Rates of reductive elimination of substituted nitrophenols from the (indol-3-yl)methyl position of indolequinones

Article abstract of DOI:10.1039/b101842f

A series of indolequinones bearing substituted nitrophenols on the (indol-3-yl)methyl position was synthesised. The nitrophenol leaving groups were appropriately substituted to give a wide range (4 units) in phenolic pK_a value. The rate of redu

Full text of DOI:10.1039/b101842f

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Rates of reductive elimination of substituted nitrophenols from the
(indol-3-yl)methyl position of indolequinones
Elizabeth Swann,a Christopher J. Moody,a Michael R. L. Stratford,b Kantilal B. Patel,b
Matthew A. Naylor,b Borivoj Vojnovic,b Peter Wardmanb and Steven A. Everett*b
2
a School of Chemistry, University of Exeter, Stocker Road, Exeter, UK EX4 4QD
b Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex, UK
HA6 2JR. E-mail: everett@graylab.ac.uk
Received (in Cambridge, UK) 27th February 2001, Accepted 31st May 2001
First published as an Advance Article on the web 10th July 2001
A series of indolequinones bearing substituted nitrophenols on the (indol-3-yl)methyl position was synthesised. The
nitrophenol leaving groups were appropriately substituted to give a wide range (4 units) in phenolic pKa value. The
rate of reductive elimination of phenoxide anions from the (indol-3-yl)methyl position of semiquinone radicals was
dependent upon this pKa, with a decrease in 3.8 pK units shortening the half-life from 28 to 1.5 ms. Only 2,4-
dinitrophenol (pKa = 3.9) was eliminated from an unsubstituted (indol-3-yl)methyl position at a rate that would
compete with reoxidation of the radical by oxygen. A nitrothiophenol leaving group was eliminated comparatively
slowly and only from the hydroquinone. These studies demonstrate the dependence upon leaving group pKa of the
rate of reductive elimination from the (indol-3-yl)methyl position of indolequinones.
Introduction
There has been much interest in the exploitation of indol-
equinones as bioreductively activated cytotoxins designed to
target the hypoxic sub-population of cells present in many
solid tumours.1–7 In addition to the ‘direct’ activation to cyto-
toxic species, of increasing importance is the alternative bio-
activation route where reduction of an indolequinone initiates
fragmentation of a linking bond and the release of a bioactive
agent, the biological activity of which is masked in the prodrug
form.8,9 We have demonstrated that indolequinones are capable
of efficiently eliminating a range of leaving groups, coupled
through various linkers, from the (indol-3-yl)methyl position
following one-electron reduction to the semiquinone radical
anion (Q Ϫ) or two-electron reduction to the hydroquinone
ؒ
(QH2).10 From a physico-chemical viewpoint, a key factor in the
design of these prodrugs is the ability to control rates of reduc-
tive fragmentation relative to the reactivities of intermediate
Ϫ/QH2 species with oxygen.11,12 The effects of indolequinone
ؒ
Q
substitution patterns on rates of elimination of the drug model
4-nitrophenol have been studied recently.13 By incorporating
radical-stabilising substituents (e.g. methyl, thienyl) at the
indolyl carbinyl position it was possible to significantly enhance
the rate of reductive fragmentation directly from the Q
radical. The use of 4-nitrophenol as a model leaving group
facilitated structural optimisation of the indolequinone moiety
but the importance of the leaving group itself in controlling
rates of fragmentation has yet to be elucidated.
Fig. 1 Structures of indolequinones 1–9 bearing substituted nitro-
phenols (or nitrothiophenol) in the (indol-3-yl)methyl position.
Ϫ
ؒ
Results
Synthesis
For this study we have synthesised a series of substituted
nitrophenols coupled to an indolequinone through a phenolic
ether bond (Fig. 1, 1–8) and the thiophenol analogue 9, and
determined the rates of reductive fragmentation by pulse
radiolysis.11 The indolequinones were reduced in a controlled
and quantifiable manner using radiolytically-produced
reducing radicals which mimic reduction by (one- and two-
electron reducing) activating enzymes such as NADPH
P450 reductase6 and NQO1 (DT-diaphorase)14,15 in biological
systems.
The 3-aryloxymethylindolequinones 1–8 were prepared from
the corresponding 3-hydroxymethylquinones 10 and 11 as out-
lined in Scheme 1. Thus, in the case of the 2,4-dinitrophenol
derivative 1, reaction of the alcohol with 2,4-dinitrofluoro-
benzene in the presence of silver() oxide gave the desired aryl-
oxy compound 1 in modest yield. The other aryloxy derivatives
2–8 were prepared from the 3-hydroxymethyl compound and
substituted phenol using the Mitsunobu reaction. The thioether
9 was prepared similarly. The 3-isopropoxymethylindolequi-
none 12 was prepared from 10 as previously described.13
1340
J. Chem. Soc., Perkin Trans. 2, 2001, 1340–1345
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b101842f

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Scheme 1 Reagents and conditions: [for X = O, Ar = 2,4-(NO2)2C6H3] i,
2,4-dinitrofluorobenzene, Ag2O, THF; [all other Ar] ii, ArOH (or
ArSH), EtO2CN᎐NCO2Et, Ph3P, THF.
