Reactivity toward thiols and cytotoxicity of 3-methylene-2-oxindoles, cytotoxins from indole-3-acetic acids, on activation by peroxidases

Article abstract of DOI:10.1021/tx025521+

Oxidation of indole-3-acetic acid and its derivatives by peroxidases such as that from horseradish produces many products, including 3-methylene-2-oxindoles. These have long been associated with biological activity, but their reactivity has not been characterized. We have previously demonstrated the potential value of substituted indole acetic acids and horseradish peroxidase as the basis for targeted cancer therapy, since the compounds are of low cytotoxicity until oxidized, when high cytotoxicity is observed; the combination of prodrug and enzyme depletes intracellular thiols. In this study, 3-methylene-2-oxindole and derivatives substituted in the 4-, 5-, or 6-position with methyl, F, or Cl have been synthesized and their reactivity toward representative thiol nucleophiles (glutathione, cysteine, and a cysteinyl peptide) measured using stopped-flow kinetic spectrophotometry. Rate constants were in the range ～2 × 10³ to 2 × 10⁴ M^-1 s^-1 at pH 7.4, 25°C, implying a lifetime of a few tens of milliseconds for these methylene oxindoles in the cellular environment and diffusion distances of a few micrometers. As expected, halogen substitution decreased the rate of production of the methylene oxindoles on treatment of horseradish peroxidase. The cytotoxicities of the compounds were measured using Chinese hamster V79 fibroblast-like cells in vitro. The halogen-substituted derivatives were much more cytotoxic than the 5-methyl analogue or the parent (unsubstituted) compound, consistent with the trends in rate constant for reaction with the thiols. The results show that the cytotoxic response in the prodrug (indole acetic acid) and enzyme (horseradish peroxidase) system reflects the reactivity of methylene oxindoles toward nucleophiles much more than the rate of generation of the oxindoles, and helps explain the possible advantages of 5-fluoroindole-3 -acetic acid compared to IAA as a lead compound for investigation in targeted cancer therapy.

Full text of DOI:10.1021/tx025521+

Chem. Res. Toxicol. 2002, 15, 877-882
877
Rea ctivity tow a r d Th iols a n d Cytotoxicity of
3-Meth ylen e-2-oxin d oles, Cytotoxin s fr om In d ole-3-a cetic
Acid s, on Activa tion by P er oxid a ses
Lisa K. Folkes, Sharon Rossiter, and Peter Wardman*
Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middx HA6 2J R, U.K.
Received February 21, 2002
Oxidation of indole-3-acetic acid and its derivatives by peroxidases such as that from
horseradish produces many products, including 3-methylene-2-oxindoles. These have long been
associated with biological activity, but their reactivity has not been characterized. We have
previously demonstrated the potential value of substituted indole acetic acids and horseradish
peroxidase as the basis for targeted cancer therapy, since the compounds are of low cytotoxicity
until oxidized, when high cytotoxicity is observed; the combination of prodrug and enzyme
depletes intracellular thiols. In this study, 3-methylene-2-oxindole and derivatives substituted
in the 4-, 5-, or 6-position with methyl, F, or Cl have been synthesized and their reactivity
toward representative thiol nucleophiles (glutathione, cysteine, and a cysteinyl peptide)
measured using stopped-flow kinetic spectrophotometry. Rate constants were in the range ∼2
× 103 to 2 × 104 M-1 s-1 at pH 7.4, 25 °C, implying a lifetime of a few tens of milliseconds for
these methylene oxindoles in the cellular environment and diffusion distances of a few
micrometers. As expected, halogen substitution decreased the rate of production of the
methylene oxindoles on treatment of horseradish peroxidase. The cytotoxicities of the
compounds were measured using Chinese hamster V79 fibroblast-like cells in vitro. The
halogen-substituted derivatives were much more cytotoxic than the 5-methyl analogue or the
parent (unsubstituted) compound, consistent with the trends in rate constant for reaction with
the thiols. The results show that the cytotoxic response in the prodrug (indole acetic acid) and
enzyme (horseradish peroxidase) system reflects the reactivity of methylene oxindoles toward
nucleophiles much more than the rate of generation of the oxindoles, and helps explain the
possible advantages of 5-fluoroindole-3-acetic acid compared to IAA as a lead compound for
investigation in targeted cancer therapy.
Sch em e 1. Str u ctu r es of In d ole-3-a cetic Acid a n d
Som e of th e P r od u cts F or m ed on Rea ction w ith
Hor ser a d ish P er oxid a se
In tr od u ction
Indole-3-acetic acid (IAA,1 Scheme 1, 1) is a plant
growth hormone (auxin) (1) that is oxidatively degraded
by peroxidases; numerous studies utilizing horseradish
peroxidase (HRP) have been reported, meriting a sepa-
rate chapter in a monograph on heme peroxidases (2).
