Novel inhibitors of NRH:Quinone oxidoreductase 2 (NQO2): Crystal structures, biochemical activity, and intracellular effects of imidazoacridin-6-ones

Article abstract of DOI:10.1021/jm200416e

Imidazoacridin-6-ones are shown to be potent nanomolar inhibitors of the enzyme NQO2. By use of computational molecular modeling, a reliable QSAR was established, relating inhibitory potency with calculated binding affinity. Further, crystal structures of

Full text of DOI:10.1021/jm200416e

ARTICLE
pubs.acs.org/jmc
Novel Inhibitors of NRH:Quinone Oxidoreductase 2 (NQO2):
Crystal Structures, Biochemical Activity, and Intracellular Effects
of Imidazoacridin-6-ones†
||
Mark S. Dunstan,‡ John Barnes,§, Matthew Humphries,§ Roger C. Whitehead,|| Richard A. Bryce,§
David Leys,‡ Ian J. Stratford,§ and Karen A. Nolan*,§
‡Manchester Interdisciplinary Biocentre, §School of Pharmacy and Pharmaceutical Sciences, and School of Chemistry,
University of Manchester and Manchester Cancer Research Centre, Manchester M13 9PT, U.K.
S Supporting Information
b
ABSTRACT: Imidazoacridin-6-ones are shown to be potent
nanomolar inhibitors of the enzyme NQO2. By use of compu-
tational molecular modeling, a reliable QSAR was established,
relating inhibitory potency with calculated binding affinity.
Further, crystal structures of NQO2 containing two of the
imidazoacridin-6-ones have been solved. To generate com-
pounds with reduced off-target (DNA binding) effects, an
N-oxide moiety was introduced into the tertiary aminoalkyl
side chain of the imidazoacridin-6-ones. This resulted in sub-
stantially less toxicity in a panel of eight cancer cell lines,
decreased protein binding, and reduced DNA binding and
nuclear accumulation. Finally, one of the N-oxides showed
potent ability to inhibit the enzymatic function of NQO2 in cells, and therefore, it may be useful as a pharmacological probe to
study the properties of the enzyme in vitro and in vivo.
’ INTRODUCTION
potent, novel inhibitors of NQO1 and NQO2.11ꢀ13 From these
studies we identified a series of imidazoacridin-6-ones that, from
computational molecular modeling, showed excellent predicted
binding characteristics to NQO2. Samples of these compounds
were supplied by NCI and evaluated for their ability to inhibit
human, recombinant NQO2. Inhibitory activity correlated
strongly with computational binding affinity, and further, the
inhibitory potency of these compounds was the highest yet
reported.13 Here, we report the synthesis of further imidazoacri-
din-6-ones in order to confirm our previous findings and provide
guidance for the development of potentially pharmacologically
active compounds. We have solved the crystal structure of NQO2
containing a parent imidazoacridin-6-one and its N-oxide ana-
logue, and this has aided in the preparation of a quantitative
structureꢀactivity relationship (QSAR) model. All the com-
pounds inhibit NQO2 at nanomolar concentrations, and their
toxicity has been determined in HCT116 colon cancer cells.
From the panel of compounds, several were selected for addi-
tional biochemical and biological evaluation in order to aid drug
development. From a comparison of a parent imidazoacridin-
6-one with an N-oxide derivative with similar NQO2 inhibitory
potency, it is shown that the N-oxide moiety confers substantially
reduced toxicity and reduced nuclear uptake and DNA binding.
The oxidoreductases NQO1 (NAD(P)H:quinone oxidoreduc-
tase 1, QR1, DT-diaphorase, EC 1.6.99.2) and NQO2 (NRH:
quinone oxidoreductase 2, QR2, EC 1.10.99.2) are cytosolic flavo-
proteins that catalyze the oxygen independent, two electron reduc-
tion of quinones to hydroquinones.1,2 The physiological role of
NQO1 is well characterized with its major function being considered
to be as a detoxification enzyme.3 A detoxification and/or chemo-
protectant role for NQO2 has also been inferred from studies on
orthoquinone reduction2 and the enhanced carcinogen activity of
benzo(a)pyrene observed in NQO2ꢀ/ꢀ mice.4 However, a com-
parable metabolic role for NQO1 and NQO2 is confounded by the
observation that genetic knockout of NQO1 increases menadione
toxicity in mice, whereas in NQO2 knockout mice there is protection
against menadione toxicity.5,6 This has led to the consideration that
pharmacological inhibition of NQO2 activity could have some
chemoprotectant potential,7ꢀ9 a notion supported by the fact that
the classical inhibitor of NQO2 is the chemoprotective agent
resveratrol.10 A variety of structurally diverse inhibitors of NQO2
have been identified,7ꢀ9,11ꢀ16 and some of these, imatinib and
melatonin, have been shown to protect against radiation induced
lung injury.17,18 However, these latter agents, as well as resveratrol,
have other substantive pharmacological properties, which make them
less than ideal for probing the cellular function of NQO2.
We have recently carried out a virtual screening of the
National Cancer Institute (NCI) chemical database to identify
Received: April 7, 2011
Published: August 22, 2011
r
2011 American Chemical Society
6597
dx.doi.org/10.1021/jm200416e J. Med. Chem. 2011, 54, 6597–6611
|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Preparation of Imidazoacridin-6-onesa
a (i) H2SO4, HNO3, 70 ꢀC, 1 h; (ii) p-R1-aniline, 24 h; (iii) POCl3, 1,2-dichloroethane, 1 h, 90ꢀC; (iv) N,N-dimethylaniline/N,N-diethylaniline, DMF,
reflux, 4 h; (v) Raney Ni, THF, 1 h, room temp; (vi) trimethyl orthoformate, reflux, 24 h; (vii) trimethyl orthoacetate, reflux, 24 h; (viii) m-CPBA,
CHCl3, 18 h.
Finally, it is demonstrated that the imidazoacridin-6-one contain-
ing the N-oxide is functionally active for inhibiting NQO2 in cells
at nontoxic concentrations.
’ RESULTS AND DISCUSSION
Chemistry. Synthesis of a range of imidazoacridin-6-ones
(Scheme 1), in which there are two loci for variation (positions
1 and 2), was carried out using an approach previously described
by Cholody et al.19 A known property of these compounds is
their ability to bind to DNA. Hence, part of the synthetic strategy
was to reduce this potentially confounding effect by introducing
an N-oxide moiety into the tertiary aminoalkyl side chains of
these molecules.
Biochemical and Biological Evaluation. Figure 1 shows the
general structure of the compounds evaluated here. All 19 com-
pounds, 11 imidazoacridin-6-ones (designated 5) and 8 corre-
sponding N-oxides (designated 6), were assayed for their ability
to inhibit purified recombinant human NQO2 (Table 1). The
IC50 values for these compounds were found to extend from 14
to 205 nM, with 6a1 being one of the most potent agents yet
reported. Our previous work on imidazoacridin-6-ones as po-
tential inhibitors of NQO2 was based on a substructure search
following hierarchical screening of the NCI chemical database.11
These compounds were supplied by the NCI, and by use of
computational molecular docking, a reliable QSAR model was
established. A number of the compounds were resynthesized for
Figure 1. General structure of the imidazoacridin-6-ones.
the present work (5c1, 5e1, 5f1, 5c2, 5e2, 5f2), and generally
there was no more than a 3-fold difference in enzyme inhibitory
potency or toxicity when comparing the fresh stocks prepared
here with compounds supplied by NCI (Table 1). The exception
was 5c1, where the inhibitory potency was 7ꢁ lower for the
NCI compound, although the toxicity against HCT116 cells was
similar. As part of the screening of the NCI database we also
identified triazoloacridin-6-ones as potential inhibitors of NQO2.12
Although these compounds were around 10-fold less efficient as
enzyme inhibitors than the imidazoacridin-6-ones, this study did
illustrate that inclusion of an N-oxide moiety into the triazoloa-
cridin-6-ones substantially reduced toxicity without compromis-
ing inhibitory potency. Introducing this functionality into these
molecules also reduced DNA binding affinity and protein bind-
ing, both of which could potentially make the compounds more
pharmacologically acceptable as inhibitors of NQO2. Hence, in
6598
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
Table 1. Calculated Computational Binding Affinities (ΔG) for Ligands in the Active Site of NQO2 (1QR2 or 5a1 Crystal
Structure), Their Experimental Enzyme Inhibitory Potency (IC50), and Toxicity towards HCT116 Colon Cancer Cells after
Exposure to Compounds for 24 or 96 ha
ΔGcalcd (kJ/mol)
toxicity IC50 (μM) at
compd
R1
R2
CH3
R3
IC50 (nM), NQO2
1QR2
5a1
24 h
96 h
5a1
5b1
OCH3
H
59 ( 7
97 ( 8
ꢀ49.8
ꢀ48.4
ꢀ47.5
ꢀ48.9
ꢀ48.2
ꢀ47.9
2.1 ( 0.35
2.9 ( 0.23
2.53 ( 0.35
0.51 ( 0.03
0.66 ( 0.03
0.60 ( 0.02
F
CH3
CH3
H
H
5c1 (NSC645833)
5e1 (NSC645808)
5f1 (NSC645809)
H
115 ( 14,
830 ( 112b
83 ( 6,
OH
OH
CH3
H
H
ꢀ48.7
ꢀ49.0
ꢀ47.7
ꢀ47.1
1.61 ( 0.18
4.16 ( 0.35
0.37 ( 0.02,
1b
296 ( 51b
126 ( 81,
148 ( 4b
46 ( 11
83 ( 11
90 ( 11,
323 ( 100b
74 ( 9
CH2CH3
1.14 ( 0.41,
0.77b
5a2
OCH3
CH3
CH3
CH3
CH3
CH3
CH3
ꢀ50.8
ꢀ50.4
ꢀ49.8
ꢀ48.9
ꢀ47.9
ꢀ47.8
1.86 ( 0.35
2.11 ( 0.19
1.94 ( 0.68,
2.27b
0.46 ( 0.16
0.51 ( 0.11
0.50 ( 0.16,
0.460b
5b2
F
5c2 (NSC645834)
H
5d2
Br
CH3
CH3
CH3
CH3
ꢀ49.5
ꢀ50.3
ꢀ48.7
ꢀ48.1
1.91 ( 0.10
3.01 ( 1.32
0.44 ( 0.11
0.95 ( 0.18,
0.650b
5e2 (NSC637991)
OH
90 ( 11,
50 ( 6b
54 ( 8,
16 ( 1.5b
14 ( 4
5f2 (NSC637992)
OH
CH2CH3
CH3
ꢀ51.4
ꢀ48.8
5.46 ( 1.67
0.93 ( 0.31,
3b
6a1
6b1
6c1
6e1
6a2
6b2
6c2
6d2
OCH3
F
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
ꢀ51.8
ꢀ46.2
ꢀ49.7
ꢀ50.3
ꢀ50.5
ꢀ49.2
ꢀ50.5
ꢀ51.2
ꢀ49.4
ꢀ46.1
ꢀ47.7
ꢀ48.6
ꢀ48
ꢀ47.5
ꢀ48.7
ꢀ48.4
40.3 ( 2.1
40.3 ( 3.2
41.0 ( 3.0
38.7 ( 11
58.2 ( 1.8
31.0 ( 1.1
34.9 ( 1.8
49.0 ( 1.8
12.8 ( 2.8
17.3 ( 3.1
12.1 ( 1.9
19.7 ( 1.8
11.4 ( 1.4
9.2 ( 0.3
16.1 ( 0.7
10.6 ( 1.0
H
205 ( 18
96 ( 5
H
H
OH
OCH3
F
H
42 ( 6
CH3
CH3
CH3
CH3
73 ( 7
97 ( 6
H
56 ( 10
47 ( 12
Br
a Values are given with (SD. b Data from ref 13.
Table 2. Activity of NQO2 in Breast Cancer Cell Lines ([(nmol of DCPIP Reduced)/min]/(mg of Protein)) and Their Sensitivity
to the Imidazoacridin-6-onesa
cell line
toxicity
MCF-7
SKBr3
BT20
BT474
MDA 468
MDA 231
T47D
5a1
6a1
5c1
6c1
0.41 ( 0.02
4.61 ( 0.28
0.43 ( 0.06
7.97 ( 0.25
8 ( 1.10
1.02 ( 0.09
9.53 ( 0.35
1.9 ( 0.10
10.47 ( 0.75
286 ( 25
0.88 ( 0.09
6.3 ( 0.3
1.04 ( 0.10
7.67 ( 0.21
1.11 ( 0.14
5.99 ( 0.42
10 ( 7
1.95 ( 0.15
6.15 ( 0.23
1.12 ( 0.10
9.42 ( 0.33
28 ( 10
0.93 ( 0.05
4.42 ( 0.24
1.02 ( 0.03
8.84 ( 0.3
292 ( 28
0.34 ( 0.05
3.48 ( 0.18
0.44 ( 0.02
7.35 ( 0.31
468 ( 132
0.99 ( 0.04
7.42 ( 0.49
11 ( 5.5
NQO2 activity
a The compounds designated 5 are parent compounds, and those designated 6 are the corresponding N-oxides. Values given are with (SD.
the current study we included synthesis of N-oxide derivatives of
the imidazocridin-6-ones (designated 6). Clearly, the introduc-
tion of the N-oxide group shows little or no systematic effect
on enzyme inhibitory potency (Table 1). However, it is appa-
rent that the toxicity of the N-oxides toward HCT116 colon
cancer cells is consistently around 20-fold less compared to their
parent (5) analogues. The HCT116 cells contain significant
levels of NQO2 ((493 nmol of DCPIP reduced/min)/(mg of
protein)).12 Therefore, in order to confirm this toxicity trend and
also determine whether there was likely to be a dependence on
NQO2 for toxicity, two pairs of compounds (5a1 and 6a1, and
5c1 and 6c1) were evaluated in a panel of seven breast cancer cell
lines with varying NQO2 activities (from 8 to 468 (nmol of
DCPIP reduced/min)/(mg of protein); Table 2). In each cell
line, the N-oxide always showed lower toxicity than its parent
compound. Further, there is clearly no relationship between the
level of cellular NQO2 activity and toxicity. Consistent with our
previous observations,12 the inclusion of the N-oxide group
decreases the ability of the compounds to interact with DNA.
This is shown by the DNA melting curves in Figure 2. The
control Tm in these experiments was 85 ꢀC; in the presence of
the parent imidazoacridin-6-one, 5a1, this increases to 88.6 ꢀC.
6599
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
In contrast, the N-oxide 6a1 shows little if any increase in melting
temperature. In light of these differences in DNA binding affinity,
the subcellular distribution of the imidazoacridin-6-ones was
explored by fluorescent imaging. MDA468 cells were treated
with 5 μM 5a1 or 25 μM 6a1 (to reflect the differences in toxicity
of the two compounds), then harvested and treated with
phalloidin to visualize the cellular architecture. Figure 3 shows
the fluorescent images of cells treated with the two compounds.
Nuclei of cells treated with 5a1 are clearly visible which is con-
sistent with the DNA binding affinity of this compound. In
contrast, little nuclear binding is observed with 6a1, even though
it is present at a 5-fold higher concentration; however, a high level
of cytoplasmic fluorescence is observed. Interestingly, this could
point to a different mechanism of toxicity for the two classes of
compound. This difference in nuclear uptake/binding is recapi-
tulated in a second cell line (HT29 colon cancer cells), and this is
given in the Supporting Information (Figure 1).