᎐
Free radical chemistry
Reductive fragmentation at the (indol-3-yl)methyl position. In
agreement with previous studies,7,10,11,13 we observed that the
ؒ
reaction of the propan-2-ol radical ((CH3)2C OH) with all the
indolequinones 1–11 in aqueous solution at pH > 6 yields tran-
sients with absorption spectra characterised by two maxima,
one in the UV (λmax ca. 350 nm), and a considerably weaker one
in the visible (λmax ca. 600 nm). These transients have been
identified as the semiquinone radicals (Q Ϫ),7,10,11,13 which are
ؒ
fully formed (k1 ∼109 dm3 molϪ1 Ϫ1) by ∼200 µs after the
s
electron pulse according to reaction (1).
Q ϩ (CH3)2C OH → Q Ϫ ϩ (CH3)2CO ϩ Hϩ
(1)
ؒ
ؒ
From the pH dependence of the absorption at 350 nm, a
pKa (QH /Q Ϫ) = 5.5 0.1 for 10 was obtained and was typical
for the semiquinone radicals of all the indolequinones in the
present study. Traditionally the hydroxymethyl compounds
exhibit poor leaving group ability and as expected the semi-
quinone radicals decayed by pure second-order kinetics via the
disproportionation reaction (2).
ؒ
ؒ
Fig. 2 Changes in the concentration of substituted nitrophenols as
measured by pulse radiolysis of indolequinones (50 µmol dmϪ3) in an
N2O-saturated propan-2-ol–water mixture (50%, v/v) at pH 9. Panels
(a–c) show the reductive elimination of substituted nitrophenols from
Ϫ
ؒ
ؒ
Q
ϩ Q Ϫ ϩ 2Hϩ
QH2 ϩ Q
(2)
indolequinones 3 (10.8 Gy ∼7.2 µmol dmϪ3 (CH3)2C OH radicals
ؒ
recorded at 434 nm), 1 (3.1 Gy ∼2.1 µmol dmϪ3 (CH3)2C OH radicals
ؒ
recorded at 400 nm), and 9 (10 Gy ∼6.7 µmol dmϪ3 (CH3)2C OH
ؒ
In contrast, the decay of the semiquinone radical of 3 was
associated with an increase in absorption between 350–500 nm
ascribed to the 2-methoxy-4-nitrophenoxide anion. In propan-
2-ol–water (50%, v/v) 2-methoxy-4-nitrophenol has a pKa (LG–
radicals recorded at 410 nm).
OH
LG–OϪ ϩ Hϩ) = 7.56 0.1 (LG = leaving group) and
when deprotonated exhibits a ground-state absorption maxi-
mum at ∼420 nm. As previously observed for 8 under similar
experimental conditions (where 4-nitrophenol has a slightly
higher pKa = 7.71 0.03), the reductive elimination of 2-
methoxy-4-nitrophenol from 3 is biphasic (see Fig. 2a): the first
phase of release complete in ∼100 ms and a second slower phase
which is complete 10 s after reduction. This biphasic release of
the 2-methoxy-4-nitrophenoxide anion reflects complex kinetics
Ϫ
ؒ
associated with reductive elimination initially from the Q
radical via reaction (3) and then from the hydroquinone via
reaction (4). The latter is formed when reaction (2) begins to
compete with reaction (3).
Ϫ
(Q–LG) → Q ϩ ϩ LG
(3)
(4)
ؒ
ؒ
(QH2–LG) → QH2ϩ ϩ LGϪ
Fig.
3
HPLC chromatogram showing typical product profiles
obtained by reductive elimination from the (indol-3-yl)methyl position
of indolequinone 3. Conditions: γ-radiolysis (124 Gy) of 3 (40 µM) in
an N2O-saturated propan-2-ol–water mixture (50%, v/v) containing
phosphate buffer (4 mmol dmϪ3) at pH 7.4.
The rates of reductive elimination of 2-methoxy-4-
nitrophenoxide anion from both the Q Ϫ radical and QH2 were
ؒ
derived using a data-fitting model in FACSIMILE. A model
comprising reactions (2)–(4) was used to give ‘best’ fits to kin-
etic traces (see Fig. 2 for an example of a fitted kinetic trace)
obtained by pulse radiolysis and gave k3 = 25.5 0.1 sϪ1
and k4 = 4.1 0.1 sϪ1 for 3. Table 1 contains the rate constants
for the reductive elimination of substituted nitrophenols from
the (indol-3-yl)methyl position of indolequinones 1–8. Steady-
state γ-radiolysis confirmed that in all cases reduction of the
parent indolequinone generated stoichiometric amounts of
the corresponding leaving group, with the reactive species
formed from the indolequinone, presumably an iminium ion,
being intercepted by water or propan-2-ol solvents to give the
J. Chem. Soc., Perkin Trans. 2, 2001, 1340–1345
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Table 1 Rate constants for the reductive elimination of substituted nitrophenols and nitrothiophenol leaving groups (LGs) from both semiquinone
radical and hydroquinone species, ground-state pKa of the LGs, and radiation chemical yields for loss of parent indolequinone (Q) and release of
LGs
Ϫ
G(ϪQ)b/
G(LGϪ)b/
ؒ
k3(Q–LG
Q
k4(QH2–LG
Q
ϩ ϩ LGϪ)/sϪ1
pKa(LG–OH
ϩ ϩ LG)/sϪ1
LG–OϪ ϩ Hϩ)a
µmol JϪ1
µmol JϪ1
ؒ
Q
1
2
3
4
5
6
7
8
9
465
—
25.5 0.1
49.9 0.2
31.2 0.1
41.3 0.2
520
25.2 0.1
—
7
—
∼0.3
4.1 0.1
—
2.2 0.1
—
—
1.7 0.1
∼0.3
3.87 0.02
9.20 0.10
7.56 0.02
5.42 0.07
6.82 0.06
6.33 0.05
6.33 0.05
7.71 0.03
4.31 0.07
0.71 0.01
1.57 0.01
1.21 0.01
∼0.65
0.70 0.01
1.10 0.01
2.12 0.01
1.60 0.01
0.32 0.01
0.58 0.01
1.43 0.01
1.22 0.01
∼0.61
0.71 0.01
1.11 0.01
2.10 0.01
1.50 0.01
0.27 0.01
5
a Excluding indolequinone 9 which contains a thioether linking bond. b Propan-2-ol–water (50%, v/v); G(CH3)2C OH = 0.67 µmol JϪ1; dose rate ∼3.9
ؒ
Gy minϪ1
.
