DNA-Targeted 1,2,4-Benzotriazine 1,4-Dioxides: Potent Analogues of the Hypoxia-Selective Cytotoxin Tirapazamine

Article abstract of DOI:10.1021/jm030399c

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pKa of the linker chain. The association constants (K _DNA) for calf thymus DNA ranged from 1 × 10² to 5.6 × 10⁵ M^-1 (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK_a. The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C₁₀), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C₁₀(aerobic)/C₁₀(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (< 167 and 400) values. There was a strong correlation between K_DNA and hypoxic cytotoxicity but no correlation between K_DNA and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC₅₀ assays, showed similar relationships with a correlation between K_DNA and hypoxic cytotoxicity but no correlation between K_DNA and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.

Full text of DOI:10.1021/jm030399c

J . Med. Chem. 2004, 47, 475-488
475
DNA-Ta r geted 1,2,4-Ben zotr ia zin e 1,4-Dioxid es: P oten t An a logu es of th e
Hyp oxia -Selective Cytotoxin Tir a p a za m in e
Michael P. Hay,*,† Frederik B. Pruijn,† Swarna A. Gamage,† H. D. Sarath Liyanage,† Mary S. Kovacs,‡
Adam V. Patterson,† William R. Wilson,† J . Martin Brown,‡ and William A. Denny†
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland, New Zealand, and Department of Radiation Oncology, Stanford University School of Medicine,
Stanford, California 94305-5152
Received August 15, 2003
Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective
cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with
cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide
(BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via
a flexible linker. DNA binding affinity was modified through variation of the chromophore or
the pKa of the linker chain. The association constants (KDNA) for calf thymus DNA ranged from
1 × 102 to 5.6 × 105 M-1 (ionic strength of 0.01 M). DNA binding affinity was dependent on the
presence of a positive charge, either in the linker chain or in the chromophore, and (for a series
of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pKa.
The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro
was determined by clonogenic survival. Cytotoxicity was measured as the concentration required
to reduce plating efficiency to 10% of controls (C10), and the hypoxic cytotoxicity ratio (HCR)
for each BTO was calculated as C10(aerobic)/C10(hypoxic). BTOs bearing a positive charge showed
increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-
51), but some excellent (>167 and 400) values. There was a strong correlation between KDNA
and hypoxic cytotoxicity but no correlation between KDNA and HCR. Cytotoxicity in HT-29
human colon carcinoma cells, determined using IC50 assays, showed similar relationships with
a correlation between KDNA and hypoxic cytotoxicity but no correlation between KDNA and HCR.
In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to
that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency
while maintaining hypoxic selectivity and provide a direction for the further development of
DNA-targeted analogues of TPZ.
In tr od u ction
potentially be ameliorated if more potent analogues
were available.
Tirapazamine (1, TPZ, 1,2,4-benzotriazin-3-amine 1,4-
dioxide) (Chart 1) is a bioreductive agent that is
selectively toxic to hypoxic cells.1,2 Hence, it is a useful
adjunct to radiotherapy and chemotherapy, which often
fail to eliminate hypoxic cells within tumors.3,4 TPZ
undergoes metabolic activation by intracellular reduc-
tases to a cytotoxic radical species which, under hypoxia,
reacts with DNA to form DNA single- and double-strand
breaks.5-7 Formation of the cytotoxic radical species is
inhibited by oxygen, leading to hypoxia-selective cyto-
toxicity.8 Metabolism of TPZ may be effected by many
intracellular reductases,9,10 but there is evidence11,12
that activation in the nucleus is predominantly respon-
sible for the DNA double-strand breaks that mediate
its hypoxic toxicity.5
Improving the potency of bioreductive drugs by tar-
geting them to DNA has been the subject of a number
of studies,18 which have used various DNA-binding units
to target 2-nitroimidazoles19-23 or nitroacridines24 and
nitroquinolines.25 However, the presence of a DNA-
binding chromophore and basic side chain in a drug may
limit extravascular transport through increased DNA
binding or entrapment in acidic compartments within
cells.26-28 Identification of this limitation provided the
impetus for a search for so-called “minimal” DNA-
intercalating agents that would penetrate the extra-
vascular compartment of tumors more effectively.29-35
Lowering the pKa of the chromophore or reducing the
size of the intercalating chromophore reduced DNA
binding, culminating in the development of the anti-
tumor drug DACA (2). DACA showed superior tissue
diffusion compared to its 9-aminoacridine analogue
DAPA (3), due to its lower overall charge-state and DNA
binding affinity,36 and received a phase II clinical
trial.37,38 A similar pathway of refinement has been
followed in the development of the DNA-targeted radio-
sensitizer NLCQ-1 (4)39 from the initial analogues NLP-
140,41 and NLA-1 (5).42,43
TPZ is currently in phase II/III clinical trial in
combination with radiotherapy and with cisplatin.13-17
In conjunction with radiotherapy, fatigue and neutro-
penia are the dose-limiting toxicities,17 which could
* Corresponding author. Phone: 64-9-3737599, ext. 86598. Fax: 64-
9-3737502. E-mail: m.hay@auckland.ac.nz.
† The University of Auckland.
‡ Stanford University School of Medicine.
10.1021/jm030399c CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/17/2003

476 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Hay et al.
Ch a r t 1
Sch em e 1a
a
Reagents: (a) Et3N, DCM; (b) (CF3CO)2O, DCM; (c) CF3CO3H, DCM; (d) NH4OH, MeOH; (e) acid 22-27, CDI, DMF; then 21, DCM.
In contrast, TPZ has received little attention as a
subject for such targeting strategies. The objective of
targeting TPZ to DNA is to increase the proportion of
“productive” metabolism occurring sufficiently close to
nuclear DNA targets to contribute to cytotoxicity. Such
targeting is expected to reduce wasteful extranuclear
consumption of drug (which has been shown to limit
extravascular transport of TPZ44) and increase potency,
thereby increasing therapeutic efficacy relative to TPZ.
DNA targeting also has the potential to compromise
extravascular transport into hypoxic regions45,46 and to
induce additional toxicities. For these reasons, one of
the objectives of the present study is to identify DNA-
targeted analogues with moderate binding affinities
allowing efficient extravascular transport yet providing
enhancement of cytotoxic potency.
Hypoxic areas in tumor tissue are located at the end
of a diffusion gradient, often distant from the micro-
vasculature. Thus, efficient extravascular transport to
the site of metabolic activation is required for hypoxia-
selective bioreductive drugs to be effective. This is a
particular issue for BTO analogues, since recent studies
have shown that inefficient transport of TPZ itself
through multicellular layer (MCL) cultures results in
resistance.44 Recently, we demonstrated that a DNA-
targeted BTO analogue showed much improved in vitro
efficacy relative to TPZ.47 Thus compound 6, a BTO
linked to a 9-aminoacridine unit, showed a 20-400-fold
increase in hypoxic cytotoxicity relative to TPZ in HeLa
and HT-29 cells while retaining similar hypoxic selec-
tivity. However, 6 was not active in potentiating tumor
cell kill by irradiation in vivo, probably because of
restricted diffusion through tumor tissue.47 Thus, the
optimization of extravascular transport properties, while
retaining the benefits of DNA targeting, is one of the
prime requirements for this new class of targeted
bioreductive drug, exemplified by BTO 6.
The objective of this study is to design and synthesize
a range of DNA-targeted BTO analogues 7-16, in which
the basicity of the amine side chain and the binding
strength of the DNA-affinic chromophore are varied, and
to quantitate the effect of these structural modifications
on the DNA-binding affinity and in vitro cytotoxicity
under oxic and hypoxic conditions.
Ch em istr y
Syn th esis. Chloride 1748 was coupled to N1-(3-amino-
propyl)-N1-methyl-1,3-propanediamine (18) and pro-
tected as the trifluoracetamide 19 to facilitate purifica-
tion (Scheme 1). Oxidation of 19 with trifluoroperacetic
acid under acidic conditions resulted in selective aro-
matic N-oxidation to give 1,4-dioxide 20 (27%) and

DNA-Targeted 1,2,4-Benzotriazine 1,4-Dioxides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 477
Sch em e 2a
a
Reagents: (a) Et3N, DCM; (b) MsCl, Et3N, DCM; (c) NaN3, DMF; (d) propane-1,3-dithiol, Et3N, MeOH; (e) BOC2O, THF; (f) MCPBA,
NaHCO3, DCM; (g) CF3CO2H, DCM; (h) 25, CDI, DMF; then 33, DCM.
Sch em e 3a
a
Reagents: (a) BOC2O, THF; (b) 17 + 35, Et3N, DME; (c) HCl, MeOH; (d) CF3CO2Et, H2O, MeCN; (e) CF3CO3H, DCM; (f) 25, CDI,
DMF; (g) 39, THF.
recovered starting material 19 (24%). Extended reaction
times gave increased conversion to 20 but was ac-
companied by the formation of byproducts that compli-
cated purification. The use of other oxidants, e.g.,
peracetic acid, MCPBA, and methyltrioxorhenium, gave
inferior yields. Deprotection of trifluoroacetamide 20
gave amine 21, which was coupled with the imidazolide
of 8-quinolinecarboxylic acid (22), formed by reaction
of acid 22 with CDI, to give carboxamide 7. Similarly,
reaction of 21 with imidazolides of 1-phenazinecarbox-
ylic acid (23),32 2-(4-pyridyl)-8-quinolinecarboxylic acid
(24),35 4-acridinecarboxylic acid (25),49 9-methylphen-
azine-4-carboxylic acid (26),32 and 5-methylacridine-4-
carboxylic acid (27)30 gave carboxamides 8-12, respec-
tively.
Reaction of chloride 17 with 2-(aminoethoxy)ethanol
(28) gave alcohol 29 in 63% yield, which was converted
to the mesylate and displaced with sodium azide to give
azide 30 in 89% yield (Scheme 2). Selective reduction
of the azide group rather than the 1-oxide of 30 could
not be effected by hydrogenation using palladium on
charcoal or Lindlar catalyst.50 Other chemoselective
methods for reducing azides such as NaBH4 under
PTC,51 BH3‚DMS,52 or Staudinger conditions using
P(OEt)353 were ineffective. However, treatment of azide
30 with propane-1,3-dithiol and Et3N in refluxing
methanol54 provided the intermediate amine which was
protected with di-tert-butyl dicarbonate to give carbam-
ate 31 in 93% yield for the two steps. Oxidation of 31
with MCPBA gave 1,4-dioxide 32 in 40% yield as well
as recovered starting material 31 (50%). Deprotection
of 32 with trifluoroacetic acid gave amine 33 in 91%
yield. Reaction of 33 with the imidazolide of acid 25 gave
carboxamide 13 in 97% yield.
Coupling of the monoprotected diamine 35, prepared55
from diamine 34, gave the 1-oxide 36 in 52% yield as
well as recovered starting material (25%) (Scheme 3).
Deprotection and reprotection of 36 gave trifluoro-
acetamide 38 in 88% yield for the two steps. Oxidation
of 38 with trifluoroperacetic acid gave 1,4-dioxide 39
(47%), which was deprotected, and the intermediate
amine was coupled to the imidazolide of acid 25 to give
carboxamide 14 in high yield.
Reaction of chloride 17 with amine 41, prepared from
N1-(2-aminoethyl)-1,2-ethanediamine (40), and protec-
tion of the intermediate amine to facilitate isolation gave
the 1-oxide 42 in 52% yield (Scheme 4). Compound 42
was oxidized with MCPBA to give 1,4-dioxide 43.

478 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Hay et al.