Another significant difference between the parent imidazoa-
cridin-6-ones and their N-oxide derivatives is their apparent
protein binding ability. This is determined indirectly by measur-
ing the ability of the compounds to inhibit NQO2 in the presence
or absence of 2 μM bovine serum albumin (BSA). Two pairs of
compounds were evaluated (5a1/6a1 and 5c1/6c1). For 6a1 the
presence of BSA reduced enzyme inhibitory potency from 14 to
98 nM (a 7-fold change). In contrast, BSA reduced the activity
of 5a1 from 59 to 3200 nM (a 54-fold change). Similarly, the
fold change for the N-oxide, 6c1, was 6.5, whereas for the parent
compound, 5c1, the change was 38. Thus, it is likely that the
N-oxide derivatives are likely to be less protein bound and
therefore may have some advantage for probing for the function-
ality of NQO2 in cells. To test this, we evaluated the ability of 6a1
to protect MDA468 cells against the cytotoxic effects of 5--
(aziridin-1-yl)-2,4-dinitrobenzamide, 7 (CB1954).20 This agent
is exquisitely dependent upon the presence of NQO2 in human
cells for it to be metabolically activated to give a potent
cytotoxin.20 Thus, inhibition of the toxicity of 7 in air can be
regarded as a surrogate measure of the inhibitory potency of
the imidazoacridin-6-ones in cells. Figure 4 shows the toxicity
of 7 in MDA468 cells treated for 3 h with varying concentra-
tions of 6a1 in the presence of 100 μM N-ribosylnicotinamide
(NRH), the cofactor necessary to facilitate metabolic activa-
tion by NQO2. It is clear that the presence of 6a1 can inhibit
the toxicity of 7 and that inhibition occurs at concentrations
of 6a1 that are nontoxic. Hence, this provides evidence for
the first time that an agent like 6a1, an N-oxide substituted
imidazoacridein-6-one, can inhibit NQO2 in cells and there-
fore can be used as a pharmacological probe for the function of
this enzyme.
Figure 2. DNA melting curves for calf thymus DNA (10 μM) in the
presence of 10 μM 5a1 or 6al. Experiments were repeated on three
separate occasions and values of Tm ( SD are 85.0 and 85.0 for
controland6a1,respectively(threeexperimentsgiving
identical results), and 88.6 ( 0.5 for 5a1. 10 μM
adriamycin is used as a positive control in these experi-
ments (Tm = 90.8 ( 0.9).
Figure 4. Functional activity of 6al as an inhibitor of NQO2 in
MDA468 cells. The activity of NQO2 in cells is shown by the ability
of the enzyme to activate 7 (CB1954) to give a cytotoxic metabolite.
Cells were treated with 10 μM 7 and varying concentrations of 6a1: (b)
7 alone; (9) 7 plus 10 μM 6a1; (2) 7 plus 100 μM 6a1; (1) 7 plus 500
μM 6a1.
Figure 3. Subcellular localization of 5al and 6al as revealed by fluorescence microscopy. MDA468 cells exposed to 5 μM 5al (A) and 25 μM 6a1 (B).
Red staining indicates the binding of phalloidin to β-actin which illuminates cellular architecture. Nuclear binding of 5al is apparent in part A, whereas
much lower fluorescence in the nucleus is observed for 6al in part B.
6600
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
Figure 5. X-ray crystal structures of the imidazoacridin-6-one ligands 5a1 and 6a1, showing detailed views of (a) 5a1 and (b) 6a1 bound in the NQO2
active site. The individual monomers and key active site residues are colored red and blue. The cofactor FAD is shown in green with hydrogen bonds
indicated by black dotted lines. Parts c and d show the ligands 5a1 and 6a1 in omitted 2Fo ꢀ Fc electron density contoured at 1.2σ.
Molecular Modeling and Crystallography. The imidazoa-
cridin-6-ones were docked into the active site of NQO2 using the
crystal structure of the apoenzyme with bound FAD as a starting
placed unambiguously, the N-oxide moiety of 6a1 appears
disordered and was placed to match that of 5a1.
The dimerization of NQO2 is functionally critical to its activity,
with the resulting interface forming two active sites made up of
residues from both monomers. The 5a1 and 6a1 imidazoacridin-
6-one ligands interact primarily through hydrophobic interactions
with residues from both monomers and stacking interactions via
the conjugate ring system of FAD. Figure 5 shows that the
isoalloxazine ring of FAD provides the floor of the active site and
stacks with the imidazoacridin-6-one moiety of both 5a1 and
6a1. Hydrophobic residues F126, F131, and Y132 form the top of
the active site, while W1050 (primes denote residues from the
other NQO2 monomer) sits at the back of the binding pocket
and forms a hydrophobic interaction with the ring system of both
5a1 and 6a1. Q122 and E1930 form an electrostatic environment
for the tail of 5a1 and the N-oxide moiety of 6a1. The apparent
disorder seen in the N-oxide tail of 6a1 is likely due to the
model (PDB code 1QR2, resolution at 2 Å).21 The 5 series were
ꢀ
docked in their protonated form. As previously described,13 the
docking calculations predicted only one energetically favorable
binding mode for this series of compounds (see Supporting
Information Figure 2). After docking, the calculated binding affinities
were compared with the newly measured experimental IC50 values
for enzyme inhibition (see Supporting Information Figure 3) and a
correlation coefficient of R2 = 0.82 was established.
To further investigate the binding of imidazoacridin-6-one
ligands to NQO2, 5a1 and the corresponding N-oxide 6a1 were
cocrystallized with the enzyme (Figure 5). Both NQO2ꢀligand
complexes crystallized in the space group P212121, forming a
single dimer in the asymmetric unit. While both imidazoacridin-
6-one ligands are well-defined in the electron density and can be
6601
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
’ CONCLUSIONS
In this work it has been shown that imidazoacridin-6-ones can
be potent nanomolar inhibitors of recombinant human NQO2
and further, one of the N-oxides, 6a1, shows the ability to inhibit
the enzymatic function of NQO2 in cells. Compounds repre-
sentative of a variety of structural classes have been shown to
inhibit isolated NQO2,7ꢀ9,11ꢀ16 but this is the first demonstra-
tion that a novel inhibitor of NQO2 can be functionally active as
an inhibitor in cells at nontoxic concentrations. Since NQO2 has
been shown to have other properties in cells such as acting as a
chaperone protein, the development of agents like 6a1 provides a
pharmacological basis to study these, and other, cellular func-
tions of the enzyme.
’ EXPERIMENTAL SECTION
Chemistry. Chemicals were purchased from Aldrich Chemical Co.
Column chromatography was carried out using silica gel grade 60
Figure 6. Relationship between the calculated binding affinity (ΔG) of
the imidazoacridin-6-ones to NQO2 using the 5a1 crystal structure and
their ability to act as inhibitors of the enzyme (IC50).
1
(230ꢀ400 mesh). The H and 13C NMR spectra were recorded on
Bruker Avance spectrometers operating at 300, 400, and 500 MHz.
Chemical shifts are reported as δH or δC in parts per million (ppm)
downfield from tetramethylsilane (TMS) for samples run in CDCl3.
Mass spectra were carried out at the School of Chemistry, University of
Manchester, U.K., using a Micromas Trio 2000 (CI, EI, FAB) and
Micromas Platform 2 (electrospray). CI mass spectra were recorded
using ammonia as a carrier gas. Melting points were determined using a
Gallenkamp MPD.350.BM2.5 machine and remain uncorrected. The
purity of all compounds was judged to be greater than or equal to 95%
based on assessment of TLC, melting point, and 1H NMR data.
Synthesis of 2,6-Dichloro-3-nitrobenzoic Acid. 2,6-Dichlor-
obenzoic acid (5.00 g, 26.2 mmol) was added gradually to concentrated
H2SO4 (200 mL) with stirring, and the mixture was warmed to 50 ꢀC
until all solid had completely dissolved. Concentrated HNO3 (2.0 mL)
was added gradually, and the mixture was stirred for 1 h. The resulting
solution was allowed to cool before it was poured onto ice (800 g) with
stirring. The solution was kept cool until crystallization was complete, and the
resulting solid was collected by filtration to give 2,6-dichloro-3-nitrobenzoic
acid (5.87 g, 95%). Mp 143ꢀ144 ꢀC; νmax/cmꢀ1 3380w (OꢀH), 2884w
(CꢀH), 1671s (CdO), 1644s; δH (500 MHz, CDCl3) 7.92 (1 H, d, J 8.8,
C(4)H), 7.55 (1 H, d, J 8.8, C(5)H), 6.84 (1H, br s, OH); δC (100 MHz,
CDCl3) 166.6 (q), 146.6 (q), 135.9 (q), 135.4 (q), 128.9 (q), 126.9
(C(4)H), 125.5 (C(5)H); m/z (ꢀES) 237.0 (6, [M ꢀ H]ꢀ, 35Cl37Cl),
235.0 (10, [M ꢀ H]ꢀ, 35Cl35Cl), 194 (15, [M ꢀ NO2]ꢀ, 37Cl37Cl),
192 (70, [M ꢀ NO2]ꢀ, 35Cl37Cl), 190 (100, [M ꢀ NO2]ꢀ, 35Cl35Cl);
234.9454 ([M ꢀ H]ꢀ, 35Cl35Cl) found by ꢀES, required 234.9445, error
4.0 ppm.
Synthesis of 2-(4-Methoxyphenylamino)-6-chloro-3-
nitrobenzoic Acid (1a). A solution of 4-methoxyaniline (0.700 g,
5.68 mmol) in N,N-dimethylaniline (1.5 mL) was added to a flask
containing 2,6-dichloro-3-nitrobenzoic acid (1.00 g, 4.24 mmol) in N,-
N-dimethylaniline (3.0 mL). The mixture was warmed with stirring
under nitrogen for 18 h. Once cooled, it was diluted with CHCl3
(20 mL), extracted with 1 M NaOH (20 mL), and washed twice with 1
M NaOH (2 ꢁ 10 mL). The combinedaqueousextractswereacidifiedwith
dilute HCl to pH 3, resulting in formation of a yellow precipitate which was
collected by filtration and dried to give 1a (0.779 g, 57%). Mp 211ꢀ213 ꢀC;
νmax/cmꢀ1 3297w (NꢀH), 2848w (CꢀH), 1698s (CdO), 1589s,
1574s, 1510s (CdC); δH (300 MHz, DMSO-d6) 8.69 (1H, s, NH),
8.04 (1H, d, J 8.9, C(4)H), 7.17 (1H, d, J 8.9, C(5)H), 6.91 (2H, d, J 8.9,
C(10)H, C(12)H), 6.79 (2H, d, J 8.9, C(9)H, C(13)H), 3.70 (3H, s,
C(14)H3); δC (75 MHz, DMSO-d6) 165.0 (q), 155.6 (q), 153.3 (q),
138.4 (q), 136.5 (q), 134.7 (q), 134.0 (q), 127.6 (C(4)H), 122.6 (CH,
Ph), 120.9 (C(5)H), 114.0 (CH, Ph), 55.2 (OCH3); m/z (ꢀES) 323
predominately negative charge from the single bonded oxygen
that would repel the negatively charged side chain of Q122.
These repulsive effects would allow the 6a1 tail moiety to explore
more of the binding cavity leading to numerous low occupancy
conformations and ultimately the loss of any definitive high
occupancy site. Although there are no direct H-bonds to any
main chain or side chain residues, the OMe group of both ligands
forms a water-mediated hydrogen bond contact with N161. The
side chain of N161 is held in place via a hydrogen bond to the OH
of Y132. A similar water-mediated interaction to N161 is also
seen in the imatinib bound structure of NQO2,22 which might
suggest a conserved mode of binding for some of these inhibitors.
Molecular docking of 5a1 and 6a1 initially suggested that
these ligands interacted with NQO2 in an alternative orientation
with the tail moiety making a direct hydrogen bond contact with
the ND2 of N161. The removal of all water molecules prior to
modeling simulations may explain these differences. To investi-
gate this further, the imidazoacridine-6-ones were docked into
the active site of the 5a1 crystal structure and the conserved crystal
water was retained. The docked orientation of 5a1 and 6a1 was
superimposed onto the crystal ligands with a root-mean-square
deviation (rmsd) of 0.98 and 1.12, respectively (see Supporting
Information Figure 4). Interestingly, the docking calculations
suggest that ligands with a methoxy (5a1, 5a2, 6a1, and 6a2)
and hydroxy (5e1, 5f1, 5e2, 5f2, and 6e1) substituent at the R1
position bind in a similar orientation to the crystal pose. However,
ligands with a halide substituent (5b1, 5b2, 5c2, 5d2, 6b1, 6b2,
and 6d2) and hydrogen (5c1, 5c2, 6c1, and 6c2) at R1 bind in an
alternative orientation (180ꢀ rotation about the Y axis) compared
to the crystal pose. The tail moiety forms a hydrogen bond with
either G149 or a water mediated hydrogen bond with N161
(see Supporting Information Figure 5 for examples of 5b1 and
6b1). The calculated binding affinities derived from docking into
the crystal structure of 5a1 are shown in Table 1 and have been
plotted against the experimental IC50 values in Figure 6, giving a
correlation coefficient of 0.87. Since it is not clear how easily
the conserved water molecule would be displaced upon ligand
binding, any future modeling simulations with NQO2 inhibitors
should perhaps take into account this ordered water molecule
during its calculations.
6602
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
(10, [M ꢀ H]ꢀ, 37Cl), 321 (30, [M ꢀ H]ꢀ, 35Cl); 321.0290 ([M ꢀ
H]ꢀ) found by ꢀES, required 321.0278, error 3.7 ppm.
35Cl81Br), 369 (80, [M ꢀ H]ꢀ, 35Cl79Br); 368.9282 ([M ꢀ H]ꢀ)
found by ꢀES, required 368.9283, error 0.3 ppm.
Synthesis of 2-(4-Fluorophenylamino)-6-chloro-3-nitro-
benzoic Acid (1b). A solution of 4-fluoroaniline (0.54 mL, 5.68
mmol) in N,N-dimethylaniline (1.5 mL) was added to a flask containing
2,6-dichloro-3-nitrobenzoic acid (1.00 g, 4.24 mmol) in N,N-dimethy-
laniline (3.0 mL). The mixture was warmed with stirring under nitrogen
for 18 h. Once cooled, it was diluted with CHCl3 (20 mL), extracted with
1 M NaOH (20 mL), and washed twice with 1 M NaOH (2 ꢁ 10 mL).
The combined aqueous extracts were acidified with dilute HCl to pH 3,
resulting in formation of a yellow precipitate which was collected by
filtration and dried to give 1b (0.786 g, 60%). Mp 176.5ꢀ177.0 ꢀC;
νmax/cmꢀ1 3348w (NꢀH), 2980br (OꢀH), 2881w, (CꢀH), 1704s
(CdO), 1588s, 1569s, 1530m, 1504s (CdC); δH (300 MHz, DMSO-
d6) 10.73 (1H, s, C(7)O2H), 8.57 (1H, s, NH), 8.05 (1H, d,
J 8.9, C(4)H), 7.35 (1H, d, J 8.9, C(5)H), 7.02 (2H, app t, J 8.8,
C(10)H, C(12)H), 6.88 (2H, dd, J 8.7, 4.8, C(9)H, C(13)H); δC (75
MHz, DMSO-d6) 165.1 (q), 157.7 (d, J 237.9, C(11)F), 140.9 (q), 139.2
(q), 136.5 (q), 135.8 (q), 131.1 (q), 127.5 (C(4)H), 123.1 (C(5)H),
120.6 (d, J 8.1, C(9)H, C(13)H), 115.3 (d, J 22.7, C(10)H, C(12)H);
δF (376 MHz, DMSO-d6) ꢀ121.60, ꢀ121.52 (m); m/z (ꢀES) 311
(10, [M ꢀ H]ꢀ, 37Cl), 309 (30, [M ꢀ H]ꢀ, 35Cl). 309.0090 ([M ꢀ H]ꢀ)
found by ꢀES, required 309.0084, error 2.0 ppm.