was not possible to determine the rate of reductive elimination
from the hydroquinone. Fig. 4 shows the linear correlation
between the half-life for the reductive elimination of the substi-
Ϫ
ؒ
tuted nitrophenols from the Q radicals (t1/2 = 0.7/k3) in milli-
seconds, versus the pKa of the phenolic group. Lowering the pKa
of the leaving group by ∼4 pH units shortened the half-life for
reductive elimination by (t1/2 = (0.7/k3)) ∼26 ms. Also shown in
Fig. 4 is a test of a linear relationship between log k3 and pKa
and a comparison with elimination rates of the leaving groups
shown in the radical anions of 4-nitrobenzyl halides and
toluene-p-sulfonates (see Discussion).16,17
Our previous studies have shown that incorporation of a
methyl or heterocyclic substituent at the (indol-3-yl)methyl pos-
ition significantly enhanced the rate of reductive elimination
from the Q Ϫ radical.13 The rate of reductive fragmentation of 6
ؒ
is k3 = 41 sϪ1 and substitution with methyl at the R3Ј position to
give 7 increases the rate 10-fold to k3 = 520 sϪ1
.
Reductive fragmentation of a thioether linker. Fig. 2c shows a
typical kinetic trace showing the reductive elimination of
nitrothiophenol from 9. Despite the fact that the nitrothiophe-
nol has
a
relatively low pKa (LG–SH
LG–
SϪ ϩ Hϩ) = 3.87 0.02, no evidence was obtained for reductive
Ϫ
ؒ
elimination directly from the Q–LG radical. Instead, the Q–
Fig. 4 Linear correlation between the half-lives (open circles) and log10
k3 (solid circles) for the reductive elimination of the substituted
nitrophenols from the semiquinone radical vs. pKa of the phenolic
hydroxy group. For comparative purposes the plot also contains a log10
k vs. pKa plot (solid squares) for the reductive elimination of leaving
groups from 4-nitrobenzyl halides and toluene-p-sulfonate (data taken
from ref. 17).
Ϫ
ؒ
LG radical decays by second-order kinetics to the hydro-
quinone which then fragments to release the nitrothiophenol (k4
∼0.3 sϪ1). Steady-state γ-radiolysis confirmed that the radiation
chemical yield for the loss of 9 G(–Q–LG) = 0.37 0.01 µmol
JϪ1 approximately equals that for the generation of the leaving
group (LG = nitrothiophenol) G(LG) = 0.28 0.1 µmol JϪ1
.
However, both yields were about one-half that expected
(G(CH3)2C OH = 0.67 µmol JϪ1), indicating that reductive elim-
ؒ
alcohol 10 and the isopropyl ether 12 (Fig. 3). For example,
the radiation chemical yield for the loss of 3 G(–Q–LG) =
1.02 0.1 µmol JϪ1 equals that for the generation of the leaving
group (LG = 2-methoxy-4-nitrophenol) G(LG) = 1.02 0.1
µmol JϪ1. There is a strong correlation between the pKa of the
phenol moiety and the rate of reductive elimination from the
ination occurs primarily from the hydroquinone. This slow
elimination from 9 contrasts with the fast elimination from 1
(for leaving groups of comparable pKa) and highlights the dra-
matic differences between thioether and phenolic ether ‘linkers’
in terms of reductive elimination from the (indol-3-yl)methyl
position of indolequinones.