Sch em e 4a
a
Reagents: (a) CF3CO2Et, ether; (b) BOC2O, THF; (c) aq NH3, MeOH; (d) Et3N, DME; (e) BOC2O, DCM; (f) MCPBA, DCM; (g) HCl,
MeOH; (h) 25, CDI, DMF; (i) 43, HCl, MeOH, then DMF.
Sch em e 5a
a
Reagents: (a) Et3N, DCM; (b) (CF3CO)2O, DCM; (c) MCPBA, NaHCO3, DCM; (d) K2CO3, aq MeOH; (e) 25, CDI, DMF; (f) 47, DCM;
(g) HCl, MeOH.
Ta ble 1. Physicochemical Data for BTO Analogues
b
pKa
no.
side chain
chromophore
log D measa
chrom
8.6
3.4
-0.1
2.9 (pyr)f
4.0
0.5
4.4
4.0
4.1
side chain
KDNAc 104 M-1
1
6
7
8
9
10
11
12
13
14
15
16
-0.34 ( 0.02d
1.69 ( 0.1
C6-alkyl
9-aminoacridine
8-quinoline
1-phenazine
2-pyridyl-8-quinoline
4-acridine
9-methyl-1-phenazine
5-methyl-4-acridine
4-acridine
1.8
8.9
8.8
8.9
8.9
8.9
8.8
14.3 ( 1.5e
0.046 ( 0.018
0.47 ( 0.12
0.84 ( 0.17
3.3 ( 0.6
C3NMeC3
C3NMeC3
C3NMeC3
C3NMeC3
C3NMeC3
C3NMeC3
C2OC2
C2NMeC2
C2NHC2
C3NHC3
0.15 ( 0.004
0.59 ( 0.04
0.66 ( 0.02
1.53 ( 0.04
1.40 ( 0.05
2.28 ( 0.03
1.84 ( 0.02
2.15 ( 0.02
1.36 ( 0.04
0.88 ( 0.03
8.5 ( 1.4
56.2 ( 6.3
0.010 ( 0.008
0.75 ( 0.34
2.1 ( 0.6
4-acridine
4-acridine
4-acridine
7.5
7.9
9.7
4.1
4.1
6.4 ( 2.0
a
b
In octanol/water at pH 7.4 (mean ( SEM). Calculated using the program ACDpKa v. 4.5. c Equilibrium association constant for
DNA binding. From ref 58. e From ref 47. Calculated for a 2-pyridyl group.
d
g
Deprotection of 43 and coupling of the intermediate
amine with the imidazolide of acid 25 gave compound
15.
was coupled to the imidazolide of acid 25, and the
carbamate was deprotected with HCl to give carbox-
amide 16.
P h ysicoch em ica l Mea su r em en ts. Octanol/water
partition coefficients at pH 7.4 of TPZ (1) and BTOs
6-16 were determined by a low-volume shake flask
method, with BTO concentrations in both the octanol
and buffer phases analyzed by HPLC as previously
described56 (Table 1). The pKa values of the BTOs were
calculated using ACD pKa prediction software (v. 4.5,
Advanced Chemistry Development Inc., Toronto, Canada)
Reaction of chloride 17 with tert-butyl bis(3-amino-
propyl)carbamate (44) and protection of the intermedi-
ate primary amine with trifluoroacetic anhydride gave
the trifluoroacetamide 45 in 39% yield for the two steps
(Scheme 5). Oxidation of 45 with MCPBA gave the 1,4-
dioxide 46 (8% with 65% recovered starting material).
Deprotection of 46 gave amine 47 in good yield, which

DNA-Targeted 1,2,4-Benzotriazine 1,4-Dioxides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 479
Ta ble 2. Cytotoxicity Data for BTO Analogues in SCCVII Cells (clonogenic) and in HT-29 Cells (SRB IC50
clonogenic assay (SCCVII) IC50 assay (HT-29)
IC50(aerobic)d (µM)
)
no.
C
10(hypoxic)a (µM)
C10(aerobic)b (µM)
HCRc
IC50(hypoxic)d (µM)
HCRf
1
6
7
8
9
10
11
12
13
14
15
16
10.2 ( 0.5 (34)g
0.61
6.5
1.07
1.79
0.82
0.35
0.18
12.4
2644 ( 266 (9)g
258 ( 21 (9)g
15.7
13.9
8.8
7.8
51.5
21.1
>167
50.8
6.2 ( 0.8
387 ( 34
0.62 ( 0.09
36.3 ( 5.4
0.59 ( 0.08
12.0 ( 0.6
5.35 ( 0.83
0.56 ( 0.07
8.4 ( 2.8
72.5 ( 10.5
15.7
62.0
39.7
61.2
4.9
107
124
31.0
88.4
12.8
87.0
98.6
87.5
9.6
90
0.016 ( 0.006
0.59 ( 0.13
0.12 ( 0.03
0.11 ( 0.02
0.043 ( 0.009
0.018 ( 0.007
0.095 ( 0.039
5.7 ( 1.4
9.4
14
42
7.4
>30
630
400
56
0.93
1.16
0.51
431
48.2
12.5
0.18
0.079 ( 0.024
0.065 ( 0.021
7.8 ( 0.2
5.7 ( 0.4
6.4
a
b
Concentration of drug for a one-log cell kill under hypoxia normalized against TPZ (1) in the same experiment. Concentration of
drug for a one-log cell kill under aerobic conditions. c Intraexperimental hypoxic cytotoxicity ratio ) C10(aerobic)/C10(hypoxic). IC50 values
d
(mean ( SEM, n g 2). f Intraexperimental hypoxic cytotoxicity ratio ) IC50(aerobic)/IC50(hypoxic). Mean ( SEM, with the number of
g
measurements in parentheses.
and the apparent (macroscopic) constants for both side
chain and chromophore moieties reported (Table 1). The
binding of 6-16 to calf thymus DNA was measured by
equilibrium dialysis, using a low ionic strength (0.01 M)
to increase sensitivity of detection of binding. Concen-
trations of compound were measured on both sides of
the dialysis membrane by HPLC.
Biologica l Assa ys
The efficacy of the BTOs in killing aerobic and hypoxic
mouse SCCVII tumor cells in vitro was determined by
clonogenic survival after 1 h drug exposure of cells
under aerobic and anoxic conditions, as previously
described.57 Cytotoxicity was measured as the concen-
tration required to reduce plating efficiency to 10% of
controls (C10), and the intraexperimental differential
between the hypoxic and aerobic cytotoxicity for each
compound was calculated as the hypoxic cytotoxicity
F igu r e 1. Cytotoxicity of targeted BTO analogues against
SCCVII cells (clonogenic assay) versus KDNA (equilibrium
dialysis): b, hypoxic cytotoxicity; O, aerobic cytotoxicity.
ratio [HCR ) C10(aerobic)/C10(hypoxic)] (Table 2). The
in vitro cytotoxicity of 1 and 6-16 was also evaluated
against HT-29 human colon carcinoma cells using a 96-
well proliferation assay (sulforhodamine B; SRB)57 to
determine IC50 values under aerobic and anoxic condi-
tions (Table 2). For each experiment, TPZ was included
as an internal control.
shown in Table 1. The binding site size was not well
defined (range 0.7-1.9 bp), due to insufficient data points
close to the asymptote. The binding of BTOs 6-16
ranged from the detectable limit, ca. 1 × 102 M-1 for
13, through to 5.6 × 105 M-1 for compound 12. Increas-
ing the binding strength of the chromophore from
8-quinolinecarboxamide 7 through to 5-methyl-4-acridine-
carboxamide 12, while keeping the linker chain con-
stant, gave a 1200-fold increase in KDNA. Variation of
the pKa of the side chain from 7.5 (14) to 9.7 (16), while
the 4-acridinecarboxamide chromophore remained fixed,
provided an 8.5-fold increase in KDNA. Replacement of
the amine side chain with a neutral ether-linked side
chain (13) reduced binding to close to the detectable
limit. Overall, DNA binding affinity was dependent on
the presence of a positive charge, either in the linker
chain or in the chromophore, and correlated strongly
with the pKa of the linker chain in the small series
containing the 4-acridine carboxamide chromophore.
In Vitr o Cytotoxicity. Ten BTO analogues were
sufficiently soluble to obtain aerobic cytotoxicity data
(C10 in µM) in SCCVII cells, using the clonogenic assay
(Table 2). The BTOs 6-16 were considerably more toxic
than TPZ (2644 µM) under aerobic conditions, with
aerobic C10 values ranging from 6.4 to 630 µM. There
was no clear correlation between aerobic C10 and KDNA
(r ) -0.646, P ) 0.032; Figure 1). Hypoxic C10 values
for analogues 6-16 ranged from 0.18 to 12.4 µM and
Resu lts a n d Discu ssion
log D. The hydrophobicities of TPZ and BTOs 6-16
were determined from HPLC analysis of drug distribu-
tion between octanol and aqueous buffer at pH 7.4
(Table 1). The value for TPZ using this method
(-0.34 ( 0.02) has been reported previously57 and is in
good agreement with an independent literature value
(-0.32).58 Although the presence of an aromatic chro-
mophore increases the lipophilicity of the BTOs, the
presence of a moderately basic amine in the chro-
mophore 6 or the linker chain 7-12 and 14-16 reduces
the log D significantly (range -0.56 to 1.99), suggesting
that these features should improve aqueous solubility
and reduce plasma protein binding.
DNA Bin d in g. The binding of BTOs to calf thymus
DNA at 0.01 M ionic strength was determined by
equilibrium dialysis as described previously,56 and the
association constants, KDNA, derived from direct plots
of drug bound/base pair versus free drug concentra-
tion47,59 using the neighboring site exclusion model, are

480 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Hay et al.
the E(1) of the neutral 13 was slightly lower (-466 ( 9
mV) and compound 16, possessing a more basic side
chain amine, was slightly higher (-421 ( 8 mV). It has
been shown for nontargeted BTOs that reduction po-
tential strongly correlates with cytotoxic potency.57
The lack of activity of SN 26955 (6) in potentiating
tumor cell kill by irradiation in vivo has been postulated
to be a result of its relatively strong DNA binding
limiting extravascular transport.47 The importance of
extravascular transport in the efficacy of bioreductive
drugs has been emphasized in a number of studies.61-63
In particular, the resistance to TPZ in 3D cell culture
has been ascribed wholly to inefficient transport rather
than changes in intrinsic sensitivity.44 The effects of
DNA binding and pKa on drug transport have been
examined in detail36 for two basic DNA intercalators,
DACA (2) and DAPA (3), which bear structural simi-
larities to the targeting units of the BTO analogues
7-16 and 6, respectively. We determined the KDNA of
DACA to be (15.7 ( 5.6) × 104 M-1 (0.01 M ionic
strength), while the KDNA of DAPA was estimated by
extrapolation to be ca. 6 × 107 M-1 (0.01 M ionic
strength).56 DACA was found to diffuse through multi-
cellular layer cultures faster than DAPA as a result of
its weaker basicity and lower DNA-binding affinity.
These properties were also suggested to be responsible
for the superior antitumor activity of related acridine-
4-carboxamides relative to corresponding 9-amino-
acridine derivatives.29 The extravascular transport of
the DNA-targeted BTOs is currently under investigation
using the multicellular layer model, but if the same
principles apply as for the above aminoacridines, then
relatively high DNA-binding affinity may compromise
diffusion in this series. If this is the case, then moder-
ately DNA-affinic BTOs such as 10, 14, and 15, which
still possess good hypoxic selectivity, may be more
promising candidates for further development than the
more tightly binding analogues 6 and 12.
