Indolequinone antitumor agents: Reductive activation and elimination from (5-methoxy-1-methyl-4,7-dioxoindol-3-yl)methyl derivatives and hypoxia- selective cytotoxicity in vitro

Article abstract of DOI:10.1021/jm970744w

A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone, and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins. The elimination of a range of functional groups-carboxylate, phenol, and thiol- was demonstrated upon reductive activation under both chemical and quantitative radiolytic conditions. Only those compounds which eliminated such groups under both sets of conditions exhibited significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and HPLC analysis indicated that efficient elimination of the leaving group occurred following one-electron reduction of the parent compound. The active species in leaving group elimination was predominantly the hydroquinone rather than the semiquinone radical. The resulting iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol following chemical reduction and by either water or 2-propanol following radiolytic reduction. A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity ratios.

Full text of DOI:10.1021/jm970744w

2720
J . Med. Chem. 1998, 41, 2720-2731
In d olequ in on e An titu m or Agen ts: Red u ctive Activa tion a n d Elim in a tion fr om
(5-Meth oxy-1-m eth yl-4,7-d ioxoin d ol-3-yl)m eth yl Der iva tives a n d
Hyp oxia -Selective Cytotoxicity in Vitr o
Matthew A. Naylor,*,† Elizabeth Swann,§ Steven A. Everett,† Mohammed J affar,‡ J ohn Nolan,†
Naomi Robertson,‡ Stacey D. Lockyer,‡ Kantilal B. Patel,† Madeleine F. Dennis,† Michael R. L. Stratford,†
Peter Wardman,† Gerald E. Adams,† Christopher J . Moody,*,§ and Ian J . Stratford*,‡
Gray Laboratory Cancer Research Trust, P.O. Box 100, Mount Vernon Hospital, Northwood,
Middlesex HA6 2J R, United Kingdom, School of Pharmacy and Pharmaceutical Sciences, University of Manchester,
Oxford Road, Manchester M13 9PL, United Kingdom, and Department of Chemistry, University of Exeter, Stocker Road,
Exeter EX4 4QD, United Kingdom
Received November 3, 1997
A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position
was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone,
and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins.
The elimination of a range of functional groupsscarboxylate, phenol, and thiolswas demon-
strated upon reductive activation under both chemical and quantitative radiolytic conditions.
Only those compounds which eliminated such groups under both sets of conditions exhibited
significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the
exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and
HPLC analysis indicated that efficient elimination of the leaving group occurred following one-
electron reduction of the parent compound. The active species in leaving group elimination
was predominantly the hydroquinone rather than the semiquinone radical. The resulting
iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol
following chemical reduction and by either water or 2-propanol following radiolytic reduction.
A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler
benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds
that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins
than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity
ratios.
In tr od u ction
There is currently much interest in the antitumor
potential of indolequinone bioreductively activated cy-
totoxic drugs.1-8 Compounds of note include the diol
EO9 (1; Figure 1),7,8 recently evaluated in clinical trials,9
and related mitosenes3,4,10,11 which show improved
properties over the naturally occurring mitomycins such
as the archetypal quinone bioreductive anticancer agent
mitomycin C (2; Figure 1)12-15 and the large number of
analogues reported in the past 20 years.16-20 The fact
that these compounds require reductive activation to
form electrophilic species toxic to cells is well-known,
and the drugs act as substrates for one or more of the
reductases present in most cells and can be targeted
toward both solid tumors with defined hypoxic fractions
and tumor tissues rich in the required activating
enzymes. Thus, bioreductive drugs are assuming in-
creasing importance as agents effective against these
targets.21-28
F igu r e 1. Structures of archetypal bioreductive quinones EO9
and mitomycin C.
Sch em e 1. Reductive Elimination and Nucleophilic
Trapping with Cyclopropamitosenes
the alkylation and cross-linking of DNA after bioreduc-
tive activation. Our own studies have focused on the
role of C-10 in the alkylation process by the design and
synthesis of the cyclopropamitosenes such as 3 (Scheme
1) in which the electrophilicity at C-1 is reduced by the
presence of a cyclopropane ring in place of the naturally
occurring aziridine.1,2,5,30 In particular we established
that the carbamate group could be eliminated from C-10
of the cyclopropamitosenes upon chemical reduction
The mechanism of action of mitomycin C and the
mitosenes has been the subject of numerous studies,29
and these have established the role of C-1 and C-10 in
* Authors for correspondence.
† Mount Vernon Hospital.
‡ University of Manchester.
§ University of Exeter.
S0022-2623(97)00744-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/18/1998

Indolequinone Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2721
Sch em e 2. Synthetic Route to Critical Parent
Compound and Precursor 6a
with sodium dithionite and the resulting intermediate
trapped by added nucleophiles such as potassium ethyl
xanthate or 4-toluidine (4a ,b; Scheme 1).5 No displace-
ment of the carbamate by the nucleophile occurs in the
absence of the reducing agent.
Our subsequent studies centered on further 2-alkyl
derivatives more closely related to 1, including the
2-cyclopropylindolequinones.6 The most effective com-
pounds, in terms of both hypoxic potency and hypoxia
selectivity in vitro as established by anoxic:oxic cyto-
toxicity ratio (commonly referred to as hypoxic cytotox-
icity ratio (HCR)) and in model tumor systems in vivo,
were either 5-aziridinyl-3-hydroxymethyl derivatives or
5-methoxy-3-carbamoyloxy(methyl) derivatives.6 Some
of the hydroxymethyl derivatives were more effective
than corresponding carbamates, which are normally the
more potent species. The carbamate group is also
present in naturally occurring mitomycins. Therefore,
since variation in the 5-substituent of indolequinones
and the equivalent positions of some mitosenes has been
studied extensively1,2,6,8,16,18 and only a limited variation
in C-10 leaving group in mitosenes and EO9 (principally
carbamates and acetoxy derivatives) has been exam-
ined,1-4,6-8,10,11,16,17 there is an urgent need to clarify
the role of both the 2- and 3-substituents in the
indolequinones and their contribution toward the anti-
tumor effects of these pharmacologically active classes
of drugs. Of particular importance is the ability of
3-indolylcarbinyl substituents to undergo elimination,
well-known in indole chemistry.31 Such reactivity is
only observed upon reductive activation of the indole-
quinone, through the participation of the 1-nitrogen lone
pair electrons which are deactivated in the quinone
parent prodrug where they are partially delocalized into
the quinone carbonyl at C-4. The resulting iminium
species is then a potential electrophilic DNA-alkylating
or other cellular-damaging species.
a
Reagents: (i) KH/MeI/DMF (81%); (ii) HNO3/AcOH (63% +
14% 6-nitro isomer); (iii) Sn/HCl (80%); (iv) (KO3S)2NO (97%); (v)
Na2S2O4 then DIBAL-H then FeCl3 (71%); (vi) LiAlH4/THF (77%)
then (KO3S)2NO (75%).
stituted in the (indol-3-yl)methyl position with hydroxy,
halo, acyloxy, arylalkyloxy, aroyloxy, aryloxy, arylthio,
or aryl substituents.
Syn th etic Ch em istr y
A more convenient route to our previously reported
3-(hydroxymethyl)indolequinone precursor compound 61
has now been developed. Thus ethyl 5-hydroxy-2-
methylindole-3-carboxylate, which is commercially avail-
able or readily obtained by the Nenitzescu reaction, was
dimethylated to give 11, nitration and reduction of
which gave 13 (Scheme 2). Oxidation of the 4-aminoin-
dole 13 with Fremy’s salt gave the quinone 14, which
was reduced to the hydroquinone, and the ester group
was immediately further reduced with DIBAL-H, to
give, on oxidative workup, the desired 3-(hydroxymeth-
yl)indolequinone 6. In an alternative method, 13 was
reduced with LiAlH4 prior to oxidation with Fremy’s salt
to give 6. The corresponding 2-desmethyl analogue 5
and the cyclopropane 7 were prepared according to our
previously published method.6 A range of indolequino-
nes bearing different groups was prepared from the
hydroxymethyl compounds 5-7. The corresponding
3-chloromethyl derivatives 8 and 9 were obtained by
treatment with SOCl2 at room temperature. The car-
boxylic esters 15-23 and 25 were readily obtained from
the appropriate (hydroxymethyl)indolequinone by direct
coupling with either the corresponding acyl chloride or
with the carboxylic acids using Mitsunobu or carbodi-
imide methodology. Aryl ethers 26-28 and 31 were
obtained directly from the appropriate chloromethyl
derivatives. Treatment of the (chloromethyl)indole-
quinone 9 with the less acidic 2- or 4-fluoro and
unsubstituted phenols gave either C-alkylated products
29 and 30 or the required O-alkylated products 27 and
28 depending on the solvent used, with DMF favoring
O-alkylation. The thioether 32 was obtained by DMF-
acetal-mediated coupling to 2-mercaptopyrimidine. The
chloromethyl compound 8 was treated directly with
It is not known whether water may be able to act as
the leaving group in 3-hydroxymethyl analogues, al-
though there is some evidence for the involvement of
this substituent in cytotoxicity.6 Compounds with more
efficient leaving groups such as carbamate and acetoxy
derivatives have generally shown greater potency and
DNA cross-linking abilities,3,4 but there have been no
studies on the chemical and biological properties of
compounds with more diverse leaving groups. Thus, the
useful range of leaving groups is still unclear for
mitosene analogues and completely unknown for 2-alkyl-
and 2-cycloalkylindolequinones. To date the 2-substitu-
ent and (indol-3-yl)methyl substituent of the indole-
quinones have not yet been optimized for hypoxia-
selective cytotoxicity.
We therefore report a study of the effect of (indol-3-
yl)methyl leaving group variation upon hypoxia selec-
tivity and potency of this series of indolequinones. The
mechanism and scope of (indol-3-yl)methyl leaving
group elimination has also been studied. The effect of
the 2-substituent on leaving group-mediated activation
has also been examined following preliminary evidence
that this substituent has a pronounced effect on poten-
cies of related compounds.6
The compounds evaluated were 5-methoxy-1,2-dial-
kylindolequinones and 2-unsubstituted analogues, sub-

