Halogenated indole-3-acetic acids as oxidatively activated prodrugs with potential for targeted cancer therapy

Article abstract of DOI:10.1016/S0960-894X(02)00505-X

Substituted indole-3-acetic acid (IAA) derivatives, plant auxins with potential for use as prodrugs in enzyme-prodrug directed cancer therapies, were oxidised with horseradish peroxidase (HRP) and toxicity against V79 Chinese hamster lung fibroblasts was determined. Rate constants for oxidation by HRP compound I were also measured. Halogenated IAAs were found to be the most cytotoxic, with typical surviving fractions of <10^-3 after incubation for 2 h with 100 μM prodrug and HRP.

Full text of DOI:10.1016/S0960-894X(02)00505-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2523–2526
Halogenated Indole-3-acetic Acids as Oxidatively Activated
Prodrugs with Potential for Targeted Cancer Therapy
Sharon Rossiter,* Lisa K. Folkes and Peter Wardman
Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex HA62JR, UK
Received 22 March 2002; revised 12 June 2002; accepted 28 June 2002
Abstract—Substituted indole-3-acetic acid (IAA) derivatives, plant auxins with potential for use as prodrugs in enzyme-prodrug
directed cancer therapies, were oxidised with horseradish peroxidase (HRP) and toxicity against V79 Chinese hamster lung fibro-
blasts was determined. Rate constants for oxidation by HRP compound I were also measured. Halogenated IAAs were found to be
À3
the most cytotoxic, with typical surviving fractions of <10 after incubation for 2 h with 100 mM prodrug and HRP. # 2002
Elsevier Science Ltd. All rights reserved.
Introduction
Methods
The oxidation of the plant auxin indole-3-acetic acid
Substituted IAAs were synthesised using standard
literature procedures or adaptations of published
methodologies. 1-Methyl-, 5-fluoro-, 5-bromo-, and
5-benzyloxy- indole-3-acetic acids were purchased from
Sigma-Aldrich, Poole, UK. A number of indole-3-acetic
acids with mono- or disubstitution in positions 4–7 (2a–
h) were synthesised via a Fischer cyclisation (Scheme
(
IAA) 1 (Fig. 1) with horseradish peroxidase (HRP) has
been extensively studied, and the mechanism is known
1À5
to be complex.
The products have been shown to be
6
inhibitory towards bacterial growth and it has recently
been shown that HRP oxidation of IAA produces a
mammalian cell cytotoxin which causes damage to
plasmid DNA. The low toxicity of IAA and the fact
that it is not readily oxidised by endogenous mamma-
lian peroxidases make it a suitable candidate for use in
an enzyme-directed anticancer prodrug therapy.9
7
,8
13À15
1).
Other substituted IAAs, which could not be unambig-
uously synthesised or gave poor yields by the Fischer
method, were obtained by the direct 3-alkylation of the
appropriate substituted indole. 7-Chloroindole was
purchased from Lancaster Synthesis, Lancaster, UK, 5-
iodoindole was purchased from Avocado, Port Heyn-
sham, UK. Other haloindoles were synthesised by the
A previous study of some commercially available sub-
stituted IAA derivatives showed that, although it was
possible to predict the relative rates of oxidation by
HRP, there was no correlation between rate of oxida-
1
0
16
tion and cytotoxicity. Recently it was found that
-fluoroindole-3-acetic acid, although a very poor sub-
strate for HRP and slow to oxidise, gave very high
Leimgruber-Batcho method. Arylindoles were obtained
1
7
5
by a Suzuki coupling of 5-bromoindole. The intro-
duction of the acetic acid side chain was carried out via
alkylation of the zinc salt followed by saponification of
the ester to give the substituted IAAs 3a–3h (Scheme
1
1,12
cytotoxicity upon activation.
A number of other
substituted indole-3-acetic acids were then synthesised
in order to investigate further this unexpected result and
develop a lead prodrug for in vivo studies, with the
working hypothesis that prodrugs substituted with
halogens or other electron-withdrawing groups would
exhibit improved cytotoxicity compared to IAA itself.
1
8
2). N-Methylated compounds 4a and 4b were also
prepared using sodium hydride/iodomethane (Scheme 3).
*Corresponding author at current address: The Wolfson Institute for
Biomedical Research, University College London, The Cruciform
Building, Gower Street, London WC1E 6BT, UK. Tel.: +44-20-7679-
6865; fax: +44-20-7679-6799; e-mail: s.rossiter@ucl.ac.uk
Figure 1. Indole-3-acetic acid.
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00505-X

