Hypoxia-selective antitumor agents. 13. Effects of acridine substitution on the hypoxia-selective cytotoxicity and metabolic reduction of the bis- bioreductive agent nitracrine N-oxide

Article abstract of DOI:10.1021/jm9600104

A series of nuclear-substituted derivatives of nitracrine N-oxide (2; a bis-bioreductive hypoxia-selective cytotoxin) were prepared and evaluated, seeking analogues of lower nitroacridine reduction potential. Disubstitution with Me or OMe groups at the 4-

Full text of DOI:10.1021/jm9600104

2508
J . Med. Chem. 1996, 39, 2508-2517
Hyp oxia -Selective An titu m or Agen ts. 13. Effects of Acr id in e Su bstitu tion on
th e Hyp oxia -Selective Cytotoxicity a n d Meta bolic Red u ction of th e
Bis-bior ed u ctive Agen t Nitr a cr in e N-Oxid e
Ho H. Lee,† William R. Wilson,‡ Dianne M. Ferry,‡ Pierre van Zijl,‡ Susan M. Pullen,† and William A. Denny*,†
Cancer Research Laboratory and Section of Oncology, Department of Pathology, The University of Auckland School of
Medicine, Private Bag 92019, Auckland, New Zealand
Received J anuary 3, 1996X
A series of nuclear-substituted derivatives of nitracrine N-oxide (2; a bis-bioreductive hypoxia-
selective cytotoxin) were prepared and evaluated, seeking analogues of lower nitroacridine
reduction potential. Disubstitution with Me or OMe groups at the 4- and 5-positions did not
provide analogues with one-electron reduction potentials significantly lower than those of the
corresponding monosubstituted derivatives (E(1) ca. -350 mV for both the 4-OMe and 4,5-
diOMe compounds). This appears not to be due to a concomitant raising of the acridine pKa
but to a lack of direct electronic effect of substituents in the ring not bearing the nitro group.
Conversely, placing two OMe groups in the nitro-bearing ring does result in a substantial further
lowering of reduction potential (the 2,4-diOMe analogue has an E(1) of -401 mV). The mono-
and disubstituted N-oxides have substantially lower cytotoxicities than the parent nitracrine
N-oxide 2 but generally retain very high hypoxic selectivity. The OMe-substituted N-oxides
all showed greater metabolic stability than 2 in hypoxic AA8 cell cultures, and the 4-OMe
compound 6 had improved activity in EMT6 multicellular spheroids suggesting that this
metabolic stabilization may allow more efficient diffusion in tumor tissue. The parent compound
2 was selectively toxic to hypoxic cells in KHT tumors in vivo and clearly superior to nitracrine
itself (although only at doses which would eventually be lethal to the host). The analogues of
lower E(1), including 6, were not superior to 2 in vivo, indicating that metabolic stabilization
of the nitro group is not alone sufficient to improve therapeutic utility.
The acridine derivative 9-[(3-(dimethylamino)propyl)-
amino]-1-nitroacridine (nitracrine, 1), originally devel-
oped as a classical anticancer drug,1,2 is also a potent
hypoxic cell radiosensitizer3,4 and hypoxia-selective cy-
totoxin5 in cell culture. However, it does not show
significant activity against hypoxic cells in solid tumors.
Primary reasons for this appear to be a sufficiently high
nitro group reduction potential (-303 mV)6 to allow
rapid metabolic reduction of the nitro group, together
with a DNA binding constant high enough5,7 to mark-
edly limit its rate of diffusion into hypoxic areas.8,9
The very high selectivity of 2 appears to be due to a
requirement for reduction of both the nitro and N-oxide
moieties for full activation. This compound was the first
reported example of such a “bis-bioreductive” agent,
with two independent oxygen-sensitive redox centers
(Scheme 1).7 Reduction of the nitro group appears to
generate reactive intermediates responsible for DNA
alkylation, as deduced earlier for nitracrine,11,12 while
oxygen-inhibitable reduction13 of the N-oxide of 2 (to
give 1), generating a cationic side chain, increases
intercalative DNA binding affinity by 15-fold at low ionic
strength.7 The potential of N-oxides to act as hypoxia-
activated prodrugs of DNA intercalators has since been
demonstrated by the in vitro hypoxic selectivity of the
anthraquinone di-N-oxide AQ4N (15)14,15 and the acri-
dine mono-N-oxide DACA-NO (16)15 and the activity of
AQ4N against hypoxic cells in some tumors.15,16
The tertiary amine N-oxide derivative 2 of this
compound was therefore evaluated, in the expectation
that the dipolar but formally neutral side chain would
provide a prodrug form with weaker DNA binding and
therefore lower toxicity and improved extravascular
drug transport properties.7 Since the metabolic reduc-
tion of tertiary amine N-oxides has long been known to
be inhibited by oxygen,10 such a prodrug was also
expected to be activated selectively in hypoxic regions.
This compound was found to show exceptional (ca. 1300-
fold) hypoxic selectivity toward AA8 cells in culture.7
The ability of nitracrine N-oxide (2) to kill cells in
EMT6 multicellular spheroids has been shown7 to be
improved relative to that of 1. However, intact sphe-
roids were still appreciably more resistant than single-
cell suspensions, suggesting that extravascular diffusion
of 2 is still restricted, possibly because of high rates of
drug metabolism.7 An avenue for further development
of this interesting lead was considered to be stabilization
† Cancer Research Laboratory.
‡ Section of Oncology.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(96)00010-6 CCC: $12.00 © 1996 American Chemical Society

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2509
Sch em e 1. Alternative Reductive Pathways for the
Bis-bioreductive Agent Nitracrine N-Oxide (2)
Sch em e 3a
a
(i) Cu(OAc)2/DMF/90 °C/7 h; (ii) CDI/DMF/40 °C/10 min, then
H2N(CH2)3Me2; (iii) PPE/100 °C/2 h.
Sch em e 2a
Sch em e 4a
a
(i) 2-(Phenylsulfonyl)-3-phenyloxaziridine/CH2Cl2/20 °C/30 min.
of these conditions resulted in contamination with
considerable amounts of products resulting from meth-
oxy group displacement (a problem noted previously in
a similar synthesis of methoxynitroquinolines).20 To
prepare these compounds, the side chain was attached
before ring closure by condensation of the N-phenylan-
thranilic acids with N,N-dimethylpropane-1,3-diamine
in the presence of 1,1′-carbonyldiimidazole21 to give the
amides (e.g., 25, Scheme 3). Subsequent cyclodehydra-
tion of these with polyphosphate ester (PPE) at 100 °C
gave the desired dimethoxynitracrine analogues 9 and
11 in moderate yields.
a
(i) K2CO3/CuI/Cu/HMPA/170 °C/2 h, then aqueous KOH/reflux/
45 min; (ii) 90% H2SO4/80-85 °C/3.5 h; (iii) SOCl2/DMF/reflux/1
h; (iv) H2N(CH2)3NMe2/DMF/80 °C/3.5 h.
of one or both of the nitroaromatic and aliphatic N-oxide
redox centers against reductive metabolism. In this
paper we report the preparation and evaluation of a
series of analogues of nitracrine substituted in the
acridine ring with electron-donating Me and OMe
groups and their corresponding N-oxides (compounds
3-14). Previous work6,17,18 has shown that monosub-
stitution of nitracrine (1) in the 4-position with such
groups does provide analogues with lower reduction
potentials and improves metabolic stability of the nitro
group, with the 4-methoxy derivative 5 showing limited
activity against hypoxic cells in EMT6 tumors in vivo.17
3-Methoxy-2-[(2-methoxy-5-nitrophenyl)amino]ben-
zoic acid (26) for the preparation of 11 was made via
the classical J ourdan-Ullman synthesis, and 2-[(2,4-
dimethoxy-5-nitrophenyl)amino]benzoic acid (24) was
prepared by coupling of diphenyliodonium-2-carboxy-
late22 (22) and 1-amino-2,4-dimethoxy-5-nitrobenzene23
(23) (Scheme 3).