2722 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Naylor et al.
Ta ble 1. Chemical Reductive Release from Indolequinones
Sch em e 3. General Synthetic Routes to
(4,7-Dioxoindol-3-yl)methyl Carboxylate Esters and
Aryl Ethersa
yield (%)
indolequinone
X
33
XH
6
15
16
19
20
27
28
31
32
OH
58
72
78
73c
91
55
81
66
58
a
67
52
46
b
b
b
33
b
OCOCH(NHCbz)CHMe2
OCOCH2C6H4-p-Ph
OCOC6H4-p-F
OCOC6H4-p-F
OC6H5
OC6H4-p-F
OC6H4-m-NHAc
S-2-pyrimidyl
a
Product is water; not detected. b Product identified by TLC but
not isolated. c Product is O-ethyl (5-methoxy-1-methyl-4,7-dioxo-
indol-3-yl)methyl dithiocarbonate (34).
indolequinone was carried out using a large excess of
sodium dithionite in the presence of the thiol (5 equiv)
in degassed aqueous THF. In all cases, the elimination
of X- occurred and was observed by the appearance of
XH in the TLC analysis of the reaction mixture.
Concurrent with the elimination of X-, the formation
of the thiol-trapped product 33 (34 in the case of the
2-desmethyl analogue) was also observed by TLC in all
cases. Oxidative workup of the reaction mixture al-
lowed the isolation of 33, the identity of which was
confirmed by independent synthesis, and, in most cases,
the released compound, XH.
a
Reagents: (i) SOCl2; (ii) R1COCl/pyridine/EtOAc or CH2Cl2,
or R1CO2H/DEAD/Ph3P/THF, or R1CO2H/1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide/4-(dimethylamino)pyridine; (iii) XH/
EtOAc/K2CO3, or XH/NaH, or K2CO3/DMF; (iv) XH/DEAD/Ph3P/
THF, or XH/(Me)2NCH[OCH2C(Me)3]2/CH3CN, or XH/MsCl/CH2Cl2/
Et3N.
2,4,6-trichlorophenol to give 26, and 9 was treated with
benzyl alcohol to give 24 (Scheme 3).
The results are summarized in Table 1 and show that
a range of functional groupsscarboxylic acids, phenols,
and a thiolscan be eliminated from the (4,7-dioxoindol-
3-yl)methyl derivatives upon reductive activation. The
yields of thiol-trapped product 33 (55-91%, also syn-
thesized independently) and the released XH are rea-
sonably consistent (33-67%). Blank experiments es-
tablished that the dithiocarbamate 33 was not formed
in the absence of the reducing agent, i.e., no direct
nucleophilic substitution occurs; the starting quinone
was recovered (ca. 90%) on workup with exposure to air.
The fact that water can function as a leaving group
under these conditions (compound 6) has implications
for the cytotoxicity of indolequinones containing the
3-hydroxymethyl substituent, although interestingly
this poorer leaving group was not efficiently eliminated
under the more controlled radiolytic reducing conditions
(Table 2). The HCR data (Table 3) also suggests that
water may not be as effective a leaving group following
cellular reduction, although compound 6 showed some
hypoxia selectivity (HCR ) 4) as do some other 3-(hy-
droxymethyl)indolequinones and certain mitosenes.3,4,6
Representative compounds of the series were evalu-
ated for hypoxia-selective cytotoxicity, and the 3-[(aroyl-
oxy)methyl]-2-methylindole-4,7-diones 18, 20, and 22
exhibited hypoxic cytotoxicity ratios (HCR) of ∼80-90.
The HCR was generally in the range ∼10-90 when the
leaving group was a carboxylic acid and also in the case
of aryl ethers with the best compound (31) having a
HCR of 93. Structurally similar analogues with poorer
leaving groups showed lower or no hypoxia selectivity
(compounds 5, 6, 7, 24, 29, and 30). This indicates that
Resu lts a n d Discu ssion
A variety of chemical reductive conditions (catalytic
hydrogenation, electrochemical, Cr(II), sodium dithion-
ite) have been used to activate mitomycin C, and sodium
dithionite had emerged as a convenient method.32,33
Thus, the activation of the indolequinones was initially
carried out using this reagent. However, radiolytic
reduction methods have also been used so that the
efficiency of leaving group loss could be quantified
following reduction by radiolytically generated radicals
and the kinetic properties of putative radical intermedi-
ates in reduction characterized. In particular, the
reactivity toward oxygen of the semiquinone radicals,
potentially formed under hypoxic conditions following
reduction by one-electron reducing enzymes, and the
effect of oxygen on leaving group release were studied.
Thus, to address the key question as to whether the
diverse leaving groups X (Table 1) in some representa-
tive compounds of the series 15-32 are eliminated upon
reductive activation, the activation of the indolequi-
nones 6, 15, 16, 19, 20, 27, 28, and 31-32 was first
carried out using sodium dithionite as reducing agent.
The reduction was carried out in the presence of a thiol
to trap the electrophilic iminium intermediate; not only
are thiols good nucleophiles, but the quenching of the
reactive electrophile by a thiol would mimic the scav-
enging role of cellular thiols such as glutathione in
biological systems. Initially tert-butylthiol was used as
nucleophile, but the water-soluble potassium O-ethyl
xanthate proved more satisfactory. The reduction of the

Indolequinone Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2723
Ta ble 2. Rate Constants, One-Electron Reduction Potentials, Semiquinone Radical pKa’s, and Relative Leaving Group Ability of
Representative Indolequinones at pH 7.4
E(Q/Q•-
)
10-8k3 (Q•- + O2)
(M-1 s-1
10-72k2 (Q•- + Q•-
(M-1 s-1
)
QH•
pKa
G(-Q)
G(LG)
c
c
Q
K
(mV)a
)
)
(µmol J -1
)
(µmol J -1
)
6
17.5 ( 1.4
4.8 ( 1.6
6.0 ( 1.2
48.5 ( 3.9
17.3 ( 5.4
25.8 ( 6.6
5.9 ( 0.4
3.1 ( 0.2
4.6 ( 0.1
-376 ( 9
5.2 ( 0.1
6.6 ( 0.1
6.8 ( 0.1
6.5 ( 0.1
6.7 ( 0.1
8.2 ( 0.1
5.3 ( 0.1
4.4 ( 0.1
7.4 ( 0.1
0.3 ( 0.1
0.8 ( 0.1
1.7 ( 0.1
0.6 ( 0.1
1.6 ( 0.1
0.6 ( 0.1
1.1 ( 0.1
1.3 ( 0.1
0.9 ( 0.1
4.51 ( 0.07
5.09 ( 0.07
5.32 ( 0.08
5.03 ( 0.05
5.09 ( 0.10
5.07 ( 0.02
4.97 ( 0.07
5.13 ( 0.03
5.93 ( 0.03
0.25 ( 0.01d
0.85 ( 0.10
1.65 ( 0.01
0.90 ( 0.12
1.47 ( 0.07
3.05 ( 0.10
1.34 ( 0.10
1.59 ( 0.10
0.00
19
20
21
22
26
27
28
29
-397 ( 25
-387 ( 12
-350 ( 9
-376 ( 15
-447 ( 14b
-403 ( 9
-420 ( 9
-410 ( 8
0.65 ( 0.02
1.71 ( 0.15
0.73 ( 0.04
1.56 ( 0.08
2.64 ( 0.10
1.32 ( 0.10
1.54 ( 0.10
0.00
a
b
Redox potentials vs E(MV2+/MV•+) ) -442 ( 7 mV (corrected for 2 M 2-propanol). Potential vs E(TQ2+/TQ•+) ) -523 ( 7 mV.
c 2-Propanol/water (50%, v/v); G(CH3)2C•OH ) 0.67 µmol J -1; dose rate ∼ 6.0-6.5 Gy min-1
quantity of the isopropyl ether 35, indicative of the elimination of water.
.
Loss of quinone did not generate a significant
d
Ta ble 3. In Vitro Cytotoxicity of Indolequinones
Sch em e 4. Reductive Activation Pathways Leading to
Elimination of Leaving Group Acid and Alkylation by
the Resulting Iminium Derivative
IC50(air) IC50(N2)
compd
R
X
(µM)a
(µM)a
HCR
5
6
7
H
Me
c-Pr OH
H
Me
H
Me
H
Me
H
Me
OH
OH
220
1077
540c
304
14.1
0.5
164
0.7
27
1.4
16
198
225
27
240
285
820c
33
0.66
0.04
2
0.05
0.3
0.04
0.2
1
4
0.7c
9
8
Cl
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
33
36
37
OCOCH2C6H4-p-Ph
OCOC6H4-o-F
OCOC6H4-o-F
OCOC6H4-p-F
OCOC6H4-p-F
OCOC6H5
21
13
82
14
90
35
80
198
0.6
11
9
OCOC6H5
c-Pr OCOC6H5
1
Me
Me
H
Me
Me
Me
Me
Me
Me
H
OCH2C6H5
OAc
OC6H3-2,4,6-Cl
OC6H5
OC6H4-p-F
C6H3-2-OH-5-F
C6H3-3-F-4-OH
OC6H4-m-NHAc
SCSOEt
410
2.5
0.08
0.7
0.7
12
31
17
36
2
0.85
110
280
0.04
3
200
470
3.7
67
2
93
22
75c
83b
OCONH2
OCONH2
3c
0.04c
0.3b
Figure 2 shows the absorption spectra of the semi-
quinone radical generated by the reduction of 20 by
(CH3)2C•OH in the pH range of 5-11. These spectra
are typical for the indolequinones in this study, all of
which exhibited differential absorption maxima in the
340-400 nm region. The radical spectra were similar
at pH 7.4 and 10 suggesting that the semiquinone
radical is deprotonated at pH 7.4 and above (typical
semiquinones have pKa ∼4-6).34 The insert in Figure
2 displays the variation with pH of the semiquinone
radical absorption at 360 nm, which corresponds to pKa
) 5.32 ( 0.08 for the semiquinone radical of 22.
Semiquinone radical pKa’s for the range of indolequino-
nes under study are displayed in Table 2 and show little
variation, falling in a narrow range of pKa ) 5-6.
At pH 3.9 the semiquinone radical absorption (345
nm) decayed via second-order kinetics (Figure 3) with
a half-life which decreased with increasing radiation
dose (i.e., with initial concentration of radicals, [(CH3)2C•
OH] ∼ 3-18 µM) indicating that the semiquinone
radical decays predominantly by a radical-radical reac-
tion to generate the hydroquinone via reaction 2.
Me
25b
a
b
Mean of three experiments. Typical errors (10%. Reference
1. c Reference 6.
bioreductively induced elimination of the leaving group
at C-3 is likely to be central to the cytotoxic action of
the drugs and that in the compounds evaluated to date,
biologically useful leaving groups are carboxylic acids
and phenols. Thiols remain unevaluated in vitro but
would be expected to be effective based on the data
obtained thus far.
Elimination of the leaving group acid or phenol could
feasibly occur either from the one-electron-reduced
semiquinone radical (Q•-; Scheme 4) produced by single-
electron reduction or via the hydroquinone (QH2; Scheme
4) produced directly by two-electron reduction or the
disproportionation of Q•- radicals. Radiolytic reducing
conditions using a one-electron reductant were ap-
propriate for probing this mechanism. The indolequi-
nones were rapidly reduced by the (CH3)2C•OH radical
(k1 ∼ 109 M-1 s-1) to generate the semiquinone radicals
via reaction 1.
Q + (CH3)2C•OH f Q•- + (CH3)2CO + H+ (1)
Q
•- + Q•- + 2H+ f QH2 + Q
(2)

2724 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Naylor et al.
tron transfer in the semiquinone species. However, for
all of the indolequinones studied, direct elimination of
the leaving group from the Q•- radical must be slow (k
, 100 s-1) based on the small intercepts obtained from
plots exemplified by Figure 3. Such processes are
unknown in other classes of quinones, such as 1,4-
naphthoquinones,36 but have been reported in the
dehalogenation of nitroaromatic anion radicals.37 The
one-electron reduction potentials (E(Q/Q•-)) at pH 7.4
for the indolequinones are also displayed in Table 2. The
nature of the (indol-3-yl)methyl substituent had little
effect on the electron affinity of benzoate esters, which
exhibited redox potentials similar to that of 6, whereas
the phenols were markedly less electron affinic. The
one-electron reduction potentials fell within a good
range (-350 to -447 mV) for reduction by enzymes such
as NADPH cytochrome c (P450) reductase.
In the presence of oxygen the semiquinone radicals
decayed faster with increasing oxygen concentrations
(ca. 2-20 µM O2), presumably via electron-transfer
according to reaction 3.
F igu r e 2. Absorption spectra of the semiquinone radicals of
20 obtained by pulse radiolysis of an N2O-saturated 2-pro-
panol/water mixture (50%, v/v) containing phosphate buffer
(4 mM, pH 10, 7.4, and 4). Insert: pKa curve showing the effect
of pH on the absorption of the semiquinone radical of 22 (40
µM 22 in 2 M 2-propanol after a dose of 3 Gy).
Q
•- + O2 f Q + O2
(3)
•-
Absolute rate constants for k3 were determined from the
slope of the linear plots of the observed first-order rate
constants for radical decay versus oxygen concentration
and are shown in Table 2. Values for k3 were found to
be in the order of 108 M-1 s-1, and hence the new
compounds exhibit similar reactivity toward oxygen as
related indolequinones determined previously.6
The marked pH dependence of the radical stability
in anoxia is important in the possible competition
between semiquinone disproportionation (reaction 2)
and oxygen inhibition (reaction 3) in reductive activa-
tion. It may thus be possible to modify pKa(QH•) to alter
the sensitivity to oxygen, of toxicity through one-electron
reduction. Further work is needed to ascertain whether
pKa(QH•) and 2k2 are linked to the variation in toxicity
with oxygen tension.
F igu r e 3. Transient absorption at 345 nm observed on the
reaction of (CH3)2C•OH with 20 (50 µM) in an N2O-saturated
2-propanol/water mixture (50%, v/v) containing phosphate
buffer (4 mM, pH 7.4) initiated by a pulse of 18.7 Gy,
corresponding to [(CH3)2C•OH] ∼ 12.5 µM. Inset: dependence
of the reciprocal of the first half-life of the semiquinone radical
on initial radical concentration at pH 3.9 and 7.4.
Elim in a tion follow in g Ra d iolytic Red u ction to
th e Sem iqu in on e Ra d ica l. The leaving group chem-
istry of the indolequinones was investigated by product
analysis (HPLC) following γ-radiolysis of N2O-saturated
solutions containing quinones (100 µM) and 2-propanol
(8.3 M, 50%, v/v) at pH 7.4. The radiation chemical
yield (G) of the (CH3)2C•OH radical in N2O-saturated
2-propanol/water mixtures was determined by ferricya-
nide reduction38 to be G((CH3)2C•OH) ) 0.67 ( 0.02
µmol J -1 in 2-propanol/water (50%, v/v) and 0.72 ( 0.03
µmol J -1 in 1 M 2-propanol, respectively. Figure 4
shows the product profile obtained on the reduction of
20 by the (CH3)2C•OH radical. Loss of the parent
quinone 20 (G(-Q) ) 1.65 ( 0.01 µmol J -1) paralleled
the formation of the fluorobenzoate leaving group (LG)
with G(LG) ) 1.71 ( 0.15 µmol J -1, both more than
double the input of reducing (single-electron) equiva-
lents. The Q•- radicals decay rapidly via reaction 2;
therefore, since the first-order elimination route is slow,
the fluorobenzoic acid is predominantly derived from the
hydroquinone (QH2; Scheme 4) and not via electron
transfer within the Q•- radicals.
The reciprocal of the first-half-life of the semiquinone
radical of 20 varied linearly with the initial radical
concentration, and from the slope of the fitted straight
line, the rate constant 2k2 ) 7.4 ( 0.1 × 107 M-1 s-1
was obtained at pH 3.9 (see insert Figure 3). At pH
7.4, where the semiquinone radical is almost fully
deprotonated, the rate of decay decreases to 2k2 ) 1.7
( 0.1 × 107 M-1 s-1. In the case of the drug EO9 (1),
reaction 2 is a reversible equilibrium;35 however, the
semiquinone radicals of all the indolequinones in this
study decay completely (even at pH > 9) suggesting that
any possible equilibrium lies well to the side of hydro-
quinone formation. Rate constants for the bimolecular
decay of semiquinone radicals (2k2) at pH 7.4 are
displayed in Table 2. Some evidence was obtained for
a first-order component attributable to unimolecular
elimination of a leaving group via intramolecular elec-
The two remaining major peaks in Figure 4 were
derived from the reaction of the resultant iminium