Unusually, the usual cofactor for oxidations catalyzed by
HRP, hydrogen peroxide, is not required. One explana-
tion may be that oxidation involves as a first step the
formation of a radical cation (Scheme 1, 2) that fragments
in microseconds (3) to release CO2 from the side chain,
forming a carbon-centered skatolyl radical (Scheme 1, 3).
This radical reacts rapidly with oxygen to form a peroxyl
radical (4) that is reduced by IAA or cellular reductants
to the hydroperoxide (5), which may substitute for H2O2
in forming the reactive peroxidase intermediate, com-
pound I from HRP. (Trace peroxides in biological media
may also be sufficient to initiate the catalytic cycle.)
a subsequent degradation product, 3-methylene-2-oxin-
dole (8) (4-13). Our interest in the IAA/HRP combination
arose from the possible application of IAA as a prodrug
in cancer therapy, with HRP generating a cytotoxin (14-
17). Targeting the activating enzyme, using, e.g., antibody-
directed [ADEPT (18)] or gene-directed [GDEPT (19)]
approaches, could provide a basis by which the combina-
tion of IAA or an analogue with HRP or another suitable
peroxidase could form an alternative prodrug/enzyme
The alcohol (6) and aldehyde (7) may also be formed
via the peroxyl radical (4), but most interest in respect
of the biological activity of IAA in plants has focused on
* To whom correspondence should be addressed. Telephone: + 44
1923 828 611. Fax: +44 1923 835 210. E-mail: wardman@gci.ac.uk.
1 Abbreviations: CysSH, cysteine; EMEM, Eagle’s modified medium;
GSH, glutathione; HRP, horseradish peroxidase; IAA, indole-3-acetic
acid; MOI, 3-methylene-2-oxindole; SMEM, spinner-modified EMEM.
10.1021/tx025521+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/21/2002

878 Chem. Res. Toxicol., Vol. 15, No. 6, 2002
Folkes et al.
strategy for cancer therapy, meriting comparison with
those already being evaluated (20, 21). Demonstration
of the potential application of transfection of the HRP
gene has been reported (22, 23). One strategy toward
tumor selectivity with gene therapy seeks to place the
HRP gene under the control of hypoxia-responsive pro-
moters (23), exploiting the different oxygen status of
tumors and normal tissues (24). Mammalian peroxidases
catalyze the formation of oxidized products from IAA
much less efficiently than HRP, and hydrogen peroxide
is an essential cofactor with the mammalian peroxidases
(25, 26) and several other fungal and plant peroxidases
(27) but not HRP. [The reactivities of the enzyme
intermediates, compounds I and II, toward some indoles
are, however, lower for HRP than for myeloperoxidase
or lactoperoxidase (28).] Although the skatolyl-type radi-
cal from the easily oxidized 2-methyl IAA derivative was
detected by spin trapping in myeloperoxidase-rich human
leukemia HL-60 cells without added HRP, very high
concentrations of the prodrug were needed. Further,
these high concentrations resulted in much less cytotox-
icity in this cell line than observed using 3 orders of
magnitude lower concentrations of the prodrugs with
HRP in fibroblasts and cell lines from solid human or
murine tumors (14, 17).
Exp er im en ta l P r oced u r es
Ma t er ia ls. All chemicals unless otherwise stated were
obtained from Sigma, U.K. The peptide Lys-Cys-Thr-Cys-Cys-
Ala (trifluoroacetate salt) was obtained from Bachem (UK) Ltd.,
St. Helens, Merseyside, U.K. 4-Cl-IAA was synthesized as
described previously (23). 3-Methylene-2-oxindole and analogues
were synthesized using the Peterson olefination procedure (34);
full details and methods for the synthesis of 5-Me-IAA, 5-Cl-
IAA, and 6-Cl-IAA are described elsewhere (35).
Rea ctivity of Meth ylen e Oxin d ole a n d An a logu es w ith
Th iols. Spectral changes were examined using a Hewlett-
Packard HP8452A spectrophotometer and a tandem cell (Hellma
type 238, with two chambers each with 4.375 mm optical path
length permitting spectra to be obtained before and after
mixing). For kinetic measurements, ethanolic stock solutions
of MOI were diluted in phosphate buffer (50 mM, pH 7.4) to
give MOI concentrations of 0.5-1 mM (ethanol 2.5-10% v/v),
and these were mixed in a Hi-Tech SF-61 stopped-flow spec-
trophotometer with equal volumes of freshly prepared GSH or
CysSH (5-10 mM, pH adjusted to 7.4 using NaOH). Similar
experiments involving MOI and the peptide Lys-Cys-Thr-Cys-
Cys-Ala involved mixing a solution of MOI (0.05 mM) with equal
volumes of peptide (0.25-1 mM). Loss of MOI was followed at
370 nm; five experiments were averaged and fitted to first-order
kinetics. Rate constants were calculated from the linear plots
of first-order rate constants against thiol concentrations.
