Indolequinone antitumour agents: Correlation between quinone structure and rate of metabolism by recombinant human NAD(P)H:quinone oxidoreductase

Article abstract of DOI:10.1039/b703370b

A series of indolequinones bearing a range of substituents at the (indol-2-yl)methyl position has been synthesized. The ability of these indolequinones to act as substrates for recombinant human NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reduct

Full text of DOI:10.1039/b703370b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Indolequinone antitumour agents: correlation between quinone structure
and rate of metabolism by recombinant human NAD(P)H:quinone
oxidoreductase†
Jeffery J. Newsome,a Elizabeth Swann,a Mary Hassani,b Kurtis C. Bray,b Alexandra M. Z. Slawin,c
Howard D. Beallb and Christopher J. Moody*a,d
Received 6th March 2007, Accepted 29th March 2007
First published as an Advance Article on the web 20th April 2007
DOI: 10.1039/b703370b
A series of indolequinones bearing a range of substituents at the (indol-2-yl)methyl position has been
synthesized. The ability of these indolequinones to act as substrates for recombinant human
NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumour cells, was
determined, along with their toxicity to an isogenic tumour cell line pair that is differentiated as either
NQO1-expressing cells (BE-NQ) or NQO1-null cells (BE-WT). Overall, the 2-substituted
indolequinones were relatively poor substrates for NQO1. Hydroxymethyl groups at C-2 led to higher
rates of reduction, a finding that was observed previously with 3-hydroxymethylated indolequinones.
Predictably, the best substrate had an electron-withdrawing ester group at the indole-2-position. The
indolequinones were generally non-toxic to both cell lines with the exception of those quinones that had
methylaziridine groups at the indole-5-position. These compounds could form DNA cross-links when
activated by reduction and were up to 3-fold more toxic to the BE-NQ cells than the BE-WT cells.
Scheme 1. This sequential generation of electrophilic centres can
hence lead to cross-linking of DNA. Likewise, the indolequinone
Introduction
The term bioreductive drug describes a range of anticancer
diol EO9 3 is also a potential bi-functional alkylating agent after
agents that are inactive in their own right, but upon metabolic
loss of water from both the indole 3-carbinyl and vinylogous indole
2-carbinyl positions (Scheme 1).6,7 The compound has recently re-
entered phase I clinical trials.8
reduction are transformed into a cytotoxic species. The drugs act as
substrates for one or more of the reductases that are present in most
cells, and could be effective against tumours in which the activating
reductase enzymes are upregulated. Bioreductive drugs based on
N-oxides and on nitroarenes are known, but it is the quinones
that form the best studied group of compounds. Quinones are
readily reduced in vitro and in vivo, and the selective activation
of agents such as mitomycin C 1 (MMC), a clinically used
antitumour antibiotic and the archetypical quinone bioreductive
alkylating agent, has been widely studied.2–5 Upon enzymatic
reduction the quinone forms a semiquinone or hydroquinone that
can subsequently form a reactive electrophilic intermediate by
elimination of an appropriate leaving group. In the case of MMC
1, the loss of methanol occurs first to form, after reoxidation,
the indolequinone (mitosene) derivative 2, which upon further
reductive activation can generate electrophilic centres at C-1 and
C-10, the indole-2-carbinyl and 3-carbinyl positions as outlined in
aDepartment of Chemistry, University of Exeter, Stocker Road, Exeter,
UK EX4 4QD. E-mail: c.j.moody@nottingham.ac.uk; Fax: +44 115 951
3564
Scheme 1
bDepartment of Biomedical and Pharmaceutical Sciences, The University of
Montana, 32 Campus Drive #1552, Missoula, MT 59812-1552, U.S.A.
As a result of their structural relationship to MMC, the
indolequinones have been widely studied, particularly because of
the ability of 3-indolyl carbinyl substituents X in such compounds
to undergo an elimination process upon either one- or two-
electron reductive-activation. The resulting iminium species is
then a potential electrophile capable of DNA-alkylation or other
cellular-damaging events (Scheme 2).6,7,9–12 However, bioreductive
cSchool of Chemistry, University of St. Andrews, Fife, Scotland, UK KY16
9ST
dSchool of Chemistry, University of Nottingham, University Park, Notting-
ham, UK NG7 2RD
† Part 3. For part 2, see reference 1. Electronic supplementary informa-
tion (ESI) available: experimental details and characterization data for
synthetic intermediates. See DOI: 10.1039/b703370b
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Scheme 2
drugs such as indolequinones are also of interest since they could
act as reductively activated drug delivery vehicles by releasing a
variety of leaving groups X in a reductive environment.13 Thus
they may have secondary biological effects due to the eliminated
molecule XH, in addition to the cytotoxic iminium derivative
formed on reduction and elimination (Scheme 2).14–17
As outlined in Scheme 2, indolequinones Q are capable of
efficiently eliminating a range of leaving groups X from the
(indolyl-3-yl)methyl position following two-electron reduction
to the hydroquinone (QH2) or one-electron reduction to the
semiquinone radical (Q•−).15 Such one- or two-electron reduc-
tions would be catalyzed in biological systems by, for example,
NADPH:cytochrome c (P450) reductase11 or NAD(P)H:quinone
oxidoreductase 1 (NQO1, DT-diaphorase) respectively.1,18–21
To date the focus has been the fragmentation of groups from
the (indol-3-yl)methyl position of indolequinones (Scheme 2),
notwithstanding the fact that in both MMC 1 and EO9 3
elimination from (indol-2-yl)methyl position also occurs. The role
of 2-indolyl substituents in other indolequinones is less clear, since
relatively few studies have been reported thus far. For example, no
elimination of the carboxylate leaving group – 2-acetoxybenzoate
(aspirin) – occurred upon (one-electron) radiolytic reduction of the
2-substituted indolequinone 4 or the corresponding ‘vinylogous’
derivative 5. On the other hand, the 3-substituted analogue 6
underwent efficient fragmentation upon reduction (Scheme 3).22
Similar results were observed with indolequinone phosphorami-
date prodrugs: upon one-electron reduction, elimination from
the 2-substituted compound 7 was slow in comparison to the
3-substituted analogue 8 (Scheme 3). In contrast, the phospho-
ramidate was rapidly released from both the 2- and 3-substituted
indolequinones 7 and 8 upon two-electron reduction suggesting
that potential drug delivery mechanisms from the indolequinone
2-position proceed better from the hydroquinone QH2.23,24 In order
to investigate this suggestion more fully, we have prepared a series
Scheme 3
of indolequinones bearing a range of substituents at the 2-position
and investigated their metabolism by the two-electron reductase
NQO1.
As already mentioned, NQO1 (EC 1.6.99.2) is an obligate
2-electron reductase that is characterized by its ability to use
either NADH or NADPH as cofactor,25,26 and is involved the
reductive activation of anticancer agents such as mitomycin C
(MMC).19,27–29 Recently the 3-dimensional structure of human
NQO1 has been solved by two research groups, and resolved to
2.3 A and 1.7 A respectively. The structures of the complex
of enzyme with duroquinone31 and potentially chemotherapeutic
quinones32 have also been solved. We have investigated the
correlation between quinone structure and rate of metabolism by
NQO1 and toxicity towards human tumour cell lines, and have
studied both quinolinequinones,33,34 and indolequinones bearing
a range of substituents at the 3-position.1,20 The present studies on
indolequinones bearing a range of substituents at the 2-position
extend and complement our earlier work.
30
31
˚
˚
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using our modified version of the Bischler indole synthesis.36,37
Nitration occurred selectively at the indole-4-position to give the
nitroindoles 16, reduction of which gave the 4-aminoindoles 17.