Indolequinone Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2725
reduction of mitomycin C by radiolytically produced
formate radicals.39
A comparison of the leaving group abilities of repre-
sentative indolequinones in Table 2 with HCR in Table
3 shows that compounds which eliminated leaving
groups upon reduction also exhibited the greatest HCRs,
in the range 10-100. Structurally similar analogues
with poorer leaving groups such as 5-7 and 24 showed
little or no hypoxia selectivity and no leaving group
chemistry (under radiolysis conditions) together with
substantially lower potency. Similarly, the compounds
in which the fluorophenyl moiety was linked through
the aromatic ring rather than through the phenolic
oxygen (29 and 30) exhibited no hypoxia selectivity and
no leaving group chemistry. Although the aerobic
cytotoxicity of these compounds that do not possess
leaving groups can be explained by redox cycling (reac-
tion), their residual hypoxic cytotoxicity (100-300 µM)
must be attributed to other unknown mechanisms.
F igu r e 4. HPLC chromatogram showing a typical product
profile obtained by the γ-radiolysis (5.2 Gy) of 100 µM 20, in
an N2O-saturated 2-propanol/water mixture (50%, v/v) con-
taining phosphate buffer (4 mM, pH 7.4).
The aerobic and anoxic cytotoxic potencies (Table 3)
of this series of indolequinones were markedly influ-
enced by 2-substitution, with 2-unsubstituted com-
pounds being up to 300 times more cytotoxic against
V79-379A cells in some cases (Table 3, cf. 17 and 18,
19 and 20). The data for the benzoate esters 21-23
(Table 3) show that there is an order of magnitude
decrease in aerobic cytotoxicity upon progressive sub-
stitution at the 2-position (2-unsubstituted to 2-methyl
and again to 2-cyclopropyl substitution). With the
exception of the known carbamates 36 and 37, which
show the same trend, the differences under hypoxia are
slightly less dramatic, and therefore HCR also increases
across the series. Similar trends in related indolequi-
nones have been observed previously, as has the excep-
tional hypoxic potency of some 2-cyclopropanes also seen
for compound 23 (HCR ) 198).6 An increase in C-2 side-
chain length is known to reduce the potencies of
compounds closely related to those in this study,6
although the differences between methyl and unsubsti-
tuted analogues were not as great as has been seen in
the present study. The reported increased rate of
reduction by NAD(P)H:quinone oxidoreductase (DT-
diaphorase, E.C. 1.6.99.2, NQO1) of 2-unsubstituted
compounds compared with 2-substituted analogues may
be a significant factor leading to the observed trends in
cytotoxicity for such compounds.40 For example, if R is
a substituent that results in slower enzymatic reduction,
then low oxygen tension would be required for hydro-
quinone buildup. This might explain increasing HCR
with certain C-2 substituent (R) groups and clearly
merits further study.
derivative (Scheme 4) with water to generate 6 and with
the 2-propanol to generate the isopropyl ether 35 (also
synthesized independently). Both of these quinones are
generated by autoxidation of their respective hydro-
quinones via reaction 4 following the unavoidable
introduction of oxygen during HPLC sampling.
QH2 + O2 f Q + H2O2
(4)
As expected, the relative yields of 6 and 35 were
dependent on the alcohol concentration, with the alky-
lation product virtually disappearing when radiolysis
was performed in 1 M 2-propanol. Other indolequinone
benzoate esters including 19, 21, and 22 also exhibited
equally efficient leaving group ability. The aryl ethers
26-28 and 31 which contained the phenoxy moieties
also eliminated from their hydroquinones. The genera-
tion of the isopropyl ether 35 following the radiolytic
reduction of 6 was indicative of the formation of the
iminium derivative following the elimination of water.
As expected, the loss of 6 (G(-Q) ) 0.25 ( 0.01 µmol
J -1) was lower than for other indolequinones exhibiting
leaving group chemistry since the iminium derivative
would also trap water to regenerate 6. Nevertheless,
the relatively low yield of 35 reflected an inefficient
elimination of water from 6 on reduction. Under the
more forcing chemical reducing conditions of excess
sodium dithionite, water was effectively eliminated from
6 and the iminium derivative trapped with xanthate to
give 33. This poorer leaving group behavior is reflected
in the poorer hypoxia selectivity of the hydroxymethyl
compounds (HCR ranging from 1 to 4, Table 3). From
Table 2, for the indolequinones which exhibited leaving
group chemistry, both G(-Q) and G(LG) were signifi-
cantly greater (G(-Q) ) 0.9-3.05 µmol J -1) than
expected from the bimolecular decay of Q•- radicals via
reaction 2 where the expected G(-Q) ) 0.33 µmol J -1
(i.e., one-half of G((CH3)2C•OH) ) 0.67 µmol J -1 deter-
mined by ferricyanide reduction). Reduction of 2,6-
dimethylbenzoquinone to its hydroquinone under the
same experimental conditions gave the expected
G(-Q) ) 0.37 ( 0.01 µmol J -1 and G(QH2) ) 0.32 (
0.03 µmol J -1 and confirmed the presence of a chain
reaction in the reduction of the indolequinones. A pH-
dependent chain reaction has also been reported in the
The present study has established that a range of
groups can be eliminated from the (indol-3-yl)methyl
position upon reductive activation of the quinone moiety.
The reduction-initiated release of a range of compounds
from the indolequinone parallels the work of Denny and
co-workers who have used the reduction of aromatic
nitro groups to initiate the release of cytotoxic leaving
groups.41-43 However unlike the work of Denny and co-
workers in which the releasing moiety is unreactive and
only the released fragment in cytotoxic, the present
work results in the formation of a highly reactive
electrophilic species from the releasing moiety that is
cytotoxic, together with a leaving group that can be