F igu r e 2. Cytotoxicity of targeted BTO analogues against HT-
29 cells (proliferation assay) versus KDNA (equilibrium di-
alysis): b, hypoxic cytotoxicity; O, aerobic cytotoxicity.
showed a strong correlation with KDNA (r ) -0.975, P
< 0.0001; Figure 1). The strongest binder in the series,
12, was 56 times more potent than TPZ under hypoxia.
Intraexperimental hypoxic cell selectivity [HCR;
C10(aerobic)/C10(hypoxic)] spanned a 55-fold range from
7.8 to 431 and showed no correlation with KDNA. Most
compounds had modest hypoxic selectivity (7.8-51), but
the strongest binder 12 had an HCR of >167 (aerobic
cytotoxicity limited by solubility), which compares fa-
vorably with TPZ (HCR ) 258). Of particular interest
was the observation that the moderate binder 14 had
excellent hypoxic selectivity (431-fold), while being 11-
fold more potent under hypoxia than TPZ.
BTOs 6-16 were all considerably more toxic than
TPZ (387 µM) against HT-29 cells in the SRB prolifera-
tion assay under aerobic conditions, with IC50 values
ranging from 0.6 to 72 µM (Table 2). Hypoxic IC50 values
ranged from 0.016 µM for BTO 6 to 5.7 µM for the
“nontargeted” neutral BTO 13. The strongest binder, 12,
was not the most toxic compound under hypoxia, with
BTOs 6 and 11 giving the largest increase in hypoxic
cytotoxicity. As with SCCVII C10 values, a correlation
between HT-29 IC50 values and KDNA (r ) -0.861, P )
0.0007; Figure 2) was seen under hypoxic but not under
oxic exposure conditions (r ) -0.592, P ) 0.055; Figure
2). Moderate to excellent hypoxic selectivity was seen
for BTOs 6-16, with seven compounds showing selec-
tivities greater than or equal to that of TPZ. There was
again no relationship between HCR and KDNA. As would
be anticipated,57 SCCVII and HT-29 hypoxic and oxic
cytotoxicity values combined showed a strong correla-
tion (r ) 0.927, P < 0.0001), suggesting that the SAR
for BTOs 6-16 is essentially cell line and end-point
independent.
Con clu sion s
We have prepared a series of BTO analogues of TPZ
in which variation of the DNA-affinic chromophore or
linker chain has provided a range in DNA association
constant (KDNA) of ca. 5600-fold (from 1 × 102 to 5.6 ×
105 M-1 at an ionic strength of 0.01 M). DNA-binding
affinity was dependent on the presence of a positive
charge, either in the linker chain or in the chromophore.
For BTOs with a fixed (acridine-4-carboxamide) chro-
mophore, binding correlated strongly with the pKa of
the linker chain. Most DNA-targeted BTOs showed
increased hypoxic cytotoxicity in both clonogenic and
IC50 assays, and a correlation between DNA binding and
hypoxic cytotoxicity was observed, but not between
cytotoxicity under aerobic conditions (and consequently
HCR). Despite the lack of correlation between KDNA and
HCR, a number of DNA-targeted BTOs showed excel-
lent hypoxic selectivity. This study has confirmed the
initial observation47 that targeting a BTO unit to DNA
by conjugation to a DNA-affinic moiety not only in-
creases cytotoxicity relative to TPZ but also retains
selective toxicity to hypoxic cells. Further development
of this class of compounds, which involves optimizing
extravascular transport properties (i.e., diffusion in
relation to metabolic consumption),44 is currently in
progress.
The mechanism of the increase in cytotoxic potency
of the DNA-targeted analogues is not yet fully under-
stood. The data presented here and elsewhere47 strongly
suggest that the main reason for the increase in hypoxic
potency is localization of the compounds and their toxic
radical metabolites in the cell nucleus. However, we
cannot exclude the possibility that increased metabo-
lism, either by nuclear or other reductive enzymes, is
also involved. In addition, while the one-electron reduc-
tion potential, E(1), of compound 10 was found60 to be
-444 ( 8 mV, similar to that of TPZ (-456 ( 8 mV),57

DNA-Targeted 1,2,4-Benzotriazine 1,4-Dioxides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 481
organic fraction dried, and the solvent evaporated on to silica
(Ca u tion : safety shield). The residue was purified by chro-
matography, eluting with 10% MeOH/DCM, to give (i) starting
material 19 (275 mg, 24%) and (ii) 1,4-dioxide 20 (319 mg, 27%)
as a red gum: 1H NMR [(CD3)2SO] δ 9.44 (br s, 1 H, NH), 8.45
(t, J ) 5.9 Hz, 1 H, NH), 8.20 (d, J ) 8.8 Hz, 1 H, H-8′), 8.12
(d, J ) 8.6 Hz, 1 H, H-5′), 7.93 (ddd, J ) 8.6, 7.1, 1.2 Hz, 1 H,
H-6′), 7.57 (ddd, J ) 8.8, 7.1, 1.3 Hz, 1 H, H-7′), 3.42-3.47 (m,
2 H, CH2N), 3.21-3.25 (m, 2 H, CH2N), 2.39 (t, J ) 6.7 Hz, 2
H, CH2N), 2.32 (t, J ) 6.9 Hz, 2 H, CH2N), 2.16 (s, 3 H, NCH3),
1.72-1.80 (m, 2 H, CH2), 1.61-1.68 (m, 2 H, CH2); 13C NMR
[(CD3)2SO] δ 155.9 (q, J ) 36 Hz), 149.7, 138.1, 135.4, 129.8,
126.7, 121.0, 116.7, 115.9 (q, J ) 288 Hz), 54.9, 54.6, 41.4,
39.5, 37.6, 25.9, 25.8; MS m/z 403 (MH+, 25%), 387 (5); HRMS
calcd for C16H22F3N6O3 (MH+) m/z 403.1706, found 403.1695.
N1-(3-Am in op r op yl)-N3-(1,4-d ioxid o-1,2,4-ben zotr ia zin -
3-yl)-N1-m eth yl-1,3-p r op a n ed ia m in e (21). A solution of
trifluoroacetamide 20 (175 mg, 0.44 mmol) and NH4OH (5 mL)
in MeOH (20 mL) was stirred at 30 °C for 4 h. The solvent
was evaporated and the residue dried to give amine 21 (131
mg, 98%) as a red gum: 1H NMR [(CD3)2SO] δ 8.43 (br s, 1 H,
NH), 8.21 (d, J ) 8.5 Hz, 1 H, H-8′), 8.13 (d, J ) 8.4 Hz, 1 H,
H-5′), 7.94 (ddd, J ) 8.4, 7.1, 1.2 Hz, 1 H, H-6), 7.75 (br s, 2
H, NH2), 7.57 (ddd, J ) 8.7, 7.2, 1.3 Hz, 1 H, H-7′), 3.45 (t, J
) 6.8 Hz, 2 H, CH2N), 3.20-3.25 (m, 2 H, CH2N), 2.88 (dd, J
) 7.4, 7.2 Hz, 2 H, CH2N), 2.40-2.46 (m, 2 H, CH2N), 2.20 (s,
3 H, NCH3), 1.77-1.83 (m, 2 H, CH2), 1.68-1.75 (m, 2 H, CH2);
MS m/z 307 (MH+, 2%), 291 (5); HRMS calcd for C14H23N6O3
(MH+) m/z 307.1883, found 307.1883.
Exp er im en ta l Section
Analyses were carried out in the Microchemical Laboratory,
University of Otago, Dunedin, New Zealand. Melting points
were determined on an Electrothermal 2300 melting point
apparatus. NMR spectra were obtained on a Bruker Avance
400 spectrometer at 400 MHz for 1H and 100 MHz for 13C
spectra. Spectra were obtained in CDCl3, unless otherwise
specified, and are referenced to Me4Si. Chemical shifts and
coupling constants were recorded in units of ppm and Hz,
respectively. Assignments were determined using COSY,
HSQC, and HMBC two-dimensional experiments. Mass spec-
tra 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. All spectra were
obtained using FAB with positive ionization unless otherwise
stated. Solutions in organic solvents were dried with anhy-
drous Na2SO4. Solvents were evaporated under reduced pres-
sure on a rotary evaporator. Thin-layer chromatography was
carried out on aluminum-backed silica gel plates (Merck 60
F
254) with visualization of components by UV light (254 nm)
or exposure to I2. Column chromatography was carried out on
silica gel (Merck 230-400 mesh). All compounds designated
for testing were analyzed at >99% purity by reverse phase
HPLC using an Agilent 1100 liquid chromatograph, an Alltima
C
18 (5 µm) stainless steel column (150 mm × 3.2 mm i.d.), and
an Agilent 1100 diode array detector. Chromatograms were
run using various gradients of aqueous (0.045 M ammonium
formate and formic acid at pH 3.5) and organic (80% MeCN/
MilliQ water) phases. DCM refers to dichloromethane; DME
refers to dimethoxyethane; DMF refers to dry dimethylform-
amide; ether refers to diethyl ether; EtOAc refers to ethyl
acetate; EtOH refers to ethanol; MeOH refers to methanol;
pet. ether refers to petroleum ether, boiling range 40-60 °C;
and THF refers to tetrahydrofuran dried over sodium benzo-
phenone ketyl. All solvents were freshly distilled.
N-{3-[{3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r o p y l}(m e t h y l)a m in o ]p r o p y l}-8-q u in o lin e c a r b o x -
a m id e (7). A solution of 8-quinolinecarboxylic acid (22) (90
mg, 0.5 mmol) and CDI (97 mg, 0.6 mmol) in DMF (5 mL) was
stirred at 55 °C for 24 h. The solution was diluted with dry
benzene (10 mL), Sephadex LH-20 (300 mg) was added, and
the mixture was stirred at 20 °C for 1 h. The mixture was
filtered, the solvent was evaporated, and the residue was
dissolved in dry THF (5 mL). A solution of amine 21 (80 mg,
0.25 mmol) in THF (5 mL) was added and the solution stirred
at 20 °C for 70 h. The solvent was evaporated and the residue
purified by chromatography, eluting with a gradient (0-2%)
of aqueous NH3/(0-8%) MeOH/DCM, to give compound 7 (110
mg, 91%) as a red powder: mp (DCM/pet. ether) 119-121 °C;
1H NMR δ 11.39 (br s, 1 H, CONH), 8.96 (dd, J ) 4.3, 1.8 Hz,
1 H, ArH), 8.74 (dd, J ) 7.3, 1.5 Hz, 1 H, ArH), 8.34 (dd, J )
8.8, 1.8 Hz, 1 H, ArH), 8.25 (d, J ) 8.3 Hz, 1 H, ArH), 8.17 (d,
J ) 8.6 Hz, 1 H, ArH), 7.98 (br s, 1 H, NH), 7.92 (dd, J ) 8.1,
1.5 Hz, 1 H, ArH), 7.78 (dd, J ) 8.1, 1.1 Hz, 1 H, ArH), 7.62 (t,
J ) 7.7 Hz, 1 H, ArH), 7.48 (dd, J ) 8.3, 4.0 Hz, 1 H, ArH),
7.43 (dd, J ) 7.9, 1.0 Hz, 1 H, ArH), 3.68-3.73 (m, 4 H, 2 ×
CH2), 3.02-3.07 (m, 4 H, 2 × CH2), 2.67 (s, 3 H, CH3), 2.25-
2.17 (m, 4 H, 2 × CH2); 13C NMR δ 166.4, 149.7, 149.6, 145.4,
138.2, 137.7, 135.6, 133.6, 132.0, 130.3, 128.5, 128.4, 127.1,
126.4, 121.5, 121.0, 117.3, 54.7, 54.5, 40.6, 39.4, 37.2, 25.5, 24.5;
MS m/z 462 (MH+, 25%), 446 (5); HRMS calcd for C24H28N7O3
(MH+) m/z 462.2254, found 462.2249. Anal. (C24H27N7O3) C,
H, N.
