cmꢃ1); pKa 4.9.
4.1.17. N-(3-(400-Formylphenyl)-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)benzenesulfonamide (24)
4.1.14. N-(3-(300-Chlorophenyl)-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)benzenesulfonamide (21)
Following Representative Procedure 6, using naphthoquinone 8
(200 mg, 0.58 mmol), 4-formylbenzeneboronic acid (174 mg,
1.14 mmol), Pd(PPh3)2Cl2 (41 mg, 0.06 mmol) and satd aq NaHCO3
(5 mL) in THF (22 mL). Purification via column chromatography on
silica gel (eluent pet ether/acetone 90:10 to 70:30) and subsequent
recrystallization from toluene gave 3-arylnaphthoquinone 24 as a
yellow solid (59 mg, 24%). Mp 200–202 °C (toluene); HPLC
(method B) tR 9 min, 99%; mmax (KBr) 3234 (N–H), 1698 (C@O),
1668 (C@O), 1650 (C@O); dH (400 MHz, DMSO-d6) 7.40–7.47 (4H,
Following Representative Procedure 6, using naphthoquinone 8
(200 mg, 0.58 mmol), 3-chlorophenylboronic acid (181 mg,
1.14 mmol), Pd(PPh3)2Cl2 (41 mg, 0.06 mmol) and satd aq NaHCO3
(5 mL) in THF (22 mL). Purification via column chromatography on
silica gel (eluent pet ether/acetone 75:25) and subsequent recrys-
tallisation from toluene gave 3-arylnaphthoquinone 21 as a yellow
solid (87 mg, 35%). Mp 168-172 °C (toluene); HPLC (method B) tR
11 min, 99%; mmax (KBr) 3229 (N–H), 1670 (C@O), 1651 (C@O); dH
0
0
00
00
00
00
m, H3 , H5 and H2 and H6 or H3 and H5 ), 7.52–7.58 (1H, m,
00
00
00
0
0
0
00
(400 MHz, Acetone-d6) 7.26–7.31 (3H, m, H4 , H5 and H6 ), 7.33
H4 ), 7.58–7.63 (2H, m, H2 and H6 ), 7.76–7.84 (2H, m, H2 and
00
0
0
00
00
00
(1H, s, H2 ), 7.44–7.51 (2H, m, H3 and H5 ), 7.57–7.63 (1H, m,
H6 or H3 and H5 ), 7.85–7.96 (2H, m, H6 and H7), 7.98–8.08 (2H,
m, H5 and H8), 10.02 (2H, s, CHO and NH); dC (100 MHz, DMSO-
d6) 126.7, 127.0, 127.2, 129.3, 129.5, 131.5, 132.1, 132.4, 133.1,
135.1, 135.6, 136.5, 138.7, 139.1, 140.0, 142.5, 182.0, 183.9,
193.8; m/z (ESIꢃ) 416 ([MꢃH]ꢃ, 100%); HRMS (ESIꢃ) C23H14NO5Sꢃ
([MꢃH]ꢃ) requires 416.0598, found 416.0600; kmax (pH 8)
<400 nm, kmax (pH 13.75) 477 nm (eeCB 5880 Mꢃ1 cmꢃ1); pKa 4.6.
0
0
0
H4 ), 7.63–7.69 (2H, m, H2 and H6 ), 7.84–7.94 (2H, m, H6 and
H7), 8.04–8.13 (2H, m, H5 and H8), 8.53 (1H, br s, NH); dC
(100 MHz, Acetone-d6) 126.6, 126.7, 126.9, 128.9, 129.1, 129.6,
129.7, 130.9, 131.2, 132.5, 133.1, 133.3, 134.3, 134.4, 135.1,
136.1, 139.7, 141.7, 181.6, 183.4; m/z (ESIꢃ) 422 ([MꢃH]ꢃ, 100%);
HRMS (ESIꢃ) C22H13ClNO4Sꢃ ([MꢃH]ꢃ) requires 422.0259, found
422.0258; kmax (pH 8) <400 nm, kmax (pH 13.75) 473 nm (eCB
3890 Mꢃ1 cmꢃ1); pKa 4.5.
4.1.18. N-(3-(Furan-200-yl)-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
benzenesulfonamide (25)
4.1.15. N-(3-(400-Chlorophenyl)-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)benzenesulfonamide (22)
Following Representative Procedure 6, using naphthoquinone 8
(200 mg, 0.58 mmol), furan-2-ylboronic acid (130 mg, 1.14 mmol),
Pd(PPh3)2Cl2 (41 mg, 0.06 mmol) and satd aq NaHCO3 (5 mL) in
THF (22 mL). Purification via column chromatography on silica
gel (eluent pet ether/acetone 90:10 to 70:30) and subsequent
recrystallisation from toluene gave 3-arylnaphthoquinone 25 as a
purple solid (100 mg, 45%). Mp 144–146 °C (toluene); HPLC
(method B) tR 10 min, 99%; mmax (KBr) 3242 (N–H), 1668 (C@O),
1648 (C@O); dH (400 MHz, Acetone-d6) 6.66–6.70 (1H, dd, J 3.5,
Following Representative Procedure 6, using naphthoquinone 8
(200 mg, 0.58 mmol), 4-chlorophenylboronic acid (181 mg,
1.14 mmol), Pd(PPh3)2Cl2 (41 mg, 0.06 mmol) and satd aq NaHCO3
(5 mL) in THF (22 mL). Purification via column chromatography on
silica gel (eluent pet ether/acetone 80:20) and subsequent recrys-
tallisation from toluene gave 3-arylnaphthoquinone 22 as a yellow
solid (115 mg, 47%). Mp 222–223 °C (toluene); HPLC (method B) tR
11 min, 95%; mmax (KBr) 3240 (N–H), 1627 (C@O), 1655 (C@O); dH
00
00
0
0
1.7, H4 ), 7.45 (1H, d, J 3.5, H5 ), 7.53–7.62 (2H, m, H3 and H5 ),
7.62–7.68 (1H, m, H4 ), 7.72 (1H, d, J 1.7, H3 ), 7.81–7.92 (2H, m,
00
00
00
0
00
(400 MHz, DMSO-d6) 7.21–7.27 (2H, m, H2 and H6 or H3 and
00
00
00
00
00
0
0
H5 ), 7.30–7.38 (2H, m, H2 and H6 or H3 and H5 ), 7.43–7.51
H6 and H7) 7.93–8.01 (3H, m, H2 , H6 and H5 or H8), 8.09–8.14
(1H, m, H5 or H8), 8.98 (1H, s, NH); dC (100 MHz, Acetone-d6)
114.0, 120.6, 125.5, 127.5, 127.9, 128.4, 130.2, 132.4, 133.6,
134.1, 135.5, 135.9, 137.4, 143.5, 146.2, 147.4, 181.9, 183.8; m/z
(ESIꢃ) 378 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C20H13NNaO5S+
([M+Na]+) requires 402.0407, found 402.0407; kmax (pH 8)
0
0
0
0
0
(2H, m, H3 and H5 ), 7.54–7.66 (3H, m, H2 , H4 and H6 ), 7.80–
7.93 (2H, m, H6 and H7), 7.97–8.05 (2H, m, H5 and H8), 9.93 (1H,
br s, NH); dC (100 MHz, DMSO-d6) 126.4, 126.7, 127.0, 127.2,
128.4, 129.5, 129.8, 131.2, 131.5, 132.4, 133.1, 134.2, 135.1,
135.5, 140.0, 142.7, 182.0, 184.0; m/z (ESIꢃ) 422 ([MꢃH]ꢃ, 100%);
HRMS (ESIꢃ) C22H13ClNO4Sꢃ ([MꢃH]ꢃ) requires 422.0259, found
422.0260; kmax (pH 8) <400 nm, kmax (pH 13.75) 477 nm (eCB
6810 Mꢃ1 cmꢃ1); pKa 5.0.
458 nm
( (eCB
eN 11320 Mꢃ1 cmꢃ1), kmax (pH 13.75) 502 nm
7180 Mꢃ1 cmꢃ1); pKa 5.1.
4.1.19. N-(3-(Furan-300-yl)-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
benzenesulfonamide (26)
4.1.16. N-(3-(300-Formylphenyl)-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)benzenesulfonamide (23)
Following Representative Procedure 6, using naphthoquinone 8
(200 mg, 0.58 mmol), furan-3-ylboronic acid (130 mg, 1.14 mmol),
Pd(PPh3)2Cl2 (41 mg, 0.06 mmol) and satd aq NaHCO3 (5 mL) in
THF (22 mL). Purification via column chromatography on silica
gel (eluent pet ether/acetone 75:25) and subsequent recrystallisa-
tion from toluene gave 3-arylnaphthoquinone 26 as a yellow solid
(51 mg, 23%). Mp 211–214 °C (toluene); HPLC (method B) tR 9 min,
Following Representative Procedure 6, using naphthoquinone 8
(200 mg, 0.58 mmol), 3-formylphenylboronic acid (174 mg,
1.14 mmol), Pd(PPh3)2Cl2 (41 mg, 0.06 mmol) and satd aq NaHCO3
(5 mL) in THF (22 mL). Purification via column chromatography on
silica gel (eluent pet ether/acetone 90:10 to 70:30) and subsequent
recrystallisation from toluene gave 3-arylnaphthoquinone 23 as a

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3045
99%; mmax (KBr) 3231 (N–H), 1664 (C@O), 1651 (C@O); dH
(400 MHz, DMSO-d6) 6.95 (1H, app. s, H5 ), 7.46–7.57 (2H, m, H3
mide (445 mg, 2.20 mmol) and Cs2CO3 (287 mg, 0.88 mmol) in
DMF (5 mL). Purification via column chromatography on silica gel
(eluent pet ether/acetone 75:25) gave sulfonamide 34 as a yellow
solid (378 mg, 44%). Mp >280 °C; mmax (neat) 3396 (N–H), 1677
00
0
0
0
00
and H5 ), 7.58–7.66 (1H, m, H4 ), 7.73–7.75 (1H, m, H4 ), 7.76–
0
0
7.80 (2H, m, H2 and H6 ), 7.82–7.93 (3H, m, H6, H7 and H5 or H8),
00
0
7.99–8.09 (1H, m, H5 or H8), 8.18 (1H, app. s, H2 ), 10.04 (1H, br
(br, C@O); dH (500 MHz, DMSO-d6) 7.65–7.82 (5H, m, H6, H7, H4 ,
0
0
s, NH); dC (100 MHz, DMSO-d6) 112.5, 116.8, 126.8, 127.2, 127.3,
129.6, 131.3, 132.6, 133.3, 134.5, 135.1, 135.3, 137.8, 142.7,
143.5, 146.8, 181.5, 184.3; m/z (ESIꢃ) 378 ([MꢃH]ꢃ, 100%); HRMS
(ESI+) C20H13NNaO5S+ ([M+Na]+) requires 402.0407, found
402.0403; kmax (pH 8) 444 nm (eN 15220 Mꢃ1 cmꢃ1), kmax (pH
13.75) 509 nm (eCB 7840 Mꢃ1 cmꢃ1); pKa 5.0.
H5 and H6 ), 7.86 (1H, dd, J 7.9, 1.0, H5 or H8), 7.97 (1H, dd, J 7.6,
0
1.0, H5 or H8), 8.13 (1H, dd, J 7.9, 1.3, H3 ); dC (125 MHz, DMSO-
d6) 123.4, 125.7, 126.4, 128.7, 130.8, 131.5, 131.6, 131.9, 132.9,
134.1, 138.7, 146.8, 149.1, 176.7, 179.6; m/z (ESIꢃ) 391
([M(35Cl)ꢃH]ꢃ, 100%); HRMS (ESIꢃ) C16H8ClN2O6Sꢃ ([M(35Cl)ꢃH]ꢃ)
requires 390.9797, found 390.9797.
4.1.24. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-20,
60-difluorobenzenesulfonamide (35)
4.1.20. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
methanesulfonamide (31)
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (500 mg, 2.20 mmol), methanesulfonamide
(209 mg, 2.20 mmol) and Cs2CO3 (287 mg, 0.88 mmol) in DMF
(5 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 90:10) gave sulfonamide 31 as a yellow solid
(246 mg, 39%). Mp 195–200 °C; mmax (neat) 3241 (N–H), 1680
(C@O), 1659 (C@O); dH (500 MHz, DMSO-d6) 3.44 (3H, s, Me),
7.89–7.94 (2H, m, H6 and H7), 8.04–8.10 (2H, m, H5 and H8),
10.10 (1H, br s, NH); dC (125 MHz, DMSO-d6) 43.3, 126.6, 126.8,
130.7, 130.9, 133.7, 134.5, 134.7, 141.0, 177.5, 179.3; m/z (ESIꢃ)
284 ([M(35Cl)ꢃH]ꢃ, 100%); HRMS (ESI+) C11H9ClNNaO4S+
([M(35Cl)+Na]+) requires 307.9755, found 307.9755.
