1H NMR studies on the reductively triggered release of heterocyclic and steroid drugs from 4,7-dioxoindole-3-methyl prodrugs

Article abstract of DOI:10.1016/S0040-4020(03)00482-4

Hypoxia is a feature of several disease states, including cancer and rheumatoid arthritis. Prodrug systems which, after bioreduction, selectively release active drugs in these tissues may be important in therapy. An improved preparation of 1,2-dimethyl-3-hydroxymethyl-5-methoxyindole-4,7-dione was developed. Mitsunobu coupling with (5-substituted) isoquinolin-1-ones (potent inhibitors of poly(ADP-ribose)polymerase) gave 1-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethoxy)isoquinolines and N-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethyl)isoquinolin-1-ones. Similar coupling with the anticancer drug pentamethylmelamine gave its potential prodrug 1,2-dimethyl-3-(N-(4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)-N- methylaminomethyl)-5-methoxyindole-4,7-dione. Treatment of sodium prednisolone hemisuccinate with 3-chloromethyl-1,2-dimethyl-5-methoxyindole-4,7-dione gave an analogous candidate prodrug of the anti-inflammatory drug prednisolone. In a chemical model system for bioreduction, SnCl₂ in CDCl₃/CD₃OD triggered rapid stoichiometric release of isoquinolin-1-ones from the O-linked prodrugs but not from the N-linked analogues. Use of this system allowed the release process to be monitored in situ by ¹H NMR spectroscopy. Diethyl hydrazine-1,2-dicarboxylate was found to reduce Sn^IV to Sn^II, making the overall reductive release catalytic in tin. The reduced (hydroquinone) prodrug may have a short lifetime under these reductive conditions, meaning that only good leaving groups are expelled. Thus 1-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethoxy)isoquinolines and analogues may be useful as reductively triggered prodrugs.

Full text of DOI:10.1016/S0040-4020(03)00482-4

Tetrahedron 59 (2003) 3445–3454
1H NMR studies on the reductively triggered release
of heterocyclic and steroid drugs from
4,7-dioxoindole-3-methyl prodrugs
Sandra Ferrer,a Declan P. Naughtonb and Michael D. Threadgillb
,
*
aDepartment of Medical Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK
bDepartment of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
Received 30 January 2003; accepted 21 March 2003
Abstract—Hypoxia is a feature of several disease states, including cancer and rheumatoid arthritis. Prodrug systems which, after
bioreduction, selectively release active drugs in these tissues may be important in therapy. An improved preparation of 1,2-dimethyl-3-
hydroxymethyl-5-methoxyindole-4,7-dione was developed. Mitsunobu coupling with (5-substituted) isoquinolin-1-ones (potent inhibitors of
poly(ADP-ribose)polymerase) gave 1-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethoxy)isoquinolines and N-(1,2-dimethyl-4,7-dioxo-
5-methoxyindol-3-ylmethyl)isoquinolin-1-ones. Similar coupling with the anticancer drug pentamethylmelamine gave its potential prodrug
1,2-dimethyl-3-(N-(4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)-N-methylaminomethyl)-5-methoxyindole-4,7-dione. Treatment of sodium
prednisolone hemisuccinate with 3-chloromethyl-1,2-dimethyl-5-methoxyindole-4,7-dione gave an analogous candidate prodrug of the anti-
inflammatory drug prednisolone. In a chemical model system for bioreduction, SnCl2 in CDCl3/CD3OD triggered rapid stoichiometric
release of isoquinolin-1-ones from the O-linked prodrugs but not from the N-linked analogues. Use of this system allowed the release process
to be monitored in situ by 1H NMR spectroscopy. Diethyl hydrazine-1,2-dicarboxylate was found to reduce SnIV to SnII, making the overall
reductive release catalytic in tin. The reduced (hydroquinone) prodrug may have a short lifetime under these reductive conditions, meaning
that only good leaving groups are expelled. Thus 1-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethoxy)isoquinolines and analogues may
be useful as reductively triggered prodrugs. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
release of drugs following reduction of the 4,7-dioxoindole-
3-methyl prodrugs 1. When 1 is reduced by two electrons to
the dihydroxyindole 2, the electron-density at the indole
nitrogen increases markedly, triggering expulsion of the
leaving group 3 (‘Drug’), in a reverse-Michael-like process.
This expulsion generates an alkenyliminium electrophile 4,
Hypoxia, whether acute or chronic, is a feature of several
disease states, including cancer and rheumatoid arthritis.1–4
In the hypoxic regions of solid tumours, viable cells are
relatively resistant to radiotherapy and to many chemo-
therapeutic strategies.1,2 We5–8 and others9–13 have pro-
posed the concept of prodrug systems in which the
pharmacophores of drugs are masked by reductively
cleaved groups. Thus, in hypoxic tissue, bioreduction of
appropriate functional groups triggers release of active
drugs selectively in that tissue. These masking groups have
included nitroheterocycles5–8 and indole-4,7-diones (indole-
quinones).9,10 In particular, Naylor et al.10,14 carried out
a study on reductively triggered release of a variety of
oxygen-based and sulfur-based leaving groups from
their (substituted) 4,7-dioxoindole-3-methyl derivatives.
Radiolysis was used for the reduction of the quinones in
this study; elimination of the leaving groups was found10 to
be predominantly from the two-electron reduction inter-
mediate, the hydroquinone (4,7-dihydroxyindole-3-methyl)
derivative. Scheme 1 shows a proposed mechanism for the
1
Keywords: prodrugs; indoledione; H NMR spectrum; isoquinolin-1-one.