N-{3-[{3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r o p y l}(m e t h y l)a m in o ]p r o p y l}-1-p h e n a zin e c a r b o x -
a m id e (8). To a solution of trifluoroacetamide 20 (283 mg,
0.7 mmol) in MeOH (10 mL) was added aqueous NH3 (6 mL)
and reaction mixture was stirred at 20 °C for 18 h. The solvent
was evaporated and the residue was dissolved in DMF (5 mL).
1-(1H-Imidazol-1-ylcarbonyl)phenazine (283 mg, 1.0 mmol),
prepared from 1-phenazinecarboxylic acid (23)32 and CDI, was
added and the mixture stirred at 20 °C for 48 h. The solvent
was evaporated and the residue was purified by chromatog-
raphy, eluting with a gradient (0-1%) of aqueous NH3/(0-
3%) MeOH/DCM to give compound 8 (293 mg, 82%) as a red
solid: mp (DCM/hexane) 129-130 °C; 1H NMR δ 10.85, (br s,
1 H, NH), 8.93 (dd, J ) 7.1, 1.4 Hz, 1 H, ArH), 8.52 (br s, 1 H,
NH), 8.33 (dd, J ) 8.7, 1.4 Hz, 1 H, ArH), 8.21-8.27 (m, 2 H,
ArH), 8.11 (d, J ) 8.7 Hz, 1 H, ArH), 7.93 (dd, J ) 8.6, 6.5 Hz,
1 H, ArH), 7.86-7.90 (m, 3 H, ArH), 7.70 (t, J ) 7.8 Hz, 1 H,
TPZ64 and BTO 647 were synthesized as previously de-
scribed.
N1-(3-Am in op r op yl)-N3-(1,4-d ioxid o-1,2,4-ben zotr ia zin -
3-yl)-N1-m eth yl-1,3-p r op a n ed ia m in e (21). 2,2,2-Tr iflu or o-
N-[3-(m eth yl{3-[(1-oxid o-1,2,4-ben zotr ia zin -3-yl)a m in o]-
pr opyl}am in o)pr opyl]acetam ide (19). A solution of chloride
1748 (2.07 g, 11.4 mmol), N1-(3-aminopropyl)-N1-methyl-1,3-
propanediamine (18) (3.31 g, 22.8 mmol), and Et3N (3.2 mL,
22.8 mmol) in DCM (200 mL) was stirred at 20 °C for 2 days.
The solvent was evaporated and the residue dissolved in MeCN
(150 mL). Ethyl trifluoroacetate (5.4 mL, 45.6 mmol) and water
(0.8 mL, 45.6 mmol) were added, and the solution was heated
at reflux temperature for 16 h. The solvent was evaporated
and the residue purified by chromatography, eluting with a
gradient (0-1%) of Et3N/(0-10%) MeOH/DCM, to give 1-oxide
19 (1.89 g, 43%) as a yellow solid: mp (DCM) 111-115 °C; 1H
NMR δ 9.04 (br s, 1 H, NH), 8.25 (dd, J ) 8.7, 1.4 Hz, 1 H,
H-8′), 7.70 (ddd, J ) 8.4, 7.1, 1.4 Hz, 1 H, H-6′), 7.57 (d, J )
8.4 Hz, 1 H, H-5′), 7.29 (ddd, J ) 8.7, 7.1, 1.1 Hz, 1 H, H-7′),
6.17 (br s, 1 H, NH), 3.58 (dd, J ) 6.6, 5.8 Hz, 2 H, CH2N),
3.49 (br t, J ) 6.0 Hz, 2 H, CH2N), 2.52-2.58 (m, 4 H, 2 ×
CH2N), 2.27 (s, 3 H, NCH3), 1.84-1.90 (m, 2 H, CH2), 1.75-
1.82 (m, 2 H, CH2); 13C NMR δ 158.9, 157.3 (q, J ) 36 Hz),
148.8, 135.6, 130.8, 126.4, 124.9, 120.4, 116.1 (q, J ) 288 Hz),
57.1, 56.4, 41.3, 40.3 (2), 26.3, 24.4; MS m/z 387 (MH+, 100%),
371 (8), 338 (30); HRMS calcd for C16H22F3N6O2 (MH+) m/z
387.1756, found 387.1765. Anal. (C16H21F3N6O2‚1/2MeOH) C,
H, N.
N-{3-[{3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r o p y l}(m e t h y l)a m in o ]p r o p y l}-2,2,2-t r iflu o r o a c e t -
a m id e (20). Trifluoroacetic anhydride (4.1 mL, 29.2 mmol)
was added to a stirred solution of 1-oxide 19 (1.13 g, 2.9 mmol)
in CHCl3 (50 mL) and the solution stirred at 20 °C for 30 min.
The solution was cooled to -10 °C and 70% H2O2 (2 mL)
(Ca u tion : vigorous reaction) added dropwise. The solution was
stirred at 20 °C for 3 days and partitioned between CHCl3 (50
mL) and saturated aqueous KHCO3 (50 mL). The aqueous
fraction was extracted with CHCl3 (3 × 30 mL), the combined

482 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Hay et al.
ArH), 7.42 (t, J ) 7.8 Hz, 1 H, ArH), 3.77 (q, J ) 6.4 Hz, 2 H,
CH2), 3.66 (q, J ) 5.7 Hz, 2 H, CH2), 2.68 (t, J ) 7.3 Hz, 2 H,
CH2), 2.62 (t, J ) 6.1 Hz, 2 H, CH2), 2.36 (s, 3 H, CH3), 2.10
(quin, J ) 7.1 Hz, 2 H, CH2), 1.89 (quin, J ) 6.2 Hz, 2 H, CH2);
13C NMR δ 165.0, 149.8, 143.4, 142.9, 141.4, 140.8, 138.2,
135.4, 135.1, 135.0, 133.4, 131.5, 130.9, 130.1, 130.1, 129.8,
129.0, 126.7, 121.6, 117.1, 56.7, 55.9, 42.1, 41.5, 38.2, 27.5, 25.6;
DCM, to give compound 11 (130 mg, 90%) as a red powder:
mp (DCM/pet. ether) 138-142 °C; 1H NMR δ 11.23 (br s, 1 H,
CONH), 8.84 (d, J ) 6.6 Hz, 1 H, ArH), 8.29 (d, J ) 7.6 Hz, 1
H, ArH), 8.07 (d, J ) 8.5 Hz, 1 H, ArH), 8.04 (d, J ) 8.5 Hz,
1 H, ArH), 7.98 (d, J ) 8.6 Hz, 1 H, ArH), 7.85 (t, J ) 7.8 Hz,
1 H, ArH), 7.71-7.78 (m, 3 H, ArH, NH), 6.48 (t, J ) 7.6 Hz,
1 H, ArH), 7.31 (t, J ) 7.7 Hz, 1 H, ArH), 3.71-3.78 (m, 4 H,
2 × CH2), 3.12-3.18 (m, 4 H, 2 × CH2), 2.88 (s, 3 H, CH3),
2.73 (br s, 3 H, CH3), 2.30-2.36 (m, 2 H, CH2), 2.19-2.24 (m,
2 H, CH2); 13C NMR δ 165.6, 149.6, 143.2, 142.9, 140.7, 139.4,
137.9, 136.4, 135.4, 135.1, 133.7, 131.3, 131.2, 130.1, 129.7,
128.5, 127.7, 127.0, 121.3, 116.9, 54.9, 54.2, 40.2, 38.9, 37.3,
25.7, 24.3, 18.1; HRMS calcd for C28H31N8O3 (MH+) m/z
527.2519 found 527.2533. Anal. (C28H30N8O3‚13/4H2O) C, H, N.
HRMS calcd for
513.2365. Anal. (C27H28N8O3) C, H, N.
C
27H29N8O3 (MH+) m/z 513.2363, found
N-{3-[{3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r op yl}(m eth yl)a m in o]p r op yl}-2-(4-p yr id yl)-8-qu in olin e-
ca r b oxa m id e (9). A solution of 2-(4-pyridyl)-8-quinoline-
carboxylic acid (24)35 (160 mg, 0.6 mmol) and CDI (150 mg,
0.9 mmol) in DMF (10 mL) was stirred at 55 °C for 24 h. The
solution was cooled to 20 °C, diluted with dry benzene (15 mL),
Sephadex LH-20 (300 mg) was added, and the mixture was
stirred at 20 °C for 1 h. The mixture was filtered and the
solvent evaporated. The residue was dissolved in dry THF (5
mL), a solution of amine 21 (90 mg, 0.3 mmol) in THF (5 mL)
added, and the solution stirred at 20 °C for 4 days. The solvent
was evaporated and the residue purified by chromatography,
eluting with a gradient (0-2%) of aqueous NH3/(0-8%) MeOH/
DCM, to give compound 9 (160 mg, 94%) as a red powder: mp
(DCM/pet. ether) 179-181 °C; 1H NMR δ 11.08 (br s, 1 H,
CONH), 8.86 (dd, J ) 4.5, 1.6 Hz, 2 H, ArH), 8.78 (dd, J )
7.4, 1.5 Hz, 1 H, ArH), 8.37 (d, J ) 8.6 Hz, 1 H, ArH), 8.21 (d,
J ) 8.6 Hz, 1 H, ArH), 8.10 (d, J ) 8.6 Hz, 1 H, ArH), 7.95
(dd, J ) 8.2, 1.4 Hz, 1 H, ArH), 7.92-7.90 (m, 4 H, NH, 3 ×
ArH), 7.98 (ddd, J ) 8.6, 7.5, 1.3 Hz, 1 H, ArH), 7.66 (t, J )
7.7 Hz, 1 H, ArH) 7.40 (ddd, J ) 8.6, 7.2, 1.2 Hz, 1 H, ArH),
3.74 (q, J ) 6.4 Hz, 2 H, CH2), 3.59-3.63 (m, 2 H, CH2), 2.83-
2.87 (m, 2 H, CH2), 2.79-2.84 (m, 2 H, CH2), 2.45 (s, 3 H, CH3),
2.17 (q, J ) 7.2 Hz, 2 H, CH2), 1.96-2.00 (m, 2 H, CH2); 13C
NMR δ 166.1, 154.5, 150.9 (2), 149.7, 146.2, 145.3, 139.0, 138.2,
135.5, 134.4, 131.5, 130.2, 129.4, 127.9, 127.2, 126.9, 121.7,
121.5, 118.7 (2), 117.2, 55.3, 55.2, 41.0, 40.1, 37.7, 26.6, 24.7;
N-{3-[{3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r op yl}(m e t h yl)a m in o]p r op yl}-5-m e t h yl-4-a cr id in e -
ca r boxa m id e (12). A solution of 5-methylacridine-4-carbox-
ylic acid (27)30 (0.13 g, 0.55 mmol) and CDI (0.21 g, 1.3 mmol)
in DMF (5 mL) was stirred at 55 °C for 24 h. The solution
was diluted with dry benzene (10 mL), Sephadex LH-20 (300
mg) was added, and the mixture stirred at 20 °C for 1 h. The
mixture was filtered and the solvent evaporated. The residue
was dissolved in dry THF (5 mL), a solution of 21 (80 mg, 0.27
mmol) in THF (5 mL) added, and the solution stirred at 20 °C
for 70 h. The solvent was evaporated and the residue purified
by chromatography, eluting with a gradient (0-2%) of aqueous
NH3/(0-8%) MeOH/DCM, to give compound 12 (0.13 g, 88%)
1
as a red powder: mp (DCM/pet. ether) 158-162 °C; H NMR
δ 12.08 (br s, 1 H, CONH), 8.83 (d, J ) 6.9 Hz, 1 H, ArH),
8.76 (s, 1 H, NH), 8.06 (t, J ) 8.9 Hz, 2 H, ArH), 7.97 (br d, J
) 8.4 Hz, 2 H, ArH), 7.83 (d, J ) 8.4 Hz, 1 H, ArH), 7.66 (d,
J ) 6.7 Hz, 1 H, ArH), 7.56-7.63 (m, 2 H, ArH), 7.46 (dd, J )
7.6, 6.5 Hz, 1 H, ArH), 7.30 (d, J ) 7.9 Hz, 1 H, ArH), 3.77 (q,
J ) 6.3 Hz, 2 H, CH2), 4.80-4.85 (m, 2 H, CH2), 3.06-3.10
(m, 4 H, 2 × CH2), 2.83 (s, 3 H, CH3), 2.67 (br s, 3 H, CH3),
2.30-2.35 (m, 2 H, CH2), 2.13-2.17 (m, 2 H, CH2); 13C NMR
δ 166.5, 149.6, 146.9, 145.1, 137.9 (2), 135.8, 135.3, 135.1,
132.4, 131.2, 130.0, 127.9, 126.8, 126.4, 126.3, 126.2, 125.8,
125.2, 121.3, 117.0, 55.1, 54.5, 40.5, 39.2, 37.4, 26.1, 24.5, 19.0;