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (500 mg, 2.20 mmol), 2,6-difluorobenzenesulf-
onamide (425 mg, 2.20 mmol) and Cs2CO3 (1000 mg, 3.08 mmol)
in DMF (5 mL). Purification via column chromatography on silica
gel (eluent pet ether/acetone 70:30) gave sulfonamide 35 as a yel-
low solid (620 mg, 67%). Mp 261–267 °C; mmax (neat) 3444 (N–H),
1657 (C@O), 1611 (C@O); dH (400 MHz, DMSO-d6) 7.32–7.40 (2H,
0
0
0
m, H3 and H5 ), 7.74–7.84 (1H, m, H4 ), 7.87–7.97 (2H, m, H6 and
H7), 7.98–8.03 (1H, m, H5 or H8), 8.09–8.14 (1H, m, H5 or H8); dF
(470 MHz, DMSO-d6) ꢃ108.8; dC (100 MHz, DMSO-d6) 113.1 (dd, J
19.8, 3.0), 120.6 (m), 126.7, 126.8, 130.3, 130.9, 134.5, 134.7,
135.0 (m), 135.4, 140.8, 158.3 (dd, J 256.1, 3.8), 177.4, 179.0; m/z
(ESIꢃ) 382 ([M(35Cl)ꢃH]ꢃ, 100%), 384 ([M(37Cl)ꢃH]ꢃ, 50%); HRMS
(ESIꢃ) C16H7ClF2NO4Sꢃ ([M(35Cl)ꢃH]ꢃ) requires 381.9758, found
381.9765.
4.1.21. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-cyclo-
hexanesulfonamide (32)
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (400 mg, 1.76 mmol), cyclohexanesulfona-
mide 29 (287 mg, 1.76 mmol) and Cs2CO3 (804 mg, 2.46 mmol) in
DMF (5 mL). Purification via column chromatography on silica gel
(eluent pet ether/acetone 90:10 to 75:25 and then pet ether/EtOAc
80:20 to 50:50) gave sulfonamide 32 as a yellow solid (396 mg,
64%). Mp 178–179 °C; mmax (neat) 3254 (N–H), 1679 (C@O), 1595
4.1.25. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-30-
nitrobenzenesulfonamide (36)
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (500 mg, 2.20 mmol), 3-nitrobenzenesulfona-
mide (445 mg, 2.20 mmol) and Cs2CO3 (1004 mg, 3.08 mmol) in
DMF (5 mL). Purification via column chromatography on silica gel
(eluent pet ether/acetone 80:20 to 50:50) gave sulfonamide 36 as
a yellow solid (837 mg, quant.). Mp 258–263 °C; mmax (neat) 3430
(N–H), 1678 (C@O), 1588 (C@O); dH (500 MHz, DMSO-d6) 7.71
(1H, app. td, J 7.6, 1.3, H6 or H7), 7.76–7.82 (2H, m, H6 or H7 and
0
(C@O); dH (500 MHz, CDCl3) 1.24–1.34 (1H, m, H4 ), 1.44 (2H, m,
0
0
0
0
0
H3 and H5 ), 1.71 (2H, m, H2 and H6 ), 1.76–1.82 (1H, m, H4 ),
0
0
0
0
1.99 (2H, m, H3 and H5 ), 2.37 (2H, app. d, J 12.6, H2 and H6 ),
0
4.14 (1H, m, H1 ), 6.93 (1H, s, NH), 7.76–7.82 (2H, m, H6 and H7),
8.12–8.19 (2H, m, H5 and H8); dC (125 MHz, CDCl3) 25.0, 25.0,
26.4, 64.0, 127.3, 127.4, 128.8, 130.4, 131.0, 134.2, 134.8, 141.1,
177.0, 178.6; m/z (ESIꢃ) 352 ([M(35Cl)ꢃH]ꢃ, 100%), 354
([M(37Cl)ꢃH]ꢃ, 33%); HRMS (ESI+) C16H16ClNNaO4S ([M(35Cl)+Na]+)
requires 376.0381, found 376.0366.
0
H5 ), 7.88 (1H, dd, J 7.6, 1.3, H5 or H8), 7.96 (1H, dd, J 7.6, 1.3, H5
0
0
or H8), 8.28 (1H, ddd, J 8.1, 2.0, 0.9, H4 or H6 ), 8.32 (1H, ddd, J
0
0
0
8.1, 2.0, 0.9, H4 or H6 ), 8.66 (1H, t, J 2.0, H2 ); dC (125 MHz,
DMSO-d6) 120.1, 124.5, 124.5, 125.5, 126.3, 130.1, 130.8, 131.7,
132.1, 132.5, 134.0, 147.3, 149.7, 151.0, 176.3, 179.8; m/z (ESIꢃ)
391 ([M(35Cl)ꢃH]ꢃ, 100%), 393 ([M(37Cl)ꢃH]ꢃ, 33%); HRMS (ESIꢃ)
4.1.22. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-20-
methylbenzenesulfonamide (33)
C
16H8ClN2O6Sꢃ ([M(35Cl)ꢃH]ꢃ) requires 390.9797, found 390.9801.
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (500 mg, 2.20 mmol), ortho-toluenesulfon-
amide (377 mg, 2.20 mmol) and Cs2CO3 (1000 mg, 3.08 mmol) in
DMF (5 mL). Purification via column chromatography on silica gel
(eluent pet ether/acetone 80:20) gave sulfonamide 33 as a yellow
solid (555 mg, 70%). Mp 157–162 °C; mmax (neat) 3370 (N–H),
1666 (C@O), 1589 (C@O); dH (500 MHz, DMSO-d6) 2.65 (3H, s,
4.1.26. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-40-
methylbenzenesulfonamide (37)
2,3-Dichloronaphthalene-1,4-dione
7 (5.00 g, 22.00 mmol),
para-toluenesulfonamide (4.90 g, 28.60 mmol) and Cs2CO3
(9.30 g, 28.60 mmol) were stirred at reflux in anhydrous toluene
(100 mL) for 18 h under argon. The reaction mixture was concen-
trated in vacuo and re-suspended in water (40 mL). The suspension
was filtered and washed with hot water (2 ꢁ 10 mL). The precipi-
tate was dried under vacuum to give sulfonamide 37 as a red pow-
der (12.10 g, 14.50 mmol, 66%). Mp 292–293 °C; mmax (neat) 1681
(C@O), 1625 (C@O); dH (400 MHz, DMSO-d6) 2.34 (3H, s, Me),
0
0
Me), 7.39 (1H, app. t, J 7.4, H5 ), 7.44 (1H, d, J 7.4, H3 ), 7.56 (1H,
0
0
app. td, J 7.4, 1.3, H4 ), 7.84–7.93 (3H, m, H6, H7 and H6 ), 7.95–
7.98 (1H, m, H5 or H8), 8.04–8.08 (1H, m, H5 or H8); dC (125 MHz,
DMSO-d6) 20.0, 125.9, 126.6, 126.7, 128.3, 130.4, 131.0, 132.2,
132.6, 134.5, 134.7, 136.2, 140.5, 141.1, 177.4, 178.8; m/z (ESI+)
384 ([M(35Cl)+Na]+, 100%); HRMS (ESI+) C17H13ClNNaO4S+
([M(35Cl)+Na]+) requires 384.0068, found 384.0057.
0
0
7.21–7.26 (2H, m, H3 and H5 ), 7.64–7.70 (1H, m, H6 or H7), 7.72–
0
0
7.77 (3H, m, H2 , H6 and H6 or H7), 7.83–7.87 (1H, m, H5 or H8),
7.90–7.94 (1H, m, H5 or H8); dC (100 MHz, DMSO-d6) 21.7, 126.0,
126.3, 127.0, 129.2, 132.0, 132.7, 133.4, 134.6, 140.0, 146.6,
153.4, 176.4, 180.9; m/z (ESIꢃ) 360 ([M(35Cl)ꢃH]ꢃ, 100%), 362
([M(37Cl)ꢃH]ꢃ, 40%); HRMS (ESI+) C17H12ClNO4SNa+ ([M(35Cl)+Na]+)
requires 384.0068, found 384.0061.
4.1.23. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-20-
nitrobenzenesulfonamide (34)
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (500 mg, 2.20 mmol), 2-nitrobenzenesulfona-

3046
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
4.1.27. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-40-
fluorobenzenesulfonamide (38)
136.6, 138.3, 141.4, 179.5, 182.3; m/z (ESI+) 393 ([M(35Cl)+Na]+,
100%); HRMS (ESI+) C19H19N2NaO4S+ ([M(35Cl)+Na]+) requires
393.0879, found 393.0868; kmax (pH 8) 491 nm (eN 10060 Mꢃ1
cmꢃ1), kmax (pH 13.75) 578 nm (eCB 6770 Mꢃ1 cmꢃ1); pKa 9.5.
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (500 mg, 2.20 mmol), 4-fluorobenzenesulfona-
mide (386 mg, 2.20 mmol) and Cs2CO3 (1000 mg, 3.08 mmol) in
DMF (5 mL). Purification via column chromatography on silica gel
(eluent pet ether/acetone 75:25) gave sulfonamide 38 as a yellow
solid (515 mg, 63%). Mp > 280 °C; mmax (neat) 1676 (C@O), 1591
4.1.31. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)cyclohexanesulfonamide (42)
Following Representative Procedure 2, using naphthoquinone 32
0
(C@O); dH (500 MHz, DMSO-d6) 7.31 (2H, app. t, J 9.0, H3 and
(100 mg, 0.28 mmol), 3,5-dimethylaniline (106 lL, 0.85 mmol) and
0
H5 ), 7.73 (1H, app. td, J 7.6, 1.4, H6 or H7), 7.79 (1H, app. td, J 7.6,
CeCl3ꢂ7H2O (105 mg, 0.28 mmol) in MeOH (5 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
90:10) and subsequent recrystallization from toluene gave 3-anili-
nonaphthoquninone 42 as a red solid (40 mg, 32%). Mp 192–197 °C
(toluene); HPLC (method A) tR 20 min, 97%; mmax (neat) 3280 (N–
H), 1711 (C@O), 1670 (C@O); dH (500 MHz, CDCl3) 0.98–1.17 (3H,
1.4, H6 or H7), 7.89 (1H, dd, J 7.6, 1.4, H5 or H8), 7.92–7.97 (3H,
0
0
m, H2 , H6 and H5 or H8); dF (470 MHz, DMSO-d6) ꢃ110.4; dC
(125 MHz, DMSO-d6) 115.1 (d, J 21.9), 125.6, 126.3, 128.3 (d, J
8.6), 128.3, 130.9, 132.1, 132.6, 132.6, 134.0, 143.5, 162.9 (d, J
248.0), 176.1, 179.7; m/z (ESIꢃ) 364 ([M(35Cl)ꢃH]ꢃ, 100%), 366
([M(37Cl)ꢃH]ꢃ, 55%); HRMS (ESIꢃ) C16H8ClFNO4Sꢃ ([M(35Cl)ꢃH]ꢃ)
requires 363.9852, found 363.9853.
0
0
0
0
0
m, H3 , H4 and H5 ), 1.41 (2H, m, H2 and H6 ), 1.62 (1H, app. d, J
0
0
0
12.6, H4 ), 1.77–1.83 (2H, m, H3 and H5 ), 2.02 (2H, app. d, J 12.1,
0
0
0
H2 and H6 ), 2.14 (1H, m, H1 ), 2.32 (6H, s, 2 ꢁ Ar-Me), 6.53 (1H,
00
00
00
4.1.28. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)furan-20-sulfonamide (39)
s, sulfonamide-NH), 6.69 (2H, s, H2 and H6 ), 6.85 (1H, s, H4 ),
7.69 (1H, app. td, J 7.6, 1.3, H6), 7.76 (1H, app. td, J 7.6, 1.3, H7),
7.81 (1H, s, aniline-NH), 8.10 (1H, dd, J 7.6, 1.3, H5), 8.15 (1H, dd,
J 7.6, 1.3, H8); dC (125 MHz, CDCl3) 21.3, 25.0, 25.0, 25.8, 61.4,
115.0, 121.1, 126.7, 126.8, 126.9, 130.5, 131.7, 133.1, 134.8,
136.8, 137.0, 138.1, 180.1, 182.1; m/z (ESIꢃ) 303 (40%), 437
([MꢃH]ꢃ, 100%), 438 (40%); HRMS (ESIꢃ) C24H25N2O4Sꢃ ([MꢃH]ꢃ)
requires 437.1541, found 437.1536; kmax (pH 8) 505 nm (eN
19850 Mꢃ1 cmꢃ1), kmax (pH 13.75) 558 nm (eCB 6790 Mꢃ1 cmꢃ1);
pKa 10.6.
Following Representative Procedure 1, using 2,3-dichloronaph-
thalene-1,4-dione 7 (500 mg, 2.20 mmol), furan-2-sulfonamide
30 (324 mg, 2.20 mmol) and Cs2CO3 (1004 mg, 3.08 mmol) in
DMF (5 mL). Purification via column chromatography on silica gel
(eluent pet ether/acetone 90:10 to 50:50) gave sulfonamide 39 as
a yellow solid (634 mg, 85%). Mp 258–259 °C; mmax (neat) 3388
(N–H), 1668 (C@O), 1652 (C@O); dH (500 MHz, DMSO-d6) 6.57
0
0
(1H, dd, J 3.1, 1.7, H4 ), 6.91 (1H, d, J 3.1, H5 ), 7.75–7.84 (3H, m,
0
H6, H7 and H3 ), 7.94 (1H, dd, J 7.6, 1.3, H5 or H8), 7.99 (1H, dd, J
7.6, 1.3, H5 or H8); dC (125 MHz, DMSO-d6) 110.7, 111.8, 125.8,
126.4, 130.8, 132.0, 133.0, 133.0, 134.1, 144.6, 154.0, 176.5,
179.4; m/z (ESIꢃ) 336 ([M(35Cl)ꢃH]ꢃ, 100%), 338 ([M(37Cl)ꢃH]ꢃ,
33%); HRMS (ESIꢃ) C14H7ClNO5Sꢃ ([M(35Cl)ꢃH]ꢃ) requires
335.9739, found 335.9748.