2726 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Naylor et al.
cooled to -10 °C, was added a mixture of concentrated HNO3
(11 mL) and AcOH (41 mL). The mixture was stirred at room
temperature for 2 h. A yellow suspension was obtained which
was poured onto an ice/water mixture, and the crystals
obtained were filtered off and dried. The crude product was
purified by column chromatography (EtOAc/light petroleum)
to yield (i) the title compound (3.8 g, 63%): mp 189-192 °C;
IR (KBr) 2942, 1696, 1625, 1573, 1540 cm-1; 1H NMR (CDCl3)
δ 7.30 (1H, d, J ) 9.1 Hz, 6/7-H), 6.93 (1H, d, J ) 9.1 Hz,
7/6-H), 4.29 (2H, q, J ) 7.1 Hz, CO2CH2CH3), 3.91 (3H, s,
OCH3), 3.66 (3H, s, NCH3), 2.67 (3H, s, CH3), 1.36 (3H, t, J )
7.1 Hz, CO2CH2CH3); 13C NMR (DMSO-d6) δ 163.2 (CO), 146.1,
145.6, 132.1, 131.4, 116.5, 110.6 (CH), 106.7 (CH), 101.5, 58.7
(OCH2), 56.5 (OCH3), 28.7 (NCH3), 13.0 (CH3), 10.7 (CH3);
HRMS found M+ 292.1059, C14H16N2O5 requires M 292.1059.
Also obtained was (ii) ethyl 5-methoxy-1,2-dimethyl-6-nitroin-
dole-3-carboxylate (0.8 g, 14%): mp 139-141 °C; IR (KBr)
varied at will either to be cytotoxic in its own right or
to exhibit other biological properties. This concept of
bioreductive release from indolequinones is being ac-
tively investigated, and further results will be reported
in due course.
Exp er im en ta l Section
NMR spectra were obtained at 60 MHz with a J EOL MY60
spectrometer, at 90 MHz with a J EOL FX90Q spectrometer,
and at 250, 300, or 400 MHz using Bruker instruments and
SiMe4 as internal standard. Elemental analyses were deter-
mined at the University of Exeter or by Butterworth Labora-
tories Ltd., Teddington, Middlesex, U.K., and all compounds
characterized by HRMS were chromatographically homoge-
neous. Solutions in organic solvents were dried by standard
procedures, and dimethylformamide (DMF), toluene, and
tetrahydrofuran (THF) were anhydrous commercial grades.
Silica gel for flash column chromatography was Merck
Kieselgel 60 H grade (230-400 mesh) or Matrex silica 60.
Melting points were determined on a Thomas-Hoover melting
point apparatus and on a Thermogallen microscope and hot
stage apparatus and are uncorrected. The 3-(hydroxymethyl)-
indolequinones 5 and 7 required as precursors were synthe-
sized as described previously.6 1,1′-Dimethyl-4,4′-bipyridinum
dichloride (methyl viologen, MV2+) and 2,6-dimethylbenzo-
quinone were obtained from Sigma-Aldrich, and 1,1′-pro-
pylene-2,2′-bipyridinum dibromide (Triquat, TQ2+) was a gift
from Dr. Peter O’Neill (MRC Radiation and Genome Stability
Unit, Chilton, U.K.).
3-(Ch lor om e t h yl)-5-m e t h oxy-1-m e t h ylin d ole -4,7-d i-
on e (8) a n d 3-(Ch lor om eth yl)-5-m eth oxy-1,2-d im eth ylin -
d ole-4,7-d ion e (9). The appropriate alcohol (5 or 6; 2 mmol)
was stirred at room temperature with SOCl2 (5 mL) for 0.5 h.
The solution was then evaporated in vacuo, redissolved in
EtOAc (25 mL), and evaporated to dryness. This procedure
was repeated twice to coevaporate SOCl2 residues, and the
crude orange solid (75-85% yield) of the corresponding
3-(chloromethyl)-5-methoxyindole-4,7-dione (8 or 9) was used
in the next step without further treatment. A small sample
of compounds 8 and 9 was recrystallized from EtOAc for NMR
and CHN analysis. 8: mp 202-204 °C dec; 1H NMR (CDCl3)
δ 6.88 (1H, s, 2-H), 5.68 (1H, s, 6-H), 4.80 (2H, s, CH2Cl), 3.94
(3H, s, OCH3), 3.80 (3H, s, NCH3). Anal. (C11H10NO3Cl) C,
H, N. 9: mp 204-205 °C dec; 1H NMR (CDCl3) δ 5.62 (1H, s,
6-H), 4.86 (2H, s, CH2Cl), 3.89 (3H, s, OCH3), 3.80 (3H, s,
NCH3), 2.28 (3H, s, 2-CH3). Anal. (C12H12NO3Cl‚0.3H2O) C,
H, N.
1
2981, 2944, 1689, 1626, 1583, 1558 cm-1; H NMR (CDCl3) δ
7.96 (1H, s, 4/7-H), 7.79 (1H, s, 7/4-H), 4.40 (2H, q, J ) 7.0
Hz, CO2CH2CH3), 4.02 (3H, s, OCH3), 3.73 (3H, s, NCH3), 2.79
(3H, s, CH3), 1.46 (3H, t, J ) 7.0 Hz, CO2CH2CH3); 13C NMR
(CDCl3) δ 167.0 (CO), 152.1, 151.5, 137.3, 133.3, 131.4, 109.5
(CH), 106.5 (CH), 106.3, 61.7 (OCH2), 58.7 (OCH3), 32.0
(NCH3), 16.4 (CH3), 14.3 (CH3); HRMS found M+ 292.1059,
C
14H16N2O5 requires M 292.1059.
E t h yl 4-Am in o-5-m et h oxy-1,2-d im et h ylin d ole-3-ca r -
boxyla te (13). To a suspension of ethyl 5-methoxy-1,2-
methyl-4-nitroindole-3-carboxylate (12) (2.18 g, 7.5 mmol) in
EtOH (190 mL) were added tin powder (4.0 g, 33.6 mmol) and
HCl (3 M, 54 mL). The mixture was heated under reflux for
30 min. Upon cooling the solution was decanted from the
excess tin and neutralized with NaHCO3 (aqueous). The
suspension obtained was added to an equal volume of water.
The precipitate and aqueous layer were stirred overnight with
CH2Cl2 and filtered through Celite to separate the layers. The
organic layer was dried (Na2SO4) and concentrated. The crude
product was purified by column chromatography (EtOAc) to
yield the title compound as an off-white crystalline solid (1.56
g, 80%): mp 96-98 °C; IR (KBr) 3459, 3336, 3289, 2928, 1663,
1
1598, 1561 cm-1; H NMR (CDCl3) δ 6.88 (1H, d, J ) 8.7 Hz,
6/7-H), 6.52 (1H, d, J ) 8.7 Hz, 7/6-H), 5.68 (2H, br s, NH2),
4.36 (2H, q, J ) 7.0 Hz, CO2CH2CH3), 3.87 (3H, s, OCH3), 3.58
(3H, s, NCH3), 2.64 (3H, s, CH3), 1.41 (3H, t, J ) 7.0 Hz, CO2-
CH2CH3); 13C NMR (CDCl3) δ 167.3 (CO), 144.3, 141.4, 134.4,
131.6, 114.8, 110.0 (CH), 104.6, 96.8 (CH), 59.5 (OCH2), 58.0
(OCH3), 30.2 (NCH3), 14.9 (CH3), 13.3 (CH3); HRMS found M+
262.1317, C14H18N2O3 requires M 262.1317. Anal. (C14H18N2O3)
C, H, N.
Eth yl 5-Meth oxy-1,2-d im eth yl-4,7-d ioxoin d ole-3-ca r -
boxyla te (14). To a solution of ethyl 4-amino-5-methoxy-1,2-
dimethylindole-3-carboxylate (13) (3.2 g, 12.2 mmol) in Me2CO
(400 mL) was added a solution of potassium nitrosodisulfonate
(5 g, excess) in sodium dihydrogen phosphate buffer (0.3 M,
400 mL). The mixture was stirred at room temperature for 1
h. The excess Me2CO was removed in vacuo. The resulting
residue was extracted with CH2Cl2 and washed with water.
The organic layer was dried (Na2SO4) and concentrated. The
crude product was recrystallized (CH2Cl2/light petroleum) to
give an orange/yellow crystalline solid (3.3 g, 97%); mp 202-
204 °C; UV (MeOH) 429 (ꢀ 1004), 328 (2614), 288 (12 673),
Eth yl 5-Meth oxy-1,2-dim eth ylin dole-3-car boxylate (11).
Ethyl 5-hydroxy-2-methylindole-3-carboxylate (10) (6.04 g, 27.5
mmol) in DMF (50 mL) was added to a stirring suspension of
KH (3.3 g, 82.5 mmol) in DMF (200 mL) at 0 °C. The mixture
was stirred at room temperature for 45 min. Iodomethane
(11.7 g, 82.4 mmol) was added dropwise at 0 °C and the
mixture allowed to warm to room temperature. The reaction
was monitored by TLC and was completed within 2 h.
Saturated ammonium chloride solution was added and the
mixture extracted with EtOAc. The EtOAc layer was washed
twice with water, dried (MgSO4), and concentrated. The crude
product was purified by column chromatography (eluting with
EtOAc) to give the title compound as a colorless solid (5.5 g,
81%): mp 119-121 °C; IR (KBr) 2979, 2947, 1680, 1621, 1584
217 nm (12 984); IR (KBr) 2980, 2942, 1686, 1639, 1608 cm-1
;
1H NMR (CDCl3) δ 5.64 (1H, s, 6-H), 4.36 (2H, q, J ) 7.1 Hz,
CO2CH2CH3), 3.91 (3H, s, OCH3), 3.81 (3H, s, NCH3), 2.44 (3H,
s, CH3), 1.40 (3H, t, J ) 7.1 Hz, CO2CH2CH3); 13C NMR (CDCl3)
δ 179.5 (CO), 175.7 (CO), 164.7 (CO), 141.8, 129.8, 121.9, 113.4
106.3 (CH), 61.3 (OCH2), 56.9 (OCH3), 32.8 (NCH3), 14.4 (CH3),
10.9 (CH3); HRMS found M+ 227.0950, C14H15NO5 requires M
277.0950.
1
cm-1; H NMR (CDCl3) δ 7.66 (1H, d, J ) 2.5 Hz, 4-H), 7.17
(1H, d, J ) 8.8 Hz, 7-H), 6.87 (1H, dd, J ) 8.8 Hz, J ) 2.5 Hz,
6-H), 4.39 (2H, q, J ) 7.1 Hz, CO2CH2CH3), 3.88 (3H, s, OCH3),
3.66 (3H, s, NCH3), 2.74 (3H, s, CH3), 1.45 (3H, t, J ) 7.1 Hz,
CO2CH2 CH3); 13C NMR (CDCl3) δ 166.0 (CO), 155.5, 145.1,
131.5, 127.3, 111.4 (CH), 109.6 (CH), 103.6 (CH), 59.1 (OCH2),
55.6 (OCH3), 29.4 (NCH3), 14.5 (CH3), 11.8 (CH3); HRMS found
3-(Hyd r oxym eth yl)-5-m eth oxy-1,2-d im eth ylin d ole-4,7-
d ion e (6). To a solution of ethyl 5-methoxy-1,2-dimethyl-4,7-
dioxoindole-3-carboxylate (14) (3.4 g, 12.3 mmol) in CHCl3 (370
mL) and EtOH (125 mL) was added a solution of sodium
dithionite (25 g) in water (160 mL). The biphasic mixture was
stirred vigorously overnight. The organic layer was separated,
M+ 247.1208, C14H17NO3 requires M 247.1208. Anal. (C14H17
NO3) C, H, N.
-
Eth yl 5-Meth oxy-1,2-d im eth yl-4-n itr oin d ole-3-ca r box-
yla te (12). To a solution of ethyl 5-methoxy-1,2-dimethylin-
dole-3-carboxylate (11) (5.1 g, 20.6 mmol) in AcOH (80 mL),