HRMS calcd for
C
29H31N8O3 (MH+) m/z 539.2519, found
539.2527. Anal. (C29H30N8O3‚1/2H2O) C, H, N.
N-{3-[{3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r op yl}(m eth yl)a m in o]p r op yl}-4-a cr id in eca r boxa m id e
(10). A solution of amine 21 in DCM (5 mL) was added to a
stirred solution of 4-(1H-imidazol-1-ylcarbonyl)acridine (125
mg, 0.46 mmol), prepared from 4-acridinecarboxylic acid (25)49
and CDI, in THF (20 mL), and the solution was stirred at 20
°C for 16 h. The solvent was evaporated and the residue
purified by chromatography, eluting with a gradient (0-1%)
of Et3N/(0-15%) MeOH/DCM, to give compound 10 (146 mg,
HRMS calcd for
C
29H32N7O3 (MH+) m/z 526.2593, found
526.2582. Anal. (C29H31N7O3‚1/2H2O) C, H, N.
N-(2-{2-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
eth oxy}eth yl)-4-a cr id in eca r boxa m id e (13). 3-{[2-(2-Hy-
d r oxyet h oxy)et h yl]a m in o}-1,2,4-b en zot r ia zin e 1-Oxid e
(29). A solution of chloride 17 (3.0 g, 16.5 mmol) in DCM (50
mL) was added to a stirred solution of 2-(aminoethoxy)ethanol
(28) (2.49 mL, 24.8 mmol) and Et3N (3.45 mL, 24.8 mmol) in
DCM (80 mL) and the solution stirred at 20 °C for 16 h. The
solvent was evaporated and the residue purified by chroma-
tography, eluting with 40% EtOAc/DCM, to give 1-oxide 29
(2.62 g, 63%) as a yellow powder: mp (DCM/EtOAc) 131-131.5
1
66%) as a red solid: mp (EtOAc/DCM) 169-171 °C; H NMR
[(CD3)2SO] δ 11.41 (t, J ) 5.3 Hz, 1 H, CONH), 9.31 (s, 1 H,
H-9), 8.69 (dd, J ) 7.0, 1 4 Hz, 1 H, H-3), 8.43 (t, J ) 5.6 Hz,
1 H, NH), 8.38 (d, J ) 7.4 Hz, 1 H, H-1), 8.32 (d, J ) 8.8 Hz,
1 H, H-5), 8.21 (d, J ) 8.4 Hz, 1 H, H-8), 8.16 (d, J ) 8.7 Hz,
1 H, H-8′), 8.09 (d, J ) 8.7 Hz, 1 H, H-5′), 7.96 (ddd, J ) 8.7,
7.1, 1.1 Hz, 1 H, H-6′), 7.91 (dd, J ) 8.8, 7.5 Hz, 1 H, H-6),
7.74 (dd, J ) 7.4, 7.0 Hz, 1 H, H-2), 7.69 (br dd, J ) 8.7, 7.1
Hz, 1 H, H-7′), 7.55 (dd, J ) 8.4, 7.5 Hz, 1 H, H-7), 3.60-3.65
(m, 2 H, CH2N), 3.42-3.48 (m, 2 H, CH2N), 3.39 (s, 3 H, NCH3),
3.00-3.08 (m, 2 H, CH2N), 2.60-2.68 (m, 2 H, CH2N), 2.02-
2.08 (m, 2 H, CH2), 1.92-1.98 (m, 2 H, CH2); MS m/z 512 (MH+,
1
°C; H NMR δ 8.25 (dd, J ) 8.7, 1.2 Hz, 1 H, H-8), 7.68 (ddd,
J ) 8.4, 7.2, 1.5 Hz, 1 H, H-6), 7.57 (d, J ) 8.4 Hz, 1 H, H-5),
7.28 (ddd, J ) 8.7, 7.2, 1.3 Hz, 1 H, H-7), 6.02 (br s, 1 H, NH),
3.74-3.80 (m, 6 H, 3 × CH2O), 3.64-3.67 (m, 2 H, CH2N), 2.71
(t, J ) 5.9 Hz, 1 H, OH); 13C NMR δ 158.9, 149.7, 135.5, 130.9,
126.4, 124.9, 120.4, 72.4, 69.5, 61.7, 41.9. Anal. (C11H14N4O3)
C, H, N.
3-{[2-(2-Azid o e t h o xy )e t h yl]a m in o }-1,2,4-b e n zo t r i-
a zin e 1-Oxid e (30). Methanesulfonyl chloride (0.82 mL, 10.6
mmol) was added dropwise to a stirred solution of alcohol 29
(2.41 g, 9.6 mmol) and Et3N (1.74 mL, 12.5 mmol) in DCM
(100 mL) at 5 °C and the solution stirred at 20 °C for 1 h. The
solution was diluted with DCM (100 mL), washed with water
(3 × 50 mL) and brine (50 mL), and dried and the solvent
evaporated. The residue was dissolved in DMF (50 mL), and
NaN3 (0.69 g, 10.6 mmol) was added. The mixture was heated
at 100 °C for 2 h and cooled to 30 °C and the solvent
evaporated. The residue was partitioned between EtOAc (100
mL) and water (100 mL). The organic fraction was washed
with brine (50 mL) and dried and the solvent evaporated. The
residue was purified by chromatography, eluting with 50%
EtOAc/pet. ether, to give azide 30 (2.35 g, 89%) as yellow
25%), 496 (10); HRMS calcd for
512.2410, found 512.2424.
C
28H30N7O3 (MH+) m/z
N-{3-[{3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r op yl}(m et h yl)a m in o]p r op yl}-9-m et h yl-4-p h en a zin e-
ca r b oxa m id e (11). A solution of 9-methylphenazine-4-
carboxylic acid (26)32 (130 mg, 0.53 mmol) and CDI (100 mg,
0.61 mmol) in DMF (5 mL) was stirred at 55 °C for 6 h. The
solution was cooled to 20 °C, diluted with dry benzene (10 mL),
Sephadex LH-20 (300 mg) was added, and the mixture was
stirred at 20 °C for 1 h. The mixture was filtered and the
solvent evaporated. The residue was dissolved in dry THF (5
mL), a solution of 21 (80 mg, 0.26 mmol) in THF (5 mL) added,
and the solution stirred at 20 °C for 24 h. The solvent was
evaporated and the residue purified by chromatography,
eluting with a gradient (0-2%) of aqueous NH3/(0-8%) MeOH/
1
crystals: mp (EtOAc/pet. ether) 102-104 °C; H NMR δ 8.27

DNA-Targeted 1,2,4-Benzotriazine 1,4-Dioxides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 483
(dd, J ) 8.7, 1.4 Hz, 1 H, H-8), 7.70 (ddd, J ) 8.6, 7.1, 1.5 Hz,
1 H, H-6), 7.59 (d, J ) 8.6 Hz, 1 H, H-5), 7.29 (ddd, J ) 8.6,
7.1, 1.4 Hz, 1 H, H-7), 5.70 (br s, 1 H, NH), 3.71-3.78 (m, 4 H,
2 × CH2O), 3.69 (dd, J ) 5.3, 4.8 Hz, 2 H, CH2N3), 3.41 (dd, J
) 5.1, 4.9 Hz, 2 H, CH2N); 13C NMR δ 158.9, 148.7, 135.5,
131.1, 126.5, 125.0, 120.4, 70.0, 69.6, 50.7, 41.1. Anal.
(C11H13N7O2) C, H, N.
dropwise to a stirred solution of 4-(1H-imidazol-1-ylcarbonyl)-
acridine (58 mg, 0.21 mmol), prepared from 4-acridinecarbox-
ylic acid (25) and CDI, in THF (5 mL) at 5 °C, and the solution
was stirred at 20 °C for 16 h. The solvent was evaporated and
the residue purified by chromatography, eluting with a gradi-
ent (0-5%) of MeOH/DCM, to give compound 13 (93 mg, 97%)
1
as a red solid: mp (EtOAc) 98-100 °C; H NMR δ 12.14 (s, 1
H, CONH), 8.96 (dd, J ) 7.1, 1.5 Hz, 1 H, H-3), 8.82 (s, 1 H,
H-9), 8.25 (d, J ) 8.4 Hz, 1 H, H-8′), 8.16 (d, J ) 8.4 Hz, 1 H,
H-5′), 8.11-8.13 (m, 2 H, H-1, H-5), 7.94 (d, J ) 8.2 Hz, 1 H,
H-8), 7.76-7.84 (m, 2 H, H-6, H-6′), 7.66 (dd, J ) 8.4, 7.1 Hz,
1 H, H-2), 7.44-7.52 (m, 2 H, H-7, H-7′), 7.36 (br s, 1 H, NH),
3.85-3.95 (m, 8 H, 2 × CH2O, 2 × CH2N); 13C NMR δ 166.1,
149.8, 147.2, 146.3, 138.1, 137.6, 135.5, 135.3, 132.4, 131.3,
130.4, 128.8, 128.3, 128.0, 127.1, 126.8, 126.2, 125.8, 125.4,
121.5, 117.3, 70.2, 68.9, 41.1, 39.5; MS m/z 471 (MH+, 5%),
455 (4); HRMS calcd for C25H23N6O4 (MH+) m/z 471.1781,
found 471.1790. Anal. (C25H22N6O4‚1/2H2O) C, H, N.
N -{2-[[2-(1,4-Dioxid o-1,2,4-b e n zot r ia zin -3-yl)e t h yl]-
(m eth yl)a m in o]eth yl}-4-a cr id in eca r boxa m id e (14). ter t-
Bu tyl 2-[(2-Am in oeth yl)(m eth yl)a m in o]eth ylca r ba m a te
(35). A solution of di-tert-butyl dicarbonate (9.60 g, 44 mmol)
in THF (50 mL) was added dropwise to a solution of bis-
(diethylamino)methylamine (34) (10.32 g, 88 mmol) in THF
(50 mL) at 5 °C, and the reaction mixture was stirred at 20
°C for 20 h. The reaction mixture was partitioned between
DCM (200 mL) and saturated brine (100 mL). The organic
layer was separated and the aqueous layer further extracted
with DCM (3 × 25 mL). The combined organic extracts were
dried, and the solvent was evaporated to give amine 35 (8.79
g, 46%) as a colorless oil, which was used directly without
further purification.