Ch em istr y
Selective N-oxidation of the side chain tertiary ali-
phatic amine was achieved in high yield using 2-(phe-
nylsulfonyl)-3-phenyloxaziridine,24 enabling synthesis of
the N-oxide derivatives essentially free of the starting
amines (<0.02% amine by HPLC) (Scheme 4). Such
high purity was essential because the corresponding
amines are the products of hypoxia-selective reduction
and are much more potent than the N-oxides (Table 1).
Contamination of the N-oxides with very small amounts
of the corresponding amines (<0.5%) markedly lowered
their hypoxic selectivity.7 Reaction of 9-chloroacridines
with N1,N1-dimethylpropane-1,3-diamine N1-oxide25 failed
to provide compounds of sufficient purity, due to dif-
ficulties in rigorously purifying the starting N-oxide.
The new compounds of Table 1 were prepared by one
of two different methods. The classical synthesis of
9-(aminoalkyl)acridines from 9-chloroacridine and alky-
lamines, in the presence of excess phenol as reagent and
solvent, proceeds through 9-phenoxy intermediates.19 In
the present case, the nitro group was sufficiently
activating for the methyl-substituted chloroacridines to
react directly with excess N,N-dimethylpropane-1,3-
diamine at 80 °C in DMF to give derivatives 7 and 13
(Scheme 2). The monomethoxy compounds 3 and 5 gave
only low yields under these conditions and required
reaction via the phenoxy compounds. However, treat-
ment of the dimethoxy-9-chloroacridines under either

2510 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Lee et al.
Ta ble 1. Structural, Physicochemical, and Biological Data for Acridine-Substituted Nitracrine Derivatives and Their Tertiary Amine
N-Oxides
aerobic growth inhibitionc
clonogenic assay (AA8)d
E(1)b
(mV)
IC50(AA8)
(µM)
HF(UV4)
ratio
C10(air)
(µM)
air/N2
ratio
mouse toxicitye
(MTD) (µmol/kg)
a
no.
formula
R
pKa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
A
B
A
B
A
B
A
B
A
B
A
B
A
B
H
H
6.21
7.10
5.48
-297
-283
-352
0.024 ( 0.001
2.7 ( 0.5
15 ( 1
0.40
61
>60
>300
16
5.1
820
200
0.3
15
15 ( 1
2-OMe
2-OMe
4-OMe
4-OMe
4-Me
4-Me
2,4-diOMe
2,4-diOMe
4,5-diOMe
4,5-diOMe
4,5-diMe
4,5-diMe
2.9 ( 0.3
4.6 ( 0.3
84 ( 19
0.47 ( 0.07
130 ( 20
0.053 ( 0.009
5.4 ( 0.6
300
15
100
0.2
15
300
800
100
450
20
6.61
6.29
5.85
6.53
6.01
-355
-315
-401
-344
-317
14 ( 5
43 ( 14
13
29 ( 2
1.3 ( 0.1
0.9
11 ( 3
14 ( 1
22 ( 12
32 ( 6
11
1250
6.9
150
2.3
1.2
14
2770
11
54
15 700
1.5
47
500
20 000
47
59 000
9.6
47 ( 17
1960
0.51 ( 0.02
150 ( 20
0.19 ( 0.0300
6.8 ( 1.5
60
15
a
b
Acridine pKa values were literature values (ref 6) or determined in aqueous solution at 25 °C by spectrophotometry (see ref 36). E(1)
values (mV) for the ArNO2/ArNO2•- were literature values (ref 6) or determined at pH 7 by pulse radiolysis, using benzylviologen as the
reference standard (see ref 6). c Growth inhibition assay as described in the text. IC50 value determined against aerobic AA8 or UV4
cells, using an exposure time of 18 h. HF (hypersensitivity factor ) IC50(AA8)/IC50(UV4)), using 18 h drug exposures under aerobic
d
conditions. Clonogenic assay as described in the text. C10 is the concentration needed to reduce cell survival to 10% of control values,
using 1 h exposure of plateau-phase AA8 cells at 106 cells/mL. Air/N2 ratio ) C10(air)/C10(nitrogen). e Maximum tolerated ip dose for
male C3H/HeN mice.
Resu lts a n d Discu ssion
The reason for this does not appear to be pKa-related
but may be due to a considerably different conformation
of the side chain due to the additional steric compression
provided by the 2-OMe substituent.
Physicochemical and in vitro biological data for
substituted nitracrines and their corresponding N-
oxides are given in Table 1. One-electron reduction
potentials for the non-N-oxides were determined by
pulse radiolysis. As reported previously,6 substitution
of 1 with a 4-Me group lowers E(1) by about 15 mV,
compared to about 55 mV for either a 2- or 4-OMe group.
However, a second electron-donating group on the other
ring had no additional effect on E(1) as demonstrated
by the 4,5-diOMe and 4,5-diMe compounds 11 and 13
which have E(1) values similar to those of the corre-
sponding 4-substituted derivatives 5 and 7. Determi-
nation of the direct electronic effects of ring substituents
on reduction potential in this series is complicated by
the concomitant effects on the acridine pKa.6 These
determine the relative proportions of cationic and free
base forms, which have different reduction potentials.12
However, in the present study the second substituents
have no net effect on pKa, presumably because their
additional electron-donating properties are counteracted
by steric inhibition of protonation, a phenomenon previ-
ously noted with 4,5-disubstituted acridines.26
The 2,4-diOMe derivative 9 was therefore prepared
to investigate the effects of disubstitution in the same
ring as the nitro group. This compound had an E(1)
100 mV lower than that of 1, suggesting essentially full
electronic contributions by each substituent, although
its pKa (5.85) did not reflect any additive effects of the
two groups, being intermediate between those of the
2-OMe and 4-OMe compounds 3 and 5. Unfortunately
both this compound and the related monosubstituted
2-OMe derivative 3, together with their respective
N-oxides 4 and 9, proved to be comparatively insoluble.