Indolequinone Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2727
washed with brine, dried (Na2SO4), and concentrated to give
ethyl 4,7-dihydroxy-5-methoxy-1,2-dimethylindolecarboxylate
as a solid that was used directly.
mmol) were stirred overnight in THF (15 mL) at 50 °C. Excess
solvent was removed and the majority of the product triturated
out with ether and filtered off. The residue was dissolved in
CH2Cl2, washed with water, dried (Na2SO4), and concentrated.
The crude residue was purified by column chromatography (1:1
light petroleum/EtOAc) to give an orange crystalline solid (0.1
g, 87%): mp 163-165 °C; UV (MeOH) 454 (ꢀ 846), 348 (1683),
243 nm (6876); IR (KBr) 3244, 3042, 2990, 1750, 1721, 1699,
1673, 1633, 1598 cm-1; 1H NMR (CDCl3) δ 7.56 (5H, m, ArH),
7.46 (2H, m, ArH), 7.33 (2H, m, ArH), 5.61 (1H, s, 6-H), 5.30
(2H, s, IndCH2O-), 3.88 (3H, s, OCH3), 3.97 (3H, s, NCH3), 3.65
(2H, s, OCOCH2), 2.22 (3H, s, CH3); 13C NMR (CDCl3) δ 179.2
(CO), 177.9 (CO), 171.9 (CO), 160.1, 141.2, 140.3, 138.3, 133.5,
130.1 (CH), 129.5, 129.1 (CH), 127.6 (CH), 127.4 (CH), 122.1,
116.1, 107.0 (CH), 57.5 (CH2), 56.8 (OCH3), 41.2 (CH2), 32.7
(NCH3), 9.9 (CH3); HRMS found M+ 429.1574, C26H23NO5
requires M 429.1576.
To a stirred suspension of the hydroquinone in CH2Cl2 (500
mL) was added DIBAL-H (1 M solution in CH2Cl2; 13.9 g, 97.9
mmol), keeping the temperature below -30 °C. The mixture
was stirred at this temperature for a further 2 h. The reaction
was quenched by dropwise addition of an iron(III) chloride
solution (1 M FeCl3/0.1 M HCl, 125 mL) while keeping the
temperature below -30 °C. The mixture was filtered through
Celite and the residue washed with hot CH2Cl2. The organic
layer was separated, washed with saturated ammonium
chloride, dried (Na2SO4), and concentrated. The crude product
was recrystallized (CH2Cl2/light petroleum) to yield the title
compound as an orange/red crystalline solid (2.0 g, 71%): mp
200-202 °C (lit.1 mp 199-200 °C); 1H NMR (CDCl3) δ 5.60
(1H, s, 6-H), 4.59 (2H, d, J ) 6.7 Hz, CH2OH), 3.87 (1H, t, J
) 6.7 Hz, OH), 3.85 (3H, s, OCH3), 3.80 (3H, s, NCH3), 2.21
(3H, s, CH3).
3-(Hyd r oxym eth yl)-5-m eth oxy-1,2-d im eth ylin d ole-4,7-
d ion e (6) (Alter n a tive Meth od ). To a suspension of LiAlH4
(1.28 g, 33.7 mmol) in THF (50 mL) at 0 °C was added a
solution of ethyl 4-amino-5-methoxy-1,2-dimethylindole-3-car-
boxylate (13) (2.21 g, 8.4 mmol) in THF (25 mL). The mixture
was allowed to warm to room temperature and stirred for 0.5
h. The mixture was cooled to 0 °C and the reaction quenched
by the addition of water (1 mL), 1 M sodium hydroxide (1 mL),
and silica gel (10 g). The granular precipitate was filtered off
through a pad of Celite. The filtrate was dried (MgSO4) and
concentrated in vacuo to yield 4-amino-3-(hydroxymethyl)-5-
methoxy-1,2-methylindole (1.42 g, 77%) as a dark-brown solid,
which was used directly in the next step without further
purification: 1H NMR (CDCl3) δ 6.86 (1H, d, J ) 8.7 Hz, ArH),
6.59 (1H, d, J ) 8.7 Hz, ArH), 4.78 (2H, s, 3-CH2), 3.88 (3H, s,
OCH3), 3.50 (3H, s, NCH3), 2.25 (3H, s, CH3).
(5-Meth oxy-1-m eth yl-4,7-d ioxoin d ol-3-yl)m eth yl 2-F lu -
or oben zoa te (17). The 3-(hydroxymethyl)-5-methoxyindole-
4,7-dione (5) (0.47 g, 2 mmol) was dissolved in CH2Cl2 (50 mL)
together with pyridine (5 mL) and 2-fluorobenzoyl chloride
(0.87 g, 5 mmol) added. The solution was then stirred at room
temperature for 2 h and EtOAc (150 mL) added followed by
HCl (aqueous, 0.1 M, 150 mL). The organic layer was
separated, washed again with HCl (aqueous, 0.1 M, 100 mL),
NaHCO3 (aqueous, 5%, 100 mL), and brine (aqueous, 100 mL),
dried, and evaporated in vacuo. The residue was purified by
column chromatography (1:1 hexane/EtOAc) to give an orange
solid (328 mg, 46%), recrystallized from EtOAc: mp 175-176
1
°C; H NMR (CDCl3) δ 7.95 (2H, m, ArH), 7.23 (2H, m, ArH),
6.96 (1H, s, 2-H), 5.69 (1H, s, 6-H), 5.56 (2H, s, CH2OAr), 3.96
(3H, s, OCH3), 3.83 (3H, s, NCH3). Anal. (C18H14FNO5) C, H,
N.
(5-Met h oxy-1,2-d im et h yl-4,7-d ioxoin d ol-3-yl)m et h yl
2-F lu or oben zoa te (18). The 3-(hydroxymethyl)-5-methoxy-
indole-4,7-dione (6) (0.47 g, 2 mmol) was dissolved in CH2Cl2
(50 mL) together with pyridine (5 mL) and 2-fluorobenzoyl
chloride (0.87 g, 5 mmol) added. The solution was then stirred
at room temperature for 1.5 h and EtOAc (150 mL) added
followed by HCl (aqueous, 0.1 M, 150 mL). The organic layer
was separated, washed again with HCl (aqueous, 0.1 M, 100
mL), NaHCO3 (aqueous, 5%, 100 mL), and brine (aqueous, 100
mL), dried, and evaporated in vacuo. The residue was purified
by column chromatography (EtOAc) to give an orange solid
To a solution of 4-amino-3-(hydroxymethyl)-5-methoxy-1,2-
methylindole (1.42 g, 5.4 mmol) in Me2CO (400 mL) was added
a solution of potassium nitrosodisulfonate (5.8 g, 21.6 mmol)
in sodium dihydrogen phosphate buffer (0.3 M, 400 mL). The
mixture was stirred at room temperature for 1 h. The excess
Me2CO was removed in vacuo. The resulting residue was
extracted with CH2Cl2 and washed with water. The organic
layer was dried (Na2SO4) and concentrated. The crude product
was purified by column chromatography (EtOAc/CH2Cl2, 4:1)
and recrystallized (CH2Cl2/light petroleum) to yield the title
compound as an orange/red solid (0.96 g, 75%); data given
above.
1
(0.6 g, 84%), recrystallized from EtOAc: mp 166-168 °C; H
NMR (CDCl3) δ 7.92 (2H, m, ArH), 7.25 (2H, m, ArH), 5.63
(1H, s, 6-H), 5.51 (2H, s, CH2OAr), 3.90 (3H, s, OCH3), 3.81
(3H, s, NCH3), 2.35 (3H, s, CH3). Anal. (C19H16FNO5‚0.3H2O)
C, H, N.
(S)-(5-Meth oxy-1,2-d im eth yl-4,7-d ioxoin d ol-3-yl)m eth -
yl 2-[(Ben zyloxycar bon yl)am in o]-3-m eth ylbu tan oate (15).
3-(Hydroxymethyl)-5-methoxy-1,2-dimethylindole-4,7-dione (6)
(0.1 g, 0.42 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide (0.098 g, 0.51 mmol), N-Cbz-L-valine (0.128 g, 0.51
mmol), and a catalytic quantity of 4-(dimethylamino)pyridine
were stirred overnight in CH2Cl2 (10 mL) at room temperature.
The crude mixture was washed with water, dried (Na2SO4),
and concentrated. The residue was purified by column chro-
matography (EtOAc) to yield the title compound (0.14 g, 70%);
mp 102-104 °C; UV (MeOH) 452 (ꢀ 1430), 347 nm (2955); IR
(5-Meth oxy-1-m eth yl-4,7-d ioxoin d ol-3-yl)m eth yl 4-F lu -
or oben zoa te (19). The 3-(hydroxymethyl)-5-methoxyindole-
4,7-dione (5) (0.44 g, 2 mmol) was dissolved in CH2Cl2 (50 mL)
together with pyridine (5 mL) and 4-fluorobenzoyl chloride
(0.87 g, 5 mmol) added. The solution was then stirred at room
temperature for 2 h and EtOAc (150 mL) added followed by
HCl (aqueous, 0.1 M, 150 mL). The organic layer was
separated, washed again with HCl (aqueous, 0.1 M, 100 mL),
NaHCO3 (aqueous, 5%, 100 mL), and brine (aqueous, 100 mL),
dried, and evaporated in vacuo. The residue was purified by
column chromatography (hexane/EtOAc, 1:1) to give an orange
solid (316 mg, 46%), recrystallized from EtOAc: mp 210-212
(KBr) 3347, 2964, 1724, 1673, 1642, 1601 cm-1 1H NMR
;
(CDCl3) δ 7.32 (5H, m, ArH), 5.62 (1H, s, 6-H), 5.29 (3H, m,
NH, OCH2Ph), 5.09 (2H, s, IndCH2O), 4.27 (1H, dd, J ) 9.1
Hz, J ) 4.5 Hz, OCOCH), 3.89 (3H, s, OCH3), 3.79 (3H, s,
NCH3), 2.26 (3H, s, CH3), 2.14 (1H, m, CH(CH3)2), 0.92 and
0.85 (6H, 2 × d, J ) 6.9 Hz, CH(CH3)2); 13C NMR (CDCl3) δ
179.2 (CO), 177.9 (CO), 172.3 (CO), 160.1 (CO), 156.6, 138.5,
136.8, 129.5, 128.9 (CH), 128.5 (CH), 128.4 (CH), 122.1, 115.5,
107.1 (CH), 67.3 (CH2), 59.5 (CH), 57.7 (CH2), 56.8 (OCH3),
32.8 (NCH3), 31.7 (CH), 19.4 (CH3), 17.7 (CH3), 9.9 (CH3);
HRMS found M+ 468.1881, C25H28N2O7 requires M 468.1896.
(5-Meth oxy-1,2-d im eth yl-4,7-d ioxoin d ol-3-yl)m eth yl (4-
P h en yl)p h en yla ceta te (16). 3-(Hydroxymethyl)-5-methoxy-
1,2-dimethylindole-4,7-dione (6) (0.085 g, 0.36 mmol), triphen-
ylphosphine (0.19 g, 0.72 mmol), diethyl azodicarboxylate
(0.125 g, 0.718 mmol), and 4-biphenylacetic acid (0.1 g, 0.47
1
°C; H NMR (CDCl3) δ 8.0 (2H, m, ArH), 7.19 (2H, m, ArH),
6.91 (1H, s, 2-H), 5.70 (1H, s, 6-H), 5.52 (2H, s, CH2OAr), 3.96
(3H, s, OCH3), 3.83 (3H, s, NCH3). Anal. (C18H14FNO5) C, H,
N.
(5-Met h oxy-1,2-d im et h yl-4,7-d ioxoin d ol-3-yl)m et h yl
4-F lu or oben zoa te (20). 3-(Hydroxymethyl)-5-methoxy-1,2-
dimethylindole-4,7-dione (6) (0.051 g, 0.22 mmol), triphen-
ylphosphine (0.113 g, 0.43 mmol), diethyl azodicarboxylate
(0.075 g, 0.43 mmol), and 4-fluorobenzoic acid (0.039 g, 0.27
mmol) were stirred overnight in THF (10 mL) at 50 °C. The
solvent was evaporated and residue dissolved in CH2Cl2 and
washed with 1 M HCl (20 mL) and NaOH (aqueous, 1 M, 20
mL). The CH2Cl2 layer was dried (Na2SO4) and concentrated

