Synthesis and evaluation of 3-aryloxymethyl-1,2-dimethylindole-4,7-diones as mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1) activity

Article abstract of DOI:10.1021/jm070396q

NAD(P)H:quinone oxidoreductase 1 is a proposed target in pancreatic cancer. We describe the synthesis of a series of indolequinones, based on the 5- and 6-methoxy-1,2-dimethylindole-4,7-dione chromophores with a range of phenolic leaving groups at the (in

Full text of DOI:10.1021/jm070396q

5780
J. Med. Chem. 2007, 50, 5780-5789
Synthesis and Evaluation of 3-Aryloxymethyl-1,2-dimethylindole-4,7-diones as Mechanism-Based
Inhibitors of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Activity
Marie A. Colucci,† Philip Reigan,‡ David Siegel,‡ Aure´lie Chilloux,† David Ross,*,‡ and Christopher J. Moody*,†
School of Chemistry, UniVersity of Nottingham, UniVersity Park, Nottingham NG7 2RD, United Kingdom, and Department of Pharmaceutical
Sciences and Cancer Center, School of Pharmacy, UniVersity of Colorado at DenVer and Health Sciences Center, DenVer, Colorado 80262
ReceiVed April 4, 2007
NAD(P)H:quinone oxidoreductase 1 is a proposed target in pancreatic cancer. We describe the synthesis of
a series of indolequinones, based on the 5- and 6-methoxy-1,2-dimethylindole-4,7-dione chromophores with
a range of phenolic leaving groups at the (indol-3-yl)methyl position. The ability of these indolequinones
to function as mechanism-based inhibitors of purified recombinant human NQO1 was evaluated, as was
their ability to inhibit both NQO1 and cell growth in human pancreatic MIA PaCa-2 tumor cells. The inhibition
of rhNQO1 was related to the pKa of the leaving group: compounds with poorer phenolic leaving groups
were poor inhibitors whereas those with more acidic leaving groups were more efficient inhibitors. These
inhibition data also correlated with the inhibition NQO1 in MIA PaCa-2 cells. However, the data demonstrate
that NQO1 inhibition does not correlate with growth inhibitory activity, at least in the MIA PaCa-2 cell
line, suggesting that targets in addition to NQO1 need to be considered to explain the potent growth inhibitory
activity of this series of indolequinones in human pancreatic cancer cells.
Scheme 1. Proposed Mechanism-Based Inhibition of NQO1 by
the 3-(4-Nitrophenoxy)methylindolequinone 1a
Introduction
The enzyme NQO1 (EC 1.6.99.2), also known as DT-
diaphorase, is an obligate two-electron reductase characterized
by its ability to use either NADH or NADPH as cofactor. NQO1
catalyzes the two-electron reduction of quinones and, hence,
can protect cells against the toxic effects of quinones.1-5
However, NQO1 is also involved in the reductive activation of
anticancer agents, such as mitomycin C and other cytotoxic
quinones that operate by the so-called bioreductive mechan-
ism,4,6-8 and continues to generate interest because of its
elevated levels in many tumors and tumor cell lines.3 During
our long-standing interest in NQO1, we have undertaken a
detailed study on the substrate specificity of the enzyme and
the correlation of quinone structure with rate of metabolism by
the enzyme and toxicity toward human tumor cell lines.9-13
As part of our studies on quinone substrates for NQO1, we
have recently developed a highly specific, potent, mechanism-
based (suicide substrate) inhibitor 5-methoxy-1,2-dimethyl-3-
(4-nitrophenoxymethyl)indole-4,7-dione 1, a compound also
known as ES936, and have fully characterized its interaction
with the enzyme using biochemistry, mass spectrometry, and
crystallography.14 In cellular studies using human breast and
colon cancer cell lines, it inhibits the enzyme at low nanomolar
concentrations.15 We rationalize the action of 1 by the mech-
anism shown in Scheme 1, whereby two-electron reduction of
the quinone by NQO1 gives the corresponding hydroquinone
2. In this compound, the indole nitrogen lone pair is no longer
conjugated with the quinone carbonyl group and, hence,
“normal” indole reactivity takes over, resulting in the elimination
of the aryloxy group, in the case of 1 4-nitrophenoxide, from
the 3-indolylmethyl position to generate a highly electrophilic
iminium ion 3. Because this occurs in the enzyme active site,
the generation of 3 leads to irreversible alkylation of the enzyme,
a Ar ) 4-nitrophenyl.
shown by our mass spectrometric studies to be at Tyr-127 or
Tyr-12911 and, hence, its inhibition.
It was of considerable interest when it was recently reported
that inhibition of NQO1 in pancreatic tumor cells using
dicumarol resulted in inhibition of cell growth of the malignant
in vitro phenotype of cells.16 Although it only accounts for less
than 10% of all cancers, pancreatic cancer has one of the worst
prognoses. It is rapidly fatal; data from Cancer Research UK
show that one-year survival rates are less than 15% (five-year
survival, ca. 2%). Surgery and radiation are the common
treatments because, to date, chemotherapy has made little or
no impact. It was proposed that the effect of dicumarol on
pancreatic tumor cell growth was mediated by inhibition of
NQO1, causing a rise in superoxide levels.16 We have recently
shown that NQO1 can directly scavenge superoxide, and in cells
containing high levels of the enzyme (i.e., most solid tumors)
scavenging by NQO1 competes with superoxide dismutase
(SOD).17 These data provide support for the hypothesis that
inhibition of NQO1 could lead to increased levels of superoxide
and, given the low levels of SOD in pancreatic cancer cell lines,
* To whom correspondence should be addressed for biology (D.R.)
and chemistry (C.J.M.). Phone: +1 303 315 7732 (D.R.); +44 115 846
8500 (C.J.M.). Fax: +1 303 315 6281 (D.R.); +44 115 951 3564 (C.J.M.).
E-mail: david.ross@uchsc.edu (D.R.); c.j.moody@nottingham.ac.uk (C.J.M.).
† University of Nottingham.
‡ University of Colorado.
10.1021/jm070396q CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/18/2007

Indolequinones as Inhibitors of Human NQO1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5781
described,9 and nine further analogs 5-13 were synthesized,
in which the 4-nitrophenoxy leaving group (4-nitrophenol pKa
) 7.2) was replaced by a range of other phenolic leaving groups.
These were chosen to explore both the effect of leaving group
ability, as evidenced by the pKa of the corresponding phenol,
and the steric effects of the 3-aryloxymethyl group. We were
also conscious of the need to identify possible alternative
electron-withdrawing groups on the phenolic moiety, because
nitro (NO2) groups are not without problems in potential drug
molecules due to their metabolism. Hence, the choice of 2,4,6-
trifluorophenoxy group was made because 2,4,6-trifluorophenol
has a similar pKa to 4-nitrophenol (7.5 and 7.2, respectively).
Of these nine compounds, the phenoxy-,21 the 2,4-dinitrophe-
noxy-,22 and the 2-fluoro-4-nitrophenoxy-compounds,22 5, 8, and
9, had been prepared previously. In general, two methods were
used to prepare the new 3-aryloxylmethylindolequinones, both
starting from the known 3-hydroxymethyl-5-methoxy-1,2-dim-
ethylindole-4,7-dione.23 First, the primary alcohol was treated
with thionyl chloride, and the resulting chloride reacted with a
phenol in the presence of a base, usually potassium carbonate.
The 3-aryloxylmethylindolequinones 6, 7, and 10 were prepared
in this manner. Alternatively, the 3-hydroxymethyl compound
was reacted with the phenol under the conditions of the
Mitsunobu reaction in the presence of a triarylphosphine and a
dialkyl azodicarboxylate. The indolequinones 11-13 were thus
obtained: in the case of 2-hydroxypyridine, both N- and
O-linked products were formed. The last analog in this series
of compounds was the 3-[1-(4-nitrophenoxy)ethyl]indolequinone
14;24 in principle, the additional methyl group should stabilize
the intermediate iminium ion and, thereby, affect rates of
fragmentation of the hydroquinone 2 (Scheme 1).
We have also investigated an isomeric series of NQO1
inhibitors based on the 6-methoxyindole-4,7-quinone ring
system. Although this may appear a trivial change, the switch
of the electron-releasing methoxy group from the 5- to the
6-position has an effect on the electronic properties of the
quinone ring system and, hence, on the reductive elimination
of phenoxide as required for NQO1 inhibition (cf. Scheme 1).
Hence, it was by no means obvious that such analogs would be
potent inhibitors because the position of the methoxy group
affects the electrophilicity of the alkylating species formed upon
loss of the leaving groupsthe 6-methoxy group, but not the
5-methoxy, can donate electron density toward the key exocyclic
carbon. The synthesis of the 6-methoxyindolequinone series is
summarized in Scheme 2. Thus, 2-benzyloxy-4-methoxyben-
zaldehyde 3025 was condensed with methyl azidoacetate, and
the resulting azidocinnamate 31 was cyclized to the indole 32
in a Hemetsberger indole synthesis.26-28 Treatment of the ester
32 with lithium aluminum hydride under forcing conditions
resulted in reduction to the 2-methylindole 33 in satisfactory
yield. Formylation under Vilsmeier-Haack conditions gave
the indole-3-carbaldehyde 34 in excellent yield, and this
was followed by N-methylation to give the indole 35. There-
after, the indole-3-carboxaldehyde was progressed to the cor-
responding 3-hydroxymethylindolequinone 37, as outlined in
Scheme 2.