Reduction of the ester group with lithium aluminium hydride gave
the 4-amino-3-hydroxymethylindoles that, without purification,
were oxidized to the quinones 11a and 11b in good yield
(Scheme 5). The 2-aryloxymethyl derivatives were prepared in
an analogous manner to their 3-substituted analogues. Thus
Mitsunobu reaction of the alcohols with 4-nitrophenol gave the
2-(4-nitrophenoxymethyl) quinones 13a and 13b.
Results and discussion
Synthetic chemistry
The key indolequinones for the present study are shown in Fig. 1.
Indolequinone 9 was obtained from the known 4-benzyloxy-5-
methoxy-1-methylindole-2-methanol10 by hydrogenolysis of the
benzyl protecting group followed by oxidation with potassium
nitrosodisulfonate (Fremy’s salt) (Scheme 4). Similarly indole-
quinone 10 was prepared from the known 4-benzyloxy-5-methoxy-
1-methylindole-2-carboxaldehyde35 by addition of methylmagne-
sium bromide, hydrogenolysis of the benzyl group and Fremy’s
salt oxidation (Scheme 4).
Fig. 1
Scheme 5
The synthesis of quinones 12 and 14 started with the known 5-
methoxy-1,3-dimethylindole 18, readily available from a Bischler
indole synthesis.38 Vilsmeier formylation at the 2-position was
followed by nitration under controlled conditions to prevent over
reaction to give the 4-nitroindole 19 (Scheme 6). Wittig reaction
of 19 gave the trans-alkene 20 in excellent yield. Reduction of
the nitro and ester groups with tin and hydrochloric acid and
di-isobutylaluminium hydride (DIBAL) respectively gave the 4-
aminoindole 21 which was oxidized to the indolequinone 12
using Fremy’s salt (Scheme 6), although some over-oxidation
(21%) of the allylic alcohol to the corresponding aldehyde also
occurred. The allylic alcohol 12 was selected for study because
of its resemblance to EO9 3. Indolequinone 14 was also prepared
from the 4-nitroindole-2-carboxaldehyde 19 as shown in Scheme 6.
Wittig reaction to give the vinyl derivative 22 was followed by a
Scheme 4
The 3-alkyl indolequinones 11 were obtained from the corre-
sponding 3-alkyl-5-methoxy-1-methylindole-2-esters 15, prepared
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Scheme 6
2-step reduction to give the 4-aminoindole 23, and oxidation to
the quinone 14 with Fremy’s salt.
the corresponding 3-substituted derivative 33 was also prepared
from the known indolequinone 3020 by way of the Mitsunobu
product 31 and the alcohol 32 as shown in Scheme 8. Finally
three further indolequinones were prepared starting from ethyl 5-
methoxy-1-methylindole-2-carboxylate 34. Vilsmeier formylation
to give the 3-carboxaldehyde 35 was followed by nitration at C-
4, both steps proceeding in high yield (Scheme 9). Reduction
of the nitro compound 36 gave the corresponding amine 37
the structure of which was confirmed by X-ray crystallography
(Fig. 2).41 Reduction of both aldehyde and ester groups using
lithium aluminium hydride was followed by oxidation to the 2,3-
bis(hydroxymethyl)indolequinone 38, a compound that has been
prepared previously by a different route.42 Displacement of the
Compounds 24–26 were prepared from the quinone 11a
by standard transformations as outlined in Scheme 7. The
quinone 29 bearing an extended linker that can eliminate
−OC6H4CH2OCONH2 (that can further fragment to give a p-
quinonemethide, CO2 and ammonia)39 was also prepared from
quinone 11a as shown in Scheme 8. Thus Mitsunobu reaction
with 4-(tert-butyldimethylsiloxymethyl)phenol40 to give 27, was
followed by removal of the silicon protecting group and conversion
of the alcohol 28 into the carbamate 29. As a comparison,
Fig. 2 X-ray crystal structure of ethyl 4-amino-3-formyl-5-methoxy-
1-methylindole-2-carboxylate 37.
Scheme 7
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Scheme 8
5-methoxy group with 2-methylaziridine gave the corresponding
aziridinylindolequinone 39. Amine 37 was also converted into the
indolequinone 40 by reduction of the aldehyde and oxidation to
the quinone (Scheme 9).
Enzyme studies
In related work, using physical chemical kinetic studies, we
have demonstrated that reductive elimination from the (indol-2-
yl)methyl position occurs most readily via the hydroquinone,43
which in biological systems is most readily accessed by two-
electron reductive activation by the enzyme NQO1. Therefore we
have examined the ability of a number of our novel 2-substituted
indolequinones to act as substrates for NQO1. Previous studies
have demonstrated that indolequinones bearing good leaving
groups at the (indol-3-yl)methyl position are poor substrates for
the two-electron reducing enzyme NQO1.20,21 In fact the indole-
quinone analogous to compound 13a with the 4-nitrophenoxy
group at the (indol-3-yl)methyl position, i.e. 5-methoxy-1,2-
dimethyl-3-(4-nitrophenoxy)methylindole-4,7-dione, a compound
known as ES936, is a potent mechanism-based inhibitor of the
enzyme.44–46
In our earlier studies on indolequinone metabolism by recom-
binant human NQO1, we used an HPLC system that is capable
of quantifying both NADH oxidation and quinone reduction.1,20
To simplify comparisons with these earlier compounds, we have
also used this method in the present study. Quinone reduction is
reversible due to redox cycling of the hydroquinone, so results
(Table 1) are reported as lmol NADH oxidized min−1 mg−1
NQO1. This HPLC method gives average rates of reduction over a
30–40 minute period. An alternative spectrophotometric method
for determining quinone metabolism uses cytochrome c as the
terminal electron acceptor and gives initial rates of reduction.34
This assay generally gives higher reduction rates than the HPLC
Scheme 9
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Table 1 Metabolism of indolequinones by recombinant human NQO1 and cytotoxicity of representative indolequinones to BE-NQ (NQO1-rich) and
BE-WT (NQO1-deficient) human colon carcinoma cell lines
Metabolism
BE-NQ
IC50 (lM)
BE-WT
IC50 (lM)
Cpd
X
Y
R2
R3
(lmol min−1 mg−1
)
9
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
MeAzc
MeAzc
OMe
OMe
OMe
MeAzc
OMe
H
Me
H
H
H
Me
Et
Me
Me
Et
Me
20.4 2.7
0.44 0.08
2.49 1.27
0.19 0.02
0.35 0.06
0.23 0.04
0.09 0.02
0.41 0.08
0.30 0.02
0.27 0.03
0.70 0.09
0.49 0.08
0.53 0.08
12.0 2.0
0.61 0.14
65.8 5.0
10
11a
11b
12
>50
>50
OH
H
a
a
13a
13b
14
OArb
OArb
H
OCONH2
OH
H
H
Me
H
H
H
H
H
H
24
Me
Me
>50
29.8 8.5
17.6 2.1
>50
>50
>50
25
26
OCONH2
H
Me
29
CH2OArd
Me
33
OArd
OH
3b8
39
CH2OH
CH2OH
CH2OH
>50
4.93 1.34
>50
15.4 3.8
OH
H
e
e
40
a
Indole 2-substituent is CH CHCH2OH. b Ar = 4-nitrophenyl. c MeAz = 2-methylaziridinyl. d Ar = 4-carbamoylmethylphenyl. e Indole 2-substituent is
=
COOEt.
method, but the relative order of metabolism is essentially the
same with two methods (data not shown). Cytotoxicity studies
were also performed on representative quinones with cell survival
being measured using the MTT colorimetric assay. As in our
previous work,1 we have used the BE human colon carcinoma
cell line stably transfected with human NQO1 cDNA.47 The wild
type BE cells have no measurable NQO1 activity whereas activity
in the transfected cells was 664 49 nmol min−1 mg−1 total cell
protein using dichlorophenolindophenol as the standard electron
acceptor. Hence, we have compared quinone toxicity (Table 1) in
the wild-type BE cells (BE-WT) and the NQO1-transfected BE
cells (BE-NQ).
