Hypoxia-selective antitumor agents. 14. Synthesis and hypoxic cell cytotoxicity of regioisomers of the hypoxia-selective cytotoxin 5-[N,N- bis(2-chloroethyl)amino]-2,4-dinitrobenzamide

Article abstract of DOI:10.1021/jm960057p

A series of regioisomers of the novel hypoxia-selective cytotoxin (HSC) 5- [N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (2a) have been prepared by displacement of the chloro group from methyl chlorodinitrobenzoates or the corresponding carboxamides

Full text of DOI:10.1021/jm960057p

2518
J . Med. Chem. 1996, 39, 2518-2528
Hyp oxia -Selective An titu m or Agen ts. 14. Syn th esis a n d Hyp oxic Cell
Cytotoxicity of Regioisom er s of th e Hyp oxia -Selective Cytotoxin
5-[N,N-Bis(2-ch lor oeth yl)a m in o]-2,4-d in itr oben za m id e
Brian D. Palmer,† William R. Wilson,‡ Robert F. Anderson,§ Maruta Boyd,† and William A. Denny*,†
Cancer Research Laboratory, Department of Pathology, and Department of Chemistry, The University of Auckland School of
Medicine, Private Bag 92019, Auckland, New Zealand
Received J anuary 17, 1996X
A series of regioisomers of the novel hypoxia-selective cytotoxin (HSC) 5-[N,N-bis(2-chloroethyl)-
amino]-2,4-dinitrobenzamide (2a ) have been prepared by displacement of the chloro group from
methyl chlorodinitrobenzoates or the corresponding carboxamides with diethanolamine, followed
by dimesylation and mesylate displacement with LiCl. The compounds fall into two classes,
where the two nitro groups have either a meta or an ortho (or para) disposition to each other.
The four meta derivatives had one-electron reduction potentials in the range -340 to -375
mV, similar to that of the known isomer 2a , while the other isomers had much higher values
(-262 to -285 mV). The meta derivatives were much less cytotoxic to AA8 cells under aerobic
conditions (IC50s from 75 to 470 µM) than were the other compounds (IC50s from 1.6 to 20 µM).
However, the ratios of IC50s of the compounds in repair-proficient (AA8) and repair-deficient
(UV4) cell lines varied, indicating differing contributions of DNA alkylation to aerobic toxicity
between the isomers, with no clear relationship between this and nitro group disposition. The
hypoxic selectivities of the (dimethylamino)ethylcarboxamide analogues for each isomer were
determined by clonogenic assay against both AA8 and UV4 cells. With one exception, the meta
derivatives showed excellent hypoxic selectivities (ca. 45-115-fold) against UV4 cells, while
the ortho or para isomers had little selectivity (ca. 2-7-fold). A possible reason may be that
the latter compounds, with higher reduction potentials, undergo rapid bioreduction even under
aerobic conditions. None showed hypoxic selectivities greater than 2-3-fold against AA8 cells.
The 3-[N,N-bis(2-chloroethyl)amino]-2,6-dinitrobenzamide isomer (5b), which showed the
highest hypoxic selectivity for UV4 cells in this series, was active against both hypoxic and
aerobic cells in KHT tumors in mice at well-tolerated doses, and showed superior in vivo activity
to the previously studied 2,4-dinitro isomer 2b.
In tr od u ction
ents on the aromatic ring.8,9 An example of this
approach to HSCs is the nitro-deactivated mustard
5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (2a;
SN 23862). We have shown10 that 2a undergoes ef-
ficient cellular bioreduction of the 4-nitro function to an
amino group under hypoxia, thus activating the mus-
tard which is in a resonant position by electron release.
While 2a has an IC50 of 1600 µM against AA8 cells in
culture (4 h exposure), the 4-amino derivative 45 has
an IC50 under similar conditions of 180 µM. The
hydroxylamino derivative (44) can be prepared by
radiolytic or chemical reduction of 2a , but its instability
precluded an exact determination of its cytotoxicity.10
We have also shown7 that 2a is an effective HSC in
culture, being 60-fold more potent against anoxic than
aerobic Chinese hamster UV4 cells. Unlike its aziridi-
nyl analogue (1; CB 1954),11 2a is not a substrate for
the two-electron reductase DT-diaphorase,12 which can
activate nitroaromatics in an oxygen-insensitive man-
ner.
Bioreductive drugs which can undergo selective meta-
bolic reduction in the absence of oxygen to generate
cytotoxic agents (hypoxia-selective cytotoxins, HSCs) are
of particular interest as potential anticancer drugs
because of increasing evidence of the existence of
severely hypoxic cells in human solid tumors.1-4 Al-
though examples of three classes of bioreductive drug
(nitro compounds, quinones and aromatic N-oxides) are
currently in clinical trial as hypoxia-selective cytotoxins,
there are still relatively few examples of such com-
pounds with activity against hypoxic cells in murine
tumors, and the available examples appear to have
limited therapeutic ratios. One possible improvement
of this general concept is to have bioreductive drugs on
activation release cytotoxins with sufficiently long half-
lives that they can diffuse several cell diameters and
thereby kill relatively well-oxygenated cells surrounding
chronically hypoxic regions.5
We have suggested5-7 that activated aromatic nitro-
gen mustards possess the required stability for such
diffusion and that appropriately deactivated bioreduc-
tive prodrug forms of these can be made, since the
cytotoxicity of aromatic nitrogen mustards is very
dependent on the electronic properties of the substitu-
† Cancer Research Laboratory.
‡ Department of Pathology.
§ Department of Chemistry.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(96)00057-X CCC: $12.00 © 1996 American Chemical Society

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2519
Ta ble 1. Structural and Physicochemical Properties of Dinitrocarboxamide Mustards
no.
type
R
mp (°C)
ref 11
ref 11
ref 11
123-124
115-118
ncc
113-116
145-148
nc
141-141.5
180-182
nc
153
90 dec
nc
138
60 dec
nc
sol (mM)a
E(1) (mV)b
-371
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
A
A
A
B
B
B
C
C
C
D
D
D
E
E
E
F
CONH2
0.17
>44
11
CONH(CH2)2NMe2
CONHCH2CH(OH)CH2OH
CONH2
CONH(CH2)2NMe2
CONHCH2CH(OH)CH2OH
CONH2
CONH(CH2)2NMe2‚HCl
CONHCH2CH(OH)CH2OH
CONH2
CONH(CH2)2NMe2‚HCl
CONHCH2CH(OH)CH2OH
CONH2
CONH(CH2)2NMe2‚HCl
CONHCH2CH(OH)CH2OH
CONH2
CONH(CH2)2NMe2‚HCl
CONHCH2CH(OH)CH2OH
CONH2
0.16
0.32
6.25
0.14
29.7
0.91
0.19
>45
12
0.08
>48
10
0.16
>47
1.40
0.04
45
-341
-375
-343
-263
-268
-286
F
F
G
G
G
193-195
88-92
nc
CONH(CH2)2NMe2‚HCl
CONHCH2CH(OH)CH2OH
7.9
a
b
Solubility (mM) in water at 20 °C, measured spectrophotometrically. One-electron reduction potential (mV), measured by pulse
radiolysis in aqueous solutions containing 2-propanol (0.1 M) buffered at pH 7.0 (1 mM phosphate), by measuring the equilibrium constant
for electron transfer between the radical anions of the compounds and benzyl viologen (see ref 18). Standard error is ( 10 mV in each
case. c Noncrystalline.
Studies with analogues of 2a have shown12 that the
use of alternate mustard leaving groups, and modifica-
tion or substitution of the carboxamide moiety, can be
used to influence potency, in vitro hypoxic selectivity,
and solubility. With regard to these properties, the best
compound in the series overall was the (dimethylamino)-
ethyl derivative 2b, which DNA elution studies show
is activated under hypoxia to a DNA cross-linking
agent.12 This analogue has some activity in vivo against
both aerobic and hypoxic cells in KHT tumors,12 al-
though it is not yet clear whether this reflects efficient
killing of aerobic cells as a result of diffusion of reduced
metabolites from hypoxic regions, or whether its cyto-
toxicity in tumors is independent of hypoxia. The
dinitrobenzamide mustards are also of interest as pro-
drugs for ADEPT (antibody-directed enzyme prodrug
therapy),13 using a nitroreductase (NR2) from Escheri-
chia coli B14 as the enzyme. Both 2a and 2c (but not
the cationic (dimethylamino)ethyl derivative 2b) are
efficiently activated by this enzyme,15 with the more
than 10-fold better solubility of 2c giving it an advan-
tage in this application.
Ch em istr y
The dinitrobenzamide mustards of Table 1 were
prepared from the corresponding chloro dinitro acids or
esters, the majority of which were known compounds.
The chloro groups of the amides 11-13 of 2-chloro-3,5-
dinitrobenzoic acid (9) were sufficiently reactive to
undergo efficient displacement by bis(2-chloroethyl)-
amine at 50 °C, giving the required compounds 3a -3c
directly (Scheme 1). The carboxamide mustards 4a -
4c, 5a -5c, 6a -6c, and 7a -7c were obtained from the
corresponding methyl chlorodinitrobenzoates (14, 22,
28, and 33, respectively) by displacement of the chlo-
rides by diethanolamine at 20-50 °C to give the diols
(15, 23, 29, and 34), followed by dimesylation and
mesylate displacement with LiCl in DMF at 100-120
°C (Schemes 2-5). The resulting mustard esters (16,
24, 30, and 35) were cleanly hydrolyzed to the corre-
sponding acids (17, 26, 31, and 36) by 3 N KOH in
p-dioxane at room temperature. These were converted
to the acid chlorides using SOCl2, followed by reaction
of these with the appropriate amines to give the desired
compounds.
Following these results, and the structure-activity
relationship (SAR) studies12,15 which indicated the
importance of the dinitrobenzamide motif, we now
report the synthesis and biological evaluation of a series
of six regioisomers of 2a . Because of the positive results
obtained previously with the soluble cationic and diol
side chain analogues 2b and 2c, as discussed above,
these analogues were prepared in addition to the parent
benzamides.