The aerobic cytotoxicities of the substituted nitra-
crines (IC50 values) in AA8 cells show a trend toward
decreasing cytotoxic potency with lower E(1). The 4-Me
derivative 7 was 2-fold less potent, and the 4-OMe and
4,5-diOMe compounds 5 and 11 were 10-20-fold less
potent. The 2-OMe derivative was very much less
potent than 1 (120-fold). The reduction potential of this
compound was similar to that of the 4-OMe derivative,
so the further decrease in potency may be due to the
additional structural distortion caused by the 2-sub-
stituent. Thus, although the 2,4-diOMe derivative 9
does have the lowest E(1), the very marked drop in
potency seen (2000-fold less than that of 1) is likely not
due entirely to this factor. This observation is remi-
niscent of the steric inhibition of nitroreduction by
substituents ortho to the nitro group in the structurally-
related 4-(alkylamino)-5-nitroquinolines.27
The relative potencies of the compounds against AA8
cells and a DNA repair-defective mutant (UV4) selected
from AA8 were determined as hypersensitivity values
(HF ) IC50(AA8)/IC50(UV4)). The UV4 mutant is defec-
tive in the incision step of excision repair of DNA
interstrand crosslinks or bulky monoadducts28 and is
consequently hypersensitive to the cytotoxic effects of
DNA-alkylating or -arylating agents. We have shown
previously17 that nitracrine (1) is significantly more
cytotoxic (11-13-fold) in this line than in AA8, suggest-
ing that the principal cytotoxic lesions it forms are DNA
adducts. All of the derivatives (both the amines and
corresponding N-oxides) had high HF values (10-30-

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2511
fold) except for the much less potent 2-OMe and 2,4-
diOMe derivatives 3 and 9. The similarity of the HF
values of the N-oxides and corresponding amines in each
case is consistent with cytotoxicity being due to reduc-
tion of the N-oxides to the amines. The low HF values
for the relatively nontoxic 2-OMe-substituted com-
pounds 3 and 9 indicate that, when nitroreduction is
severely depressed, a nonbioreductive mechanism of
toxicity becomes dominant.
Cytotoxic potencies determined in clonogenic assays
with aerobic AA8 cell suspensions, assessed as the
concentration required to lower cell survival to 10%
(C10), showed similar trends to the IC50 data (Table 1)
with the substituted compounds all less potent than 1
and the 2-OMe derivatives 3 and 9 being the least
potent. The N-oxides, as expected,7 were considerably
less cytotoxic than the parent amines in both the growth
inhibition and clonogenic assays, but there were sig-
nificant differences between chromophore substitution
patterns (Table 1). Since the N-oxides are designed as
nontoxic prodrugs which will liberate the tertiary
amines on reduction, the preferred compounds are those
showing a large deactivation relative to the correspond-
ing amine. By this criterion the best are the 4-OMe and
4,5-diOMe 1-nitroacridines (C10 ratio N-oxide/amine ca.
1000) followed by the parent 1-nitroacridine (ratio 150)
with the 2,4-diOMe- and methyl-substituted compounds
showing lower ratios.
The hypoxic selectivities of all compounds were
determined by comparing C10 values in AA8 cell sus-
pensions gassed with 5% CO2 in air or N2. The survival
curves for the key N-oxide prodrugs are shown in Figure
1, and the results are summarized for all compounds
in Table 1. The C10 ratios for nitracrine itself (1) and
its 4-OMe and 4-Me derivatives 5 and 7 were similar
to the differentials reported previously using slightly
different methods.6 The 4,5-diOMe and 4,5-diMe com-
pounds 11 and 13 had ratios of 11-14, similar to those
for the corresponding monosubstituted derivatives. The
2,4-diOMe derivative 9 showed only low selectivity (2-
fold), and a value could not be determined for the 2-OMe
compound 3.
F igu r e 1. Survival curves for treatment of plateau-phase AA8
cells with acridine-subsituted 1-nitroacridine N-oxides for 60
min at the indicated concentrations. Open symbols: aerobic.
Filled symbols: hypoxic. Different symbol shapes refer to
separate experiments.
the parent nitracrine N-oxide 2 (Figure 2B) was slower
than that of nitracrine (1) itself (Figure 2A) but was
extensive. Since tissue cell densities are 100-1000-fold
higher than those used in the in vitro experiments (106
cells/mL), very rapid metabolism would be expected in
the hypoxic regions of tumors. The observed loss from
the extracellular compartment is primarily due to
metabolism (rather than uptake of drugs into cells) since
little loss was observed under aerobic conditions except
during the first 30 min (data not shown), and loss of
the parent compounds in hypoxic cultures was ac-
companied by appearance of reductive metabolites.
Thus incubation with nitracrine (1) under the conditions
shown in Figure 2A gave the nitroreduction product
previously tentatively identified as the hydroxylamine,9
and the N-oxide 2 gave the amine 1 as an intermediate
as reported previously7 (see also Figure 4).
All four OMe-substituted tertiary amines showed
dramatically slower rates of metabolism (Figure 2A)
with the compound of lowest E(1) value (the 2,4-diOMe
compound 9) being the most stable. The corresponding
OMe-substituted N-oxides also showed slower rates of
metabolism than the parent 2 (Figure 2B), although the
extent of protection was not as marked. The lesser
With the exception of the 2-OMe-substituted com-
pounds, the N-oxide derivatives all showed very high
selectivity for hypoxia (Figure 1), with differentials
much greater than those of the corresponding tertiary
amines (Table 1). The hypoxic differential for the
parent compound 2 (820) was similar to that reported
previously,7 while the 4-OMe derivative 6 gave a ratio
of 1250 and the 4,5-diOMe derivative 12 a ratio of 2800.
The 4-Me (8) and 4,5-diMe (14) analogues were less
selective than the parent, with ratios of 150 and 54,
respectively. Surprisingly, the 2,4-diOMe compound 10
showed essentially no hypoxic selectivity. Excluding the
2-OMe-substituted compounds, these hypoxic differen-
tials parallel the differences in aerobic potency between
N-oxide and tertiary amine, indicating that a key design
feature for hypoxic selectivity in this series is that the
prodrug (N-oxide) and active effector (tertiary amine)
show a large difference in cytotoxic potency.
The ability of electron-donating OMe substituents to
stabilize the 1-nitroacridine tertiary amines, and their
corresponding N-oxide prodrugs, against reductive me-
tabolism in hypoxic AA8 cultures was assessed by HPLC
of samples of extracellular medium (Figure 2). Loss of

2512 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Lee et al.
F igu r e 2. Loss of acridine-substituted nitracrine derivatives (initial concentration 30 µM) from extracellular medium (RMEM
with 5% FBS) under hypoxic conditions as determined by HPLC. A: Tertiary amine derivatives in AA8 cell cultures (106 cells/
mL). B: Tertiary amine N-oxides in AA8 cell cultures (106 cells/mL). C: Tertiary amine N-oxides in medium without cells.
F igu r e 3. Reduction of DACA N-oxide (16) in RMEM culture
medium (without cells) as determined by HPLC. Left panel:
loss of parent N-oxide. Right panel: formation of DACA free
amine (O, 20% O2, 5% FBS; 0, N2, 5% FBS; 4, N2, no FBS).
F igu r e 4. Reduction of the parent N-oxide 2 (left panel) and
its 2,4-diOMe derivative 10 (right panel) in hypoxic culture
effect in the N-oxides was expected, since reduction of
medium (RMEM with 5% FBS) alone (open symbols) and with
the N-oxide moiety itself provides an alternate route for
AA8 cells at 106 cells/mL (filled symbols) as determined by
compound loss which would not be expected to be
HPLC. Circles: N-oxide prodrug. Squares: tertiary amine
influenced by substitution of the acridine nucleus.
reduction product.