The enzyme data show that the new indolequinones are
generally quite poor substrates for NQO1, although compounds
with leaving groups are substrates as opposed to inhibitors. Com-
pounds 13a and 24 with leaving groups at the (indol-2-yl)methyl
position had no inhibitory effect on NQO1 (data not shown)
whereas analogous 3-substituted indolequinones (including the
aforementioned ES936) completely inactivated the enzyme.20 Still,
these two compounds, 13a and 24, were relatively poor substrates
for NQO1. The NQO1 active site can accommodate a range of
substituents at the indole-2-position since these substituents are
normally oriented towards the entrance to the active site.21,32 Other
factors appear to be more important than steric bulk at C-2 to
the metabolism of these compounds. For example, most of the
better substrates have hydroxymethyl groups at C-2, a finding
that was also observed with the C-3 substituted indolequinones20
suggesting the possibility that hydrogen bonding with active site
residues may be important. The best substrate, 40, has an electron-
withdrawing ester group at C-2, demonstrating the importance of
electronic effects on quinone reduction by NQO1. With regard to
theindole-3-position, stericeffectsdoappeartobemoreimportant
here since ethyl substituents lead to much lower rates of reduction
than methyl substituents.
A selection of indolequinones was chosen for cytotoxicity
evaluation in the isogenic cell lines BE-NQ and BE-WT. As
expected, only indolequinones 25, 26 and 39 showed detectable
toxicity to either cell line, since they contain methylaziridine
alkylating groups at C-5. With potential leaving groups at C-2
and/or C-3, they also have the ability to form DNA cross-links, a
more toxic lesion than simple alkylation. All three methylaziridinyl
indolequinones were selectively toxic to the NQO1-transfected
BE-NQ cells. Indolequinones 26 and 39 were >3 times more toxic
to the BE-NQ cells than the BE-WT cells whereas 25 was ca.
1.6 times more toxic. Selectivity was related to substrate efficiency
as 26 and 39 were also better substrates for NQO1 than 25.
The results presented here complement our previous work on
bioreductive activation of indolequinone antitumour agents by
NQO1.1,20 Novel indolequinones have been synthesized, character-
ized and studied biologically as substrates for recombinant human
NQO1 and for their toxicity to NQO1-rich tumour cells. These
data will be valuable for the design and development of NQO1-
directed antitumour agents that are either intended to act directly
as cytotoxic agents or to release a secondary cytotoxic metabolite
by reductive fragmentation.
Experimental
Chemistry
General procedures. Commercially available reagents were
used throughout without purification unless otherwise stated.
Light petroleum refers to the fraction with bp 40–60 ◦C and was
distilled before use. Ether refers to diethyl ether. Reactions were
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routinely carried out under a nitrogen or argon atmosphere. Ana-
lytical thin layer chromatography was carried out on aluminium-
backed plates coated with Merck Kieselgel 60 GF254, and visual-
ized under UV light at 254 and/or 360 nm. Flash chromatography
was carried out using Merck Kieselgel 60 H silica or Matrex silica
60. Fully characterized compounds were chromatographically
homogeneous. Infrared spectra were recorded in the range 4000–
600 cm−1 using Nicolet Magna FT-550 or Perkin Elmer FT-1600
spectrometers. NMR spectra were carried out on Bruker 300
and 400 MHz instruments (1H frequencies, corresponding 13C
frequencies are 75 and 100 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 a Micromass
GCT TDF High Resolution mass spectrometer, or at the EPSRC
Mass Spectrometry Service (Swansea).
carboxaldehyde35 (0.135 g, 0.46 mmol) in THF (20 ml) at −10 ◦C
was added methylmagnesium bromide (3 M; 0.4 ml, 1.2 mmol).
The mixture was stirred for 1 h, quenched with saturated am-
monium chloride solution and extracted with ethyl acetate. The
organic layer was dried (MgSO4) and concentrated. The crude
product was used directly in the next step without purification.
To a solution of the benzyl ether in ethyl acetate was added a
catalytic quantity of palladium on carbon (5%; 15 mg) and stirred
overnight under an atmosphere of hydrogen. The crude mixture
was filtered through a pad of Celite. The solvent was removed
in vacuo and used directly in the next step without purification.
To a solution of the phenol in acetone (20 ml) was added a
solution of potassium nitrosodisulfonate (0.35 g, 1.3 mmol) in
sodium dihydrogen phosphate buffer (0.3 M; 20 ml). The mixture
was stirred at room temperature overnight. The acetone was
removed in vacuo, and the residue was extracted with ethyl acetate
and washed with saturated ammonium chloride solution. The
organic layer was dried (MgSO4) and concentrated. The residue
was purified by chromatography (ethyl acetate) and recrystallized
(ethyl acetate/hexane) to yield the title compound (0.76 g, 71%),
as an orange solid, mp 166–168 ◦C; (Found: C, 60.3; H, 5.5;
N, 5.6. C12H13NO4.0.2H2O requires C, 60.3; H, 5.6; N, 5.9%);
(Found: MH+, 236.0924. C12H13NO4 + H requires 236.0923);
kmax (DMSO)/nm 444 (log e 3.18), 336 (3.52), 280 (4.04); mmax
(KBr)/cm−1 3483, 2976, 2930, 1671, 1634, 1588; dH (300 MHz;
CDCl3) 6.48 (1 H, s, 3-H), 5.55 (1 H, s, 6-H), 4.85 (1 H, q, J 6.5,
CHMe), 3.97 (3 H, s, OMe), 3.78 (3 H, s, NMe), 1.60 (3 H, d,
J 6.5, CHMe); OH not observed; dC (100 MHz; CDCl3) 179.2,
177.1, 160.0, 143.5, 130.5, 123.5, 107.2 (CH), 105.0 (CH), 61.9
(CH), 56.4 (Me), 32.9 (Me), 22.1 (Me); m/z (EI) 235 (M+, 33%),
220 (40), 192 (17).
The synthesis of the 16 compounds described in Table 1 is
included below. The experimental details and characterization
data for all other compounds are in the Electronic Supplementary
Information†.
2-Hydroxymethyl-5-methoxy-1-methylindole-4,7-dione 9. (a) A
catalytic quantity of palladium/carbon (10%; ca. 25 mg) was
added to a solution of 4-benzyloxy-5-methoxy-1-methylindole-2-
methanol10 (0.235 g, 0.79 mmol) in methanol (30 ml). The reaction
mixture was stirred under an atmosphere of hydrogen for 12 h.
The catalyst was removed by filtering the crude mixture through
a pad of Celite. The filtrate was evaporated in vacuo and the
residue purified by chromatography (9/1 dichloromethane/ethyl
acetate) to yield 4-hydroxy-5-methoxy-1-methylindole-2-methanol
as a◦n off white crystalline solid, (0.095 g, 58%), mp 168–
171 C; (Found: M+, 207.0898. C11H13NO3 requires 207.0895);
mmax (KBr)/cm−1 3447, 2947, 1514; dH (300 MHz; d6-acetone) 7.60
(1 H, s, ArOH), 6.94 (1 H, d, J 8.7, ArH), 6.80 (1 H, dd, J 8.7, 0.8,
ArH), 6.42 (1 H, d, J 0.6, 3-H), 4.74 (2 H, d, J 5.5, CH2OH), 4.14
(1 H, t, J 5.5, CH2OH), 3.82 (3 H, s, OMe), 3.75 (3 H, s, NMe);
dC (100 MHz; d6-acetone) 139.5, 139.4, 139.2, 135.7, 117.8, 110.6
(CH), 99.6 (CH), 97.3 (CH), 58.0 (Me), 56.4 (CH2), 29.2 (Me);
m/z (EI) 207 (M+, 67%), 192 (98), 164 (34).