Synthesis of 2-(4-(Benzyloxy)phenylamino)-6-chloro-
3-nitrobenzoic Acid (1e). A solution of 4-benzoxyaniline (2.03 g,
10.2 mmol) in N,N-dimethylaniline (3.0 mL) was added to a flask
containing 2,6-dichloro-3-nitrobenzoic acid (2.00 g, 8.47 mmol) in N,-
N-dimethylaniline (6.0 mL). The mixture was warmed with stirring
under nitrogen for 18 h. Once cooled, it was diluted with CHCl3
(40 mL), extracted with 1 M NaOH (40 mL), and washed twice with 1
M NaOH (2 ꢁ 20 mL). The combined aqueous extracts were acidified
with dilute HCl to pH 3, resulting in formation of a yellow precipitate
which was collected by filtration and dried to give 1e (1.27 g, 38%). Mp
271.5ꢀ271.8 ꢀC; νmax/cmꢀ1 3314m (NꢀH), 3037m (CꢀH), 16577s
(CdO), 1618s, 1594s; δH (300 MHz, DMSO-d6) 9.18 (1H, s, NH),
7.62 (1H, d, J 8.9, C(4)H), 7.45ꢀ7.43 (5H, m, Ph), 6.95 (1H, d, J 8.9,
C(5)H), 6.85 (2H, d, J 8.6, C(10)H, C(12)H), 6.69 (2H, d, J 8.6,
C(9)H, C(13)H), 5.00 (2H, s, CH2); δC (75 MHz, DMSO-d6) 165.3
(q), 153.6 (q), 137.3 (q), 137.2 (q), 136.2 (q), 135.8 (q), 135.6 (q),
135.2 (q), 128.4 (CH), 127.7 (CH), 127.5 (CH), 124.1 (C(4)H), 120.6
(C(5)H), 118.2 (C(9)H, C(13)H), 115.2 (C(10)H, C(12)H), 69.5
(CH2); m/z (ꢀES) 397 (100, [M ꢀ H]ꢀ); 397.0593 ([M ꢀ H]ꢀ)
found by ꢀES, required 397.0596, error 0.8 ppm.
Synthesis of 1-Chloro-7-methoxy-4-nitroacridin-9(10H)-
one (2a). A mixture of 1a (248 mg, 0.80 mmol), POCl3 (0.50 mL,
5.25 mmol), and N,N-dimethylaniline (0.16 mL) in 1,2-dichloroethane
(25 mL) was heated under reflux for 2 h. The resulting red precipitate
was collected by filtration, washed with ethanol (2.0 mL), and dried to
give 2a (186 mg, 58%) as red needles. Mp 263ꢀ264 ꢀC; νmax/cmꢀ1
3288m (NꢀH), 3113w, 2839w (CꢀH), 1644s (CdO), 1606s, 1589s,
1565s; δH (300 MHz, DMSO-d6) 11.64 (1H, s, NH), 8.55 (1H, d,
J 8.9, C(3)H), 8.02 (1H, d, J 9.1, C(5)H), 7.59 (1H, d, J 2.9, C(8)H),
7.47 (1H, dd, J 9.1, 2.9, C(6)H), 7.38 (1H, d, J 8.9, C(2)H), 3.16
(3H, s, C(14)H3); m/z (ꢀES) 305 (35, [M ꢀ H]ꢀ, 37Cl), 303
(100, [M ꢀ H]ꢀ, 35Cl); (+ES) 329 (20, [M + Na]+, 37Cl), 327 (60,
[M + Na]+, 35Cl); 303.0180 ([M ꢀ H]ꢀ) found by ꢀES, required
303.0173, error 2.4 ppm.
Synthesis of 6-Chloro-3-nitro-2-(phenylamino)benzoic Acid
(1c). A solution of aniline (0.879 g, 8.47 mmol) in N,N-dimethylaniline
(50 mL) was added to a flask containing 2,6-dichloro-3-nitrobenzoic
acid (1.00 g, 4.23 mmol) in N,N-dimethylaniline (50 mL). The mixture
was warmed with stirring under nitrogen for 18 h. Once cooled, it was
diluted with CHCl3 (100 mL), extracted with 1 M NaOH (50 mL), and
washed twice with 1 M NaOH (2 ꢁ 30 mL). The combined aqueous
extracts were acidified with dilute HCl to pH 3, resulting in formation of
a yellow precipitate which was collected by filtration and dried to give 1c
(0.552 g, 45%). Mp 203ꢀ205 ꢀC (lit., 199ꢀ204 ꢀC); νmax/cmꢀ1 3333w
(NꢀH), 3040w (CꢀH), 1704s (CdO), 1583s, 1568s; δH (500 MHz,
DMSO-d6) 8.47 (1H, s, NH), 8.02 (1H, d, J 8.8, C(4)H), 7.41 (1H, d,
J 8.8, C(5)H), 7.14 (2H, app t, J 7.9 C(10)H, C(12)H), 6.85 (1H, app t,
J 7.4, C(9)H, C(11)H), 6.77 (2H, d, J 7.6, C(9)H, C(13)H); δC (125
MHz, DMSO-d6) 165.1 (q), 143.4 (q), 142.1 (q), 135.3 (C(4)H), 132.5
(q), 129.0 (q), 128.6 (C(10)H, C(12)H), 127.7 (C(11)H), 124.1 (q),
121.0 (C(9), C(13)H), 116.9 (C(5)H); m/z (ꢀES) 293 (8, [M ꢀ H]ꢀ,
37Cl), 291 (24, [M ꢀ H]ꢀ, 35Cl), 249 (41, M ꢀ CO2H, 37Cl), 247
(100, M ꢀ CO2H, 37Cl); 291.0178 ([M ꢀ H]ꢀ) found by ꢀES,
required 291.0178, error 0.0 ppm.
Synthesis of 2-(4-Bromophenylamino)-6-chloro-3-nitro-
benzoic Acid (1d). 4-Bromoaniline (7.29 g, 42.4 mmol, 5 equiv)
was heated to 110 ꢀC until completely melted. 2,6-Dichloro-3-nitro-
benzoic acid (2.00 g, 8.47 mmol, 1 equiv) was then added in small
portions over 20 min with stirring. Heating was continued, and after 3 h a
solid mass had formed which was kept at 110 ꢀC for a further 4 h. Once
cooled to room temperature, this solid was thoroughly crushed in 2 M
NaOH (30 mL) and the mixture left to stir for 30 min. Unreacted
4-bromoaniline was collected by filtration and washed once with 2 M
NaOH (5 mL) and twice with water (2 ꢁ 5 mL). The filtrate was then
acidified with dilute HCl to pH 3, which resulted in formation of a yellow
precipitate which was collected by filtration and dried to give 1d (1.89 g,
60%) as a yellow powder. Mp 198.8ꢀ202.0 ꢀC; νmax/cmꢀ1 3305w
(NꢀH), 3090w, 2818m (CꢀH), 1703s (CdO), 1596s, 1574s, 1528m;
δH (500 MHz, DMSO-d6) 13.87 (1H, s, C(7)O2H), 8.54 (1H, s, NH),
8.06 (1H, d, J 8.9, C(4)H), 7.51 (1H, d, J 8.9, C(5)H), 7.30 (2H, d, J 8.8,
C(10)H, C(12)H), 6.71 (2H, d, J 8.8, C(9)H, C(13)H); δC (125 MHz,
DMSO-d6) 165.0 (q), 143.4 (q), 143.0 (q), 135.2 (q), 134.5
(q), 133.5 (q), 131.5 (C(10)H, C(12)H), 127.3 (C(4)H), 125.2
(C(5)H), 118.7 (C(9)H, C(13)H), 112.0 (q, C(11)); m/z (ꢀES)
373 (25, [M ꢀ H]ꢀ, 37Cl81Br), 371 (100, [M ꢀ H]ꢀ, 37Cl79Br,
Synthesis of 1-Chloro-7-fluoro-4-nitroacridin-9(10H)-one
(2b). A mixture of 1b (248 mg, 0.80 mmol), POCl3 (0.50 mL, 5.25
mmol), and N,N-dimethylaniline (0.16 mL) in 1,2-dichloroethane
(25 mL) was heated under reflux for 2 h. The resulting red precipitate
was collected by filtration, washed with ethanol (2.0 mL), and dried to
give 2b (135 mg, 58%) as a red powder. Mp 269.5ꢀ271.0 ꢀC; νmax
/
cmꢀ1 3305m (NꢀH), 3078w (CꢀH), 1647s (CdO), 1624m, 1589s,
1569s, 1525w, 1484s; δH (300 MHz, DMSO-d6) 11.68 (1H, NH), 8.55
(1H, d, J 8.8, C(3)H), 8.12 (1H, dd, J 8.9, 4.6, C(8)H), 7.82 (1H, dd,
J 8.9, 3.0, C(5)H), 7.73 (1H, app dt, J 8.9, 3.0, C(6)H), 7.41 (1H, d, J 8.8,
C(2)H); δF (376 MHz, DMSO-d6) ꢀ117.65, ꢀ117.59 (m); m/z
(ꢀES) 293 (35, [M ꢀ H]ꢀ, 37Cl), 291 (100, [M ꢀ H]ꢀ, 35Cl);
(+ES) 317 (35, [M + Na]+, 37Cl), 315 (100, [M + Na]+, 35Cl); 314.9960
([M + Na]+) found by +ES, required 314.9943, error 5.3 ppm.
Synthesis of 1-Chloro-4-nitroacridin-9(10H)-one (2c). A
mixture of 1c (1.60 g, 5.47 mmol), POCl3 (3.28 mL, 36 mmol), and
N,N-dimethylaniline (0.16 mL) in dichloroethane (16 mL) was heated
under reflux for 2 h. The resulting red precipitate was collected by
filtration, washed with ethanol (2.0 mL), and dried to give 2c as a red
powder (1.23 g, 82%). Mp 248ꢀ249 ꢀC; νmax/cmꢀ1 3307m (NꢀH),
1644s (CdO); δH (500 MHz, DMSO-d6) 11.47 (1H, s, NH), 8.55 (1H,
d, J 8.8, C(3)H), 8.18 (1H, d, J 8.0, C(5)H), 7.99 (1H, d, J 8.2, C(8)H),
7.89ꢀ7.82 (1H, m, C(6)H), 7.40 (1H, d, J 8.8, C(2)H), 7.38ꢀ7.41 (1H,
m, C(7)H); δC (100 MHz, DMSO-d6) 174.9 (q), 141.8 (q), 138.4 (q),
137.0 (q), 133.9 (C(3)H), 130.1 (C(6)H), 125.6 (C(8)H), 123.2
(C(7)H), 122.5 (C(5)H), 121.8 (q), 118.6 (q), 118.3 (C(2)H), 99.2
(q); m/z (ꢀES) 275 (32, [M ꢀ H]ꢀ, 37Cl), 273 (100, [M ꢀ H]ꢀ, 35Cl);
297.0051 ([M + Na]+) found by +ES, required 297.0054, error 2.7 ppm.
6603
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
Synthesis of 7-Bromo-1-chloro-4-nitroacridin-9(10H)-one
(2d). A mixture of1d (1.80 g, 4.84 mmol), POCl3 (3.00 mL, 31.8 mmol),
and N,N-dimethylaniline (0.14 mL) in 1,2-dichloroethane (14 mL) was
heated under reflux for 1 h. The resulting red precipitate was collected by
filtration, washed with 1,2-dichloroethane (2.0 mL), and dried to give 2d
as a bright red powder (1.61 g, 94%). Mp 310.0ꢀ310.8 ꢀC; νmax/cmꢀ1
3291m (NꢀH), 3074m (CꢀH), 1658s (CdO), 1613, 1571s; δH
(500 MHz, DMSO-d6) 8.50 (1H, d, J 8.8, C(3)H), 8.24 (1H, s, C(8)H),
7.98 (1H, d, J 8.6, C(5)H), 7.93 (1H, d, J 8.6, C(6)H), 7.39 (1H, d,
J 8.8, C(2)H); m/z (ꢀES) 355 (25, [M ꢀ H]ꢀ, 37Cl81Br), 353 (100,
[M ꢀ H]ꢀ, 37Cl79Br, 35Cl81Br), 351 (80, [M ꢀ H]ꢀ, 35Cl79Br); (+ES)
378 (3, [M + Na]+, 37Cl81Br), 376 (12, [M + Na]+, 37Cl79Br, 35Cl81Br),
374 (9, [M + Na]+, 35Cl79Br); 350.9178 ([M ꢀ H]ꢀ) found by ꢀES,
required 350.9178, error 0.0 ppm.
was collected by filtration and washed with ethanol (2.0 mL) to give
3c (1.10 g, 67%). Mp 211.0ꢀ211.8 ꢀC; νmax/cmꢀ1 3233m (NꢀH),
3065m, 2977m, 2941m, 2862m, 2815m, 2768m (CꢀH), 1615s
(CdO); δH (500 MHz, CDCl3) 12.64 (1H, s br, NH), 11.96 (1H, s
br, NH), 8.46 (1H, d, J 9.9, C(3)H), 8.40 (1H, d, J 8.2, C(8)H), 7.71
(1H, app t, J 7.2, C(6)H), 7.46 (1H, d, J 8.2, C(5)H), 7.38 (1H, app t,
J 7.8, C(7)H), 6.38 (1H, d, J 9.9, C(2)H), 3.54 (2H, app q, J 5.9,
C(11)H2), 2.73 (2H, t, J 6.5, C(12)H2), 2.38 (6H, s, C(13)H3,
C(14)H3); δC (75 MHz, CDCl3) 180.0 (q), 157.9 (q), 140.6 (q),
138.1 (q), 133.8 (C(3)H, C(6)H), 126.3 (C(8)H), 123.8 (C(7)H),
122.8 (q), 121.2 (q), 117.7 (C(5)H), 104.7 (q), 102.5 (C(2)H),
57.5 (C(11)H3), 45.5 (C(13)H3, C(14)H3), 41.2 (C(12)H3); m/z
(ꢀES) 325 (100, [M ꢀ H]ꢀ); (+ES) 327 (100, [M + H]+); 327.1463
([M + H]+) found by +ES, required 327.1452, error 3.5 ppm.