Ϫ
ؒ
Q
radical. For example, 2-methoxy-5-nitrophenol has a high
pKa (LG–OH
LG–OϪ ϩ Hϩ) = 9.2 0.1 and as a con-
Semiquinone and hydroquinone reactivities with oxygen. In
order to put the rates of reductive fragmentation of the indole-
quinones 1–9 into context, both semiquinone radical and
hydroquinone reactivities towards oxygen were also deter-
sequence elimination of this leaving group following reduction
of indolequinone 2 is only observed from the hydroquinone (k4
∼0.3 sϪ1). In contrast, 2,4-dinitrophenol, with a much lower pKa
(LG–OH
LG–OϪ ϩ Hϩ) = 3.87 0.02, is rapidly released
mined. The one-electron reduction potentials [E(Q/Q Ϫ)] for the
ؒ
following the reduction of indolequinone 1. The formation of
indolequinone alcohols 10 and 11 are Ϫ376 and Ϫ302 mV
the 2,4-dinitrophenoxide anion (see Fig. 2b) follows first-order
respectively.10,13 This is reflected in the corresponding rates of
Ϫ
ؒ
kinetics and matches the decay of the corresponding Q–LG
radical and is consistent with reaction (3) being the predom-
inant decay pathway (k3 ∼465 sϪ1). Even at high doses per pulse
electron transfer from the Q Ϫ radical to oxygen in reaction (5),
ؒ
Ϫ
Ϫ
ؒ
ؒ
(ca. 30 Gy) the radiation chemical yields G(Q–LG) Ϫ = G(2,4-
ؒ
Q
ϩ O2 → Q ϩ O2
(5)
dinitrophenoxide anion) are stoichiometric. Poor solubility of
the parent indolequinone 1 precluded the use of higher doses
per pulse and since reaction (3) out-competed reaction (2) it
which are k5 = 5.2 × 108 dm3 molϪ1 sϪ1 and k5 = 1.7 × 108 dm3
molϪ1 sϪ1 for 10 and 11 respectively.
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The indolequinone alcohols exhibit much poorer leaving
group behaviour than their corresponding substituted nitro-
phenol conjugates 1–9, and were therefore utilised for the study
of hydroquinone autoxidation without the added complication
of reductive elimination of leaving groups.
the same concentration of oxygen the half-life for the elimin-
ation of the leaving group from the semiquinone radical of 11
is t1/2 = 0.8 ms. Reaction (5) represents the primary competing
reaction to the reductive elimination reaction (3) and the bal-
ance between these reactions is likely to control release of a
bioactive agent as leaving group in hypoxic tumour cells. In this
work we have studied elimination of substituted nitrophenols
as representative models for bioactive agents that might be
released following fragmentation of a phenolic ether linker. It is
predicted that the half-lives for the reductive elimination from
indolequinones 3–6, 8 which fall in the range t1/2 = 28–14 ms,
would be too long to compete with the very short half-lives of
their semiquinone radicals on reaction with oxygen (t1/2 < 1
Discussion
The rates of reductive elimination from the (indol-3-yl)methyl
position of indolequinones 1–8 are dependent upon the pKa of
the hydroxy group of the substituted nitrophenol. The span of
the line in Fig. 4 (6.8 0.4 pHϪ1) indicates that a decrease in
pKa by four units shortens the half-life for reductive elimination
Ϫ
from the Q radical by ca. 27 ms. A similar effect has previ-
ously been achieved by substitution with a methyl or hetero-
cyclic moiety at the (indol-3-yl)methyl position in 8. This sub-
stitution also shortens the half-life for reductive elimination by
ca. 26 ms.
ms), since even at a low oxygen concentration of 5 µmol dmϪ3
,
ؒ
elimination of the leaving group would be efficiently inhibited.
The half-life for 1 is t1/2 ∼1.5 ms which begins to approach a
level where reductive fragmentation can begin to compete with
Ϫ
ؒ
Q
reactivity with oxygen. Many biologically active com-
A number of studies have established relationships between
rates of cleavage of radical anions and free energy changes.18
Over a limited range an approximately linear relationship
between log k and free energy may be found, and in the case of
cleavage of radical anions of α-aryloxyacetophenones in DMF,
a linear relationship was established between log k and the pKa
values of the phenols corresponding to the leaving groups, with
a slope of the regression line equal to Ϫ0.53.19 In Fig. 4 the
corresponding plot covers a limited range and the linear fit is
less satisfactory, but the slope equals Ϫ0.31 0.06. Also shown
in Fig. 4 is an analysis of the rates of elimination of leaving
groups from 4-nitrobenzyl halides and toluene-p-sulfonate,
where the slope of the plot of log k vs. pKa equals Ϫ0.50 0.05.
If this relationship for 4-nitrobenzyl radical anions extends to
other leaving groups such as tertiary amines, i.e. alkylating
mustard pro-drugs that are nitrobenzyl derivatives linked to
substituted trialkylamines or dimethylanilines [O2NC6H4CH2-
NϩR3],20,21 then it is no surprise that the radical anions dis-
proportionate before release of the amine leaving group can be
observed.22 The corresponding aliphatic or aromatic tertiary
amines have pKa values around 6.4 or 5 respectively. The
relationship in Fig. 4, if applicable, would predict half-lives for
elimination of these amines in excess of 10 s (of the order of 70
s for elimination of MeN(CH2CH2Cl)2 from the radical anion
of the 4-nitrobenzyl prodrug).
pounds with phenolic hydroxy groups available for coupling to
the (indol-3-yl)methyl position of indolequinones are unlikely
to have as sufficiently low a pKa as 2,4-dinitrophenol (pKa
∼3.87). Consequently, a significant lowering of the pKa of the
leaving group or active drug (without compromising biological
activity) to facilitate faster rates of reductive fragmentation is
one possible strategy for targeting delivery to hypoxic tissues.