2-Hydroxymethyl-5-methoxy-1,3-dimethylindole-4,7-dione 11a.
To a suspension of lithium aluminium hydride (0.174 g, 4.58 mmol)
in THF (30 ml) at 0 ◦C was added a solution of methyl 4-
amino-5-methoxy-1,3-dimethylindole-2-carboxylate 17a (0.379 g,
1.52 mmol) in THF (15 ml). The reaction was allowed to warm
to room temperature and stirred for 30 min. The mixture was
◦
cooled to 0 C and quenched by the addition of water (0.5 ml),
sodium hydroxide (1 M; 0.5 ml) and silica gel (5 g). The granular
precipitate was filtered off through a pad of Celite. The filtrate
was dried (MgSO4) and concentrated in vacuo to give the alcohol,
which was used directly in the next step without purification or
characterization. To a solution of the above 4-aminoindole in
acetone (100 ml) was added a solution of potassium nitrosodisul-
fonate (1.23 g, 4.59 mmol) in sodium dihydrogen phosphate buffer
(0.3 M; 100 ml). The mixture was stirred at room temperature for
1 h. The acetone was removed in vacuo, the residue was extracted
with dichloromethane and washed with water. The organic layer
was dried (Na2SO4) and concentrated. The crude product was
purified by chromatography, eluting with dichloromethane/ethyl
acetate (3:1), to give the title compound as an orange solid
(0.210 g, 58%), mp 223–224 ◦C (from ethyl acetate/hexane);
(Found: C, 59.9; H, 5.7; N, 5.8. C12H13NO4.0.3H2O requires C,
59.9; H, 5.7; N, 5.8%); (Found: M+, 235.0845. C12H13NO4 requires
235.0844); kmax (MeOH)/nm 448 (log e 3.26), 356 (3.43), 280 (4.05);
mmax (KBr)/cm−1 3452, 3053, 2935, 1665, 1642, 1606, 1503; dH
(300 MHz; CDCl3) 5.63 (1 H, s, 6-H), 4.65 (2 H, d, J 5.4, CH2OH),
4.02 (3 H, s, OMe), 3.81 (3 H, s, NMe), 2.33 (3 H, s, Me), 1.67
(b) To a solution of the above phenol (0.078 g, 0.37 mmol) in
acetone (10 ml) was added a solution of potassium nitrosodisul-
fonate (0.31 g, 1.1 mmol) in sodium dihydrogen phosphate buffer
(0.3 M; 10 ml). The mixture was stirred at room temperature for
1 h, extracted with ethyl acetate, the organic layer washed with
water, dried (MgSO4) and concentrated. The crude product was
purified by chromatography (9/1 dichloromethane/ethyl acetate)
to yield the title compound as an orange crystalline solid (0.46 g,
55%), mp 208–210 ◦C (from dichloromethane/ether); (Found: C,
59.6; H, 4.9; N, 6.2. C11H11NO4 requires C, 59.7; H, 5.0; N, 6.3%);
kmax (MeOH) 440 (log e 2.98), 336 (3.32), 280 (3.87) nm; mmax
(KBr)/cm−1 3380, 2930, 1679, 1632, 1587; dH (300 MHz; CDCl3)
6.53 (1 H, s, 3-H), 5.64 (1 H, s, 6-H), 4.66 (2 H, d, J 4.2, CH2OH),
4.00 (3 H, s, OMe), 3.82 (3 H, s, NMe), 2.01 (1 H, br t, CH2OH); dC
(100 MHz; CDCl3) 179.2, 177.1, 160.1, 139.6, 130.8, 123.5, 107.6
(CH), 107.2 (CH), 56.5 (Me), 56.3 (CH2), 32.8 (Me); m/z (EI) 221
(M+, 8%), 150 (16), 122 (24), 69 (100).
2-(1-Hydroxyethyl)-5-methoxy-1-methylindole-4,7-dione 10.
To
a
solution of 4-benzyloxy-5-methoxy-1-methylindole-2-
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(1 H, t, J 5.4, CH2OH); dC (75 MHz; CDCl3) 179.1, 178.0, 160.1,
136.2, 129.6, 121.4, 120.7, 107.0 (CH), 56.5 (Me), 53.1 (CH2), 32.8
(Me), 9.9 (Me); m/z (CI) 236 (MH+, 96%), 220 (22), 206 (40),
200 (100).
11.3 (Me); m/z (EI) 261 (M+, 100%), 244 (31), 230 (53), 218 (80),
187 (29), 176 (32), 158 (34).
Mitsunobu reactions: general method. Diethyl azodicarboxy-
late (64 ll, 0.41 mmol) was added to a stirred solution of the
2-hydroxyalkylindolequinone (0.12 mmol), 4-nitrophenol (44 mg,
0.32 mmol) and triphenylphosphine (84 mg, 0.32 mmol) in dry
THF (10 ml) at 0 ◦C. The reaction mixture was stirred at
room temperature overnight. The mixture was evaporated and
the residue was dissolved in ethyl acetate and washed with
hydrochloric acid (1 M; 10 ml) and sodium hydroxide (1 M; 10 ml),
dried (MgSO4) and concentrated. The crude material was purified
by chromatography.
3-Ethyl 2-hydroxymethyl-5-methoxy-1-methylindole-4,7-dione 11b.
To a suspension of lithium aluminium hydride (0.056 g,
1.5 mmol) in THF (20 ml) at 0 ◦C was added a solution of
methyl 4-amino-3-ethyl-5-methoxy-1-methylindole-2-carboxylate
17b (0.15 g, 0.59 mmol) in THF (15 ml). The mixture was allowed
to warm to room temperature and stirred for 30 min. The mixture
was cooled to 0 ◦C and quenched by the addition of water (0.5 ml),
sodium hydroxide (1 M; 0.5 ml) and silica gel (5 g). The granular
precipitate was filtered off through a pad of Celite. The filtrate
was dried (MgSO4) and concentrated in vacuo to give the alcohol,
which was used directly in the next step without purification. To
a solution of the amino indole in acetone (40 ml) was added a
solution of potassium nitrosodisulfonate (0.47 g, 1.7 mmol) in
sodium dihydrogen phosphate buffer (0.3 M; 40 ml). The mixture
was stirred at room temperature for 1 h. The acetone was removed
in vacuo, the residue was extracted with dichloromethane and
washed with water. The organic layer was dried (Na2SO4) and
concentrated. The crude product was purified by chromatography,
eluting with ethyl acetate/dichloromethane (1 : 4), to yield the
title compound as an orange solid (0.081 g, 54%), mp 192–194 ◦C
(from ethyl acetate/hexane); (Found: C, 62.2; H, 6.0, N, 5.4.
C13H15NO4·0.1H2O requires C, 62.2; H, 6.1, N, 5.6%); (Found: M+,
249.1002. C13H15NO4.requires 249.1001); kmax (MeOH)/nm 448
(log e 3.21), 360 (3.38), 280 (4.07); mmax (KBr)/cm−1 3406, 2966,
2925, 1667, 1644, 1601; dH (300 MHz; CDCl3) 5.60 (1 H, s, 6-H),
4.63 (2 H, d, J 4.4, CH2OH), 4.00 (3 H, s, OMe), 3.79 (3 H, s,
NMe), 2.75 (2 H, q, J 7.5, CH2Me), 1.99 (1 H, br t, CH2OH), 1.12
(3 H, t, J 7.5, CH2Me); dC (75 MHz; CDCl3) 179.2, 177.6, 160.1,
135.8, 129.8, 127.7, 120.7, 106.9 (CH), 56.5 (Me), 53.0 (CH2), 32.8
(Me), 17.9 (CH2), 15.7 (Me); m/z (EI) 250 (MH+, 100%), 236 (27),
234 (48), 220 (47), 206 (52).