*
Corresponding author. Tel.: þ44-1225-386840; fax: þ44-1225-386114;
Scheme 1. Proposed mechanism of reductively triggered release of drugs
from general 4,7-dioxoindole-3-methyl prodrugs 1.
e-mail: m.d.threadgill@bath.ac.uk
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00482-4

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S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
In our previous paper,8 we reported on the successful release
of (5-substituted) isoquinolin-1-ones from 1-(5-nitrothien-
2-ylmethoxy)isoquinolines upon reductive triggering with
a sodium borohydride/palladium/aqueous propan-2-ol sys-
tem. These 5-substituted isoquinolin-1-ones are potent
inhibitors of poly(ADP-ribose) polymerase (PARP),15 an
enzyme which is important in the control of repair of DNA
and inhibition of which is potentially therapeutic in
cancer,16 inflammation,17,18 haemorrhagic shock,19 stroke20
and HIV infection.21 Isoquinolin-1-ones are also released
from their 2-(5-nitrofuran-2-ylmethyl)- and 2-(1-methyl-2-
nitroimidazol-5-ylmethyl)- derivatives5,7 by treatment with
the same reductant and with zinc/ammonium chloride,
respectively. It is notable that all the reductively triggered
release studies reported to date have used HPLC, TLC or ex
situ NMR analysis of the products to monitor the course of
the release process. In this paper, we report the results of a
series of biomimetic reductively triggered release studies on
4,7-dioxoindole-3-methyl prodrugs of isoquinolin-1-ones
and of an anti-inflammatory steroid, in which the release of
the parent drugs is monitored in situ by 1H NMR
spectroscopy.
Scheme 2. Investigation of a synthetic route to 1,2-dimethyl-3-(hydroxy-
methyl)-5-methoxyindole-4,7-dione 11 from the ester 6. Reagents and
conditions: (i) KH, MeI, DMF, N2, 08C; (ii) HNO3, AcOH, 2108C!208C;
(iii) Sn, aq. HCl, EtOH, reflux.
2. Results
which is trapped by water (giving the hydroxymethylindole
5) or by other ambient nucleophiles. In this process, the rate
of release of the drug 3 is predicted to depend on its leaving
group ability (as reported by the pKa of its conjugate acid)10,14
and on the lifetime of the reduced species 2.
The first aspect of the present work was to develop an
efficient synthesis of the indole-4,7-dione trigger unit in a
form suitable for attachment to the drug moieties. Synthesis
of the initial target, 1,2-dimethyl-3-(hydroxymethyl)-5-
methoxyindole-4,7-dione 11 has been reported by Naylor
Scheme 3. An efficient synthetic route to 11 and Mitsunobu couplings giving the isoquinolinone-derived prodrugs 21–24 and the pentamethylmelamine-
derived prodrugs 25,26. Reagents and conditions: (i) NaH, MeI, DMF, Ar, 608C; (ii) POCl3, DMF, reflux; (iii) fuming HNO3, AcOH, 58C!208C; (iv) Sn, aq.
HCl, EtOH, reflux; (v) K2ON(SO3)2, NaH2PO4, Na2HPO4, water, acetone; (vi) NaBH4, MeOH, Ar, then air; (vii) Ph3P, EtO2CNNCO2Et, THF, Ar.

S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
3447
Scheme 4. Synthesis of 4,7-dioxoindol-3-methyl prodrug 28 of prednisolone. Reagents and conditions: (i) SOCl2; (ii) DMF, reflux.
et al.10 from ethyl 1,2-dimethyl-4,7-dioxo-5-hydroxyindole-
3-carboxylate 6, in which most of the required substituents
are in the required locations on the indole. However, several
problems were encountered when this sequence was used
(Scheme 2). The phenolic hydroxyl of 6 was deprotonated
with potassium hydride and methylated with iodomethane
in high yield, as expected. Optimisation of the conditions for
nitration of 7 led to a maximum yield of 63% of the required
4-nitro isomer 8, which could be separated only by careful
chromatography from its 6-nitro regioisomer 9 (14% yield).
Reduction of the nitro group of 8 with tin under acidic
conditions gave the amine 10; this aminoindole, bearing an
ortho-electron-donating group, was highly unstable as the
free base and decomposed rapidly. Thus, although the later
steps of Naylor’s route10 to 11 were found to be efficient, the
difficult separation of 8 from 9 and the instability of 10 led
to low and unreliable overall yields from this sequence.
Scheme 4 shows the approach to the synthesis of the
prodrug 29 for the anti-inflammatory steroid prednisolone.
The 3-hydroxymethylindole-4,7-dione 11 was converted to
the 3-chloromethyl analogue 27 in high yield with thionyl
chloride. This compound did not alkylate any of the three
hydroxy groups of prednisolone under a variety of basic
conditions, an observation which correlates with the lack of
reactivity of this steroid with chloromethylthiophenes.8
Prednisolone hemisuccinate 28, the monoester of predniso-
lone at the primary alcohol with butanedioic acid, is a
known prodrug of the therapeutic steroid in its own right,26
being cleaved efficiently to 11 in vivo by esterases.
Alkylation of the sodium salt of 28 with the chloromethyl-
1
indole 27 gave the required candidate prodrug 29. The H
NMR spectrum of 29 was somewhat complex. However, it
provided confirmation that the dioxoindol-3-ylmethyl group
had become attached as an ester at the carboxylate, rather
than as an ether at either of the two hydroxyls of 28. The
signal for the indole-3-CH2 protons moved downfield from
d 4.86 to 5.16 in the spectrum of samples in (CD3)2SO. In
the spectra of samples of 29 in (CD3)2SO and in CDCl3,
these CH2 protons resonated as a singlet; in contrast, the
spectrum run in CD3OD showed separate doublet signals for
these protons, indicating that they are magnetically
inequivalent and demonstrating that the indoledione unit
was attached to a chiral molecule.