However, a more productive strategy, as exemplified by indole-
quinone 7, would involve manipulation of leaving group pKa
coupled with the required substituent at the indole carbinyl
position.
In conclusion, this study demonstrates the dependence upon
leaving group pKa of the rate of reductive elimination from
the (indol-3-yl)methyl position of indolequinones. Therefore,
in addition to α-substitution of these positions, manipulation
of the pKa of potential agents to be reductively-eliminated
may be a strategy for enhancing hypoxic targeting if this can
be achieved without compromising the activity of the drug to
be delivered.
Experimental
General procedures and materials
NMR spectra: J values are given in Hz. Elemental analyses
were determined at the University of Exeter and all compounds
characterized by HRMS were chromatographically homo-
geneous. Solutions in organic solvents were dried by standard
procedures, and dimethylformamide, toluene and tetrahydro-
furan were anhydrous commercial grades. Silica gel for flash
column chromatography was Merck Kieselgel 60 H grade (230–
400 mesh) or Matrex silica 60. Propan-2-ol was obtained from
Sigma-Aldrich Chemical Company Ltd (Gillingham, Dorset,
UK), and nitrous oxide was obtained from the British Oxygen
Company (Gillingham, Kent, UK).
Substitution by methyl on the linker (e.g. 7 vs. 6) increases the
rate of elimination of the leaving group by a factor of ∼12. This
is of the same order as that found in cleavage of radical anions
of 4-nitrobenzyl halides substituted on the exocyclic carbon
with methyl (∼11–24).16,17
Clearly both lowering the pKa of the leaving group and
incorporation of suitable radical-stabilising substituents at the
indolyl carbinyl position can have a dramatic effect on rates of
elimination. This is exemplified by indolequinone 7, the semi-
quinone of which has the shortest half-life (t1/2 = (0.7/k3) ∼1.3
ms) of the indolequinones studied to date. However pKa is
clearly not the only factor since the correlation appears to apply
only to the elimination of phenoxides since a thioether linker, as
exemplified by indolequinone 9, fragments only slowly from the
hydroquinone despite nitrothiophenol having a low pKa (∼4.3)
which would normally favour the elimination of substituted
nitrophenols. There have been a number of studies that com-
pare the relative leaving ability of RSϪ and ROϪ groups in uni-
molecular and bimolecular processes.23 For leaving groups of
the same pKa, it has been shown that ROϪ (or ArOϪ) can be a
better leaving group than RSϪ (or ArSϪ) by over 3 orders of
magnitude, the difference reflecting the intrinsic nature of the
heteroatom.23c
Pulse radiolysis
The redox properties of the indolequinones and the kinetic
characteristics of their semiquinone radicals (Q Ϫ) were investi-
ؒ
gated by pulse radiolysis. Semiquinone radicals were generated
following reduction of the parent indolequinone by the propan-
ؒ
2-ol radical [(CH3)2C OH]. Kinetic spectrophotometry with
sub-microsecond time resolution was used to monitor the reac-
tions involving the Q Ϫ radical and the reductive elimination of
ؒ
substituted nitrophenols. Experiments were performed using a
6 MeV linear accelerator as described previously.10 The
absorbed radiation dose per electron pulse (typically 1–30 Gy)
was determined by the thiocyanate dosimeter.24
The semiquinone radical derived from 10 reacts with oxygen
For measurements of the reductive elimination of substituted
nitrophenols from indolequinones over longer time-scales up to
10 s, a solid-state light source was developed to minimize pos-
sible sample photobleaching. The source uses a number of
with a first order rate constant of k5 = 5.2 × 108 dm3 molϪ1 sϪ1
.
This value indicates a half-life (0.7/k5[O2]) of ∼0.3 ms at a
tumour relevant oxygen concentration ([O2] ∼5 µmol dmϪ3 ). At
J. Chem. Soc., Perkin Trans. 2, 2001, 1340–1345
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narrow-band (15–30 nm) light-emitting diodes (LEDs) which
cover the range ∼430–900 nm. Thus 12 LEDs were positioned in
front of an optical fiber and positioning was achieved using a
rotating wheel servo system. The output end of the fiber was at
the focus of an aspheric lens, producing a highly collimated
beam to illuminate the sample cell. This novel system was util-
ized in combination with a tungsten lamp and photodiode
detector to determine the rates of substituted nitrophenol
release. For indolequinones where the disproportionation
of semiquinone radicals could compete with the release
of substituted nitrophenols a simulated data fitting model
(FACSIMILE for Windows version 2.00, AEA Technology)
provided estimates of rate constants from experimental data.
indolequinone 11 (0.2 mmol), triphenylphosphine (3 equiv.)
and the nitrophenol (3 equiv.) in THF (15 cm3). The solution
was stirred for 1 h. The solvent was removed in vacuo and the
residue dissolved in ethyl acetate and washed with sodium
hydroxide (1 mol dmϪ3), hydrochloric acid (1 mol dmϪ3), water,
dried (MgSO4) and concentrated. The residue was purified by
column chromatography.
5-Methoxy-3-(2-methoxy-5-nitrophenoxymethyl)-1,2-
dimethylindole-4,7-dione 2. Chromatography (80% dichloro-
methane–20% ethyl acetate elution), to yield the title compound
(31%) as an orange crystalline solid, mp 253–255 ЊC (from
dichloromethane–ethyl acetate) (Found: C, 57.6; H, 4.5; N, 6.9.