Cell Su r viva l Exp er im en ts. Chinese hamster lung fibro-
blast V79 cells, obtained from the European Collection of Cell
Cultures, Salisbury, U.K., were maintained in Eagle’s modified
medium (EMEM) supplemented with 10% FCS, 2 mM L-
glutamine, 100 units/mL penicillin, and 100 µg/mL streptomy-
cin, and with spinner modified EMEM (SMEM) supplemented
with 7.5% FCS. Cells were subcultured by trypsin (1 × 0.5%
porcine trypsin, 0.2% EDTA) removal of the cells. For cytotox-
icity experiments, V79 cells were allowed to attach to 6 cm Petri
dishes from spinner culture for at least 1 h in a humidified 37
°C 5% CO2/air incubator before administration of the drug. MOI
stock solutions (0.5-1 mM) were made in 1-6% ethanol/phenol
red free Hanks’ balanced salt solution. MOI dilutions (2 mL,
0.25-10 µM) were added to the cells for 2 h, and then washed
with 2 mL of Hanks’ solution and left to form colonies in EMEM
for 7 days. After growth, colonies were fixed with 75% methanol
and stained with 1% (w/v) crystal violet. Colonies containing
>50 cells were counted and surviving fractions (SF) calculated
relative to untreated controls.
While our original hypothesis was based on enhanced
oxidative stress from the peroxyl radical intermediate
(29), involving (for example) lipid peroxidation (14-16),
it soon became evident that the biologically active
intermediate was much longer-lived (14-16). Our inter-
est naturally turned to the methylene oxindole (8) as a
putative cytotoxin in mammalian cells, but it is poorly
characterized. Although it is known to be reactive toward
thiols (7) or histones (8), and depletion of intracellular
thiols after exposure to IAA/HRP has been demonstrated
(17), reactivity has not been quantified. In addition to
the mechanistic relevance, it is important to know the
rate constants of reaction with key ‘sinks’ for the reactive
cytotoxin, as these will determine the diffusion distance
and hence the potential for ‘bystander’ effects. An ad-
ditional reason for characterizing some of the properties
of methylene oxindoles was our unexpected observation
that substitution of IAA with fluorine in the 5-position
significantly enhanced the cytotoxicity of the combination
with HRP compared to IAA (17). This was despite the
expectedsand foundsdecrease in the ease of oxidation
by the peroxidase intermediates (30, 31) conferred by the
electron-withdrawing substituent, since peroxidase-
catalyzed oxidations have remarkably high redox de-
pendencies (2, 32, 33).
F or m a tion of Meth ylen e Oxin d oles fr om Oxid a tion of
In d ole Acetic Acid s by HRP . Indole stock solutions (1 mM)
were freshly prepared in Hanks’ solution with 1% v/v ethanol
and protected from light. IAA or analogues (0.1 mM, 2 mL) were
added to a 6 cm Petri dish with 1.2 µg/mL HRP and incubated
at 37 °C using the same conditions as in the cell survival
experiments. Samples were removed over 2 h for HPLC analysis
using a Waters 2690 HPLC with a model 996 photodiode array
detector. Products were eluted on a reversed-phase RPB column
(100 × 3.2 mm) with ammonium acetate (25 mM, pH 5.1), with
a gradient of 25-75% of 75% acetonitrile over 10 min. MOI
formation was measured at 250 nm, calibrating with synthe-
sized standards.
In this study, we have synthesized 3-methylene-2-
oxindole (MOI, Scheme 1, 8) and five derivatives substi-
tuted with methyl, F, or Cl in the benzenoid ring, to vary
the redox properties. Rate constants for reaction of MOI
or derivatives with glutathione (GSH) and cysteine
(CysSH) have been measured using stopped-flow kinetic
spectrophotometry, the cytotoxicities of the compounds
have been measured using mammalian fibroblasts, and
the production of MOI or derivatives by treatment of the
corresponding IAA derivatives with HRP has been com-
pared. The results help explain the unexpected high
cytotoxicity of some halogenated IAA derivatives when
activated by HRP.