The hitherto unreported 3-chloro-2,6-dinitrobenzoic
acid (21) was obtained as a minor product from nitration
of 3-chlorobenzoic acid. Thus while nitration of 3-chloro-
benzoic acid (19) with fuming HNO3/concentrated H2SO4
at 120 °C gave predominantly the known 5-chloro-2,4-
dinitro acid16,17 20 in high yield, reaction at 140 °C gave
a mixture of 20 together with ca. 30% of the isomeric
3-chloro-2,6-dinitrobenzoic acid (21) (Scheme 3). The
mixture could be readily separated by fractional

2520 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Palmer et al.
Sch em e 1a
Sch em e 3a
a
(i) SOCl2/reflux/5 h; (ii) concentrated NH4OH/20 °C; (iii)
H2N(CH2)2NMe2/0 °C/15 min; (iv) H2NCH2CH(OH)CH2OH/0 °C/
15 min; (v) HN(CH2CH2Cl)2‚HCl/Et3N/p-dioxane/50 °C/18 h.
Sch em e 2a
a
(i) Concentrated H2SO4/fuming HNO3 (d 1.42)/140 °C/6 h; (ii)
MeOH/concentrated H2SO4/reflux/8 h; (iii) NH(CH2CH2OH)2/p-
dioxane/50 °C/5 h; (iv) MsCl/Et3N/CH2Cl2/0 °C/15 min, then LiCl/
DMF/120 °C/5 min; (v) KOH/p-dioxane/H2O/20 °C/18 h; (vi) SOCl2/
reflux/5 h; (vii) RNH2.
Sch em e 4a
a
(i) NH(CH2CH2OH)2/p-dioxane/50 °C/5 h; (ii) MsCl/Et3N/
CH2Cl2/0 °C/15 min, then LiCl/DMF/120 °C/5 min; (iii) KOH/p-
dioxane/H2O/20 °C/3 h; (iv) SOCl2/reflux/5 h; (v) RNH2.
crystallization, providing the acid 21 in 9% yield. The
regiochemistry of the nitro groups in 21 was confirmed
by X-ray crystallographic analysis of a derivative, ethyl
3-[N,N-bis(2-chloroethyl)amino]-2,6-dinitrobenzoate (25)
(see Supporting Information).
In the case of the 4,5-dinitro carboxamides 8a -c,
reaction of methyl 2-chloro-4,5-dinitrobenzoate with
diethanolamine resulted in preferential displacement of
the 5-nitro group. However, a similar reaction on the
primary carboxamide (40) gave a mixture resulting from
displacement of both nitro groups and chloride, from
which the desired 41 was isolated in 65% yield (Scheme
6). The derived mustard 8a was hydrolyzed slowly but
cleanly by 90% H2SO4 at 70 °C to give the acid 42, from
which the carboxamides 8b and 8c were obtained as
above, via the acid chloride 43.
a
(i) NH(CH2CH2OH)2/p-dioxane/20 °C/48 h; (ii) MsCl/Et3N/
CH2Cl2/0 °C/15 min, then LiCl/DMF/100 °C/15 min; (iii) KOH/p-
dioxane/MeOH/H2O/20 °C/30 min; (iv) SOCl2/1,2-dichloroethane/
reflux/6 h; (v) RNH2.
para relationship between the mustard and nitro group,
this study was restricted to isomers having at least one
of the nitro groups in such a position. Seven of these
proved to be synthetically accessible and were exam-
ined, including all three where both nitro groups are
ortho or para to the mustard. The structures of the
compounds and their physicochemical properties are
given in Table 1. The solubility of the compounds in
water were determined by spectrophotometry. Solubili-
ties in tissue culture medium (RMEM) containing 5%
fetal calf serum were also measured and were similar
to those in water in most cases. The exceptions were
some of the cationic derivatives, which were less soluble
in the chloride ion-containing culture medium as the
Resu lts a n d Discu ssion
Because both theoretical6 and experimental9 studies
suggested that the highest differential electron release
(and thus the largest hypoxic selectivities) in mononitro
aromatic mustards occurs when there is an ortho or

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2521
Sch em e 5a
concentrations, and are presented in Table 1 (see
Supporting Information for additional details). The
main determinant of E(1) appeared to be the relative
disposition of the two nitro groups. The four derivatives
where these are meta to each other (2b-5b) had E(1)
values in the range -340 to -375 mV, while those
compounds with ortho- or para-disposed nitro groups
(6b-8b) had much higher values (-260 to -285 mV).
Beyond this, the relative disposition of the other groups
on the ring seemed to have a much lesser effect.
The aerobic cytotoxicities of the compounds were
determined (as IC50 values) in two Chinese hamster
lines (AA8 and UV4) and the murine mammary carci-
noma EMT6, using a growth inhibition microassay.19
The UV4 cell line is a repair-defective mutant of AA8
which is hypersensitive to alkylating agents whose
cytotoxicity is due to bulky DNA adducts or cross-
links.20 IC50 values for the compounds following 18 h
exposures in air are given in Table 2. Within each
series, the parent benzamides and the (dimethylamino)-
ethyl derivatives had broadly similar IC50 values, with
the diol derivatives generally being significantly less
cytotoxic. Between series, the compounds fell into two
well-defined classes. For the parent benzamides, those
compounds (2a -5a ) with meta-disposed nitro groups
(and lower reduction potentials) were less cytotoxic
(IC50s from 73 to 470 µM), while those (6a -8a ) with
ortho/para-disposed nitro groups (and higher reduction
potentials) were much more potent (IC50s from 1.6 to
20 µM). However, this pattern did not hold for the
ratios of their aerobic cytotoxicities in the repair-
proficient and -deficient cell lines, with cationic deriva-
tives from both classes (compounds 3b and 6b) showing
the highest hypersensitivity factors [HF ) IC50(AA8)/
IC50(UV4)] of about 13-fold. Overall, there were no
obvious relationships between HF values and either the
structures of the compounds or their absolute aerobic
cytotoxicities.
a
(i) NH(CH2CH2OH)2/p-dioxane/20 °C/72 h; (ii) MsCl/pyridine/
20 °C/10 min, then LiCl/DMF/100 °C/15 min; (iii) KOH/MeOH/
H2O/20 °C/1 h; (iv) SOCl2/1,2-dichloroethane/reflux/5 h; (v) RNH2.
Sch em e 6a
a
(i) SOCl2/1,2-dichloroethane/reflux/24 h/N2; (ii) concentrated
NH4OH/Et2O/0 °C/15 min; (iii) NH(CH2CH2OH)2/p-dioxane/20 °C/
18 h; (iv) MsCl/pyridine/20 °C/15 min, then LiCl/DMF/130 °C/15
min; (v) 90% H2SO4/70 °C/5 days; (vi) SOCl2/1,2-dichloroethane/
reflux/6 h; (vii) RNH2.
The hypoxic selectivities of the (dimethylamino)ethyl
derivatives were determined by clonogenic assay of
stirred plateau-phase cultures of both AA8 and UV4
cells, continuously gassed with 5% CO2 in air or N2, as
described previously.21-23 The cytotoxic potency in this
assay was determined as C10, the concentration of drug
required for 10% cell survival after a 1 h exposure.
Against the more sensitive UV4 line, the compounds fell
into the same two groups as in the growth inhibition
assay, with the m-dinitro derivatives having much
higher aerobic C10s and high hypoxic selectivities (45-
115 fold), with the exception of 4b, which was nonselec-
tive for unknown reasons. In contrast, the ortho/ para
isomers had much higher aerobic potencies but lower
selectivities (2-7-fold). Illustrative data are shown in
Figure 1 for one m-dinitro compound of high selectivity
(5b) and one o-dinitro compound of low selectivity (7b).
The hypoxic selectivities of both were much lower in the
repair-competent AA8 line, with 7b being slightly more
toxic under aerobic than hypoxic conditions (Figure 1).
The weaker hypoxic selectivity in AA8 cells was a
general finding (Table 2). Although there was no clear
correlation between the hypoxic selectivity in the two
cell lines, all compounds giving an air/N2 ratio of <5 in
the UV4 line were devoid of hypoxic selectivity in AA8
cells.
hydrochloride salts, presumably due to common ion
effects (e.g., 4b; 29.7 mM in water, 1.8 mM in culture
medium). The parent compound 2a has only moderate
solubility in water (0.17 mM), and most of the corre-
sponding isomers showed very similar values, with two
(6a and 8a ) being even less soluble. The cationic
(dimethylamino)ethyl derivatives were much more
soluble than the parent compounds, with the exception
of 3b. The carboxamide diols generally had intermedi-
ate solubilities, but showed large and unpredictable
variations between isomers (from 0.9 mM for 4c to 12
mM for 5c).
One-electron reduction potentials [E(1)] for the more
soluble (dimethylamino)ethyl derivatives of each regio-
isomer were measured by pulse radiolysis, using a
modified Dynaray 4 (4 MeV) linear accelerator (200 ns
pulse length and a custom-built optical radical detection
system). Potentials were determined in aqueous solu-
tions containing 2-propanol (0.1 M) buffered at pH 7.0
(1 mM phosphate) by measuring the equilibrium con-
stant18 for electron transfer between the radical anions
of the compounds and benzyl viologen as reference
standard. Data were obtained at three or more different

2522 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Palmer et al.
Ta ble 2. Biological Activities of the Dinitro Carboxamide Mustards of Table 1
aerobic growth inhibition
Clonogenic assay
hypoxic C10 (µM)c
hypersensitivity factorb
air/N2 ratiod
a
no.