Much more effective in reliably delivering good overall
yields of 11 was a route modified significantly from an
earlier published sequence22 (Scheme 3). 5-Methoxy-2-
methylindole 12 was deprotonated at nitrogen then
methylated with iodomethane under moderately forcing
conditions to give the intermediate 13. Vilsmeier formyla-
tion with the dimethylformamide/phosphorus oxychloride
reagent was found to give a higher yield of the 3-carb-
oxaldehyde 14 with an easier work-up than did the
N-methylformanilide/phosphorus oxychloride reagent.22
Nitration with fuming nitric acid in acetic acid was now
selective for the 4-position and the intermediate 15 was
reduced with tin to 16. This aminoindole was much more
stable and tractable than the analogous ester 10 and could be
isolated and stored satisfactorily. Oxidation with Fremy’s
salt gave the quinone 17, then reduction of the aldehyde
(and, simultaneously, the quinone), followed by air
oxidation in situ of the intermediate hydroquinone gave
the required 3-hydroxymethylindole quinone 11. As
reported previously,23 Mitsunobu coupling of 11 with the
isoquinolin-1-ones 18–20 gave the 1-(4,7-dioxoindolyl-
methoxy)isoquinolines 21 and 22 and the 2-(4,7-dioxo-
indolylmethyl)isoquinolin-1-ones 23 and 24. A similar
coupling of 11 with pentamethylmelamine 25a, an anti-
cancer drug,24 gave the potential prodrug 26a, in which the
4,7-dioxoindolylmethyl unit is attached to an exocyclic
nitrogen, in much higher yield than was obtained for the
trideuterio isotopomer 26b.25
Several reductant systems have been used in ‘biomimetic’
studies of the reductively triggered release of drugs and
other leaving groups from prodrugs. These include NaBH4/
Pd in aqueous propan-2-ol,5–7 Na2S2O4,14 Zn/NH4Cl7 and
SnCl2,7 in addition to radiolytic reduction.14 In particular,
Naylor and co-workers have used radiolysis and sodium
dithionite in their studies on reductively triggered release
from indole-4,7-dione derivatives.10,22,27 However, all of
these studies of release from indolediones and nitrohetero-
cycles have employed either HPLC analysis or ex situ NMR
spectroscopy to follow the release profile.
In the present study, SnCl2, which has not previously been
used to reduce indolequinones, was used as the reductant;
initial experiments used chromatographic methods to report
the outcomes of the reductions. Treatment of the potential
prodrug 21 with SnCl2 in dilute solution in methanol
(Method A) gave a major fully resolved peak in the HPLC
chromatogram (Fig. 1) with a retention time (Rt) 4.9 min,

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S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
limited solubility in methanol, a solution of 21 in CDCl3
was prepared and SnCl2 was added in portions as a solution
in CDCl3. 1H NMR spectra were recorded after each
addition. These spectra showed conversion of the prodrug
21 to the isoquinolin-1-one 18 and material which had
signals very similar to 3-hydroxymethylindole-4,7-dione
11. The spectra run after addition of less than 1 equiv. of
the reductant showed mixtures of 21,18 and this material
with integral ratios corresponding to the amount of tin(II)
added. Figure 2 shows three spectra from this experiment,
corresponding to the original sample of 21, the sample after
addition of 0.5 equiv. of SnCl2 and that after addition of
1 equiv. of the reductant. Upon reduction, the signals for the
isoquinoline 3-H and 4-H were changed most markedly,
with upfield shifts of 1.1 and 0.8 ppm, respectively. A
smaller upfield shift was seen for 5-H (0.3 ppm) whereas the
signals for 6-H, 7-H and 8-H were moved by less than
0.1 ppm. The greater effect in the heterocyclic ring probably
reflects the change from the ‘alkoxypyridine’ structure in
the prodrug 21 to the bioactive carbonyl ‘pyridinone’
tautomer in 18. The signal for the methylene group at
position-3 of the indole ring, which was also the linking
point between the two subunits of 21, shifted from d 5.7
to 4.4, corresponding to 4,7-dioxoindole-3-CH2OH(D) or
4,7-dioxoindole-3-CH2O alkyl and giving further evidence
that release of the drug moiety 18 had occurred. The signals
for the ring protons of the indole unit formed indicated
clearly that it was a quinone, with 6-H at d 5.4, rather than a
4,7-dihydroxyindole.
Figure 1. HPLC chromatogram of the reaction mixture after treatment of
prodrug 21 with SnCl2 (Method A). The main trace shows the reaction
mixture and the inset shows the control peak corresponding to authentic 21
without addition of SnCl2. The major product peak at Rt¼4.9 min
corresponds to 18.
after 10 min reaction time, and 21 (Rt¼6.0 min) had been
completely consumed. This product peak corresponded to
the released isoquinolin-1-one 18. Under the same HPLC
conditions, the 3-hydroxymethylindole-4,7-dione 11 gave a
peak at Rt¼4.6 min.
Having established for the first time in this preliminary
experiment that tin(II) chloride is capable of reducing the
indole-4,7-dione unit in 21 and hence triggering rapid
release of 18, a series of experiments was devised to study
1
this release in situ by H NMR spectroscopy. Owing to its
Figure 2. 1H NMR spectra of samples of 21 treated with SnCl2 in CDCl3/CD3OD, showing stoichiometric and quantitative release of 18, by Method B. The top
spectrum shows 21 in CDCl3 before addition of SnCl2, the upper middle trace shows the spectrum after addition of 0.1 equiv. SnCl2 in CD3OD and illustrates
the effect of the solvent on the chemical shift, the lower middle trace shows the spectrum after addition of 0.5 equiv. SnCl2 and the bottom trace shows the
spectrum after addition of 1.0 equiv. SnCl2. The peak at d 7.3 corresponds to CHCl3, the peak at d 4.3 corresponds to CD3OH and the peak at d 3.3 corresponds
to CD2HOD.