Figure 1. Structures of NQO1 inhibitors.
renders the contribution of NQO1, the levels of which are known
to be elevated, potentially extremely important in regulating
superoxide in these cells. Although the interaction of dicumarol
with NQO1 has been characterized crystallographically,18 the
major problem with this inhibitor, however, is its lack of
selectivity, because it is known to inhibit many other enzymes.6
The indolequinone 1, on the other hand, is a mechanism-based
inhibitor of NQO1, and we have confirmed that, like dicumarol,
it is a potent inhibitor of human pancreatic cancer cell growth
in vitro. The compound is also effective in vivo; MIA PaCa-2
xenografts in mice grow significantly slower after treatment with
1.19 However, in the case of 1, our studies showed that the
cytotoxicity was independent of superoxide generation, and
therefore raises the question of whether NQO1 inhibition is the
only mechanism operating in pancreatic cancer cells.
To gain additional insights, we initiated a much wider study
of quinone based inhibitors of NQO1, and we now describe
the synthesis of a number of new analogs of 1, their biological
evaluation as mechanism-based inhibitors of NQO1 and their
toxicity toward pancreatic tumor cell lines. Some preliminary
data on six of these quinones have been included in our previous
publications.11,19,20
The attachment of phenolic leaving groups to the 3-hy-
droxymethyl-6-methoxyindolequinone 37 was for the most part
accomplished by conversion to the 3-chloromethylindole, fol-
lowed by reaction with the corresponding phenol in the presence
of base, as described above for the isomeric 5-methoxy series.
The indolequinones 15, 16, 18, 19, 21-26, and 28 were all
prepared in this manner. The 2,4-dinitrophenoxy compound 20
was prepared by reaction of the hydroxymethyl compound 37
Results and Discussion
Chemistry. The structures of the new NQO1 inhibitors are
shown in Figure 1. 5-Methoxy-1,2-dimethyl-3-(4-nitrophe-
noxymethyl)indole-4,7-dione 1 was prepared as previously

5782 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Colucci et al.
Scheme 2. Preparation of
3-Hydroxymethyl-6-methoxy-1,2-dimethylindole-4,7-dione
concentrations of indolequinones used in this assay, only minor
inhibition of NQO1 activity was observed in the absence of
NADH, indicating that inhibition of NQO1 by these indole-
quinones occurred following catalytic turnover of the indole-
quinone by NQO1. Representative examples of NADH-
dependent inhibition of NQO1 by indolequinones are shown in
Figure 2.
The dependence on NADH for inhibition was taken as
preliminary evidence for mechanism-based inactivation of
NQO1,14 and compounds showing such properties were studied
further. An important term used to describe the efficiency of
inactivation for mechanism-based enzyme inhibitors is the
partition ratio. The partition ratio is defined as the number of
catalytic cycles required to inhibit one molecule of enzyme. In
these experiments, partition ratios were determined by incubating
defined molar ratios of indolequinone and rhNQO1 in the
presence of NADH and measuring the resultant NQO1 activity.
Partition ratios for the inhibition of NQO1 by this series of
indolequinones are shown in Table 1. Many of the indolequino-
nes in this study had partition ratios near 1, indicating very
efficient inactivation of NQO1, and 13 out of 26 indolequinones
in this series had partition ratios less than 5. The 2,4-
dinitrophenoxy derivatives 8 and 20 displayed poor solubility
characteristics and precipitated out of solution, so partition ratios
could not be determined.
The ability of these indolequinones to inhibit NQO1 in cells
was examined using the human pancreatic cancer cell line MIA
PaCa-2 (NQO1 activity; 1000 nmol 2,6-dichlorophenol-in-
dophenol (DCPIP)/min/mg). For these studies, MIA PaCa-2 cells
were treated with indolequinones (1-10 000 nM) for 1 h, the
cells were then washed free of drug and NQO1 activity was
measured in cell sonicates. Results from these experiments
clearly demonstrated that these indolequinones could enter cells
and inactivate NQO1 (Figure 3 and Table 1). For most
indolequinones in this study greater than 95% of NQO1 activity
could be inhibited at indolequinone concentrations between 10
and 100 nM (Table 1). However, indolequinones with large
partition ratio such as 23 or 24 required much higher concentra-
tions to achieve 95% inhibition of NQO1 (Figure 3, Table 1).
Finally, the ability of these indolequinones to induce cyto-
toxicity was measured in MIA PaCa-2 cells using the 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
growth inhibition assay (Table 1). Because previous studies with
mechanism-based inhibitors 1,15, 23, and 29 have demonstrated
near complete inhibition of NQO1 catalytic activity after 4 h,
this time point, as well as a longer time point of 72 h, were
selected for growth inhibition studies in the MIA PaCa-2 cells.
The IC50 values are reported for this extended series of
compounds for both 4 and 72 h treatments with the appropriate
indolequinone (Table 1). The growth inhibitory potency of these
compounds after 4 h of incubation indicates that compounds 1,
15, 17, 6, 18, 7, 19, 21, 22, 23, 25, 10, and 26 were effective
inhibitors of the growth of MIA PaCa-2 cells (IC50, 4 h, <650
nM), while compounds 5, 16, 8, 20, 9, 24, 11, 27, 12, 28, 13,
29, and 14 were relatively ineffective at inducing growth
inhibition (IC50, 4 h, >900 nM).
Scheme 3. Preparation of 3-Aryloxymethylindolequinones from
Corresponding 5- or 6-Methoxy-3-hydroxymethyl Derivatives
with 2,4-dinitrofluorobenzene, and the 2- and 4-pyridyl deriva-
tives 27 and 29 were obtained using the Mitsunobu reaction
(Scheme 3). The 3-(4-aminophenoxy)methyl compound 17 was
prepared from 4-azidophenol29 by coupling to the hydroxy-
methyl compound 37 followed by reduction of the azide group.
Biology. All 26 indolequinones 1 and 5-29 were assayed
for their ability to inhibit the quinone reductase NQO1 using a
series of assays employing both purified recombinant human
NQO1 and NQO1-rich cells. Initially, experiments were carried
out to determine whether members of this series of indole-
quinones were mechanism-based inhibitors of NQO1 (Table 1,
Figure 2). For these experiments, indolequinones were incubated
with rhNQO1a in the absence and presence of NADH. At the
The phenol and pyridine-based leaving groups derived from
each of the indolequinones tested were also examined for growth
inhibitory potency. The compounds tested included phenol, 2-,
3-, and 4-nitrophenol, 2-fluoro-4-nitrophenol, 4-fluorophenol,
a Abbreviations: rhNQO1, recombinant human NAD(P)H:quinone oxi-
doreductase 1; DCPIP, 2,6-dichlorophenol-indophenol; MTT, 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TBAF, tetra-n-
butylammonium fluoride.

Indolequinones as Inhibitors of Human NQO1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5783
Table 1. Inhibition of hNQO1 by Indolequinones 1 and 5-29 and Inhibition of Cell Growth in the MIA PaCa-2 Cell Line
>90% inhibition
of NQO1
IC50 MIA
PaCa-2
4 h
IC50 MIA
PaCa-2
72 h
mechanism-
based
in MIA
PaCa-2 cellsb
nM
partition
ratiob
No.
R3
R5
R6
Ar
inhibitiona
nMc
nMc
1
15
5
16
17
6
18
7
19
8
20
9d
21
22
23
24
25
10
26
11
27
12
28
13
29
14
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
OMe
H
OMe
H
H
OMe
H
OMe
H
OMe
H
OMe
H
H
H
H
H
OMe
H
OMe
H
OMe
H
OMe
H
OMe
H
OMe
H
OMe
OMe
H
OMe
H
OMe
H
OMe
H
OMe
OMe
OMe
OMe
OMe
H
OMe
H
OMe
H
OMe
H
OMe
H
4-NO2-C6H4
4-NO2-C6H4
C6H5
yes
yes
yes
yes
no
3.5
3.7
4000
3800
nd
2.3
3.5
1.0
1.0
nd
nd
0.8
0.8
10-100
10-100
nd
nd
nd
10-100
10-100
nd
10-100
nd
nd
nd
10-100
nd
5000-10 000
5000-10 000
10-100
nd
10-100
<10
629 ( 17
638 ( 15
1385 ( 24
4563 ( 26
504 ( 4
352 ( 16
351 ( 25
345 ( 20
363 ( 9
NC
508 ( 5
355 ( 3
962 ( 18
409 ( 30
160 ( 4
258 ( 21
257 ( 22
104 ( 18
178 ( 4
NC
C6H5
4-NH2-C6H4
3-NO2-C6H4
3-NO2-C6H4
2-NO2-C6H4
2-NO2-C6H4
2,4-(NO2)2-C6H3
2,4-(NO2)2-C6H3
2-F-4-NO2-C6H3
2-F-4-NO2-C6H3
4-CN-C6H4
4-CF3-C6H4
4-F-C6H4
2,4-F2-C6H4
2,4,6-F3-C6H4
2,4,6-F3-C6H4
2-pyridyl
2-pyridyl
3-pyridyl
yes
yes
yes
yes
see text
see text
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
NC
NC
4654 ( 48
529 ( 5
463 ( 6
496 ( 3
905 ( 25
255 ( 5
427 ( 5
452 ( 4
NC
9579 ( 48
904 ( 26
2475 ( 40
2007 ( 16
2560 ( 7
1829 ( 14
3556 ( 31
417 ( 5
192 ( 3
311 ( 5
493 ( 32
75 ( 4
37
652
>100 000
21.3
1.9
1.7
nd
1.1
6.1
nd
1.3
86 ( 3
212 ( 3
NC
10-100
10-100
nd
nd
10-100
nd
2393 ( 28
562 ( 24
1824 ( 19
2172 ( 25
3271 ( 15
1696 ( 23
3-pyridyl
4-pyridyl
4-pyridyl
4-NO2-C6H4
0.9
9.1
a Classification of mechanism-based inhibition refers to dependence (or not) on NADH. b nd ) not determined. c NC denotes no convergence. The IC50
value cannot be determined as profile is a straight line. d Compound 9 was found to be unstable in DMSO and was tested immediately for NQO1 mechansim-
based inhibition.