IC50 (µM) AA8
UV4
EMT6
AA8
UV4
AA8
UV4
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
470 ( 54
220 ( 33
1930 ( 140
73 ( 27
150 ( 8
500 ( 66
97 ( 16
62 ( 14
320 ( 70
>244g
6.4 ( 0.3
3.1 ( 0.6
3.4 ( 0.7
4.3 ( 0.5
13 ( 1
4.9 ( 1.0
1.5 ( 0.1
1.8 ( 0.1
0.9 ( 0.1
>2.6g
2.7 ( 0.3
2.7 ( 0.1
2.8 ( 0.1
13 ( 2
12 ( 0.4
1.0 ( 0.1
1.0 ( 0.2
2.3 ( 0.1
0.88 ( 0.05
1.4
1.1
3400e
400
42e
44
6.7 ( 4.0
2.3 ( 0.3
0.7
2.5 ( 0.7
1.8 ( 0.2
1.3 ( 0.3
2.1 ( 0.4
2.3 ( 1.0
1300
3.1
3600e
8.6e
>1500f
270
1100f
250
2200
12
>2.35f
0.96
2.2
68f
1.04
426 ( 27
652 ( 13
11 ( 1
3.0 ( 0.2
1.7
2200
120
116
1.9 ( 0.3
5.8 ( 1.5
3.5 ( 1.3
3.1 ( 1.7
1.2
3.0 ( 0.15
0.43 ( 0.05
3.0
12 ( 3
3.2
7.5
4.0
1.8
81 ( 26
20 ( 4
13 ( 5
140
160
18
0.82
0.98
108 ( 13
1.6 ( 0.2
4.0 ( 0.1
73 ( 2
44
1.5 ( 0.1
1.9
a
IC50 determined against aerobic AA8 cells as described in the text, using an exposure time of 18 h. Values are means ( SEM.
b
Values without SEM are for a single determination only. Hypersensitivity factor ) IC50 (AA8)/IC50 (named cell line). Values are
c
intraexperiment means ( SEM. C10: the drug concentration (µM) required to reduce cell survival to 10% of controls under hypoxic
conditions, using the indicated cell line at 106/mL in the clonogenic assay (see text). Ratio of C10 values in air and N2 [C10 (air)/C10
d
(nitrogen)]. e Estimated from data in ref 12. f Inactive using 1 h exposure at the solubility limit. The estimates of C10 are from experiments
using up to 8 h exposure; equivalent C10 values (for 1 h exposure) were calculated assuming that concentration × time for 10% survival
g
is constant. Inactive against AA8 cells at the solubility limit.
The major factor determining differences in hypoxic
selectivity between the isomeric dinitrobenzamides ap-
pears to be reduction potential, rather than the genera-
tion of species of different absolute cytotoxicity following
reduction. Compounds in the first group, with meta-
disposed nitro groups and relatively low reduction
potentials, presumably undergo less reductive metabo-
lism under aerobic conditions, leading to relatively low
aerobic cytotoxicities and high hypoxic selectivities. The
higher potencies and lower selectivities of the isomers
in the second group, with ortho- or para-disposed nitro
groups and higher reduction potentials, are then likely
to be due to more extensive reduction, even under
aerobic conditions.
The cationic derivative 2b was previously evaluated
against the KHT tumor in vivo in conjunction with
irradiation, using an excision assay.12 This compound
caused substantial tumor cell killing, but gave signifi-
cant effects only at doses above the maximum tolerated
F igu r e 1. Survival curves for 1 h exposure of early plateau-
dose (MTD) for long-term survival of the host. It proved
phase AA8 and UV4 cells to compounds 5b and 7b under
equally proficient at killing cells in the absence of
radiation, indicating activity against oxic as well as
hypoxic cells. Of the isomers studied here, 5b showed
the highest level of hypoxic selectivity against UV4 cells
in vitro and was therefore evaluated against the KHT
tumor in vivo. The MTD of 5b was 450 µmol/kg (cf. 240
for 2b), consistent with the lower toxicity of 5b against
aerobic cells in culture (Table 2). When 5b was given
either before or after radiation (15 Gy) at a dose
corresponding to 75% of the MTD, significant additional
cell killing was observed (Figure 2). Activity was also
seen without radiation (Figure 2), indicating that this
drug (like 2b at supratoxic doses) kills both aerobic and
hypoxic tumor cells. At an equivalent fraction of the
MTD, compound 2b showed only marginal activity
either with or without radiation (Figure 2). The supe-
aerobic (open symbols) or hypoxic (filled symbols) conditions.
Different symbol shapes refer to separate experiments.
The in vitro biological data can be rationalized by
proposing at least two mechanisms of toxicity in the
dinitrobenzamide mustards. One, due to net nitro-
reduction (which is suppressed in aerobic cells), forms
activated nitrogen mustards which are responsible for
hypoxia-selective toxicity. The greater hypoxic selectiv-
ity in UV4 cells then reflects failure to repair the
resulting DNA alkylation lesions. The second mecha-
nism of toxicity is unrelated to DNA alkylation and is
presumably due to redox cycling of the prodrug. This
mechanism is relatively important in cells resistant to
alkylating agents, such as AA8, and is enhanced in
oxygenated cells, thereby counteracting the ability of
oxygen to inhibit cytotoxicity.

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2523
In the parent series, where studies with 2a have
shown10 that metabolism in AA8 cells occurs exclusively
at the 4-nitro group ortho to the mustard, the resulting
4-amino (and presumed 4-hydroxylamino) metabolites
undergo quite rapid intramolecular cyclization (t1/2 ca.
30 min at 37 °C for the amine, more rapidly for the
hydroxylamine) to the much less cytotoxic monofunc-
tional tetrahydroquinoxalines.10 It is possible that this
rapid deactivation detracts from the cytotoxicity of the
reduction products. The only compound clearly superior
to 2b in the UV4 clonogenic assay was the 3-[N,N-bis-
(2-chloroethyl)amino]-2,6-dinitrobenzamide (5b), which
also possesses a nitro group ortho to the mustard, but
nothing is yet known about the hypoxic metabolism of
this regioisomer. Compound 5b showed activity supe-
rior to 2b against both the hypoxic and aerobic sub-
fraction of cells in KHT tumors in vivo when the two
compounds were compared at equivalent toxicity. How-
ever, it is not yet clear whether this activity is due to
hypoxia-selective bioreductive activation in tumors.
F igu r e 2. Activity of compounds 2b and 5b against the KHT
tumor as determined by excision and clonogenic assay 18 h
after treatment. Open symbols: unirradiated. Filled sym-
bols: 15 Gy whole-body γ irradiation. Circles: no drug treat-
ment. Triangles: ip drug at 180 (2b) or 338 (5b) µmol/kg. The
drug doses correspond to 75% of the MTD (as determined in
non-tumor-bearing mice using a 28-day observation period).
Each point represents two or three pooled tumors from
separate mice. Multiple points at each dose are from separate
experiments. The horizontal lines show the 95% confidence
intervals for untreated tumors (top band) and tumors treated
with radiation only at 15 Gy (lower band) based on historical
data.
Exp er im en ta l Section
Ch em istr y. Analyses were carried out in the Microchemi-
cal Laboratory, University of Otago, Dunedin, NZ. Melting
points were determined on an Electrothermal 9200 melting
point apparatus. NMR spectra were obtained on Bruker AC-
200 or AM-400 spectrometers, and are referenced to Me4Si for
organic solutions and 3-(trimethylsilyl)propanesulfonic acid,
sodium salt for D2O solutions. Thin-layer chromatography was
carried out on aluminum-backed silica gel plates (Merck 60
F254). Flash column chromatography was carried out on Merck
silica gel (230-400 mesh). Petroleum ether refers to the
fraction boiling at 40-60 °C. Benzamides containing the 2,3-
dihydroxypropyl side chain were all viscous oils or foams which
retained trace amounts of EtOAc tenaciously, making suc-
cessful combustion analysis difficult. Satisfactory high-resolu-
tion mass spectral data were obtained for these compounds
using desorption electron impact ionization at 70 eV, or
chemical ionization using NH3 as carrier gas. All final
products were judged to be >98% pure by reverse-phase HPLC
analysis with diode array detection.
riority of 5b over 2b was confirmed in dose-response
experiments in which the drugs were administered 5
min after irradiation (data not shown).
Con clu sion s
The 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenz-
amides (2a -c) have confirmed activities as hypoxia-
selective cytotoxins7,12 and as prodrugs for ADEPT.15
Because SAR studies12 suggested that significant fur-
ther improvements in these properties were not likely
to be achieved by alteration of side chains, we have
studied six additional regioisomers of these dinitrobenz-
amide mustards, including the two further isomers
where both nitro groups are in resonant (ortho or para)
positions with respect to the mustard. The results show
that the reduction potentials for addition of the first
electron are dominated by the positioning of the nitro
groups with respect to each other; those isomers with
ortho- or para-disposed nitro groups have higher reduc-
tion potentials than those where the two nitro groups
are meta-positioned. The compounds fall into the same
broad groups with respect to biological activity, with the
former showing higher absolute cytotoxicities but lower
hypoxic selectivities. These results are consistent with
the compounds of higher reduction potential undergoing
more rapid bioactivation by reduction under aerobic
conditions.
The results suggest that a necessary (but not suf-
ficient, given the data for 4) requirement for significant
hypoxic selectivity in the UV4 assay for the dinitro-
benzamide mustard derivatives is positioning the two
nitro groups meta to each other. The importance (if any)
of the positioning of the nitro groups with respect to the
mustard is less clear. However, the compounds showed
much lower hypoxic selectivity against repair-competent
AA8 cells, with no clear superiority for the meta
analogues.
2-[N ,N -B is (2-c h lo r o e t h y l)a m in o ]-3,5-d in it r o b e n z-
a m id e (3a ). 2-Chloro-3,5-dinitrobenzoic acid (9) (10.0 g of
75%, 0.03 mol) was treated with SOCl2 (10 mL) containing 1
drop of DMF under reflux for 5 h. Evaporation of reagent
followed by azeotroping with benzene gave the crude acid
chloride 10, which was dissolved in Me2CO (30 mL) and
treated with concentrated NH4OH (10 mL) to give a quantita-
tive yield of 2-chloro-3,5-dinitrobenzamide (11). A solution of
11 (1.00 g, 4.07 mmol) and Et3N (1.42 g, 10 mmol) in p-dioxane
(30 mL) was treated with N,N-bis(2-chloroethyl)amine hydro-
chloride (1.45 g, 8.14 mmol) at 50 °C for 18 h. The mixture
was poured into water and extracted with EtOAc to give an
oil, which was chromatographed on silica gel. Elution with
EtOAc/petroleum ether (1:1) gave 2-[N,N-bis(2-chloroethyl)-
amino]-3,5-dinitrobenzamide (3a ) (1.15 g, 80%): mp (CHCl3/
1
petroleum ether) 123-124 °C; H NMR [(CD3)2SO] δ 8.70 (d,
J ) 2.7 Hz, 1 H, H-4), 8.49 (d, J ) 2.7 Hz, 1 H, H-6), 7.68,
7.35 (2 × br, 2H, CONH2), 3.80, 3.61 (2 × t, J ) 6.9 Hz, 8 H,
NCH2CH2Cl); 13C NMR δ 167.94 (CONH2), 147.27 (C-2),
147.20, 142.84 (C-3,5), 137.70 (C-1), 128.40, 123.24 (C-4,6),
55.84 (CH2N), 42.28 (CH2Cl). Anal. (C11H12Cl2N4O5) C, H, N,
Cl.