The analysis of the reduction of the N-oxide moiety
is complicated by the finding that the N-oxides are also
reduced in hypoxic (but not aerobic) culture medium
(Figure 2C), although at lower rates than when AA8
cells are present (Figure 2B). The corresponding ter-
tiary amines were stable under these conditions (data
not shown). To explore this phenomenon, a variety of
other tertiary amine N-oxides, including AQ4N (15),14
the desnitro analogue of 2, and the acridine carboxamide
derivative DACA N-oxide (16),15 were also examined in
hypoxic culture medium. All the compounds were
reduced to the corresponding amines, at broadly similar
rates. As illustrated for DACA N-oxide (16), the loss of
the N-oxide was accompanied by quantitative formation
of the corresponding amine DACA, a reaction which was
inhibited by O2 and completely dependent on the fetal
bovine serum (FBS) component of the medium (Figure
3). The rate of reduction of 16 was ca. 3-fold higher
using a batch of FBS with a hemoglobin concentration
of 0.16 mg/mL than it was with a batch containing only
0.11 mg/mL, suggesting that the known29 reduction of
tertiary amine N-oxides by denatured hemoglobin may
partially account for this phenomenon.
1-nitro group) in Figure 4. The N-oxide 2 is slowly
reduced to 1 in hypoxic culture medium. The loss of 2
is faster in the presence of cells, but the concentrations
of 1 are lowered because the latter is reduced further
by cellular nitroreduction (Figure 4A). In contrast, 10
is lost at essentially the same rate in the presence or
absence of cells, and the concentrations of the product
9 are unaffected by cells (Figure 4B). The other OMe-
substituted compounds gave results intermediate be-
tween those of 2 and 10 but more similar to the latter
(data not shown). The above data are consistent with
two major hypoxia-selective routes of reduction of the
1-nitroacridine tertiary amine N-oxides: reduction of
the N-oxide moiety to provide the tertiary amine (prob-
ably mainly by hemoglobin in the extracellular medium)
and intracellular reduction of the 1-nitro group. When
the latter process is slowed (e.g., in the 2,4-diOMe
derivative 10), only the N-oxide reduction is significant
in cell culture. Thus the use of electron-donating
functionality can be used to slow nitroreduction as
expected.
The relative ability of the metabolically-stabilized
4-OMe N-oxide 6, and its unsubstituted parent 2, to
diffuse into spheroids was evaluated by comparing
killing in intact EMT6 multicellular spheroids with
The interplay of cellular and extracellular reduction
is illustrated by comparison of 2 (with a readily-
metabolized 1-nitro group) and 10 (with a stabilized

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2513
Ta ble 2. Sensitivity of EMT6 Cells in Intact and Dissociated
Spheroidsa to Selected Nitracrine N-Oxides
SRFc
b
no.
C10 (µM)
1
2
5
6
0.11 ( 0.04 (9)d
1.9 ( 0.6 (3)
10.7 ( 0.9 (9)
7.0 ( 1.0 (3)
7.7 ( 2.0 (5)
1.9 ( 0.4 (3)
1.7 ( 0.3 (5)
300 ( 10 (3)
a
Spheroids of ca. 1200 µm diameter were exposed to drugs
under aerobic conditions (5% CO2 in air) at 37 °C with stirring
for 60 min, either before or immediately after dissociation with a
pronase/DNAase enzyme cocktail (see the Experimental Section).
b
Concentration resulting in 10% survival in dissociated spheroid
cell suspensions. c SRF, spheroid resistance factor ) C10(intact)/
C
10(dissociated). See text for further definition and comments.
Values are means ( SEM for the number of independent
d
experiments indicated in parentheses.
killing of single cells from freshly-dissociated spheroids
exposed under equivalent ambient conditions (Table 2).
The differential was quantitated as the spheroid resis-
tance factor (SRF), defined as the ratio of C10 values
for intact and dissociated spheroids. As reported previ-
ously7 (although now based on a larger data set), the
SRF of 2 against intact spheroids is somewhat less than
that of nitracrine itself (7.0 compared to 10.7), but for
the 4-OMe compounds the SRF for the N-oxide 6 is
4-fold lower than that for the amine 5 (SRF ) 1.9
compared with 7.7). These ratios cannot be directly
related to drug penetration since the SRF will also
depend on the ability of the drug to kill cells under the
microenvironmental conditions pertaining in spheroids.
However, since both the N-oxides show similar hypoxic
selectivity in AA8 single-cell suspensions and have
otherwise similar physicochemical properties, it is likely
that the low SRF for the 4-OMe compound reflects
improved distributive properties because of lowered
rates of metabolic reduction.
F igu r e 5. Activity of 1-nitroacridine tertiary amines (left
panels) and their N-oxide derivatives (right panels) against
the KHT tumor as determined by excision and clonogenic assay
18 h after treatment. Drugs were administered ip alone (O)
or 5 min after γ-irradiation at 15 Gy (b). In each case the
highest drug dose tested corresponds to 3 times the maximum
tolerated dose (as determined using a 28-day observation
period). Each point represents two pooled tumors from sepa-
rate mice. Multiple points at each dose are from separate
experiments.
compound 6, and there appears to be no advantage of
this derivative relative to 2.
Con clu sion s
The main aims of this work were to provide analogues
of nitracrine N-oxide with lower nitroacridine reduction
potentials, and consequently lowered rates of metabolic
activation, and to test whether this would lead to
improved distributive properties and enhanced thera-
peutic activity against hypoxic cells in tumors. The
results show that 4,5-disubstitution of nitracrine (1)
with Me or OMe groups does not provide analogues with
lower reduction potentials than the corresponding mono-
substituted compounds. This appears not to be due to
a concomitant raising of the acridine pKa but to a lack
of direct electronic effect of substituents not in the ring
bearing the nitro group. However, placing two OMe
groups in the nitro-bearing ring does result in a
substantial further lowering of E(1) (from -355 mV in
5 to -401 mV in 9). With all of the Me- or OMe-
substituted compounds, a significant metabolic stabili-
zation was observed in hypoxic AA8 cell cultures. This
effect was greatest for the tertiary amine 9, which had
the lowest E(1), although lesser protection against
overall metabolic loss was achieved with the corre-
sponding N-oxide 10 (presumably because reduction of
the N-oxide moiety now becomes significant).
While the 2,4-diOMe-substituted N-oxide 10 showed
poor hypoxic selectivity in culture, the N-oxides of the
4-substituted and 4,5-disubstituted derivatives of ni-
tracrine generally retained the very high selectivity of
the parent N-oxide 2. For the 4-OMe derivative 6 and
the 4,5-diOMe derivative 12, this selectivity was >1000-
fold, although absolute potency was much lower than
that of 2. The 4-OMe compound also showed activity
in intact multicellular spheroids almost as great as that
in single-cell suspensions, with a SRF of only 1.9 (cf. 7
The host toxicities of the N-oxide prodrugs and their
tertiary amines were compared in mice, using the
maximum tolerated (nonlethal) dose as the end point
(Table 1). The parent N-oxide 2 was 50-fold less toxic
than nitracrine (1). Of the analogues, only the 4-Me
derivatives 5 and 6 provided a differential as large as
this. The 4-OMe- and 4,5-diOMe-substituted chro-
mophores also gave substantial differences (ca. 5-fold)
between prodrug and tertiary amine toxicity, but the
other compounds showed little differential. The activity
of compounds 2, 6, 8, 10, 12, and 14 was examined
against the KHT tumor, at a dose corresponding to 75%
of the MTD, by excising tumors 18 h after treatment
and determining clonogen number in vitro. None of
these compounds showed activity when administered
alone or provided increased killing when given either
before or after radiation (data not shown). At doses
above the MTD, mouse deaths due to drug occurred
sufficiently late with 2, 6, and 14 so that it was possible
to evaluate doses up to 3 times the MTD. Under these
conditions, both the parent N-oxide 2 and the 4-OMe
derivative 6 (but not the 4,5-diMe derivative 14) showed
activity when administered after irradiation (Figure 5).