Resu lts
Kin etics of Rea ction of Meth ylen eoxin d oles w ith
Th iols. The absorption spectra of MOI and analogues
were obtained before and after mixing with thiols. Figure
1 shows spectral changes on mixing 5-Me-MOI with GSH
in a two-compartment or ‘tandem’ spectrophotometer cell;
changes occurred immediately on mixing. This methylene
oxindole exhibited an absorbance maximum at ∼250 nm
with shoulders at ∼290 and 370 nm, and the absorbances
decreased immediately on reaction with GSH, similar to
the changes reported for MOI and GSH (7). The kinetics

Reaction of 3-Methylene-2-oxindoles with Thiols
Chem. Res. Toxicol., Vol. 15, No. 6, 2002 879
F igu r e 1. Absorption spectra of 5-methyl-3-methylene-2-
oxindole and GSH (pH 7.4) in a tandem cell before and after
mixing. 5-Me-MOI (0.05 mM) with GSH (0.5 mM): (a, s) before
mixing; (b, ---) after mixing. 5-Me-MOI (0.25 mM) with GSH
(2.5 mM): (c, - ‚ -) before mixing; d (‚‚‚) after mixing. (Con-
centrations are those in each chamber of the tandem optical cell,
both with 4.375 mm optical path, before mixing.)
F igu r e 3. Loss of substituted indole-3-acetic acids (upper
graph) or formation of substituted 3-methylene-2-oxindoles
(lower graph) on reaction of indole-3-acetic acids (100 µM) with
HRP (1.2 µg/mL) at 37 °C. Substituents: (O) none; (b) 5-methyl;
(0) 5-F; (9) 4-Cl; (4) 5-Cl; (2) 6-Cl. Inset: HPLC chromatogram
(absorbance at 250 nm) of IAA before (‚‚‚) and after (s)
treatment with HRP after 2 h. Peaks: 1, oxindole-3-carbinol;
2, IAA; 3, indole-3-carbinol; 4, MOI.
Ta ble 1. Secon d -Or d er Ra te Con sta n ts for Rea ction s of
Su bstitu ted Meth ylen e Oxin d oles w ith Th iols a t p H 7.4,
25 °C
k/103 M-1 s-1
substituent
GSH
CysSH
none
5-Me
5-F
4-Cl
5-Cl
6-Cl
2.46 ( 0.03
2.07 ( 0.02
9.03 ( 0.08
8.44 ( 0.14
12.4 ( 0.2
6.03 ( 0.04
4.50 ( 0.07
14.4 ( 0.1
10.9 ( 0.1
17.8 ( 0.2
11.0 ( 0.1
5.95 ( 0.06
F igu r e 2. Dependence on thiol concentration of first-order rate
constants for reaction of 3-methylene-2-oxindole (0.25 mM, or
0.05 mM for the peptide experiments) with excess GSH (O),
cysteine (b), or the peptide Lys-Cys-Thr-Cys-Cys-Ala (0) at pH
7.4, 25 °C. Inset: absorbance (370 nm)/time profiles for MOI
(0.25 mM) with 2.5 mM GSH (a, s) or cysteine (b, ‚‚‚).
Transients and data points are the means of 5 experiments;
uncertainties are smaller than the symbol size.
Loss of In d ole Acetic Acid s a n d F or m a tion of
Meth ylen e Oxin d oles on Tr ea tm en t w ith Hor ser a d -
ish P er oxid a se. The various indole-3-acetic acid deriva-
tives (100 µM) were incubated with HRP (1.2 µg/mL) for
up to 2 h at 37 °C. Figure 3 shows loss of the indoles,
together with the amounts of the corresponding meth-
ylene oxindole formed. A typical chromatogram is inset.
Cytotoxicity of Meth ylen e Oxin doles towar d Mam -
m a lia n Cells in Vitr o. Clonogenic survival of Chinese
hamster V79 fibroblast-like cells was measured after
exposure to varying concentrations of methylene oxin-
doles for 2 h in an incubator at 37 °C. Figure 4 shows
survival curves for all the compounds studied.
of the reactions were measured by mixing MOI or
analogues with at least a 10-fold excess of GSH or CysSH
in milliseconds in a stopped-flow spectrophotometer,
monitoring the spectral changes at 370 nm and fitting
an exponential function to obtain first-order rate con-
stants. Representative absorbance/time traces are shown
in Figure 2, along with typical dependences of the first-
order rate constants on thiol concentration. From the
slopes of such plots, values of second-order rate constants
for reaction between MOI or analogues with the thiols
were estimated, shown in Table 1.
To compare the reactivities of free cysteine with
cysteinyl residues in a peptide, the rate constant for
reaction of MOI with the peptide Lys-Cys-Thr-Cys-Cys-
Ala was measured similarly. Figure 2 includes the
dependence on peptide concentration of the first-order
rate constant characterizing the absorbance change, from
which a second-order rate constant of (1.67 ( 0.04) × 104
M-1 s-1 was determined.
Discu ssion
Reactivity of methylene oxindoles toward thiols is not
unexpected. As an R,â-unsaturated ketone, Michael ad-
dition of the thiol to the exocyclic double bond should
occur (36), presumably forming the thioether (Scheme 1,
9). Reaction between MOI and GSH was reported many
years ago (7), although rate constants have not been
reported, nor have the effects of substituents upon
reactivity.