Treatment of the crude acid chloride 10 (prepared as above
from 10.0 g of acid 9 of 75% purity, 0.03 mol) in Et2O (80 mL)
with a solution of N,N-dimethylethylenediamine (4.9 mL, 0.06
mol) in water (15 mL) at 0 °C for 15 min, followed by workup
in EtOAc, gave an oil which was chromatographed on silica
gel. Elution with EtOAc/MeOH (9:1) gave N-[2-(N,N-dimethyl-
amino)ethyl]-2-chloro-3,5-dinitrobenzamide (12) as a viscous
oil (5.6 g, 59%). The hydrochloride salt crystallised from
MeOH/isopropyl ether: mp 220-222 °C; 1H NMR (D2O) δ 8.99

2524 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Palmer et al.
(d, J ) 2.3 Hz, 1 H, ArH-4), 8.74 (d, J ) 2.3 Hz, 1 H, H-6),
3.89 (t, J ) 6.3 Hz, 2 H, CH2N+Me2), 3.51 (t, J ) 6.3 Hz, 2 H,
CONHCH2), 3.04 (s, 6 H, N+Me2); 13C NMR δ 169.32 (CONH),
150.88 (C-2), 148.70, 141,02 (C-3,5), 133.23 (C-1), 129.45,
125.27 (C-4,6), 58.64 (CH2N+Me2), 45.88 (N+Me2), 37.94 (CON-
HCH2). Anal. (C11H13ClN4O5‚HCl) C, H, N, Cl. Reaction of
12 with bis(2-chloroethyl)amine hydrochloride and Et3N as
above, followed by chromatography on silica gel and elution
with EtOAc/MeOH (9:1), gave N-[2-(N,N-dimethylamino)-
ethyl]-2-[N,N-bis(2-chloroethyl)amino]-3,5-dinitrobenzamide (3b)
(56% yield): mp (EtOAc/petroleum ether) 115-118 °C; 1H
NMR (CDCl3) δ 8.63 (s, 2 H, H-4,6), 7.41 (br, 1 H, CONH),
3.74 (t, J ) 6.3 Hz, 4 H, CH2Cl), 3.59 (t, J ) 5.8 Hz, 2 H,
CONHCH2), 3.55 (t, J ) 6.3 Hz, 4 H, NCH2CH2Cl), 2.56 (t, J
) 5.8 Hz, 2 H, CH2NMe2), 2.27 (s, 6 H, NMe2); 13C NMR δ
164.70 (CO), 145.93 (C-2), 144.86, 141.94 (C-3,5), 135.68 (C-
1), 129.63 (C-4), 123.16 (C-6), 57.51 (CONHCH2), 54.82
(NCH2CH2Cl), 45.12 (NMe2), 41.39 (NCH2CH2Cl), 37.67 (CON-
HCH2). Anal. (C15H21Cl2N5O5) C, H, N, Cl. The hydrochloride
salt was a hygroscopic foam.
Treatment of the crude acid chloride 10 in Me2CO with a
solution of 3-aminopropane-1,2-diol (2 equiv) in water, as
above, followed by chromatography of the product on silica gel
and elution with EtOAc, gave N-(2,3-dihydroxypropyl)-2-
chloro-3,5-dinitrobenzamide (13) (40% yield) as a colorless
gum: 1H NMR [(CD3)2SO] δ 8.97 (d, J ) 2.6 Hz, 1 H, H-4),
8.80 (t, J ) 5.6 Hz, 1 H, CONH), 8.55 (d, J ) 2.6 Hz, 1 H,
H-6), 4.00 (br s, 2 H, OH), 3.70-3.63 (m, 1 H, CHOH), 3.50-
3.42 (m, 1 H, CONHCHH), 3.41-3.37 (m, 2 H, CH2OH), 3.22-
3.15 (m, 1 H, CONHCHH); 13C NMR δ 163.19 (CONH), 148.48,
145.89, 140.44 128.49 (4s), 126.10 (C-4), 120.55 (C-6), 70.01
(CHOH), 63.79 (CH2OH), 42.93 (CONHCH2); CIMS found [M
+ H]+ 322.0253, 320.0274; C10H11Cl2N3O7 requires 322.0256,
320.0285.
Reaction of 13 with bis(2-chloroethyl)amine hydrochloride
and Et3N as above, followed by chromatography on silica gel
and elution with EtOAc, gave N-(2,3-dihydroxypropyl)-2-[N,N-
bis(2-chloroethyl)amino]-3,5-dinitrobenzamide (3c) as a hy-
groscopic, yellow foam (65% yield): 1H NMR [(CD3)2SO] δ 8.73
(d, J ) 2.8 Hz, 1 H, H-4), 8.70 (t, J ) 5.5 Hz, 1 H, CONH),
8.35 (d, J ) 2.8 Hz, 1 H, H-6), 4.91, 4.65 (2 × br s, 2 H, OH),
3.72 (t, J ) 7.5 Hz, 4 H, CH2Cl), 3.66 (br s, 1 H, CHOH), 3.43
(t, J ) 7.5 Hz, 4 H, CH2N), 3.41-3.31 (m, 3 H, CONHCHH
and CH2OH), 3.21-3.12 (m, 1 H, CONHCHH); 13C NMR δ
165.41 (CONH), 145.79, 145.30, 140.92, 136.34 (4s), 127.54 (C-
4), 122.11 (C-6), 69.86 (CHOH), 63.88 (CH2OH), 54.07 (CH2N),
42.98 (CONHCH2), 41.52 (CH2Cl). CIMS found [M + H]+
429.0587, 427.0612, 425.0637; C14H19Cl2N4O7 requires 429.0572,
427.0601, 425.0631.
NMR δ 162.90 (COOCH3), 147.83 (C-4), 140.53 (C-3,5), 129.85
(C-2,6), 126.70 (C-1), 55.47 (CH2N), 53.23 (OCH3), 41.18
(CH2Cl). Anal. (C12H13Cl2N3O6) C, H, N, Cl. Hydrolysis of
16 with excess 3 N aqueous KOH in p-dioxane at 20 °C for 3
h gave a quantitative yield of 4-[N,N-bis(2-chloroethyl)amino]-
3,5-dinitrobenzoic acid (17): mp (EtOAc/petroleum ether) 164-
166 °C; 1H NMR [(CD3)2SO] δ 8.61 (s, 2 H, H-2,6), 3.78 (t, J )
6.9 Hz, 4 H, CH2Cl), 3.52 (t, J ) 6.9 Hz, 4 H, CH2N) (COOH
not visible); 13C NMR δ 164.12 (COOH), 149.12 (C-4), 141.40
(C-3,5), 130.70 (C-2,6), 128.19 (C-1), 56.14 (CH2N), 42.46
(CH2Cl). Anal (C11H11Cl2N3O6) C, H, N, Cl.
A solution of 17 (2.00 g, 5.46 mmol) in 1,2-dichloroethane
(40 mL) containing DMF (1 drop) was treated with SOCl2 (10
mL) as above to give the crude acid chloride 18, which was
dissolved in dry Et2O (100 mL), cooled to 5 °C, and treated
dropwise with concentrated NH4OH (20 mL). After 10 min
the solution was worked up and the residue was chromato-
graphed on silica. Elution with EtOAc/petroleum ether (3:7)
gave foreruns, while EtOAc/petroleum ether (7:3) gave 4-[N,N-
bis(2-chloroethyl)amino]-3,5-dinitrobenzamide (4a ) (80% over-
all yield), mp (EtOAc/petroleum ether) 113-116 °C; 1H NMR
[(CD3)2CO] δ 8.61 (s, 2 H, H-2,6), 7.98, 7.24 (2 × br, 2 H,
CONH2), 3.75 (t, J ) 7.0 Hz, 4 H, CH2Cl), 3.50 (t, J ) 7.0 Hz,
4 H, CH2N); 13C NMR δ 164.91 (CONH2), 149.32 (C-4), 140.07
(C-3,5), 132.18 (C-1), 128.91 (C-2,6), 56.37 (CH2N), 42.55 (CH2-
Cl). Anal. (C11H12Cl2N4O5) C, H, N, Cl.
Similar reaction of crude 18 in CH2Cl2 with neat N,N-
dimethylethylenediamine (2 equiv) and chromatography of the
product on silica gel, eluting with EtOAc/MeOH (9:1), gave
N-[2-(N,N-dimethylamino)ethyl]-4-[N,N-bis(2-chloroethyl)-
amino]-3,5-dinitrobenzamide (4b) (62% yield) as a yellow oil.
The hydrochloride salt crystallized from MeOH/isopropyl
1
ether: mp 135-140 °C; H NMR [(CD3)2SO] δ 10.65 (br, 1 H,
HCl), 9.49 (t, J ) 5.4 Hz, 1 H, CONH), 8.75 (s, 2 H, Ar-H2,6),
3.71 (t, J ) 6.9 Hz, 4 H, NCH2CH2Cl), 3.44-3.28 (m, 8 H,
NCH2CH2Cl and CH2CH2N+Me2), 2.83 (s, 6 H, N+Me2); 13C
NMR δ 162.63 (CO), 147.43 (C-3,5), 138.75 (C-4), 130.22 (C-
1), 128.31 (C-2,6), 55.44 (CH2N+Me2), 54.75 (NCH2CH2Cl),
42.22 (N+Me2), 42.18 (NCH2CH2Cl), 34.75 (CONHCH2). Anal.
(C15H21Cl2N5O5‚HCl) C, H, N, Cl.