4.1.32. N-(3-(300,500-Dimethylphenylamino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-20-methylbenzenesulfonamide (43)
Following Representative Procedure 2, using naphthoquinone 33
(100 mg, 0.28 mmol), 3,5-dimethylaniline (103 lL, 0.83 mmol) and
CeCl3ꢂ7H2O (103 mg, 0.28 mmol) in MeOH (10 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
80:20) gave 3-anilinonaphthoquninone 43 as a red solid (61 mg,
4.1.29. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-1-
phenylmethanesulfonamide (40)
49%). Mp 213–217 °C; HPLC (method A) tR 20 min, 98%; mmax (neat)
Following Representative Procedure 1, using 2,3-dichloronaph-
3293 (N–H), 1671 (C@O), 1613 (C@O); dH (500 MHz, DMSO-d6)
thalene-1,4-dione 7 (500 mg, 2.20 mmol),
a-toluenesulfonamide
2.19 (6H, s, 2 ꢁ aniline-Me), 2.34 (3H, s, sulfonamide-Me), 6.42
00
00
00
0
(317 mg, 2.20 mmol) and Cs2CO3 (1000 mg, 3.08 mmol) in DMF
(5 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 80:20) gave sulfonamide 40 as a yellow solid
(643 mg, 81%). Mp 176–179 °C; mmax (neat) 3259 (N–H), 1709
(C@O), 1663 (C@O); dH (400 MHz, CDCl3) 5.07 (2H, s, CH2Ph),
(2H, s, H2 and H6 ), 6.61 (1H, s, H4 ), 7.09 (1H, d, J 7.4, H3 ), 7.16
0
0
(1H, app. t, J 7.4, H5 ), 7.36 (1H, app. td, J 7.5, 0.9, H4 ), 7.53 (1H,
0
d, J 7.5, H6 ), 7.78 (1H, app. td, J 7.6, 1.3, H6 or H7), 7.84 (1H, app.
td, J 7.6, 1.3, H6 or H7), 7.92 (1H, m, H5 or H8), 8.02 (1H, m, H5 or
H8), 8.60 (1H, s, –NH), 9.03 (1H, s, –NH); dC (125 MHz, DMSO-d6)
20.1, 21.0, 114.4, 120.1, 125.0, 125.4, 125.8, 126.2, 127.8, 130.2,
131.6, 131.8, 131.8, 133.0, 135.0, 136.4, 137.0, 137.7, 139.3,
141.6, 179.4, 182.5; m/z (ESI+) 469 ([M(35Cl)+Na]+, 100%); HRMS
(ESI+) C25H23N2NaO4S+ ([M(35Cl)+Na]+) requires 469.1192, found
469.1179; kmax (pH 8) 512 nm (eN 11250 Mꢃ1 cmꢃ1), kmax (pH
13.75) 529 nm (eCB 6430 Mꢃ1 cmꢃ1); pKa 9.3.
0
0
0
6.78 (1H, br s, NH), 7.39–7.46 (3H, m, H3 , H4 and H5 ), 7.51–7.58
0
0
(2H, m, H2 and H6 ), 7.77–7.85 (2H, m, H6 and H7), 8.14–8.22
(2H, m, H5 and H8); dC (100 MHz, CDCl3) 61.2, 127.4, 127.5,
128.3, 128.9, 129.2, 129.6, 130.5, 131.0, 131.2, 134.3, 134.9,
141.0, 176.9, 178.6; m/z (ESIꢃ) 360 ([M(35Cl)ꢃH]ꢃ, 100%), 362
([M(37Cl)ꢃH]ꢃ,
33%);
HRMS
(ESI+)
C17H12ClNNaO4S+
([M(35Cl)+Na]+) requires 384.0068, found 384.0068.
4.1.33. N-(3-(300,500-Dimethylphenylamino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-20-nitrobenzenesulfonamide (44)
Following Representative Procedure 2, using naphthoquinone 34
4.1.30. N-(3-(30,50-Dimethylphenylamino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)methanesulfonamide (41)
Following Representative Procedure 2, using naphthoquinone 31
(100 mg, 0.26 mmol), 3,5-dimethylaniline (95 lL, 0.76 mmol) and
(100 mg, 0.35 mmol), 3,5-dimethylaniline (131
l
L, 1.05 mmol) and
CeCl3ꢂ7H2O (95 mg, 0.26 mmol) in MeOH (10 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
80:20) gave 3-anilinonaphthoquninone 44 as a red solid (24 mg,
CeCl3ꢂ7H2O (131 mg, 0.35 mmol) in MeOH (10 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
80:20) gave 3-anilinonaphthoquninone 41 as a red solid (84 mg,
65%). Mp 84–92 °C; HPLC (method A) tR 24 min, >99%; mmax (neat)
3294 (N–H), 1710 (C@O), 1673 (C@O); dH (500 MHz, DMSO-d6)
20%). Mp 257-263 °C; HPLC (method A) tR 20 min, 96%;
mmax (neat)
3297 (N–H), 1674 (C@O), 1613 (C@O); dH (500 MHz, CDCl3) 2.32
00
00
00
(6H, s, 2 ꢁ Ar-Me), 6.74 (2H, s, H2 and H6 ), 6.83 (1H, s, H4 ),
0
0
0
2.24 (6H, s, 2 ꢁ Ar-Me), 2.63 (3H, s, –SO2Me), 6.71 (3H, s, H2 , H4
7.52–7.55 (1H, m, H5 ), 7.56 (1H, s, sulfonamide-NH), 7.63–7.73
0
0
0
and H6 ), 7.81 (1H, app. td, J 7.6, 1.3, H6), 7.88 (1H, app. td, J 7.6,
1.3, H7), 8.01–8.06 (2H, m, H5 and H8), 8.54 (1H, s, sulfonamide-
NH), 8.83 (1H, s, aniline-NH); dC (125 MHz, DMSO-d6) 20.9, 41.1,
114.9, 121.2, 125.4, 125.9, 126.2, 130.5, 131.8, 133.1, 134.9,
(3H, m, H6, H7 and H4 ), 7.78 (1H, dd, J 7.9, 1.6, H6 ), 7.90–7.93
0
(2H, m, H8 and H3 ), 7.94 (1H, s, aniline-NH), 8.13 (1H, dd, J 7.4,
1.4, H5); dC (125 MHz, CDCl3) 21.3, 111.9, 121.9, 125.6, 126.6,
126.9, 127.7, 130.2, 130.2, 131.9, 132.7, 132.9, 133.3, 134.5,

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3047
135.1, 136.5, 138.2, 140.3, 147.3, 179.4, 182.1; m/z (ESIꢃ) 476
dC (125 MHz, CDCl3) 21.4, 21.5, 113.8, 121.0, 126.4, 126.9, 126.9,
127.3, 129.3, 130.6, 131.4, 133.0, 134.7, 135.9, 137.5, 138.0,
139.0, 143.9, 179.1, 181.9; m/z (ESIꢃ) 445 ([MꢃH]ꢃ, 100%); HRMS
(ESI+) C25H22N2O4SNa+ ([M+Na]+) requires 469.1192, found
469.1190; kmax (pH 8) 505 nm (eN 14040 Mꢃ1 cmꢃ1), kmax (pH
13.75) 578 nm (eCB 7170 Mꢃ1 cmꢃ1); pKa 9.7.
([M(35Cl)ꢃH]ꢃ,
100%);
HRMS
(ESIꢃ)
C24H20N3NaO6Sꢃ
([M(35Cl)ꢃH]ꢃ) requires 476.0922, found 476.0903; kmax (pH 8)
528 nm
( (eCB
eN 12380 Mꢃ1 cmꢃ1), kmax (pH 13.75) 560 nm
7610 Mꢃ1 cmꢃ1); pKa 7.9.
4.1.34. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-20,60-difluorobenzenesulfonamide
(45)
4.1.37. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-40-fluorobenzenesulfonamide (48)
Following Representative Procedure 2, using naphthoquinone 38
Following Representative Procedure 2, using naphthoquinone 35
(200 mg, 0.52 mmol), 3,5-dimethylaniline (195 L, 1.57 mmol) and
l
(200 mg, 0.55 mmol), 3,5-dimethylaniline (205 lL, 1.64 mmol) and
CeCl3ꢂ7H2O (194 mg, 0.52 mmol) in MeOH (5 mL). Purification via
column chromatography on silica gel (eluent pet ether/EtOAc
85:15) and subsequent recrystallization from toluene gave 3-anili-
nonaphthoquninone 45 as a red solid (81 mg, 33%). Mp 222–225 °C
(toluene); HPLC (method A) tR 22 min, 98%; mmax (neat) 3347 (N–
H), 3249 (N–H), 1679 (C@O), 1613 (C@O); dH (500 MHz, CDCl3)
CeCl3ꢂ7H2O (204 mg, 0.55 mmol) in MeOH (10 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
80:20) and subsequent recrystallization from toluene gave 3-anili-
nonaphthoquninone 48 as a red solid (146 mg, 59%). Mp 211–
217 °C (toluene); HPLC (method A) tR 22 min, 96%; mmax (neat)
3278 (N–H), 1674 (C@O), 1595 (C@O); dH (500 MHz, CDCl3) 2.32
00
00
00
00
2.30 (6H, s, 2 ꢁ Ar-Me), 6.69 (2H, s, H2 and H6 ), 6.72 (1H, s,
(6H, s, 2 ꢁ Ar-Me), 6.67 (2H, s, H2 and H6 ), 6.77 (1H, s, sulfon-
00
0
0
00 0 0
amide-NH), 6.84 (1H, s, H4 ), 6.98–7.03 (2H, m, H3 and H5 ),
H4 ), 6.87–6.92 (2H, m, H3 and H5 ), 7.03 (1H, s, sulfonamide-
0
0
0
NH), 7.37–7.43 (1H, m, H4 ), 7.67 (1H, app. td, J 7.5, 1.4, H6), 7.71
7.65–7.73 (4H, m, H6, H7, H2 and H6 ), 7.91–7.94 (1H, m, H8),
7.97 (1H, s, aniline-NH), 8.07–8.10 (1H, m, H5); dF (470 MHz, CDCl3)
ꢃ104.5; dC (125 MHz, CDCl3) 21.3, 112.8, 115.9 (d, J 11.5), 121.1,
126.4, 126.9, 127.0, 130.1 (d, J 9.5), 130.4, 131.4, 133.1, 134.9,
135.0 (d, J 2.8), 137.1, 138.0, 139.1, 165.2 (d, J 255.1), 179.2,
181.9; m/z (ESIꢃ) 449 ([MꢃH]ꢃ, 100%); HRMS (ESIꢃ) C24H18FN2O4-
Sꢃ ([MꢃH]ꢃ) requires 449.0977, found 449.0967; kmax (pH 8)
(1H, app. td, J 7.5, 1.4, H7), 7.87 (1H, s, aniline-NH), 8.00 (1H, dd,
J 7.5, 1.4, H8), 8.11 (1H, dd, J 7.5, 1.4, H5); dF (470 MHz, CDCl3)