In the cellular environment, the steady-state concen-
tration of MOI or analogues will be much less than that
of the thiol pool, the decay of MOI by thiol reactions will
be exponential, and so the half-life of MOI can be
Addition of equine glutathione-S-transferase (0.59 unit/
mL after mixing) did not accelerate the reaction between
5-F-MOI and GSH detectably.

880 Chem. Res. Toxicol., Vol. 15, No. 6, 2002
Folkes et al.
of GSH (44), so that at pH 7.4, the fraction of the thiol
present as thiolate is ∼2.5-fold higher for CysSH com-
pared to GSH, about the ratio of reactivity shown in
Table 1 for MOI. The rate constant for reaction between
MOI and the peptide Lys-Cys-Thr-Cys-Cys-Ala was found
to be over twice the rate constant for free cysteine (Table
1), but the peptide has three cysteinyl residues. Reactiv-
ity of thiol groups in proteins is thus expected not to be
significantly lower than that of free thiols.
Methyl substitution decreased, and F or Cl substitution
increased, the reactivity of MOI toward both GSH and
CysSH. While insufficient analogues were studied to
establish a Hammett-type relationship, the results were
consistent with the electron-donating and -withdrawing
properties of the substituents. It is noteworthy that 5-F-
MOI was ∼4-fold more reactive toward GSH than MOI,
whereas fluorine substitution resulted in a full order of
magnitude reduction in reactivity toward the key inter-
mediate in peroxidase catalysis, HRP Compound I (17).
The latter result was as expected from the dependence
of rate constants on the electronic properties of the
substituents (30, 31), which show a marked redox rela-
tionship, with electron-donating substituents increasing
ease of oxidation by Compound I. It was expected that
halogen substitution should result in lower rates of
formation of oxidative breakdown products on treatment
with HRP, including methylene oxindoles. This was
observed (Figure 3). Although the rate of production of
5-Me-MOI from 5-Me-IAA was unexpectedly slower than
that of MOI from IAA (Figure 3, lower plot), the initial
rate of loss of 5-Me-IAA was, as expected, greater than
that of IAA (upper plot). The turnover of IAA and
derivatives on treatment with HRP in the absence of
hydrogen peroxide (the usual cofactor for oxidations
catalyzed by HRP) is a complex subject discussed else-
where (2, 33). The present results indicate the extent of
oxidative breakdown cannot simply be predicted from
rate constants with Compound I.
While our early work in the area of HRP-catalyzed
effects of IAA derivatives had demonstrated well-behaved
structure-activity relationships for the effects of sub-
stituents on both Compound I reactivity (30, 31) and
peroxidation of liposomes (45), later studies failed to
demonstrate clear-cut substituent effects on the cytotox-
icity of IAA/HRP (14, 15). In fact, the markedly enhanced
cytotoxicity of the combination of 5-F-IAA/HRP compared
to IAA/HRP was quite unexpected (17). The results of
the present study go some way toward explaining the
relative cytotoxic effects of IAA derivatives. Figure 4
shows that the halogenated derivatives of MOI are all
significantly more toxic than MOI itself, but that the
5-methyl analogue is similar in behavior to MOI. This is
consistent with the thiol reactivity shown in Table 1, and
suggests that the effects of substituents on the reactivity
of methylene oxindoles toward nucleophiles are more
important than the effects on oxidative breakdown. The
cytotoxicities of MOI or 5-F-MOI were about 3 orders of
magnitude greater than those of the parent IAA or 5-F-
IAA in the absence of HRP (17).
F igu r e 4. Clonogenic survival of V79 cells after exposure of
varying concentrations of substituted 3-methylene-2-oxindoles
for 2 h. Substituents: (O) none; (b) 5-methyl; (0) 5-F; (9) 4-Cl;
(4) 5-Cl; (2) 6-Cl. Means and standard errors of 3 independent
experiments.
calculated as (ln 2)/(sum of products of rate constant ×
thiol concentration). Although CysSH is consistently
more reactive toward MOI and analogues than GSH
(Table 1), the latter is the dominant biological thiol, and
except in the plasma [where the concentration of CysSH
exceeds that of GSH (37)], the GSH concentration will
provide an estimate of the maximum lifetime of MOI or
analogues. Typical levels of intracellular nonprotein thiol
(NPSH, principally GSH) are of the order of 5 mM,
although values of around 10 mM have been reported
(38). In the presence of ∼5 mM GSH at 25 °C, pH 7.4,
the half-life of MOI is calculated as ∼60 ms, a value
reduced by a factor of ∼4 or ∼5 on substitution with, e.g.,
F or Cl, respectively, at the 5-position (Table 1).