3-{[2-(2-ter t-Bu tyloxyca r ba m oyleth oxy)eth yl]a m in o}-
1,2,4-ben zotr ia zin e 1-Oxid e (31). Propane-1,3-dithiol (5.7
mL, 57.0 mmol) was added dropwise to a stirred solution of
azide 30 (1.57 g, 5.7 mmol) and Et3N (8.0 mL, 57.0 mmol) in
MeOH (100 mL) under N2 and the solution heated at reflux
temperature for 8 h. The solution was cooled to 20 °C and
partitioned between 1 M HCl (100 mL) and Et2O (100 mL).
The aqueous fraction was adjusted to pH 12 with 7 M NaOH
solution and extracted with DCM (3 × 50 mL). The organic
fraction was dried and the solvent evaporated. The residue
was dissolved in THF (100 mL), and a solution of di-tert-butyl
dicarbonate (1.87 g, 8.6 mmol) in THF (50 mL) was added
dropwise. The solution was stirred at 20 °C for 16 h, the solvent
evaporated, and the residue purified by chromatography,
eluting with 40% EtOAc/pet. ether, to give carbamate 31 (1.85
g, 93%) as a yellow solid: mp (EtOAc/pet. ether) 134-137 °C;
1H NMR δ 8.26 (dd, J ) 8.4, 0.9 Hz, 1 H, H-8), 7.71 (ddd, J )
8.3, 7.1, 1.4 Hz, 1 H, H-6), 7.59 (d, J ) 8.3 Hz, 1 H, H-5), 7.29
(ddd, J ) 8.4, 7.1, 1.3 Hz, 1 H, H-7), 5.74 (br s, 1 H, NH), 4.93
(br s, 1 H, NH), 3.67-3.73 (m, 4 H, 2 × CH2O), 3.56 (t, J ) 5.2
Hz, 2 H, CH2N), 3.29-3.36 (m, 2 H, CH2N), 1.45 [s, 9 H,
C(CH3)3]; 13C NMR δ 159.9, 155.9, 148.7, 135.5, 131.0, 126.5,
125.0, 120.4, 79.4, 70.2, 69.2, 41.1, 40.4, 28.4 (3). Anal.
(C16H23N5O4) C, H, N.
3-{[2-(2-ter t-Bu tyloxyca r ba m oyleth oxy)eth yl]a m in o}-
1,2,4-ben zotr ia zin e 1,4-Dioxid e (32). A solution of MCPBA
(1.57 g, 6.4 mmol) in DCM (50 mL) was added dropwise to a
stirred solution of carbamate 31 (1.85 g, 5.3 mmol) in DCM
(100 mL) and NaHCO3 (0.89 g, 10.6 mmol), and the mixture
was stirred at 20 °C for 6 h. The suspension was filtered
through Celite, the solvent evaporated, and the residue
purified by chromatography, eluting with a gradient (0-5%)
of MeOH/(40-0%) EtOAc/DCM, to give (i) starting material
31 (926 mg, 50%) and (ii) 1,4-dioxide 32 (702 mg, 40%) as a
ter t-Bu tyl 2-[[2-(1-Oxid o-1,2,4-ben zotr ia zin -3-yl)eth yl]-
(m eth yl)a m in o]eth ylca r ba m a te (36). A solution of chloride
17 (2.0 g, 11.0 mmol), amine 35 (2.90 g, 13.3 mmol), and
triethylamine (3.0 mL, 22.1 mmol) in DME (20 mL) was heated
at 90 °C for 3 h. The solvent was evaporated and the residue
partitioned between DCM (100 mL) and aqueous NH3 (50 mL).
The aqueous layer was extracted further with DCM (3 × 30
mL), the combined organic fraction dried, and the solvent
evaporated. The residue was purified by chromatography,
eluting with a gradient (0-1%) of aqueous NH3/(0-5%) MeOH/
DCM, to give (i) starting material 35 (500 mg, 25%) and (ii)
1-oxide 36 (2.1 g, 52%) as a yellow solid: mp (DCM/hexane)
1
red solid: mp (EtOAc) 139-140 °C; H NMR δ 8.33 (d, J )
8.7 Hz, 1 H, H-8), 8.30 (d, J ) 8.7 Hz, 1 H, H-5), 7.88 (ddd, J
) 8.7, 7.2, 1.2 Hz, 1 H, H-6), 7.43-7.50 (m, 2 H, H-7, NH),
5.06 (br s, 1 H, NH), 3.78-3.83 (m, 2 H, CH2O), 3.69 (dd, J )
5.1, 5.0 Hz, 2 H, CH2O), 3.56 (dd, J ) 5.1, 5.0 Hz, 2 H, CH2N),
3.29-3.36 (m, 2 H, CH2N), 1.43 [s, 9 H, C(CH3)3]; 13C NMR δ
156.0, 149.8, 138.5, 135.9, 130.6, 129.5, 121.6, 117.4, 79.4, 70.3,
68.9, 41.3, 40.3, 28.3 (3); MS m/z 366 (MH+, 40%), 350 (5) 310
(20); HRMS calcd for C16H24N5O5 (MH+) m/z 366.1777, found
366.1767. Anal. (C16H23N5O5‚1/2H2O) C, H, N.
1
122-124 °C; H NMR [(CD3)2SO] δ 8.13 (dd, J ) 8.6, 1.0 Hz,
1 H, H-8), 7.78 (ddd, J ) 8.4, 7.0, 1.6 Hz, 1 H, H-6), 7.71 (br
s, 1 H, NH), 7.52 (d, J ) 8.4 Hz, 1 H, H-5), 7.33 (ddd, J ) 7.8,
7.1, 1.2 Hz, 1 H, H-7), 6.61 (br s, 1 H, NH), 3.43 (q, J ) 6.0
Hz, 2 H, CH2), 3.01 (q, J ) 6.2 Hz, 2 H, CH2), 2.57 (t, J ) 6.6
Hz, 2 H, CH2), 2.42 (t, J ) 6.7 Hz, 2 H, CH2), 2.23 (s, 3 H,
CΗ3), 1.35 [s, 9 H, C(CH3)3]; 13C NMR [(CD3)2SO] δ 158.8,
155.4, 148.2, 135.6, 129.9, 126.0, 124.4, 119.8, 77.4, 56.4, 55.
5, 41.8, 38.5, 37.8, 28.1 (3); HRMS (CI+) calcd for C17H27N6O3
(MH+) m/z 363.2145, found 363.2144. Anal. (C17H26N6O3) C,
H, N.
3-{[2-(2-Am in oe t h oxy)e t h yl]a m in o}-1,2,4-b e n zot r i-
a zin e 1,4-Dioxid e (33). Trifluoroacetic acid (1.66 mL, 34.6
mmol) was added dropwise to a stirred solution of 1,4-dioxide
32 (632 mg, 1.7 mmol) in DCM (50 mL) and the solution stirred
at 20 °C for 16 h. The solvent was evaporated and the residue
partitioned between saturated aqueous KHCO3 solution (100
mL) and CHCl3 (100 mL). The aqueous phase was extracted
with CHCl3 (8 × 50 mL), the combined organic fraction dried,
and the solvent evaporated. The residue was recrystallized to
give the amine 33 (406 mg, 91%) as a red solid: mp (CHCl3)
124 °C (dec); 1H NMR δ 8.26 (d, J ) 8.9 Hz, 1 H, H-8), 8.23 (d,
J ) 8.9 Hz, 1 H, H-5), 7.79 (dd, J ) 8.8, 7.8 Hz, 1 H, H-6),
7.45 (dd, J ) 8.9, 7.7 Hz, 1 H, H-7), 3.75 (dd, J ) 5.0, 4.8 Hz,
2 H, CH2O), 3.66 (dd, J ) 5.0, 4.9 Hz, 2 H, CH2O), 3.47 (dd, J
) 5.1, 5.0 Hz, 2 H, CH2N), 2.82 (dd, J ) 5.1, 5.0 Hz, 2 H,
CH2N), NH and NH2 not observed; 13C NMR δ 149.8, 138.3,
135.8, 130.5, 127.2, 121.6, 117.4, 73.0, 68.9, 41.7, 41.3; MS m/z
266 (MH+, 20%), 250 (5); HRMS calcd for C11H16N5O3 (MH+)
m/z 266.1253, found 266.1230. Anal. (C11H15N5O3‚1/4H2O) C,
H, N.
N1-[2-(1-Oxid o-1,2,4-ben zotr ia zin -3-yl)eth yl]-N1-m eth -
yl-1,2-eth a n ed ia m in e (37). Carbamate 36 (2.14 g, 5.9 mmol)
was dissolved in methanolic HCl (30 mL) and stirred for 20 h
at 20 °C. The solvent was evaporated and the residue
partitioned between DCM (100 mL) and dilute aqueous NH3
(100 mL), and the aqueous layer was further extracted with
DCM (4 × 30 mL). The combined organic fraction was dried,
and the solvent evaporated to give amine 37 (1.55 g, 100%) as
yellow solid which was used directly without further purifica-
tion: 1H NMR [(CD3)2SO] δ 8.13 (dd, J ) 8.6, 1.3 Hz, 1 H,
H-8), 7.76-7.80 (m, 2 H, H-6, NH), 7.57 (d, J ) 8.4 Hz, 1 H,
H-5), 7.33 (ddd, J ) 7.8, 7.1, 1.3 Hz, 1 H, H-7), 3.42-3.46 (m,
2 H, CH2), 3.27-3.30 (m, 2 H, CH2), 2.58 (q, J ) 6.9 Hz, 2 H,
CH2), 2.37 (t, J ) 6.5 Hz, 2 H, CH2), 2.22 (s, 3 H, CH3), 1.40-
1.45 (m, 2 H, NH2).
N-{2-[[2-(1-Oxid o-1,2,4-ben zotr ia zin -3-yl)eth yl](m eth -
yl)a m in o]eth yl}-2,2,2-tr iflu or oa ceta m id e (38). Ethyl tri-
fluoroacetate (2.1 mL, 17.2 mmol) and water (0.3 mL, 17.2
mmol) were added to a solution of amine 37 (1.5 g, 5.7 mmol)
N-(2-{2-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
eth oxy}eth yl)-4-a cr id in eca r boxa m id e (13). A solution of
the amine 33 (54 mg, 0.20 mmol) in THF (2 mL) was added

484 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Hay et al.
in CH3CN (50 mL), and the reaction mixture was heated at
reflux temperature for 48 h. The solvent was evaporated and
the residue partitioned between DCM (100 mL) and aqueous
NaHCO3 solution (100 mL). The aqueous layer was further
extracted with DCM (3 × 30 mL), the combined organic
fraction dried, and the solvent evaporated to give trifluoro-
acetamide 38 (1.80 g, 88%) as a yellow solid: mp (DCM/
N-[2-({2-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
et h yl}a m in o)et h yl]-4-a cr id in eca r b oxa m id e (15). ter t-
Bu tyl Bis-(2-a m in oeth yl)ca r ba m a te (41). Diethylenetri-
amine 40 (9.9 mL, 96 mmol) was added to a solution of ethyl
trifluoroacetate (22.8 mL, 192 mmol) in dry ether (80 mL) at
5 °C, and the reaction mixture was stirred at 20 °C for 20 h.