S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
3449
Figure 3. 1H NMR spectra of samples of 22 treated with SnCl2 in CDCl3/CD3OD, showing stoichiometric and quantitative release of 19, by Method B. The
upper spectrum shows 22 in the solvent mixture before addition of SnCl2 and the lower trace shows the spectrum after addition of 1.0 equiv. SnCl2. The peak at
d 7.3 corresponds to CHCl3 and that at d 3.3 corresponds to CD2HOD.
1
At this point, two control experiments were conducted.
First, since the chemical shifts of the protons in the mixture
of 11 and 18 produced by the treatment of 21 with SnCl2 did
not exactly match those of authentic samples in CDCl3, the
spectra were recorded of individual samples of 11 and 18 in
a mixture of CDCl3 and CD3OD of composition corre-
sponding to the solvent mixture after addition of 1 equiv. of
reductant in CD3OD in the reductively triggered release
experiment. The chemical shifts now corresponded exactly,
indicating that the previous small discrepancies had been
due to solvent polarity and dipole effects. Second, it may be
postulated that Sn2þ, being a Lewis acid, may have
catalysed the cleavage of the carbon–oxygen bond by
nucleophilic attack of chloride, water or methanol at the
methylene, rather than the desired quinone reduction
triggering the release of the drug moiety. As a control, a
similar NMR-monitored experiment was then carried out
using SnCl4, which is a stronger Lewis acid but exhibits no
reductive properties. No release of 18 was observed,
demonstrating that the release by SnCl2 is, indeed,
reductively triggered.
with SnCl2 in a mixture of CDCl3 and CD3OD. The H
NMR spectra (Fig. 3) of the reaction mixture after additions
of the reductant corresponded to mixtures of the starting
material 22, 5-iodoisoquinolin-1-one 19 and the material
which had signals very similar to those of 11. Again, the
reduction showed 1:1 (22: Sn(II)) stoichiometry. No
reductive deiodination took place, in that no signals
corresponding to 18 were observed, in contrast with the
reductive debromination which takes place during treatment
of 5-bromo-2-(1-methyl-2-nitroimidazol-5-ylmethyl)iso-
quinolin-1-one with an alternative ‘biomimetic’ reductant
system, NaBH4/Pd/aqueous propan-2-ol.7
Application of the SnCl2/CDCl3/CD3OD reductant system
to the N-linked dioxoindol-3-ylmethyl isoquinolin-1-one
candidate prodrugs 23 and 24 gave a markedly different
outcome. No changes in the 1H NMR spectra were seen after
addition of 1 equiv. of SnCl2, indicating that release of
19 and 20, respectively, does not occur. More forcing
reaction conditions (5 equiv. of SnCl2 and 24 h reaction
time; Method C) also gave no release of the isoquinolinones,
as shown by the NMR spectra.
Thus the reductively triggered release from the indolequi-
none prodrug 21 occurs with 1:1 stoichiometry, implying
that a two-electron reduction is required for expulsion of the
leaving group under these conditions. No paramagnetic
effects were seen in any of the NMR spectra.
Treatment of the potential prodrug 26a of pentamethyl-
melamine with SnCl2 by Method B gave mixed results. The
1H NMR spectrum obtained after addition of SnCl2 in
CD3OD comprised only broad peaks. Addition of deuterium
oxide caused the spectrum to sharpen enough to allow
the identification of individual peaks, by breaking up tin
complexes and allowing extraction of the tin salts to the
upper aqueous phase. The spectrum of the lower (largely
CDCl3 and CD3OD) phase now showed the presence of
unreacted 26a but also contained signals corresponding to
free pentamethylmelamine 26a and to the 4,7-dioxoindole-
3-CH2OR derivative observed in the release experiments on
21 and 22, above. However, the spectrum was of too poor
resolution to allow quantification of the species present.
Reduction of the trideuterio analogue 26b gave similar
results.
In stark contrast with the above strictly stoichiometric
reductively triggered release from 21 with tin(II), an
analogous NMR experiment in which 21 was treated with
SnCl2 in the presence of ca. 3 equiv. of diethyl hydrazine-
1,2-dicarboxylate showed complete release of 18 after
the addition of only 0.1 equiv. of the tin reagent. Repetition
of the experiment with other ratios of SnCl2 to 21 (0.2–1.0)
gave similar NMR evidence of complete reaction. In a
control experiment, no reaction was observed in the absence
of SnCl2. Thus, in these experiments, the process is
apparently catalytic in tin.
The 5-iodo analogue 22 gave similar results when treated
Subjection of the indoledione–prednisolone potential

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S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
Scheme 5. Proposed mechanism of release of drugs from prodrugs 21–24, 26 and 29, reductively triggered by stoichiometric SnCl2 in a solvent mixture of
CDCl3 and CD3OD, and illustration of the natures of the respective leaving groups.
prodrug 29 (containing the succinate linker) to the same
conditions (Method B) gave spectra in which the chemical
shifts were not changed upon addition of the SnCl2.