This activity was somewhat variable, but the N-oxides
were clearly more active than the corresponding tertiary
amines 1 and 5 at equivalent multiples of the MTD
(Figure 5). The parent N-oxide 2 was more active in
combination with radiation than when administered
alone, indicating selective toxicity for hypoxic cells.
Selectivity for hypoxic cells was less clear for the 4-OMe

2514 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Lee et al.
resulting mixture was stirred at 80-85 °C for 3.5 h, cooled,
and poured onto ice. The precipitate was filtered, washed with
water, and dried to give 4,5-dimethyl-1-nitro-9-oxoacridan (20)
(1.26 g, 58%), mp (CH2Cl2/MeOH) 349-351 °C: 1H NMR
[(CD3)2SO] δ 8.87 (br s, 1 H, exchangeable with D2O, NH), 8.03
(dd, J ) 8.0, 1.0 Hz, H-8), 7.75 (d, J ) 8.0 Hz, 1 H, H-2), 7.69
(d, J ) 8.0 Hz, 1 H, H-6), 7.43 (d, J ) 8.0 Hz, 1 H, H-3), 7.28
(t, J ) 8.0 Hz, 1 H, H-7), 2.74 (s, 3 H, CH3), 2.69 (s, 3 H, CH3).
Anal. (C15H12N2O3) C, H, N.
The above acridone 20 (1.19 g, 4.44 mmol) was suspended
in SOCl2 (5 mL) and DMF (1 drop), and the mixture was
heated under reflux for 1 h. Excess reagent was evaporated,
and the residue was coevaporated with toluene and then
dissolved in CH2Cl2 and washed with ice-cold aqueous Na2-
CO3. Workup gave a quantitative yield of crude 9-chloro-4,5-
dimethyl-1-nitroacridine (21), which was dissolved in dry DMF
(5 mL) and treated with N,N-dimethylpropane-1,3-diamine
(0.83 mL, 6.7 mmol) at 80 °C for 3.5 h. The cooled mixture
was partitioned between CH2Cl2 and cold aqueous Na2CO3.
Workup of the organic layer followed by chromatography of
the residue on silica gel (elution with EtOAc) gave 4,5-
dimethyl-9-[(3-(dimethylamino)propyl)amino]-1-nitroacri-
dine (13) (0.35 g, 23%). This was converted to the dihydro-
chloride salt, mp (MeOH) 170 °C dec: 1H NMR (D2O) δ 8.15
(d, J ) 8.2 Hz, 1 H, H-2), 8.02 (d, J ) 8.2 Hz, 1 H, H-3), 7.87
(d, J ) 7.8 Hz, 1 H, H-8), 7.81 (d, J ) 7.8 Hz, 1 H, H-6), 7.49
(t, J ) 7.8 Hz, 1 H, H-7), 3.77 (t, J ) 7.6 Hz, 2 H, NHCH2),
3.11 (t, J ) 7.6 Hz, 2 H, CH2N(CH3)2), 2.88 (s, 6 H, N(CH3)2),
2.80 (s, 3 H, CH3), 2.70 (s, 3 H, CH3), 2.20 (quintet, J ) 7.6
Hz, 2 H, CH2CH2CH2). Anal. (C20H24N4O2‚2HCl‚H2O) C, H,
N, Cl.
for the parent N-oxide 2), suggesting that it is better
able to diffuse in a multicellular environment. These
apparent improvements in vitro did not translate into
improved activity against hypoxic cells in KHT tumors.
However, this work has shown that the unsubstituted
nitracrine N-oxide 2 and its 4-methoxy derivative 6 have
sufficient selectivity as hypoxia-selective cytotoxins to
warrant further development. Significant improvement
of therapeutic activity in vivo may require lowering of
the rates of reduction of the N-oxide center as well as
the nitro group, and future work in this series will
therefore focus on stabilization of the N-oxide center
toward hypoxic metabolism.
Exp er im en ta l Section
Analyses indicated by symbols of the elements were within
(0.4% of theoretical. Analyses were carried out in the
Microchemical Laboratory, University of Otago, Dunedin, New
Zealand. Melting points were determined on an Electrother-
mal 2300 melting point apparatus. pKa values were deter-
mined by UV spectrophotometry. NMR spectra were obtained
on a Bruker AC-200 or AM-400 spectrometer and are refer-
enced to Me4Si (organic solvents) or sodium 2,2-dimethyl-2-
silapentane-5-sulfonate (D2O solvent). Mass spectra were
determined on a VG-70SE mass spectrometer using an ionizing
potential of 70 eV at a nominal resolution of 1000. High-
resolution spectra were obtained at nominal resolutions of
3000, 5000, or 10 000 as appropriate. Spectra were obtained
using the ionization mode specified, with PFK as the reference
unless otherwise stated. Thin-layer chromatography was
carried out on aluminum-backed silica gel plates (Merck, 60
F254) and preparative column chromatography on silica gel
(Merck, 230-400 mesh).
Compounds for biological evaluation were analyzed by
HPLC using a Waters µBondapak C18 column (8 × 100 mm)
with a mobile phase comprising a 25 min linear gradient of
MeCN (typically 14-50% MeCN) in 0.45 M ammonium for-
mate, pH 4.5, at a flow rate of 1.8 mL/min. All compounds
were >99% pure based on absorbance at 254 nm. The
1-nitroacridine N-oxides contained <0.02% of the correspond-
ing tertiary amines as impurities. The absence of biologically
significant amine impurities was confirmed in each case by
bioassay of the column eluate after injecting 0.6-40 µmol of
the N-oxide. Fractions (1 mL) were collected, diluted 200-fold
into 96-well plate cultures of UV4 cells (360 cells/well in 0.2
mL of culture medium) and grown for 4 days before staining
with methylene blue to assess cell growth.30 In all cases the
only bioactive peak corresponded to the parent N-oxide.
4,5-Dim et h yl-9-[(3-(d im et h yla m in o)p r op yl)a m in o]-1-
n itr oa cr id in e (13) (Sch em e 2): Exa m p le of Gen er a l
Meth od A. A mixture of methyl 2-bromo-3-methylbenzoate
(17) (prepared from 2-bromo-3-methylbenzoic acid31 with
SOCl2/MeOH) (3.46 g, 15.1 mmol), 2-methyl-5-nitroaniline (18)
(4.59 g, 30.2 mmol), anhydrous K2CO3 (6.25 g, 45.3 mmol), CuI
(0.75 g, 3.93 mmol), and copper powder (0.23 g, 3.62 mmol) in
HMPA (15 mL) was stirred at 170 °C under N2 for 2 h. The
cooled mixture was filtered, and the solids were washed with
aqueous MeOH. The combined filtrates were basified with
KOH, heated under reflux for 45 min, and then concentrated
under reduced pressure, diluted with 1 N aqueous KOH, and
filtered. The filtrate was acidified to pH 1-2 with concen-
trated HCl to give 3-methyl-2-[(2-methyl-5-nitrophenyl)amino]-
benzoic acid (19) (2.7 g, 63%), sufficiently pure for the next
step. A sample crystallized from MeOH had mp 203-205 °C:
1H NMR (CDCl3) δ 8.36 (br s, 1 H, exhangeable with D2O,
COOH), 8.01 (dd, J ) 7.7, 1.0 Hz, 1 H, H-6), 7.69 (dd, J ) 8.2,
2.3 Hz, 1 H, H-4′), 7.50 (dd, J ) 7.7, 1.0 Hz, 1 H, H-4), 7.29 (d,
J ) 8.2 Hz, 1 H, H-3′), 7.17 (t, J ) 7.7 Hz, 1 H, H-5), 7.12 (d,
J ) 2.3 Hz, 1 H, H-6′), 3.5 (s, 1 H, exchangeable with D2O,
NH), 2.50 (s, 3 H, CH3), 2.05 (s, 3 H, CH3). Anal. (C15H14N2O4)
C, H, N.