ꢃ106.6; dC (125 MHz, CDCl3) 21.3, 112.0, 112.6 (dd, J 22.9, 3.9),
113.1 (dd, J 23.1, 3.7), 121.4, 126.6, 126.9, 127.4, 130.3, 131.6,
133.0, 134.2 (m), 135.0, 136.3, 137.9, 138.9, 159.3 (dd, J 258.5,
3.8), 179.4, 181.9; m/z (ESI+) 491 ([M+Na]+, 100%); HRMS (ESI+) C24-
H19FN2NaO4S+ ([M+Na]+) requires 491.0848, found 491.0842; kmax
(pH 8) 509 nm (eN 7420 Mꢃ1 cmꢃ1), kmax (pH 13.75) 548 nm (eCB
7780 Mꢃ1 cmꢃ1); pKa 8.4.
497 nm
( (eCB
eN 6580 Mꢃ1 cmꢃ1), kmax (pH 13.75) 546 nm
3490 Mꢃ1 cmꢃ1); pKa 9.9.
4.1.38. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
4.1.35. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-30-nitrobenzenesulfonamide (46)
Following Representative Procedure 2, using naphthoquinone 36
dihydronaphthalen-2-yl)furan-20-sulfonamide (49)
Following Representative Procedure 2, using naphthoquinone 39
(200 mg, 0.49 mmol), 3,5-dimethylaniline (222 L, 1.78 mmol) and
l
(175 mg, 0.43 mmol), 3,5-dimethylaniline (162
l
L, 1.23 mmol) and
CeCl3ꢂ7H2O (222 mg, 0.49 mmol) in MeOH (5 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
90:10) and subsequent recrystallization from toluene gave 3-anili-
nonaphthoquninone 49 as a red solid (164 mg, 66%). Mp 205–
206 °C (toluene); HPLC (method A) tR 23 min, 96%; mmax (neat)
3273 (N–H), 1674 (C@O), 1596 (C@O); dH (500 MHz, DMSO-d6)
CeCl3ꢂ7H2O (162 mg, 0.43 mmol) in MeOH (5 mL). Purification via
column chromatography on silica gel (eluent pet ether/EtOAc
80:20) gave 3-anilinonaphthoquninone 46 as a red solid (59 mg,
26%). Mp 216–221 °C; HPLC (method A) tR 22 min, 96%;
3290 (N–H), 1708 (C@O), 1675 (C@O); dH (500 MHz, CDCl3) 2.30
mmax (neat)
00
00
0
0
(6H, s, 2 ꢁ Ar-Me), 6.63 (2H, s, H2 and H6 ), 6.78 (1H, s, H4 ), 6.90
2.25 (6H, s, 2 ꢁ Ar-Me), 6.46–6.48 (1H, dd, J 3.5, 1.8, H4 ), 6.69
0
00
00
00
0
(1H, s, sulfonamide-NH), 7.55 (1H, app. t, J 8.0, H5 ), 7.68 (1H,
(2H, s, H2 and H6 ), 6.72 (1H, s, H4 ), 6.79 (1H, dd, J 3.5, 0.9, H5 ),
0
app. td, J 7.6, 1.3, H6), 7.72 (1H, app. td, J 7.6, 1.3, H7), 7.89 (1H, s,
aniline-NH), 7.93 (1H, dd, J 7.6, 1.3, H8), 7.98–8.01 (1H, ddd, J 8.0,
7.75–7.81 (2H, m, H6 and H3 ), 7.84 (1H, app. td, J 7.5, 1.3, H7),
7.86–7.89 (1H, m, H8), 8.03 (1H, dd, J 7.5, 1.3, H5), 8.94 (1H, s, ani-
line-NH), 9.31 (1H, s, sulfonamide-NH); dC (125 MHz, DMSO-d6)
21.0, 111.0, 112.2, 115.0, 121.5, 125.7, 125.7, 126.2, 130.3, 131.6,
133.0, 135.0, 136.4, 138.1, 142.8, 146.4, 149.1, 178.9, 182.3; m/z
(ESI+) 445 ([M+Na]+, 100%); HRMS (ESI+) C22H18N2NaO5S+
([M+Na]+) requires 445.0829, found 445.0819; kmax (pH 8)
0
1.9, 0.9, H6 ), 8.12 (1H, dd, J 7.6, 1.3, H5), 8.30 (1H, ddd, J 8.0, 1.9,
0
0
0.9, H4 ), 8.45 (1H, app. t, J 1.9, H2 ); dC (125 MHz, CDCl3) 21.3,
112.0, 121.0, 122.5, 126.5, 127.0, 127.1, 127.3, 129.7, 130.2,
131.5, 132.6, 133.2, 135.2, 136.5, 138.2, 139.2, 141.6, 147.7,
179.5, 181.8; m/z (ESIꢃ) 476 ([MꢃH]ꢃ, 100%); HRMS (ESIꢃ) C24H18-
N3O6Sꢃ ([MꢃH]ꢃ) requires 476.0922, found 476.0922; kmax (pH 8)
509 nm
( (eCB
eN 7770 Mꢃ1 cmꢃ1), kmax (pH 13.75) 541 nm
509 nm
(
eN 8730 Mꢃ1 cmꢃ1), kmax (pH 13.75) 551 nm
(
eCB
8440 Mꢃ1 cmꢃ1); pKa 8.3.
5670 Mꢃ1 cmꢃ1); pKa 8.6.
4.1.39. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-1-phenylmethanesulfonamide (50)
Following Representative Procedure 2, using naphthoquinone 40
(100 mg, 0.28 mmol), 3,5-dimethylaniline (103 L, 0.83 mmol) and
4.1.36. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-40-methylbenzenesulfonamide (47)
Following Representative Procedure 2, using naphthoquinone 37
l
(200 mg, 0.58 mmol), 3,5-dimethylaniline (214
l
L, 1.73 mmol) and
CeCl3ꢂ7H2O (103 mg, 0.28 mmol) in MeOH (5 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
85:15) and subsequent recrystallization from toluene gave 3-anili-
nonaphthoquninone 50 as a red solid (66 mg, 53%). Mp 152–156 °C
(toluene); HPLC (method A) tR 25 min, 96%; mmax (neat) 3279 (N–
H), 1708 (C@O), 1671 (C@O); dH (500 MHz, DMSO-d6) 2.23 (6H, s,
CeCl3ꢂ7H2O (214 mg, 0.58 mmol) in toluene (10 mL) at 110 °C.
Purification via column chromatography on silica gel (eluent pet
ether/EtOAc 80:20) gave 3-anilinonaphthoquninone 47 as a red
solid (64 mg, 25%). Mp 172–173 °C; HPLC (method A) tR 22 min,
96%; mmax (neat) 3260 (N–H), 1672 (C@O), 1597 (C@O); dH
(500 MHz, CDCl3) 2.32 (6H, s, 2 ꢁ aniline-Me), 2.33 (3H, s, sulfon-
00
00
2 ꢁ Ar-Me), 4.06 (2H, s, –CH2Ph), 6.71 (1H, s, H4 ), 6.73 (2H, s, H2
00
00
00
0
0
0
amide-Me), 6.68 (2H, s, H2 and H6 ), 6.80 (1H, s, sulfonamide-
and H6 ), 7.24–7.27 (2H, m, H2 and H6 ), 7.27–7.33 (3H, m, H3 ,
00
0
0
0
0
NH), 6.83 (1H, s, H4 ), 7.14 (2H, d, J 8.0, H3 and H5 ), 7.56 (2H, d, J
H4 and H5 ), 7.82 (1H, app. td, J 7.6, 1.3, H6), 7.90 (1H, app. td, J
7.6, 1.3, H7), 8.04 (1H, dd, J 7.6, 1.3, H5), 8.08 (1H, dd, J 7.6, 1.3,
H8), 8.55 (1H, s, sulfonamide-NH), 8.86 (1H, s, aniline-NH); dC
0
0
8.0, H2 and H6 ), 7.63–7.71 (2H, m, H6 and H7), 7.91 (1H, dd, J
7.4, 1.3, H8), 8.04 (1H, s, aniline-NH), 8.06 (1H, dd, J 7.4, 1.3, H5);

3048
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
(125 MHz, DMSO-d6) 20.9, 58.6, 115.1, 121.3, 125.4, 126.0, 126.2,
128.0, 128.2, 129.6, 130.5, 130.9, 131.8, 133.1, 134.9, 136.7,
138.3, 141.1, 179.6, 182.3; m/z (ESIꢃ) 445 ([MꢃH]ꢃ, 100%); HRMS
(ESI+) C25H22N2NaO4S+ ([M+Na]+) requires 469.1192, found
469.1190; kmax (pH 8) 507 nm (eN 17580 Mꢃ1 cmꢃ1), kmax (pH
13.75) 568 nm (eCB 6660 Mꢃ1 cmꢃ1); pKa 10.0.
to give the crude product. Purification via column chromatography
on silica gel (eluent pet ether/EtOAc 75:25) gave phenylacetamide
54 as a beige solid (13 mg, 9%).
Method C: 2-amino-3-chloro-1,4-naphthoquinone 51 (2.00 g,
9.63 mmol) was stirred in toluene (25 mL) in a 2-necked round-
bottomed flask at 90 °C. To a pressure-equalised dropping funnel
was added phenylacetyl chloride (5.97 mL, 38.53 mmol) and boron
trifluoride diethyl etherate (1.16 mL, 7.11 mmol) in toluene
(15 mL). This solution was added dropwise to the reaction flask
over a period of 30 min and the reaction mixture was stirred at
90 °C for a further 4 h. The reaction mixture was cooled to rt and
then concentrated in vacuo. Acetone (20 mL) was added to the
reaction mixture and the beige precipitate that formed was col-
lected by suction filtration. This precipitate was washed three fur-
ther times with cold acetone (3 ꢁ 10 mL) to give phenylacetamide
54 as a beige solid (1.781 g, 57%). Mp 205–209 °C (lit. 207–
208 °C);36adH (400 MHz, CDCl3) 3.86 (2H, s, –CH2Ph), 7.29–7.48
4.1.40. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)acetamide (52)36a
2-Amino-3-chloronaphthalene-1,4-dione 51 (500 mg, 2.41 mmol)
and 3 drops concd H2SO4 were added to a microwave vial containing
acetyl chloride (5 mL). The vial was sealed and the solution stirred at
50 °C for 1 h. The solution was then cooled to 0 °C and quenched by
the dropwise addition of EtOH until gas liberation ceased, before
being partitioned between 1 M HCl (30 mL) and EtOAc (30 mL).
The organic layer was collected, washed with brine (3 ꢁ 20 mL),
dried, filtered and concentrated in vacuo to give the crude reaction
mixture. Purification via column chromatography on silica gel (elu-
ent pet ether/EtOAc 90:10) gave amide 52 as a yellow solid (489 mg,
81%). Mp 215–218 °C (lit. 219–220 °C);36adH (500 MHz, DMSO-d6)
2.14 (3H, s, CO-Me), 7.87–7.94 (2H, m, H6 and H7), 8.02–8.06 (1H,
m, H8), 8.06–8.10 (1H, m, H5), 10.17 (1H, s, NH); m/z (ESIꢃ) 248
([M(35Cl)ꢃH]ꢃ, 100%).
00
00
00
(3H, m, H2 , H3 and H4 ), 7.62 (1H, br s, NH), 7.76 (2H, m, H6 and
H7), 8.05–8.09 (1H, m, H5), 8.15–8.19 (1H, m, H8); m/z (ESI+) 348
([M(35Cl)+Na]+, 100%).
4.1.43. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)hydrocinnanamide (55)
2-Amino-3chloro-1,4-naphthoquinone 51 (1.70 g, 8.19 mmol)
and hydrocinnamoyl chloride (5.0 mL, 30.13 mmol) were stirred
in BF3ꢂOEt2 (1.0 mL) in a round bottomed flask and heated to reflux
for 30 min. The reaction mixture was then cooled and toluene
(10 mL) was added, before being heated to reflux for a further
24 h. The crude reaction mixture was concentrated in vacuo and
the subsequent addition of acetone (20 mL) resulted in the precip-
itation of a yellow solid. This solid was collected by suction filtra-
tion and recrystallized from boiling acetone to give
hydrocinnanamide 55 as a yellow solid (1.39 g, 50%). Mp 162–
167 °C; mmax (neat) 3284 (N–H), 1662 (C@O); dH (500 MHz, CDCl3)
2.84 (2H, t, J 7.6, CO–CH2–CH2Ph), 3.09 (2H, t, J 7.6, Ph–CH2–CH2-
4.1.41. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)benzamide (53)36a
2-Amino-3-chloronaphthalene-1,4-dione
51
(1.00 g,
4.82 mmol) was stirred in a microwave vial under N2 with sodium
hydride (as a 60% dispersion in oil, 636 mg, 15.89 mmol) in anhy-
drous THF (10 mL) at rt for 30 min Benzoyl chloride (721 lL,
6.26 mmol) was added and stirring continued under N2 at rt for
1 h. EtOH was added dropwise until gas liberation ceased, and
the solution partitioned between satd aq NH4Cl (30 mL) and EtOAc
(30 mL). The organic layer was collected, washed with brine
(3 ꢁ 20 mL), dried, filtered and concentrated in vacuo to give the
crude product. Purification via column chromatography on silica
gel (eluent pet ether/acetone 95:5) gave amide 53 as a beige solid
(1.042 g, 69%). Mp 183–185 °C (lit. 254–256 °C);36adH (400 MHz,
0
0
0
0
CO), 7.21–7.28 (3H, m, H2 , H4 and H6 ), 7.30–7.34 (2H, m, H3
0
and H5 ), 7.61 (1H, s, NH), 7.74–7.81 (2H, m, H6 and H7), 8.10
(1H, dd, J 7.3, 1.5, H8), 8.19 (1H, dd, J 7.3, 1.5, H5); dC (125 MHz,
CDCl3) 31.1, 38.9, 126.5, 127.0, 127.5, 128.4, 128.7, 130.2, 131.5,
133.2, 134.1, 134.8, 138.9, 139.9, 168.8, 177.6, 179.8; m/z (ESIꢃ)
113 (30%), 249 (40%), 338 ([M(35Cl)ꢃH]ꢃ, 100%); HRMS C19H14ClN-
0
0
0
DMSO-d6) 7.55–7.60 (2H, m, H3 and H5 ), 7.65–7.69 (1H, m, H4 ),
0
0
7.91–7.96 (2H, m, H6 and H7), 8.01–8.05 (2H, m, H2 and H6 ),
8.07–8.10 (1H, m, H5 or H8), 8.12–8.15 (1H, m, H5 or H8), 10.49
(1H, s, NH); m/z (ESIꢃ) 310 ([M(35Cl)ꢃH]ꢃ, 100%).
NaO3 ([M(35Cl)+Na]+) requires 362.0554, found 362.0554.
+
4.1.42. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)phenylacetamide (54)36a
4.1.44. N-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-1-
phenylcyclopropanecarboxamide (58)
Method A: 2-Amino-3-chloronaphthalene-1,4-dione 51 (2.50 g,
12.04 mmol) was stirred in phenyl acetyl chloride (12.5 mL). HCl
gas, produced in situ by the dropwise addition of concd H2SO4 onto
NaCl (20 g), was bubbled through the reaction mixture for 15 min
After the removal of the gas inlet, the reaction mixture was heated
to reflux under a gaseous HCl atmosphere for 2 h. The solution was
cooled to rt and EtOH was added dropwise until gas liberation
ceased. The solution was then partitioned between satd aq NH4Cl
(100 mL) and EtOAc (100 mL). The organic layer was collected,
washed with brine (3 ꢁ 50 mL), dried, filtered and concentrated
in vacuo. Purification via column chromatography on silica gel
(eluent pet ether/acetone 90:10) gave phenylacetamide 54 as a
beige solid (549 mg, 14%).