C19H18N2O7ؒ0.5H2O requires C, 57.7; H, 4.8; N, 7.1%); νmax
(KBr)/cmϪ1 3099, 2925, 2848, 1669, 1631, 1601; λmax (DMF)/nm
456 (log ε 3.30), 340 (4.08), 292 (4.40); δH (300 MHz; CDCl3)
7.91 (2H, m, ArH), 6.89 (1H, dd, J 7.3, J 2.2, ArH), 5.62 (1H, s,
6-H), 5.36 (2H, s, CH2), 3.92 (3H, s, OMe), 3.90 (3H, s, OMe),
3.81 (3H, s, NMe), 2.32 (3H, s, Me); δC (100 MHz; CDCl3)
178.8, 177.9, 159.7, 155.2, 147.9, 141.4, 138.1, 129.0, 121.6,
118.1 (CH), 115.8, 110.1 (CH), 109.2 (CH), 106.7 (CH), 61.9
(CH2), 56.44 (OMe), 56.37 (OMe), 32.4 (NMe), 9.8 (Me);
m/z (CI, relative intensity) 404 ([M ϩ NH4]ϩ, 100%).
Steady-state ꢀ-radiolysis
HPLC analysis was carried out using indolequinone solutions
(40 µmol dmϪ3) which were saturated with N2O gas in gas-tight
syringes before irradiation in a 60Co source. An absorbed dose
of 1 Gy equals 0.67 µmol dmϪ3 (CH3)2C OH radicals in the
ؒ
radiolysis of an N2O-saturated propan-2-ol–water (50%, v/v)
solution as determined by ferricyanide reduction. A dose rate
of 3.9 Gy minϪ1 was used, as determined by Fricke
dosimetry.25
5-Methoxy-3-(2-methoxy-4-nitrophenoxymethyl)-1,2-
High-performance liquid chromatography
dimethylindole-4,7-dione 3. Chromatography (80% dichloro-
methane–20% ethyl acetate), to yield the title compound
(75%) as an orange crystalline solid, mp 237–239 ЊC (from
dichloromethane–ethyl acetate) (Found: C, 58.1; H, 4.6; N, 6.9.
C19H18N2O7ؒ0.3H2O requires C, 58.2; H, 4.8; N, 7.2%); νmax
(KBr)/cmϪ1 3088, 2945, 2914, 1669, 1633, 1602; λmax (MeOH)/
nm 456 (log ε 2.89), 336 (3.70), 292 (4.02); δH (300 MHz;
CDCl3) 7.86 (1H, dd, J 8.9, J 2.6, ArH), 7.71 (1H, d, J 2.6,
ArH), 7.12 (1H, d, J 8.9, ArH), 5.62 (1H, s, 6-H), 5.44 (2H, s,
CH2), 3.90 (3H, s, OMe), 3.87 (3H, s, OMe), 3.81 (3H, s, NMe),
2.32 (3H, s, Me); δC (75 MHz; CDCl3) 178.7, 178.3, 159.5,
153.5, 149.3, 141.5, 138.4, 128.8, 121.3, 117.8 (CH), 115.8,
112.0 (CH), 106.8 (CH), 106.6 (CH), 61.7 (CH2), 56.5 (OMe),
56.3 (OMe), 32.4 (NMe), 9.9 (Me); m/z (EI, relative intensity)
386 (MHϩ, 96%), 369 (62), 324 (47).
Product analysis following γ-radiolysis of indolequinone sol-
utions was performed by gradient separation on a 100 mm ×
3.2 mm base-deactivated reverse-phase column (Hichrom RPB,
Hichrom, Reading, UK) at a flow rate of 1 cm3 minϪ1. The
eluents were A: 5 mmol dmϪ3 KH2PO4, 5 mmol dmϪ3 H3PO4
and B: 75% acetonitrile, 25% water with a gradient of 35–95%
for 5–8 min. Detection was at 292 nm using a Waters 616 pump,
717 detector, 996 photodiode array detector and Millennium
data acquisition (Watford, UK).
Synthesis
The synthesis of the following indolequinones has previously
been described in the literature: 5-methoxy-1,2-dimethyl-3-
(4-nitrophenoxymethyl)indole-4,7-dione 8,10 3-hydroxymethyl-
5-methoxy-1,2-dimethylindole-4,7-dione 10,10 3-(1-hydroxy-
3-(2-Formyl-4-nitrophenoxymethyl)-5-methoxy-1,2-
ethyl)-5-methoxy-1,2-dimethylindole-4,7-dione
11,13
and
dimethylindole-4,7-dione 4. Chromatography (5% ethyl acetate–
dichloromethane), to yield the title compound (46%) as a
yellow–orange crystalline solid, mp 225–227 ЊC (from ether–
hexane) (Found: C, 55.7; H, 4.1; N, 6.4. C19H16N2O7ؒ1.4H2O
requires C, 55.7; H, 4.6; N, 6.8%) (Found: MHϩ 385.1042.