Synthesis of 1-(2-(Dimethylamino)ethylamino)-7-bromo-
4-nitroacridin-9(10H)-one (3d). N,N-Dimethylethylenediamine
(0.54 mL, 5.00 mmol) was added to a suspension of 2d (0.708 g, 2.00
mmol) in anhydrous DMF (6.0 mL), and the reaction mixture was
heated to reflux for 4 h. Ethanol (10 mL) was added and the resulting
yellow crystals were collected by filtration and washed with ethanol
(2.0 mL) to give 3d (792 mg, 98%). Mp 242.0ꢀ242.5 ꢀC; νmax/cmꢀ1
3201m (NꢀH), 3065m, 2943w, 2859w (CꢀH), 1611s (CdO), 1592s,
1566s; δH (300 MHz, CDCl3) 12.63 (1H, s br, NH), 11.82 (1H, s br,
NH), 8.51 (1H, d, J 2.3, C(8)H), 8.44 (1H, d, J 10.0, C(3)H), 7.77 (1H,
dd, J 8.7, 2.3, C(6)H), 7.34 (1H, d, J 8.7, C(5)H), 6.38 (1H, d, J 10.0,
C(2)H), 3.52 (2H, app q, J 5.8, C(11)H2), 2.73 (2H, t, J 6.3, C(12)H2),
2.39 (6H, s, C(13)H2, C(14)H2); δC (75 MHz, CDCl3) 178.6 (q),
157.7 (q), 140.5 (q), 136.8 (q), 136.8 (C(6)H), 134.0 (C(3)H), 128.9
(C(8)H), 123.9 (q), 121.2 (q), 119.4 (C(5)H), 117.1 (q), 104.7
(q), 102.8 (C(2)H), 57.4 (C(12)H2), 45.5 (C(13)H2, C(14)H2),
41.2 (C(11)H2); m/z (ꢀES) 405 (100, [M ꢀ H]ꢀ, 81Br), 403
(100, [M ꢀ H]ꢀ, 79Br); (+ES) 407 (100, [M + H]+, 81Br), 405 (100,
[M + H]+, 79Br); 405.0558 ([M + H]+, 79Br) found by +ES, required
405.0557, error 0.3 ppm.
Synthesis of 1-(2-(Dimethylamino)ethylamino)-7-hydroxy-
4-nitroacridin-9(10H)-one (3e). N,N-Dimethylethylenediamine
(1.09 mL, 10.0 mmol) was added to a suspension of 2e (1.45 g, 5.00
mmol) inanhydrousDMF(5.0mL), andthereactionmixturewasheated to
reflux for 4 h. Ethanol (10 mL) was added and the resulting precipitate
was was collected by filtration and washed with ethanol (2.0 mL) to give
3e (1.60 g, 94%). Mp 215.0ꢀ215.8 ꢀC; νmax/cmꢀ1 3204m (NꢀH),
3093m, 2946m (CꢀH), 1608s (CdO), 1568s, 1543s; δH (300 MHz,
DMSO-d6) 12.46 (1H, s br, NH), 11.95 (1H, s br, NH), 8.37 (1H, d,
J 10.0, C(3)H), 7.86 (1H, d, J 9.0, C(5)H), 7.53 (1H, d, J 2.7, C(8)H),
7.28 (1H, dd, J 9.0, 2.7, C(6)H), 6.56 (1H, d, J 10.0, C(2)H), 3.54 (2H,
app q, J 5.5, C(11)H2), 2.61 (2H, t, J 6.0, C(12)H2), 2.26 (6H, s,
C(13)H3, C(14)H3); δC (75 MHz, DMSO-d6) 178.2 (q), 157.4 (q),
154.2 (q), 139.1 (q), 133.3 (C(3)H), 131.4 (q), 123.8 (C(6)H), 123.2
(q), 120.5 (C(5)H), 120.1 (q), 107.8 (C(8)H), 103.1 (C(2)H), 102.7
(q), 57.0 (C(12)H2), 45.0 (C(13)H3, C(14)H3), 36.3 (C(11)H2); m/z
(ꢀES) 341 (100, [M ꢀ H]ꢀ); (+ES) 343 (100, [M + H]+); 343.1399
([M + H]+) found by +ES, required 343.1401, error 0.7 ppm.
Synthesis of 1-Chloro-7-hydroxy-4-nitroacridin-9(10H)-
one (2e). A mixture of 1e (1.20 g, 3.01 mmol) and POCl3 (1.80 mL,
20 mmol) in dichloroethane (16 mL) was heated under reflux for 2 h.
The resulting red precipitate was collected by filtration, washed with
ethanol (2.0 mL), and dried to give 2e as a red powder (0.841 g, 96%).
Mp >400 ꢀC; νmax/cmꢀ1 3326m (NꢀH), 3078m (CꢀH), 16308s
(CdO), 1587, 1567s; δH (300 MHz, DMSO-d6) 11.59 (1H, s, OH),
8.54 (1H, d, J 8.8, C(3)H), 7.92 (1 H, d, J 9.0, C(5)H), 7.52 (1 H, d, J 2.7,
C(8)H), 7.36ꢀ7.30 (2 H, m, C(6)H, C(2)H); m/z (ꢀES) 289 (100,
[M ꢀ H]ꢀ); 289.0015 ([M ꢀ H]ꢀ) found by ꢀES, required 289.0011,
error 1.1 ppm.
Synthesis of 1-(2-(Dimethylamino)ethylamino)-7-meth-
oxy-4-nitroacridin-9(10H)-one (3a). N,N-Dimethylethylenedia-
mine (1.09 mL, 10.0 mmol) was added to a suspension of 2a (1.53 g,
5.00 mmol) in anhydrous DMF (5.0 mL), and the reaction mixture was
heated to reflux for 4 h. Ethanol (10 mL) was added and the resulting
yellow precipitate was collected by filtration and washed with ethanol
(2.0 mL) to give 3a (1.64 g, 92%). Mp 239.5 ꢀ240.0 ꢀC; νmax/cmꢀ1
3380m (NꢀH), 3068m, 2944m (CꢀH), 1666s (CdO), 1629s, 1606s,
1572s, 1543s; δH (500 MHz, CDCl3) 12.69 (1H, s br, NH), 12.03 (1H, s
br, NH), 8.46 (1H, d, J 9.8, C(3)H), 7.77 (1H, d, J 2.9, C(8)H), 7.41
(1H, d, J 8.8, C(5)H), 7.35 (1H, dd, J 8.8, 2.9, C(6)H), 6.36 (1H, d, J 9.8,
C(2)H), 3.94 (3H, s, OCH3), 3.54 (2H, app q, J 5.9, C(11)H2), 2.74
(2H, t, J 6.3, C(12)H2), 2.39 (6H, s, C(13)H3, C(14)H3); δC (75 MHz,
CDCl3) 179.3 (q), 158.0 (q), 156.5 (q), 139.8 (q), 133.9 (C(3)H),
132.7 (q), 124.8 (C(6)H), 123.6 (q), 121.1 (q), 119.3 (C(5)H), 105.3
(C(8)H), 104.3 (q), 102.2 (C(2)H), 57.5 (C(12)H2), 55.8 (OCH3),
45.5 (C(13)H3, C(14)H3), 41.2 (C(11)H2); m/z (ꢀES) 355 (100, [M
ꢀ H]ꢀ); (+ES) 357 (100, [M + H]+); 357.1562 ([M + H]+) found by
+ES, required 357.1557, error 1.3 ppm.
Synthesis of 1-(2-(Dimethylamino)ethylamino)-7-fluoro-4-ni-
troacridin-9(10H)-one (3b). N,N-Dimethylethylenediamine (1.09 mL,
10.0 mmol) was added to a suspension of 2b (1.47 g, 5.00 mmol) in
anhydrous DMF (5.0 mL), and the reaction mixture was heated to reflux for
4 h. Ethanol (10 mL) was added, and the resulting yellow precipitate was
collected by filtration and washed with ethanol (2.0 mL) to give 3b (1.43 g,
83%). Mp 236.5ꢀ237.0 ꢀC; νmax/cmꢀ1 3196m (NꢀH), 3053m, 2947m
(CꢀH), 1622s (CdO), 1602s; δH (500 MHz, CDCl3) 12.70 (1H, s br,
NH), 11.88 (1H, s br, NH), 8.46 (1H, d, J 9.8, C(3)H), 8.04 (1H, d, J 8.8,
C(5)H), 7.46ꢀ7.47 (2H, m, C(6)H, C(8)H), 6.38 (1H, d, J 9.8, C(2)H),
3.52 (2H, app q, 5.9, C(11)H2), 2.72 (2H, t, J 6.3, C(12)H2), 2.38 (6H, s,
C(13)H3, C(14)H3); δF (376 MHz, DMSO-d6) ꢀ117.81, ꢀ116.84 (m);
m/z (ꢀES) 343 (100, [M ꢀ H]ꢀ); (+ES) 367 (5, [M + Na]+), 345
(100, [M + H]+); 345.1364 ([M + H]+) found by +ES, required 345.1358,
error 1.8 ppm.
Synthesis of 1-(2-(Diethylamino)ethylamino)-7-hydroxy-
4-nitroacridin-9(10H)-one (3f). N,N-Diethylethylenediamine
(1.16 mL, 10.0 mmol) was added to a suspension of 2e (1.45 g,
5.00 mmol) in anhydrous DMF (5.0 mL), and the reaction mixture
was heated to reflux for 4 h. Ethanol (10 mL) was added and the
resulting yellow precipitate was was collected by filtration and washed
with ethanol (2.0 mL) to give 3f (1.68 g, 91%). Mp 218ꢀ220 ꢀC;
νmax/cmꢀ1 3216m (NꢀH), 2969m, 2808m (CꢀH), 1599s (CdO),
1548s; δH (300 MHz, DMSO-d6) 12.46 (1H, s, NH), 11.97 (1H,
s br, NH), 9.93 (1H, s, OH), 8.36 (1H, d, J 10.0, C(3)H), 7.85 (1H, d,
J 9.0, C(5)H), 7.55 (1H, d, J 2.7, C(8)H), 7.28 (1H, dd, J 9.0, 2.7,
C(6)H), 6.55 (1H, d, J 10.0, C(2)H), 3.50 (2H, app q, J 5.7,
Synthesis of 1-(2-(Dimethylamino)ethylamino)-4-nitroa-
cridin-9(10H)-one (3c). N,N-Dimethylethylenediamine (1.09 mL,
10.0 mmol) was added to a suspension of 2c (1.38 g, 5.00 mmol) in
anhydrous DMF (5.0 mL), and the reaction mixture was heated to reflux
for 4 h. Ethanol (10 mL) was added and the resulting yellow precipitate
6604
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
C(11)H2), 2.73 (2H, t, 5.7, C(12)H2), 2.58 (4 H, q, J 7.1, 2 ꢁ CH2),
1.02 (6H, t, J 7.1, 2 ꢁ CH3); δC (75 MHz, DMSO-d6) 178.2 (q),
157.5 (q), 154.2 (q), 139.3 (q), 133.3 (C(3)H), 131.5 (q), 129.5 (q),
123.9 (C(6)H), 120.6 (C(5)H), 120.1 (q), 107.8 (C(8)H), 103.2
(C(2)H), 102.9 (q), 50.5 (C(12)H2), 40.9 (C(11)H2), 46.3 (CH2),
11.7 (CH3); m/z (ꢀES) 369 (100, [M ꢀ H]ꢀ); (+ES) 393 (100,
[M + Na]+), 371 (50, [M + H]+); 371.1712 ([M + H]+) found by
+ES, required 371.1714, error 0.6 ppm.
8.17 (1H, d, J 7.8, C(8)H), 7.82 (1H, d, J 7.8, C(5)H), 7.73 (1H, app t,
J 7.8, C(6)H), 7.61 (1H, d, J 8.8, C(3)H), 7.29 (1H, app t, J 7.8, C(7)H),
6.51 (1H, d, J 8.8, C(2)H), 3.74 (2H, t, J 6.5, C(11)H2), 3.32 (2H, t,
J 6.5, C(12)H2), 2.82 ((6H, s, C(13)H3, C(14)H3); δC (75 MHz,
DMSO-d6) 179.5 (q), 150.6 (q), 140.0 (q), 136.8 (q), 133.7 (C(4)H),
130.2 (C(3)H), 125.5 (C(8)H), 122.0 (C(7)H), 120.8 (q), 117.0
(C(5)H), 106.9 (q), 105.5 (q), 99.4 (C(2)H), 54.3 (C(12)H2),
42.4 (C(13)H3, C(14)H3), 37.2 (C(11)H2); m/z (+ES) 297 (100,
[M + H]+); 297.1706 ([M + H]+) found by +ES, required 297.1710,
error 1.3 ppm.
Synthesis of 1-(2-(Dimethylamino)ethylamino)-4-amino-
7-bromoacridin-9(10H)-one Hydrochloride (4d). Raney Ni
(285 mg) was added to a suspension of 3d (0.770 g, 1.90 mmol) and
hydrazine monohydrate (0.34 mL) in THF (20 mL), and the reaction
mixture was stirred for 1 h. The resulting solution was filtered through
Celite, washed with THF (2.0 mL), and the filtrate was adjusted with
concentrated HCl to pH 3 and stirred for 30 min. The resulting brown
precipitate was collected by filtration and purified by recrystallization
from MeOHꢀdioxane (9:1) (acidified with HCl to ∼pH 2). This gave
4d (0.630 g, 88%) as a green powder. νmax/cmꢀ1 3301m (NꢀH),
3077m, 3024m, 2998sm, 2956m (CꢀH), 1649s (CdO), 1614m, 1598s,
1562s, 1548s, 1508s; δH (300 MHz, DMSO-d6) 12.09 (1H, s, NH),
10.64 (1H, s br, NH), 10.13 (3H, s br, NH3), 8.21 (1H, s, C(8)H),
7.89ꢀ7.80 (2H, m, C(5)H, C(6)H), 7.62 (1H, d, J 8.9, C(3)H), 6.53
(1H, d, J 8.9, C(2)H), 3.74 (2H, t, J 6.5, C(12)H2), 3.31 (2H, t, J 6.5,
C(11)H2), 2.83 (6H, s, C(13)H3, C(14)H3); δC (75 MHz, DMSO-d6)
178.2 (q), 150.5 (q), 138.9 (q), 136.5 (C(6)H), 136.2 (q), 130.4
(C(3)H), 127.4 (C(8)H), 122.1 (q), 119.5 (C(5)H), 114.0 (q), 106.9
(q), 105.8 (q), 99.9 (C(2)H), 54.2 (C(11)H2), 42.4 (C(13)H3,
C(14)H3), 37.2(C(12)H2); (+ES) 377 (100, [M + H]+, 81Br), 375
(100, [M + H]+, 79Br); 375.0805 ([M + H]+) found by +ES, required
375.0815, error 2.7 ppm.
Synthesis of 4-Amino-1-(2-(dimethylamino)ethylamino)-
7-hydroxyacridin-9(10H)-one Hydrochloride (4e). Raney Ni
(750 mg) was added to a suspension of 3e (1.07 g, 3.00 mmol) and
hydrazine monohydrate (0.6 mL) in THF (15 mL), and the reaction
mixture was stirred for 24 h. The resulting solution was filtered through
Celite, washed with THF (20 mL), and the filtrate was adjusted with
concentrated HCl to pH 3 and stirred for 30 min. The resulting brown
precipitate was collected by filtration and purified by recrystallization
from MeOHꢀdioxane (9:1) (acidified with HCl to ∼pH 2). This gave
4e (0.751 g, 72%) as a brown powder. Mp 230.8ꢀ231.4 ꢀC; νmax/cmꢀ1
3323m (NꢀH), 3025m, 2958m, 2819 (CꢀH), 1655s (CdO); δH (500
MHz, DMSO-d6) 11.49 (1H, s, NH), 10.54 (1H, s, OH), 10.34 (1H, s
br, NH), 10.21 (3H, s br, NH3), 9.73 (1H, s br, NH), 7.68 (1H, d, J 8.8,
C(5)H), 7.52ꢀ7.30 (2H, m, C(3)H, C(8)H), 7.29 (1H, dd, J 8.8, 2.8,
C(6)H), 6.39 (1H, d, J 8.8, C(2)H), 3.70 (2H, s br, C(11)H2), 3.33
(2H, s br, C(12)H2), 2.84 (6H, s, C(13)H3, C(14)H3); m/z (+ES) 313
(100, [M + H]+); 313.1648 ([M + H]+) found by +ES, required
313.1659, error 3.1 ppm.