Method B: 2-Amino-3-chloro-1,4-naphthoquinone 51 (100 mg,
0.44 mmol), 2-phenylacetamide (71 mg, 0.53 mmol), Pd(OAc)2
(1 mg, 0.004 mmol), Xantphos (4 mg, 0.007 mmol) and KOtBu
(74 mg, 0.66 mmol) were stirred in 1,4-dioxane (1 mL) in a sealed
microwave vial under argon and heated to 80 °C for 16 h. The solu-
tion was cooled to rt and partitioned between EtOAc (30 mL) and
satd aq NH4Cl (30 mL). The organic layer was collected, washed
with brine (3 ꢁ 20 mL), dried, filtered and concentrated in vacuo
1-Phenyl-1cyclopropanecarboxylic acid 56 (3.12 g, 19.27 mmol)
and DMF (3 drops) were stirred in anhydrous CH2Cl2 (10 mL) at rt.
Oxalyl chloride (1.83 mL, 19.27 mmol) was added to this solution
and stirring continued at rt for 3 h. The reaction mixture was then
concentrated in vacuo. 2-amino-3-chloro-1,4-naphthoquinone 51
(1.00 g, 4.82 mmol), boron trifluoride diethyl etherate (0.81 mL,
4.82 mmol) and toluene (15 mL) were added and the mixture
was stirred at 90 °C for 30 min and subsequently at 110 °C for
1 h. The crude reaction mixture was concentrated in vacuo and
the subsequent addition of acetone (20 mL) resulted in the precip-
itation of a yellow solid. This solid was collected by suction filtra-
tion and washed with cold acetone (2 ꢁ 10 mL) to give
phenylcyclopropane-carboxamide 58 as a yellow solid (1.354 g,
80%). Mp 214–215 °C; mmax (neat) 3338 (N–H), 1739 (C@O), 1713
(C@O), 1665 (C@O); dH (500 MHz, DMSO-d6) 1.22–1.25 (2H, m,
2 ꢁ cyclopropyl-CH), 1.51–1.54 (2H, m, 2 ꢁ cyclopropyl-CH),
0
0
0
7.33–7.38 (1H, m, H4 ), 7.40–7.45 (2H, m, H3 and H5 ), 7.49–7.53
0
0
(2H, m, H2 and H6 ), 7.87–7.92 (2H, m, H6 and H7), 7.99–8.03
(1H, m, H8), 8.05–8.08 (1H, m, H5), 8.55 (1H, s, NH); dC (125 MHz,
DMSO-d6) 16.0, 31.0, 126.6, 126.8, 127.8, 128.9, 130.1, 130.5,
130.9, 134.4, 134.6, 134.7, 138.9, 140.9, 170.9, 177.6, 178.8; m/z

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3049
(ESI+) 199 (100%), 352 ([M(35Cl)+H]+, 40%), 374 ([M(35Cl)+Na]+,
(125 MHz, CDCl3) 21.3, 43.1, 114.8, 120.4, 126.2, 126.2, 126.7,
127.5, 129.0, 129.6, 130.5, 131.8, 132.9, 133.8, 134.6, 134.7,
136.9, 137.6, 167.3, 179.9, 182.1; m/z (ESIꢃ) 409 ([MꢃH]ꢃ, 100%);
HRMS (ESI+) C26H22N2NaO+3 ([M+Na]+) requires 433.1523, found
433.1502.
+
60%), 376 ([M(37Cl)+Na]+, 20%); HRMS (ESI+) C20H14ClNNaO3
([M(35Cl)+Na]+) requires 374.0554, found 374.0549.
4.1.45. N-(3-(300,500-Dimethylphenylamino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)acetamide (59)
Following Representative Procedure 2, using naphthoquinone 52
(101 mg, 0.41 mmol), 3,5-dimethylaniline (151 lL, 1.21 mmol) and
4.1.48. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)hydrocinnanamide (62)
CeCl3ꢂ7H2O (151 mg, 0.41 mmol) in MeOH (5 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
80:20) gave 3-anilinonaphthoquinone 59 as a dark red solid
(57 mg, 42%). Mp 231–234 °C; HPLC (method A) tR 23 min, 97%;
mmax (neat) 3291 (N–H), 1667 (C@O), 1638 (C@O); dH (400 MHz,
DMSO-d6) 1.36 (3H, s, CO-Me), 2.22 (6H, s, 2 ꢁ Ar-Me), 6.56 (2H,
Following Representative Procedure 2, using naphthoquinone 55
(100 mg, 0.30 mmol), 3,5-dimethylaniline (110 lL, 0.89 mmol) and
CeCl3ꢂ7H2O (110 mg, 0.30 mmol) in methanol (5 mL). Purification
via column chromatography on silica gel (eluent pet ether/acetone
90:10 to 75:25) gave 3-anilinonaphthoquinone 62 as a red solid
(67 mg, 54%). Mp 186–190 °C; HPLC (method A) tR 23 min, 95%;
mmax (neat) 3305 (N–H), 1669 (C@O), 1596 (C@O); dH (500 MHz,
CDCl3) 2.19–2.24 (2H, m, –CH2CH2Ph), 2.30 (6H, s, 2 ꢁ Ar-Me),
0
0
0
s, H2 and H6 ), 6.69 (1H, s, H4 ), 7.78 (1H, app. td, J 7.5, 1.4, H6),
7.85 (1H, app. td, J 7.3, 1.3, H7), 8.00 (1H, dd, J 7.6, 1.0, H8), 8.03
(1H, dd, J 7.6, 1.3, H5), 8.84 (1H, s, aniline-NH), 9.19 (1H, br s,
amide-NH); dC (100 MHz, DMSO-d6) 20.9, 21.7, 115.4, 120.9,
124.9, 125.7, 126.1, 130.2, 131.9, 133.0, 134.9, 136.2, 137.3,
165.9, 179.1, 182.5; m/z (ESI+) 357 ([M+Na]+, 100%); HRMS (ESI+)
00
00
2.46–2.51 (2H, m, –CH2CH2Ph), 6.57 (2H, s, H2 and H6 ), 6.78
00
0
0
(1H, s, H4 ), 7.06 (2H, app. d, J 7.3, H2 and H6 ), 7.19 (1H, app. t, J
0
0
0
7.3, H4 ), 7.24–7.29 (2H, m, H3 and H5 ), 7.60 (1H, s, amide-NH),
7.67 (1H, app. td, J 7.6, 1.3, H6 or H7), 7.73 (1H, app. td, J 7.6, 1.3,
H6 or H7), 7.84 (1H, s, aniline-NH), 8.09–8.12 (2H, m, H5 and H8);
dC (125 MHz, CDCl3) 21.3, 30.8, 37.7, 114.9, 120.1, 125.9, 126.2,
126.3, 126.7, 128.1, 128.5, 130.6, 131.8, 133.0, 134.2, 134.6,
136.9, 137.6, 140.6, 168.4, 180.1, 182.1; m/z (ESI+) 425 ([M+H]+,
45%), 447 ([M+Na]+, 80%), 871 ([2 M+H]+, 100%); HRMS C27H24N2-
NaO3S+ ([M+Na]+) requires 447.1679, found 447.1672.
C
20H18N2NaO+3 ([M+Na]+) requires 357.1210, found 357.1198.
4.1.46. N-(3-(300,500-Dimethylphenylamino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzamide (60)
tBuOH (0.63 mL) and H2O (0.2
lL, 0.013 mmol) were sealed in a
vessel under argon. Pd(OAc)2 (0.9 mg, 0.003 mmol) and XPhos
(5.5 mg, 0.01 mmol) were added and the resulting light yellow
mixture was heated under argon to 110 °C for 3 min until it
became dark brown. This solution was then syringed into a vessel
containing naphthoquinone 53 (100 mg, 0.32 mmol), 3,5-dimeth-
4.1.49. N-(3-((300,500-Dimethylphenyl)amino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-1-phenylcyclopropanecarboxamide
(63)
ylaniline (48
lL, 0.39 mmol) and K2CO3 (62 mg, 0.45 mmol) under
Following Representative Procedure 2, using naphthoquinone 58
argon; this vessel was sealed and heated to 110 °C for 16 h. The
solution was cooled to rt, diluted with EtOAc (10 mL) and filtered
through CeliteÒ. The filtrate was then partitioned between EtOAc
(30 mL) and satd aq NH4Cl (20 mL); the organic layer was col-
lected, washed with brine (3 ꢁ 20 mL), dried, filtered and concen-
trated in vacuo. Purification via column chromatography on silica
gel (eluent pet ether/EtOAc 80:20) gave 3-anilinonaphthoquinone
60 as a purple solid (104 mg, 82%). Mp 198–204 °C; HPLC (method
A) tR 23 min, 95%; mmax (neat) 3303 (N–H), 1667 (C@O), 1595
(C@O); dH (400 MHz, CDCl3) 2.16 (6H, s, 2 ꢁ Ar-Me), 6.53 (1H, s,
(150 mg, 0.43 mmol), 3,5-dimethylaniline (160 lL, 1.28 mmol) and
CeCl3ꢂ7H2O (159 mg, 0.43 mmol) in MeOH (5 mL). Purification via
column chromatography on silica gel (eluent pet ether/EtOAc
95:5 to 75:25) gave 3-anilinonaphthoquinone 63 as a red solid
(107 mg, 58%). Mp 241–242 °C; HPLC (method A) tR 23 min.,
>99%; mmax (neat) 3356 (N–H), 1734 (C@O), 1669 (C@O); dH
(500 MHz, CDCl3) 0.83–0.86 (2H, m, 2 ꢁ cyclopropyl-CH), 1.07–
1.10 (2H, m, 2 ꢁ cyclopropyl-CH), 2.35 (6H, s, 2 ꢁ Ar-Me), 6.52
00
00
00
0
(2H, s, H2 and H6 ), 6.87 (1H, s, H4 ), 7.25–7.29 (2H, m, H2 and
0
0
0
0
H6 ), 7.35–7.38 (1H, m, H4 ), 7.39–7.44 (2H, m, H3 and H5 ), 7.46
(1H, s, amide-NH), 7.62 (1H, app. td, J 7.5, 1.3, H6), 7.65–7.70
(2H, m, aniline-NH and H7), 8.00 (1H, dd, J 7.5, 1.3, H8), 8.05 (1H,
dd, J 7.5, 1.3, H5); dC (125 MHz, CDCl3) 16.3, 21.4, 30.4, 115.5,
120.4, 126.0, 126.1, 126.6, 128.3, 129.1, 130.5, 131.0, 131.8,
132.8, 134.3, 134.5, 137.2, 137.6, 139.0, 170.8, 179.9, 182.2; m/z
(ESI+) 437 ([M+H]+, 60%), 459 ([M+Na]+, 100%); HRMS (ESI+) C28H24-
N2NaO+3 ([M+Na]+) requires 459.1679, found 459.1668.
00
00
00
0
0
H4 ), 6.59 (2H, s, H2 and H6 ), 7.30–7.36 (2H, m, H3 and H5 ),
0
0
0
7.42–7.47 (1H, m, H4 ), 7.50–7.55 (2H, m, H2 and H6 ), 7.68 (1H,
app. td, J 7.3, 1.5, H6 or H7), 7.74 (1H, app. td, J 7.8, 1.3, H6 or H7),
7.94 (1H, s, aniline-NH), 8.10–8.15 (2H, m, H5 and H8), 8.34 (1H,
br s, amide-NH); dC (100 MHz, CDCl3) 21.1, 115.4, 120.0, 126.0,
126.4, 126.8, 127.3, 128.2, 130.7, 131.7, 131.9, 133.0, 133.5,
134.2, 134.6, 137.0, 137.6, 163.8, 180.1, 182.1; m/z (ESIꢃ) 395
([MꢃH]ꢃ, 100%); HRMS (ESI+) C25H20N2NaO+3 ([M+Na]+) requires
419.1366, found 419.1347.