The resulting white precipitate was filtered, washed with
cold ether (100 mL), and dried under vacuum to give 2,2,2-
trifluoro-N-[2-({2-[(trifluoroacetyl)amino]ethyl}amino)ethyl]-
acetamide (17.26 g, 61%): 1H NMR [(CD3)2SO] δ 7.26 (br s, 2
H, 2 × CÃΝΗ), 3.43 (br s, 4 H, 2 × CH2), 2.86 (br t, J ) 5.8
Hz, 4 H, 2 × CH2); 13C NMR [(CD3)2SO] δ 157.7 (q, J ) 37
Hz), 115.8 (q, J ) 288 Hz), 47.3 (2), 39.3 (2).
Di-tert butyl dicarbonate (8.26 g, 37.8 mmol) was added to
a solution of acetamide (10.15 g, 34.4 mmol) in THF (100 mL)
at 0 °C, and the mixture was stirred at 20 °C for 20 h.
Saturated aqueous NH4Cl (80 mL) was added and the mixture
stirred at 20 °C for 5 h. The mixture was extracted with DCM
(3 × 50 mL) and dried, and the solvent was evaporated to give
tert-butyl bis{2-[(trifluoroacetyl)amino]ethyl}carbamate (13.5
g, 100%), which was used without further purification: 1H
NMR [(CD3)2SO] δ 9.47 (br s, 1 H, CONH), 9.40 (br s, 1 H,
CONH), 3.27-3.33 (m, 8 H, 4 × CH2), 1.38 [s, 9 H, C(CΗ3)3];
13C NMR [(CD3)2SO] δ 156.4 (q, J ) 36 Hz), 154.7, 115.8 (q, J
) 288 Hz), 78.9, 45.4, 45.0, 37.7, 37.4, 27.7 (3).
Aqueous NH3 (50 mL) was added to a solution of carbamate
(14.0 g, 35.5 mmol) in MeOH (100 mL) and heated at reflux
temperature for 20 h. The solvent was evaporated to give
diamine 41 as a yellow foam: 1H NMR [(CD3)2SO] δ 3.39 (t, J
) 6.4 Hz, 4 H, 2 × CH2), 2.94 (t, J ) 6.4 Hz, 4 H, 2 × CH2),
1.42 [s, 9 H, C(CH3)3]; 13C NMR [(CD3)2SO] δ 154.9, 79.9, 45.1
(2), 37.4 (2), 27.9 (3).
Di-ter t-b u t yl 2-Am in oet h yl{2-[(1-oxid o-1,2,4-b en zo-
tr ia zin -3-yl)a m in o]eth yl}d ica r ba m a te (42). A solution of
chloride 17 (1.0 g, 5.5 mmol), diamine 41 (4.47 g, 22.0 mmol),
and Et3N (3.07 mL, 22.0 mmol) in DME (20 mL) was heated
at 90 °C for 3 h. The solvent was evaporated and the residue
purified by chromatography, eluting with a gradient (0-4%)
of MeOH/DCM, to give (i) tert-butyl bis{2-[(1-oxido-1,2,4-
benzotriazin-3-yl)amino]ethyl}carbamate (0.34 g, 25%) [1H
NMR δ 8.17 (br d, J ) 8.5 Hz, 2 H, H-8, H-8′), 7.62-7.71 (m,
2 H, H-6, H-6′), 7.52 (br d, J ) 8.3 Hz, 2 H, H-5, H-5′), 7.22-
7.26 (m, 2 H, H-7, H-7′), 6.15 (br s, 1 H, NH), 5.95 (br s, 1 H,
NH), 3.71 (q, J ) 5.8 Hz, 4 H, 2 × CH2), 3.37 (br s, 4 H,
2 × CH2), 1.50 [s, 9 H, C(CΗ3)3]; 13C NMR δ 158.9, 156.3 (2),
148.6 (2), 135.5 (2), 130.9 (2), 126.4 (2), 124.9 (2), 120.3 (2),
80.7, 47.7 (2), 40.9 (2), 28.4 (3).] and (ii) crude tert-butyl
2-aminoethyl{2-[(1-oxido-1,2,4-benzotriazin-3-yl)amino]ethyl}-
carbamate (1.38 g, 72%) as a yellow foam.
1
hexane) 141-143 °C; H NMR [(CD3)2SO] δ 9.29 (br s, 1 H,
NH), 8.13 (dd, J ) 8.6, 1.3 Hz, 1 H, H-8), 7.78 (ddd, J ) 8.4,
7.0, 1.5 Hz, 1 H, H-6), 7.68 (br s, 1 H, NH), 7.57 (d, J ) 8.4
Hz, 1 H, H-5), 7.33 (ddd, J ) 8.5, 7.1, 1.3 Hz, 1 H, H-7), 3.42
(q, J ) 6.3 Hz, 2 H, CH2), 3.30 (q, J ) 6.8 Hz, 2 H, CH2), 2.61
(t, J ) 6.7 Hz, 2 H, CH2), 2.56 (t, J ) 6.7 Hz, 2 H, CH2), 2.27
(s, 3 H, CH3); 13C NMR [(CD3)2SO] δ 158.8, 156.1 (q, J ) 36
Hz) 148.3, 135.6, 130.0, 125.9, 124.4, 119.8, 115.8 (q, J ) 288
Hz), 55.4, 55.1, 41.7, 38.4, 37.2; HRMS calcd for C14H18F3N6O2
(MH+) m/z 359.1443, found 359.1451. Anal. (C14H17F3N6O2) C,
H, N, F.
N -{2-[[2-(1,4-Dioxid o-1,2,4-b e n zot r ia zin -3-yl)e t h yl]-
(m eth yl)a m in o]eth yl}-2,2,2-tr iflu or oa ceta m id e (39). Hy-
drogen peroxide (70%, 2.0 mL, 48.8 mmol) was added dropwise
to a stirred solution of trifluoroacetic anhydride (6.78 mL, 48.8
mmol) in DCM (20 mL) at 5 °C and the solution stirred at 5
°C for 15 min. The solution was added to a solution of
trifluoroacetamide 38 (1.75 g, 4.9 mmol) and trifluoroacetic
acid (0.80 mL, 9.8 mmol) in DCM (20 mL) and the reaction
mixture stirred at 20 °C for 5 h. The reaction mixture was
slowly added to a cooled solution of aqueous NaHCO3 (100 mL).
The organic layer was separated and the aqueous layer was
further extracted with DCM (5 × 30 mL). The combined
organic fraction was dried, the solvent was evaporated, and
the residue was purified by chromatography, eluting with a
gradient (0-4%) of MeOH/DCM, to give (i) starting material
38 (100 mg, 6%) and (ii) 1,4-dioxide 39 (859 mg, 47%) as a red
1
solid: mp (DCM/hexane) 141-144 °C; H NMR [(CD3)SO] δ
9.28 (br s, 1 H, NH), 8.20 (d, J ) 9.1 Hz, 1 H, H-5), 8.12 (d, J
) 8.6 Hz, 1 H, H-8), 8.03 (t, J ) 5.8 Hz, 1 H, NH), 7.96 (ddd,
J ) 8.6, 7.2, 1.3 Hz, 1 H, H-6), 7.56 (ddd, J ) 8.6, 7.1, 1.3 Hz,
1 H, H-7), 3.48 (q, J ) 6.3 Hz, 2 H, CH2), 3.31 (q, J ) 6.3 Hz,
2 H, CH2), 2.63, (t, J ) 6.6 Hz, 2 H, CH2), 2.54 (t, J ) 6.8 Hz,
2 H, CH2), 2.27 (s, 3 H, CH3); 13C NMR [(CD3)2SO] δ 156.1 (q,
J ) 36 Hz), 149.7, 138.0, 135.4, 129.9, 126.9, 121.0, 115.8 (q,
J ) 288 Hz), 116.7, 55.4, 55.0, 41.6, 38.3, 37.1; HRMS calcd
for C14H17F3N6O3 (MH+) m/z 375.1393, found 375.1392. Anal.
(C14H17F3N6O3) C, H, N.
N -{2-[[2-(1,4-Dioxid o-1,2,4-b e n zot r ia zin -3-yl)e t h yl]-
(m eth yl)a m in o]eth yl}-4-a cr id in eca r boxa m id e (14). Aque-
ous NH3 (6 mL) was added to a solution of dioxide 39 (125
mg, 0.33 mmol) in MeOH (6 mL) and the reaction mixture
stirred at 20 °C for 16 h. The solvent was evaporated, the
residue was dissolved in THF (5 mL) and 4-(1H-imidazol-1-
ylcarbonyl)acridine (180 mg, 0.66 mmol), prepared from 4-acri-
dinecarboxylic acid (25) and CDI, and the reaction was stirred
at 20 °C for 48 h. The solvent was evaporated, and the residue
was dissolved in DCM (20 mL) and washed with water (3 ×
15 mL). The organic fraction was dried, the solvent evaporated,
and the residue purified by chromatography, eluting with a
gradient (0-1%) of aqueous NH3/(0-3%) MeOH/DCM, to give
compound 14 (132 mg, 94%) as a red solid: mp (DCM/hexane)
Di-tert-butyl dicarbonate (2.7 g, 12.4 mmol) was added to a
solution of the crude carbamate (1.38 g, 4.0 mmol) in THF (50
mL) and the solution stirred at 20 °C for 36 h. Water (100
mL) was added and the mixture stirred at 20 °C for 1 h. The
mixture was extracted with DCM (3 × 50 mL), the organic
fraction dried, and the solvent evaporated. The residue was
purified by chromatography, eluting with a gradient (0-1%)
of aqueous NH3/(0-7%) MeOH/DCM, to give carbamate 42
(0.94 g, 52%) as a yellow powder: mp (DCM/hexane) 160-
1
163 °C; H NMR [(CD3)2SO] δ 8.14 (dd, J ) 8.3, 0.7 Hz, 1 H,
H-8), 8.00 and 7.92 (2 × br s, 1 H, CONH), 7.79 (dd, J ) 7.5,
1.2 Hz, 1 H, H-6), 7.58 (br d, J ) 7.5 Hz, 1 H, H-5), 7.34 (dd,
J ) 7.7, 1.2 Hz, 1 H, H-7), 6.81 (br s, 1 H, NH), 3.43-3.47 (m,
2 H, CH2), 3.37 (m, 2 H, CH2), 3.22 (t, J ) 6.2 Hz, 2 H, CH2),
3.03-3.07 (m, 2 H, CH2), 1.34 [s, 9 H, C(CH3)3], 1.34 and 1.27
[2 × s, 9 H, C(CH3)3]; 13C NMR [(CD3)2SO] δ 158.9, 155.5,
154.7, 148.2, 135.7, 130.1, 126.0, 124.6, 119.8, 78.4, 77.5, 47.0,
46.2, 38.5, 38.1, 28.1 (3), 27.8 (3). Anal. (C21H32N6O5) C, H, N.