Figure 2. Mechanism based inhibition of NQO1 by representative
Figure 3. Inhibition of NQO1 activity in MIA PaCa-2 cells treated
indolequinones. NQO1 activity was assayed following the incubation
with indolequinones. NQO1 activity was measured in MIA PaCa-2 cells
of indolequinone (100 nM) with rhNQO1 (1 µg) in the absence (solid
following treatment with indolequinones (1 nM-10 µM) for 1 h. Results
bars) and presence (striped bars) of 0.2 mM NADH. Incubations (0.5
are the mean ( standard deviation of three separate determinations.
mL) were performed for 5 min at 32 °C. Results are the mean (
standard deviation of three separate determinations.
Open circles, 24; open diamonds, 23; open squares, 22; open triangles,
12; closed circles, 6; closed squares, 19.
2,4-difluorophenol, 2,4,6-trifluorophenol, 4-trifluoromethylphe-
nol, 4-cyanophenol, 4-aminophenol, 2-, 3-, and 4-hydroxypy-
ridine. All compounds were relatively nontoxic (data shown in
Supporting Information). For example, all IC50 values obtained
by MTT analysis were greater than 12 900 nM after 72 h of
incubation of these compounds with MIA PaCa-2 cells. These
data indicate that growth inhibitory properties of the indole-
quinones are unlikely to be related to the liberation of phenols
or pyridines from the parent molecule.
Interestingly, the indolequinones 27, 12, 28, 13, 29, and 14
were potent mechanism-based inhibitors of NQO1 activity,
however displayed poor growth inhibitory profiles. This finding
indicates dissociation of NQO1 inhibitory activity and the ability
to induce growth inhibition in MIA PaCa-2 cells for these
indolequinone compounds and implies a function via an NQO1-
independent mechanism to inhibit cell proliferation.
In addition, compounds 5, 16, and 24, which are very poor
inhibitors of NQO1 as indicated by high partition ratios

5784 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Colucci et al.
demonstrated decreases in IC50 value after 72 h relative to 4 h
of incubation with cells and the change with compound 4 was
dramatic from 4563 nM to 409 nM. This observation also argues
for an NQO1-independent mechanism of toxicity in MIA PaCa-2
cells.
shown that the indolequinones 1, 6, 15, 21, 25, 26, 27, and 29
all caused >95% inhibition of enzyme activity in the human
pancreatic MIA PaCa-2 solid tumor cell line after 1 h. The data
for compounds 1, 15, 29, 5, 6, 13, and 29 were included in our
previous paper,20 and the data for the remaining compounds
are shown in Figure 3.
Discussion. To make a more detailed comparison of the
efficiency of NQO1 inactivation, the partition ratios of the
indolequinones were examined. Within the series of analogs of
1 bearing a 5-methoxy group, there was a loose correlation of
the partition ratio with the leaving group ability of the aryloxy
group at the 3-indolylmethyl position, as evidenced by the pKa
of the corresponding phenol. Certainly, compound 9 with the
most acidic phenol leaving group, 2-fluoro-4-nitrophenol (pKa
) 5.7), has the lowest partition ratio, while the higher are
exhibited by the indolequinones 5 and 24, which have the
poorest leaving group (phenol pKa ) 9.9, 4-fluorophenol pKa
) 9.9). It is also noteworthy that a nitrophenolate leaving group
is not essential because the indolequinone 10 bearing a 2,4,6-
trifluorophenoxy group (2,4,6-trifluorophenol pKa ) 7.5) is a
highly efficient inhibitor. Likewise, the compounds 12 and 13
with 3-hydroxypyridine and 4-hydroxypyridine (4-pyridone)
leaving groups (3-hydroxypyridine pKa ) 8.51, 4-hydroxypy-
ridine pKa ) 5.2) were also efficient inhibitors of NQO1.
Finally, compound 14 bearing the additional methyl group at
the 3-indolylmethyl position is a poorer inhibitor and less
cytotoxic than its des-methyl analog 1, notwithstanding the fact
that the additional methyl group should stabilize the intermediate
iminium ion and, thereby, affect rates of fragmentation of the
hydroquinone 2 (Scheme 1).
In previous studies, we showed that 1 inhibited the growth
of human pancreatic MIA PaCa-2 cancer cells, and therefore,
it was of interest to evaluate this indolequinone series in the
same cell line. Although greater than 95% inhibition of NQO1
occurs after a 1 h exposure to indolequinones, MIA PaCa-2
cells were treated with indolequinones for 4 h to maintain a
prolonged period of NQO1 inhibition because, once these
inhibitors are removed, NQO1 activity slowly returns due to
the synthesis of new NQO1 protein. A longer exposure period
(72 h) was also used for comparative purposes, and the IC50
values are reported for both 4 and 72 h treatments in Table 1.
The 26 indolequinones tested exhibited IC50 values in the range
of about 0.1-9.5 µM (100-9500 nM) with the 2- and
3-nitrophenyl (6, 7, 18, 19), with the di- and tri-fluorophenyl
(10, 25, 26) derivatives being among the most potent growth
inhibitors, and the pyridyloxy derivatives (13, 27-29) the least
effective. Hence, it is clear that potent NQO1 inhibitors such
as the pyridyloxy derivatives do not exhibit the highest levels
of cytotoxicity. Conversely, relatively poor inhibitors such as
the 4-aminophenoxy 17 and 4-trifluoromethylphenoxy 23
compounds demonstrated potent cell growth inhibition at both
time periods. In addition, the relatively poor inhibitors, the
phenoxy derivatives 5 and 16 and the 4-fluorophenoxy 24
compound, demonstrated potentiated cell growth inhibition with
increasing drug exposure. These data on a large set of indole-
quinones reinforce our earlier observations20 that NQO1 inhibi-
tion can be divorced from cell growth inhibition in the human
pancreatic MIA PaCa-2 cell line at least.
In summary, we have examined a series of 26 indolequinones,
most of them previously unreported, and evaluated their capacity
to inhibit NQO1 in cell-free and cell-based systems, together
with their ability to inhibit cell proliferation in the human
pancreatic MIA PaCa-2 cancer cell line. We have characterized
new mechanism-based inhibitors of NQO1 and shown that their
partition ratios reflect their ability to inhibit NQO1 in tumor
cells. Our data demonstrate that NQO1 inhibition does not
correlate with growth inhibitory activity, at least in the MIA
PaCa-2 cell line, suggesting that targets in addition to NQO1
need to be considered to explain the potent activity of this series
of indolequinones in human pancreatic cancer cells.
Although it is not obvious that the isomeric series of NQO1
inhibitors based on the 6-methoxyindole-4,7-quinone ring system
would be inhibitors of NQO1 due to the electronic effect of the
switching of the methoxy group from the 5- to the 6-position,
the indolequinones 15, 16, and 18-29 inactivated the enzyme.
In fact, the 6-methoxy analog 15 of 1 is equipotent with the
5-methoxy isomer. Likewise, in the five pairs of compounds
where the leaving groups are phenoxide (compounds 5 and 16),
2-nitrophenoxide (7 and 19), 2-fluoro-4-nitrophenoxide (com-
pounds 9 and 21), 2,4,6-trifluorophenoxide (compound 10 and
26), or the 4-pyridyloxy (compounds 13 and 29), the 5- and
6-methoxy isomers exhibit very similar biological profiles as
inhibitors of NQO1, as evidenced by their similar partition ratios.
Therefore, surprisingly, the position of the electron releasing
methoxy group appears to have little effect on the ability of the
indolequinones to undergo reduction by NQO1, followed by
elimination of the aryloxy group from the 3-indolylmethyl
position with the ensuing inactivation of the enzyme.
Experimental Section
As with the 5-methoxy series of indolequinones, there also
appears to be a relationship between the pKa of the aryloxy
leaving group at the 3-indolylmethyl position and the efficiency
of enzyme inhibition, as measured by the partition ratio, in the
6-methoxyindolequinones. This is nicely illustrated by the series
of fluorophenoxy compounds 24-26, where the compounds
become more potent inhibitors as the pKa of the leaving group
decreases as the degree of fluorine substitution increases (4-
fluorophenol pKa ) 9.9; 2,4-difluorophenol pKa ) 8.7; 2,4,6-
trifluorophenol pKa ) 7.5).
Next, the inhibition of NQO1 in cellular systems by repre-
sentative indolequinones was examined to verify inhibition of
NQO1, the proposed intracellular target of these compounds,
and ensure that quinones were taken up into cells. Importantly,
there was a general relationship between the partition ratio
measured using purified NQO1 and the ability of the indole-
quinones to inhibit NQO1 in MIA PaCa-2 cells. Hence, it was
Chemistry. General Experimental Details. Commercially
available reagents were used throughout without purification unless
otherwise stated. Light petroleum refers to the fraction with a bp
40-60 °C and was distilled before use. Ether refers to diethyl ether.
Reactions were routinely carried out under a nitrogen or argon
atmosphere. Analytical thin layer chromatography was carried out
on aluminum-backed plates coated with Merck Kieselgel 60 GF254
and visualized under UV light at 254 and/or 360 nm. Chromatog-
raphy was carried out using Merck Kieselgel 60 H silica or Matrex
silica 60. Fully characterized compounds were chromatographically
homogeneous.