Whereas little change in the chemical shifts of most of the
protons in the indole and steroid units would be expected,
cleavage of the CH2–O linkage should change the chemical
shift of the indole-3-CH2 protons and change their
multiplicity, since this methylene is no longer in a chiral
molecule once cleavage has taken place (note that CD3OD
was the solvent required for this magnetic inequivalence of
the CH2 protons to be evident). A similar lack of reductively
triggered release was observed when an alternative
reductant, Na2S2O4 (a reductant previously used for
triggering reductive release from 4,7-dioxoindole-based
prodrugs14), was used (Method D). Interestingly, hydroly-
sis/methanolysis of the esters in the linker butanedioate unit
was also not observed under both sets of conditions, in
contrast to the effects of the SnCl2/MeOH system on the
analogous nitrothiophene-based candidate prodrug for
prednisolone ((5-nitrothien-2-yl)methyl prednisolone-21-yl
butanedioate).
whereas the release of isoquinolin-1-ones from N-linked
candidate prodrugs 23 and 24 would need the corresponding
nitrogen-centred isoquinolin-1-one anions to act as leaving
groups. However, the isoquinoline-1-alkoxides and nitro-
gen-centred isoquinolin-1-one anions are only resonance
forms of one another and must therefore, be of the same
energy. Hence, the difference in the fate of the two types of
reduced prodrug cannot have a thermodynamic explanation
but must be kinetic; the precise origin of the difference in
outcome remains unclear. The indole moiety forms the
electrophilic intermediate 4, which is trapped by an ambient
nucleophile. In this case, the ambient nucleophile will either
be adventitious D2O or, more likely, CD3OD, leading to
4,7-dihydroxyindole intermediate 31. This, in turn, is
oxidised by air to the corresponding indole 4,7-dione 32.
Thus the indole co-product observed in the 1H NMR
experiments is likely to be 1,2-dimethyl-5-methoxy-3-
(trideuteriomethoxymethyl)indole-4,7-dione.
Thus the ability of the O-linked prodrugs 21 and 22 to
release isoquinolin-1-ones upon reductive triggering,
whereas the N-linked analogues 23 and 24 do not under
these conditions, can be rationalised. Comparison with the
fates of nitroheterocycle-based prodrugs of isoquinolin-1-
ones can be made. Reduction of the O-linked prodrugs
1-(5-nitrothien-2-ylmethoxy)isoquinoline, 5-iodo-1-(5-
nitrothien-2-ylmethoxy)isoquinoline and 5-bromo-1-(5-
nitrothien-2-ylmethoxy)isoquinoline with NaBH4/Pd trig-
gers rapid release of the corresponding isoquinolin-1-ones.8
However, selective reduction of the nitro groups of the
N-linked prodrugs N-(5-nitrofuran-2-ylmethyl)isoquinolin-
1-one and 5-bromo-N-(1-methyl-2-nitroimidazol-5-yl-
methyl)isoquinolin-1-one also leads8 to release of the
corresponding isoquinolin-1-ones. This latter observation
can be rationalised in that the reduction of the nitro group to
3. Discussion
Schemes 5 and 6 show the rationalisation and proposed
mechanisms underlying these results. In the first experi-
ments, the tin(II) reduces the substrates 21–24, 26a,b or 29
(Scheme 5) to the corresponding dihydroxyindole 30.
This can have two possible fates: if the expulsion of the
drug-based anion 2R1 is rapid, it will leave to give the
intermediate 31 and 2R1; if not, intermediate 30 may have
sufficient lifetime to be re-oxidised by atmospheric oxygen
back to the candidate indole-4,7-dione prodrug. Prodrugs 21
and 22 lead to the isoquinoline-1-alkoxide anions 33 and 34,

S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
3451
Scheme 6. Proposed mechanism of release of 18 from prodrug 21 in a process which is apparently catalytic in SnCl2, in which 39 is the stoichiometric
reductant.
an amine or a hydroxylamine is effectively irreversible.
Thus the reduced intermediates have a long lifetime and
even poor leaving groups can be released. This is
particularly evident in the case of 5-nitrofuran-2-ylmethyl
ethers which release even unstabilised alkoxides (notably
poor leaving groups) upon reductive triggering.6
than are the radiolysis and sodium dithionite systems in
which a large excess of reductant is present, effectively
prolonging the lifetime of the reduced intermediate and
allowing weaker leaving groups to leave. Finally, the
O-linked prodrugs 21 and 22 are shown to undergo very
rapid and quantitative release of the therapeutically useful
PARP inhibitory isoquinolin-1-ones, triggered by reduction.
Further work in our laboratory will seek to exploit and adapt
this finding to other pharmacologically active agents.
The apparently catalytic role of tin in the reductively
triggered release of 18 from 21 in the presence of excess
diethyl hydrazine-1,2-dicarboxylate 39 is rationalised as
shown in Scheme 6. In this process, the tin(II) reduces the
prodrug 21 to the dihydroxyindol-3-ylmethoxyisoquinoline
38, as before. This fragments in the usual way, giving the
isoquinolin-1-one 18 and the electrophilic intermediate 4
which captures (deuterated) water or methanol to give
31, which is in turn autooxidised to give the observed
4,7-dioxoindole co-product 32. The tin(IV) produced is
reduced by 39 back to Sn(II) which is then available for
another catalytic cycle; diethyl azodicarboxylate 40 is the
product of the oxidation of 39. Tin(IV) has not previously
been reported to oxidise hydrazine-1,2-dicarboxylates but
this oxidation has been carried out with the analogous
lead(IV).29
5. Experimental
5.1. General
NMR spectra were recorded using CDCl3 as solvent, except
where noted. Melting points were determined using a
Reichert-Jung Thermo Galen Koffler block and are
uncorrected. Infra-red spectra were recorded as KBr discs.