Synthesis of 4-Amino-1-(2-(diethylamino)ethylamino)-7-
hydroxyacridin-9(10H)-one Hydrochloride (4f). Raney Ni (750
mg) was added to a suspension of 3f (1.07 g, 3.00 mmol) and hydrazine
monohydrate (0.6 mL) in THF (15 mL), and the reaction mixture was
stirred for 24 h. The solution was filtered through Celite, washed with
THF (2.0 mL), and the filtrate was adjusted with concentrated HCl to
pH 3 and stirred for 30 min. The resulting brown precipitate was collected
by filtration and purified by recrystallization from MeOHꢀdioxane (9:1)
(acidified with HCl to ∼pH 2). This gave 4f (0.645 g, 57%) as a brown
powder. Mp 337.5ꢀ339.5 ꢀC; νmax/cmꢀ1 3385m (OꢀH), 3212m
(NꢀH), 2967m (CꢀH), 1667s (CdO), 1641s, 1584s; δH (300 MHz,
DMSO-d6) 11.26 (1H, s, NH), 10.97 (1H, s, OH), 10.31 (1H, s br, NH),
7.81 (1H, d, J 8.9, C(5)H), 7.51 (1H, d, J 2.0, C(8)H), 7.22 (1H, m,
C(6)H), 7.06 (1H, d, J 8.2, C(3)H), 6.35 (1H, d, J 8.2, C(2)H), 4.10
Synthesis of 4-Amino-1-(2-(dimethylamino)ethylamino)-
7-methoxyacridin-9(10H)-one Hydrochloride (4a). Raney Ni
(750 mg) was added to a suspension of 3a (1.07 g, 3.00 mmol) and
hydrazine monohydrate (0.6 mL) in THF (15 mL), and the reaction
mixture was stirred for 1 h. The resulting solution was filtered through
Celite and washed with THF (2.0 mL). The filtrate was treated with
concentrated HCl to pH 3 and stirred for 30 min. The resulting brown
precipitate was filtered and purified by recrystallization from MeOHꢀ
dioxane (9:1) (acidified with HCl to ∼pH 2). This gave 4a (0.888 g,
81%) as a brown powder. Mp 279.0ꢀ280.0 ꢀC; νmax/cmꢀ1 3290m
(NꢀH), 3078m (CꢀH), 1644s (CdO), 1560s, 1534s, 1508s; δH (500
MHz, DMSO-d6) 11.91 (1H, s br, NH), 10.74 (1H, s br, NH), 10.33
(3H, s br, NH3), 7.80 (1H, d, J 9.2, C(5)H), 7.59 (1H, d, J 8.9, C(3)H),
7.56 (1H, d, J 2.9, C(8)H), 7.43 (1H, dd, J 9.2, 2.9, C(6)H), 6.45 (1H, d,
J 8.9, C(2)H), 3.84 (3H, s, OCH3), 3.73 (2H, t, J 6.7, C(11)H3), 3.32
(2H, t, J 6.7, C(12)H3), 2.83 (6H, s, C(13)H3, C(14)H3); δC (75 MHz,
DMSO-d6) 178.8 (q), 154.6 (q), 150.5 (q), 136.4 (q), 134.7 (q), 129.8
(C(3)H), 124.1 (C(6)H), 121.4 (q), 118.8 (C(5)H), 106.5 (q), 105.4
(q), 104.8 (C(8)H), 98.7 (C(2)H), 55.4 (OCH3), 54.3 (C(12)H3),
42.4 (C(13)H3, C(14)H3), 37.3 (C(11)H3); m/z (+ES) 327 (100,
[M + H]+); 327.1819 ([M + H]+) found by +ES, required 327.1816,
error 1.1 ppm.
Synthesis of 4-Amino-1-(2-(dimethylamino)ethylamino)-
7-fluoroacridin-9(10H)-one Hydrochloride (4b). Raney Ni
(750 mg) was added to a suspension of 3b (1.03 g, 3.00 mmol) and
hydrazine monohydrate (0.6 mL) in THF (15 mL), and the reaction
mixture was stirred for 1 h. The solution was filtered through Celite,
washed with THF (2.0 mL), and the filtrate was adjusted with concen-
trated HCl to pH 3 and stirred for 30 min. The resulting brown precipitate
was collected by filtration and recrystallized from MeOHꢀdioxane (9:1)
(acidified with HCl to ∼pH 2). This gave 4b (0.968 g, 91%) as a green
powder. Mp 259.0ꢀ260.0 ꢀC; νmax/cmꢀ1 3439m (NꢀH), 3192m,
2966m (CꢀH), 1647s (CdO); δH (500 MHz, DMSO-d6) 12.08 (1H, s
br, NH), 10.69 (1H, s br, NH), 10.18 (3H, s br, NH3), 7.92 (1H, dd,
J 8.8, 4.4, C(5)H), 7.80 (1H, dd, J 8.8, 2.2, C(8)H), 7.68 (1H, app dt,
J 8.8, 2.2, C(6)H), 7.62 (1H, d, J 8.8, C(3)H), 6.51 (1H, d, J 8.8, C(2)H),
3.74 (2H, t, J 6.5, C(11)H2), 3.31 (2H, t, J 6.5, C(12)H2), 2.82 (6H, s,
C(13)H3, C(14)H3); δC (125 MHz, DMSO-d6) 178.7 (q), 158.5
(d, J 236.6, C(7)F),150.6 (q), 136.8 (q), 136.5 (q), 130.6 (C(3)H),
122.6 (d, J 24.5, C(6)H), 121.5 (d, J 6.3, q), 119.6 (d, J 8.2, C(5)H),
109.5 (d, J 22.7, C(8)H), 106.5 (q), 105.4 (q), 99.5 (C(2)H), 54.2
(C(12)H2), 42.5 (C(13)H3, C(14)H3), 37.3 (C(11)H2); δF (376 MHz,
CDCl3) ꢀ118.91, ꢀ117.94 (m); m/z (+ES) 315 (100, [M + H]+);
315.1603 ([M + H]+) found by +ES, required 315.1616, error 4.0 ppm.
Synthesis of 4-Amino-1-(2-(dimethylamino)ethylamino)-
acridin-9(10H)-one Hydrochloride (4c). Raney Ni (750 mg) was
added to a suspension of 3c (0.980 g, 3.00 mmol) and hydrazine
monohydrate (0.6 mL) in THF (15 mL), and the reaction mixture was
stirred for 1 h. The resulting solution was filtered through Celite, washed
with THF (2.0 mL), and the filtrate was adjusted with concentrated HCl to
pH 3 and stirred for 30 min. The resulting brown precipitate was collected
by filtration and purified by recrystallization from MeOHꢀdioxane
(9:1) (acidified with HCl to ∼pH 2). This gave 4c (0.991 g, 99%) as a
brown powder. Mp 249.0ꢀ251.0 ꢀC; νmax/cmꢀ1 3396m (NꢀH),
3064m (CꢀH), 1640s (CdO), 1611s, 1599s; δH (500 MHz, DMSO-
d6) 11.86 (1H, s br, NH), 10.28 (1H, s br, NH), 10.00 (3H, s br, NH3),
6605
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
(3H, s br, NH3), 3.64 (2H, t, J 6.4, C(11)H2), 3.32 (2 H, t, J 6.4,
C(12)H2), 3.19 (4H, q, J 7.0, C(13)H2), 1.23 (6H, t, J 7.0, 2 ꢁ CH3); δC
(75 MHz, DMSO-d6) 178.9 (q), 158.5 (q), 152.1 (q), 133.9 (q), 132.7
(q), 123.8 (C(6)H), 121.8 (q), 119.3 (C(5)H), 117.3 (C(3)H), 108.0
(C(8)H), 107.6 (q), 102.9 (C(2)H), 102.7(q), 48.8 (C(12)H2), 46.8
(CH2), 30.7 (C(11)H2), 8.5 (CH3); m/z (ꢀES) 339 (100, [M ꢀ H]ꢀ);
(+ES) 341 (100, [M + H]+); 341.1982 ([M + H]+) found by +ES,
required 341.1973, error 2.8 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-methoxy-
6H-imidazo[4,5,1-de]acridin-6-one (5a1). 4a (182 mg, 0.50 mmol)
was heated under reflux in trimethyl orthoformate (2.0 mL) for 24 h. The
solvent was removed under vacuum and the solid residue was purified
by recrystallization from ethanol to give 5a1 (150 mg, 89%). Mp 219.0ꢀ
219.4 ꢀC; νmax/cmꢀ1 3264m (NꢀH), 3076m (CꢀH), 1656s (CdO),
1601, 1577s, 1535s; δH (300 MHz, CDCl3) 9.12 (1H, s br, NH), 8.51 (1H,
s, C(1)H), 8.01ꢀ7.97 (2H, m, C(3)H, C(7)H), 7.86 (1H, d, J 8.9,
C(10)H), 7.38 (1H, dd, J 8.9, 3.0, C(9)H), 6.76 (1H, d, J 9.0, C(4)H),
3.97 (3H, s, OCH3), 3.53 (2H, app q, J 6.0, C(12)H2), 2.74 (2H, t, J 6.4,
C(13)H2), 2.40 (6H, s, C(14)H3, C(15)H3); δC (75 MHz, CDCl3) 178.2
(q), 157.3 (q), 150.2 (q), 133.0 (C(1)H), 132.7 (q), 131.0 (q), 129.4
(C(3)H), 129.3 (q), 126.9 (q), 122.5 (C(9)H), 116.1 (C(10)H), 109.0
(C(7)H), 106.6 (C(4)H), 102.9 (q), 77.4 (q), 77.0 (q), 76.6 (q), 57.9
(C(13)H2), 55.9 (OCH3), 45.5 (C(14)H3, C(15)H3), 41.0 (C(12)H2);
m/z (+ES) 359 (40, [M + Na]+), 337 (100, [M + H]+); 337.1650
([M + H]+) found by +ES, required 337.1659, error 2.7 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-fluoro-6H-
imidazo[4,5,1-de]acridin-6-one (5b1). 4b (176 mg, 0.50 mmol)
was heated under reflux in trimethyl orthoformate (2.0 mL) for 24 h. The
solvent was removed under vacuum and the residue was purified by
recrystallization from ethanol to give 5b1 (150 mg, 68%). Mp 190.6ꢀ
191.8 ꢀC; νmax/cmꢀ1 3275m (NꢀH), 3074m, 2976m (CꢀH), 1657s
(CdO), 1605s, 1576s; δH (300 MHz, DMSO-d6) 9.20 (1H, s, C(1)H),
8.94 (1H, t, J 4.6, NH), 8.51 (1H, dd, J 8.9, 4.5, C(10)H), 8.04 (1H, dd,
J 9.1, 3.0, C(7)H), 8.01 (1H, d, J 9.1, C(3)H), 7.86 (1H, app dt, J 8.9, 3.0,
C(9)H), 6.83 (1H, d, 9.1, C(4)H), 3.45 (2H, app q, J 5.6, C(12)H2), 2.60
(2H, t, J 6.1, C(13)H2), 2.25 (6H, s, C(14)H3, C(15)H3); m/z (+ES) 347
(40, [M + Na]+), 325 (100, [M + H]+); 325.1459 ([M + H]+) found by
+ES, required 325.1459, error 0.0 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-6H-imidazo-
[4,5,1-de]acridin-6-one (5c1). 4c (167 mg, 0.50 mmol) was heated
under reflux in trimethyl orthoformate (2.0 mL) for 24 h. The solvent was
removed under vacuum and the residue was purified by recrystallization from
ethanol to give 5c1 (89 mg, 51%). Mp 215.8ꢀ216.6 ꢀC; νmax/cmꢀ1 3282m
(NꢀH), 3077m, 2971m, 2937m, 2863m, 2815, (CꢀH), 1660s (CdO),
1622, 1598s; δH (300 MHz, DMSO-d6) 9.23 (1H, s, C(1)H), 8.98 (1H, t,
J 4.5, NH), 8.38ꢀ8.44 (2H, m, C(7)H, C(10)H), 8.01 (1H, d, J 8.9,
C(3)H), 7.93 (1H, app t, J 7.7), 7.60 (1H, app t, J 7.6), 6.84 (1H, d, J 8.9,
C(4)H), 3.46 (2H, app q, J5.6, C(12)H2), 2.60(2H, t, J6.1, C(13)H2), 2.25
(6H, s, C(14)H3, C(15)H3); δC (125 MHz, DMSO-d6) 177.6 (q), 149.3
(C(1)H), 135.5 (q), 134.9 (q), 133.8 (CH), 132.5 (q), 130.6 (q), 129.2
(CH), 127.4 (C(7)H), 125.5 (CH), 124.9 (q), 116.3 (C(10)H), 107.0
(C(4)H), 102.4 (q), 57.4 (C(13)H2), 45.2 (C(14)H3, C(15)H3), 40.2
(C(12)H2); m/z (+ES) 329 (95, [M + Na]+), 307 (100, [M + H]+);
307.1547 ([M + H]+) found by +ES, required 307.1553, error 2.1 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-hydro-
xy-6H-imidazo[4,5,1-de]acridin-6-one (5e1). 4e (175 mg, 0.50
mmol) was heated under reflux in trimethyl orthoformate (2.0 mL) for
24 h. The solvent was removed under vacuum and the residue was
purified by recrystallization from ethanol to give 5e1 (155 mg, 96%). Mp
291.0ꢀ292.0 ꢀC; νmax/cmꢀ1 3354br (OꢀH), 3173m (NꢀH), 3072m,
2969m (CꢀH), 1668s (CdO), 1631s, 1586s, 1561s, 1530s; δH (300
MHz, DMSO-d6) 10.1 3 (1H, s br, OH), 9.12 (1H, s, C(1)H), 8.97 (1H,
t, J 4.9, NH), 8.28 (1H, d, J 9.0, C(10)H), 7.99 (1H, d, J 8.8, C(3)H),
7.72 (1H, d, J 2.9, C(7)H), 7.34 (1H, dd, J 9.0, 2.9, C(9)H), 6.82 (1H, d,
J 8.8, C(4)H), 3.45 (2H, app q, J 5.6, C(12)H2), 2.60 (2H, t, J 6.2,
C(13)H2), 2.25 (6H, s, C(14)H3, C(15)H3); δC (75 MHz, DMSO-d6)
177.6 (q), 155.8 (q), 148.8 (q), 142.1(q), 135.4 (C(1)H), 127.8 (q),
127.5 (q), 126.2 (q), 122.4 C(9)H), 120.7 (C(3)H), 117.9 (C(10)H),
111.3 (C(7)H), 107.6 (C(4)H), 102.2 (q), 54.6 (C(12)H2),
42.4 (C(13)H2), 30.7 (C(14)H3, C(15)H3); m/z (ꢀES) 321 (100,
[M ꢀ H]ꢀ); (+ES) 345 (20, [M + Na]+), 323 (100, [M + H]+);
323.1507 ([M + H]+) found by +ES, required 323.1503, error 1.4 ppm.
Synthesis of 5-(2-(Diethylamino)ethylamino)-8-hydroxy-
6H-imidazo[4,5,1-de]acridin-6-one (5f1). 4f (189 mg, 0.50 mmol)
was heated under reflux in trimethyl orthoformate (2.0 mL) for 24 h.