The half-lives of MOI or analogues may be used to
estimate the approximate diffusion distances of these
reactive intermediates in tissue. The root-mean-square
diffusion distance in three-dimensional space is (6Dt)1/2
where D is the diffusion coefficient and t the time. The
diffusion coefficient of a small molecule in water at 37
°C may be estimated from the empirical relationship: D
) 1.013 × 10-8 M-0.46 m2 s-1 (39); for MOI or 5-F-MOI,
the relationship yields D ∼ 9 × 10-10 m2 s-1. However,
the cytosol of mammalian cells is more viscous than
water: relative viscosities of 1.1-1.2 have been suggested
(40), but overall impaired translational mobility of a
small molecule in the cytoplasm compared to water by a
factor of ∼4 has been measured (41), consistent with
other high values (42, 43). If D ∼ 3 × 10-10 m2 s-1 and t
∼ 60 or ∼ 15 ms (MOI or 5-F-MOI, respectively, reacting
with 5 mM GSH), then the diffusion distance is ∼10 or
∼5 µm, respectively. Even though diffusion involves other
barriers than the cytoplasm, these calculations suffice
to show that methylene oxindoles will not diffuse much
more than cellular dimensions in tissue.
The ∼1.3-2.5-fold higher reactivity of CysSH over
GSH toward MOI or analogues probably reflects the
higher fraction of CysSH present in the thiolate form at
pH 7.4. Thiols generally react as nucleophiles via the
thiolate form, and although the higher the pKa for thiol
dissociation, the better the conjugate base as a nucleo-
phile, this is usually less important than the effect of pKa
on thiol ionization [since the Brønsted coefficient cor-
relating log k with pKa for reactions of thiolate is <1 (36)].
The microscopic ionization constant (pKa for SH dissocia-
tion of +H3N‚‚‚SH) for cysteine is ∼0.4 lower than that
Whether thiols are the key target of the IAA/HRP
treatment is not answered by this study. The relative
reactivity of, e.g., MOI and 5-F-MOI toward other nu-
cleophiles may be similar to reaction with thiols. Binding
of HRP-catalyzed degradation products of IAA to histones
was shown many years ago (8), but we demonstrated
effects of IAA/HRP on DNA plasmids (15). Adding GSH

Reaction of 3-Methylene-2-oxindoles with Thiols
Chem. Res. Toxicol., Vol. 15, No. 6, 2002 881
(17) Folkes, L. K., Greco, O., Dachs, G. U., Stratford, M. R. L., and
to IAA solutions that had been irradiated to generate the
radical cation to initiate oxidation (but added after all
radical reactions had ceased) was cytoprotective (16), and
depleting cells of GSH by treatment with buthionine
sulfoximine enhanced cytotoxicity (17). There was ∼60%
loss of intracellular GSH after treatment of V79 cells with
50 µM IAA or 5-F-IAA (17). Even if thiol centers are not
the critical target, they are likely to be important to the
overall response to the combination of IAA and deriva-
tives with HRP.
We have discussed elsewhere (16, 21) the potential for
the use of IAA and derivatives in cancer therapy involv-
ing targeted enzyme activation of prodrugs, including
gene therapy (23, 46). The present study underlines the
conclusion that the rational choice of prodrugs must
involve a quantitative understanding of the chemical
reactivity of breakdown products as much as the details
of the prodrug/enzyme interaction.
Wardman, P. (2002) 5-Fluoroindole-3-acetic acid:
a prodrug
activated by a peroxidase with potential for use in targeted cancer
therapy. Biochem. Pharmacol. 63, 265-272.
(18) Bagshawe, K. D. (1989) Towards generating cytotoxic agents at
cancer sites. Br. J . Cancer 60, 275-281.
(19) Deonarain, M. P., Spooner, R. A., and Epenetos, A. A. (1995)
Genetic delivery of enzymes for cancer therapy. Gene Ther. 2,
235-244.
(20) Melton, R. G., and Knox, R. J ., Eds. (1999) Enzyme-Prodrug
Strategies for Cancer Therapy, Kluwer Academic/Plenum Publish-
ers, New York.
(21) Wardman, P. (2002) Indole-3-acetic acids and horseradish per-
oxidase: a novel prodrug/enzyme combination for targeted cancer
therapy. Curr. Pharmaceut. Des. 8, 1363-1374.
(22) Greco, O., Dachs, G. U., Wardman, P., Folkes, L. K., and Chaplin,
D. J . (1999) Development of an enzyme/prodrug combination for
gene therapy. Proc. Am. Assn. Cancer Res. 40, 478.
(23) Greco, O., Rossiter, S., Kanthou, C., Folkes, L. K., Wardman, P.,
Tozer, G. M., and Dachs, G. U. (2001) Horseradish peroxidase-
mediated gene therapy: choice of prodrugs in oxic and anoxic
tumor conditions. Mol. Cancer Ther. 1, 151-160.
(24) Molls, M., and Vaupel, P., Eds. (1998) Blood Perfusion and
Microenvironment of Human Tumors. Implications for Clinical
Radiobiology, Springer, Berlin.