UV/vis spectra were recorded on a Phillips PU 8700 series
spectrophotometer. Infrared spectra were recorded in the range
4000-600 cm-1 using Nicolet Magna FT-550, Perkin-Elmer 1600
series, or Avatar 320 FT-IR spectrometers. NMR spectra were
carried out on Jeol EX270, Bruker AC300, AV400, and DRX500
instruments (operating at 1H frequencies of 270, 300, 400, and 500
MHz, respectively; corresponding 13C frequencies are 67.5, 75, 100,

Indolequinones as Inhibitors of Human NQO1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5785
and 125 MHz). Chemical shifts are quoted in ppm with TMS as
internal standard. J values are recorded in Hz. In the 13C spectra,
signals corresponding to CH, CH2, or CH3 groups, as assigned from
DEPT, are noted; all others are C. High and low-resolution mass
spectra were recorded on Micromass GCT TDF, Bruker MicroTof,
or VG Autospec spectrometers or at the EPSRC Mass Spectrometry
Center (Swansea).
General Method A. Coupling of 3-(Hydroxymethyl)indole-
quinones with Substituted Phenols via the 3-Chloromethylin-
dole. The following general procedure was used unless otherwise
stated: To a stirred solution of the 5-methoxy or 6-methoxy
3-hydroxymethyl-1,2-dimethylindole-4,7-dione (0.1-0.2 mmol, 1
equiv) in dichloromethane (5 mL) at 0 °C was added thionyl
chloride (50 equiv) dropwise. The reaction mixture was stirred at
room temperature for 1 h. The solvent and excess thionyl chloride
were removed in vacuo and the crude product was used directly in
the next step without further purification. A mixture of the crude
3-chloromethylindolequinone, the phenol (3 equiv), and the potas-
sium carbonate (3 equiv) was stirred in DMF (5 mL) for 16 h. The
solvent was removed in vacuo and the crude product was dissolved
in dichloromethane (50 mL), washed with NaOH (2 M; 20 mL),
HCl (2 M; 20 mL), and water (20 mL), dried (MgSO4), and filtered,
and the filtrate was evaporated in vacuo. The crude product was
purified by chromatography.
General Method B. Coupling of 3-(Hydroxymethyl)indole-
quinones with Substituted Phenols via the Mitsunobu Reaction.
The following general procedure was used unless otherwise
stated: the dialkyl azodicarboxylate (4 equiv) was added to a
solution of the 5-methoxy or 6-methoxy 3-(hydroxymethyl)-1,2-
dimethylindole-4,7-dione (100 mg, 0.4 mmol), triarylphosphine (3
equiv), and the phenol (3 equiv) in THF (15 mL). The solution
was stirred for 1 h. The solvent was removed in vacuo, and the
residue was dissolved in ethyl acetate and washed with sodium
hydroxide (1 M), hydrochloric acid (1 M), and water, dried
(MgSO4), and concentrated. The residue was purified by column
chromatography.
5-Methoxy-1,2-dimethyl-3-(2,4-dinitrophenoxymethyl)indole-
4,7-dione 8. Prepared as previously described.22
3-(2-Fluoro-4-nitrophenoxymethyl)-5-methoxy-1,2-dimethyl-
indole-4,7-dione 9. Prepared as previously described.22
5-Methoxy-1,2-dimethyl-3-(2,4,6-trifluorophenoxymethyl)in-
dole-4,7-dione 10. Using method A, 3-(hydroxymethyl)-5-methoxy-
1,2-dimethylindole-4,7-dione (30 mg, 0.13 mmol), thionyl chloride
(0.85 mL, 11.7 mmol), 2,4,6-trifluorophenol (56.7 mg, 0.38 mmol),
and potassium carbonate (52.9 mg, 0.38 mmol) gave after chro-
matography (elution with ethyl acetate) the title compound (28.9
mg, 62%) as an orange-red crystalline solid: mp 201-202 °C; 1H
NMR (400 MHz, CDCl3) δ 6.67 (2 H, t, J 8.0, ArH), 5.62 (1 H, s,
6-H), 5.33 (2 H, s, CH2), 3.93 (3 H, s, Me), 3.81 (3 H, s, Me), 2.35
(3 H, s, Me); 13C NMR (100 MHz, CDCl3) δ 177.8 (C), 176.7 (C),
156.3 (dt, JCF 245, 14, CF), 155.5 (ddd, JCF 250, 14, 8, CF), 155.0
(C), 137.9 (C), 130.8 (td, JCF 14, 6, C), 127.7 (C), 120.5 (C), 115.2
(C), 105.6 (C), 99.5 (ddd, JCF 27, 27, 8, CH), 64.9 (CH2), 55.4
(Me), 31.4 (Me), 8.5 (Me); MS (ESI) 388 (M + Na+, 100%), 363
(4); found, M + Na+, 388.0773. C18H14F3NO4 + Na requires
388.0773; Anal. (C18H14F3NO4) C, H, N.
5-Methoxy-1,2-dimethyl-3-(pyridin-2-yloxymethyl)indole-4,7-
dione 11. Using method B, 2-hydroxypyridine (25 mg, 0.26 mmol),
triphenylphosphine (101 mg, 0.39 mmol), diethyl azodicarboxylate
(0.047 mL, 0.30 mmol), and 3-hydroxymethyl-5-methoxy-1,2-
dimethylindole-4,7-dione (30 mg, 0.13 mmol) gave after chroma-
tography (ethyl acetate elution) the title compound (20 mg, 50%)
as an orange solid, mp 138-140 °C; 1H NMR (400 MHz, CDCl3)
δ 8.18 (1 H, ddd, J 0.8, 2.0, 5.2, PyrH), 7.54 (1 H, ddd, J 2.0, 7.2,
8.4, PyrH), 6.86 (1 H, ddd, J 0.8, 5.2, 6.8, PyrH), 6.72 (1 H, dt, J
0.8, 8.4, PyrH), 5.61 (1 H, s, 6-H), 5.51 (2 H, s, CH2), 3.90 (3 H,
s, Me), 3.81 (3 H, s, Me), 2.32 (3 H, s, Me); 13C NMR (100 MHz,
CDCl3) δ 178.9 (C), 177.7 (C), 163.7 (C), 159.8 (C), 146.8 (C),
138.5 (CH), 138.0 (CH), 121.9 (C), 117.2 (2 C), 116.8 (CH), 111.2
(CH), 106.6 (CH), 58.0 (CH2), 56.4 (Me), 32.4 (Me), 9.7 (Me);
MS (ESI) 335 (M + Na+, 100%), 218 (82); found, M + Na+,
335.1002. C17H16N2O4 + Na requires 335.1008.
5-Methoxy-1,2-dimethyl-3-(4-nitrophenoxymethyl)indole-4,7-
Also formed was 5-methoxy-1,2-dimethyl-3-(2-pyridon-1-yl-
methyl)indole-4,7-dione (8.4 mg, 21% yield) as an orange solid:
dione 1 (ES936). Prepared as previously described.9
1
5-Methoxy-1,2-dimethyl-3-(phenoxymethyl)indole-4,7-dione 5.
Prepared as previously described.21
mp 226-232 °C; H NMR (400 MHz, CDCl3) δ 7.95 (1 H, dd, J
2.0, 6.8, PyrH), 7.26 (1 H, ddd, J 2.0, 6.4, 8.8, PyrH), 6.50 (1 H,
d, J 8.8, PyrH), 6.10 (1 H, td, J 1.2, 6.8, PyrH), 5.62 (1 H, s, 6-H),
5.22 (2 H, s, CH2), 3.88 (3 H, s, Me), 3.82 (3 H, s, Me), 2.47 (3 H,
s, Me); 13C NMR (100 MHz, CDCl3) δ 177.5 (2 C), 158.5 (C),
138.6 (CH), 138.4 (C), 138.3 (CH), 131.2 (C), 128.1 (C), 120.4
(CH), 119.5 (C), 115.2 (C), 105.8 (CH), 104.9 (CH), 55.5 (Me),
41.4 (CH2), 31.6 (Me), 9.1 (Me); MS (ESI) 335 (M + Na+, 100%),
218 (50); found, M + Na+, 335.1006. C17H16N2O4 + Na requires
335.1008.
5-Methoxy-1,2-dimethyl-3-(3-nitrophenoxymethyl)indole-4,7-
dione 6. Using method A, 3-(hydroxymethyl)-5-methoxy-1,2-
dimethylindole-4,7-dione23 (30 mg, 0.13 mmol), thionyl chloride
(0.75 mL, 10.3 mmol), 3-nitrophenol (53 mg, 0.38 mmol), and
potassium carbonate (53 mg, 0.38 mmol) gave after chromatography
(elution with 20% ethyl acetate/light petroleum) the title compound
(33 mg, 44%) as an orange crystalline solid: mp 219-227 °C; 1H
NMR δ 7.82 (2 H, m, ArH), 7.43 (1 H, t, J 8.4, ArH), 7.29 (1 H,
m, ArH), 5.64 (1 H, s, 6-H), 5.36 (2 H, s, CH2), 3.91 (3 H, s, Me),
3.82 (3 H, s, Me), 2.32 (3 H, s, Me); 13C NMR (100 MHz, CDCl3)
δ 178.8 (C), 178.2 (C), 160.0 (C), 159.1 (C), 149.2 (C), 138.0 (C),
130.0 (CH), 129.0 (C), 121.6 (CH), 121.4 (C), 115.9 (overlapping
CH and C), 109.9 (CH), 106.8 (CH), 61.1 (CH2), 56.5 (Me), 32.4
(Me), 9.9 (Me); MS (ESI) 379 (M + Na+, 100%), 218 (71); found,
M + Na+, 379.0902. C18H16N2O6 + Na requires 379.0906.