4.1.50. N-(3-(300-Formylphenyl)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)benzamide (64)
4.1.47. N-(3-(300,500-Dimethylphenylamino)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)phenylacetamide (61)
Following Representative Procedure 2, using naphthoquinone 54
(80 mg, 0.25 mmol), 3,5-dimethylaniline (92 lL, 0.74 mmol) and
CeCl3ꢂ7H2O (92 mg, 0.25 mmol) in MeOH (3 mL). Purification via
column chromatography on silica gel (eluent pet ether/acetone
90:10 to 70:30) and subsequent recrystallisation from boiling tol-
uene gave 3-anilinonaphthoquinone 61 as a purple solid (49 mg,
Following Representative Procedure 6, using naphthoquinone 53
(100 mg, 0.32 mmol), 3-formylphenylboronic acid (96 mg,
0.64 mmol), Pd(PPh3)2Cl2 (22 mg, 0.03 mmol) and satd aq NaHCO3
(2.5 mL) in THF (11 mL). Purification via column chromatography
on silica gel (eluent pet ether/EtOAc 85:15) gave 3-arylnaphthoqui-
none 64 as a yellow–orange solid (19 mg, 15%). Mp 63–69 °C; HPLC
(method A) tR 24 min, >99%; mmax (neat) 3307 (N–H), 1694 (C@O),
0
0
1663 (C@O); dH (500 MHz, CDCl3) 7.41–7.46 (2H, m, H3 and H5 ),
0
00
0
0
49%). Mp 216–217 °C; HPLC (method A) tR 24 min, 98%;
3299 (N–H), 1670 (C@O), 1612 (C@O); dH (500 MHz, CDCl3) 2.31
m
max (neat)
7.52–7.61 (2H, m, H4 and H5 ), 7.70–7.74 (2H, m, H2 and H6 ),
00
00
7.74–7.78 (1H, m, H6 ), 7.78–7.87 (3H, m, H6, H7 and H4 ), 7.94–
00
00
00
(6H, s, 2 ꢁ Ar-Me), 3.20 (2H, s, –CH2Ph), 6.53 (2H, s, H2 and H6 ),
7.97 (1H, m, H2 ), 8.18–8.25 (2H, m, H5 and H8), 8.62 (1H, s, NH),
00
0
0
6.81 (1H, s, H4 ), 7.03 (2H, d, J 6.9, H2 and H6 ), 7.28–7.36 (3H, m,
9.98 (1H, s, CHO); dC (500 MHz, CDCl3) 126.5, 127.2, 127.6, 128.8,
128.9, 129.7, 130.1, 130.3, 132.1, 132.2, 132.8, 133.2, 133.7, 134.8,
135.1, 135.4, 136.1, 137.9, 163.7, 182.3, 183.0, 191.9; m/z (ESIꢃ)
380 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C24H15NNaO+4 ([M+Na]+) requires
0
0
0
H3 , H4 and H5 ), 7.45 (1H, s, amide-NH), 7.65 (1H, app. td, J 7.5,
1.1, H6), 7.71 (1H, app. td, J 7.5, 1.1, H7), 7.75 (1H, s, aniline-NH),
8.05 (1H, dd, J 7.5, 1.1, H8), 8.08 (1H, dd, J 7.5, 1.1, H5); dC

3050
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
404.0893, found 404.0890; kmax (pH 8) <400 nm, kmax (pH 13.75)
485 nm (
CB 2670 Mꢃ1 cmꢃ1); pKa 10.9.
4.1.54. N-(3-Chloro-5-nitro-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)benzenesulfonamide (70)
e
Following Representative Procedure 1, using 2,3-dichloro-5-
nitronaphthalene-1,4-dione 68 (1.00 g, 3.68 mmol), benzenesul-
fonamide (577 mg, 3.68 mmol) and Cs2CO3 (1.68 g, 5.15 mmol) in
DMF (15 mL). Purification and separation of regioisomers via col-
umn chromatography on silica gel (eluent pet ether/acetone
50:50) gave sulfonamide 70 as a yellow solid (556 mg, 38%). Mp
220–223 °C; mmax (KBr) 3201 (N–H), 1687 (br, C@O); dH
4.1.51. N-(3-(Furan-300-yl)-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)benzamide (65)
Following Representative Procedure 6, using naphthoquinone 54
(100 mg, 0.32 mmol), 3-furanylboronic acid (72 mg, 0.64 mmol),
Pd(PPh3)2Cl2 (22 mg, 0.3 mmol) and satd aq NaHCO3 (2.5 mL) in
THF (11 mL). Purification via column chromatography on silica
gel (eluent pet ether/EtOAc 80:20) gave 3-arylnaphthoquinone
65 as a copper brown solid (65 mg, 59%). Mp 202–205 °C; HPLC
(method A) tR 24 min, 97%; mmax (neat) 3232 (N–H), 1675 (C@O),
0
0
0
(400 MHz, DMSO-d6) 7.57–7.65 (2H, m, H2 and H6 or H3 and
0
0
0
0
0
H5 ), 7.65–7.71 (1H, m, H4 ), 7.94–7.99 (2H, m, H2 and H6 or H3
0
and H5 ), 7.99–8.06 (1H, m, H7), 8.14–8.20 (2H, m, H6 and H8); dC
(100 MHz, DMSO-d6) 122.0, 126.7, 128.1, 129.1, 129.3, 131.9,
132.9, 135.7, 142.6, 147.9, 174.7, 177.5; m/z (ESIꢃ) 391
([M(35Cl)ꢃH]ꢃ, 100%); HRMS (ESIꢃ) C16H8ClN2O6Sꢃ ([MꢃH]ꢃ)
requires 390.9797, found 390.9797.
00
1647 (C@O); dH (400 MHz, CDCl3) 6.62–6.65 (1H, m, H5 ), 7.35–
00
0
0
7.38 (1H, m, H4 ), 7.50–7.55 (2H, m, H3 and H5 ), 7.59–7.64 (1H,
0
m, H4 ), 7.75 (1H, app. td, J 7.6, 1.3, H7), 7.79 (1H, app. td, J 7.3,
0
0
1.6, H6), 7.90–7.94 (2H, m, H2 and H6 ), 8.13 (1H, dd, J 7.4, 1.1,
00
H8), 8.19 (1H, dd, J 7.6, 1.0, H5), 8.25 (1H, app. s, H2 ), 8.66 (1H,
br s, NH); dC (400 MHz, CDCl3) 109.3, 117.3, 126.3, 127.1, 127.6,
127.8, 129.0, 130.2, 132.6, 132.8, 133.3, 133.6, 134.6, 135.9,
142.5, 145.8, 164.2, 182.1, 183.2; m/z (ESIꢃ) 342 ([MꢃH]ꢃ, 100%);
HRMS (ESIꢃ) C21H12NOꢃ4 ([MꢃH]ꢃ) requires 342.0772, found
342.0782; kmax (pH 8) <400 nm, kmax (pH 13.75) 513 nm (eCB
4820 Mꢃ1 cmꢃ1); pKa 12.1.
4.1.55. N-(3-Chloro-8-nitro-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)benzenesulfonamide (73)
Following Representative Procedure 1, using 2,3-dichloro-5-
nitronaphthalene-1,4-dione 68 (1.00 g, 3.68 mmol), benzenesul-
fonamide (577 mg, 3.68 mmol) and Cs2CO3 (1.68 g, 5.15 mmol) in
DMF (15 mL). Purification and separation of regioisomers via col-
umn chromatography on silica gel (eluent pet ether/acetone
50:50) gave sulfonamide 73 as a yellow solid (405 mg, 28%). Mp
222–225 °C; mmax (KBr) 3223 (N–H), 1694 (C@O), 1675 (C@O); dH
4.1.52. N-(3-(300-Formylphenyl)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)phenylacetamide (66)
0
0
0
Following Representative Procedure 6, using naphthoquinone 53
(200 mg, 0.61 mmol), 3-formylphenylboronic acid (184 mg,
1.23 mmol), Pd(PPh3)2Cl2 (43 mg, 0.06 mmol) and Na2CO3
(194 mg, 1.84 mmol) in toluene (10 mL). Purification via column
chromatography on silica gel (eluent pet ether/EtOAc 90:10 to
70:30) gave 3-arylnaphthoquinone 66 as a yellow solid (107 mg,
44%). Mp 158–162 °C; HPLC (method A) tR 23 min, >99%; mmax
(neat) 3272 (N–H), 1695 (C@O), 1665 (C@O); dH (500 MHz, CDCl3)
(400 MHz, DMSO-d6) 7.57–7.65 (2H, m, H2 and H6 or H3 and
0
0
0
0
0
H5 ), 7.65–7.71 (1H, m, H4 ), 7.87–7.92 (2H, m, H2 and H6 or H3
0
and H5 ), 8.05 (1H, app. t, J 8.0, H6), 8.17 (1H, d, J 8.0, H5 or H7),
8.26 (1H, d, J 8.0, H5 or H7); dC (100 MHz, DMSO-d6) 122.3, 126.6,
127.9, 129.1, 129.3, 132.7, 132.9, 135.9, 142.6, 148.1, 175.9,
176.6; m/z (ESIꢃ) 391 ([M(35Cl)ꢃH]ꢃ, 100%); HRMS (ESIꢃ) C16H8-
ClN2O6Sꢃ ([M(35Cl)ꢃH]ꢃ) requires 390.9797, found 390.9796.
3.51 (2H, s, –CH2Ph), 7.12 (2H, dd, J 7.4, 1.7, H2 and H6 ), 7.31–7.37
4.1.56. N-(3-(300,500-Dimethylphenylamino)-5-nitro-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (74)
Following General Procedure 2, using naphthoquinone 70
0
0
0
0
0
00
(3H, m, H3 , H4 and H5 ), 7.53 (1H, app. t, J 7.7, H5 ), 7.58 (1H, app.
00
00
dt, J 7.7, 1.5, H6 ), 7.72 (1H, app. t, J 1.5, H2 ), 7.75 (1H, app. td, J 7.6,
1.3, H7), 7.79 (1H, app. td, J 7.6, 1.3, H6), 7.85 (1H, app. dt, J 7.7, 1.5,
(100 mg, 0.26 mmol), 3,5-dimethylaniline (95 lL, 0.77 mmol) and
00
H4 ), 7.94 (1H, s, NH), 8.11 (1H, dd, J 7.6, 1.3, H8), 8.14 (1H, dd, J 7.6,
CeCl3ꢂ7H2O (95 mg, 0.26 mmol) in toluene (10 mL) at 110 °C. Puri-
fication via column chromatography on silica gel (eluent pet ether/
acetone 75:25) and subsequent trituration using hexane gave 3-
anilinonaphthoquinone 74 as a purple solid (86 mg, 70%). Mp
242–244 °C; HPLC (method B) tR 9 min, 95%; mmax (KBr) 3272 (N–
H), 1687 (C@O), 1595 (C@O); dH (500 MHz, DMSO-d6) 2.24 (6H, s,
1.3, H5), 9.93 (1H, s, CHO); dC (125 MHz, CDCl3) 44.6, 126.4, 127.1,
127.8, 128.6, 129.1, 129.2, 129.3, 130.2, 130.2, 132.1, 133.0, 133.2,
133.8, 134.4, 134.9, 135.6, 136.0, 137.3, 166.9, 182.1, 183.0, 191.9;
m/z (ESI+) 111 (30%), 418 ([M+Na]+, 100%), 434 (25%); HRMS (ESIꢃ)
C
25H16NOꢃ4 ([MꢃH]ꢃ) requires 394.1085, found 394.1076; kmax (pH
8) <400 nm, kmax (pH 13.75) 451 nm (eCB 8490 Mꢃ1 cmꢃ1); pKa
2 ꢁ Ar-Me) 6.60 (2H, s, H2 and H6 ), 6.69 (1H, s, H4 ), 7.37–7.43
00
00
00
0
0
0
0
11.4.
(2H, m, H3 and H5 ), 7.49–7.54 (1H, m, H4 ), 7.60–7.65 (2H, m, H2
0
and H6 ), 7.97 (1H, app. t, J 7.9, H7), 8.02 (1H, dd, J 7.9, 1.3, H6 or
H8), 8.08 (1H, dd, J 7.9, 1.3, H6 or H8), 9.05 (1H, s, NH), 9.24 (1H,
s, NH); dC (100 MHz, DMSO-d6) 21.8, 115.5, 121.7, 123.1, 126.4,
127.4, 127.6, 129.1, 129.4, 133.0, 133.3, 136.6, 137.5, 139.2,
141.8, 144.4, 148.5, 177.5, 180.5; m/z (ESIꢃ) 476 ([MꢃH]ꢃ, 100%);
HRMS (ESIꢃ) C24H18N3O6Sꢃ ([MꢃH]ꢃMꢃH]ꢃ) requires 476.0922,
found 476.0917; kmax (pH 8) 555 nm (eN 5360 Mꢃ1 cmꢃ1), kmax
(pH 13.75) 578 nm (eCB 4520 Mꢃ1 cmꢃ1); pKa 8.2.
4.1.53. N-(3-(Furan-300-yl)-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)phenylacetamide (67)
Following Representative Procedure 6, using naphthoquinone 54
(80 mg, 0.25 mmol), 3-furanylboronic acid (55 mg, 0.49 mmol),
Pd(PPh3)2Cl2 (18 mg, 0.03 mmol) and satd aq NaHCO3 (2 mL) in
THF (8.8 mL). Purification via column chromatography on silica
gel (eluent pet ether/acetone 90:10 then pet ether/EtOAc 80:20)
and recrystallisation from boiling toluene gave 3-arylnaphthoqui-
none 67 as a copper brown solid (8 mg, 9%). Mp 120–124 °C; HPLC
(method A) tR 25 min, 97%; mmax (neat) 3306 (N–H), 1665 (br,
C@O); dH (400 MHz, CDCl3) 3.71 (2H, s, –CH2Ph), 6.38–6.41 (1H,
4.1.57. N-(3-(300,500-Dimethylphenylamino)-6-nitro-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (75)
Following Representative Procedure 1, using 2,3-dichloro-6-
nitronaphthalene-1,4-dione 69 (1.00 g, 3.68 mmol), benzenesul-
fonamide (577 mg, 3.68 mmol) and Cs2CO3 (1.68 g, 5.15 mmol) in
DMF (15 mL). Purification via column chromatography on silica
gel (eluent pet ether/acetone 95:5 to 80:20) gave a mixture of reg-
ioisomeric 6-nitro and 7-nitro products, 71 and 72 (788 mg, 55%).