C19H16N2O7 ϩ H requires 385.1035); νmax (KBr)/cmϪ1 2960,
2842, 1690, 1642, 1597; λmax (MeOH)/nm 456 (log ε 3.02), 320
(4.07), 292 (4.32); δH (300 MHz; CDCl3) 10.4 (1H, s, CHO),
8.67 (1H, d, J 2.9, ArH), 8.41 (1H, dd, J 9.2, J 2.9, ArH), 7.39
(1H, d, J 9.2, ArH), 5.66 (1H, s, H-6), 5.56 (2H, s, CH2), 3.92
(3H, s, OMe), 3.83 (3H, s, NMe), 2.34 (3H, s, Me); δC (100
MHz; CDCl3) 187.5, 178.6, 178.4, 166.7, 164.7, 159.6, 141.7,
138.2, 130.6 (CH), 124.9, 124.6 (CH), 121.2, 114.8, 114.1 (CH),
106.9 (CH), 61.9 (CH2), 56.5 (OMe), 32.5 (NMe), 9.9 (Me); m/z
(CI, relative intensity) 385 (MHϩ, 100%), 357 (46), 340 (30), 292
(33).
3-(isopropoxy)methyl-5-methoxy-1,2-dimethylindole-4,7-dione
12.10
5-Methoxy-1,2-dimethyl-3-(2,4-dinitrophenoxymethyl)indole-
4,7-dione 1.
A solution of 3-hydroxymethyl-5-methoxy-
1,2-dimethylindole-4,7-dione 10 (0.04 g, 0.17 mmol),
2,4-dinitrofluorobenzene (0.063 g, 0.34 mmol) and silver()
oxide (0.079 g, 0.34 mmol) in THF (15 cm3) was stirred at room
temperature for 5 days. The crude mixture was filtered through
Celite, and the filtrate concentrated and purified by column
chromatography (20% ethyl acetate–80% light petroleum
elution), to yield the title compound (0.015 g, 22%) as an
orange crystalline solid, mp 203–205 ЊC (from ethyl acetate–
light petroleum) (Found: C, 52.5; H, 3.5; N, 9.9.
C18H15N3O8ؒ0.5H2O requires C, 52.9; H, 3.9; N, 10.2%); νmax
(KBr)/cmϪ1 3114, 3047, 2925, 2848, 1686, 1643, 1604; λmax
(DMF)/nm 448 (log ε 3.21), 292 (4.38); δH (300 MHz; CDCl3)
8.68 (1H, d, J 2.8, ArH), 8.38 (1H, dd, J 9.3, J 2.8, ArH), 7.57
(1H, d, J 9.3, ArH), 5.66, 5.65 (3H, 2s, CH2, 6-H), 3.90 (3H, s,
OMe), 3.83 (3H, s, NMe), 2.36 (3H, s, Me); δC (100 MHz;
CDCl3) 178.7, 178.5, 159.5, 156.1, 140.2, 139.2, 129.0 (CH),
128.9, 121.6 (CH), 120.9, 115.9 (CH), 114.3, 106.9 (CH), 62.7
(CH2), 56.6 (OMe), 32.5 (NMe), 9.9 (Me); m/z (CI, relative
intensity) 419 ([M ϩ NH4]ϩ, 100%).
3-(3-Formyl-4-nitrophenoxymethyl)-5-methoxy-1,2-
dimethylindole-4,7-dione 5. Chromatography (5% ethyl acetate–
dichloromethane), to yield the title compound (18%) as an
orange crystalline solid, mp 214–216 ЊC (from ethyl acetate–
hexane) (Found: C, 58.7; H, 4.0; N, 7.0. C19H16N2O7ؒ0.25H2O
requires C, 58.7; H, 4.3; N, 7.2%); νmax (KBr)/cmϪ1 3068, 2924,
2848, 1735, 1693, 1671, 1643, 1595; λmax (DMF)/nm 448 (log ε
3.22), 332 (4.00), 288 (4.32); δH (300 MHz; CDCl3) 10.50 (1H, s,
CHO), 8.14 (1H, d, J 9.1, ArH), 7.40 (1H, d, J 2.8, ArH), 7.25
(1H, dd, J 9.1, J 2.8, ArH), 5.64 (1H, s, 6-H), 5.41 (2H, s, CH2),
3.91 (3H, s, OMe), 3.82 (3H, s, NMe), 2.31 (3H, s, Me); δC (100
MHz; CDCl3) 188.5, 178.7, 178.2, 163.1, 159.6, 142.2, 138.1,
General method for Mitsunobu reaction. Diethyl azodi-
carboxylate (4 equiv.) was added to a solution of the
3-hydroxymethylindolequinone 10 or the 3-(1-hydroxyethyl)-
1344
J. Chem. Soc., Perkin Trans. 2, 2001, 1340–1345

View Article Online
134.3, 129.1, 127.3 (CH), 121.3, 118.7 (CH), 115.1, 115.0 (CH),
106.8 (CH), 61.5 (CH2), 56.5 (OMe), 32.4 (NMe), 9.8 (Me); m/z
(CI, relative intensity) 402 ([M ϩ NH4]ϩ, 5%), 222 (30), 220
(20), 192 (55), 138 (100).
the EPSRC Mass Spectrometry Service at Swansea for mass
spectra.