5-Methoxy-1,3-dimethyl-2-(4-nitrophenoxy)methylindole-4,7-
dione 13a. Purified by chromatography eluting with hex-
ane/ethyl acetate (1:1) to give the title compound (61%) as a yellow
solid, mp 230–232 ◦C (from ethyl acetate/hexane); (Found: C,
60.3; H, 4.3; N, 7.5. C18H16N2O6 requires C, 60.6; H, 4.5; N, 7.9%);
kmax (MeOH) 436 (log e 3.39), 284 (4.28) nm; mmax (KBr)/cm−1 3114,
3068, 290, 2909, 2838, 1676, 1646, 1596; dH (300 MHz; CDCl3) 8.25
(2 H, d, J 9.1, ArH), 7.06 (2 H, d, J 9.1, ArH), 5.70 (1 H, s, 6-H),
5.08 (2 H, s, CH2), 4.00 (3 H, s, OMe), 3.83 (3 H, s, NMe), 2.40 (3
H, s, Me); dC (75 MHz; CDCl3) 179.2, 177.8, 162.8, 160.3, 142.2,
130.9, 130.3, 126.1 (CH), 122.5, 121.4, 114.7 (CH), 107.1 (CH),
59.1 (CH2), 56.6 (Me), 32.9 (Me), 10.2 (Me); m/z (CI) 357 (MH+,
95), 298 (22).
3-Ethyl-5-methoxy-1-methyl-2-(4-nitrophenyl)oxymethyl)indole-
4,7-dione 13b. Purified by chromatography eluting with hexane/
ethyl acetate (5:1) to yield the title compound (64%) as a yellow
solid, mp 219–221 ◦C (from ethyl acetate/hexane); (Found: C,
61.4; H, 4.8, N, 7.4. C19H18N2O6 requires C, 61.6; H, 4.9, N, 7.6%;
kmax (MeOH)/nm 440 (log e 3.26), 288 (4.45); mmax (KBr)/cm−1
3114, 3073, 2960, 2934, 1677, 1642, 1592; dH (300 MHz; CDCl3)
8.26 (2 H, d, J 9.1, ArH), 7.07 (2 H, d, J 9.1, ArH), 5.70 (1
H, s, 6-H), 5.06 (2 H, s, CH2), 4.00 (3 H, s, OMe), 3.84 (3 H, s,
NMe), 2.82 (2 H, q, J 7.5, CH2Me), 1.16 (3 H, t, J 7.5, CH2Me);
dC (75 MHz; acetone-d) 179.7, 177.5, 164.4, 161.3, 142.8, 132.3,
131.0, 129.2, 126.6 (CH), 121.4, 116.1 (CH), 107.7 (CH), 60.0
(CH2), 56.9 (Me), 33.0 (Me), 18.3 (CH2), 15.7 (Me); m/z (CI) 371
(MH+, 8%), 234 (14), 206 (14), 127 (28), 110 (100).
(E)-2-(3-Hydroxypropenyl)-5-methoxy-1,3-dimethylindole-4,7-
dione 12. To a solution of (E)-3-(4-amino-5-methoxy-1,3-
dimethylindol-2-yl)prop-2-en-1-ol 21 (416 mg, 1.69 mmol) in ace-
tone (95 ml) was added a solution of potassium nitrosodisulfonate
(1.86 g, 6.77 mmol) in sodium dihydrogen phosphate buffer (0.3 M;
76 ml). The mixture was stirred at room temperature for 1 h. The
acetone was removed in vacuo, and the residue was stirred at room
temperature in a 1 : 1 mixture of hydrochloric acid (2 M) and
acetone (200 ml) for 1 h. The acetone was removed in vacuo and
the residue was extracted with dichloromethane. The organic layer
was washed with water, dried (MgSO4) filtered and evaporated
under reduced pressure. The crude material was purified by
chromatography eluting with ethyl acetate/light petroleum (1 : 1)
to yield the title compound (262 mg, 52%) as an orange crystalline
solid, mp 206–207 ◦C; (Found: M+, 261.0991. C14H15NO4 requires
261.1001); kmax (acetonitrile)/nm 472 (log e 3.37), 351 (3.42), 285
(4.04), 258 (4.09); mmax (KBr)/cm−1 3423, 2926, 2849, 1671, 1639,
1595, 1491, 1451, 1338, 1226, 1170, 1150, 1098, 1030; dH (300 MHz;
2-Ethyl-5-methoxy-1,3-dimethylindole-4,7-dione 14. To a solu-
tion of 4-amino-2-ethyl-5-methoxy-1,3-dimethylindole 23 (32 mg,
0.15 mmol) in acetone (9 ml) was added a solution of potassium
nitrosodisulfonate (159 mg, 0.58 mmol) in sodium dihydrogen
phosphate buffer (0.3 M; 7.3 ml). The mixture was stirred at
room temperature for 1 h. The acetone was removed in vacuo,
and the residue was stirred at room temperature in a 1:1 mixture
of hydrochloric acid (3 M) and acetone for 1 h. The acetone was
removed in vacuo, and the resulting residue was extracted with
dichloromethane. The organic layer was washed with water, dried
(MgSO4), filtered and evaporated under reduced pressure. The
crude material was purified by chromatography eluting with ethyl
acetate/light petroleum (1 : 1) to yield the title compound (30 mg,
◦
CDCl3) 6.50 (1 H, dt, J 12.1, 1.3, CH), 6.21 (1 H, dt, J 12.1, 3.7,
89%) as a red crystalline solid, mp 200–201 C; (Found: M+,
=
=
CH), 5.63 (1 H, s, 6-H), 4.41 (2 H, dd, J 3.7, 1.3, CH2OH), 3.95
233.1050. C13H15NO3 requires 233.1052); kmax (acetonitrile)/nm
461 (log e 3.35), 356.5 (3.44), 273 (3.99); mmax (KBr)/cm−1 3434,
2967, 2922, 2851, 1666, 1636, 1596, 1556, 1506, 1456, 1338, 1221,
1156; dH (300 MHz; CDCl3) 5.55 (1 H, s, 6-H), 3.87 (3 H, s, OMe),
(3 H, s, NMe), 3.80 (3 H, s, OMe), 2.41 (3 H, s, Me); dC (100 MHz;
CDCl3) 179.8, 178.2, 159.9, 135.44 (CH), 135.40, 132.6, 122.1,
120.8, 117.0 (CH), 107.2 (CH), 63.4 (CH2), 56.4 (Me), 33.0 (Me),
1636 | Org. Biomol. Chem., 2007, 5, 1629–1640
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3.77 (3 H, s, NMe), 2.58 (2 H, q, J 7.6, CH2Me), 2.24 (3 H, s,
Me), 1.11 (3 H, t, J 7.6, CH2Me); dC (75 MHz; CDCl3) 178.5,
178.3, 159.6, 140.7, 128.1, 122.0, 118.3, 106.8 (CH), 56.3 (Me),
32.2 (Me), 16.7 (Me), 13.4 (CH2), 9.8 (Me); m/z (EI) 233 (M+,
80%), 218 (100), 203 (16), 190 (34).
260 (M+, 61%), 243 (35), 196 (32), 149 (100), 91 (46), 71 (48),
57 (68).
2-Carbamoyloxymethyl-5-(2-methylaziridinyl)-1,3-dimethyl-
indole-4,7-dione 26. To a solution of 2-carbamoyloxymethyl-5-
methoxy-1,3-dimethylindole-4,7-dione 24 (41 mg, 0.15 mmol) in
DMF (5 ml) was added 2-methylaziridine (517 ll, 7.3 mmol).
The reaction mixture was stirred at room temperature for 3 days.