The solvent was removed under vacuum and the residue was purified by
recrystallization from ethanol to give 5f1 (80 mg, 46%). Mp 272.0ꢀ
274.0 ꢀC; νmax/cmꢀ1 3375br (OꢀH), 3275m (NꢀH), 3041m, 2951
(CꢀH), 1667s (CdO), 1587s, 1566s, 1530s; δH (300 MHz, DMSO-d6)
10.16 (1H, s br), 10.06 (1H, s br), 9.27 (1H, s, C(1)H), 8.95 (1H, s br,
NH), 8.31 (1H, d, J 8.7, C(10)H), 8.05 (1H, d, J 8.8, C(3)H), 7.75 (1H, d,
J 2.8, C(7)H), 7.38 (1H, dd, J 8.9 2.8, C(9)H), 7.02 (1H, d, J 8.9, C(4)H),
3.90 (2H, s br, C(12)H2), 3.35 (2H, app q, C(13)H2), 3.23 (4H, s br,
C(14)H2, C(16)H2), 1.23 (6H, t, J 7.2, C(15)H2, C(17)H2); δC (75 MHz,
DMSO-d6) 177.3 (q), 163.0 (q), 157.5 (q), 156.1 (q), 149.0 (q), 135.0
(C(1)H), 127.1 (q), 126.3 (q), 122.3 (C(9)H), 120.9 (C(3)H), 118.1
(C(10)H), 111.4 (C(7)H), 108.4 (C(4)H), 101.8 (q), 49.0 (C(13)H2),
46.5 (C(14)H2, C(16)H2), 37.3 (C(12)H2), 8.3 (CH3); m/z (ꢀES) 349
(100, [M ꢀ H]ꢀ); (+ES) 351 (30, [M + H]); 351.1807 ([M + H]+) found
by +ES, required 351.1816, error 2.6 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-methoxy-
6H-imidazo[4,5,1-de]acridin-6-one N-Oxide (6a1). m-CPBA
(69.2 mg, 0.2 mmol) was added to a suspension of 5a1 (33.6 mg, 0.10
mmol) in CHCl3 (3.0 mL), and the reaction mixture was stirred for 18
h at room temperature. The mixture was then passed through a column
of alumina, eluting sequentially with CHCl3 (400 mL) followed by
CHCl3/MeOH (9:1, 400 mL) to remove 5a1 and finally by CHCl3/
MeOH (3:1, 500 mL) to elute the target compound. The final fraction
was concentrated in vacuo to give 6a1 (32.0 mg, 91%). Mp
112.6ꢀ113.6 ꢀC; νmax/cmꢀ1 3245m (NꢀH), 3089m, 2957m
(CꢀH), 1654s (CdO), 1574s, 1532s; δH (500 MHz, DMSO-d6)
9.59 (1H, s br, NH), 9.15 (1H, s, C(1)H), 8.36 (1H, d, J 8.5, C(10)H),
7.98 (1H, d, J 8.5, C(2)H), 7.76 (1H, s, C(7)H), 7.53 (1H, d, J 8.5,
C(9)H), 6.93 (1H, d, J 8.5, C(3)H), 3.94ꢀ3.92 (5H, m, CH3,
C(13)H2), 3.55 (2H, s br, C(12)H2), 3.15 (6H, s, 2 ꢁ CH3); m/z
(+ES) 375 (100, [M + Na]+), 353 (10, [M + H]+); 375.1437 ([M +
Na]+) found by +ES, required 375.1428, error 2.5 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-fluoro-
6H-imidazo[4,5,1-de]acridin-6-one N-Oxide (6b1). m-CPBA
(69.2 mg, 0.2 mmol) was added to a suspension of 5b1 (32.4 mg,
0.10 mmol) in CHCl3 (3.0 mL), and the reaction mixture was stirred at
room temperature for 18 h. The mixture was then passed through a
column of alumina, eluting sequentially with CHCl3 (400 mL), CHCl3/
MeOH (9:1, 400 mL) to remove 5b1, and finally CHCl3/MeOH (3:1,
500 mL) to elute the target compound. The final fraction was concen-
trated in vacuo to give 6b1 (29.0 mg, 85%). Mp 116ꢀ118 ꢀC; νmax/cmꢀ1
3237m (NꢀH), 3095m (CꢀH), 1652s (CdO), 1575s, 1527s; δH (300
MHz, DMSO-d6) 9.49 (1H, J 5.5, NH), 9.22 (1H, s, C(1)H), 8.53 (1H,
dd, J 8.8, 3.7, C(10)H), 8.03 (2H, m, C(3)H, C(7)H), 7.88 (1H, app dt, J
8.8, 3.0, C(9)H), 6.99 (1H, d, J 9.0, C(4)H), 3.93 (2H, app q, J 5.6,
C(12)H2), 3.54 (2H, t, J 6.0, C(13)H2), 3.12 (6H, s, C(14)H2, C-
(15)H2); m/z (+ES) 363 (100, [M + Na]+), 341 (40, [M + H]+);
363.1232 ([M + Na]+) found by +ES, required 363.1228, error 1.2 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-6H-imidazo-
[4,5,1-de]acridin-6-one N-Oxide (6c1). m-CPBA (69.2 mg, 0.2
mmol) was added to a suspension of 5c1 (30.6 mg, 0.10 mmol) inCHCl3
(3.0 mL), and the reaction mixture was stirred at room temperature for 18 h.
The mixture was then passed through a column of alumina, eluting
6606
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
sequentially with CHCl3 (400 mL), CHCl3/MeOH (9:1, 400 mL) to
remove 5c1, and finally CHCl3/MeOH (3:1, 500 mL) to elute the tar-
get compound. The final fraction was concentrated in vacuo to give 6c1 (30
mg, 93%). Mp 110.5ꢀ111.5 ꢀC; νmax/cmꢀ1 3242m (NꢀH), 3072m,
2959m (CꢀH), 1646s (CdO), 1579s; δH (300 MHz, DMSO-d6) 9.60
3314m (NꢀH), 2990m, 2964m, 2905m (CꢀH), 1652s (CdO), 1602s,
1582s, 1569s; δH (300 MHz, DMSO-d6) 9.01 (1H, t, J 4.9, NH), 8.47 (1H,
dd, J 7.9 1.7, C(7)H), 8.27 (1H, d, J 8.5, C(10)H), 7.92 (1H, app dt, 7.8, 1.7,
C(9)H), 7.88 (1H, d, J 8.8, C(3)H), 7.60 (1H, app t, J 7.6, C(8)H), 6.77
(1H, d, J 8.8, C(4)H), 3.43 (2H, app q, J 5.7, C(13)H2), 3.08 (3H, s,
C(11)H3), 2.60 (2H, t, J 6.0, C(14)H2), 2.25 (6H, s, C(15)H3, C(16)H3);
δC (75 MHz, DMSO-d6) 177.6 (q), 148.2 (q), 147.0 (q), 136.1 (C(9)H),
133.8 (C(10)H), 129.4 (q), 127.8 (q), 127.6 (q), 125.2 (C(8)H), 124.9
(q), 117.0 (C(7)H), 111.1 (C(3)H), 105.8 (C(4)H), 102.8 (q),
56.2 (C(14)H2), 44.0 (C(15)H3, C(16)H3), 43.9 (C(13)H2), 18.5
(C(11)H3); m/z (+ES) 343 (50, [M + Na]+), 321 (100, [M + H]+);
343.1536 ([M + H]+) found by +ES, required 343.1529, error 1.9 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-bromo-
1-methyl-6H-imidazo[4,5,1-de]acridin-6-one (5d2). 4d (94 mg,
0.25 mmol) was heated under reflux in trimethyl orthoacetate (2.0 mL)
for 24 h. The solvent was removed under vacuum and the residue was
purified by recrystallization from ethanol to give 5d2 (45 mg, 45%).
Mp 176.0ꢀ177.0 ꢀC; νmax/cmꢀ1 3274m (NꢀH), 3113m, 3056w, 2968w
(CꢀH), 1690s (CdO), 1651m, 1618s, 1596s; δH (300 MHz, CDCl3)
9.09 (1H, s br, NH), 8.77 (1H, d, J 2.4, C(7)H), 7.99 (1H, d, J 8.9),
7.90ꢀ7.86 (2 H, m), 6.73 (1H, d, J 8.7), 3.58 (2H, app q, J 5.3, C(13)H2),
3.11 (3H, s, C(11)H3), 2.80(2H, t, J5.4, C(14)H2), 2.45(6H, s, C(15)H3,
C(16)H3); m/z (+ES) 423 (70, [M + Na]+, 81Br), 421 (70, [M + Na]+,
79Br), 401 (100, [M + H]+, 81Br), 399 (100, [M + H]+, 79Br); 399.0825
([M + H]+) found by +ES, required 399.0815, error 2.5 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-hydroxy-
1-methyl-6H-imidazo[4,5,1-de]acridin-6-one (5e2). 4e (182 mg,
0.50 mmol) was heated under reflux in trimethyl orthoacetate (2.0 mL)
for 24 h. The solvent was removed under vacuum and the residue was
purified by recrystallization from ethanol to give 5e2 (67 mg, 40%). Mp
295.5ꢀ298.0 ꢀC; νmax/cmꢀ1 3289m (NꢀH), 2967m, 2930m, 2861m
(CꢀH), 1651s (CdO), 1604s, 1582s, 1536s, 1520s; δH (300 MHz,
DMSO-d6) 10.17 (1H, s br, NH), 10.01 (1H, s br, OH), 8.97 (1H, s br,
NH), 8.16 (1H, d, J 9.2, C(10)H), 7.93 (1H, d, J 8.7, C(3)H), 7.82 (1H,
d, J 2.9, C(7)H), 7.38 (1H, dd, J 9.2, 2.9, C(9)H), 6.92 (1H, d, J 8.7,
C(4)H), 3.86 (2H, app q, J 6.5, C(12)H2), 3.06 (3H, s, CH3), 2.50
(obscured, C(13)H2), 2.87 (6 H, d, J 4.3, 2 ꢁ CH3); m/z (ꢀES) 335
(100, [M ꢀ H]ꢀ); (+ES) 337 (20, [M + H]+); 337.1652 ([M + H]+)
found by +ES, required 337.1660, error 2.2 ppm.
Synthesis of 5-(2-(Diethylamino)ethylamino)-8-hydroxy-
1-methyl-6H-imidazo[4,5,1-de]acridin-6-one (5f2). 4f (189 mg,
0.50 mmol) was heated under reflux in trimethyl orthoacetate (2.0 mL)
for 24 h. The solvent was removed under vacuum and the residue was
purified by recrystallization from ethanol to give 5f2 (96 mg, 52%). Mp
254ꢀ255 ꢀC; νmax/cmꢀ1 3290m (NꢀH), 2967m, 2929m, 2865m
(CꢀH), 1651s (CdO), 1604s, 1582s, 1536s, 1520s; δH (500 MHz,
DMSO-d6) 10.02 (1H, s, NH), 9.04 (1H, s br, NH), 8.13 (1H, d,
J 9.1, C(10)H), 7.85 (1H, d, J 8.6, C(3)H), 7.81 (1H, d, J 2.9, C(7)H),
7.34 (1H, dd, J 9.1, 2.9, C(9)H), 6.73 (1H, d, J 8.6, C(4)H), 3.40
(obscured, C(13)H2), 3.03 (3H, s, C(11)H3), 2.74 (2H, t, J 6.0, C-
(14)H2), 2.58 (4H, q, J 7.2, 2 ꢁ CH2), 1.03 (6H, t, J 7.2, 2 ꢁ CH3); δC
(75 MHz, DMSO-d6) 177.2 (q), 154.6 (q), 148.6 (q), 146.1 (q), 133.8
(q), 129.2 (q), 128.9 (q), 127.6 (C(3)H), 126.9 (q), 121.7 (C(9)H),
118.4 (C(10)H), 111.8 (C(7)H), 105.5 (C(4)H), 102.1 (q), 50.8
(C(14)H2), 46.4 (CH2), 40.5 (C(13)H2), 18.3 (C(11)H3), 11.8
(CH3); m/z (ꢀES) 363 (100, [M ꢀ H]ꢀ; (+ES) 365 (40, [M + H]+);
365.1973 ([M + H]+) found by +ES, required 365.1972, error 0.3 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-methoxy-
1-methyl-6H-imidazo[4,5,1-de]acridin-6-one N-Oxide (6a2). m-
CPBA (69 mg, 0.2 mmol) was added to a suspension of 5a2 (35.0 mg,
0.10 mmol) in CHCl3 (3.0 mL), and the reaction mixture was stirred at
room temperature for 18 h. The mixture was then passed through a
column of alumina, eluting sequentially with CHCl3 (400 mL), CHCl3/
MeOH (9:1, 400 mL) to remove 5a2, and finally CHCl3/MeOH
(1H,
s
br, NH), 9.25 (1H, s, C(1)H), 8.19 (1H, d,
J 8.5, C(10)H), 7.97 (1H, d, J 8.4, C(2)H), 7.85 (1H, s, C(7)H),
7.63 (1H, app t, J 8.3, C(9)H), 7.54 (1H, app t, J 8.3, C(8)H), 6.93 (1H,
d, J 8.4, C(3)H), 3.90ꢀ3.86 (5H, m, CH3, C(13)H2), 3.55 (2H, s br,
C(12)H2), 3.15 (6H, s, 2 ꢁ CH3); m/z (ꢀES) 321 (40, [M ꢀ H]ꢀ);
(+ES) 345 (100, [M + Na]+), 323 (100, [M + H]+); 345.1322
([M + Na]+) found by +ES, required 345.1322, error 0.0 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-hydro-
xy-6H-imidazo[4,5,1-de]acridin-6-one N-Oxide (6e1). m-CPBA
(35.0 mg, 0.1 mmol) was added to a suspension of 5e1 (16.1 mg,
0.05 mmol) in CHCl3 (2.0 mL), and the reaction mixture was stirred at
room temperature for 18 h. The mixture was then passed through a
column of alumina, eluting sequentially with CHCl3 (400 mL), CHCl3/
MeOH (9:1, 400 mL) to remove 5e1, and finally CHCl3/MeOH (3:1,
1.0 L) to elute the target compound. The final fraction was concentrated
invacuo togive a pale yellow powderthat was dissolved inMeOH (3 mL)
and filtered to remove reidual silica. The solvent was removed to give 6e1
(5.0 mg, 30%). δH (500 MHz, DMSO-d6) 10.56 (1H, s br, OH), 9.35
(1H, s br, NH), 9.00 (1H, s, C(1)H), 8.14 (1H, d, J 8.7, C(3)H), 7.81
(1H, d, J 8.9, C(10)H), 7.65 (1H, d, J 2.5, C(7)H), 7.28 (1H, dd, J 8.9,
2.5, C(9)H), 6.76 (1H, d, J 8.7, C(4)H), 3.89 (2H, app q, J 5.6,
C(12)H2), 3.73 (2H, s br, C(13)H2), 3.26 (6H, s, C(14)H3, C(15)H3);
m/z (ꢀES) 337 (70, [M ꢀ H]ꢀ); 337.1302 ([M ꢀ H]ꢀ) found by ꢀES,
required 337.1301, error 0.1 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-methoxy-1-
methyl-6H-imidazo[4,5,1-de]acridin-6-one (5a2). 4a (182 mg, 0.50
mmol) was heated under reflux in trimethyl orthoacetate (2.0 mL) for 24 h.