2524
S. Rossiter et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2523–2526
Scheme 2. IAAs via 3-alkylation of substituted indoles.
Scheme 1. Fischer synthesis of IAAs.
kinetics. Rate constants were calculated from the linear
plots of first-order rate constants against five IAA con-
centrations.
Each IAA derivative was oxidised with HRP, and the
cytotoxicity against Chinese hamster lung fibroblasts
(
V79 cells) was determined by a clonogenic assay as
8
previously described. Cells were treated with 2 mL
indoles (50 or 100 mM) and HRP (1.2 mg/mL) for 0–2 h,
then washed and left to form colonies for 7 days. After
growth, colonies were fixed with 75% methanol and
stained with 1% (w/v) crystal violet. Colonies contain-
ing >50 cells were counted and surviving fractions (SF)
calculated relative to untreated controls.
Results and Discussion
Results are shown in Table 1, with the results for the
previously studied IAA derivatives given as a compar-
No toxicity was seen when cells were incubated
with either HRP or 100 mM prodrug alone.
1
1,19
ison.
The measurement of the rate of reaction of each IAA
with HRP compound I, formed from a 1 s premixing of
equimolar HRP and hydrogen peroxide (0.47–0.76 mM),
was carried out as previously described, at pH 7, using
double-mix stopped-flow spectrophotometry, monitor-
Rates of oxidation are broadly in line with mechanistic
predictions, i.e., the presence of electron-donating
groups enhances the rate of oxidation by HRP com-
pound I. Oxidation rates show some variation with
substituent position. There is no direct correlation
between rate of oxidation and subsequent toxicity,
although many of the monosubstituted IAAs that are
8
,10,19
ing the formation of HRP compound II at 411 nm.
Five experiments were averaged and fitted to first order
Table 1. Oxidation rates of IAA derivatives and cytotoxicity of products
Reference
Substituent
Rate constant with HRP cmp
À1 À1
Surviving fraction after 2 h
incubation, mean (ÆSEM), n=3
3
a
I Â10 M
s
(ÆSEM)
2
a
5-CF
-F
1-Me, 5-F
4-Cl, 6-Cl
1-Me, 6-Cl
7-Cl
4-F
4-Cl
5-Cl
5-Br
6-F
1-Me
None
5-I
5-Me, 7-F
5-Cl, 7-F
4-Me, 7-F
6-Cl
3
O
0.3Æ0.01
0.4Æ0.01
0.4Æ0.03
0.6Æ0.02
0.9Æ0.1
1
1
1
À5
5
<1Â10
4
2
4
3
3
3
2
a
b
b
a
b
c
1
1
1
À5b
0.9Æ0.1
4Â10
À5
À5
1.0Æ0.06
1.4Æ0.01
1.4Æ0.04
1.7Æ0.04
1.8Æ0.02
3.7Æ0.03
9Â10 (1Æ7Â10
)
À3
À5
À2
À2
À3
À5
c
3Â10 (Æ2Â10
)
)
4Â10 (Æ3Â10
À3 c
3d
1Â10 (Æ7Â10
)
À3
1Â10 (Æ5Â10
)
)
1
9
À2
À3
3.8Æ0.1
3.9Æ0.2
4.1Æ0.9
5.3Æ0.4
5.8Æ0.3
7.5Æ0.2
1Â10 (Æ5Â10
3
2
2
2
3
e
d
e
f
1
1
1
1
À5
f
<1Â10
5
5
-BnO
-MeO
21Æ0.03
0.2 (Æ0.1)
19
22Æ0.1
41Æ2
1
1
1
1
1
2g
3g
2h
3h
5-Me
5-Ph
2-Me, 5-F
46Æ3
88Æ3
5-(4-ClC
-Me
-Me, 5-MeO
6
H
4
)
103Æ0.2
1
9
À3
À2
À3
À2
2
435Æ6
4Â10 (Æ2Â10
)
)
1
9
2
1710Æ130
4Â10 (Æ1Â10
a
1
00 mM prodrug+1.2 mg/mL HRP.
b
n=1.
n=2.
c

S. Rossiter et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2523–2526
2525
Figure 3. 3-Methyleneoxindole.
substituted IAAs. We are currently investigating the
synthesis and reactivity of 5 and substituted analogues,
and have demonstrated that these compounds exhibit
Scheme 3. N-Methylation of IAAs.
cytotoxic have an oxidation rate constant in the range
À1 À1
toxicities broadly consistent with those of the corre-
22,23
2
3
5
Â10 –5Â10 M
s
. Disubstituted halogenated IAAs
sponding oxidised prodrugs.
The toxicity of the
were generally not toxic after oxidation. The results
seem to indicate that both steric and electronic effects
may be involved, as prodrugs with similar oxidation
rates show a wide variation in subsequent cytotoxicity.
For example, it is interesting to note that 4-chloro-IAA
most rapidly oxidised 2-methyl-substituted IAA analo-
gues cannot be explained in terms of an oxindole pro-
duct, and so it is expected that there is at least one other
mechanism of toxicity involved, which requires further
investigation.
3
c and 5-chloro-IAA 2c have equal rates of oxidation,
but oxidation of 3c results in no observable toxicity
whereas the products of oxidation of 2c give a cell sur-
Conclusion
À3
viving fraction of 3Â10 . Prodrugs that are very slowly
3
À1 À1
oxidised (k<1Â10 M
s ) gave little cytotoxicity,
Halogenated indole-3-acetic acids, in particular 6-chloro
IAA, have been identified as potent lead prodrugs for
peroxidase-mediated targeted cancer therapy. The par-
ent prodrugs exhibit negligible toxicity at 100 mM, and
so efficient targeting of the activating enzyme has the
potential to give a very favourable therapeutic ratio for
this enzyme-prodrug system.
with the notable exception of 5-fluoro-IAA. In these
cases, the slow turnover of the prodrug by HRP may
limit any cytotoxic effect of the products generated.
6
-Chloro IAA 3f exhibited the highest toxicity upon
HRP activation, with further experiments using 50 mM
prodrug plus HRP giving a measured cell surviving
À4
fraction after 2 h incubation of 8.5Â10 . This analogue
is now the lead prodrug for further studies of in vitro
and in vivo drug targeting strategies (Fig. 2). Such stra-
tegies include antibody, gene, or polymer targeting of
HRP to tumours (antibody/gene/polymer directed
enzyme-prodrug therapy; ADEPT, GDEPT, PDEPT)
Acknowledgements
This work has been funded by the Cancer Research
Campaign, the Association for International Cancer
Research and the Gray Laboratory Cancer Research
Trust.
for selective activation of the prodrug at the tumour
site.11,20,21
Preliminary studies suggest that 3-methyleneoxindole 5
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