The reaction mixture was diluted with dichloromethane, washed
with brine (2 × 150 ml) and deionized water (2 × 150 ml), dried
(MgSO4), filtered and evaporated under reduced pressure to yield
the title compound (30 mg, 67%) as a red crystalline solid, mp 160–
2-Carbamoyloxymethyl-5-methoxy-1,3-dimethylindole-4,7-dione
24. (a) To
a solution of 2-hydroxymethyl-5-methoxy-1,3-
dimethylindole-4,7-dione 11a (81 mg, 0.37 mmol) in dry pyridine
(6.1 ml) at 0 ◦C was added phenyl chloroformate (184 ll,
1.48 mmol). The reaction mixture was stirred at 0 ◦C for 2 h
and at room temperature overnight. The reaction mixture was
evaporated under reduced pressure and the residue was purified by
chromatography eluting with ethyl acetate/light petroleum (1 : 3)
to yield the phenyl carbonate (103 mg, 83%) as a yellow crystalline
solid, mp 124–126 ◦C, used without further purification; mmax
(KBr)/cm−1 3438, 2926, 1757, 1685, 1641, 1597, 1505, 1269, 1225,
1161; dH (300 MHz; CDCl3) 7.38 (2 H, dd, J 8.0, 7.5, ArH), 7.24
(1 H, t, J 8.0, ArH), 7.16 (2 H, d, J 7.5, ArH), 5.67 (1 H, s, 6-H),
5.27 (2 H, s, CH2), 4.03 (3 H, s, NMe), 3.81 (3 H, s, OMe), 2.40 (3
H, s, Me); dC (100 MHz; CDCl3) 179.2, 177.7, 160.4, 153.4, 150.9,
130.8, 130.2, 129.6 (CH), 126.3 (CH), 123.6, 121.4, 120.9 (CH),
107.1 (CH), 58.2 (CH2), 56.5 (Me), 32.8 (Me), 10.0 (Me).
◦
162 C; (Found: M+, 303.1214. C15H17N3O4 requires 303.1219);
kmax (acetonitrile)/nm 464 (log e 3.37), 344 (3.55), 312 (4.06); mmax
(KBr)/cm−1 3436, 3288, 2924, 1729, 1682, 1639, 1586, 1498, 1405,
1321, 1167; dH (300 MHz; CDCl3) 5.75 (1 H, s, 6-H), 5.09 (2 H, s,
CH2), 4.71 (2 H, bs, NH2), 3.96 (3 H, s, NMe), 2.37 (3 H, s, Me),
2.25 (1 H, m, CH), 2.10 (1 H, d, J 3.7, CH), 2.06 (1 H, d, J 5.8,
CH), 1.43 (3 H, d, J 5.5, CHMe); dC (100 MHz; CDCl3) 179.8,
179.2, 157.6, 156.0, 131.7, 130.2, 122.6, 121.9, 116.6 (CH), 54.9
(CH2), 36.1 (CH), 34.5 (CH2), 32.6 (Me), 17.7 (Me), 9.9 (Me); m/z
(EI) 303 (M+, 8%), 279 (16), 196 (28), 167 (30), 149 (100), 104 (16),
91 (26).
(b) A solution of the above phenyl carbonate (84 mg, 0.28 mmol)
in dry dichloromethane (14 ml) was cooled to −78 ◦C. Ammonia
gas was bubbled into the solution for 20 min. The mixture was
stirred for 6 h whilst allowing it to warm up to room temperature
The reaction mixture was evaporated under reduced pressure
and the residue was purified by chromatography eluting with
ethyl acetate to yield the title comp◦ ound (47 mg, 62%) as an
orange crystalline solid, mp 246–247 C; (Found: MH+, 279.0981.
C13H14N2O5 + H requires 279.0981); kmax (acetonitrile)/nm 428
(log e 3.15), 344 (3.34), 288 (3.96); mmax (KBr)/cm−1 3436, 3300,
2916, 1734, 1634, 1596, 1500, 1392, 1325, 1254, 1225, 1158, 1037;
dH (300 MHz; CDCl3) 6.63 (2 H, bs, NH2), 5.79 (1 H, s, 6-H), 5.00
(2 H, s, CH2), 3.89 (3 H, s, NMe), 3.74 (3 H, s, OMe), 2.23 (3
H, s, Me); dC (100 MHz; CDCl3) 179.0, 177.7, 160.2, 156.6, 134.1,
129.3, 121.1, 121.0, 107.5 (CH), 57.0 (Me), 53.8 (CH2), 32.8 (Me),
10.1 (Me); m/z (CI) 279 (MH+, 25%), 192 (55), 112 (65), 98 (100),
84 (60).
(5-Methoxy-1,3-dimethyl-4,7-dioxoindol-2-yl)methyloxyphenyl-
4-methylcarbamate 29. (a) To a stirred solution of 2-[(4-
hydroxymethyl)phenoxymethyl]-5-methoxy-1,3-dimethyl-indole-
4,7-dione 28 (33 mg, 0.097 mmol) in pyridine (5 ml) was added an
excess of phenyl chloroformate (500 ll). The reaction mixture
was stirred at room temperature overnight and quenched by
addition of water. The reaction mixture was extracted with
dichloromethane, washed twice with aqueous copper sulfate
(10%), dried over MgSO4, filtered and evaporated under reduced
pressure. The residue obtained was purified by chromatography,
elutingwithethylacetate–light petroleum (1:3) toyield the carbon-
ate intermediate, 2-(4-phenylcarbonatemethyl-phenoxymethyl)-5-
methoxy-1,3-dimethyl-indole-4,7-dione, as an orange semi solid
which was used in the next step with no further purification.
(b) The carbonate intermediate was dissolved in dry dichloro-
methane (7 ml) and the solution was cooled down to −78 ◦C.
Ammonia was bubbled into the solution until the flask is filled
up with liquid ammonia (ca. 25 ml). The reaction mixture was
left to warm up to room temperature until all the ammonia has
disappeared. The reaction mixture was evaporated off under re-
duced pressure and purified by chromatography, eluting with ethyl
acetate/dichloromethane (1 : 2) to yield◦the title compound (15 mg,
45%) as an orange solid; mp 212–213 C; kmax (acetonitrile)/nm
280 (log e 4.11), 348 (3.37), 432 (3.17); mmax (KBr)/cm−1 3413, 3258,
3207, 2915, 1666, 1692, 1637, 1592, 1493, 1333, 1222, 1175, 1160;
dH (300 MHz; CDCl3) 7.34 (2 H, d, J 8.6, ArH), 6.96 (2 H, d, J
8.6, ArH), 5.67 (1 H, s, 6-H), 5.05 (2 H, s, OCH2), 4.98 (2 H, s,
OCH2), 4.68 (2 H, brs, NH2), 3.99 (3 H, s, NMe), 3.82 (3 H, s,
OMe), 2.37 (3 H, s, Me); dC (100 Hz; CDCl3) 179.5, 178.2, 160.5,
158.3, 156.9, 132.5, 130.5 (CH), 129.9, 129.2, 122.4, 121.7, 115.1
(CH), 107.3 (CH), 66.9 (CH2), 58.8 (CH2), 56.8 (Me), 33.2 (Me),
10.4 (Me).
2-Hydroxymethyl-1,3-dimethyl-5-(2-methylaziridinyl)indole-4,7-
dione 25. To a solution of 2-hydroxymethyl-5-methoxy-1,3-
dimethylindole-4,7-dione 11a (100 mg, 0.46 mmol) in DMF
(16 ml) was added 2-methylaziridine (1.61 ml, 22.8 mmol). The
reaction mixture was stirred at room temperature for 4 days.