5-Methoxy-1,2-dimethyl-3-(2-nitrophenoxymethyl)indole-4,7-
dione 7. Using method A, 3-(hydroxymethyl)-5-methoxy-1,2-
dimethylindole-4,7-dione (30 mg, 0.13 mmol), thionyl chloride (1.2
mL, 16.5 mmol), 2-nitrophenol (124.3 mg, 0.89 mmol), and
potassium carbonate (123.5 mg, 0.89 mmol) gave after chroma-
tography (elution with ethyl acetate) the title compound (24.6 mg,
5-Methoxy-1,2-dimethyl-3-(3-pyridyloxymethyl)indole-4,7-di-
one 12. Using method B, 3-hydroxypyridine (40.8 mg, 0.43 mmol),
triphenylphosphine (169 mg, 0.64 mmol), diethyl azodicarboxylate
(0.079 mL, 0.50 mmol), and 3-hydroxymethyl-5-methoxy-1,2-
dimethylindole-4,7-dione (50 mg, 0.21 mmol) gave after chroma-
tography (ethyl acetate elution) the title compound (34 mg, 51%)
as an orange solid: mp 158-162 °C; 1H NMR (400 MHz, CDCl3)
δ 8.38 (1 H, bs, PyrH), 8.25 (1 H, d, J 0.8, PyrH), 7.45 (1 H, dd,
J 2.0, 8.4, PyrH), 7.30 (1 H, dd, J 4.0, 8.4, PyrH), 5.66 (1 H, s,
6-H), 5.39 (2 H, s, CH2), 3.93 (3 H, s, Me), 3.84 (3 H, s, Me), 2.34
(3 H, s, Me); 13C NMR (100 MHz, CDCl3) δ 178.7 (C), 178.2 (C),
159.6 (C), 154.8 (C), 141.9 (CH), 138.4 (CH), 138.1 (C), 128.9
(C), 124.1 (CH), 121.8 (CH), 121.3 (C), 116.3 (C), 106.8 (CH),
60.8 (CH2), 56.5 (Me), 32.4 (Me), 9.9 (Me); MS (ESI) 313 (MH+,
100%), 218 (76); found, MH+, 313.1174. C17H16N2O4 + H requires
313.1188.
1
54%) as an orange crystalline solid: mp 204-205 °C; H NMR
(400 MHz, CDCl3) δ 7.78 (1 H, dd, J 1.6, 8.0, ArH); 7.50 (1 H, dt,
J 1.2, 8.4, ArH), 7.41 (1 H, dd, J 0.6, 8.4, ArH), 7.00 (1 H, dt, J
0.6, 8.0, ArH), 5.63 (1 H, s, 6-H), 5.50 (2 H, s, CH2), 3.89 (3 H,
s, Me), 3.82 (3 H, s, Me), 2.26 (3 H, s, Me); 13C NMR (100 MHz,
CDCl3) δ 178.6 (C), 178.5 (C), 159.5 (C), 151.6 (C), 140.5 (C),
139.0 (C), 134.2 (CH), 128.7 (C), 125.4 (CH), 121.2 (C), 120.7
(CH), 116.4 (CH), 115.9 (C), 106.8 (CH), 62.1 (CH2), 56.2 (Me),
32.4 (Me), 9.9 (Me); MS (ESI) 379 (M + Na+, 100); found, M +
Na+, 379.0883. C18H16N2O6 + Na requires 379.0906.
5-Methoxy-1,2-dimethyl-3-(pyridin-4-yloxymethyl)indole-4,7-
dione 13. Using method B, 4-hydroxypyridine (40.8 mg, 0.43
mmol), triphenylphosphine (169 mg, 0.64 mmol), diethyl azodi-
carboxylate (0.079 mL, 0.50 mmol), and 3-hydroxymethyl-5-
methoxy-1,2-dimethylindole-4,7-dione (50 mg, 0.21 mmol) gave
after chromatography (ethyl acetate elution) the title compound
1
(25.5 mg, 38%) as an orange solid: mp 195-201 °C; H NMR

5786 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Colucci et al.
(400 MHz, CDCl3) δ 8.40 (2 H, br s, PyrH), 6.89 (2 H, d, J 4.8,
PyrH), 5.62 (1 H, s, 6-H), 5.34 (2 H, s, CH2), 3.89 (3 H, s, Me),
3.80 (3 H, s, Me), 2.29 (3 H, s, Me); 13C NMR (100 MHz, CDCl3)
δ 178.7 (C), 178.3 (C), 164.5 (C), 159.6 (C), 151.1 (CH), 138.1
(C), 128.9 (C), 121.2 (C), 115.9 (C), 110.6 (CH), 106.8 (CH), 60.1
(CH2), 56.5 (Me), 32.4 (Me), 9.9 (Me); MS (ESI) 218 (MH+,
100%), 313 (9); found, MH+, 313.1190. C17H16N2O4 + H requires
313.1188.
dole-4,7-dione (50 mg, 59%) as an impure yellow crystalline solid
(ca. 5% impurity): mp 182-184 °C (not recrystallized); 1H NMR
(500 MHz, CDCl3) δ 6.99-6.97 (2 H, m, ArH), 6.94-6.92 (2 H,
m, ArH), 5.65 (1 H, s, 5-H), 5.35 (2 H, s, CH2), 3.90 (3 H, s, Me),
3.81 (3 H, s, Me), 2.32 (3 H, s, Me); 13C NMR (125 MHz, CDCl3)
δ 184.5 (C), 171.4 (C), 160.0 (C), 155.9 (C), 140.4 (C), 138.3 (C),
132.6 (C), 127.1 (C), 124.0 (C), 120.0 (CH), 116.3 (CH), 106.8
(CH), 60.7 (CH2), 56.6 (Me), 32.7 (Me), 10.2 (Me); MS (ES) 375
(M + Na+, 100%), 218 (65); found, M + Na+, 375.1060.
C18H16N4O4 + Na requires 375.1069.
5-Methoxy-1,2-dimethyl-3-[1-(4-nitrophenoxy)ethyl]indole-
4,7-dione 14. Prepared as previously described.24
(c) To a solution of 3-(4-azidophenoxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione (25 mg, 0.07 mmol) in THF/water (4 mL,
9:1) was added polymer supported triphenylphosphine (0.047 g,
0.14 mmol). The reaction mixture was stirred at room temperature
for 48 h and then diluted with dichloromethane (25 mL), washed
with water (10 mL), dried (MgSO4), and purified by column
chromatography (gradient elution with 30-60% ethyl acetate/light
petroleum) to give a solid. Further purification by preparative
reverse phase HPLC (250 × 21.2 mm Polaris C18 column;
gradient elution 5-95% MeCN/water) gave the title compound (6
mg, 26%) as a red crystalline solid: mp 211-213 °C (from MeCN/
hexane); 1H NMR (400 MHz, CDCl3) δ 6.86-6.82 (2 H, m, ArH),
6.65-6.62 (2 H, m, ArH), 5.64 (1 H, s, 5-H), 5.23 (2 H, s, CH2),
3.90 (3 H, s, Me), 3.81 (3 H, s, Me), 3.42 (2 H, bs, NH2), 2.31 (3
H, s, Me); 13C NMR (125 MHz, CDCl3) δ 184.4 (C), 171.3 (C),
159.8 (C), 151.6 (C), 140.4 (C), 140.2 (C), 126.9 (C), 124.0 (C),
118.1 (C), 116.3 (CH), 116.2 (CH), 106.7 (CH), 61.0 (CH2), 56.5
(Me), 32.6 (Me), 10.2 (Me); MS (ES) 349 (M + Na+, 100%), 327
(45); found, M + Na+, 349.1156. C18H18N2O4 + Na requires
349.1164.
6-Methoxy-1,2-dimethyl-3-(4-nitrophenoxymethyl)indole-4,7-
dione 15. Using method A, 3-(hydroxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione 37 (0.045 g, 0.19 mmol), thionyl chloride
(0.70 mL, 9.57 mmol), 4-nitrophenol (0.080 g, 0.57 mmol), and
potassium carbonate (0.079 g, 0.57 mmol) gave after chromatog-
raphy (gradient elution with 30-60% ethyl acetate/light petroleum)
the title compound (0.057 g, 85%) as a yellow/orange crystalline
1
solid: mp 213-215 °C (decomp, from methanol); H NMR (300
MHz, CDCl3) δ 8.19 (2 H, d, J 9.3, ArH), 7.06 (2 H, d, J 9.3,
ArH), 5.68 (1 H, s, 5-H), 5.43 (2 H, s, CH2), 3.92 (3 H, s, Me),
3.83 (3 H, s, Me), 2.34 (3 H, s, Me); 13C NMR (75 MHz, CDCl3)
δ 184.9 (C), 171.8 (C), 163.9 (C), 160.4 (C), 142.0 (C), 140.8 (C),
127.5 (C), 126.3 (CH), 124.3 (C), 116.4 (C), 115.3 (CH), 107.1
(CH), 61.3 (CH2), 57.1 (Me), 33.1 (Me), 10.6 (Me); MS (FI) 356
(M+, 10%), 219 (28), 139 (100); found, M+, 356.1006. C18H16N2O6
requires 356.1008; Anal. (C18H16N2O6) H, N, C: calcd, 60.67;
found, 60.11.
6-Methoxy-1,2-dimethyl-3-(phenoxymethyl)indole-4,7-dione
16. To a solution of 3-(hydroxymethyl)-6-methoxy-1,2-dimethylin-
dole-4,7-dione 37 (0.04 g, 0.17 mmol) in dichloromethane (5 mL)
at 0 °C was added thionyl chloride (0.62 mL, 8.51 mmol) dropwise,
and the reaction mixture was stirred at room temperature for 1 h.
The solvent was removed in vacuo, and the crude product was used
directly in the next step without further purification.