2728 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Naylor et al.
and the residue purified by column chromatography (EtOAc)
and recrystallized (EtOAc/light petroleum) to yield the title
compound as an orange crystalline solid (0.048 g, 63%): mp
212-214 °C; 1H NMR (CDCl3) δ 8.00 (2H, m, ArH), 7.04 (2H,
m, ArH), 5.62 (1H, s, 6-H), 5.47 (2H, s, CH2OAr), 3.91 (3H, s,
OCH3), 3.80 (3H, s, NCH3), 2.34 (3H, s, CH3); 13C NMR (CDCl3)
δ 178.8, 177.5, 167.4, 165.6, 164.0, 159.7, 138.0, 132.3 (CH),
132.2 (CH), 129.2, 126.6, 126.5, 121.9, 115.8, 115.5 (CH), 115.2
(CH), 106.7 (CH), 57.1 (OCH2), 56.4 (OCH3), 32.4 (NCH3), 9.6
(CH3); HRMS found M+ 357.1013, C19H16FNO5 requires M
357.1012.
(5-Meth oxy-1-m eth yl-4,7-d ioxoin d ol-3-yl)m eth yl Ben -
zoa te (21). The 3-(hydroxymethyl)-5-methoxyindole-4,7-dione
(5) (0.44 g, 2 mmol) was dissolved in CH2Cl2 (50 mL) together
with pyridine (5 mL) and benzoyl chloride (0.78 g, 5 mmol)
added. The solution was then stirred at room temperature
for 16 h and EtOAc (150 mL) added followed by HCl (aqueous,
0.1 M, 150 mL). The organic layer was separated, washed
again with HCl (aqueous, 0.1 M, 100 mL), NaHCO3 (aqueous,
5%, 100 mL), and brine (aqueous, 100 mL), dried, and
evaporated in vacuo. The residue was purified by column
chromatography (hexane/EtOAc, 1:1) to give an orange solid
(312 mg, 48%), recrystallized from EtOAc: mp 134-135 °C;
1H NMR (CDCl3) δ 7.57 (5H, m, ArH), 6.90 (1H, s, 2-H), 5.69
(1H, s, 6-H), 5.53 (2H, s, CH2OAr), 3.93 (3H, s, OCH3), 3.83
(3H, s, NCH3). Anal. (C18H15NO5‚0.5H2O) C, H, N.
(5-Met h oxy-1,2-d im et h yl-4,7-d ioxoin d ol-3-yl)m et h yl
Ben zoa te (22). The 3-(hydroxymethyl)-5-methoxyindole-4,7-
dione (6) (0.47 g, 2 mmol) was dissolved in CH2Cl2 (50 mL)
together with pyridine (5 mL) and benzoyl chloride (0.78 g, 5
mmol) added. The solution was then stirred at 40 °C for 1.5
h and EtOAc (150 mL) added followed by HCl (aqueous, 0.1
M, 150 mL). The organic layer was separated, washed again
with HCl (aqueous, 0.1 M, 100 mL), NaHCO3 (aqueous, 5%,
100 mL), and brine (aqueous, 100 mL), dried, and evaporated
in vacuo. The residue was purified by column chromatography
(EtOAc) to give an orange solid (468 mg, 69%), recrystallized
from EtOAc/Me2CO: mp 168-170 °C; 1H NMR (CDCl3) δ 7.97
(2H, m, ArH), 7.38 (3H, m, ArH), 5.58 (1H, s, 6-H), 5.46 (2H,
s, CH2OAr), 3.86 (3H, s, OCH3), 3.76 (3H, s, NCH3), 2.31 (3H,
s, CH3). Anal. (C19H17NO5) C, H, N.
(2-Cyclop r op yl-5-m eth oxy-1-m eth yl-4,7-d ioxoin d ol-3-
yl)m eth yl Ben zoa te (23). The 3-(hydroxymethyl)-5-meth-
oxyindole-4,7-dione (7) (0.55 g, 2 mmol) was dissolved in
CH2Cl2 (50 mL) together with pyridine (5 mL) and benzoyl
chloride (5 mmol) added. The solution was then stirred at
room temperature for 1.5 h and EtOAc (150 mL) added
followed by HCl (aqueous, 0.1 M, 150 mL). The organic layer
was separated, washed again with HCl (aqueous, 0.1 M, 100
mL), NaHCO3 (aqueous, 5%, 100 mL), and brine (aqueous, 100
mL), dried, and evaporated in vacuo. The residue was purified
by column chromatography (hexane/EtOAc, 1:1) to give an
orange solid (496 mg, 68%), recrystallized from EtOAc/Me2-
CO: mp 221-223 °C; 1H NMR (CDCl3) δ 8.0 (2H, m, ArH),
7.43 (3H, m, ArH), 5.64 (1H, s, 6-H), 5.56 (2H, s, CH2OAr),
4.03 (3H, s, OCH3), 3.79 (3H, s, NCH3), 1.66 (1H, m, c-Pr), 1.17
(2H, m, c-Pr). Anal. (C21H19NO5) C, H, N.
(6) (0.47 g, 2 mmol) was dissolved in CH2Cl2 (50 mL) together
with pyridine (5 mL) and acetyl chloride (0.39 g, 5 mmol)
added. The solution was then stirred at 40 °C for 0.5 h and
EtOAc (150 mL) added followed by HCl (aqueous, 0.1 M, 150
mL). The organic layer was separated, washed again with HCl
(aqueous, 0.1 M, 100 mL), NaHCO3 (aqueous, 5%, 100 mL),
and brine (aqueous, 100 mL), dried, and evaporated in vacuo.
The residue was purified by column chromatography (EtOAc)
to give an orange solid (0.38 g, 69%), recrystallized from
EtOAc: mp 185-186 °C; 1H NMR (CDCl3) δ 5.62 (1H, s, 6-H),
5.24 (2H, s, CH2OAc), 3.90 (3H, s, OCH3), 3.81 (3H, s, NCH3),
2.28 (3H, s, CH3), 2.04 (s, 3H, COCH3). Anal. (C14H15NO5) C,
H, N.
3-[(2,4,6-Tr ich lor op h e n yl)oxym e t h yl]-5-m e t h oxy-1-
m eth ylin d ole-4,7-d ion e (26). The 3-(chloromethyl)-5-meth-
oxyindole-4,7-dione (8) (90 mg, 0.4 mmol) and K2CO3 (140 mg,
1 mmol) were dissolved in anhydrous EtOAc (20 mL), and
2,4,6-trichlorophenol (0.9 g, 5 mmol) was added with stirring.
Stirring was continued at room temperature for 15 h and then
water (20 mL) added. The organic layer was separated,
washed with saturated NaHCO3 (aqueous, 20 mL) and brine
(aqueous, 20 mL), dried, and evaporated to dryness. The
residue was purified by column chromatography (EtOAc) and
recrystallized from EtOAc to give an orange solid (104 mg,
1
61%): mp 222-223 °C; H NMR (CDCl3) δ 7.35 (2H, s, ArH),
7.06 (s, 1H, 2-H), 5.67 (1H, s, 6-H), 5.26 (2H, s, IndCH2O), 3.98
(3H, s, OCH3), 3.82 (3H, s, NCH3). Anal. (C18H12Cl3NO5) C,
H, N.
5-Met h oxy-3-(p h en yloxym et h yl)-1,2-d im et h ylin d ole-
4,7-d ion e (27). To a stirring solution of 3-(hydroxymethyl)-
5-methoxy-1,2-dimethylindole-4,7-dione (6) (78 mg, 0.33 mmol)
in CH2Cl2 (30 mL) was added SOCl2 (3.3 g, 27 mmol) dropwise.
The solution was stirred at room temperature overnight. The
solvent was removed and the crude material used directly in
the next step. A solution of the crude chloride in DMF (20
mL) was added to a stirring suspension of phenol (62 mg, 0.66
mmol) and NaH (16 mg, 0.66 mmol) in DMF (25 mL) at 0 °C.
After the addition the mixture was allowed to stir at room
temperature for 3 h. The reaction was quenched by the
addition of NH4Cl (aqueous, saturated, 20 mL). The mixture
was extracted with CH2Cl2, washed with NaOH (aqueous, 1
M), 1 M HCl, and water, dried (Na2SO4), and concentrated.
The crude material was purified by column chromatography
(EtOAc/petroleum ether, 1:1) and recrystallized (CH2Cl2/Et2O)
to give the title compound as an orange crystalline solid (67
mg, 65%): mp 183-185 °C; UV (MeOH) 456 (ꢀ 1696), 342
(3110), 288 (18 363), 222 nm (25 148); IR (KBr) 3420, 2967,
1
2933, 1683, 1637, 1611, 1499 cm-1; H NMR (CDCl3) δ 7.30
(2H, m, ArH), 6.97 (3H, m, ArH), 5.62 (1H, s, 6-H), 5.30 (2H,
s, IndCH2O), 3.89 (3H, s, OCH3), 3.81 (3H, s, NCH3), 2.31 (3H,
s, CH3); 13C NMR (CDCl3) δ 178.7 (CO), 178.1 (CO), 159.6,
158.5, 138.0, 129.4 (CH), 128.7, 121.3, 120.9 (CH), 117.3, 115.0
(CH), 106.7 (CH), 60.4 (CH2), 56.4 (OCH3), 32.3 (NCH3), 9.9
(CH3); HRMS found M+ 312.1236. C18H17NO4 requires M
312.1236, Anal. (C18H17NO4) C, H, N.
3-[(4-F lu or op h en yl)oxym eth yl]-5-m eth oxy-1,2-d im eth -
ylin d ole-4,7-d ion e (28). To a stirring solution of 3-(hy-
droxymethyl)-5-methoxy-1,2-dimethylindole-4,7-dione (6) (0.109
g, 0.46 mmol) in CH2Cl2 (30 mL) was added dropwise SOCl2
(3.3 g, 27 mmol). The solution was stirred at room tempera-
ture overnight. The solvent was removed and the crude
material used directly in the next step. A solution of the crude
chloride in DMF (20 mL) was added to a stirring suspension
of 4-fluorophenol (0.104 g, 0.92 mmol) and NaH (0.22 g, 0.92
mmol) in DMF (25 mL) at 0 °C. After the addition the mixture
was stirred at room temperature for 3 h. The reaction was
quenched by the addition of NH4Cl (aqueous, saturated, 20
mL). The mixture was extracted with CH2Cl2, washed with
NaOH (aqueous, 1 M), 1 M HCl, and water, dried (Na2SO4),
and concentrated. The crude material was purified by column
chromatography (EtOAc/petroleum ether, 1:1) and recrystal-
lized (CH2Cl2/Et2O) to give the title compound as an orange
crystalline solid (59 mg, 43%): mp 191-192 °C; UV (MeOH)
458 (ꢀ 1661), 342 (3043), 284 (19 691), 222 nm (22 191); IR
3-(Ben zyloxym et h yl)-5-m et h oxy-1,2-d im et h ylin d ole-
4,7-d ion e (24). The 3-(chloromethyl)-5-methoxyindole-4,7-
dione (9) (100 mg, 0.4 mmol) and K2CO3 (140 mg, 1 mmol)
were dissolved in anhydrous EtOAc (20 mL), and benzyl
alcohol (0.78 g, 5 mmol) was added with stirring. Stirring was
continued at room temperature for 12 h and then water (20
mL) added. The organic layer was separated, washed with
saturated NaHCO3 (aqueous, 20 mL) and brine (aqueous, 20
mL), dried, and evaporated to dryness. The residue was
purified by column chromatography (hexane/EtOAc, 1:1) and
recrystallized from EtOAc to give an orange solid (70 mg,
53%): mp 132-133 °C; 1H NMR (CDCl3) δ 7.32 (5H, m, ArH),
5.60 (1H, s, 6-H), 4.74 (2H, s, IndCH2O), 4.59 (2H, s, OCH2-
Ar), 3.86 (3H, s, OCH3), 3.80 (3H, s, NCH3), 2.25 (3H, s, CH3).
Anal. (C19H19NO4‚0.5H2O) C, H, N.
(5-Meth oxy-1,2-dim eth yl-4,7-dioxoin dol-3-yl)m eth yl Ac-
eta te (25). The 3-(hydroxymethyl)-5-methoxyindole-4,7-dione