The reaction mixture was diluted with dichloromethane, washed
with brine (3 × 150 ml) and deionized water (2 × 150 ml), dried
(MgSO4), filtered and evaporated under reduced pressure to
yield the title compound (140 mg, 64%) as a red crystalline solid,
◦
mp 148–150 C; (Found: M+, 260.1160. C14H16N2O3 requires
260.1161); kmax (acetonitrile)/nm 468 (log e 3.30), 356 (3.46), 308
(3.99); mmax (KBr)/cm−1 3360, 2921, 1660, 1624, 1580, 1492, 1463,
1272, 1151, 1007; dH (300 MHz; CDCl3) 5.68 (1 H, s, 6-H), 4.59
(2 H, s, CH2), 3.96 (3 H, s, NMe), 2.30 (3 H, s, Me), 2.23 (1 H, m,
CH), 2.08 (1 H, d, J 3.7, CH), 2.04 (1 H, d, J 5.9, CH), 1.42 (3 H,
d, J 5.5, CHMe); OH not observed; dC (100 MHz; CDCl3) 179.7,
178.9, 157.2, 136.1, 129.7, 121.9, 120.5, 116.8 (CH), 53.0 (CH2),
36.2 (CH), 34.5 (CH2), 32.7 (Me), 17.7 (Me), 9.9 (Me); m/z (EI)
(5-Methoxy-1,2-dimethyl-4,7-dioxoindol-3-yl)methyloxyphenyl-
4-methylcarbamate 33. (a) To a stirred solution of 5-methoxy-3-
(4-hydroxymethylphenoxy)methyl-1,2-dimethylindole-4,7-dione
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32 (74 mg, 0.22 mmol) in dry pyridine (10 ml) was added an excess
of phenyl chloroformate (1 ml). The reaction mixture was stirred
at room temperature overnight and quenched by addition of water.
The reaction mixture was extracted with dichloromethane, washed
twice with aqueous copper sulfate (10%), dried over MgSO4,
filtered and evaporated under reduced pressure. The residue
was purified by chromatography, eluting with ethyl acetate–light
petroleum (1 : 3) to yield (5-methoxy-1,2-dimethyl-4,7-dioxoindol-
3-yl)methyloxyphenyl-4-methylphenyl carbonate (66 mg, 67%) as
removed in vacuo. The residue was extracted with dichloromethane
and concentrated. The residue was stirred at room temperature in
a 1 : 1 mixture of hydrochloric acid (2 M) and acetone (120 ml) for
1 h. The acetone was removed in vacuo. The resulting residue was
extracted with dichloromethane. The organic layer was washed
with water, dried over MgSO4, filtered and evaporated under
reduced pressure to yield the title compound (165 mg, 64%) as
an orange crystalline solid, recrystallized from dichloromethane–
pentane; mp 200–202 ◦C (lit.,42 mp 191–193 ◦C); (Found: C, 57.2;
H, 5.1; N, 6.0. C12H13NO5 requires C, 57.4; H, 5.2; N, 5.6%);
kmax (acetonitrile)/nm 280 (log e 3.97), 344 (3.38), 440 (3.11); mmax
(KBr)/cm−1 3318, 2927, 1672, 1634, 1596, 1507, 1473, 1335, 1224,
1143, 1070; dH (400 MHz; d6-DMSO) 5.81 (1 H, s, 6-H), 5.20 (1 H,
t, J 5.4, OH), 4.71 (1 H, t, J 5.4, OH), 4.62 (2 H, d, J 5.4, OCH2),
4.57 (2 H, d, J 5.4, OCH2), 3.94 (3 H, s, NMe), 3.79 (3 H, s, OMe);
dC (100 MHz; d6-DMSO) 179.1, 177.8, 160.1, 139.7, 128.9, 123.4,
120.5, 107.3 (CH), 57.0 (Me), 53.5 (CH2), 51.9 (CH2), 32.9 (Me);
m/z (EI) 251 (M+, 10), 233 (65), 218 (20), 204 (10), 190 (25), 176
(25), 162 (20), 120 (25), 69 (100).
◦
+
an orange solid; mp 41–43 C; (Found: M + NH4 , 479.1818.
C26H23NO7 + NH4 requires 479.1818); kmax (acetonitrile)/nm 280
(log e 4.21), 336 (3.55), 452 (3.30); mmax (KBr)/cm−1 3426, 2922,
2853, 1764, 1675, 1637, 1598, 1510, 1460, 1229, 1179, 1156; dH
(400 MHz; CDCl3) 7.38 (4 H, m, ArH), 7.24 (1 H, m, ArH), 7.15
(2 H, d, J 8.0, ArH), 7.00 (2 H, d, J 8.0, ArH), 5.60 (1 H, s, 6-H),
5.30 (2 H, s, OCH2), 5.18 (2 H, s, OCH2), 3.86 (3 H, s, NMe), 3.79
(3 H, s, OMe), 2.28 (3 H, s, Me); dC (100 MHz; CDCl3) 179.1,
178.6, 160.0, 159.4, 154.1, 151.5, 138.5, 131.0 (CH), 130.0 (CH),
129.1, 127.5, 126.7, 126.4, 121.7, 121.5 (CH), 117.3, 115.5 (CH),
107.1 (CH), 70.7 (CH2), 60.8 (CH2), 56.9 (Me), 32.7 (Me), 10.3
2,3-Bis(hydroxymethyl)-1-methyl-5-(2-methylaziridinyl)indole-
+
(Me); m/z (ES) 479 (M + NH4 , 100), 232 (20), 213 (20).
4,7-dione 39. To
a solution of 2,3-bis(hydroxymethyl)-5-
(b) The above carbonate (65 mg, 0.14 mmol) was dissolved
in dry dichloromethane (10 ml) and the solution was cooled
methoxy-1-methylindole-4,7-dione 38 (50 mg, 0.20 mmol) in DMF
(7.4 ml) was added 2-methylaziridine (702 ll, 9.96 mmol). The
reaction mixture was stirred at room temperature for 3 days.
The reaction mixture was diluted with ethyl acetate and washed
with brine (150 ml). The aqueous layer was re-extracted with
ethyl acetate and was quenched with hydrochloric acid (2 M).
The combined organic layer was dried over MgSO4, filtered and
evaporated under reduced pressure. The crude material was puri-
fied by chromatography on florisil, eluting with dichloromethane,
then dichloromethane-ethyl acetate (1 : 1) to yield the title
◦
down to −78 C. Ammonia was bubbled into the solution until
the flask was filled up with liquid ammonia (ca. 40 ml). The
reaction mixture was left to warm up to room temperature until
all the ammonia had disappeared. The reaction mixture was
evaporated under reduced pressure and the residue purified by
chromatography, eluting with ethyl acetate/dichloromethane (1 :
2) to yield the title compound (51 mg, 95%) as a red solid; mp 222–
◦
+
224 C; (Found: M + NH4 , 402.1661. C20H20N2O6 + NH4 requires
402.1665); kmax (acetonitrile)/nm 280 (log e 4.11), 340 (3.41), 452
(3.17); mmax (KBr)/cm−1 3433; 2921, 1732, 1682, 1632, 1598, 1509,
1455, 1328, 1224, 1159; dH (300 MHz; CDCl3) 7.29 (2 H, d, J
6.6, ArH), 6.98 (2 H, d, J 6.6, ArH), 5.61 (1 H, s, 6-H), 5.30 (2
H, s, OCH2), 5.02 (2 H, s, OCH2), 4.62 (2 H, bs, NH2), 3.88 (3 H, s,
NMe), 3.81 (3 H, s, OMe), 2.30 (3 H, s, Me); dC (100 MHz; CDCl3)
178.8, 178.2, 159.6, 158.6, 156.7, 138.1, 130.0 (CH), 128.7, 128.5,
121.3, 117.0, 115.0 (CH), 106.7 (CH), 66.8 (CH2), 60.4 (CH2), 56.5
◦
compound (14 mg, 25%) as a red crystalline solid; mp 77–79 C;
(Found: MH+, 277.1185. C14H16N2O4 + H requires 277.1188);
kmax (acetonitrile)/nm 308 (log e 4.04), 352 (3.58), 476 (3.18); mmax
(KBr)/cm−1 3418, 2918, 1636, 1582, 1505, 1455, 1267, 1232, 1136;
dH (300 MHz; CDCl3) 5.72 (1 H, s, 6-H), 4.65 (2 H, s, OCH2),
4.58 (2 H, s, OCH2), 3.95 (3 H, s, NMe), 2.27 (1 H, m, CH), 2.10
(1 H, d, J 6.3, CH), 2.06 (1 H, d, J 11.1, CH), 1.41 (3 H, d, J
5.4, Me); OH not observed; dC (100 MHz; CDCl3) 181.0, 179.2,
157.3, 136.5, 130.8, 123.7, 122.1, 117.1 (CH), 55.6 (CH2), 53.0
(CH2), 36.6 (CH), 34.9 (CH2), 32.9 (Me), 17.9 (Me); m/z (ES) 299
(MNa+, 10%), 277 (M+, 40), 259 (10), 219 (10), 187 (10), 155 (20),
123 (40), 91 (100).