Mass spectra were obtained in the FAB positive ionisation
mode. Experiments were conducted at room temperature,
unless otherwise stated. Solutions in organic solvents were
dried with MgSO4. Solvents were evaporated under reduced
pressure. The stationary phase for flash column chroma-
tography was silica gel. HPLC was performed using a semi-
preparative column Kromasil 10C18, a Jasco PU-986
preparative pump and Jasco UV-975 detector. MeOH was
used as the eluant, with a flow rate of 5.0 mL min21, the
injection volume was 20 mL. 1,2-Dimethyl-3-(isoquinolin-
1-yloxymethyl)indole-4,7-dione 21,23 1,2-dimethyl-3-(5-
iodoisoquinolin-1-yloxymethyl)indole-4,7-dione 22,23 1,2-
dimethyl-3-(5-iodo-1-oxoisoquinolin-2-ylmethyl)-5-meth-
oxyindole-4,7-dione 23,23 1,2-dimethyl-3-(5-bromo-1-oxo-
isoquinolin-2-ylmethyl)-5-methoxyindole-4,7-dione 2423
and 1,2-dimethyl-3-(N-(4,6-bis(dimethylamino)-1,3,5-tri-
azin-2-yl)-N-trideuteriomethylaminomethyl)-5-methoxy-
indole-4,7-dione 26b25 were prepared as previously
described by us.
4. Conclusions
Several conclusions can be drawn from this study. First,
SnCl2 is a good reductant for biomimetic reduction of
indole–quinone prodrugs in chemical model systems. It
can also be used catalytically in the presence of a co-
reductant, a hydrazine-1,2-dicarboxylate ester. Second, this
reductant can be used in a chloroform/methanol solvent
system, which dissolves many candidate prodrugs and
enables the progress of the reductively triggered release of
drugs from the prodrugs to be studied in situ by NMR.
Third, the dependence of the outcome on the kinetics of
release is confirmed. This system is a much more rigorous
test of reductively triggered indolequinone-based prodrugs

3452
S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
5.1.1. Ethyl 1,2-dimethyl-5-methoxyindole-3-carboxyl-
ate 7. Ethyl 5-hydroxy-2-methylindole-3-carboxylate 6
(6.0 g, 27 mmol) in dry DMF (50 mL) was added under
N2 to a stirred suspension of KH (3.3 g, 82 mmol) in dry
DMF (200 mL) at 08C. The mixture was stirred for 45 min.
Iodomethane (11.7 g, 82 mmol) was added dropwise at 08C
and the mixture was warmed to 208C during 4 h. Saturated
aq. NH4Cl was added and the mixture was extracted thrice
with EtOAc. The combined extracts were washed with
water. Drying, evaporation and chromatography (EtOAc)
gave 7 (5.4 g, 81%) as a white solid: mp 119–1208C (lit.10
mp 119–1218C); NMR dH 1.45 (3H, t, J¼7.1 Hz, CH2CH3),
2.74 (3H, s, 2-Me), 3.66 (3H, s, NMe), 3.88 (3H, s, OMe),
4.39 (2H, q, J¼7.1 Hz, CH2), 6.87 (1H, dd, J¼8.8, 2.5 Hz,
6-H), 7.17 (1H, d, J¼8.8 Hz, 7-H), 7.66 (1H, d, J¼2.5 Hz,
4-H).
5.1.5. 1,2-Dimethyl-5-methoxyindole 13. 2-Methyl-5-
methoxyindole 12 (100 mg, 620 mmol) was added slowly
to a stirred suspension of NaH (27 mg, 60% in oil,
680 mmol) in dry DMF (15 mL) under Ar. The suspension
was heated at 458C for 10 min and cooled to 208C.
Iodomethane (750 mg, 5.3 mmol) was added during 5 min.
The mixture was heated at 608C for 1 h and poured onto cold
aq. NaHSO4 (10%). The mixture was extracted thrice with
EtOAc. Drying, evaporation and chromatography (EtOAc/
hexane 3:97) gave 13 (90 mg, 80%) as a pale buff solid:
mp 70–738C (lit.22 mp 73–748C); NMR dH 2.37 (3H, s,
2-Me), 3.59 (3H, s, NMe), 3.82 (3H, s, OMe), 6.16 (1H, s,
3-H), 6.73 (1H, dd, J¼2.4, 8.8 Hz, 6-H), 6.93 (1H, d, J¼
2.4 Hz, 4-H), 7.06 (1H, d, J¼8.8 Hz, 7-H).
5.1.6. 1,2-Dimethyl-5-methoxyindole-3-carboxaldehyde
14. POCl3 (169 mg, 1.1 mmol) was added dropwise to dry
DMF (80 mg, 1.1 mmol) and 13 (100 mg, 570 mmol) in
CH2Cl2 (3 mL) and the mixture was heated under reflux for
2 h. Aq. NaOAc (1.0 M, 10 mL) was added and the mixture
was stirred for 2.5 h before being extracted with EtOAc.
Drying, evaporation and chromatography (EtOAc/hexane
1:1) gave 14 (50 mg, 43%) as an off-white solid: mp 115–
1178C (lit.22 mp 108–1108C); NMR dH 2.62 (3H, s, 2-Me),
3.63 (3H, s, NMe), 3.89 (3H, s, OMe), 6.16 (1H, s, 3-H),
6.89 (1H, dd, J¼2.3, 8.9 Hz, 6-H), 7.17 (1H, d, J¼8.9 Hz,
7-H), 7.80 (1H, d, J¼2.3 Hz, 4-H), 10.10 (1H, s, CHO).