6-Methoxy-1,2-dimethyl-3-(3-nitrophenoxymethyl)indole-4,7-
dione 18. Using method A, 3-(hydroxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione 37 (40 mg, 0.17 mmol), thionyl chloride
(0.62 mL, 8.51 mmol), 3-nitrophenol (71 mg, 0.51 mmol), and
potassium carbonate (71 mg, 0.51 mmol) gave after chromatography
(elution with 30% ethyl acetate/light petroleum) the title compound
(0.047 g, 78%) as a yellow crystalline solid: mp 203-205 °C (from
A solution of the crude chloride in DMF (10 mL) was added
dropwise to a stirring suspension of phenol (0.032 g, 0.34 mmol)
and sodium hydride (60% in oil; 0.014 g, 0.34 mmol) in DMF (10
mL) at 0 °C. After the addition, the mixture was allowed to stir at
room temperature for 5 h. The reaction mixture was quenched with
a saturated solution of ammonium chloride (10 mL), washed with
NaOH (2 M, 10 mL), HCl (2 M, 10 mL), water (2 × 20 mL), and
dried (MgSO4). The solvent was removed in vacuo, and the crude
product was purified by chromatography (30% ethyl acetate/light
petroleum) to give the title compound (23 mg, 47%) as a yellow
crystalline solid: mp 173-175 °C (decomp, from ethyl acetate/
1
ethyl acetate/light petroleum); H NMR (500 MHz, DMSO-d) δ
7.82-7.79 (2 H, m, ArH), 7.58 (1 H, t, J 8.0, ArH), 7.44 (1 H,
ddd, J 8.0, 2.5, 1.5, ArH), 5.76 (1 H, s, 5-H), 5.32 (2 H, s, CH2)
3.86 (3 H, s, Me), 3.77 (3 H, s, Me), 2.30 (3 H, s, Me); 13C NMR
(125 MHz, DMSO-d) δ 183.8 (C), 170.6 (C), 159.8 (C), 159.0 (C),
148.8 (C), 141.1 (C), 130.7 (C), 126.6 (C), 123.3 (C), 122.2 (CH),
115.6 (CH), 115.2 (C), 108.8 (CH), 106.4 (CH), 60.5 (CH2), 56.7
(Me), 32.5 (Me), 9.5 (CH3); MS (ES) 379 (M + Na+, 100%), 218
(16); found, M + Na+, 379.0906. C18H16N2O6 + Na requires
379.0901; Anal. (C18H16N2O6) H, N, C: calcd, 60.67; found, 59.99.
6-Methoxy-1,2-dimethyl-3-(2-nitrophenoxymethyl)indole-4,7-
dione 19. Using method A, 3-(hydroxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione 37 (20 mg, 0.21 mmol), thionyl chloride
(0.78 mL, 10.64 mmol), 2-nitrophenol (89 mg, 0.64 mmol), and
potassium carbonate (88 mg, 0.64 mmol) gave after column
chromatography (elution with 30% ethyl acetate/light petroleum)
the title compound (33 mg, 44%) as an orange crystalline solid:
mp 222-224 °C (from ethyl acetate); 1H NMR (500 MHz, CDCl3)
δ 7.78 (1 H, dd, J 8.1, 1.7, ArH), 7.49 (1 H, ddd, J 8.5, 7.4, 1.7,
ArH), 7.39 (1 H, dd, J 8.5, 0.9, ArH), 7.00, (1 H, m, ArH), 5.66 (1
H, s, 5-H), 5.53 (2 H, s, CH2), 3.91 (3 H, s, Me), 3.82 (3 H, s,
Me), 2.38 (3 H, s, Me); 13C NMR (100 MHz, CDCl3) δ 185.1 (C),
171.6 (C), 160.4 (C), 151.9 (C), 141.6 (C), 140.7 (C), 134.5 (CH),
127.3 (C), 125.7 (CH), 124.1 (C), 120.9 (CH), 116.6 (C), 116.3
(CH), 106.9 (CH), 62.1 (CH2), 57.0 (Me), 33.0 (Me), 10.6 (Me);
MS (ES) 379 (M + Na+, 100%); found, M + Na+, 379.0901.
C18H16N2O6 + Na requires 379.0906; Anal. (C18H16N2O6) H, N,
C: calcd, 60.67; found, 60.07.
1
light petroleum); H NMR (500 MHz, DMSO-d) δ 7.29 (2 H, m,
ArH), 6.99 (2 H, m, ArH), 6.94 (1 H, m, ArH), 5.77 (1 H, s, 5-H),
5.20 (2 H, s, CH2) 3.87 (3 H, s, Me), 3.78 (3 H, s, Me), 2.29 (3 H,
s, Me); 13C NMR (125 MHz, DMSO-d) δ 184.4 (C), 171.0 (C),
160.3 (C), 158.9 (C), 141.5 (C), 129.9 (CH), 126.9 (C), 123.7 (C),
121.0 (CH), 116.7 (C), 115.0 (CH), 106.9 (CH), 59.9 (CH2), 57.1
(Me), 32.9 (Me), 10.0 (Me); MS (ES) 334 (M + Na+, 100%), 218
(11); found, M + Na+, 334.1056. C18H17NO4 + Na requires
334.1055.
3-(4-Aminophenoxymethyl)-6-methoxy-1,2-dimethylindole-
4,7-dione 17. (a) To a solution of DMSO (10 mL) were added
4-iodophenol (1 g, 4.55 mmol), sodium azide (355 mg, 5.46 mmol),
copper(I) iodide (87 mg, 0.46 mmol), L-proline (105 mg, 0.91
mmol), and sodium hydroxide (36.4 mg, 0.91 mmol). The reaction
mixture was degassed and then stirred under an argon atmosphere
at 60 °C for 20 h. On cooling, the reaction mixture was diluted
with water (10 mL) and extracted into ethyl acetate (2 × 25 mL).
The solvent was removed in vacuo to give 4-azidophenol as a
brown/red oil (300 mg, 49%; lit.,29 pale red oil) that was taken
through to the next step without further purification.
(b) Using method A, 3-(hydroxymethyl)-6-methoxy-1,2-dimethyl-
indole-4,7-dione 37 (60 mg, 0.26 mmol), thionyl chloride (0.93
mL, 12.77 mmol), 4-azidophenol (103 mg, 0.77 mmol), and
potassium carbonate (106 mg, 0.77 mmol) gave after column
chromatography (gradient elution with 20-30% ethyl acetate/light
petroleum) 3-(4-azidophenoxymethyl)-6-methoxy-1,2-dimethylin-
6-Methoxy-1,2-dimethyl-3-(2,4-dinitrophenoxymethyl)indole-
4,7-dione 20. To a solution of 3-(hydroxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione 37 (40 mg, 0.17 mmol) and 2,4-
dinitrofluorobenzene (128 µL, 1.02 mmol) in THF (5 mL) at 0 °C
was added a solution of TBAF (1 M in THF, 1.02 mmol). The
reaction mixture was stirred for 18 h at room temperature. The

Indolequinones as Inhibitors of Human NQO1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5787
solvent was removed in vacuo, and the crude product was purified
by chromatography (elution with dichloromethane then 50%
dichloromethane/ethyl acetate) to give the title compound (54 mg,
79%) as a yellow crystalline solid: mp 184-186 °C (from ethyl
acetate/light petroleum); 1H NMR (400 MHz, DMSO-d) δ 8.68 (1
H, d, J 2.8, ArH), 8.37 (1 H, dd, J 2.8, 9.2, ArH), 7.60 (1 H, d, J
9.2, ArH), 5.69 (1 H, s, 5-H), 5.67 (2 H, s, CH2) 3.91 (3 H, s, Me),
3.84 (3 H, s, Me), 2.38 (3 H, s, Me); 13C NMR (100 MHz, CDCl3)
δ 184.9 (C), 171.3 (C), 160.1 (C), 156.0 (C), 141.4 (C), 140.1 (C),
139.3 (C), 129.0 (CH), 127.0 (C), 123.6 (C), 121.7 (CH), 115.9
(CH), 114.7 (C), 106.6 (CH), 62.5 (CH2), 56.8 (Me), 32.8 (Me),
10.3 (Me); MS (ES) 424 (M + Na+, 100%); found, M + Na+,
424.0735. C18H15N3O8 + Na requires 424.0756; Anal. (C18H15N3O8)
H, C: calcd, 53.87; found, 53.43; N: calcd, 10.47; found, 9.75.
3-(2-Fluoro-4-nitrophenoxymethyl)-6-methoxy-1,2-dimethyl-
indole-4,7-dione 21. Using method A, 3-(hydroxymethyl)-6-
methoxy-1,2-dimethylindole-4,7-dione 37 (0.040 g, 0.17 mmol),
thionyl chloride (0.62 mL, 8.51 mmol), 2-fluoro-4-nitrophenol
(0.080 g, 0.51 mmol), and potassium carbonate (0.071 g, 0.51
mmol) gave after chromatography (elution with 20% ethyl acetate/
light petroleum) the title compound (0.040 g, 63%) as a yellow
(0.78 mL, 10.6 mmol), 4-fluorophenol (71.6 mg, 0.64 mmol), and
potassium carbonate (88 mg, 0.64 mmol) gave after column
chromatography (gradient elution with 20-40% ethyl acetate/
hexane) the title compound (30 mg, 43%) as a yellow crystalline
solid: mp 171 °C (from ethyl acetate); 1H NMR (400 MHz, CDCl3)
δ 6.96-6.93 (4 H, m, ArH), 5.65 (1 H, s, 5-H), 5.28 (2 H, s, CH2),
3.91 (3 H, s, Me), 3.82 (3 H, s, Me), 2.32 (3 H, s, Me); 13C NMR
(100 MHz, CDCl3) δ 184.4 (C), 171.4 (C), 159.9 (C), 157.4 (d,
JCF 238, CF), 154.6 (C), 140.3 (C), 127.0 (C), 124.0 (C), 117.4
(C), 116.0 (d, JCF 8.1, CH), 115.8 (d, JCF 23.0, CH), 106.7 (CH),
60.9 (CH2), 56.6 (Me), 32.6 (Me), 10.2 (Me); MS (ES) 352 (M +
Na+, 100%), 218 (56); found, M + Na+, 352.0959. C18H16FNO4
+ Na requires 352.0961.