+
(Me), 32.4 (Me), 9.9 (Me); m/z (ES) 402 (M + NH4 , 100), 276
(15).
2,3-Bis(hydroxymethyl)-5-methoxy-1-methylindole-4,7-dione 38.
(a) To a suspension of lithium aluminium hydride (200 mg,
5.22 mmol) in THF (7.7 ml) at 0 ◦C was added a solution of
ethyl 4-amino-3-formyl-5-methoxy-1-methylindole-2-carboxylate
37 (360 mg, 1.31 mmol) in THF (3.5 ml). The mixture was allowed
to warm up to room temperature, stirred for 30 min, and cooled
to 0 ◦C before being quenched by the addition of water (1 ml),
NaOH (1 M; 1 ml) and silica gel. The granular precipitate was
filtered off through a pad of Celite. The filtrate was dried over
MgSO4, and concentrated in vacuo to yield 4-amino-5-methoxy-
1-methylindole-2,3-dimethanol (249 mg, 81%) as a brown solid,
which was used in the next step without any further purification.
(b) To a solution of 4-amino-5-methoxy-1-methylindole-2,3-
dimethanol (242 mg, 1.03 mmol) in acetone (62 ml) was added
a solution of potassium nitrosodisulfonate (1.10 g, 3.42 mmol) in
sodium dihydrogen phosphate buffer (0.3 M; 49 ml). The reaction
was stirred at room temperature for 1 h. The excess acetone was
Ethyl 3-hydroxymethyl-5-methoxy-1-methyl-4,7-dioxoindole-2-
carboxylate 40. (a) Sodium borohydride (18 mg, 0.45 mmol)
was added portionwise to a solution of ethyl 4-amino-3-formyl-5-
methoxy-1-methylindole-2-carboxylate 37 (50 mg, 0.18 mmol) in
dry methanol (2 ml) at 0 ◦C. The reaction mixture was allowed to
warm up to room temperature and stirred for 1 h. The mixture
was cooled to 0 ◦C, quenched by the addition of water (1 ml),
extracted with ether, dried over MgSO4, filtrated and concentrated
in vacuo to yield ethyl 4-amino-3-hydroxymethyl-5-methoxy-1-
methylindole-2-carboxylate (44 mg, 86%) as a light orange solid;
mp 136–139 ◦C; mmax (KBr)/cm−1 3359, 3124, 2980, 2875, 1705,
1521, 1403, 1255, 1224, 1128, 1001; dH (400 MHz; CDCl3) 7.04 (1
H, d, J 6.6, ArH), 6.64 (1 H, d, J 6.6, ArH), 5.19 (2 H, s, CH2OH),
4.39 (2 H, q, J 5.4, OCH2Me), 3.86 (3 H, s, NMe), 3.84 (3 H, s,
OMe), 1.42 (3 H, t, J 5.4, OCH2Me); dC (100 MHz; CDCl3) 162.4,
1638 | Org. Biomol. Chem., 2007, 5, 1629–1640
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140.1, 136.2, 131.2, 124.9, 121.7, 117.0, 114.1 (CH), 98.8 (CH),
60.9 (CH2), 58.0 (Me), 56.6 (CH2), 32.1 (Me), 14.3 (Me); m/z (EI)
278 (M+, 40%), 260 (30), 247 (25), 232 (50), 217 (100), 189 (15),
145 (25).
a density of 1–2 × 104 cells ml−1 and allowed to attach overnight
(16 h). Indolequinone solutions were applied in medium for 2 h.
Indolequinone solutions were removed and replaced with medium
alone, and the 96 well plates were incubated for 5–7 days. MTT
(Sigma) was added to each well (50 lg), and the cells were
incubated for another 4 h. Medium–MTT solutions were removed
carefully by aspiration, the MTT formazan crystals were dissolved
in 100 ll DMSO and absorbance was determined on a plate reader
at 550 nm. IC50 values (concentration at which cell survival equals
50% of control) were determined from plots of percent of control
vs. concentration.
(b) To a solution of ethyl 4-amino-3-hydroxymethyl-5-methoxy-
1-methylindole-2-carboxylate (321 mg, 1.16 mmol) in acetone
(70 ml) was added a solution of potassium nitrosodisulfonate
(1.24 g, 4.62 mmol) in sodium dihydrogen phosphate buffer
(0.3 M, 56 ml). The mixture was stirred at room temperature
for 1 h. The excess acetone was removed in vacuo. The resulting
residue was extracted with dichloromethane and evaporated. The
residue was stirred at room temperature in a 1 : 1 mixture of
hydrochloric acid (2 M) and acetone (140 ml) for 1 h. The acetone
was removed in vacuo. The resulting residue was extracted with
dichloromethane. The organic layer was washed with water, dried
over MgSO4, filtered and evaporated under reduced pressure.
The residue was purified by chromatography, eluting with ethyl
acetate/dichloromethane (1 : 20) to yield the title compound
(181 mg, 54%) as a light orange crystalline solid, recrystallized
from dichloromethane–pentane; mp 149–151 ◦C; (Found: C,
57.1; H, 5.0; N, 4.6. C14H15NO6 requires C, 57.3; H, 5.2; N,
Acknowledgements
This work was supported by the Cancer Research Campaign
(now Cancer Research UK) [CUK], the FORCE Cancer Charity
and NIH grant P20 RR17670. We also thank the EPSRC Mass
Spectrometry Centre at Swansea for mass spectra, and Professor
David Ross (University of Colorado) for generous gifts of enzyme
and cells.
+
4.8%); (Found: M + NH4 , 311.1246. C14H15NO6 + NH4 requires
References
311.1243); kmax (acetonitrile)/nm 250 (log e 4.10), 336 (3.37), 408
(3.15); mmax (KBr)/cm−1 3433, 2949, 1708, 1673, 1646, 1604, 1493,
1435, 1374, 1266, 1178, 1047, 1016; dH (400 MHz; CDCl3) 5.80 (1
H, s, 6-H), 5.00 (2 H, s, CH2O), 4.40 (2 H, q, J 7.2, OCH2Me), 4.26
(3 H, s, NMe), 3.86 (3 H, s, OMe), 1.41 (3 H, t, J 7.2, OCH2Me);
OH not observed; dC (100 MHz; CDCl3) 179.1, 178.5, 160.7, 160.5,
132.8, 132.2, 126.3, 121.3, 108.2 (CH), 61.8 (CH2), 56.8 (Me), 56.0
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265 (45), 250 (50), 236 (25).
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