Similar reaction of crude 18 in Me2CO with 3-aminopro-
pane-1,2-diol (2 equiv) in water at 0 °C as above, followed by
chromatography on silica gel, eluting with EtOAc, gave N-(2,3-
dihydroxypropyl)-4-[N,N-bis(2-chloroethyl)amino]-3,5-di-
nitrobenzamide (4c) (66% yield) as a yellow oil: 1H NMR
[(CD3)2SO] δ 8.84 (t, J ) 5.5 Hz, 1 H, CONH), 8.63 (s, 2 H,
H-2,6) 4.85 (d, J ) 5.0 Hz, 1 H, CHOH), 4.59 (t, J ) 5.7 Hz, 1
H, CH2OH), 3.70 (t, J ) 6.7 Hz, 4 H, CH2Cl), 3.50-3.35 (m, 8
H, CH2N and CHHCHOHCH2OH), 3.20 (m, 1 H, CHH); 13C
NMR δ 162.41 (CONH), 147.50 (s), 138.60 (s), 130.89 (s),
128.17 (C-2,6), 70.11 (CHOH), 63.87 (CH2OH), 54.85 (CH2N),
43.33 (CONHCH2), 42.21 (CH2Cl). EIMS found M+ 428.0507,
426.0533, 424.0552; C14H18Cl2N4O7 requires 428.0493, 426.0523,
424.0552).
4-[N ,N -B is (2-c h lo r o e t h y l)a m in o ]-3,5-d in it r o b e n z-
a m id e (4a ). A solution of methyl 4-chloro-3,5-dinitrobenzoate
(14) (10.0 g, 0.036 mol) and diethanolamine (7.66 g, 0.073 mol)
in p-dioxane (60 mL) was stirred at 50 °C for 5 h. Volatiles
were removed under reduced pressure, and the residue was
adsorbed directly onto silica gel and chromatographed. Elution
with EtOAc/petroleum ether (1:10) gave foreruns, while elution
with EtOAc/petroleum ether (3:2) gave methyl 4-[N,N-bis(2-
hydroxyethyl)amino]-3,5-dinitrobenzoate (15) (9.61 g, 80%):
3-[N ,N -B is (2-c h lo r o e t h y l)a m in o ]-2,6-d in it r o b e n z-
a m id e (5a ). A solution of 3-chlorobenzoic acid (19) (60 g, 0.38
mol) in concentrated H2S04 (600 mL) was treated portionwise
with fuming nitric acid (d 1.42; 150 mL), and the solution was
warmed gradually with stirring to 140 °C in an open flask and
held at this temperature for 6 h. After cooling overnight, ice-
water (4 L) was added cautiously and the mixture was kept
at 5 °C for 3 h. The precipitated product was removed by
filtration, washed with water (4 × 200 mL), and crystallized
from aqueous EtOH to give 5-chloro-2,4-dinitrobenzoic acid
(20) (62 g, 66%): mp 180-183 °C (lit.16 mp 182-183 °C). The
original filtrate and washings were combined and allowed to
stand for several hours, depositing crystals of pure 3-chloro-
2,6-dinitrobenzoic acid (21) (8.4 g, 9%): mp (H2O) 162-163.5
1
mp (CHCl3/petroleum ether) 101-103 °C; H NMR (CDCl3) δ
8.52 (s, 2 H, ArH-2,6), 3.97 (s, 3 H, COOCH3), 3.76 (d × t, J )
6.5, 4.7 Hz, 4 H, CH2OH), 3.26 (t, J ) 4.7 Hz, 4 H, NCH2),
2.76 (t, J ) 6.5 Hz, 2 H, OH); 13C NMR δ 163.14 (COOCH3),
145.50 (C-4), 142.54 (C-3,5), 131.19 (C-2,6), 123.59 (C-1), 59.16
(CH2OH)1, 54.58 (CH2N), 53.14 (OCH3). Anal. (C12H15N3O8)
C, H, N.
MsCl (3.93 mL, 0.049 mol) was added dropwise at 0 °C to a
solution of 15 (7.30 g, 0.022 mol) and Et3N (7.72 mL, 0.055
mol) in CH2Cl2 (100 mL). After 15 min, the solution was
washed several times with water and worked up to give the
crude dimesylate, which was treated with LiCl (15.0 g) in DMF
(80 mL) at 120 °C for 5 min. Workup and chromatography on
silica gel, eluting with EtOAc/petroleum ether (1:4), gave
methyl 4-[N,N-bis(2-chloroethyl)amino]-3,5-dinitrobenzoate (16)
1
°C; H NMR [CD3)2SO] δ 10.50 (br, 1 H, COOH), 8.82 (d, J )
8.8 Hz, 1 H, H-5), 8.62 (d, J ) 8.8 Hz, 1 H, H-4); 13C NMR δ
162.03 (COOH), 146.34 (C-2), 145.57 (C-6), 133.69 (C-5), 130.01
(C-1), 128.26 (C-4), 125.19 (C-3). Anal. (C7H3ClN2O6) C, H,
N, Cl.
1
(6.21 g, 77%): mp (CHCl3/petroleum ether) 93-95 °C; NMR
The methyl ester 22, prepared from 21 using MeOH/
concentrated H2SO4 (reflux, 8 h), crystallized from MeOH as
needles: mp 110-112 °C; 1H NMR (CDCl3) δ 8.22 (d, J ) 8.9
(CDCl3) δ 8.54 (s, 2 H, H-2,6), 3.99 (s, 3 H, COOCH3), 3.63 (t,
J ) 6.9 Hz, 4 H, CH2Cl), 3.43 (t, J ) 6.9 Hz, 4 H, CH2N); 13C

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2525
Hz, 1H, H-5), 7.82 (d, J ) 8.9 Hz, 1H, H-4), 3.96 (s, 3H,
COOCH3); 13C NMR δ 161.30 (COOCH3), 145.22, 143.74 (C-
2,6), 133.20 (C-5), 132.31 (s), 127.06 (C-4), 125.28 (s), 54.61
(COOCH3). Anal. (C8H5ClN2O6) C, H, N. The ester 22 was
converted into the diol 23 with diethanolamine in p-dioxane
at 50 °C, as above (83% yield), and this was reacted with MsCl/
LiCl as above to give methyl 3-[N,N-bis(2-chloroethyl)amino]-
2,6-dinitrobenzoate (24) (67% yield): mp (EtOAc/petroleum
methyl 4-[N,N-bis(2-hydroxyethyl)amino]-2,5-dinitrobenzoate
(29) (0.75 g, 99%): mp (EtOAc/petroleum ether) 102 °C; 1H
NMR [(CD3)2SO] δ 8.25 (s, 1 H, H-6), 7.86 (s, 1 H, H-3), 4.79
(t, J ) 5.3 Hz, 2 H, OH), 3.80 (s, 3 H, OCH3), 3.56 (dt, J ) 5.5,
5.3 Hz, 4 H, CH2OH), 3.40 (t, J ) 5.5 Hz, 4 H, NCH2); 13C
NMR δ 162.13 (COOCH3), 151.86 (s), 147.54 (s), 147.54 (s),
137.68 (s), 130.10 (C-6), 115.21 (C-3), 109.15 (s), 58.14 (CH2OH),
54.15 (CH2N), 52.80 (COOCH3); mass spectrum m/ z (rel int)
329 (M+, 7), 298 (100), 282 (5), 268 (20), 254 (18), 251 (9), 222
(19). Anal. (C12H15N3O8) C, H, N.
1
ether) 118-119.5 °C; H NMR (CDCl3) δ 8.30 (d, J ) 9.1 Hz,
H-5), 8.00 (d, J ) 9.1 Hz, H-4), 3.95 (s, 3H, COOCH3), 3.88-
3.75 (m, 8H, NCH2CH2Cl); 13C NMR δ 163.10 (COOCH3),
148.21 (C-3), 140.98, 138.71 (C-2,6), 128.76, 124.58 (C-4,5),
127.34 (C-1), 54.77 (COOCH3), 54.37 (CH2N), 42.16 (CH2Cl).
Anal. (C12H13Cl2N3O6) C, H, N.
A stirred solution of 29 (14.47 g, 0.044 mol) and Et3N (15.31
mL, 0.110 mol) in CH2Cl2 (270 mL) was treated dropwise with
MsCl (7.79 mL, 0.097 mol) at 0 °C. After a further 15 min at
0 °C, the solution was washed with 1 N Na2CO3 and worked
up to give the crude dimesylate as an oil. This was dissolved
in DMF (100 mL), LiCl (20.00 g, 0.471 mol) was added, and
the mixture was stirred at 100 °C for 15 min. After removal
of the DMF under reduced pressure, the residue was parti-
tioned between EtOAc and water, and the organic portion was
worked up to give an oil which was chromatographed on silica.
Elution with EtOAc/petroleum ether (3:7) gave methyl 4-[N,N-
bis(2-chloroethyl)amino]-2,5-dinitrobenzoate (30) (15.08 g,
An identical series of reactions beginning with ethyl 3-chloro-
2,6-dinitrobenzoate gave ethyl 3-[N,N-bis(2-chloroethyl)amino]-
2,6-dinitrobenzoate (25): mp (EtOAc/petroleum ether) 84-87
1
°C; H NMR [CD3)2CO] δ 8.32 (d, J ) 9.4 Hz, 1 H, H-5), 7.79
(d, J ) 9.4 Hz, 1 H, H-4) 4.39 (q, J ) 7.1 Hz, 2 H, OCH2CH3),
3.85-3.77 (m, 8 H, NCH2CH2Cl), 1.34 (t, J ) 7.1 Hz, 3 H,
OCH2CH3); 13C NMR δ 163.24 (COOEt), 148.30 (C-3), 141.04,
138.80 (C-2,6), 128.84, 124.65 (C-4,5), 127.39 (C-1), 63.94
(OCH2CH3), 54.39 (NCH2), 42.00 (CH2Cl) 13.89 (OCH2CH3).
Anal. (C13H15Cl2N3O6) C, H, N, Cl. An X-ray crystallographic
determination was carried out on 25 (see below) for confirma-
tion of structure.
1
86%): mp (EtOAc/petroleum ether) 108 °C; H NMR (CDCl3)
δ 8.29 (s, 1 H, H-6), 7.49 (s, 1 H, H-3), 3.91 (s, 3 H, COOCH3),
3.64 (br s, 8 H, NCH2CH2Cl); 13C NMR δ 162.52 (COOCH3),
151.85 (s), 146.30 (s), 129.91 (C-6), 116.97 (C-3), 116.23 (s),
53.70 (CH2N), 53.31 (COOCH3), 40.71 (CH2Cl). Anal. (C12H13
Cl2N3O6) C, H, N, Cl.