5.1.2. Ethyl 1,2-dimethyl-5-methoxy-4-nitroindole-3-
carboxylate 8 and ethyl 1,2-dimethyl-5-methoxy-6-
nitroindole-3-carboxylate 9. Conc. HNO3 (11 mL) in
AcOH (41 mL) was added to 7 (5.1 g, 21 mmol) in AcOH
(80 mL) at 2108C. The mixture was stirred for 2 h, while
warming to 208C. The suspension was poured onto ice/water
and, after 15 min, the solid was collected by filtration and
dried. Chromatography (EtOAc/hexane 1:1) yielded 9
(800 mg, 14%) as a pale yellow solid: mp 138–1398C
(lit.28 mp 139–1418C); NMR dH 1.46 (3H, t, J¼7.0 Hz,
CH2CH3), 2.79 (3H, s, 2-Me), 3.73 (3H, s, NMe), 4.02 (3H,
s, OMe), 4.40 (2H, q, J¼7.0 Hz, CH2), 7.79 (1H, s, 4-H),
7.96 (1H, s, 7-H). Further elution gave 8 (3.8 g, 63%) as a
pale yellow solid: mp 187–1888C (lit.10 mp 189–1928C);
NMR dH 1.36 (3H, t, J¼7.1 Hz, CH2CH3), 2.67 (3H, s,
2-Me), 3.66 (3H, s, NMe), 3.91 (3H, s, OMe), 4.29 (2H, q,
J¼7.1 Hz, CH2), 6.93 (1H, d, J¼9.1 Hz, 6-H), 7.30 (1H, d,
J¼9.1 Hz, 7-H).
5.1.7. 1,2-Dimethyl-5-methoxy-4-nitroindole-3-carbox-
aldehyde 15. Fuming HNO3 (90%, 2.0 mL) in AcOH
(8.2 mL) was added to 14 (700 mg, 3.4 mmol) in AcOH
(55 mL) at 58C during 5 min. The temperature was allowed
to rise to 208C during 3 h, the mixture was poured onto
ice and the precipitate was collected by filtration. Chroma-
tography (EtOAc/hexane 2:1) gave 15 (430 mg, 60%) as a
pale yellow solid: mp 235–2378C (lit.22 mp 236–2388C);
NMR dH ((CD3)2SO) 2.71 (3H, s, 2-Me), 3.76 (3H, s, NMe),
3.89 (3H, s, OMe), 7.25 (1H, d, J¼9.0 Hz, 6-H), 7.75 (1H, d,
J¼9.0 Hz, 7-H), 9.90 (1H, s, CHO).
5.1.3. Ethyl 4-amino-1,2-dimethyl-5-methoxyindole-3-
carboxylate 10. Compound 8 (2.18 g, 7.5 mmol) was
stirred under reflux with Sn powder (4.0 g, 34 mmol) in
EtOH (190 mL) and aq. HCl (9 M, 10 mL) for 30 min. The
cooled solution was decanted off and was neutralised with
aq. NaHCO3. The suspension was added to an equal volume
of water, was stirred overnight with CH2Cl2 (200 mL) and
was filtered (Celitew). The organic layer was dried.
Evaporation and chromatography (EtOAc) yielded 10
(1.56 g, 80%) as pale yellow crystals: mp 96–978C (lit.10
mp 96–988C); NMR dH 1.41 (3H, t, J¼7.0 Hz, CH2CH3),
2.64 (3H, s, 2-Me), 3.58 (3H, s, NMe), 3.87 (3H, s, OMe),
4.36 (2H, q, J¼7.0 Hz, CH2), 5.68 (2H, br s, NH2), 6.52
(1H, d, J¼8.7 Hz, 6-H), 6.88 (1H, d, J¼8.7 Hz, 7-H).
5.1.8. 4-Amino-1,2-dimethyl-5-methoxyindole-3-carbox-
aldehyde 16. Compound 15 (150 mg, 0.60 mmol) was
heated under reflux with Sn powder (350 mg, 2.9 mmol) in
EtOH (15 mL) and aq. HCl (3.0 M, 5 mL) for 1 h. The
solution was neutralised with aq. NaHCO3 and was
extracted thrice with CHCl3. Drying, evaporation and
chromatography (EtOAc/hexane 1:1) gave 16 (100 mg,
62%) as a pale yellow solid: mp 155–1578C (lit.22 mp 152–
1538C); NMR dH ((CD3)2SO) 2.61 (3H, s, 2-CH3), 3.59 (3H,
s, NCH3), 3.75 (3H, s, OCH3), 6.0 (2H, s, NH2), 6.56 (1H, d,
J¼9.0 Hz, 7-H), 6.85 (1H, d, J¼9.0 Hz, 6-H), 9.71 (1H, s,
CHO).
5.1.4. 1,2-Dimethyl-3-(hydroxymethyl)-5-methoxy-
indole-4,7-dione 11. NaBH4 (160 mg, 4.3 mmol) was
added to a suspension of 17 (100 mg, 0.43 mmol) in dry
MeOH (40 mL, degassed) under Ar. The mixture was stirred
for 1 h and aerated prior to the addition of water (20 mL).
The mixture was extracted thrice with CH2Cl2. Drying,
evaporation, chromatography (EtOAc) and recrystallisation
(EtOAc) afforded 11 (50 mg, 33%) as an orange-red solid:
mp 200–2018C (lit.30 mp 199–2008C); NMR dH 2.21 (3H,
s, 2-Me), 3.81 (3H, s, NMe), 3.88 (3H, s, OMe), 3.87 (1H, t,
J¼6.7 Hz, OH), 4.59 (2H, d, J¼7.0 Hz, CH2OH), 5.60 (1H,
s, 6-H).
5.1.9. 1,2-Dimethyl-3-formyl-5-methoxyindole-4,7-dione
17. Potassium nitrosodisulfonate (920 mg, 3.4 mmol) in aq.
NaH2PO4/Na2HPO4 buffer (30.5 mL, 0.3 M, pH 6.0) was
added to 16 (150 mg, 680 mmol) in acetone (30.5 mL) and
the mixture was stirred for 1 h. The acetone was evaporated.
The precipitate was washed with water and cold MeOH
and dried to give 17 (120 mg, 75%) as an orange solid: mp
240–2428C (lit.30 mp 246–2478C); NMR ((CD3)2SO) dH
2.50 (3H, s, 2-Me), 3.82 (3H, s, NMe), 3.88 (3H, s, OMe),
5.89 (1H, s, 6-H), 10.37 (1H, s, CHO).