3-(2,4-Difluorophenoxymethyl)-6-methoxy-1,2-dimethylindole-
4,7-dione 25. Using method A, 3-(hydroxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione 37 (60 mg, 0.26 mmol), thionyl chloride
(0.93 mL, 12.77 mmol), 2,4-difluorophenol (99.5 mg, 0.77 mmol),
and potassium carbonate (106 mg, 0.77 mmol) gave after column
chromatography (gradient elution with 20-40% ethyl acetate/
hexane) the title compound (42 mg, 47%) as a yellow crystalline
solid: mp 173 °C (from ethyl acetate); 1H NMR (400 MHz, CDCl3)
δ 7.11 (1 H, td, J 9.2, 5.4, ArH), 6.82 (1 H, ddd, J 11.2, 8.3, 2.8,
ArH), 6.79-6.73 (1 H, m, ArH), 5.65 (1 H, s, 5-H), 5.34 (2 H, s,
1
crystalline solid: mp 229-231 °C (from chloroform/hexane); H
NMR (400 MHz, CDCl3) δ 8.04 (1 H, ddd, J 8.8, 2.8, 1.6, ArH),
7.96 (1 H, dd, J 10.8, 2.8, ArH), 7.34 (1 H, dd, J 8.8, 8.4, ArH),
5.68 (1 H, s, 5-H), 5.53 (2 H, s, CH2) 3.92 (3 H, s, Me), 3.83 (3 H,
s, Me), 2.36 (3 H, s, Me); 13C NMR (100 MHz, CDCl3) δ 184.6
(C), 171.4 (C), 160.0 (C), 151.5 (d, JCF 249, CF), 152.2 (d, JCF
11.3, C), 140.9 (C), 140.7 (C), 127.2 (C), 123.9 (C), 121.0 (d, JCF
3.8, CH), 115.6 (C), 114.1 (CH), 112.3 (d, JCF 23.8, CH), 106.7
(CH), 61.8 (CH2), 56.7 (Me), 32.7 (Me), 10.2 (Me); MS (ES) 397
(M + Na+, 79%), 218 (100); found, M + Na+, 397.0788. C18H15-
FN2O6 + Na requires 397.0812; Anal. (C18H15FN2O6) C, H, N:
calcd, 7.44; found, 6.92.
CH2), 3.91 (3 H, s, Me), 3.81 (3 H, s, Me), 2.35 (3 H, s, Me); 13
C
NMR (100 MHz, CDCl3) δ 184.4 (C), 171.4 (C), 159.9 (C), 156.7
(dd, JCF 242, 10.3, CF), 152.9 (dd, JCF 249, 12.1, CF), 142.8 (dd,
JCF 10.8, 3.3, C), 140.7 (C), 127.0 (C), 124.0 (C), 117.0 (dd, JCF
9.5, 2.9, CH), 116.9 (C), 110.4 (dd, JCF 22.4, 3.8, CH), 106.6 (CH),
104.7 (dd, 2J 26.7, 2J 22.3, CH), 62.3 (CH2), 56.6 (Me), 32.6 (Me),
10.1 (Me); MS (ES) 370 (M + Na+, 80%), 218 (100); found, M +
Na+, 370.0863. C18H15F2NO4 + Na requires 370.0867; Anal.
(C18H15F2NO4) C, H, N.
6-Methoxy-1,2-dimethyl-3-(2,4,6-trifluorophenoxymethyl)in-
dole-4,7-dione 26. Using method A, 3-(hydroxymethyl)-6-methoxy-
1,2-dimethylindole-4,7-dione 37 (60 mg, 0.26 mmol), thionyl
chloride (0.93 mL, 12.77 mmol), 2,4,6-trifluorophenol (113 mg,
0.77 mmol), and potassium carbonate (106 mg, 0.77 mmol) gave
after column chromatography (gradient elution with 20-40% ethyl
acetate/hexane) the title compound (70 mg, 75%) as a yellow
crystalline solid: mp 221 °C (from ethyl acetate/hexane); 1H NMR
(400 MHz, CDCl3) δ 6.67 (2 H, t, J 8.2, ArH), 5.60 (1 H, s, 5-H),
5.32 (2 H, s, CH2), 3.92 (3 H, s, Me), 3.79 (3 H, s, Me), 2.35 (3 H,
s, Me); 13C NMR (100 MHz, CDCl3) δ 183.9 (C), 171.5 (C), 159.7
(C), 157.3 (dt, JCF 246, 14.3, CF), 156.5 (ddd, JCF 250, 14.8, 7.7,
CF), 141.0 (C), 131.9 (dd, JCF 15.3, 5.0, C), 127.0 (C), 124.2 (C),
116.5, (C), 106.6 (CH), 100.6 (t, JCF 26.7, CH), 65.9 (CH2), 56.5
(Me), 32.6 (Me), 9.7 (Me); MS (ES) 388 (M + Na+, 57%), 218
(100); found, M + Na+, 388.0766. C18H14F3NO4 + Na requires
388.0773; Anal. (C18H14F3NO4) C, H, N.
3-(4-Cyanophenoxymethyl)-6-methoxy-1,2-dimethylindole-
4,7-dione 22. Using method A, 3-(hydroxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione 37 (60 mg, 0.26 mmol), thionyl chloride
(0.93 mL, 12.77 mmol), 4-hydroxybenzonitrile (91.1 mg, 0.77
mmol), and potassium carbonate (106 mg, 0.77 mmol) gave after
column chromatography (gradient elution with 20-30% ethyl
acetate/light petroleum) the title compound (53 mg, 62%) as a
yellow crystalline solid: mp 184 °C (from ethyl acetate); 1H NMR
(400 MHz, CDCl3) δ 7.57 (2 H, dd, J 7.0, 1.8, ArH), 7.05 (2 H,
dd, J 7.0, 1.8, ArH), 5.67 (1 H, s, 5-H), 5.38 (2 H, s, CH2), 3.92 (3
H, s, Me), 3.83 (3 H, s, Me), 2.32 (3 H, s, Me); 13C NMR (100
MHz, CDCl3) δ 184.4 (C), 171.4 (C), 161.7 (C), 159.9 (C), 140.3
(C), 134.0 (CH), 127.1 (C), 123.9 (C), 119.2 (C), 116.2 (C), 115.6
(CH), 106.7 (CH), 104.1 (C), 60.5 (CH2), 56.6 (Me), 32.7 (Me),
10.2 (Me); MS (ES) 359 (M + Na+, 80%), 218 (100); found, M +
Na+, 359.1017. C19H16N2O4 + Na requires 359.1007; Anal.
(C19H16N2O4) H, N, C: calcd, 67.85; found, 66.67.
6-Methoxy-1,2-dimethyl-3-[4-(trifluoromethyl)phenoxymethyl]-
indole-4,7-dione 23. Using method A, 3-(hydroxymethyl)-6-
methoxy-1,2-dimethylindole-4,7-dione 37 (60 mg, 0.26 mmol),
thionyl chloride (0.93 mL, 12.77 mmol), 4-trifluoromethylphenol
(124 mg, 0.77 mmol), and potassium carbonate (106 mg, 0.77
mmol) gave after column chromatography (gradient elution with
20-40% ethyl acetate/hexane) the title compound (75 mg, 78%)
as an orange crystalline solid: mp 150 °C (from ethyl acetate/
hexane); 1H NMR (400 MHz, CDCl3) δ 7.52 (2 H, d, J 8.8, ArH),
7.05 (2 H, d, J 8.8, ArH), 5.66 (1 H, s, 5-H), 5.37 (2 H, s, CH2),
3.91 (3 H, s, Me), 3.82 (3 H, s, Me), 2.32 (3 H, s, Me); 13C NMR
(100 MHz, CDCl3) δ 184.4 (C), 171.4 (C), 160.9 (C), 159.9 (C),
140.3 (C), 127.1 (C), 126.8 (q, JCF 3.7, CH), 124.4 (q, JCF 271,
CF3), 123.0 (q, JCF 32.7, C), 123.9 (C), 116.7 (C), 114.8 (CH),
106.7 (CH), 60.4 (CH2), 56.6 (Me), 32.6 (Me), 10.2 (Me); MS (ES)
402 (M + Na+, 49%), 218 (100); found, M + Na+, 402.0930.
C19H16F3NO4 + Na requires 402.0929; Anal. (C19H16F3NO4) C, H,
N.