Indolequinone Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2729
(KBr) 3401, 2934, 2848, 1677, 1637, 1604, 1505 cm-1; 1H NMR
(CDCl3) δ 6.94, 6.93 (4H, 2s, ArH), 5.61 (1H, s, 6-H), 5.24 (2H,
s, IndCH2O), 3.88 (3H, s, OCH3), 3.80 (3H, s, NCH3), 2.29 (3H,
s, CH3); 13C NMR (CDCl3) δ 178.7 (CO), 178.1 (CO), 159.6,
158.6, 156.2, 154.6, 138.0, 128.8, 121.3, 117.1, 116.3 (CH),
116.2 (CH), 115.8 (CH), 115.6 (CH), 106.7, 61.2 (CH2), 56.4
(OCH3), 32.3 (CH3), 9.8 (CH3); HRMS found M+ 329.1063,
(3353); IR (KBr) 3031, 1940, 1664, 1599, 1564, 1548 cm-1; 1H
NMR (CDCl3) δ 8.51 (2H, d, J ) 5.0 Hz, NCH, pyrimidine),
6.95 (1H, t, J ) 5.0 Hz, CH, pyrimidine), 5.60 (1H, s, 6-H),
4.59 (2H, s, CH2S), 3.88 (3H, s, OCH3), 3.80 (3H, s, NCH3),
2.34 (3H, s, CH3); 13C NMR (CDCl3) δ 178.7 (CO), 177.7 (CO),
172.8, 159.7, 157.1 (CH), 136.6, 128.8, 121.3 (CH), 117.0, 116.3
(CH), 106.8 (CH), 56.4 (OCH3), 32.4 (NCH3), 25.2 (SCH2), 9.8
(CH3); HRMS found M+ 329.0838, C16H15N3O3S requires M
329.0834.
C
18H16FNO4 requires M 329.1063. Anal. (C18H16FNO4) C, H,
N.
Typ ica l Red u ction P r oced u r e. The indolequinone drug
(20-30 mg) and potassium ethyl xanthate (5 equiv) in THF/
H2O (3:1) (20 mL) were degassed thoroughly by bubbling
nitrogen directly into the stirring solution. Sodium dithionite
(Na2S2O4, sodium hydrosulfite; 10 equiv) was added in one
portion with the nitrogen needle still in the stirring solution.
After a few minutes the orange coloration was lost from the
solution. The formation of released compound XH and the
xanthate-captured product was followed by TLC, and the
reaction was usually completed within 30 min. The solvent
was removed and the residue dissolved in CH2Cl2 and washed
with NH4Cl (aqueous, saturated). The organic layer was dried
(Na2SO4) and concentrated, and after analysis by TLC, the
mixture was separated by column chromatography to give the
trapped indolequinone 33 (or 34) and the product of elimina-
tion.
3-(5-F lu or o-2-h yd r oxyb en zyl)-5-m et h oxy-1,2-d im et h -
ylin d ole-4,7-d ion e (29). The 3-(chloromethyl)-5-methoxyin-
dole-4,7-dione (6) (94 mg, 0.4 mmol) and K2CO3 (140 mg, 1
mmol) were dissolved in anhydrous EtOAc (20 mL), and
4-fluorophenol (0.56 g, 5 mmol) was added with stirring.
Stirring was continued at room temperature for 12 h and then
water (20 mL) added. The organic layer was separated,
washed with saturated NaHCO3 (aqueous, 20 mL) and brine
(aqueous, 20 mL), dried, and evaporated to dryness. The
residue was purified by column chromatography (EtOAc) and
recrystallized from EtOAc to give an orange solid (66 mg,
50%): mp 228-230 °C; 1H NMR (CDCl3) δ 6.86 (3H, m, ArH),
5.62 (1H, s, 6-H), 4.99 (1H, br, ArOH), 4.01 (2H, s, IndCH2-
Ar), 3.89 (3H, s, OCH3), 3.81 (3H, s, NCH3), 2.33 (3H, s, CH3).
Anal. (C18H16FNO4) C, H, N.
3-(3-F lu or o-4-h yd r oxyb en zyl)-5-m et h oxy-1,2-d im et h -
ylin d ole-4,7-d ion e (30). The 3-(chloromethyl)-5-methoxyin-
dole-4,7-dione (6) (94 mg, 0.4 mmol) and K2CO3 (140 mg, 1
mmol) were dissolved in anhydrous EtOAc (20 mL), and
2-fluorophenol (0.56 g, 5 mmol) was added with stirring.
Stirring was continued at room temperature for 12 h and then
water (20 mL) added. The organic layer was separated,
washed with saturated NaHCO3 (aqueous, 20 mL) and brine
(aqueous, 20 mL), dried, and evaporated to dryness. The
residue was purified by column chromatography (EtOAc) and
recrystallized from EtOAc to give an orange solid (65 mg,
49%): mp 212-213 °C; 1H NMR (CDCl3) δ 6.89 (3H, m, ArH),
5.59 (1H, s, 6-H), 4.92 (1H, br, ArOH), 4.02 (2H, s, IndCH2-
Ar), 3.89 (3H, s, OCH3), 3.79 (3H, s, NCH3), 2.21 (3H, s, CH3).
Anal. (C18H16FNO4) C, H, N.
O-Eth yl (5-m eth oxy-1,2-d im eth yl-4,7-d ioxoin d ol-3-yl)-
m eth yl Dith ioca r bon a te (33): data given below.
O-Eth yl (5-m eth oxy-1-m eth yl-4,7-dioxoin dol-3-yl)m eth -
yl d ith ioca r bon a te (34): mp 153-154 °C; UV (MeOH) 351
(ꢀ 4000), 280 (33 500), 223 nm (28 750); IR (film) 3059, 2993,
1
1683, 1663, 1593, 1518 cm-1; H NMR (CDCl3) δ 6.85 (1H, s,
2-H), 5.64 (1H, s, 6-H), 4.64 (2H, q, J ) 7.1 Hz, CH2CH3), 4.51
(2H, s, IndCH2), 3.91 (3H, s, OCH3), 3.81 (3H, s, NCH3), 1.40
(3H, t, J ) 7.1 Hz, CH2CH3); 13C NMR (CDCl3) δ 214.8 (CS),
178.8 (CO), 177.6 (CO), 160.3, 129.7, 129.4, 121.2, 120.3, 106.8
(CH), 70.0 (CH2), 56.5 (OCH3), 36.2 (NCH3), 30.6 (CH2), 13.8
(CH3); HRMS found M+ 325.0443, C14H15NO4S2 requires M
325.0442.
In d ep en d en t Syn th esis of O-Eth yl (5-Meth oxy-1,2-
dim eth yl-4,7-dioxoin dol-3-yl)m eth yl Dith iocar bon ate (33).
Methanesulfonyl chloride (0.033 g, 0.28 mmol) was added
dropwise to a stirring solution of 3-(hydroxymethyl)-5-meth-
oxy-1,2-dimethylindole-4,7-dione (6) (0.053 g, 0.22 mmol) in
CH2Cl2 (10 mL) and Et3N (0.029 g, 0.28 mmol) at 0 °C. The
mixture was stirred at room temperature for 30 min. The
crude material was concentrated and the residue used directly
in the next step without purification. To a stirring solution
of the mesylate in CH2Cl2 (10 mL) was added potassium ethyl
xanthate (0.11 g, 0.68 mmol). The mixture was stirred at room
temperature for 3 h. The crude material was concentrated
and the residue purified by column chromatography (EtOAc)
to give the title compound as an orange crystalline solid (0.053
g, 85%): mp 205-207 °C; UV (MeOH) 459 (ꢀ 2381), 348 (3907),
270 (17 187), 229 nm (13 662); IR (KBr) 2986, 1667, 1638, 1597,
3-[(3-Aceta m id op h en yl)oxym eth yl]-5-m eth oxy-1,2-d i-
m eth ylin d ole-4,7-d ion e (31). To a stirring solution of 3-(hy-
droxymethyl)-5-methoxy-1,2-dimethylindole-4,7-dione (6) (0.307
g, 1.3 mmol) in CH2Cl2 (20 mL) was added dropwise SOCl2
(7.77 g, 65 mmol). The solution was stirred at room temper-
ature for 1 h. The solvent was removed and the crude material
used directly in the next step. The crude chloride, 3-acet-
amidophenol (0.59 g, 3.9 mmol), and K2CO3 (0.54 g, 3.9 mmol)
were stirred in DMF (15 mL) overnight. The DMF was
evaporated in vacuo, and the residue was dissolved in CH2-
Cl2. The CH2Cl2 was washed with NaOH (aqueous, 1 M), 1 M
HCl, and water, dried (Na2SO4), and concentrated. The crude
material was purified by column chromatography (EtOAc/CH2-
Cl2, 9:1) to give the title compound as a yellow/orange crystal-
line solid (0.29 g, 62%): mp 264-266 °C; UV (MeOH) 444
(ꢀ 1161), 433 (2102), 288 (14 597), 216 nm (29 064); IR (KBr)
3302, 3256, 3138, 2954, 1683, 1637, 1611, 1518 cm-1; 1H NMR
(DMSO-d6) δ 9.84 (1H, s, NH), 7.28 (1H, t, J ) 2.0 Hz, ArH),
7.16 (1H, t, J ) 8.1 Hz, ArH), 7.08 (1H, d, J ) 8.8 Hz, ArH),
6.65 (1H, dd, J ) 8.1 Hz, J ) 1.8 Hz, ArH), 5.75 (1H, s, 6-H),
5.10 (2H, s, IndCH2O), 3.85 (3H, s, OCH3), 3.74 (3H, s, NCH3),
2.24 (3H, s, CH3), 2.01 (3H, s, CH3); 13C NMR (DMSO-d6) δ
178.7, 177.8, 168.7, 159.7, 159.2, 140.9, 139.0, 129.8 (CH),
128.5, 121.2, 116.4, 111.9 (CH), 109.4 (CH), 107.2 (CH), 106.2
(CH), 60.3 (CH2), 57.0 (OCH3), 32.6 (NCH3), 24.5 (NCOCH3),
9.7 (CH3). Anal. (C20H20N2O5) C, H, N.
1
1507 cm-1; H NMR (CDCl3) δ 5.60 (1H, s, 6-H), 4.66 (2H, q,
J ) 7.7 Hz, OCH2Me), 4.58 (2H, s, CH2S), 3.88 (3H, s, OCH3),
3.80 (3H, s, NCH3), 2.29 (3H, s, CH3), 1.43 (3H, t, J ) 7.0,
OCH2CH3); 13C NMR (CDCl3) δ 215.2 (CS), 178.9 (CO), 178.0
(CO), 160.0, 137.0, 129.3, 121.6, 116.3, 107.2 (CH), 70.3 (CH2),
56.8 (OCH3), 32.8 (NCH3), 30.9 (CH2), 14.2 (CH3), 10.2 (CH3);
HRMS found M+ 339.0606, C15H17NO4S2 requires M 339.0599.
Anal. (C15H17NO4S2) C, H, N.
In d ep en d en t Syn th esis of 3-[(2-Meth yleth oxy)m eth yl]-
5-m eth oxy-1,2-d im eth ylin d ole-4,7-d ion e (35). The 3-(chlo-
romethyl)-5-methoxyindole-4,7-dione (6) (94 mg, 0.4 mmol) and
K2CO3 (140 mg, 1 mmol) were dissolved in anhydrous EtOAc
(20 mL), and 2-propanol (5 mL) was added with stirring.
Stirring was continued at 90 °C for 1 h and then water (20
mL) added. The organic layer was separated, washed with
saturated NaHCO3 (aqueous, 20 mL) and brine (aqueous, 20
mL), dried, and evaporated to dryness. The residue was
purified by column chromatography (hexane/EtOAc, 1:1) and
recrystallized from EtOAc to give an orange solid (83 mg,
5-Meth oxy-1,2-d im eth yl-3-[(p yr im id ylth io)m eth yl]in -
d ole-4,7-d ion e (32). 3-(Hydroxymethyl)-5-methoxy-1,2-di-
methylindole-4,7-dione (6) (90 mg, 0.38 mmol), 2-mercaptopy-
rimidine (0.085 g, 0.75 mmol), and dimethylformamide
dineopentyl acetal (0.32 g, 1.38 mmol) in CH3CN (50 mL) were
heated for 2 h at 80 °C. The solvent was removed under
reduced pressure and the crude mixture purified on silica gel
to give the desired product as an orange crystalline solid (90
mg, 71%): mp 238-239 °C; UV (MeOH) 464 (ꢀ 2202), 348 nm
1
75%): mp 174-176 °C; H NMR (CDCl3) δ 5.54 (1H, s, 6-H),