References
3-(2-Fluoro-4-nitrophenoxymethyl)-5-methoxy-1,2-
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dimethylindole-4,7-dione 6. Chromatography (5% ethyl acetate–
dichloromethane), to yield the title compound (68%) as an
orange crystalline solid, mp 219–221 ЊC (from ethyl acetate–
hexane) (Found: C, 57.4; H, 3.8; N, 7.4. C18H15FN2O6 requires
C, 57.7; H, 4.0; N, 7.5%); νmax (KBr)/cmϪ1 3073, 2945, 2837,
1675, 1642, 1598; λmax (DMF)/nm 448 (log ε 3.19), 316 (4.10),
288 (4.25); δH (300 MHz; CDCl3) 8.02 (1H, m, ArH), 7.94 (1H,
dd, J 10.6, J 2.7, ArH), 7.27 (1H, m, ArH), 5.64 (1H, s, 6-H),
5.48 (2H, s, CH2), 3.89 (3H, s, OMe), 3.82 (3H, s, NMe), 2.33
(3H, s, Me); δC (100 MHz; CDCl3) 178.6, 178.4, 159.6, 152.2
(d, JCF 10), 151.5 (d, JCF 249), 141.0 (d, JCF 7), 138.4, 129.0,
121.2, 121.0 (d, JCF 4), 115.2, 114.2 (d, JCF 2), 112.4 (d, JCF 23),
106.8 (CH), 62.0 (CH2), 56.5 (OMe), 32.4 (NMe), 9.8 (Me);
m/z (CI, relative intensity) 392 ([M ϩ NH4]ϩ, 3%), 235 (18),
220 (Mϩ Ϫ C6H3FNO2, 100).
3-[1-(2-Fluoro-4-nitrophenoxy)ethyl]-5-methoxy-1,2-
dimethylindole-4,7-dione 7. Chromatography (5% ethyl acetate–
dichloromethane), to yield the title compound (84%) as an
orange crystalline solid, mp 175–177 ЊC (from ethyl acetate–
hexane) (Found: C, 58.7; H, 4.2; N, 7.1. C19H17FN2O6 requires
C, 58.7; H, 4.4; N, 7.2%); νmax (KBr)/cmϪ1 3109, 3006, 2945,
1671, 1619, 1593; λmax (MeOH)/nm 460 (log ε 3.11), 292 (4.16);
δH (300 MHz; CDCl3) 7.92 (2H, m, ArH), 7.00 (1H, apparent t,
ArH), 6.45 (1H, q, J 6.5, CH(Me)OAr), 5.65 (1H, s, 6-H), 3.85
(3H, s, OMe), 3.82 (3H, s, NMe), 2.33 (3H, s, Me), 1.73 (3H, d,
J 6.5, Me); δC (100 MHz; CDCl3) 178.7, 178.4, 159.5, 152.7,
151.4 (d, JCF 249), 151.3 (d, JCF 10), 140.6 (d, JCF 8), 135.8,
128.5, 121.6, 120.9 (d, JCF 4), 120.0, 114.4 (d, JCF 2), 112.2
(d, JCF 23), 106.9 (CH), 70.2 (CH), 56.5 (OMe), 32.1 (NMe),
21.9 (Me), 10.4 (Me); m/z (CI, relative intensity) 406 ([M ϩ
NH4]ϩ, 28%), 389 (MHϩ, 73), 317 (70), 290 (60).
16 M. Meot-Ner, P. Neta, R. K. Norris and K. Wilson, J. Phys. Chem.,
1986, 90, 168.
5-Methoxy-1,2-dimethyl-3-(4-nitrophenylthiomethyl)indole-
4,7-dione 9. Chromatography (5% ethyl acetate–dichloro-
methane elution) to yield the title compound (16%) as an
orange crystalline solid, mp 215–217 ЊC (from dichloro-
methane–ethyl acetate) (Found: C, 57.5; H, 4.0; N, 7.3.
C18H16N2O5Sؒ0.2H2O requires C, 57.5; H, 4.4; N, 7.4%); νmax
(KBr)/cmϪ1 2851, 1665, 1639, 1601; λmax (MeOH)/nm 460 (log ε
3.09), 340 (3.94), 292 (4.05); δH (300 MHz; CDCl3) 8.10 (2H,
J 7.0, ArH), 7.46 (2H, J 7.0, ArH), 5.62 (1H, s, 6-H), 4.49 (2H,
s, CH2), 3.89 (3H, s, OMe), 3.81 (3H, s, NMe), 2.25 (3H, s, Me);
δC (100 MHz; CDCl3) 178.5, 178.0, 159.6, 147.0, 145.4, 136.8,
128.8, 127.7 (CH), 123.8 (CH), 121.0, 116.0, 106.8 (CH), 56.5
(OMe), 32.5 (NMe), 27.0 (CH2), 9.6 (Me); m/z (CI, relative
intensity) 390 ([M ϩ NH4]ϩ, 83%), 373 (MHϩ, 100).
17 J. P. Bays, S. T. Blumer, S. Baral-Tosh, D. Behar and P. Neta, J. Am.
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Acknowledgements
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This work is supported by the Cancer Research Campaign
[CRC] and the Gray Cancer Institute [GCI]. We also thank
J. Chem. Soc., Perkin Trans. 2, 2001, 1340–1345
1345