(25) Kobayashi, S., Sugioka, K., Nakano, M., Takyu, C., Yamagishi,
A., and Inaba, H. (1980) Excitation of indole acetate in myelop-
eroxidase-H2O2 system: possible formation of indole acetate cation
radical. Biochem. Biophys. Res. Commun. 93, 967-973.
(26) Kanofsky, J . R. (1988) Singlet oxygen production from the
peroxidase-catalysed oxidation of indole-3-acetic acid. J . Biol.
Chem. 263, 14171-14175.
(27) Savitsky, P. A., Gazaryan, I. G., Tishkov, V. I., Lagrimini, L. M.,
Ruzgas, T., and Gorton, L. (1999) Oxidation of indole-3-acetic acid
by dioxygen catalysed by plant peroxidases: specificity for the
enzyme structure. Biochem. J . 340, 579-583.
(28) J antschko, W., Furtmu¨ller, P. G., Allegra, M., Livrea, M. A.,
J akopitsch, C., Regelsberger, G., and Obinger, C. (2002) Redox
intermediates of plant and mammalian peroxidases: a compara-
tive transient-kinetic study of their reactivity toward indole
derivatives. Arch. Biochem. Biophys. 398, 12-22.
(29) Candeias, L. P., Folkes, L. K., and Wardman, P. (1995) Amplifica-
tion of oxidative stress by decarboxylation: a new strategy in anti-
tumour drug design. Biochem. Soc. Trans. 23, 262S.
(30) Candeias, L. P., Folkes, L. K., Porssa, M., Parrick, J ., and
Wardman, P. (1996) Rates of reaction of indoleacetic acids with
horseradish peroxidase compound I and their dependence on the
redox potentials. Biochemistry 35, 102-108.
(31) Candeias, L. P., Folkes, L. K., and Wardman, P. (1997) Factors
controlling the substrate specificity of peroxidases: kinetic and
thermodynamics of the reaction of horseradish peroxidase com-
pound I with phenols and indole-3-acetic acids. Biochemistry 36,
7081-7085.
(32) J ob, D., and Dunford, H. B. (1976) Substituent effect on the
oxidation of phenols and aromatic amines by horseradish peroxi-
dase compound I. Eur. J . Biochem. 66, 607-614.
(33) Dunford, H. B. (1995) One-electron oxidations by peroxidases.
Xenobiotica 25, 725-733.
(34) Peterson, D. J . (1968) A carbonyl olefination reaction using silyl-
substituted organometallic compounds. J . Org. Chem. 33, 780-
784.
(35) Rossiter, S. (2002) A convenient synthesis of 3-methyleneoxin-
doles: cytotoxic metabolites of indole-3-acetic acids. Tetrahedron
Lett. (in press).
(36) Douglas, K. T. (1988) Reactivity of glutathione in model systems
for glutathione S-transferases and related enzymes. In Glu-
tathione Conjugation. Mechanisms and Biological Significance
(Sies, H., and Ketterer, B., Eds.) pp 1-41, Academic Press,
London.
Ack n ow led gm en t. This work was supported by the
Association for International Cancer Research and The
Cancer Research Campaign.
Refer en ces
(1) Bandurski, R. S., Cohen, J . D., Slovin, J . P., and Reinecke, D. M.
(1995) Auxin biosynthesis and metabolism. In Plant Hormoness
Physiology, Biochemistry and Molecular Biology (Davies, P. J .,
Ed.) 2nd ed., pp 39-65, Kluwer Academic Publishers, Dordrecht,
The Netherlands.
(2) Dunford, H. B. (1999) Heme Peroxidases, Wiley-VCH, New York.
(3) Candeias, L. P., Folkes, L. K., Dennis, M. F., Patel, K. B., Everett,
S. A., Stratford, M. R. L., and Wardman, P. (1994) Free-radical
intermediates and stable products in the oxidation of indole-3-
acetic acid. J . Phys. Chem. 98, 10131-10137.
(4) Hinman, R. L., Bauman, C., and Lang, J . (1961) The conversion
of indole-3-acetic acid to 3-methyleneoxindole in the presence of
peroxidase. Biochem. Biophys. Res. Commun. 5, 250-254.
(5) Fukuyama, T. T., and Moyed, H. S. (1964) Inhibition of cell growth
by photooxidation products of indole-3-acetic acid. J . Biol. Chem.
239, 2392-2397.
(6) Hinman, R. L., and Lang, J . (1965) Peroxidase-catalyzed oxidation
of indole-3-acetic acid. Biochemistry 4, 144-158.
(7) Still, C. C., Fukuyama, T. T., and Moyed, H. S. (1965) Inhibitory
oxidation products of indole-3-acetic acid. J . Biol. Chem. 240,
2612-2618.