Subsequently, following Representative Procedure 2, using this reg-
ioisomeric mixture (788 mg, 2.01 mmol), 3,5-dimethylaniline
00
0
0
0
0
0
00
m, H5 ), 7.33–7.47 (6H, m, H2 , H3 , H4 , H5 , H6 and H4 ), 7.69–
00
7.79 (2H, m, H6 and H7), 7.86 (1H, s, NH), 8.05–8.09 (2H, m, H2
and H5 or H8), 8.13 (1H, dd, J 7.5, 1.4, H5 or H8); dC (500 MHz, CDCl3)
44.8, 109.5, 117.0, 126.2, 127.0, 127.9, 128.4, 129.3, 129.5, 130.3,
132.4, 133.5, 133.6, 134.5, 135.6, 142.3, 145.6, 167.6, 181.8,
183.2; m/z (ESIꢃ) 356 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C22H15NNaO+4
([M+Na]+) requires 380.0893, found 380.0883; kmax (pH 8)
<400 nm, kmax (pH 13.75) 512 nm (eCB 2450 Mꢃ1 cmꢃ1); pKa 12.5.
(751
lL, 6.02 mmol) and CeCl3ꢂ7H2O (748 mg, 2.01 mmol) in

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3051
00
00
00
00
MeOH (5 mL). Purification and separation of regioisomers via col-
umn chromatography (eluent pet ether/EtOAc 95:5 to 85:15) gave
3-anilinonaphthoquinone 75 as a purple solid (115 mg, 12% over 2
steps). Mp 250–253 °C; HPLC (method A) tR 23 min, >99%; mmax
(neat) 3460 (N–H), 1653 (br, C@O); dH (500 MHz, CDCl3) 2.27
7.03–7.12 (3H, m, H2 , H4 and H6 ), 7.22–7.29 (2H, m, H3 and
00
0
0
0
H5 ), 7.34–7.41 (2H, m, H3 and H5 ), 7.48–7.57 (3H, m, H2, H4
0
and H6 ), 7.91–7.96 (1H, m, H6), 8.03 (1H, dd, J 7.8, 1.0, H7), 8.23
(1H, dd, J 7.8, 1.0, H5), 9.04 (1H, s, sulfonamide-NH), 9.28 (1H, s,
aniline-NH); dC (125 MHz, DMSO-d6) 113.1, 122.0, 123.9, 124.1,
126.5, 127.4, 128.1, 128.5, 128.5, 131.6, 132.2, 134.2, 138.1,
140.8, 142.6, 147.7, 175.0, 180.7; m/z (ESIꢃ) 448 ([MꢃH]ꢃ, 100%);
HRMS (ESIꢃ) C22H14N3O6Sꢃ ([MꢃH]ꢃ) requires 448.0609, found
448.0591; kmax (pH 8) 504 nm (eN 3510 Mꢃ1 cmꢃ1), kmax (pH
13.75) 578 nm (eCB 3010 Mꢃ1 cmꢃ1); pKa 8.4.
00
00
(6H, s, 2 ꢁ Ar-Me), 6.63 (2H, s, H2 and H6 ), 6.67 (1H, s, NH),
00
0
0
6.81 (1H, s, H4 ), 7.26–7.32 (2H, m, H3 and H5 ), 7.39–7.46 (1H,
0
0
0
m, H4 ), 7.58–7.62 (2H, m, H2 and H6 ), 8.03 (1H, d, J 8.4, H8),
8.06 (1H, s, NH), 8.42 (1H, dd, J 8.4, 2.3, H7), 8.80 (1H, d, J 2.3,
H5); dC (125 MHz, CDCl3) 20.3, 113.1, 120.4, 121.0, 126.3, 126.8,
127.1, 127.8, 127.9, 130.5, 132.2, 134.3, 135.7, 137.2, 137.6,
138.6, 149.5, 175.9, 179.1; m/z (ESIꢃ) 476 ([MꢃH]ꢃ, 100%); HRMS
(ESI+) C24H19N3NaO6S ([M+Na]+) requires 500.0887, found
500.0876; kmax (pH 8) 527 nm (eN 4500 Mꢃ1 cmꢃ1), kmax (pH
13.75) 600 nm (eCB 2240 Mꢃ1 cmꢃ1); pKa 7.5.
4.1.61. N-(5-Amino-3-(300,500-dimethylphenylamino)-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (79)
Following Representative Procedure 7, using naphthoquinone 74
(22 mg, 0.05 mmol) and 10% Pd/C (1.0 mg, 0.01 mmol) in MeOH
(2 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 90:10 to 75:25) gave 5-aminonaphthoqui-
none 79 as an orange solid (20 mg, quant.). Mp 232–234 °C;
HPLC (method B) tR 9 min, 95%; mmax (KBr) 3464 (N–H), 3300 (N–
H), 1573 (br, C@O); dH (400 Hz, DMSO-d6) 2.21 (6H, s, 2 ꢁ Ar-
4.1.58. N-(3-(300,500-Dimethylphenylamino)-7-nitro-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (76)
Following an identical procedure to the synthesis of 75 above,
the other regioisomeric product, 3-anilinonaphthoquinone 76,
00
00
00
was obtained by column chromatography as
a
purple solid
Me), 6.58 (2H, s, H2 and H6 ), 6.64 (1H, s, H4 ), 7.01–7.08 (2H, m,
0
0
(121 mg, 13% over 2 steps). Mp 253–256 °C; HPLC (method A) tR
24 min, 97%; mmax (neat) 3390 (N–H), 1656 (br, C@O); dH
H6 and H8), 7.32–7.43 (3H, m, H7, H3 and H5 ), 7.46–7.52 (3H, m,
0
0
0
H2 , H4 and H6 ), 7.88 (2H, br s, NH2), 8.60 (1H, s, aniline-NH),
8.86 (1H, s, sulfonamide-NH); dC (100 MHz, DMSO-d6) 21.9,
109.0, 113.3, 115.9, 121.7, 123.0, 126.0, 127.2, 129.2, 132.6,
133.3. 136.0, 137.2, 138.8, 142.2, 143.1, 152.5, 179.6, 182.9; m/z
(ESI+) 470 ([M+Na]+, 100%); HRMS (ESI+) C24H21N3NaO4S+,
([M+H]+) requires 448.1326; found 448.1326; kmax (pH 8) 474 nm
00
(500 MHz, DMSO-d6) 2.23 (6H, s, 2 ꢁ Ar-Me), 6.63 (2H, s, H2 and
00
00
0
0
H6 ), 6.70 (1H, s, H4 ), 7.36–7.41 (2H, m, H3 and H5 ), 7.49–7.54
0
0
0
(1H, m, H4 ), 7.57 (2H, dd, J 8.4, 1.1, H2 and H6 ), 8.25 (1H, d, J
8.5, H5), 8.40 (1H, d, J 2.2, H8), 8.53 (1H, dd, J 8.5, 2.2, H6), 9.05
(1H, s, NH), 9.18 (1H, br s, NH); dC (125 MHz, DMSO-d6) 21.0,
114.2, 120.0, 121.1, 125.6, 126.4, 127.2, 128.2, 128.4, 131.9,
132.8, 134.4, 136.4, 138.0, 141.4, 142.7, 150.9, 176.7, 181.0; m/z
(ESIꢃ) 476 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C24H19N3NaO6S
([M+Na]+) requires 500.0887, found 500.0863; kmax (pH 8)
(
eN 22420 Mꢃ1 cmꢃ1),
15800 Mꢃ1 cmꢃ1).
kmax
(pH
13.75)
490 nm
(eCB
4.1.62. N-(6-Amino-3-(300,500-dimethylphenylamino)-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (80)
529 nm
( (eCB
eN 7720 Mꢃ1 cmꢃ1), kmax (pH 13.75) 616 nm
3880 Mꢃ1 cmꢃ1); pKa 8.0.
Following Representative Procedure 7, using naphthoquinone 75
(26 mg, 0.06 mmol) and 10% Pd/C (1.2 mg, 0.01 mmol) in MeOH
(2 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 80:20) gave 6-aminonaphthoquinone 80 as a
brown solid (24 mg, quant.). Mp 282–288 °C; HPLC (method A) tR
24 min, >99%; mmax (neat) 3475 (N–H), 3372 (N–H), 1589 (br,
C@O); dH (500 MHz, DMSO-d6) 2.21 (6H, s, 2 ꢁ Ar-Me), 6.36 (2H,
4.1.59. N-(3-(300,500-Dimethylphenylamino)-8-nitro-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (77)
Following Representative Procedure 2, using naphthoquinone 73
(60 mg, 0.15 mmol), 3,5-dimethylaniline (57 L, 0.46 mmol) and
l
CeCl3ꢂ7H2O (58 mg, 0.15 mmol) in toluene (10 mL) at 110 °C. Puri-
fication via column chromatography on silica gel (eluent pet ether/
acetone 75:25) and subsequent recrystallisation from toluene gave
3-anilinonaphthoquinone 77 as a purple solid (8 mg, 11%). Mp
218–220 °C (toluene); HPLC (method B) tR 9 min, 99%; mmax (KBr)
3298 (N–H), 3219 (N–H), 1678 (C@O), 1641 (C@O); dH (400 MHz,
00
00
00
s, NH2), 6.51 (2H, s, H2 and H6 ), 6.59 (1H, s, H4 ), 6.81 (1H, dd, J
8.5, 2.5, H7), 7.12 (1H, d, J 2.5, H5), 7.34–7.39 (2H, m, H3 and
0
0
0
0
0
H5 ), 7.47–7.55 (4H, m, H8, H2 , H4 and H6 ), 8.32 (1H, s, aniline-
NH), 8.94 (1H, br s, sulfonamide-NH); dC (125 MHz, DMSO-d6)
21.0, 109.7, 115.5, 117.8, 119.3, 119.8, 124.3, 126.3, 128.1, 128.3,
131.9, 132.0, 136.4, 138.7, 140.0, 141.2, 153.4, 178.2, 183.2; m/z
(ESI+) 470 ([M+Na]+, 100%); HRMS (ESI+) C24H21N3NaO4S+
([M+Na]+) requires 470.1145, found 470.1142; kmax (pH 8)
00
00
DMSO-d6) 2.22 (6H, s, 2 ꢁ Ar-Me), 6.60 (2H, s, H2 and H6 ), 6.68
00
0
0
0
(1H, s, H4 ), 7.32–7.40 (2H, m, H3 and H5 ), 7.46–7.55 (3H, m, H2 ,
0
0
H4 and H6 ), 7.94 (1H, app. t, J 7.9, H6), 8.05 (1H, dd, J 7.9, 1.0, H5
or H7), 8.22 (1H, dd, J 7.9, 1.0, H5 or H7), 9.07 (1H, s, NH), 9.08
(1H, s, NH); dC (100 MHz, DMSO-d6) 21.8, 114.1, 122.2, 123.0,
126.6, 127.2, 129.0, 129.3, 129.4, 132.4, 132.8, 135.0, 137.2,
138.6, 141.8, 143.4, 148.6, 176.2, 181.6; m/z (ESIꢃ) 476 ([MꢃH]ꢃ,
100%); HRMS (ESI+) C24H19N3NaO6S+ ([M+Na]+) requires
398 nm
( (eCB
eN 9830 Mꢃ1 cmꢃ1), kmax (pH 13.75) 389 nm
12540 Mꢃ1 cmꢃ1).
4.1.63. N-(7-Amino-3-(300,500-dimethylphenylamino)-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (81)
500.0887, found 500.0882; kmax (pH 8) 515 nm (eN 5500 Mꢃ1
cmꢃ1), kmax (pH 13.75) 573 nm (eCB 2740 Mꢃ1 cmꢃ1); pKa 8.6.
-
Following Representative Procedure 7, using naphthoquinone 76
(40 mg, 0.08 mmol) and 10% Pd/C (1.8 mg, 0.02 mmol) in MeOH
(2 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 85:15) gave 7-aminonaphthoquinone 81 as
an orange solid (29 mg, 77%). Mp 255–258 °C; HPLC (method A)
tR 24 min, 97%; mmax (neat) 3364 (N–H), 1579 (br, C@O); dH
4.1.60. N-(8-Nitro-1,4-dioxo-3-(phenylamino)-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide (78)
Following Representative Procedure 2, using naphthoquinone 73
(100 mg, 0.26 mmol), aniline (70
00
l
L, 0.77 mmol) and CeCl3ꢂ7H2O
(500 MHz, DMSO-d6) 2.22 (6H, s, 2 ꢁ Ar-Me), 6.58 (2H, s, H2 and
00
00
(95 mg, 0.26 mmol) in toluene (10 mL) at 110 °C. Purification via
column chromatography on silica gel (eluent pet ether/acetone
75:25) and subsequent recrystallisation from toluene gave 3-anili-
nonaphthoquinone 78 as a purple solid (30 mg, 26%). Mp 231–
233 °C (toluene); HPLC (method A) tR 24 min, 98%; mmax (KBr)
3330 (N–H), 1673 (C@O), 1649 (C@O); dH (500 MHz, DMSO-d6)
H6 ), 6.64 (1H, s, H4 ), 6.71 (2H, s, NH2), 6.74 (1H, dd, J 8.5, 2.2,
0
0
H6), 6.96 (1H, d, J 2.2, H8), 7.33–7.39 (2H, m, H3 and H5 ), 7.46–
0
0
7.53 (3H, m, H2, H4 and H6 ), 7.73 (1H, d, J 8.5, H5), 8.60 (1H, s, ani-
line-NH), 8.80 (1H, s, sulfonamide-NH); dC (125 MHz, DMSO-d6)
21.0, 109.8, 111.8, 115.7, 117.6, 121.0, 125.2, 126.3, 128.3, 129.4,
131.8, 134.2, 136.3, 137.9, 141.4, 142.2, 155.4, 179.1, 179.3; m/z

3052
J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
(ESIꢃ) 446 ([MꢃH]ꢃ, 100%); HRMS (ESI+) C24H21N3NaO4S+
([M+Na]+) requires 470.1145, found 470.1139; kmax (pH 8)