2730 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Naylor et al.
4.57 (2H, s, IndCH2O), 3.82 (3H, s, OCH3), 3.73 (3H, s, NCH3),
3.62 (1H, m, i-Pr), 2.24 (3H, s, CH3), 1.11 (6H, d, J ) 6 Hz,
i-Pr). Anal. (C15H19NO4) C, H, N.
Refer en ces
(1) Cotterill, A. S.; Moody, C. J .; Mortimer, R. J .; Norton, C. L.;
O’Sullivan, N.; Stephens, M. A.; Stradiotto, N. R.; Swann, E.;
Stratford, I. J . Cyclopropamitosenes, novel bioreductive anti-
cancer agents. Synthesis, electrochemistry, and biological activ-
ity of 7-substituted cyclopropamitosenes and related indole-
quinones. J . Med. Chem. 1994, 37, 3834-3843.
(2) Cotterill, A. S.; Hartopp, P.; J ones, G. B.; Moody, C. J .; Norton,
C. L.; O’Sullivan, N.; Swann, E. Cyclopropamitosenes, novel
bioreductive anticancer agents. Synthesis of 7-methoxycyclopro-
pamitosene and related indolequinones. Tetrahedron 1994, 50,
7657-7674.
Biologica l Eva lu a tion in Vitr o. Selective toxicity to
hypoxic V79-379A cells was determined for all compounds
using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide] assay as has been described previous-
ly.44-46 These results are presented in Table 3, where C50(air),
the concentration required to kill 50% of the aerobic cells under
the conditions of the assay, is divided by C50(N2), the corre-
sponding concentration for hypoxic cell killing, to give hypoxic
cytotoxicity ratios (HCR).
(3) Maliepaard, M.; de Mol, N. J .; J anssen, L. H. M.; Hoogvliet, J .
C.; van der Neut, W.; Verboom, W.; Reinhoudt, D. N. Reductive
activation of potential antitumor mitosene compounds. J . Med.
Chem. 1993, 36, 2091-2097.
P u lse Ra d iolysis. The redox properties of the quinones
and the kinetic characteristics of the associated semiquinone
radicals (Q•-) were investigated by pulse radiolysis.47 Semi-
quinone radicals were generated following reduction of the
parent indolequinone by the 2-propanol radical ((CH3)2C•OH).
Kinetic spectrophotometry with sub-microsecond resolution
was used to monitor the reactions of quinone radicals. Experi-
ments were performed using a 6-MeV linear accelerator as
described previously.48 The redox potentials of the one-electron
couple E(Q/Q•-) were determined by establishing redox equi-
libria with a viologen (V2+) of known redox potential according
to methods described previously.49 Solutions contained 2-pro-
panol (1 M) and phosphate buffer (NaH2PO4/Na2HPO4, 4 mM,
pH 7.4) with Q (0-60 µM) and MV2+ (0-4 mM). Absorbances
were measured at 600 nm at a dose per pulse of 3 Gy. The
alcohol converts the radiolytically produced •OH and H•
radicals in <2 µs to the reductant (CH3)2C•OH which reduces
the indolequinones and the MV2+ to the corresponding semi-
quinone (Q•-) radicals or viologen radical-cation (V•+). Redox
equilibration usually occurred within ∼100 µs (during which
time there was negligible decay of semiquinone radicals via
bimolecular decay), and it was therefore possible to calculate
the equilibrium constant K from absorbances at equilibrium.
Values for E(Q/Q•-) are quoted relative to E(MV2+/MV•+) )
(4) Maliepaard, M. Ph.D. Thesis, Utrecht, 1994.
(5) Moody, C. J .; O’Sullivan, N.; Stratford, I. J .; Stephens, M. A.;
Workman, P.; Bailey, S. M.; Lewis, A. Cyclopropamitosenes:
novel bioreductive anticancer agents - mechanism of action and
enzymic reduction. Anti-Cancer Drugs 1994, 5, 367-372.
(6) Naylor, M. A.; J affar, M.; Nolan, J .; Stephens, M. A.; Butler, S.;
Patel, K. B.; Everett, S. A.; Adams, G. E.; Stratford, I. J .
2-Cyclopropylindoloquinones and their analogues as bioreduc-
tively activated antitumor agents: structure-activity in vitro
and efficacy in vivo. J . Med. Chem. 1997, 40, 2335-2346.
(7) Oostveen, E. A.; Speckamp, W. N. Mitomycin analogues I.
Indoloquinones as (potential) bisalkylating compounds. Tetra-
hedron 1987, 43, 255-262.
(8) Speckamp, W. N.; Oostveen, E. A. Indoloquinone compounds.
Int. Patent WO 87/06227, 1987.
(9) Dirix, L. Y.; Tonnesen, F.; Cassidy, J .; Epelbaum, R.; ten Bokkel
Huinink, W. W.; Pavlidis, N.; Sorio, R.; Gamucci, T.; Wolff, I.;
Te Velde, A.; Lan, J .; Verweij, J . EO9 Phase II Study in
Advanced Breast, Gastric, Pancreatic and Colorectal Carcinoma
by the EORTC Early Clinical Studies Group. Eur. J . Cancer
1996, 32A, 2019-2022.
(10) Verboom, W.; Reinhoudt, D. N.; Lammerink, B. H. M.; Orlemans,
E. O. M.; van Veggel, F. C. J . M.; Lelieveld, P. Novel bioreductive
mitosene and mitosane analogues of mitomycin c. Anti-Cancer
Drug Des. 1987, 2, 271-277.
(11) Orlemans, E. O. M.; Verboom, W.; Scheltinga, M. W.; Reinhoudt,
D. N.; Lelieveld, P.; Fiebig, H. H.; Winterhalter, B. R.; Double,
J . A.; Bibby, M. C. Synthesis, mechanism of action, and biological
evaluation of mitosenes. J . Med. Chem. 1989, 32, 1612-1620.
(12) Franck, R. W.; Tomasz, M. In Chemistry of Antitumor Agents;
Wilman, D. E. V., Ed.; Blackie and Son Ltd.: Glasgow, 1990;
pp 379-393.
(13) Carter, S. K.; Crooke, S. T. Mitomycin C; Current Status and
New Developments; Academic Press: New York, 1979.
(14) Remers, W. A.; Dorr, R. T. In Alkaloids: Chemical and Biological
Perspectives; Pelletier, S. W., Ed.; Wiley: New York, 1988; Vol.
6, pp 1-74.
(15) Danishefsky, S. J .; Schkeryantz, J . M. Chemical explorations
driven by an enchantment with mitomycinoids - a twenty year
account. Synlett 1995, 475-490.
(16) Casner, M. L.; Remers, W. A.; Bradner, W. T. Synthesis and
biological activity of 6-substituted mitosene analogues of the
mitomycins. J . Med. Chem. 1985, 28, 921-926.
(17) Hodges, J . C.; Remers, W. A. Synthesis and antineoplastic
activity of mitosene analogues of the mitomycins. J . Med. Chem.
1981, 24, 1184-1191.
(18) Iyengar, B. S.; Remers, W. A.; Bradner, W. T. Preparation and
antitumor activity of 7-substituted 1,2-aziridinomitosenes. J .
Med. Chem. 1986, 29, 1864-1868.
-442 ( 7 mV corrected for 1 M 2-propanol50 and E(TQ2+/TQ•+
) -523 ( 7 mV corrected for 2 M 2-propanol.
)
The reactivities of semiquinone radicals with oxygen were
determined by gassing solutions with N2O/O2 mixtures (British
Oxygen Co., U.K.) to give solutions which contained 0.1-0.5%
O2. In the presence of oxygen the semiquinone radicals
decayed faster with increasing oxygen concentrations. Abso-
lute rate constants were determined from the slopes of the
linear plots of the observed first-order rate constants versus
oxygen concentration. The latter was corrected for oxygen
solubility in 2 M 2-propanol from published data.51
Stea d y-Sta te γ-Ra d iolysis. Indolequinone solutions were
saturated with N2O gas in gastight vials before irradiation in
a
60Co source. An absorbed dose of 1 Gy ) 0.67 µM (CH3)2C•
OH radicals in N2O-saturated 2-propanol/water (50%, v/v). A
dose rate of 6-6.5 Gy min-1 was used, as determined by Fricke
dosimetry,52 and radiation chemical yields were corrected for
the absorbed dose in the various alcohol-water mixtures
employed.
High -P er for m a n ce Liqu id Ch r om a togr a p h y (HP LC).
Product analysis following γ-radiolysis was performed by
gradient HPLC separation on a 100 mm × 4.6 mm base-
deactivated reverse-phase column (Hichrom RPB, Hichrom,
Reading, U.K.). The eluents were (A) KH2PO4 (5 mM), H3-
PO4 (5 mM); (B) CH3CN/H2O (3:1, v/v), with a flow rate of 2
cm3 min-1. One of two linear gradients was used for each com-
pound: (1) 35-80% B in 8 min or (2) 20-50% B in 5 min.
Detection was at 232 nm using a Waters 486 detector (Watford,
U.K.), and concentrations were determined from peak areas
using Waters Maxima software.
(19) Kunz, K. R.; Iyengar, B. S.; Dorr, R. T.; Alberts, D. S.; Remers,
W. A. Structure-activity relationships for mitomycin c and
mitomycin a analogues. J . Med. Chem. 1991, 34, 2281-2286.
(20) Leadbetter, G.; Frost, D. L.; Ekwuribe, N. N.; Remers, W. A.
Mitomycin antibiotics. Synthesis of 1-substituted 7-methoxymi-
tosenes. J . Org. Chem. 1974, 39, 3580-3583.
(21) Kennedy, K. A. Hypoxic cells as specific drug targets for
chemotherapy. Anti-Cancer Drug Des. 1987, 2, 181-194.
(22) Coleman, C. N. Hypoxia in tumors: A paradigm for the approach
to biochemical and physiological heterogeneity. J . Natl. Cancer
Inst. 1988, 80, 310-317.
(23) Workman, P.; Walton, M. I. In Selective Activation of Drugs by
Redox Processes; Adams, G. E., Ed.; Plenum Press: New York,
1990; pp 173-191.
(24) Robertson, N.; Stratford, I. J .; Houlbrook, S.; Carmichael, J .;
Adams, G. E. The sensitivity of human tumor cells to quinone
bioreductive drugs - what role for DT-diaphorase. Biochem.
Pharmacol. 1992, 44, 409-412.
(25) Walton, M. I.; Suggett, N.; Workman, P. The role of human and
rodent DT-diaphorase in the reductive metabolism of hypoxic
cell cytotoxins. Int. J . Radiat. Oncol. Biol. Phys. 1992, 22, 643-
647.
Ack n ow led gm en t. This work was funded by finan-
cial support of the U.S. National Cancer Institute under
Grant No. CA55165-02, the BBSRC, the U.K. Medical
Research Council (MRC), and the Cancer Research
Campaign (CRC). We thank the EPSRC Mass Spec-
trometry Centre at Swansea, U.K., for mass spectra.

Indolequinone Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2731
(26) Workman, P. In New Applications in Cancer Pharmacology:
Drug Design and Development; Workman, P., Ed.; Springer-
Verlag: New York, 1992; pp 63-74.
(41) Denny, W. A.; Wilson, W. R. Bioreducible mustards - a paradigm
for hypoxia-selective prodrugs of diffusible cytotoxins (HPDCs).
Cancer Metastasis Rev. 1993, 12, 135-151.
(42) Tercel, M. T.; Wilson, W. R.; Anderson, R. F.; Denny, W. A.
Hypoxia-selective antitumor agents. 12. Nitrobenzyl quaternary
salts as bioreductive prodrugs of the alkylating agent mechlor-
ethamine. J . Med. Chem. 1996, 39, 1084-1094.
(43) Denny, W. A.; Wilson, W. R.; Hay, M. P. Recent developments
in the design of bioreductive drugs. Br. J . Cancer 1996, 74
(Suppl. XXVII), S32-S38.
(44) Stratford, I. J .; Stephens, M. A. The differential hypoxic cyto-
toxicity of bioreductive agents determined in vitro by the MTT
assay. Int. J . Radiat. Oncol. Biol. Phys. 1989, 16, 973-976.
(45) Naylor, M. A.; Stephens, M. A.; Stratford, I. J .; Keohane, A.;
O’Neill, P.; Threadgill, M. D.; Webb, P.; Fielden, E. M.; Adams,
G. E. Aziridinyl nitropyrroles and nitropyrazoles as hypoxia-
selective cytotoxins and radiosensitizers. Anti-Cancer Drug Des.
1991, 6, 151-167.
(46) Naylor, M. A.; Adams, G. E.; Haigh, A.; Cole, S.; J enner, T.;
Robertson, N.; Siemann, D.; Stephens, M. A.; Stratford, I. J .
Fused pyrazine mono-N-oxides as bioreductive drugs III. Char-
acterization of RB 90740 in vitro and in vivo. Anti-Cancer Drugs
1995, 6, 259-269.
(47) Patel, K. B.; Willson, R. L. Semiquinone free radicals and oxygen.
J . Chem. Soc., Faraday Trans. 1 1973, 69, 814-825.
(48) Candeias, L. P.; Folkes, L. K.; Porssa, M.; Parrick, J .; Wardman,
P. Rates of reaction of indoleacetic acids with horseradish
peroxidase compound I and their dependence on the redox
potentials. Biochemistry 1996, 35, 102-108.
(27) Bailey, S. M.; Suggett, N.; Walton, M. I.; Workman, P. Structure-
activity relationships for DT-diaphorase reduction of hypoxic cell
directed agents-indoloquinones and diaziridinyl benzoquinones.
Int. J . Radiat. Oncol. Biol. Phys. 1992, 22, 649-653.
(28) Workman, P.; Stratford, I. J . The experimental development of
bioreductive drugs and their role in cancer therapy. Cancer
Metastasis Rev. 1993, 12, 73-82.
(29) Li, V. S.; Choi, D.; Wang, Z.; J imenez, L. S.; Tang, M.; Kohn, H.
The role of the C-10 substituent in mitomycin C-1 DNA bonding.
J . Am. Chem. Soc. 1996, 118, 2326-2331.
(30) J ones, G. B.; Moody, C. J . Structurally modified antitumour
agents. Part 2. Total synthesis of a cyclopropamitosene. J . Chem.
Soc., Perkin Trans. 1 1989, 2455-2462.
(31) Sundberg, R. J . The Chemistry of Indoles; Academic Press: New
York, 1970.
(32) Schiltz, P.; Kohn, H. Studies on the use of Na2S2O4 for the
reductive activation of mitomycin c. J . Am. Chem. Soc. 1993,
115, 10497-10509.
(33) Hong, Y. P.; Kohn, H. Studies on the use of chromium(II)
perchlorate for the reductive activation of mitomycin c. J . Am.
Chem. Soc. 1991, 113, 4634-4644.
(34) Swallow, A. J . In Function of Quinones in Energy Conserving
Systems; Trumpower, B., Ed.; Academic Press: London, 1982;
pp 59-72.
(35) Butler, J .; Spanswick, V. J .; Cummings, J . The autoxidation of
the reduced forms of EO9. Free Radical Res. 1996, 25, 141-
148.
(49) Wardman, P. Reduction potentials of one-electron couples
involving free radicals in aqueous solution. J . Phys. Chem. Ref.
Data 1989, 18, 1637-1755.
(50) Wardman, P.; Clarke, E. D. Redox properties and rate constants
in free-radical mediated damage. Br. J . Cancer 1987, 55 (Suppl.
VIII), 172-177.
(51) Tokunaga, J . Solubilities of oxygen, nitrogen and carbon dioxide
in aqueous alcohol solutions. J . Chem. Eng. Data 1975, 20, 41-
46.
(52) Sehested, K.; Rasmussen, O. L.; Fricke, H. Rate constants of
•OH with HO2•, O2•- and H2O2+ from hydrogen peroxide forma-
tion in pulse-irradiated oxygenated water. J . Phys. Chem. 1968,
72, 626-631.
(36) Wilson, I.; Wardman, P.; Lin, T.; Sartorelli, A. One-electron
reduction of 2- and 6-methyl-1,4-naphthoquinone bioreductive
alkylating agents. J . Med. Chem. 1986, 29, 1381-1384.
(37) Bays, J . P.; Blumer, S. T.; Baral-Tosh, S.; Behar, D.; Neta, P.
Intramolecular electron transfer and dehalogenation of nitroaro-
matic anion radicals. J . Am. Chem. Soc. 1983, 105, 320-324.
(38) Adams, G. E.; Willson, R. L. Pulse radiolysis studies on the
oxidation of organic radicals in aqueous solution. Trans. Faraday
Soc. 1969, 65, 2981-2987.
(39) Hoey, B. M.; Butler, J .; Swallow, A. J . Reductive activation of
mitomycin C. Biochemistry 1988, 27, 2608-2614.
(40) J affar, M.; Naylor, M. A.; Lockyer, S. D.; Phillips, R. M.; Everett,
S. A.; Adams, G. E.; Stratford, I. J . 5-Substituted analogues of
3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]-
prop-2-en-1-ol (EO9, NSC 382459) and their regioisomers as
hypoxia-selective agents: structure-cytotoxicity in vitro. Anti-
Cancer Drug Des. 1997, 13, 105-123.
J M970744W