-
Hydrolysis of 24 or 25 in excess 3 N KOH/p-dioxane at 20
°C for 18 h gave 3-[N,N-bis(2-chloroethyl)amino]-2,6-dinitro-
benzoic acid (26) which was converted to the crude acid
chloride 27 with SOCl2 as above. A solution of 27 in Et2O at
0 °C was treated with excess concentrated NH4OH as described
above, and the product was chromatographed on silica gel,
EtOAc/petroleum ether (1:1), eluting 3-[N,N-bis(2-chloroethyl)-
amino]-2,6-dinitrobenzamide (5a ) (56% overall yield): mp
A solution of 30 (13.10 g, 0.033 mol) and 2 N aqueous KOH
(60 mL, 0.12 mol) in p-dioxane/MeOH (1:1; 200 mL) was stirred
at 20 °C for 30 min, then acidified with concentrated HCl, and
extracted with EtOAc. Workup of the organic portion gave
4-[N,N-bis(2-chloroethyl)amino]-2,5-dinitrobenzoic acid (31)
(11.81 g, 93%): mp (EtOAc/petroleum ether) 188 °C; 1H NMR
[(CD3)2SO] δ 8.31 (s, 1 H, H-6), 8.00 (s, 1 H, H-3), 4.18 (br s, 1
H, COOH), 3.80 (t, J ) 5.8 Hz, 4 H, CH2Cl), 3.65 (t, J ) 5.8
Hz, 4 H, CH2N); 13C NMR δ 162.99 (COOH), 152.26 (s), 146.95
(s), 139.92 (C-6), 116.24 (C-3), 113.92 (s), 52.56 (CH2N), 41.85
(CH2Cl). Anal. (C11H11Cl2N3O6) C, H, N, Cl.
1
(EtOAc/petroleum ether) 140-141.5 °C; H NMR [(CD3)2CO]
δ 8.26 (d, J ) 9.3 Hz, 1 H, H-5), 7.71 (d, J ) 9.3 Hz, 1 H, H-4),
7.63, 7.23 (2 × br, 2 H, CONH2), 3.75 (s, 8 H, NCH2CH2Cl);
13C NMR δ 163.99 (CONH2), 147.38 (C-3), 142.92, 139.76 (C-
2,6), 130.48 (C-1), 128.22, 124.23 (C-4,5), 54.74 (NCH2), 42.00
(CH2Cl). Anal. (C11H12Cl2N4O5) C, H, N, Cl.
A mixture of 31 (2.00 g, 5.21 mmol) and SOCl2 (10.00 mL)
in 1,2-dichloroethane (150 mL) containing DMF (1 drop) was
refluxed with protection from moisture for 6 h and then
concentrated to dryness to give crude 4-[N,N-bis(2-chloroethyl)-
amino]-2,5-dinitrobenzoyl chloride (32), which was used di-
rectly. Concentrated NH4OH (4 mL) was added in one portion
to a vigorously stirred solution of 32 (2.00 g, 4.78 mmol) in
Me2CO (30 mL) at 0 °C. After 10 min, the mixture was diluted
with EtOAc, washed with 2 N NaHCO3, and worked up to give
an oil which was chromatographed on silica gel. Elution with
EtOAc/petroleum ether (2:3) gave 4-[N,N-bis(2-chloroethyl)-
amino]-2,5-dinitrobenzamide (6a ) (1.38 g, 82%): mp (EtOAc/
Reaction of the crude acid chloride 27 in CH2Cl2 with N,N-
dimethylethylenediamine (2 equiv) as described above, fol-
lowed by chromatography of the product on silica gel and
elution with EtOAc/MeOH (10:1), gave N-[2-(N,N-dimethyl-
amino)ethyl]-3-[N,N-bis(2-chloroethyl)amino]-2,6-dinitro-
benzamide (5b) as a yellow oil (58% yield). The hydrochloride
salt crystallized from MeOH/isopropyl ether: mp 180-182 °C;
1H NMR (D2O) δ 8.37 (d, J ) 9.6 Hz, 1 H, H-5), 7.63 (d, J )
9.6 Hz, 1 H, H-4), 3.80-3.71 (m, 10 H, NCH2CH2Cl and
CH2N+Me2), 3.44 (t, J ) 6.4 Hz, 2 H, CONHCH2), 3.00 (s, 6
H, N+Me2); 13C NMR δ 168.75 (CONH), 150.91 (C-3), 141.55,
138,72 (C-2,6), 131.61 (C-4), 130.45 (C-1), 125.85 (C-5), 58.31
(CH2N+Me2), 55.74 (NCH2CH2Cl), 45.97 (N+Me2), 44.16
(NCH2CH2Cl), 37.91 (CONHCH2). Anal. (C15H21Cl2N5O5‚HCl)
C, H, N, Cl.
Reaction of the crude acid chloride 27 in Me2CO with
3-aminopropane-1,2-diol (2 equiv) in water at 0 °C as described
above, followed by chromatography of the product on silica gel
and elution with EtOAc, gave N-(2,3-dihydroxypropyl)-3-N,N-
bis(2-chloroethyl)amino]-2,6-dinitrobenzamide (5c) (62% yield)
as a yellow foam: 1H NMR [(CD3)2SO] δ 8.72 (t, J ) 5.8 Hz,
1H, CONH), 8.32 (d, J ) 9.3 Hz, 1H, H-5), 7.68 (d, J ) 9.3 Hz,
1H, H-4), 4.84, 4.58 (2 × br, 2H, OH), 3.83 (t, J ) 6.0 Hz, 4H,
CH2Cl), 3.68 (t, J ) 6.0 Hz, 4H, CH2N), 3.65 (m, 1H, CHOH),
3.43 (m, 1H, CONHCHH), 3.39 (m, 2H, CH2OH), 3.14 (m, 1H,
CONHCHH); 13C NMR δ 167.71 (CONH), 147.16 (C-3), 142.17,
138.61 (C-2,6), 131.27, 130.48 (C-1,4), 125.47 (C-5), 70.09
(CHOH), 63.78 (CH2OH), 52.55 (CH2N), 42.80 (CONHCH2),
41.75 (CH2Cl); CIMS found [M + H]+ 429.0584, 427.0602,
425.0630; C14H19Cl2N4O7 requires 429.0572, 427.0601, 425.0631.
4-[N ,N -B is (2-c h lo r o e t h y l)a m in o ]-2,5-d in it r o b e n z-
a m id e (6a ). A mixture of methyl 4-chloro-2,5-dinitrobenzoate
(28)24 (0.60 g, 2.30 mmol) and diethanolamine (0.48 g, 4.60
mmol) in p-dioxane (25 mL) was stirred at room temperature
for 48 h and then adsorbed directly onto silica gel by concen-
tration and chromatographed. Elution with EtOAc gave
1
petroleum ether) 153 °C; H NMR [(CD3)2SO] δ 8.22, 7.70 (2
× br, 2 H, CONH2), 8.15 (s, 1 H, H-6), 7.97 (s, 1 H, H-3), 3.77
(t, J ) 6.0 Hz, 4 H, CH2Cl), 3.61 (t, J ) 6.0 Hz, 4 H, CH2N);
13C NMR δ 164.40 (CONH2), 151.05 (s), 145.04 (s), 140.74 (s),
127.11 (C-6), 121.17 (s), 117.63 (C-3), 52.64 (CH2N), 41.88
(CH2Cl). Anal. (C11H12Cl2N4O5) C, H, N, Cl.
Reaction of 32 in CH2Cl2 with neat N,N-dimethylethylene-
diamine (2 equiv) as above gave N-[2-(N,N-dimethylamino)-
ethyl]-4-[N,N-bis(2-chloroethyl)amino]-2,5-dinitrobenzamide (6b)
(64%) as a yellow oil. The hydrochloride salt crystallized from
MeOH/Et2O as a hygroscopic orange foam: mp 70 °C; 1H NMR
(D2O) δ 8.20, 8.12 (2 × s, 2 H, H-3,6), 3.85-3.67 (m, 10 H,
NCH2CH2Cl and CH2N+Me2), 3.45 (t, J ) 6.1 Hz, 2 H,
CONHCH2), 3.00 (s, 6H, +NMe2); 13C NMR δ 168.04 (CONH),
149.58 (s), 146.92 (s), 141.85 (s), 128.28 (d), 120.94 (s), 119.96
(d), 56.41 (CH2N+(CH3)2), 53.23 (CH2N), 43.50 (CH2N+(CH3)2),
42.05 (CH2Cl), 35.55 (CONHCH2). Anal. (C15H22Cl2N5O5) C,
H, N, Cl.
Reaction of 32 in Me2CO with 3-aminopropane-1,2-diol (2
equiv) in water as above, followed by chromatography of the
product on silica gel and elution with EtOAc, gave N-(2,3-
dihydroxypropyl)-4-[N,N-bis(2-chloroethyl)amino]-2,5-di-
nitrobenzamide (6c) (83%) as an orange gum: 1H NMR
[(CD3)2SO] δ 8.76 (t, J ) 5.5 Hz, 1 H, CONH), 8.15, 7.98 (2 ×
s, 2 H, H-3,6), 3.77 (t, J ) 5.8 Hz, 4 H, CH2Cl), 3.62 (t, J ) 5.8

2526 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Palmer et al.
Hz, 4 H, CH2N), 3.48-3.29 (m, 6 H, CH(OH)CH2OH and
CONHCH), 3.12 (m, 1 H, CONHCH); 13C NMR δ 163.10
(CONH), 150.79 (s), 144.88 (s), 141.06 (s), 127.35 (C-6), 121.66
(s), 117.74 (C-3), 70.19 (CHOH), 63.81 (CH2OH), 52.73 (CH2N),
42.96 (CONHCH2), 41.92 (CH2Cl); CIMS found [M + H]+
429.0569, 427.0595, 425.0616; C14H19Cl2N4O7 requires 429.0572,
427.0601, 425.0631.