The solvent was removed under vacuum and the residue was purified by
recrystallization from ethanol to give 5a2 (170 mg, 97%). Mp 205.0ꢀ
206.0 ꢀC; νmax/cmꢀ1 3270w (NꢀH), 3002w, 2975w, 2946w, 2885w,
2848m, 2811m, 2760m (CꢀH), 1756s (CdO), 1652s, 1608s; δH (300
MHz, CDCl3) 9.14 (1H, s br, NH), 8.09 (1H, d, J 3.0, C(7)H), 8.04 (1H, d, J
9.1, C(10)H), 7.89 (1H, d, J 8.8, C(3)H), 7.38 (1H, dd, J 9.1, 3.0, C(9)H),
6.71 (1H, d, J 8.8, C(4)H), 3.99 (3H, s, OC(17)H3), 3.55 (2H, app q,
C(13)H2), 3.12 (3H, s br, C(11)H3), 2.78 (2H, t, C(14)H2), 2.42 (6H, s,
C(15)H3, C(16)H3); δC (75 MHz, CDCl3) 178.2 (q), 156.7 (q), 149.5 (q),
145.8 (q), 134.5 (q), 130.9 (q), 129.4 (q), 128.1 (C(3)H), 127.6 (q), 122.0
(C(9)H), 117.3 (C(10)H), 109.4 (C(7)H), 105.6 (C(4)H), 103.1 (q), 58.0
(C(14)H2),55.8(OC(17)H3), 45.6 (C(15)H3,C(16)H3),41.0(C(13)H2),
19.0 (C(11)H3); m/z (+ES) 373 (40, [M + Na]+), 351 (100, [M + H]+);
351.1811 ([M + H]+) found by +ES, required 351.1816, error 1.3 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-fluoro-
1-methyl-6H-imidazo[4,5,1-de]acridin-6-one (5b2). 4b (176 mg,
0.50 mmol) was heated under reflux in trimethyl orthoacetate (2.0 mL) for
24 h. The solvent was removed under vacuum and the residue was purified
by recrystallization from ethanol to give 5b2 (150 mg, 89%).
Mp 189.2ꢀ198.0 ꢀC; νmax/cmꢀ1 3265m (NꢀH), 3037m, 2968m,
2935m (CꢀH), 1652s (CdO), 1604s, 1581s, 1540s, 1525s; δH (300
MHz, CDCl3) 9.09 (1H, s br, NH), 8.31 (1H, dd, J 9.0, 3.2, C(7)H), 8.11
(1H, dd, J 9.3, 4.3, C(10)H), 7.91 (1H, d, J 8.9, C(3)H), 7.53 (1H, app dt,
J 8.1 3.2, C(9)H), 6.76 (1H, d, J 8.9, C(4)H), 3.62 (2H, s br, C(13)H2),
3.13 (3H, s, C(11)H3), 2.84 (2 H, s br, C(14)H2), 2.48 (6H, s, C(15)H3,
C(16)H3); m/z (+ES) 361 (20, [M + Na]+), 339 (100, [M + Na]+);
339.1616 ([M + H]+) found by +ES, required 339.1616, error 0.1 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-1-methyl-6H-
imidazo[4,5,1-de]acridin-6-one (5c2). 4c (182 mg, 0.50 mmol) was
heated under reflux in trimethyl orthoacetate (2.0 mL) for 24 h. The solvent
was removed under vacuum and the residue was purified by recrystallization
from ethanol to give 5c2 (48 mg, 28%). Mp 258.0 ꢀ 260.0 ꢀC; νmax/cmꢀ1
6607
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
(3:1, 500 mL) to elute the target compound. The final fraction was
concentrated in vacuo to give 6a2 (25.0 mg, 69%). Mp 117.6ꢀ120.0 ꢀC;
νmax/cmꢀ1 3245m (NꢀH), 3089m, 2957m (CꢀH), 1654s (CdO),
1574s, 1532s; δH (500 MHz, DMSO-d6) 9.60 (1H, s br, NH), 8.36 (1H,
d, J 8.5, C(10)H), 7.98 (1H, d, J 8.5, C(2)H), 7.76 (1H, s, C(7)H), 7.38
(1H, d, J 8.5, C(9)H), 6.93 (1H, d, J 8.5, C(3)H), 3.94ꢀ3.92 (5H, m,
CH3, C(13)H2), 3.55 (2H, s br, C(12)H2), 3.15 (6H, s, 2 ꢁ CH3), 2.40
(3H, s, CH3); m/z (ꢀES) 365 (60, [M ꢀ H]ꢀ); (+ES) 389 (20,
[M + Na]+), 367 (10, [M + H]+; 365.1610 ([M ꢀ H]ꢀ) found by ꢀES,
required 365.1608, error 0.2 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-fluoro-
1-methyl-6H-imidazo[4,5,1-de]acridin-6-one N-Oxide (6b2).
m-CPBA (69.2 mg, 0.2 mmol) was added to a suspension of 5b2 (33.8
mg, 0.10 mmol) in CHCl3 (3.0 mL), and the reaction mixture was stirred
at room temperature for 18 h. The mixture was then passed through a
column of alumina, eluting sequentially with CHCl3 (400 mL), CHCl3/
MeOH (9:1, 400 mL) to remove 5b2, and finally CHCl3/MeOH (3:1,
500 mL) to elute the target compound. The final fraction was concen-
appropriate ionization at physiological pH. The protonated complex was
then minimized within Sybyl 7.3 (Tripos Ltd., St. Louis, MO) while
holding all heavy atoms stationary. Inspection of the active site showed a
reasonable internal hydrogen bond network within the protein.
For preparation of ligand structures, fragments from Sybyl 7.3 were
used to construct the compounds. Ligands were subject to 1000 itera-
tions of energy minimization using the steepest descent algorithm by
means of the Tripos force field. For computational docking, the GOLD
4.1 software was used in combination with the ChemScore scoring
function. GOLD uses a genetic algorithm (GA), whereby the molecular
features of a proteinꢀligand complex are encoded as a chromosome.
Since GOLD’s internal definition of “lipophilic atoms” are nonaccepting
sulfurs, nonpolar carbon atoms, and nonionic Cl, Br, or I, adoption of the
default setup seemed likely to underestimate the aromatic/lipophilic
nature of the receptor in the immediate vicinity of the isoalloxazine
fragment of FAD, consequently causing a systematically low prediction
of binding affinities for known active aromatic ligands. To avoid this
probability, we re-atom-typed the FAD fragment as previously described.23
The active site was defined as being any volume within 15 Å of N5 of
the FAD cofactor. Each GA run comprised 100 000 genetic operations
on an initial population of 100 members divided into five subpopula-
tions. Operator weights for crossover, mutation, and migration were set
to 95, 95, and 10, respectively. GOLD allows a user-definable number of
GA runs per ligand, each of which starts from a different orientation. For
these experiments, the number of GA runs was set to 10, and scoring of
the docked poses was performed with the ChemScore scoring function.
The top 10 solutions for each ligand were retained and analyzed for
favorable interactions within the active site of NQO2, including low
proteinꢀligand clash, ligand distortion energies, lipophilic contacts, and
hydrogen bonding interactions.
The 5a1 crystal structure was prepared as described above for 1QR2.
The active site for the crystal complex was defined as being any volume
within 15 Å of the 5a1 crystal ligand. The ligands were docked using the
same parameters as for the preceding experiments. The top 25 poses for
each ligand were retained and compared to the 5a1 crystal ligand. In
addition, each solution was analyzed within the active site of the enzyme
as previously discussed.
Protein Expression and Purification. The overexpression of
NQO2 was performed in BL21 codon+ cells induced with 1 mM
isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 20 ꢀC.
The protein was purified to homogeneity by HisTrap HP Ni2+ affinity
chromatography followed by size exclusion chromatography on a Super-
dex S200 10/300 GL column. Samples were concentrated to 15 mg/mL
and supplemented with 5 μM FAD prior to crystallization experiments.
Crystallization, Refinement, and Model Building. Crystals of
NQO2 ligand complex were achieved by cocrystallization in the presence
of both 5a1 and 6a1. Conditions were identified using the PACT premier
matrix screen (Molecular Dimensions) on a Mosquito nanodrop crystal-
lization robot. Crystals suitable for diffraction experiments were obtained
by sitting drop vapor diffusion in 4 μL drops containing equal volumes of
protein and a solution containing 0.2 M sodium formate and 20%
polyethylene glycol 3350. Data were collected on beamline I04 at the
Diamond Light Source Facility and reduced and scaled with the X-ray
Detector Software suite (XDS).24
trated in vacuo to give 6b2 (33.0 mg, 93%). Mp 130.5ꢀ132.5 ꢀC; νmax
/
cmꢀ1 3247m (NꢀH), 3095m, 2976m (CꢀH), 1651s (CdO); δH (300
MHz, DMSO-d6) 9.49 (1H, J 5.5, NH), 8.53 (1H, dd, J 8.4, 3.7,
C(10)H), 7.99 (2H, m, C(3)H, C(7)H), 7.78 (1H, app dt, J 8.4, 3.0,
C(9)H), 6.99 (1H, d, J 9.0, C(4)H), 3.93 (2H, app q, J 5.6, C(12)H2),
3.54 (2H, t, J 6.0, C(13)H2), 3.12 (6 H, s, C(14)H2, C(15)H2) 2.41 (3H,
s, CH3); m/z (+ES) 377 (60, [M + Na]+), 355 (55, [M + H]+);
377.1378 ([M + Na]+) found by +ES, required 377.1384, error 1.7 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-1-methyl-
6H-imidazo[4,5,1-de]acridin-6-one N-Oxide (6c2). m-CPBA
(69.2 mg, 0.2 mmol) was added to a suspension of 5c2 (34.2 mg, 0.10
mmol) in CHCl3 (3.0 mL), and the reaction mixture was stirred for 18 h.
The mixture was then passed through a column of alumina, eluting
sequentially with CHCl3 (400 mL), CHCl3/MeOH (9:1, 400 mL) to
remove 5c2, and finally CHCl3/MeOH (3:1, 500 mL) to elute the target
compound. The final fraction was concentrated in vacuo to give 6c2
(32.0 mg, 95%). Mp 125.0ꢀ127.8 ꢀC; νmax/cmꢀ1 3199m (NꢀH),
3098m, 2960m (CꢀH), 1646s (CdO); δH (300 MHz, DMSO-d6) 9.01
(1H, t, J 4.9, NH), 8.47 (1H, dd, J 7.9, 2.0, C(7)H), 8.27 (1H, d, J 8.5,
C(10)H), 7.92 (1H, app dt, J 7.8. 2.0, C(9)H), 7.88 (1H, d, J 8.8,
C(3)H), 7.60 (1H, app t, J 7.3, C(8)H), 6.77 (1H, d, J 8.8, C(4)H),
3.94ꢀ3.92 (5H, m, CH3, C(13)H2), 3.55 (2 H, s br, C(12)H2), 3.15
(6H, s, 2 ꢁ CH3), 3.08 (3H, s, CH3); m/z (+ES) 359 (100, [M + Na]+),
337 (90, [M + H]+); 337.1655 ([M + H]+) found by +ES, required
337.1660, error 1.3 ppm.
Synthesis of 5-(2-(Dimethylamino)ethylamino)-8-bromo-
1-methyl-6H-imidazo[4,5,1-de]acridin-6-one N-Oxide (6d2).
m-CPBA (35 mg, 0.1 mmol) was added to a suspension of 5d2 (22 mg,
0.055 mmol) in CHCl3 (3.0 mL), and the reaction mixture was stirred
for 18 h. The mixture was then passed through a column of alumina,
eluting sequentially with CHCl3 (400 mL), CHCl3/MeOH (9:1,
400 mL) to remove 5d2, and finally CHCl3/MeOH (3:1, 500 mL) to
elute the target compound. The final fraction was concentrated in
vacuo to give 6d2 (16.0 mg, 70%). Mp 127.0ꢀ129.0 ꢀC; νmax/cmꢀ1
3259m (NꢀH), 3039m, 2977m, (CꢀH), 1656s (CdO); δH (300
MHz, DMSO-d6) 9.52 (1H, s br, NH), 8.43 (1H, s br, C(7)H), 8.19
(1H, d, J 9.0, C(10)H), 8.05 (1 H, dd, J 9.0, 1.7, C(9)H), 7.87 (1H, d,
J 8.3, C(3)H), 6.88 (1H, d, J 8.3, C(4)H), 3.89 (2 H, app q, J 5.7,
C(12)H2), 3.52 (2H, s br, C(13)H2), 3.13 (6H, s, C(14)H2, C-
(15)H2), 3.04 (3H, s, CH3); m/z (+ES) 439 (100, [M + Na]+, 81Br),
437 (100, [M + Na]+, 79Br); 415.0764 ([M + H]+) found by +ES,
required 415.0765, error 0.1 ppm.
The NQO2ꢀ5a1 and NQO2ꢀ6a1 crystal structures were deter-
mined by molecular replacement using PHASER from PHENIX25 and
the human NQO2 structure (1QR2)21 as a starting model. Structures
were completed by iterative cycles of manual model building and real
space refinement using the program Coot26 and crystallographic refine-
ment using phenix.refine. Structure validation was preformed with
Molprobity. The processing and final refinement statistics are presented
in Table 3.
Molecular Modeling. For docking purposes, the crystallographic
coordinates of human NQO2 (PDB code 1QR2, resolution 2 Å)21 was
obtained from the Brookhaven database. Hydrogen atoms were added
to the structure, which included the protein and FAD, allowing for
Cell Lines. The breast cancer cell lines MDA-MB-468, BT474,
MCF-7, SKBr3, BT20, MDA231, and T47D were chosen because they
6608
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
Table 3. Crystallographic Data and Refinement Statistics
bicinchoninic acid protein assay. The assay for enzyme activity was
determined by adding 10 μL of cell lysate to 490 μL of the reaction
mixture described above. The difference in the rate of reaction in the
presence and absence of 1 mM resveratrol was used to define the NQO2
activity in the cellular lysates.
NQ02ꢀ6A1
NQ02ꢀ5A1
P212121
38.8ꢀ1.7
(1.6ꢀ1.7)
space group
P212121
resolution (Å)
28.2ꢀ1.45
(1.51ꢀ1.45)
Assays of Toxicity. Cells were seeded at 2.5 ꢁ 103 (HCT116) and
between 4.0 ꢁ 103 and 10 ꢁ 103 (breast cancer cells) into 96-well
plates, left overnight to attach, then exposed to the various imidazoa-
cridin-6-ones in full growth medium for 24 or 96 h. For the shorter
exposure time, drug was removed and replaced with fresh medium and
cells were allowed to grow for a total of 96 h. The number of surviving
cells was then determined using the MTT assay.23 Briefly, MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
dissolved in sterile PBS and added to the wells at a final concentration
of 1.5 mM. Cells were incubated with MTT for 4 h, medium was
removed, and the remaining formazan crystals were dissolved in
DMSO. Absorbance at 540 nm was then measured using a multiwell
scanning spectrophotometer. Data were analyzed and curves drawn
using the GraphPad Prism5 software package. Values of IC50 were
calculated as the concentration of compound required to reduce optical
density by 50% relative to vehicle treated control cells. Since optical
density is directly proportional to cell density, these values of IC50 can
be regarded as the concentration of compound(s) required to reduce
proliferation by 50%. All toxicity experiments were repeated on at least
three separate occasions. To determine the potency at which the
imidazoacridin-6-ones 5a1 and 6a1 could inhibit the cellular activity
of NQO2, cells were treated for 3 h with the putative inhibitors together
with varying concentration of 7 plus 100 μM NRH. The cells were then
washed with PBS, and fresh medium was added. The plates were
incubated for a total of 96 h, and the MTT assay was used to determine
cell viability.
unit cell a, b, c (Å)
redundancy
56.7, 83.5, 106.2 56.9, 84.0, 106.55
3.7 (3.7)
277 321
74 838
3.7 (3.3)
410 461
111 557
95.0 (91.7)
5.1 (50.6)
16.3 (3.28)
16.95
total reflections
unique reflections
completeness (%)
Rsym a (%)
99.1 (98.3)
5.7 (60.0)
14.9 (2.76)
14.9
ÆI/σIæ
Rworkb (%)
Rfree c (%)
16.5
20.6
Wilson B-factor (Å2)
Ramachandran plot (%)
favorable
14.37
18.4
95.9
3.88
0
96.9
2.88
0
allowed
generous
disallowed
0.22
0.22
rms deviations from ideal geometry
bond length (Å)
bond angle (deg)
0.012
1.496
0.007
1.163
a Rsym = ∑h∑i/Ih,i ꢀ Ih/∑h∑iIhi, where Ih is the mean intensity of the
observations (i) of symmetry related reflections (h). b Rwork = ∑hkl1/2Fo-
(hkl) ꢀ Fc(hkl)1/2/∑hkl1/2Fo(hkl), where Fo and Fc are observed and
calculated structure factors, respectively. c For the calculation of Rfree, 5%
of reflections were chosen at random to constitute a test set.