6-Methoxy-1,2-dimethyl-3-(pyridin-2-yloxymethyl)indole-4,7-
dione 27. Using method B, diethyl azodicarboxylate (96 µL, 0.61
mmol), 3-(hydroxymethyl)-6-methoxy-1,2-dimethylindole-4,7-dione
37 (40 mg, 0.17 mmol), tri(2-furyl)phosphine (122 mg, 0.51 mmol),
and 2-hydroxypyridine (49.5 mg, 0.51 mmol) gave after column
chromatography (gradient elution with 10-40% ethyl acetate/light
petroleum) the title compound (22 mg, 41%) as an orange crystalline
1
solid: mp 178-180 °C (from ethyl acetate/light petroleum); H
NMR (500 MHz, CDCl3) δ 8.19 (1 H, dd, J 5.0, 1.5, ArH), 7.57-
7.54 (1 H, m, ArH), 6.88 (1 H, ddd, J 7.0, 5.0, 1.0, ArH), 6.74 (1
H, d, J 8.5, ArH), 5.66 (1 H, s, 5-H), 5.54 (2 H, s, CH2), 3.92 (3
H, s, Me), 3.81 (3 H, s, Me), 2.35 (3 H, s, Me); 13C NMR (100
MHz, CDCl3) δ 183.9 (C), 171.5 (C), 163.6 (C), 159.7 (C), 146.8
(CH), 140.3 (C), 138.5 (CH), 127.3 (C), 124.5 (C), 117.5 (C), 116.8
(CH), 111.1 (CH), 106.8 (CH), 57.9 (CH2), 56.5 (Me), 32.6 (Me),
10.0 (Me); MS (ES) 335 (M+Na+, 72%), 218 (100%); found, M
+ Na+, 335.1006. C17H16N2O4 + Na requires 335.1007; Anal.
(C17H16N2O4) H, N; C: calcd, 65.38; found, 62.00.
3-Methoxy-1,2-dimethyl-3-(3-pyridyloxymethyl)indole-4,7-di-
one 28. To a solution of 3-hydroxypyridine (26.9 mg, 0.277 mmol)
in DMF (3 mL) was added sodium hydride (60% dispersion in oil;
11 mg, 0.28 mmol) at 0 °C. This solution was stirred at room
3-(4-Fluorophenoxymethyl)-6-methoxy-1,2-dimethylindole-
4,7-dione 24. Using method A, 3-(hydroxymethyl)-6-methoxy-1,2-
dimethylindole-4,7-dione 37 (60 mg, 0.21 mmol), thionyl chloride

5788 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Colucci et al.
temperature for 45 min. The crude chloride (0.213 mmol) (prepared
as in method A) in DMF (4 mL) was added dropwise at 0 °C, and
the reaction mixture was stirred at room temperature for an
additional 3 h. The solvent was concentrated in vacuo, and the crude
product was dissolved in dichloromethane (50 mL), washed with
water (2 × 25 mL) and NaOH (25 mL), and dried (MgSO4), and
the solvent removed in vacuo. The crude product was purified by
column chromatography (gradient elution with 30-80% ethyl
acetate/light petroleum) to give the title compound (28 mg, 42%)
as a yellow crystalline solid: mp 179-181 °C (from ethyl acetate/
Inactivation of NQO1 in MIA PaCa-2 cells in culture, by these
indolequinones, was determined using the following procedure.
MIA PaCa-2 cells (1 × 106) were plated in 60 mm plates with 5
mL of complete medium. The following day the growth medium
was replaced with the indolequinone containing complete medium
for 1 h, after which the medium was removed, the cells were washed
with 10 mL of phosphate buffered saline, and the cells were then
scraped into 0.5 mL of 50 mM potassium phosphate buffer, pH
7.4, containing 250 mM sucrose and 5 µM FAD and briefly
sonicated on ice. NQO1 activity was determined in MIA PaCa-2
sonicates using the reduction of DCPIP. Briefly, sonicates (20 µL)
were added to 50 mM potassium phosphate buffer, pH 7.4,
containing 1 mg/mL bovine serum albumin and 0.2 mM NADH.
The reaction was started with the addition of 40 µM DCPIP (final
volume 1 mL), and the linear decrease in absorbance was monitored
at 600 nm for 1 min at 32 °C.
Growth Inhibition Assays. Growth inhibition in the human
pancreatic MIA PaCa-2 cancer cell line was measured using the
MTT colorimetric assay.20 In these studies, the MIA PaCa-2 cells
were seeded at 2 × 103 cells/well in 96-well plates, in triplicate,
for each indolequinone, and allowed to attach for 16 h. Medium
was removed by aspiration, and the MIA PaCa-2 cells were treated
with the appropriate indolequinone (6.25-3200 nM) in complete
medium for 4 and 72 h time periods. The medium was removed,
and MTT (50 µg) in medium (50 µL) was added to each well and
incubated for an additional 4 h. Cell viability was determined by
measuring the cellular reduction of MTT to the crystalline formazan
product, dissolved by the addition of DMSO (100 µL). Optical
density was determined at 550 nm using a Molecular Devices
Thermomax microplate reader. The IC50 values were defined as
the concentration of indolequinone that resulted in 50% reduction
in cell number compared to the DMSO-treated control, determined
from semilog plots of percentage of control versus indolequinone
concentration.
1
light petroleum); H NMR (400 MHz, CDCl3) δ 8.36 (1 H, bs,
ArH), 8.21 (1 H, apparent bd, ArH), 7.36 (1H, ddd, J 8.4, 1.6, 1.2,
ArH), 7.22 (1 H, dd, J 8.4, 4.8, ArH), 5.67 (1 H, s, 5-H), 5.38 (2
H, s, CH2), 3.92 (3 H, s, Me), 3.82 (3 H, s, Me), 2.33 (3 H, s, Me);
13C NMR (100 MHz, CDCl3) δ 184.4 (C), 171.4 (C), 159.9 (C),
154.7 (C), 142.1 (CH), 140.4 (C), 138.6 (CH), 127.1 (C), 124.0
(C), 123.9 (CH), 121.4 (CH), 116.6 (C), 106.7 (CH), 60.5 (CH2),
56.6 (Me), 32.6 (Me), 10.2 (Me); MS (EI) 313 (MH+, 100%);
found, MH+, 313.1178. C17H16N2O4 + H requires 313.1188.
6-Methoxy-1,2-dimethyl-3-(pyridin-4-yloxymethyl)indole-4,7-
dione 29. Using method B, diethyl azodicarboxylate (96 µL, 0.61
mmol), 3-(hydroxymethyl)-6-methoxy-1,2-dimethylindole-4,7-dione
37 (40 mg, 0.17 mmol), tri(2-furyl)phosphine (122 mg, 0.51 mmol),
and 4-hydroxypyridine (49.5 mg, 0.51 mmol) gave after column
chromatography (gradient elution with 10-40% ethyl acetate/light
petroleum) the title compound (0.022 g, 41%) as an orange
crystalline solid: mp 161-163 °C (from ethanol/light petroleum);
1H NMR (400 MHz, CDCl3) δ 8.42 (2 H, d, J 5.0, ArH), 6.92 (2
H, d, J 5.0, ArH), 5.67 (1 H, s, 5-H), 5.39 (2 H, s, CH2), 3.92 (3
H, s, Me), 3.83 (3 H, s, Me), 2.32 (3 H, s, Me); 13C NMR (100
MHz, CDCl3) δ 184.5 (C), 171.4 (C), 164.7 (C), 160.0 (C), 150.8
(CH), 140.4 (C), 127.2 (C), 123.9 (C), 116.1 (C), 110.7 (CH), 106.7
(CH), 60.2 (CH2), 56.6 (Me), 32.7 (Me), 10.2 (Me); MS (ES) 313
(MH+, 17%), 218 (100); found, MH+, 313.1177. C17H16N2O4 + H
requires 313.1188.
Acknowledgment. We acknowledge support from the
FORCE cancer charity (Ph.D. studentship to M.A.C.), the
Association for International Cancer Research (Ph.D. studentship
to A.C.), and National Institute of Health Grant RO1 CA114441.
Biology. Materials. NADH, FAD, DCPIP, MTT, and bovine
serum albumin were obtained from Sigma Chemical (St. Louis
MO). rhNQO1 was purified from E. coli using Cibacron blue
affinity chromatography, as previously described,30 and had a
specific activity of 800 µmol DCPIP/min/mg protein. For all
biological assays, stock concentrations of indolequinones (5 mM)
were prepared in dimethyl sulfoxide and stored in the dark at
32 °C.
Cell Lines. MIA PaCa-2 human pancreatic carcinoma cells were
obtained from ATCC (Manassas VA). MIA PaCa-2 cells were
grown in Dulbecco’s modified Eagle’s medium adjusted to contain
4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose,
10% (v/v) fetal bovine serum, 2.5% (v/v) horse serum, 100 units/
mL penicillin and 100 µg/mL streptomycin in a humidified
incubator containing 5% carbon dioxide at 37 °C.
NQO1 Inhibition Studies. The mechanism-based inactivation
of rhNQO1 by this indolequinone series was assayed using the
following methods. In these reactions (0.5 mL final volume),
rhNQO1 (2 µg/mL) was incubated with 0.1-5.0 µM of the
indolequinone in the absence and presence of 0.2 mM NADH in
50 mM potassium phosphate buffer, pH 7.4, containing 125 mM
NaCl and 1 mg/mL bovine serum albumin at 32 °C. After 5 min,
a 50 µL aliquot was removed and diluted 100-fold in stop buffer
(50 mM potassium phosphate buffer, pH 7.4, containing 250 mM
sucrose, 5 µM FAD and 0.1% (v/v) Tween-20), and NQO1 activity
was measured using the reduction of DCPIP. Briefly, 960 µL was
transferred to a cuvette with 0.2 mM NADH. The reaction was
started with the addition of 40 µM DCPIP (final volume 1 mL),
and the linear decrease in absorbance was monitored at 600 nm
for 1 min at 32 °C.
Note Added after ASAP Publication. This manuscript was
released ASAP on October 18, 2007 with errors in Figure 1
and Table 1. The correct version was posted on October 31,
2007.
Supporting Information Available: General experimental
details, experimental procedures for compounds 31-37, elemental
analyses, full characterization data for compounds 6, 7, 10-13,
and 15-29, HPLC data, and cytotoxicity data on phenols and
hydroxypyridines. This material is available free of charge via the
Internet at http://pubs.acs.org.
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