4-[N ,N -B is (2-c h lo r o e t h y l)a m in o ]-2,3-d in it r o b e n z-
a m id e (7a ). A mixture of methyl 4-chloro-2,3-dinitroben-
zoate24 (33) (3.15 g, 0.012 mol) and diethanolamine (2.54 g,
0.024 mol) in p-dioxane (120 mL) was stirred at 20 °C for 3
days, adsorbed onto silica gel by concentration under reduced
pressure, and chromatographed by elution with MeOH/EtOAc
(1:19) to give methyl 4-[N,N-bis(2-hydroxyethyl)amino]-2,3-
dinitrobenzoate (34) (3.12 g, 78%): mp (EtOAc) 151-152 °C;
1H NMR [(CD3)2SO] δ 7.96 (d, J ) 9.3 Hz, 1 H, H-6), 7.61 (d,
J ) 9.3 Hz, 1 H, H-5), 4.81 (t, J ) 5.2 Hz, 2 H, OH), 3.81 (s,
3 H, COOCH3), 3.56 (dd, J ) 5.4, 5.2 Hz, 4 H, CH2OH), 3.38
(d, J ) 5.4 Hz, 4 H, CH2N); 13C NMR δ 161.96 (COOCH3),
147.48 (s), 145.51 (s), 132.92 (C-6), 131.30 (s), 122.34 (C-5),
109.53 (s), 58.13 (CH2OH), 53.82 (CH2N), 53.00 (COOCH3).
Anal. (C12H15N3O8) C, H, N.
) 5.1 Hz, 4 H, CH2Cl), 3.51 (t, J ) 5.1 Hz, 4 H, CH2N), 3.40
(t, J ) 6.0 Hz, 2 H, CONHCH2), 2.95 (s, 6 H, N+Me2); 13C NMR
δ 167.96 (CONH), 148.05 (s), 145.25 (s), 140.23 (s), 133.89 (C-
6), 130.34 (C-5), 125.14 (s), 58.51 (CH2N+(CH3)2), 56.71 (CH2N),
45.71 (N+(CH3)2), 44.12 (CH2Cl), 37.73 (CONHCH2). Anal.
(C15H21Cl2N5O5‚HCl) C, H, N, Cl.
Reaction of 37 in Me2CO with a solution of 3-aminopropane-
1,2-diol (2 equiv) in water as above, followed by chromatog-
raphy on silica gel and elution with MeOH/EtOAc (1:19), gave
N-(2,3-dihydroxypropyl)-4-[N,N-bis(2-chloroethyl)amino]-2,3-
dinitrobenzamide (7c) (63% yield) as a yellow oil: 1H NMR
[(CD3)2SO] δ 8.87 (t, J ) 5.6 Hz, 1 H, CONH), 7.88 (d, J ) 8.8
Hz, 1 H, H-6), 7.85 (d, J ) 8.8 Hz, 1 H, H-5), 3.75 (m, 1 H,
CHOH), 3.70 (t, J ) 6.3 Hz, 4 H, CH2Cl), 3.65 (br, 2 H, OH),
3.57 (t, J ) 6.3 Hz, 4 H, CH2N), 3.40-3.30 (m, 3 H, CONHCHH
and CH2OH), 3.14-3.08 (m, 1 H, CONHCHH); 13C NMR δ
162.85 (CONH), 144.46 (s), 142.59 (s), 136.94 (s), 131.77 (C-
6), 127.34 (C-5), 124.03 (s), 70.11 (CHOH), 63.80 (CH2OH),
53.42 (CH2N), 43.00 (CONHCH2), 41.86 (CH2Cl). Anal. (C14H18
-
Cl2N4O7) C, H, N, Cl.
2-[N ,N -B is (2-c h lo r o e t h y l)a m in o ]-4,5-d in it r o b e n z-
a m id e (8a ). A mixture of 2-chloro-4,5-dinitrobenzoic acid25
(38) (6.00 g, 0.024 mol) and SOCl2 (15 mL) in 1,2-dichloro-
ethane (60 mL) containing DMF (1 drop) was refluxed under
nitrogen for 24 h and then concentrated to dryness. The
residue of crude acid chloride 39 was dissolved in Et2O (100
mL), cooled to 0 °C, and treated with concentrated NH4OH
(3.30 mL, 0.049 mol). The resulting precipitate was filtered
off and recrystallized from MeOH to give 2-chloro-4,5-dini-
trobenzamide (40) (4.89 g, 82%): mp 206-209 °C; 1H NMR
[(CD3)2SO] δ 8.54 (s, 1 H, H-6), 8.40 (s, 1 H, H-3), 8.24, 8.08 (2
× br, 2 H, CONH2); 13NMR δ 169.84 (CONH), 147.11, 147.02,
145.20, 140.69 (4 × s), 132.02, 130.59 (C-3,6). Anal. (C7H4-
MsCl (1.59 mL, 0.020 mol) was added dropwise to a solution
of 34 (2.96 g, 8.99 mmol) in pyridine (30 mL) at 20 °C. After
10 min, the pyridine was removed under reduced pressure,
and the residue was partitioned between EtOAc and water.
Workup of the organic layer gave the crude dimesylate, which
was dissolved in DMF (50 mL) containing LiCl (10 g) and
warmed at 100 °C for 15 min. The DMF was removed under
reduced pressure, the residue was partitioned between EtOAc
and water, and the oily residue from the organic layer was
chromatographed on silica gel. Elution with EtOAc/petroleum
ether (1:1) gave methyl 4-[N,N-bis(2-chloroethyl)amino]-2,3-
dinitrobenzoate (35) (3.05 g, 93%) as yellow needles: mp
ClN3
O3) C, H, N, Cl.
1
(EtOAc/petroleum ether) 90-92 °C; H NMR (CDCl3) δ 8.05
A mixture of 40 (3.70 g, 0.015 mol) and diethanolamine (3.33
g, 0.032 mol) in p-dioxane (100 mL) was stirred at 20 °C for
18 h, then adsorbed directly onto silica by concentration, and
chromatographed. Elution with EtOAc gave foreruns, while
EtOAc/MeOH (9:1) gave 2-[N,N-bis(2-hydroxyethyl)amino]-4,5-
dinitrobenzamide (41) (3.10 g, 65%): mp (EtOAc/petroleum
(d, J ) 8.9 Hz, 1 H, H-6), 7.45 (d, J ) 8.9 Hz, 1 H, H-5), 3.90
(s, 3 H, COOCH3), 3.62 (br t, J ) 3.1 Hz, 8 H NCH2CH2Cl);
13C NMR δ 162.07 (COOCH3), 146.09 (s), 144.12 (s), 133.49
(C-6), 131.20 (s), 124.61 (C-5), 116.51 (s), 54.18 (CH2N), 53.35
(COOCH3), 40.87 (CH2Cl). Anal. (C12H13Cl2N4O5) C, H, N, Cl.
A solution of 35 (3.00 g, 8.19 mmol) and 3 N KOH (20 mL)
in MeOH (100 mL) was stirred at 20 °C for 1 h. MeOH was
removed under reduced pressure, and the residue was acidified
with 3 N HCl, diluted with water, and extracted with EtOAc
to give 4-[N,N-bis(2-chloroethyl)amino]-2,3-dinitrobenzoic acid
(36) (2.81 g, 90%) as yellow plates: mp (EtOAc/petroleum
ether) 190 °C; 1H NMR [(CD3)2SO] δ 8.05 (d, J ) 8.5 Hz, 1 H,
H-6), 7.74 (d, J ) 8.5 Hz, 1 H, H-5), 3.85 (br, COOH), 3.76,
3.62 (2 × br, 8 H, NCH2CH2Cl); 13C NMR δ 162.97 (COOH),
146.30 (s), 144.37 (s), 134.32 (s), 133.82 (C-6), 124.87 (C-5),
1
ether) 146-148 °C; H NMR [(CD3)2SO] δ 8.13, 7.84 (2 × br,
2 H, CONH2), 7.82 (s, 1 H, H-6), 7.60 (s, 1 H, H-3), 4.87 (br, 2
H, OH), 3.60-3.20 (m, 8 H, NCH2CH2OH); 13NMR δ 168.42
(CONH2), 153.14, 145.82 (2 × s), 128.44 (C-6), 126.92, 125.55
(2 × s), 112.89 (C-3), 58.20 (CH2OH), 54.45 (CH2N). Anal.
(C11H14N4O7) C, H, N.
The diol 41 was treated sequentially with MsCl/pyridine (20
°C, 15 min) and LiCl/DMF (130 °C, 15 min) as above, and the
product was chromatographed on silica gel. Elution with
EtOAc/petroleum ether (1:1) gave foreruns, while EtOAc/
petroleum ether (2:1) gave 2-[N,N-bis(2-chloroethyl)amino]-4,5-
dinitrobenzamide (8a ) (1.08 g, 60%): mp (EtOAc/petroleum
ether) 193-195 °C (yellow cubes); 1H NMR [(CD3)2SO] δ 8.15,
7.94 (2 × br, 2 H, CONH2), 8.06 (s, 1 H, H-6), 7.74 (s, 1 H,
H-3), 3.84-3.80 (m, 8 H, CH2CH2Cl); 13NMR δ 168.06 (CONH2),
151.32, 145.55, 128.53 (3 × s), 128.08 (C-6), 126.63 (s), 112.51
(C-3), 53.05 (CH2N), 41.15 (CH2Cl); MS m/ z (rel int) 354, 352,
350 (M+, 1), 316,314 (19), 303, 301 (32), 285, 283 (38), 278 (54),
265 (31), 63 (90), 43 (100). Anal. (C11H12Cl2N4O5) C, H, N.
A solution of 8a (3.6 g, 0.010 mol) in 90% H2SO4 (100 mL)
was warmed at 70 °C for 5 days, poured into crushed ice, and
extracted with EtOAc. The organic layer was extracted with
saturated aqueous NaHCO3, and the aqueous extract was
acidified with concentrated HCl, extracted into EtOAc, and
worked up to give 2-[N,N-bis(2-chloroethyl)amino]-4,5-dini-
trobenzoic acid (42) (2.78 g, 79%) as a gum, which was used
directly. The neutral portion from the reaction was worked
up to give starting material (8a ) (0.80 g, 22%).
115.43 (s), 52.70 (CH2N), 41.76 (CH2Cl). Anal. (C11H11
Cl2N3O6) C, H, N, Cl.