S. Ferrer et al. / Tetrahedron 59 (2003) 3445–3454
3453
5.1.10. 1,2-Dimethyl-3-(N-(4,6-bis(dimethylamino)-1,3,5-
triazin-2-yl)-N-methylaminomethyl)-5-methoxyindole-
4,7-dione 26a. Diethyl azodicarboxylate (52 mg, 300 mmol)
was added dropwise to 2,4-bis(dimethylamino)-6-methyl-
amino-1,3,5-triazine 25a24 (59 mg, 300 mmol) and Ph3P
(79 mg, 300 mmol) in dry THF (15 mL) under dry Ar.
The mixture was stirred for 15 min. Compound 11
(71 mg, 300 mmol) was added and the mixture was stirred
for 16 h. Evaporation and chromatography (EtOAc)
afforded 26a (49 mg, 40%) as an orange glass: IR nmax
1680, 1690, 1470 cm21; NMR dH 2.21 (3H, s, indole
2-Me), 2.94 (3H, s, melamine NMe), 3.11 (12H, s,
2£NMe2), 3.81 (3H, s, indole 1-Me), 3.88 (3H, s, OMe),
5.12 (2H, m, CH2), 5.61 (1H, s, indole 6-H); dC 9.9, 29.8,
32.8, 35.9, 39.6, 56.4, 106.6, 120.3, 122.5, 137.8, 159.4,
165.2, 178.6; MS m/z 414.2253 (MþH) (C20H27N7O3
requires 414.2256).
SnCl2 (10% in CD3OD) was added in portions of 10 mol%.
1
A H NMR spectrum was run after each addition.
Method C. Compound 24 (10 mg) was dissolved in CDCl3
(0.6 mL) and transferred to an NMR tube and the 1H NMR
spectrum was recorded. SnCl2 (5.0 equiv., 10% in CD3OD)
1
1
was added and a H NMR spectrum was run. A further H
NMR spectrum was recorded after the homogeneous
mixture had been allowed to stand for 24 h.
Method D. Compound 29 (10 mg) was dissolved in CDCl3
1
(0.6 mL) and transferred to an NMR tube. The H NMR
spectrum of each sample was recorded. Na2S2O4 (10% in
1
CD3OD) was added in portions of 10 mol%. A H NMR
spectrum was run after each addition.
Acknowledgements
5.1.11. 3-(Chloromethyl)-1,2-dimethyl-5-methoxyindole-
4,7-dione 27. Compound 11 (500 mg, 2.4 mmol) was stirred
with SOCl2 (5 mL) for 30 min. Evaporation and recrystal-
lisation (EtOAc) gave 27 (420 mg, 79%) as an orange solid:
mp 203–2048C (lit.10 mp 204–2058C); NMR dH 2.28 (3H,
s, 2-Me), 3.80 (3H, s, NMe), 3.89 (3H, s, OMe), 4.86 (2H, s,
CH2), 5.62 (1H, s, 6-H).
We thank Mr R. R. Hartell and Mr D. Wood (University of
Bath) for the NMR spectra and Mr C. Cryer (University of
Bath) for the mass spectra. We are grateful to the Arthritis
Research Campaign for part financial support. S. F. held a
studentship from the Department of Medical Sciences,
University of Bath.
5.1.12. (1,2-Dimethyl-4,7-dioxo-5-methoxyindol-3-yl)-
methyl prednisolone-21-yl butanedioate 29. Predniso-
lone-21-hemisuccinate sodium salt 28 (380 mg, 790 mmol)
was heated under reflux with 27 (200 mg, 790 mmol) in
DMF (50 mL) for 24 h. Evaporation and chromatography
(EtOAc/CH2Cl2 4:1) gave 29 (210 mg, 40%) as a bright
orange glass: IR nmax 3450, 1730, 1650 cm21; NMR
((CD3)2SO) dH 0.76 (3H, s, prednisolone 18-H3), 1.05
(1H, m, prednisolone 14-H), 1.13 (1H, m, prednisolone
8-H), 1.38 (3H, s, prednisolone 19-H3), 1.45 (2H, m,
prednisolone 7-H2), 1.75 (2H, m, prednisolone 12-H2), 2.05
(1H, m, prednisolone 9-H), 2.15 (2H, m, prednisolone
15-H2), 2.24 (3H, s, indole 2-Me), 2.40 (2H, m, predniso-
lone 16-H2), 3.65 (2H, m, prednisolone 6-H2), 3.85 (3H, s,
indole 1-Me), 3.98 (3H, s, OMe), 4.27 (1H, s, prednisolone
11-H), 4.71 (1H, d, J¼17.5 Hz, prednisolone 21-H), 5.04
(1H, d, J¼17.5 Hz, prednisolone 21-H), 5.16 (2H, s, indole-
CH2), 5.92 (1H, s, prednisolone 4-H), 5.77 (1H, s, indole
6-H), 6.17 (1H, d, J¼10.6 Hz, prednisolone 1-H), 7.32 (1H,
d, J¼10.5 Hz, prednisolone 2-H); dC 9.9, 17.7, 21.4, 24.3,
29.4, 31.0, 32.5, 34.0, 34.6, 39.8, 44.6, 48.0, 51.7, 55.8,
56.7, 57.3, 68.5, 70.3, 89.9, 106.8, 115.7, 121.8, 122.3,
127.7, 129.3, 138.2, 157.1, 159.7, 170.9, 172.0, 178.0,
186.1, 204.9; MS m/z 678 (MþH). An HRMS determination
could not be made owing to the extremely low abundance of
the MþH ion.
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