Similarly prepared, from 4-methyl-1-nitro-9-oxoacridan,6
was 9-[(3-(dimethylamino)propyl)amino]-4-methyl-1-nitroacri-
dine (7) (63%), dihydrochloride salt, mp 259-261 °C dec (lit.12
mp 256-257 °C): 1H NMR (D2O) δ 7.50-8.20 (m, 6 H, ArH),
3.73 (t, J ) 7.2 Hz, 2 H, NHCH2), 3.10 (t, J ) 7.9 Hz, 2 H,
CH2N(CH3)2), 2.88 (s, 6 H, N(CH3)2), 2.71 (s, 3 H, CH3), 2.20
(m, 2 H, CH2CH2CH2).
9-[(3-(Dimethylamino)propyl)amino]-4-methoxy-1-nitroacri-
dine (5) was prepared from 4-methoxy-1-nitro-9-oxoacridan,6
via phenol-mediated reaction of the corresponding 9-chloro
compound, in 48% yield, dihydrochloride salt, mp 190-192 °C
dec (lit.12 mp 190-191 °C): 1H NMR [(CD3)2SO] δ 7.40-8.80
(m, 6 H, ArH), 4.25 (s, 3 H, OCH3), 3.60 (br s, 2 H, NHCH2),
3.00 (br s, 2 H, CH2N(CH3)2), 2.65 (s, 6 H, N(CH3)2), 2.20 (br
s, 2 H, CH2CH2CH2).
9-[(3-(Dimethylamino)propyl)amino]-2-methoxy-1-nitroacri-
dine (3) was similarly prepared from 9-chloro-2-methoxy-1-
nitroacridine32 in 82% yield: 1H NMR (CDCl3) δ 7.30-8.30 (m,
6 H, ArH), 6,10 (br s, 1 H, exchangeable with D2O, NH), 3.45
(br s, 2 H, NHCH2), 2.48 (t, J ) 6 Hz, 2 H, NHCH2CH2CH2),
2.30 (s, 6 H, N(CH3)2), 1.80 (quintet, J ) 6.5 Hz, 2 H,
NHCH2CH2). The dihydrochloride salt crystallized from EtOAc/
MeOH, mp 170-172 °C dec (lit.24 mp 167-168 °C).
2,4-Dim eth oxy-9-[(3-(d im eth yla m in o)p r op yl)a m in o]-1-
n itr oacr idin e (9) (Sch em e 3): Exam ple of Gen er al Meth od
B. A suspension of 1,5-dimethoxy-2,4-dinitrobenzene33 (20.1
g, 90.0 mmol) in water (117 mL) was stirred under reflux and
treated dropwise (over 30 min) with a solution of sodium
polysulfide, prepared by heating Na2S‚9H2O (28.6 g, 119 mmol)
and sulfur (6.8 g, 213 mmol) in water (120 mL). The reaction
mixture was stirred under reflux for a further 3 h and then
cooled and evaporated under reduced pressure at 45 °C to give
a dark residue. This was extracted several times with warm
EtOAc, and the combined extracts were filtered through a
short column of silica gel. The eluates were washed with water
and evaporated to dryness, and the residue was dissolved in
1 N HCl, filtered, and basified with aqueous NaOH to give
1-amino-2,4-dimethoxy-5-nitrobenzene (23) (11.0 g, 63%), mp
126-128 °C (lit.19 mp 136-137 °C): 1H NMR (CDCl3) δ 7.40
(s, 1 H, H-6), 6.49 (s, 1 H, H-3), 3.95 (s, 3 H, OCH3), 3.93 (s, 3
H, OCH3), 3.73 (br s, 2 H, exchangeable with D2O, NH2).
A mixture of 23 (6.0 g, 30.9 mmol), diphenyliodonium-2-
carboxylate22 (22) (20.0 g, 61.4 mmol), and copper(II) acetate
(0.4 g, 2.2 mmol) in dry DMF (180 mL) was stirred at 90 °C
(bath temperature) for 7 h and then at 20 °C overnight. Most
The above acid 19 (2.34 g, 8.18 mmol) was powdered and
added slowly to stirred, ice-cold 90% sulfuric acid (50 mL). The

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2515
of the DMF was evaporated under reduced pressure, and the
residue was taken up into CH2Cl2, washed twice with water,
and extracted into 0.5 N KOH. The alkaline layer was back-
extracted with CH2Cl2 and filtered, and the filtrate was
acidified at 0 °C to give 2-[(2,4-dimethoxy-5-nitrophenyl)amino]-
benzoic acid (24) (9.7 g, 99%), mp (CHCl3/MeOH) 283-285
°C: 1H NMR [(CD3)2SO] δ 9.50 (s, 1 H, exchangeable with D2O,
COOH), 7.96 (s, 1 H, H-6′), 7.91 (d, J ) 7.3 Hz, 1 H, H-6), 7.41
(t, J ) 7.3 Hz, 1 H, H-4), 7.07 (d, J ) 7.3 Hz, 1 H, H-3), 6.97
(s, 1 H, H-3′), 6.80 (t, J ) 7.3 Hz, 1 H, H-5), 4.00 (s, 6 H, 2 ×
OCH3). Anal. (C15H14N2O6) C, H, N.
A suspension of 24 (5.0 g, 15.9 mmol) in dry DMF (25 mL)
was treated with CDI21 (3.87 g, 23.9 mmol) at 40 °C for 10
min and then cooled and treated with N,N-dimethylpropane-
1,3-diamine (6.0 mL, 47.7 mmol) at 20 °C for 17 h. The
mixture was partitioned between CH2Cl2 and 0.5 N aqueous
NaOH, and the organic layer was washed twice with water,
dried, and evaporated to give N-[3-(dimethylamino)propyl]-2-
[(2,4-dimethoxy-5-nitrophenyl)amino]benzenecarboxamide (25)
(5.3 g, 84%), mp (EtOAc) 88-90 °C: 1H NMR (CDCl3) δ 9.69
(s, 1 H, exchangeable with D2O, NH), 8.55 (br t, 1 H,
exchangeable with D2O, NHCO), 8.08 (s, 1 H, H- 6′), 7.39 (d,
J ) 7.8 Hz, 1 H, H-6), 7.32 (m, 2 H, H-3,4), 6.81 (ddd, J ) 7.8,
5.6, 2.8 Hz, 1 H, H-5), 6.57 (s, 3 H, H-3′), 3.99 (s, 6 H, 2 ×
OCH3), 3.54 (q, J ) 5.9 Hz, collapsed to t after D2O, 2 H,
CONHCH2), 2.50 (t, J ) 5.9 Hz, 2 H, NHCH2CH2CH2), 2.30
(s, 6 H, N(CH3)2), 1.76 (quintet, J ) 5.9 Hz, 2 H, CH2CH2-
CH2). Anal. (C20H26N4O5) C, H, N.