Immunofluorescent Staining of Cells. MDA-MB-468 cells
were grown on 13 mm coverslips in 6 cm Petri dishes. When cells
had reached approximately 70% confluency, they were treated
with varying concentrations of 5a1 or 6a1 for 3 h. Cells were then
fixed on the coverslips with 3.6% formaldehyde plus 0.2% glutaralde-
hyde in PBS for 10 min. The coverslips were washed with 2ꢀ3 mL of
PBS and the cells permeablized with 0.1% Triton-X-100 (1 mL) for
10 min. The Triton-X-100 was removed and the cells were washed twice
with PBS before being immersed in a blocking agent (10% BSA in PBS)
for 30 min at room temperature. Separately, Alexa Fluor 568 phalloidin
antibody (Invitrogen, A12380) was diluted in 1% BSA solution to give
a dilution of 1:1000. Then 200 μL of this solution was placed on a sec-
tion of Parafilm onto which coverslips were inverted. The cells were
incubated for at least 20 min at room temperature, after which the
coverslips were washed 3 times with PBS. The coverslips were mounted
on poly-L-lysine microscope slides and stored at 4 ꢀC in the absence
of light until ready for viewing. Phalloidin was visualized at 568 nm,
whereas the imidazoacridin-6-ones were excited at 340 nm with emis-
sion at 520 nm.
Measurement of Melting Temperature. DNA stability was
studied by establishing the melting temperature of calf thymus DNA
(20 μg/mL) in the presence of 10 μM of the imidazoacridin-6-ones in
low salt phosphate buffer (7.5 mM NaH2PO4, 1 mM ethylenediamine-
tetraacetic acid, pH 7.0). Cuvettes were heated over the range
37ꢀ98 ꢀC, and absorbance at 260 nm was read at every degree, using
a PerkinꢀElmer Lambda 25 UV/vis spectrophotometer. Temperature
was controlled by a Perkin-Elmer Peltier temperature programmer.
Relative absorbance was plotted using Origin 8.0 software, and curve
fitting was done using the sigmoidal function which gave a Boltzmann
distribution and regression analysis of the data. Values of Tm were read
from the midpoint of the hypochromic transition curves. Values of Tm
were derived from at least three independent determinations.
express varying levels of NQO2 (Table2). HCT116 cells were used, as
these were the cells used in previous work in our laboratory,12,13 thus
allowing us to make toxicity comparisons with published work. The cell
lines were cultured and maintained in exponential phase in RPMI-1640
medium supplemented with 10% (v/v) heat-inactivated fetal calf serum
and 2 mM L-glutamine. The cellswere maintained at 37 ꢀC ina humidified
incubator in an atmosphere of air plus 5% CO2.
Assay of NQO2 Activity. Recombinant human NQO2 was diluted
in 50 mM phosphate buffer to give an enzyme activity that would result
in a change in optical absorbance of substrate of approximately 0.1 per
minute. The enzyme reaction was started by adding 5 μL of this solution
to 495 μL of 50 mM phosphate buffer at pH 7.4 containing 40 μM
dichlorophenolindophenol (DCPIP) and 200 μM NRH (synthesized
in house), together with various concentrations of the potential inhibitor
dissolved in DMSO (final concentration 1.0% v/v). The DMSO
concentration used is sufficiently small to ensure minimal perturbation
of hydrogen bonding networks in aqueous NQO2 complexes. Assays
were carried out in the presence or absence of 2 μM BSA. Reactions were
carried out at 37 ꢀC, and reduction of DCPIP was monitored at 600 nm
in a Beckman DU 650 spectrophotometer. Concentrations resulting in
50% inhibition (IC50) were determined using nonlinear curve fitting as
implemented in the program Excel for which a 50% reduction of the
initial rate was attained. Each measurement was made in triplicate,
and the experiments were carried out three times. IC50 values, given in
Table 1, are derived from each of these determinations.
To measure enzyme activity in cells, exponentially growing cultures
were washed with phosphate buffered saline (PBS) and scraped into
50 mM phosphate buffer (pH 7.4) containing 5 μM FAD and 250 mM
sucrose. The cells were sonicated twice for 5 s on ice and centrifuged at
13 000 rpm for 15 min at 4 ꢀC. The supernatants were collected and
stored at ꢀ80 ꢀC. Protein concentrations were determined using the
6609
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
’ ASSOCIATED CONTENT
(7) Maiti, A.; Reddy, P. V.; Sturdy, M.; Marler, L.; Pegan, S. D.;
Mesecar, A. D.; Pezzuto, J. M.; Cushman, M. Synthesis of casimiroin and
optimization of its quinone reductase 2 and aromatase inhibitory
activities. J. Med. Chem. 2009, 52, 1873–1884.
(8) Sun, B.; Hoshino, J.; Jermihov, K.; Marler, L.; Pezzuto, J. M.;
Mesecar, A. D.; Cushman, M. Design, synthesis, and biological evalua-
tion of resveratrol analogues as aromatase and quinone reductase 2
inhibitors for chemoprevention of cancer. Bioorg. Med. Chem. 2010,
18, 5352–5366.
(9) Conda-Sheridan, M.; Marler, L.; Park, E. J.; Kondratyuk, T. P.;
Jermihov, K.; Mesecar, A. D.; Pezzuto, J. M.; Asolkar, R. N.; Fenical, W.;
Cushman, M. Potential chemopreventive agents based on the structure
of the lead compound 2-bromo-1-hydroxyphenazine, isolated from
Streptomycesspecies, strain CNS284. J. Med. Chem. 2010, 53, 8688–8699.
(10) Buryanovskyy, L.; Fu, Y.; Boyd, M.; Ma, Y.; Hsieh, T. C.; Wu,
J. M.; Zhang, Z. Crystal structure of quinone reductase 2 in complex with
resveratrol. Biochemistry 2004, 43, 1417–1426.
(11) Nolan, K. A.; Caraher, M. C.; Humphries, M. P.; Bettley, H. A.;
Bryce, R. A.; Stratford, I. J. In silico identification and biochemical
evaluation of novel inhibitors of NRH:quinone oxidoreductase 2
(NQO2). Bioorg. Med. Chem. Lett. 2010, 20, 7331–7336.
S
Supporting Information. Figures showing microscopy
b
results, comparisons of docked ligands, and binding affinity. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Accession Codes
†The atomic coordinates and structure factors have been depos-
ited in the Protein Data Bank (PDB) for NQO2 in complex with
5a1 (PDB code 3TE7) and with 6a1 (PDB code 3TEM).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +44-(0)161-275-5634. Fax: +44-(0)161-275-8342.
E-mail: Karen.Nolan-2@manchester.ac.uk.
’ ACKNOWLEDGMENT
This work was funded by an MRC program grant to I.J.S.
(Grant G0500366) and a project grant from the Association for
International Cancer Research to K.A.N., R.A.B., R.C.W., and I.J.
S. Catherine Treslove and Katherine Scott are thanked for
excellent technical assistance. Dr. David Berk is thanked for
advice on carrying out the fluorescent imaging.
(12) Nolan, K. A.; Humphries, M. P.; Barnes, J.; Doncaster, J. R.;
Caraher, M. C.; Tirelli, N.; Bryce, R. A.; Whitehead, R. C.; Stratford, I. J.
Triazoloacridin-6-ones as novel inhbitors of the quinone oxidoreduc-
tases NQO1 and NQO2. Bioorg. Med. Chem. 2010, 18, 696–706.
(13) Nolan, K. A.; Humphries, M. P.; Bryce, R. A.; Stratford, I. J.
Imidazoacridin-6-ones as novel inhibitors of the quinine oxidoreductase
NQO2. Bioorg. Med. Chem. Lett. 2010, 20, 2832–2836.
(14) Ferry, G.; Hecht, S.; Berger, S.; Moulharat, N.; Coge, F.;
Guillaumet, G.; Leclerc, V.; Yous, S.; Delagrange, P.; Boutin, J. A. Old
and new inhibitors of quinone reductase 2. Chem.-Biol. Interact. 2010,
186, 103–109.
(15) Mailliet, F.; Ferry, G.; Vella, F.; Berger, S.; Cogꢀe, F.; Chomarat,
P.; Mallet, C.; Guꢀenin, S. P.; Guillaumet, G.; Viaud-Massuard, M. C.;
Yous, S.; Delagrange, P.; Boutin, J. A. Characterization of the melato-
ninergic MT3 binding site on the NRH:quinone oxidoreductase 2
enzyme. Biochem. Pharmacol. 2005, 71, 74–88.
(16) Rix, U.; Hantschel, O.; D€urnberger, G.; Remsing, Rix, L. L.;
Planyavsky, M.; Fernbach, N. V.; Kaupe, I.; Bennett, K. L.; Valent, P.;
Colinge, J.; K€ocher, T.; Superti-Furga, G. Chemical proteomic profiles of
the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel
kinase and nonkinase targets. Blood 2007, 110, 4055–4063.
(17) Serin, M.; G€ulbas-, H.; G€urses, I.; Erkal, H. S.; Y€ucel, N. The
histopathological evaluation of the effectiveness of melatonin as a
protectant against acute lung injury induced by radiation therapy in a
rat model. Int. J. Radiat. Biol. 2007, 83, 187–193.
(18) Li, M.; Abdollahi, A.; Gr€one, H. J.; Lipson, K. E.; Belka, C.;
Huber, P. E. Late treatment with imatinib mesylate ameliorates radiation-
induced lung fibrosis in a mouse model. Radiat. Oncol. 2009, 21, 4:66.
(19) Cholody, W. M.; Martelli, S.; Konopa, J. 8-Substituted 5-
[(aminoalkyl)amino]-6H-v-triazolo[4,5,1-de]acridin-6-ones as potential
antineoplastic agents. Synthesis and biological activity. J. Med. Chem.
1990, 33, 2852–2856.
(20) Knox, R. J.; Jenkins, T. C.; Hobbs, S. M.; Chen, S.; Melton,
R. G.; Burke, P. J. Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenza-
mide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a
novel co-substrate-mediated antitumor prodrug therapy. Cancer Res.
2000, 60, 4179–4186.
(21) Foster, C. E.; Bianchet, M. A.; Talalay, P.; Zhao, Q.; Amzel,
L. M. Crystal structure of human quinone reductase type 2, a metallo-
flavoprotien. Biochemistry 1999, 38, 9881–9886.
(22) Winger, J. A.; Hantschel, O.; Superti-Furga, G.; Kuriyan, J. The
structure of the leukemia drug imatinib bound to human quinone
reductase 2 (NQO2). BMC Struct. Biol. 2009, 9, 7.
(23) Nolan, K. A.; Doncaster, J. R.; Dunstan, M.; Scott, K. A.;
Frenkel, A. D.; Siegel, D.; Ross, D.; Barnes, J.; Levy, C.; Whitehead,
R. C.; Stratford, I. J.; Bryce, R. A. Synthesis and biological evaluation of
’ ABBREVIATIONS USED
NRH, N-ribosyldihydronicotinamide; NQO2, NRH:quinone oxido-
reductase 2;NQO1, NAD(P)H:quinone oxidoreductase 1; PDB,
Protein Data Bank; NCI, National Cancer Institute; rmsd, root-
mean-square deviation; QSAR, quantitative structureꢀactivity rela-
tionship; DCPIP, dichlorophenolindophenol; PBS, phosphate buf-
fered saline; GA, genetic algorithm; MTT, (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide); IPTG, isopropyl β-D-1-thio-
galactopyranoside; BSA, bovine serum albumin; IC50, inhibitory con-
centration at 50%
’ REFERENCES
(1) Phillips, R. M.; Mohammed, J.; Maitland, D. J.; Loadman, P. M.;
Shnyder, S. D.; Steans, G.; Cooper, P. A.; Race, A.; Patterson, A. V.;
Stratford, I. J. Pharmacological and biological evaluation of a series of
substituted 1,4-naphthoquinone bioreductive drugs. Biochem. Pharama-
col. 2004, 68, 2107–2116.
(2) Gaikwad, N. W.; Yang, L.; Rogan, E. G.; Cavalieri, E. L. Evidence
for NQO2-mediated reduction of the carcinogenic estrogen ortho-
quinones. Free Radical Biol. Med. 2009, 46, 253–262.
(3) Dinkova-Kostova, A. T.; Talalay, P. NAD(P)H:quinone accep-
tor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme
and exceptionally versatile cytoprotector. Arch. Biochem. Biophys. 2010,
501, 116–123.
(4) Iskander, K.; Paquet, M.; Brayton, C.; Jaiswal, A. K. Deficiency of
NRH:quinone oxidoreductase 2 increases susceptibility to 7,12-dime-
thylbenz(a)anthracene and benzo(a)pyrene-induced skin carcinogenesis.
Cancer Res. 2004, 64, 5925–5928.
(5) Radjendirane, V.; Joseph, P.; Lee, Y. H.; Kimura, S.; Klein-
Szanto, A. J.; Gonzalez, F. J.; Jaiswal, A. K. Disruption of the DT
diaphorase (NQO1) gene in mice leads to increased menadione toxicity.
J. Biol. Chem. 1998, 273, 7382–7389.
(6) Long, D. J., 2nd; Iskander, K.; Gaikwad, A.; Arin, M.; Roop,
D. R.; Knox, R.; Barrios, R.; Jaiswal, A. K. Disruption of dihydronico-
tinamide riboside:quinone oxidoreductase 2 (NQO2) leads to myeloid
hyperplasia of bone marrow and decreased sensitivity to menadione
toxicity. J. Biol. Chem. 2002, 277, 46131–46139.
6610
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611

Journal of Medicinal Chemistry
ARTICLE
coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1
(NQO1). J. Med. Chem. 2009, 52, 7142–7156.
(24) Kabsch, W. Evaluation of single-crystal X-ray diffraction data
from a position-sensitive detector. J. Appl. Crystallogr. 1988, 21, 916–924.
(25) Adams, P. D.; Afonine, P. V.; Bunkꢀoczi, G.; Chen, V. B.; Davis,
I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-
Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.;
Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H.
PHENIX: a comprehensive Python-based system for macromolecular
structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,
66, 213–221.
(26) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular
graphics. Acta Crystallographica D Biol Crystalogr. 2004, 60, 2126–2132.
6611
dx.doi.org/10.1021/jm200416e |J. Med. Chem. 2011, 54, 6597–6611