4.2.3. ZR-75-1 Breast Cancer Cell Line Studies
4.2.3.1. Growth of ZR-75-1 cells.
Cells from the human breast
410 nm
(
eN 37310 Mꢃ1 cmꢃ1), kmax (pH 13.75) 420 nm
(
eCB
cancer cell line ZR-75-1, which had previously been stored in liquid
N2, were grown in RMPI 1640 L-glutamine-containing growth med-
21890 Mꢃ1 cmꢃ1).
ium, with 10% (v/v) foetal calf serum and 1% (v/v) Pen/Strep/Gluta-
mine added, at 37 °C in a 5% CO2 environment. Cell growth was
monitored at regular intervals and once almost confluent (every
5–7 days) the cells were split in a 1:2 fashion, using Trypsin-EDTA
solution (0.25% (w/v)) to detach the cell monolayer from the cul-
ture flask. After 5 passages, the cells were harvested to prepare a
lysate, by resuspending the cell pellet in complete lysis buffer
4.1.64. N-(8-Amino-3-(300,500-dimethylphenylamino)-1,4-dioxo-
1,4-dihydronaphthalen-2-yl)benzenesulfonamide (82)
Following Representative Procedure 7, using naphthoquinone 77
(60 mg, 0.13 mmol) and 10% Pd/C (2.7 mg, 0.03 mmol) in MeOH
(2 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 75:25 to 60:40) gave 8-aminonaphthoqui-
none 82 as a magenta solid (56 mg, quant.). Mp 223–225 °C;
HPLC (method B) tR 9 min, 95%; mmax (KBr) 3337 (N–H), 3295 (N–
H), 1661 (C@O), 1618 (C@O); dH (400 MHz, DMSO-d6) 2.20 (6H, s,
(100
lL frozen protease inhibitors and 10
lL 100 mM dithiothrei-
tol added per 900
lL of lysis buffer (20 mM Tris, 20 mM NaCl,
0.5% (w/v) Nonidet P40 containing one EDTA-free Complete Prote-
ase Inhibitor per 2.5 mL)) to a final cell concentration of 5 ꢁ 107
cells/mL. Cell debris was removed by centrifugation (10 min, 4 °C,
14000 rpm, microfuge) and the supernatant retained. The cell
lysate was kept on ice and all studies with inhibitors were carried
out on the same day.
00
00
00
2 ꢁ Ar-Me), 6.50 (2H, s, H2 and H6 ), 6.59 (1H, s, H4 ), 7.09 (1H,
0
0
d, J 8.4, H7), 7.25 (1H, d, J 7.0, H5), 7.33–7.42 (5H, m, H6, H3 , H5
0
0
0
and NH2), 7.47–7.55 (3H, m, H2 , H4 and H6 ), 8.35 (1H, s, aniline-
NH), 8.96 (1H, s, sulfonamide-NH); dC (100 MHz, DMSO-d6) 21.9,
110.1, 116.4, 116.5, 120.9, 125.2, 125.4, 127.2, 129.2, 132.2,
132.8, 134.1, 137.2, 139.4, 140.8, 141.9, 151.3, 182.7, 183.4; m/z
(ESIꢃ) 470 ([M+Na]+, 100%); HRMS (ESI+) C24H21N3NaO4S+
([M+Na]+) requires 470.1145, found 470.1147; kmax (pH 8)
4.2.3.2. Quantification of hNAT1 in ZR-75-1 Cell Extracts by
Western and Dot Blotting.
extract samples were run on a 12% SDS–PAGE gel, using 8
ple and 2
L reduced gel loading buffer (10 mM TrisꢂHCl (pH 6.8),
For Western Blotting, ZR-75-1 cell
542 nm
(
eN 15090 Mꢃ1 cmꢃ1), kmax (pH 13.75) 479 nm
(
eCB
lL sam-
12380 Mꢃ1 cmꢃ1); pKa 8.4.
l
8 M urea, 2% (w/v) SDS, 5 mg/mL DTT) per well. Following transfer
to PVDF micro-porous membranes in a suitable buffer (48 mM
TrisꢂHCl (pH 8.3), 39 mM glycine, 20% MeOH) for 4 h using a
semi-dry blotter, membranes were blocked with 10% milk in
Tris-Buffered Saline Tween-20 (9 g/L NaCl, 6 g/L TrisꢂHCl, 0.05%
(v/v) Tween-20), washed thrice with 3% milk and incubated with
the polyclonal antibody 19525 at a dilution of 1:2000 for 1 h. Mem-
branes were subsequently incubated with horseradish peroxidise-
conjugated mouse anti-rabbit IgG at a dilution of 1:10000 for 1 h as
a secondary antibody. Bound mNat2 and hNAT1 were detected
using a chemiluminescent kit (ECL Reagent Kit, Amersham). For
4.1.65. N-(8-Amino-1,4-dioxo-3-(phenylamino)-1,4-
dihydronaphthalen-2-yl)benzenesulfonamide (83)
Following representative procedure 7, using naphthoquinone
78 (25 mg, 0.06 mmol) and 10% Pd/C (1.2 mg, 0.01 mmol) in MeOH
(2 mL). Purification via column chromatography on silica gel (elu-
ent pet ether/acetone 80:20) gave 8-aminonaphthoquinone 83 as a
magenta solid (22 mg, quant.). Mp 242–244 °C; HPLC (method A) tR
25 min, >99%;
mmax (neat) 3459 (N–H), 3335 (N–H), 1618 (br, C@O);
00
00
dH (500 MHz, DMSO-d6) 6.93–6.97 (2H, m, H2 and H6 ), 6.98–7.02
00
00
(1H, m, H4 ), 7.08 (1H, dd, J 8.5, 1.1, H7), 7.18–7.23 (2H, m, H3 and
00
0
0
H5 ), 7.26 (1H, dd, J 7.3, 1.1, H5), 7.36–7.40 (3H, m, H6, H3 and H5 ),
Dot Blotting, the samples (200 lL/well containing 100 lL of recom-
0
0
0
7.48–7.52 (1H, m, H4 ), 7.54–7.58 (2H, m, H2 and H6 ), 8.57 (1H, s,
NH), 8.90 (1H, s, NH); dC (125 MHz, DMSO-d6) 109.2, 115.1, 115.6,
122.5, 122.7, 124.2, 126.5, 127.5, 128.5, 131.4, 132.1, 133.2, 138.9,
140.0, 140.9, 150.3, 181.6, 182.5; m/z (ESI+) 442 ([M+Na]+, 100%);
HRMS (ESI+) C22H17N3NaO4S+ ([M+Na]+) requires 442.0832, found
442.0821; kmax (pH 8) 514 nm (eN 16660 Mꢃ1 cmꢃ1), kmax (pH
13.75) 486 nm (eCB 15840 Mꢃ1 cmꢃ1); pKa 8.2.
binant mNat2 or ZR-75-1 cell lysate) were directly transferred onto
a PVDF membrane using the Bio-DotÒ Microfiltration Apparatus
and a procedure similar to Western Blotting was used to detect
the relevant protein.
4.2.4. Acetyl CoA Hydrolysis Assay
The rate of production of free thiol Coenzyme A (CoA-SH) by
NATs in the presence of both an arylamine and AcCoA was mea-
sured in end-point assays using Ellman’s reagent, 5,50-dithio-bis-
(2-nitrobenzoic acid) (DTNB), as previously described.29 The final
4.2. Biology
4.2.1. General Experimental
Molecular biology reagents and competent Escherichia coli cells
were purchased from Promega (Southampton, UK).
NAT concentration in each assay well was 1.5
For a preliminary test of inhibitory potency of a compound at
M, assays were conducted in triplicate and the specific NAT
lg/mL.
30
l
activity was calculated from a graph of the OD405, measured on a
plate reader (Tecan Sunrise), against time. The% retained specific
activity was calculated against a control assay without inhibitor.
In IC50 tests, a range of ten concentrations was selected for each
4.2.2. Expression and Purification of mNat2, hNAT1 and shNat2
Recombinant mNat2 with an N-terminal hexa-His tag was
expressed from E. coli RosettaÒ(-DE3)pLysS cells containing
pET28b(+), into which the mNat2 open-reading frame had been
sub-cloned.39 The protein was purified via Ni-NTA affinity chroma-
tography (Qiagen) and thrombin cleavage of the His tag, as previ-
ously described.22 Both pure recombinant hNAT128,40 and
shNat232 were produced as previously described.
The concentrations of solutions containing purified proteins
were determined by measuring their absorption at 280 nm and
using the molar extinction coefficients calculated for each enzyme.
The purified NATs were stored at -80 °C in 20 mM TrisꢂHCl (pH 8.0)
buffer solution containing 5 mM dithiothreitol and 5% (v/v) glyc-
erol at the following concentrations: mNat2 8 mg/mL; hNAT1
1.5 mg/mL; shNat2 20 mg/mL. These solutions were diluted as
required on the day of use.
compound, most often beginning at 10, 30 or 50 lM 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 without inhibitor. KyPlotÒ software was used
to determine the IC50 value from a plot of% retained specific activ-
ity against inhibitor concentration using the IC50%FUN regression
model.
4.2.5. Colorimetric Evaluation of Inhibitors
4.2.5.1. Spectrophotometry.
pound were recorded using a U-2001 spectrophotometer (Hitachi)
and 50
L UVettesÒ (Eppendorf). The compounds were tested at
15 M in buffer solutions containing 5% (v/v) DMSO at pH 8
Visible spectra of each com-
l
l

J. E. Egleton et al. / Bioorg. Med. Chem. 22 (2014) 3030–3054
3053
2. Breast Cancer–Survival Statistics. http://info.cancerresearchuk.org/cancerstats/
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(20 mM TrisꢂHCl), pH 13.75 (4 M NaOH) and, where relevant, in the
presence of mNat2 and/or hNAT1 (30
M in 20 mM TrisꢂHCl). All
spectra were blank-corrected and normalised.
l
4. Turner, N.; Pearson, A.; Sharpe, R.; Lambros, M.; Geyer, F.; Lopez-Garcia, M. A.;
Natrajan, R.; Marchio, C.; Iorns, E.; Mackay, A.; Gillett, C.; Grigoriadis, A.; Tutt,
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Full absorbance spec-
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l
solutions containing 5% (v/v) DMSO in a flat-bottomed 96-well
plate (Corning) were recorded on a plate reader (Omega). Recorded
spectra were blank-corrected and normalised before kmax was
determined. A graph of kmax against pH was then plotted for each
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(variable slope) regression model was used to determine the pKa
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4.2.5.3. Determination of Absorption Coefficients.
absorbance spectra of selected compounds at 200 M, 100
50 M and 25 M in buffer solutions at pH 8 or 13 containing 5%
(v/v) DMSO in a 96-well flat-bottomed plate (Corning) were
recorded on a plate reader (Omega). Spectra were blank-corrected
and normalised and experiments performed in duplicate. A plot of
absorbance at kmax against concentration then yielded the absorp-
Full
l
lM,
l
l
tion coefficient, at either pH 8 (
divided by the path length.
eN) or pH 13 (eCB), as the gradient
4.2.6. 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)12e, the ground state conformation of the ligand was
predicted using the molecular editor Avogadro,41 and the protein
was protonated to be consistent with the assay conditions of pH
8. The docking simulations and the analysis of the possible interac-
tions between the protein and the ligand were performed using
GOLDÒ.30 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 GOLDÒ Score Fitness function.30 Each docking
simulation was repeated ten times to ensure the observed solu-
tions were consistent.
Acknowledgments
This work was supported by Cancer Research UK through an
Oxford Cancer Research Centre Prize D.Phil. Studentship
(C38302/A12450, JEE), a Cancer Medicinal Chemistry Studentship
(NL) and
a Small Molecule Cancer Drug Discovery Award
(C17468/A9332, PTS, AMJ and ST). We would also like to thank
Research Councils United Kingdom for a fellowship (AJR). We
would also like to thank the following for their assistance: Julien
Dairou, Jean-Marie Dupret and Fernando Rodrigues-Lima for pro-
viding us with recombinant hNAT1 and hNAT2; Akane Kawamura
for the purification of recombinant shNat2; Camilo Quevedo for
helpful discussions about in silico modeling; Jon Williams and
Adrian Harris for providing us with ZR-75-1 cells; Hilary Long
and Roderick Walker for assistance with cell culture techniques;
Hayden Peacock for assistance with HPLC; and Carole Bataille for
assistance with NMR.
26. Johnson, N. In The Role of Human Arylamine N-Acetyltransferase in Development
and Disease; University of Oxford, 2001.
27. Burgos, C. H.; Barder, T. E.; Huang, X.; Buchwald, S. L. Angew. Chem., Int. Ed.
2006, 45, 4321.
28. Wang, H.; Vath, G. M.; Kawamura, A.; Bates, C. A.; Sim, E.; Hanna, P. E.; Wagner,
C. R. Protein J. 2005, 24, 65.
29. (a) Brooke, E. W.; Davies, S. G.; Mulvaney, A. W.; Pompeo, F.; Sim, E.; Vickers, R.
J. Bioorg. Med. Chem. 2003, 11, 1227; (b) Brooke, E. W.; Davies, S. G.; Mulvaney,
A. W.; Okada, M.; Pompeo, F.; Sim, E.; Vickers, R. J.; Westwood, I. M. Bioorg.
Med. Chem. Lett. 2003, 13, 2527.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.015.
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