The above amide 25 (5.1 g, 12.8 mmol) was added to stirred,
neat polyphosphate ester (PPE), and the mixture was heated
at 100 °C for 2 h, allowing volatile solvents to evaporate. A
further portion of PPE (50 mL) was then added, and the
mixture was stirred at 100 °C for another 2 h. The cooled
mixture was poured into cold 2 N aqueous Na2CO3 and
extracted with EtOAc. The crude product from the organic
layer was chromatographed on silica gel, eluting with CH2-
Cl2/Et3N (99:1), to give 2,4-dimethoxy-9-[(3-(dimethylamino)-
propyl)amino]-1-nitroacridine (9) (2.4 g, 49%), mp (dihydro-
chloride salt from EtOAc/CH3OH) 158 °C dec: 1H NMR (D2O)
δ 8.17 (d, J ) 7.7 Hz, 1 H, H-8), 7.96 (t, J ) 7.7 Hz, 1 H, H-6),
7.90 (d, J ) 7.7 Hz, 1 H, H-5), 7.60 (t, J ) 7.7 Hz, 1 H, H-7),
7.22 (s, 1 H, H-3), 4.27 (s, 3 H, OCH3), 4.18 (s, 3 H, OCH3),
3.74 (t, J ) 7.2 Hz, 2 H, NHCH2), 2.90 (m, 8 H, CH2N(CH3)2),
2.19 (m, 2 H, CH2CH2CH2). Anal. (C20H24N4O4‚2HCl‚H2O) C,
H, N.
(dimethylamino)propyl)amino]-4-methyl-1-nitroacridine (7) (1.08
g, 3.19 mmol) in dry CH2Cl2 (40 mL) was treated dropwise
over 30 min at 20 °C with a solution of 2-(phenylsulfonyl)-3-
phenyloxaziridine20 (1.00 g, 3.83 mmol) in the same solvent.
The reaction mixture was stirred at 20 °C for a further 30 min
and then evaporated to dryness under reduced pressure. The
residue was applied directly to a column of silica gel. Elution
with EtOAc/Et3N (99:1) followed by CHCl3/EtOH/Et3N (90:9:
1) gave 9-[(3-(dimethylamino)propyl)amino]-4-methyl-1-ni-
troacridine N3-oxide (8) (1.1 g, 97%). A solution of this in a
mixture of CH2Cl2 and i-PrOH was treated with anhydrous
HCl gas, followed by addition of EtOAc, to give the dihydro-
chloride salt, mp 251 °C dec: 1H (D2O) δ 7.50-8.10 (m, 6 H,
ArH), 3.79 (t, J ) 7.2 Hz, 2 H, NHCH2), 3.60 (t, J ) 7.2 Hz, 2
H, CH2N(CH3)2), 3.48 (s, 6 H, N(CH3)2), 2.72 (s, 3 H, CH3), 2.35
(quintet, J ) 7.2 Hz, 2 H, CH2CH2CH2). Anal. (C19H22N4O3‚-
2HCl‚0.5H2O) C, H, N.
Similarly prepared, from 1, was 9-[(3-(dimethylamino)-
propyl)amino]-1-nitroacridine N3-oxide (2) (100% yield), dihy-
drochloride salt, mp (MeOH/EtOAc) 194-196 °C (lit.25 mp 196
°C): 1H NMR (D2O) δ 8.60-7.50 (m, 7 H, ArH), 3.79 (t, J )
7.2 Hz, 2 H, NHCH2), 3.62 (t, J ) 7.2 Hz, 2 H, NHCH2-
CH2CH2), 3.47 (s, 6 H, NMe2), 2.37 (quintet, J ) 7.2 Hz, 2 H,
CH2CH2CH2).
Similarly prepared, from 3, was 9-[(3-(dimethylamino)-
propyl)amino]-2-methoxy-1-nitroacridine N3-oxide (4) (92%
yield), dihydrochloride salt, mp (EtOAc/MeOH) 195 °C dec: 1H
NMR (D2O) δ 8.20 (d, J ) 8.6 Hz, 1 H, H-4), 8.03 (dd, J ) 8.0,
1.0 Hz, 1 H, ArH), 8.01 (dd, J ) 8.0, 1.0 Hz, 1 H, ArH), 7.96
(td, J ) 8.0, 1.0 Hz, 1 H, ArH), 7.79 (d, J ) 8.6 Hz, 1 H, H-3),
7.58 (td, J ) 8.0, 1.0 Hz, 1 H, ArH), 4.13 (s, 3 H, OCH3), 3.86
(t, J ) 7.3 Hz, 2 H, NHCH2), 3.63 (t, J ) 7.3 Hz, 2 H, NHCH2-
CH2CH2), 3.46 (s, 6 H, N(CH3)2), 2.35 (quintet, J ) 7.3 Hz, 2
H, NHCH2CH2). Anal. (C19H22N4O4‚2HCl‚0.5H2O) C, H, N,
Cl.
Similarly prepared, from 5, was 9-[(3-(dimethylamino)-
propyl)amino]-4-methoxy-1-nitroacridine N3-oxide (6) (92%
yield), dihydrochloride salt, mp (MeOH/EtOAc) 190 °C dec: 1H
NMR (D2O) δ 8.21 (d, J ) 8.8 Hz, 2 H, H-2), 7.47-8.05 (m, 4
H, ArH), 7.32 (d, J ) 8.8 Hz, 1 H, H-3), 4.28 (s, 3 H, OCH3),
3.78 (t, J ) 7.0 Hz, 2 H, NHCH2), 3.61 (t, J ) 7.0 Hz, 2 H,
CH2N(CH3)2), 3.49 (s, 6 H, N(CH3)2), 2.35 (quintet, J ) 7.0 Hz,
2 H, CH2CH2CH2). Anal. (C19H22N4O4‚2HCl‚0.5H2O) C, H, N.
Similarly prepared, from 9, was 2,4-dimethoxy-9-[(3-(dim-
ethylamino)propyl)amino]-1-nitroacridine N3-oxide (10) (76%
yield), dihydrochloride salt, mp (EtOAc/MeOH) 182 °C dec: 1H
NMR (D2O) δ 8.02 (d, J ) 8.0 Hz, 1 H, H-8), 7.92 (t, J ) 8.9
Hz, 1 H, H-6), 7.81 (d, J ) 8.0 Hz, 1 H, H-5), 7.53 (t, J ) 8.0
Hz, 1 H, H-7), 7.20 (s, 1 H, H-3), 4.28 (s, 3 H, OCH3), 4.21 (s,
3 H, OCH3), 3.75 (t, J ) 7.3 Hz, 2 H, NHCH2), 3.61 (t, J ) 7.3
Hz, 2 H, NHCH2CH2CH2), 3.49 (s, 6 H, N(CH3)2), 2.32 (quintet,
Similar reaction of 3-methoxy-2-[(2-methoxy-5-nitrophenyl)-
amino]benzoic acid (26) [prepared in 64% yield by J ourdan-
Ullmann condensation of methyl 2-bromo-3-methoxybenzoate
and 2-methoxy-5-nitroaniline by Method A: mp (CHCl3/
1
MeOH) 261-263 °C; H NMR (CDCl3) δ 6.90-7.98 (m, 6 H,
ArH), 4.06 (s, 3 H, OCH3), 3.81 (s, 3 H, OCH3). Anal.