-
A solution of 36 (1.00 g, 2.84 mmol) and SOCl2 (15 mL) in
1,2-dichloroethane (30 mL) containing DMF (1 drop) was
heated under reflux for 5 h and then concentrated to dryness
under reduced pressure. The crude acid chloride 37 was
dissolved in Me2CO (30 mL), and the solution was cooled to 0
°C and treated with concentrated NH4OH (5 mL). After 10
min, the mixture was diluted with water, extracted with
EtOAc, and worked up to give an oil which was chromato-
graphed on silica gel. Elution with EtOAc/petroleum ether
(1:5) gave foreruns, while EtOAc eluted 4-[N,N-bis(2-chloro-
ethyl)amino]-2,3-dinitrobenzamide (7a ) (0.87 g, 87%): mp
1
(EtOAc/petroleum ether) 138 °C; H NMR [(CD3)2SO] δ 8.35
(br s, 1 H, CONHH), 7.87 (br s, 3 H, H-5,6, CONHH), 3.71 (t,
J ) 6.3 Hz, 4 H, CH2Cl), 3.58 (t, J ) 6.3 Hz, 4 H, CH2N); 13C
NMR δ 164.32 (CONH2), 144.54 (s), 142.79 (s), 136.75 (s),
131.59 (C-6), 127.01 (C-5), 123.51 (s), 53.32 (CH2N), 41.83
(CH2Cl). Anal. (C11H12Cl2N4O5) C, H, N, Cl.
A solution of 42 (1.00 g, 2.83 mmol) and SOCl2 (5 mL) in
1,2-dichloroethane (50 mL) containing DMF (1 drop) was
heated under reflux for 6 h and then concentrated to dryness
to give the crude acid chloride (43). This was dissolved in
Me2CO (20 mL) and treated with a solution of 3-aminopropane-
1,2-diol (0.58 g, 6.42 mmol) in water (5 mL) cooled to 5 °C.
After 15 min, the solution was partitioned between EtOAc and
water, and the organic portion was worked up to give an oil
Reaction of 37 in CH2Cl2 with N,N-dimethylethylenediamine
(2 equiv) as above gave N-[2-(N,N-dimethylamino)ethyl]-4-
[N,N-bis(2-chloroethyl)amino]-2,3-dinitrobenzamide (7b) as a
yellow oil (62% yield). The hydrochloride salt crystallized from
MeOH/Et2O as hygroscopic yellow cubes: mp 60 °C dec; 1H
NMR (D2O) δ 7.84 (d, J ) 8.7 Hz, 1 H, H-6), 7.69 (d, J ) 8.7
Hz, 1 H, H-5), 3.75 (t, J ) 6.0 Hz, 2 H, CHN+Me2), 3.56 (t, J

Hypoxia-Selective Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2527
which was chromatographed on silica gel. EtOAc eluted
N-(2,3-dihydroxypropyl)-2-[N,N-bis(2-chloroethyl)amino]-4,5-
dinitrobenzamide (8c) (0.82 g, 68%) as a yellow oil: 1H NMR
[(CD3)2SO] δ 8.73 (t, J ) 5.6 Hz, 1 H, CONH), 8.09 (s, 1 H,
H-6), 7.72 (s, 1 H, H-3), 4.02 (br, 2 H, OH), 3.84-3.75 (m, 9 H,
NCH2CH2Cl and CHOH), 3.42-3.35 (m, 3 H, CONHCHH and
CH2OH), 3.16 (m, 1 H, CONHCHH); 13C NMR δ 166.39
(CONH), 151.21 (s), 145.62 (s), 128.70 (s), 128.42 (C-6), 125.32
(s), 112.35 (C-3), 69.91 (CHOH), 64.00 (CH2OH), 53.12 (CH2N),
43.05 (CONHCH2) 39.07 (CH2Cl); EIMS found M+ 428.0492,
426.0523, 424.0532; C14H18Cl2N4O7 requires 428.0493, 426.0523,
424.0552.
aerobic and hypoxic survival curves was used as the measure
of hypoxic selectivity.
In Vivo Toxicity a n d An titu m or Activity. Drugs were
formulated in phosphate-buffered saline and injected ip at 0.01
mL/g body weight using 20-25 g male C3H/HeN mice.
Toxicity was determined using groups of six non-tumor-bearing
mice with 1.33-fold dose increments and an observation time
of 28 days. The maximum tolerated dose (MTD) was defined
as the highest dose which did not cause deaths or unacceptable
morbidity. Antitumor activity was assessed using subcutane-
ous KHT tumors in the size range 0.5-1 g as determined by
caliper measurements, with excision of tumors 18 h after
treatment, pooling of tumors from two mice, and determination
of clonogenic survivors by preparation of single cell suspen-
sions and plating in agar using a modification28 of the original
method.29 Drugs were combined with whole-body γ radiation
(60Co) at a dose of 15 Gy to test activity against hypoxic
(radioresistant) tumor cells.
A solution of the crude acid chloride 43 (0.60 g, 1.70 mmol),
prepared as above, in CH2Cl2 (20 mL) was reacted with N,N-
dimethylethylenediamine (0.37 mL, 3.41 mmol) for 15 min.
The solution was diluted with CH2Cl2, washed with water, and
extracted with 3 N HCl. The extract was basified with
concentrated NH4OH, extracted with EtOAc and worked up
to give an oil which was chromatographed on silica gel.
Elution with MeOH/EtOAc (1:5) gave N-[2-(N,N-dimethyl-
amino)ethyl]-2-[N,N-bis(2-chloroethyl)amino]-4,5-dinitro-
benzamide (8b) (0.24 g, 33%) as a yellow oil. The hydrochlo-
ride salt crystallized from MeOH/Et2O as a hygroscopic orange
solid: mp 88-92 °C; 1H NMR (D2O) δ 8.27 (s, 1 H, H-6), 7.46
(s, 1 H, H-3), 7.28 (br, 1 H, CONH), 3.83-3.65 (m, 10 H, NCH2-
CH2Cl and CH2N+Me2), 3.44 (br, 2 H, CONHCH2), 2.99 (s, 6
H, N+Me2); 13C NMR δ 170.77 (CONH), 155.50 (s), 148.43 (s),
133.06 (s), 131.67 (C-6), 128.68 (s), 116.90 (C-3), 58.45
(CH2N+Me2), 55.80 (CH2N), 45.74 (N+Me2), 43.96 (CH2Cl),
37.75 (CONHCH2); CIMS found [M + H]+ 426.0944, 424.0959,
422.0999; C15H22Cl2N5O5 requires 426.0939, 424.0968, 422.0998.
Ack n ow led gm en t. This work was supported by
contract NO-1 CM 47019 from the U.S. National Cancer
Institute, by the Auckland Division of the Cancer
Society of New Zealand, and by the Health Research
Council of New Zealand. We thank Xing Z. Xu, Diane
Schultz, and Susan Pullen for technical assistance with
biological assays and Prof. P. Wardman, CRC Gray
Laboratory, for determination of some E(1) values.
Su p p or tin g In for m a tion Ava ila ble: ORTEP diagram,
X-ray bond angles, and bond lengths for compound 25 (10
pages); structure factors for compound 25 (14 pages). Ordering
information is given on any current masthead page.
X-r a y Cr ysta llogr a p h ic Deter m in a tion of 25. Ethyl
3-[N,N-bis(2-chloroethyl)amino]-2,6-dinitrobenzoate (25) was
crystallized from EtOAc/petroleum ether as yellow needles, mp
84-87 °C, space group P21/n; cell constants a ) 13.007(2) Å,
b ) 7.654(4) Å, c ) 16.770(3) Å, â ) 98.87(1)°; z ) 4; V )
1649.5(9) Å3. Lattice constants and intensity data were
measured using graphite monochromated Mo Ka radiation, λ
) 0.710 69 Å, on a Nonius CAD-4 diffractometer. The data
set consisted of 2581 unique reflections, of which 1956 were
considered observed (I > 3σ(I)). The structure was solved
using SHELXS and refined with SHELX-76.26 All atoms
including hydrogens were found from successive difference
maps and allowed to refine freely. Anisotropic temperature
factors for atoms other than H and isotropic temperature
factors for H atoms were allowed. R and Rw were 0.0408 and
0.0436. The largest shift/esd values during the final refine-
ment were less than 0.08, and maximum and minimum peaks
in the final difference map were 0.27 and 0.32 e Å-3, respec-
tively. The nitro group at N3, the ester group, and the nitro
group at N2 were rotated from the plane of the aromatic ring
by 1.5, 86.4, and 48.5°, respectively.
Refer en ces
(1) Wilson, W. R. Tumour hypoxia: challenges for cancer chemo-
therapy. In Cancer Biology and Medicine (Vol 3); Waring, M.
J ., Ponder, B. A. J ., Eds.; Kluwer Academic Publishers: Lan-
caster, 1992; pp 87-131.
(2) Denny, W. A.; Hay, M. P.; Wilson, W. R. Recent developments
in the design of bioreductive drugs. Br. J . Cancer, in press.
(3) Adams, G. E.; Stratford, I. J . Bioreductive drugs in cancer
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Cell Cu ltu r e Gr ow th In h ibition Assa y. Cell cultures
were initiated in 96-well microtiter trays to give 200 (AA8),
300 (UV4), or 75 (EMT6) cells in 0.05 mL per well and grown
for 24 h before drugs were added in culture medium (RMEM
with 5% fetal bovine serum plus antibiotics).12 After incubat-
ing under aerobic conditions for 18 h, drugs were removed by
washing cultures three times with fresh medium. Cultures
were incubated for
a further 78 h before staining with
methylene blue.27 IC50s were calculated as the drug concen-
tration providing 50% inhibition of growth relative to the
controls.
Deter m in a tion of Hyp oxia -Selective Cytotoxicity in
Vitr o. Cells were grown to early plateau phase in spinner
flasks (AA8 cells to 106/mL and UV4 to 6 × 105/mL) and
resuspended in fresh medium. Clonogenic assays were per-
formed using magnetically stirred 10 mL cell suspensions (106/
mL) after drug exposure for 1 h while continuous gassing with
5% CO2 in air or N2 as detailed elsewhere.21,22 Both cell
suspensions and drug solutions in growth medium were pre-
equilibrated under the appropriate gas phase for 1 h prior to
mixing to ensure essentially complete anoxia throughout the
period of drug contact in hypoxic cultures. The ratios of the
concentration for a surviving fraction of 10% (C10) for the
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(17) We note that under IUPAC rules 20 is named 3-chloro-4,6-
dinitrobenzoic acid rather than 5-chloro-2,4-dinitrobenzoic acid.
However, to be consistent with our previous publications and
previous literature on the related compound CB 1954 (1), we
have chosen to use the former numbering for 20 and its
analogues in this paper.
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