(8) Demorest, D. M., and Stahmann, M. A. (1972) The binding of the
peroxidase oxidation products of indole-3-acetic acid to histone.
Biochem. Biophys. Res. Commun. 47, 227-233.
(9) Ricard, J ., and J ob, D. (1974) Reaction mechanisms of indole-3-
acetate degradation by peroxidases. A stopped-flow and low-
temperature spectroscopic study. Eur. J . Biochem. 44, 359-374.
(10) Bednar, T. W., Linsmaier-Bednar, E. M., and King, C. M. (1976)
Peroxidase-H2O2 catalysed incorporation of auxin derivatives into
sRNA. Biochem. Biophys. Res. Commun. 72, 761-767.
(11) Abreu, S. L., and Lucas-Lenard, J . (1976) Inhibition of cellular
and viral protein synthesis by 3-methyleneoxindole. Antimicrob.
Agents Chemother. 11, 521-527.
(12) Kornet, M. J ., Thio, A. P., and Thorstenson, J . H. (1977) Synthesis
and cytotoxic activity of latentiated derivatives of 3-methyleneox-
indole. J . Pharm. Sci. 66, 1022-1024.
(13) Appleton, M. L., Larson, D. N., Skiles, G. L., Nichols, W. K., and
Yost, G. S. (1990) Toxicity of 3-methyleneoxindole, a proposed
reactive intermediate in the metabolism of 3-methylindole. Adv.
Exp. Med. Biol. 283, 245-248.
(14) Folkes, L. K., Candeias, L. P., and Wardman, P. (1998) Toward
targeted ‘oxidation therapy’ of cancer: peroxidase-catalysed cy-
totoxicity of indole-3-acetic acids. Int. J . Radiat. Oncol. Biol. Phys.
42, 917-920.
(15) Folkes, L. K., Dennis, M. F., Stratford, M. R. L., Candeias, L. P.,
and Wardman, P. (1999) Peroxidase-catalyzed effects of indole-
3-acetic acid and analogues on lipid membranes, DNA and
mammalian cells in vitro. Biochem. Pharmacol. 57, 375-382.
(16) Folkes, L. K., and Wardman, P. (2001) Oxidative activation of
indole-3-acetic acids to cytotoxic speciessa potential new role for
plant auxins in cancer therapy. Biochem. Pharmacol. 61, 129-
136.
(37) Wardman, P., Folkes, L. K., Bentzen, S. M., Stratford, M. R. L.,
Hoskin, P. J ., Phillips, H., and J ackson, S. (2001) Influence of
plasma glutathione levels on radiation mucositis. Int. J . Radiat.
Oncol. Biol. Phys. 51, 459-463.
(38) Wahlla˜nder, A., Soboll, S., and Sies, H. (1979) Hepatic mitochon-
drial and cytosolic glutahione content and the subcellular distri-
bution of GSH-S-transferases. FEBS Lett. 97, 138-140.
(39) Fournier, R. L. (1999) Basic Transport Phenomena in Biomedical
Engineering, Taylor & Francis, Philadelphia.
(40) Srivastava, A., and Krishnamoorthy, G. (1997) Cell type and
spatial location dependence of cytoplasmic viscosity measured by
time-resolved fluorescence microscopy. Arch. Biochem. Biophys.
340, 159-167.

882 Chem. Res. Toxicol., Vol. 15, No. 6, 2002
Folkes et al.
(41) Kao, H. P., Abney, J . R., and Verkman, A. S. (1993) Determinants
of the translational mobility of a small solute in cell cytoplasm.
J . Cell Biol. 120, 175-184.
(42) Lang, I., Scholz, M., and Peters, R. (1986) Molecular mobility and
nucleocytoplasmic flux in hepatoma cells. J . Cell Biol. 102, 1183-
1190.
scopic ionization constants of glutathione and methylmercury-
complexed glutathione. J . Am. Chem. Soc. 95, 2797-2803.
(45) Candeias, L. P., Folkes, L. K., Porssa, M., Parrick, J ., and
Wardman, P. (1995) Enhancement of lipid peroxidation by indole-
3-acetic acid and derivatives: substituent effects. Free Radical
Res. 23, 403-418.
(46) Greco, O., Folkes, L. K., Wardman, P., Tozer, G. M., and Dachs,
G. U. (2000) Development of a novel enzyme/prodrug combination
for gene therapy of cancer: horseradish peroxidase/indole-3-acetic
acid. Cancer Gene Ther. 7, 1414-1420.
(43) Seksek, O., Biwersi, J ., and Verkman, A. S. (1997) Translational
diffusion of macromolecule-sized solutes in cytoplasm and nucleus.
J . Cell Biol. 138, 131-142.
(44) Rabenstein, D. L. (1973) Nuclear magnetic resonance studies of
the acid-base chemistry of amino acids and peptides. I. Micro-
TX025521+