(C15H14N2O6) C, H, N] with CDI and N,N-dimethylpropane-
1,3-diamine gave N-[3-(dimethylamino)propyl]-3-methoxy-2-
(2-methoxy-5-nitrophenyl)benzenecarboxamide (27) (55% yield),
mp (EtOAc/MeOH) 161-162 °C: 1H NMR (CDCl3) δ 8.55 and
8.14 (2 × br s, 2 H, exchangeable with D2O, 2 × NH), 7.74
(dd, J ) 8.9, 2.7 Hz, 1 H, H-4′), 7.22 (d, J ) 2.7 Hz, 1 H, H-6′),
7.07-7.20 (m, 3 H, H-4,5,6), 6.84 (d, J ) 8.9 Hz, 1 H, H-3′),
4.03 (s, 3 H, OCH3), 3.82 (s, 3 H, OCH3), 3.45 (q, J ) 6.2 Hz,
2 H, collapsed to a t after D2O, NHCH2), 2.40 (t, 2 H, J ) 6.2
Hz, CH2N(CH3)2), 2.23 (s, 6 H, N(CH3)2), 1.67 (quintet, J )
6.2 Hz, 2 H, CH2CH2CH2). Anal. (C19H26N4O5) C, H, N.
Cyclodehydration of 27 with PPE as above gave 4,5-
dimethoxy-9-[(3-(dimethylamino)propyl)amino]-1-nitroacri-
dine (11) (0.85 g, 64%). A solution of this in a mixture of
CH2Cl2 and i-PrOH was treated with anhydrous HCl gas,
followed by addition of EtOAc, to give the dihydrochloride salt,
mp 170 °C dec: 1H NMR (D2O) δ 8.15 (d, J ) 8.8 Hz, 1 H,
H-2), 7.34-7.55 (m, 3 H, H-6,7,8), 7.27 (d, J ) 8.8 Hz, 1 H,
H-3), 4.28 (s, 3 H, OCH3), 4.18 (s, 3 H, OCH3), 3.65 (t, J ) 7.0
Hz, 2 H, NHCH2), 3.11 (t, J ) 8.1 Hz, 2 H, CH2N(CH3)2), 2.89
J
) 7.3 Hz, 2 H, NHCH2CH2CH2). Anal. (C20H24N4O5‚-
2HCl‚H2O) C, H, N, Cl.
Similarly prepared, from 11, was 4,5-dimethoxy-9-[(3-(dim-
ethylamino)propyl)amino]-1-nitroacridine N3-oxide (12) (66%
yield), dihydrochloride salt, mp (EtOAc/MeOH) 160 °C dec: 1H
NMR (D2O) δ 8.20 (d, J ) 8.7 Hz, 1 H, H-2), 7.40-7.59 (m, 3
H, H-6,7,8), 7.31 (d, J ) 8.7 Hz, 1 H, H-3), 4.27 (s, 3 H, OCH3),
4.17 (s, 3 H, OCH3), 3.78 (br s, 2 H, NHCH2), 3.60 (br s, 2 H,
CH2N(CH3)2), 3.46 (s, 6 H, N(CH3)2), 2.33 (br s, 2 H, CH2CH2-
CH2). Anal. (C20H24N4O5‚2HCl‚1.5H2O) C, H, N, Cl.
Similarly prepared, from 13, was 4,5-dimethyl-9-[(3-(dim-
ethylamino)propyl)amino]-1-nitroacridine N3-oxide (14) (100%
yield), dihydrochloride salt, mp (CH2Cl2/EtOH/EtOAc) 227 °C
dec: 1H NMR (D2O) δ 8.18 (d, J ) 7.9 Hz, 1 H, H-2), 7.93 (d,
J ) 7.8 Hz, 1 H, H-8), 7.87 (d, J ) 7.9 Hz, 1 H, H-3), 7.77 (d,
J ) 7.8 Hz, 1 H, H-6), 7.42 (t, J ) 7.8 Hz, 1 H, H-7), 3.81 (t,
J ) 7.0 Hz, 2 H, NHCH2), 3.61 (t, J ) 7.0 Hz, 2 H, CH2N-
(CH3)2), 3.47 (s, 6 H, N(CH3)2), 2.80 (s, 3 H, CH3), 2.67 (s, 3 H,
CH3), 2.36 (quintet, J ) 7.0 Hz, 2 H, CH2CH2CH2). Anal.
(C20H24N4O3‚2HCl‚0.5H2O) C, H, N.
(s,
6 H, N(CH3)2), 2.18 (m, 2 H, CH2CH2CH2). Anal.
Cytotoxicity Assa ys. AA8 and UV4 cells were maintained
in logarithmic-phase growth at 37 °C as monolayers and grown
to plateau phase in spinner flasks as described previously.34
All drug exposures were performed in RMEM with 5% fetal
calf serum under an atmosphere containing 5% CO2. Growth
(C20H24N4O4‚2HCl‚2H2O) C, H, N.
9-[(3-(Dim et h yla m in o)p r op yl)a m in o]-4-m et h yl-1-n i-
tr oa cr id in e N3-Oxid e (8) (Sch em e 4): Exa m p le of Gen -
er a l Meth od C. A stirred solution of the free base of 9-[(3-

2516 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Lee et al.
inhibition studies were performed as described in detail
elsewhere,30 by seeding 200 log-phase AA8 or 300 UV4 cells
in 96-well tissue culture dishes. One day later, cells were
exposed to drugs for 18 h under aerobic conditions. The IC50
was determined as the drug concentration needed to reduce
the cell mass (protein content, measured 72-78 h after
removal of drug by staining with methylene blue) to 50% of
the mean value for eight control cultures on the same 96-well
plate. Clonogenic assays with magnetically-stirred 10 mL
suspension cultures (plateau-phase AA8 cells, 106/mL) were
performed using continuous gassing with 5% CO2 in air or N2
as detailed elsewhere.34 Both cell suspensions and drug
solutions in growth medium were preequilibrated under the
appropriate gas phase for 60 min prior to mixing, to ensure
essentially complete anoxia (in hypoxic cultures) throughout
the drug exposure period. Cell killing was quantitated as the
drug concentration required to lower the plating efficiency to
10% of that of concurrent non-drug-treated controls.
Meta bolic Red u ction in Cell Cu ltu r e. Plateau-phase
AA8 cells were incubated with drugs (30 µM) under identical
conditions to those used for cytotoxicity (clonogenic) assays.
Cells were routinely stained with trypan blue at the end of
drug exposures; in all studies it was confirmed that the drug
did not compromise metabolic integrity of the cells (>90%
viability). The gassed and stirred cell suspensions (or solutions
of drugs in media without cells) were sampled at intervals and
centrifuged quickly, and the cell-free supernatant was stored
at -80 °C. Thawed samples were centrifuged briefly and
assayed by HPLC, without further workup, using the method
outlined above. Samples were held in a WISP autosampler
at 4 °C until injection, with interspersed standards to check
analyte stability, and samples of up to 0.25 mL were injected.
Detection was by diode array absorbance, with data capture
and analysis using a Hewlett Packard Chemstation instru-
ment. Identification of tertiary amine products was by ab-
sorbance spectrum and retention time.
Institute, the Auckland Division of the Cancer Society
of New Zealand, and the Health Research Council of
New Zealand.
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Ack n ow led gm en t. The authors thank Professor
Peter Wardman (CRC Gray Laboratory, Mt. Vernon
Hospital, London) for determining the E(1) values for
compounds 9, 11, and 13 and Alison Coleman for
technical assistance. This work was supported by
Contract NO1 CM 47019 from the US National Cancer

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2517
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