1
160-162 °C; H NMR δ 11.90 (br s, 1 H, NH), 8.95 (dd, J )
7.1, 1.5 Hz, 1 H, ArH), 8.72 (s, 1 H, ArH), 8.16 (d, J ) 8.6 Hz,
1 H, ArH), 8.12 (d, J ) 8.8 Hz, 1 H, ArH), 8.07 (dd, J ) 8.4,
1.4 Hz, 1 H, ArH), 7.84 (t, J ) 8.3 Hz, 2 H, ArH), 7.78 (ddd, J
) 7.7, 6.7, 1.5 Hz, 1 H, ArH), 7.70 (ddd, J ) 7.8, 7.1, 1.3 Hz,
1 H, ArH), 7.64 (dd, J ) 8.2, 7.1 Hz, 1 H, ArH), 7.46 (ddd, J )
7.5, 7.2, 0.7 Hz, 1 H, ArH), 7.40 (ddd, J ) 7.9, 7.1, 1.3 Hz, 1
H, ArH), 7.29 (br, 1 H, NH), 3.86 (q, J ) 6.0 Hz, 2 H, CH2),
3.70 (q, J ) 5.6 Hz, 2 H, CH2), 2.93 (t, J ) 6.4 Hz, 2 H, CH2),
2.89 (t, J ) 6.1 Hz, 2 H, CH2), 2.57 (s, 3 H, CH3); 13C NMR δ
166.0, 149.7, 147.4, 146.5, 137.8, 137.4, 135.3, 135.0, 132.1,
131.1, 130.1, 128.8, 128.6, 127.9, 126.8, 126.7, 126.1, 125.8,
125.5, 121.5, 117.2, 56.5, 55.9, 42.5, 39.1, 37.8; HRMS calcd
for C26H26N7O3 (MH+) m/z 484.2097, found 484.2102.
Di-ter t-bu tyl 2-Am in oeth yl{2-[(1,4-d ioxid o-1,2,4-ben zo-
tr ia zin -3-yl)a m in o]eth yl}d ica r ba m a te (43). MCPBA (247
mg, 1.0 mmol) was added to a solution of 1-oxide 42 (300 mg,
0.67 mmol) in DCM (10 mL) and the mixture was stirred at
20 °C for 16 h. The mixture was partitioned between dilute
aqueous NH3 (50 mL) and DCM (50 mL), and the aqueous
fraction was extracted with DCM (3 × 30 mL). The combined

DNA-Targeted 1,2,4-Benzotriazine 1,4-Dioxides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 485
organic fraction was dried and the solvent evaporated. The
(MH+, 60%), 457 (10), 373 (100); HRMS calcd for C20H28F3N6O4
(MH+) m/z 473.2124, found 473.2136. Anal. (C20H27F3N6O4) C,
H, N.
residue was purified by chromatography, eluting with
a
gradient (0-2%) of MeOH/DCM, to give (i) starting material
42 (188 mg, 62%) and (ii) 1,4-dioxide 43 (122 mg, 39%): mp
(DCM/hexane) 128-134 °C; 1H NMR [(CD3)2SO] δ 8.39 and
8.32 (2 × br s, 1 H, CONH), 8.21 (dd, J ) 8.7, 0.7 Hz, 1 H,
H-8), 8.14 (t, J ) 8.0 Hz, 1 H, H-5), 7.94 (t, J ) 7.6 Hz, 1 H,
H-6), 7.57 (t, J ) 7.9 Hz, 1 H, H-7), 6.77 (br s, 1 H, NH), 3.50-
3.54 (m, 2 H, CH2), 3.41-3.44 (m, 2 H, CH2), 3.20 (t, J ) 6.5
Hz, 2 H, CH2), 3.03 (q, J ) 5.6 Hz, 2 H, CH2), 1.33 [s, 9 H,
C(CH3)3], 1.33 and 1.26 [2 × s, 9 H, C(CH3)3]; 13C NMR [(CD3)2-
SO] δ 155.5, 154.6, 149.8, 138.1, 135.5, 129.9, 127.0, 121.0,
116.8, 78.6, 77.4, 46.8, 46.1, 38.5, 38.0, 28.8 (3), 27.7 (3); HRMS
calcd for C21H33H6O6 (MH+) m/z 465.2462, found 465.2456.
ter t-Bu tyl 3-[(1,4-Dioxid o-1,2,4-ben zotr ia zin -3-yl)a m i-
n o]p r op yl{3-[(t r iflu or oa ce t yl)a m in o]p r op yl}ca r b a m -
a te (46). A solution of MCPBA (2.12 g, 8.6 mmol) in DCM (50
mL) was added dropwise to a stirred solution of 1-oxide 45
(3.13 g, 6.6 mmol) in DCM (250 mL) and NaHCO3 (1.1 g, 13.2
mmol). The mixture was stirred at 20 °C for 16 h and
partitioned between DCM (200 mL) and saturated aqueous
KHCO3 solution (100 mL). The organic fraction was dried, the
solvent evaporated, and the residue purified by chromatogra-
phy, eluting with a gradient (0-4%) of MeOH/40% EtOAc/
DCM, to give (i) starting material 45 (2.04 g, 65%) and (ii)
1,4-dioxide 46 (252 mg, 8%) as a red solid: 1H NMR δ 8.34 (d,
J ) 8.7 Hz, 1 H, H-8), 8.30 (d, J ) 8.4 Hz, 1 H, H-5), 8.25 (br
s, 1 H, NH), 7.88 (br dd, J ) 8.4, 7.0 Hz, 1 H, H-6), 7.52 (br
dd, J ) 8.7, 7.0 Hz, 1 H, H-7), 7.20 (br s, 1 H, NH), 3.62 (dt,
J ) 6.8, 6.7 Hz, 2 H, CH2N), 3.26-3.38 (m, 6 H, 3 × CH2N),
1.92-1.98 (m, 2 H, CH2), 1.73-1.79 (m, 2 H, CH2), 1.49 [s, 9
H, C(CH3)3]; 13C NMR δ 157.3 (q, J ) 37 Hz), 156.8, 149.8,
138.2, 135.9, 130.5, 127.4, 121.7, 117.4, 116.1 (q, J ) 288 Hz),
80.9, 44.4, 43.2, 38.9, 31.9, 29.7, 28.4 (3), 22.7; MS m/z 489
(MH+, 10%), 473 (12), 373 (15); HRMS calcd for C20H28F3N6O5
(MH+) m/z 489.2073, found 489.2086.
ter t-Bu tyl 3-Am in op r op yl{3-[(1,4-d ioxid o-1,2,4-ben zo-
tr ia zin -3-yl)a m in o]p r op yl}ca r ba m a te (47). A mixture of
trifluoroacetamide 46 (541 mg, 1.1 mmol) and K2CO3 (0.77 g,
5.5 mmol) in MeOH (20 mL) and water (5 mL) was heated at
reflux temperature for 1 h. The mixture was partitioned
between CHCl3 (50 mL) and water (30 mL). The aqueous
fraction was extracted with CHCl3 (3 × 30 mL), the combined
organic fraction dried, and the solvent evaporated to give
amine 47 (322 mg, 74%) as a red oil: 1H NMR [(CD3)2SO] δ
10.50 (br s, 1 H, NH), 8.21 (d, J ) 8.7 Hz, 1 H, H-8), 8.13 (d,
J ) 8.6 Hz, 1 H, H-5), 7.94 (br dd, J ) 8.6, 7.5 Hz, 1 H, H-6),
7.56 (br dd, J ) 8.6, 7.5 Hz, 1 H, H-7), 7.20 (br s, 2 H, NH2),
3.39 (t, J ) 6.9 Hz, 2 H, CH2N), 3.11-3.21 (m, 6 H, 3 × CH2N),
1.78-1.86 (m, 2 H, CH2), 1.49-1.58 (m, 2 H, CH2), 1.39 [s, 9
H, C(CH3)3]; 13C NMR [(CD3)2SO] δ 154.7, 149.7, 138.1, 135.4,
129.8, 127.9, 121.0, 116.7, 78.3, 44.3, 43.9, 38.8, 38.4, 32.2, 31.6,
27.9 (3); MS m/z 393 (MH+, 15%), 377 (9), 338 (3); HRMS calcd
for C18H29N6O4 (MH+) m/z 393.2250, found 393.2249.
N-[2-({2-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
eth yl}a m in o)eth yl]-4-a cr id in eca r boxa m id e (15). A solu-
tion of carbamate 43 (252 mg, 0.54 mmol) in HCl saturated
MeOH (10 mL) was stirred at 20 °C for 24 h. The solvent was
evaporated and the residue partitioned between aqueous NH3
(20 mL) and DCM (50 mL). The aqueous fraction was extracted
with DCM (5 × 20 mL) and the combined organic extract dried.
The solvent was evaporated to give crude N1-(2-aminoethyl)-
N2-(1,4-dioxido-1,2,4-benzotriazin-3-yl)-1,2-ethanediamine (109
mg, 76%): 1H NMR δ 8.33 (d, J ) 8.7 Hz, 1 H, H-8), 8.30 (d,
J ) 8.8 Hz, 1 H, H-5), 7.87 (ddd, J ) 8.5, 7.1, 1.0 Hz, 1 H,
H-6), 7.50 (ddd, J ) 8.4, 7.1, 1.2 Hz, 1 H, H-7), 3.70 (t, J ) 5.9
Hz, 2 H, CH2), 2.98 (t, J ) 5.9 H, 2 H, CH2), 2.84 (t, J ) 5.6
Hz, 2 H, CH2), 2.74 (t, J ) 5.6 Hz, 2 H, CH2), 2 × NH and
NH2 not observed.
A solution of amine (96 mg, 0.36 mmol) and 4-(1H-imidazol-
1-ylcarbonyl)acridine (119 mg, 0.43 mmol), prepared from
4-acridinecarboxylic acid (25) and CDI, in DMF (5 mL) was
stirred at 20 °C for 5 h. The solvent was evaporated and the
residue purified by chromatography, eluting with a gradient
(0-2%) of aqueous NH3/(0-7%) MeOH/DCM, to give compound
15 (168 mg, 99%) as a red solid: mp (DCM/hexane) 151-154
°C; 1H NMR δ 11.98 (t, J ) 5.3 Hz, 1 H, CONH), 8.82 (dd, J )
8.2, 1.4 Hz, 1 H, ArH), 8.81 (s, 1 H, ArH), 8.08-8.17 (m, 4 H,
4 × ArH), 7.97 (d, J ) 8.3 Hz, 1 H, ArH), 7.75-7.82 (m, 2 H,
2 × ArH), 7.60 (dd, J ) 8.2, 7.2 Hz, 1 H, ArH), 7.54 (ddd, J )
8.3, 7.3, 0.9 Hz, 1 H, ArH), 7.40 (ddd, J ) 8.6, 7.2, 1.2 Hz, 1
H, ArH), 3.86 (q, J ) 5.8 Hz, 2 H, CH2), 3.70 (t, J ) 5.8 Hz, 2
H, CH2), 3.17 (q, J ) 6.3 Hz, 4 H, 2 × CH2), 2 × NH not
observed; 13C NMR δ 166.5, 149.7, 147.4, 146.2, 138.0, 137.7,
135.6, 135.3, 132.5, 131.4, 130.2, 128.9, 128.0, 126.9 (2), 126.7,
126.3, 125.9, 125.4, 121.5, 117.2, 48.6, 47.8, 40.6, 39.3; HRMS
calcd for C25H24N7O3 (MH+) m/z 470.1941, found 470.1934.
Anal. (C25H23N7O3‚11/2H2O) C, H, N.
N-[3-({3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r op yl}a m in o)p r op yl]-4-a cr id in eca r boxa m id e (16). ter t-
Bu tyl 3-[(1-Oxid o-1,2,4-ben zotr ia zin -3-yl)a m in o]p r op yl-
{3-[(t r iflu or oa cet yl)a m in o]p r op yl}ca r b a m a t e (45). A
solution of chloride 17 (1.34 g, 7.41 mmol) in DCM (50 mL)
was added dropwise to a stirred solution of tert-butyl bis(3-
aminopropyl)carbamate 44 (2.57 g, 11.1 mmol) and Et3N (1.55
mL, 11.1 mmol) in DCM (200 mL) at 20 °C. The solution was
stirred at 20 °C for 3 days. The solvent was evaporated and
the residue purified by chromatography, eluting with 50%
EtOAc/acetone, to give a crude oil. Trifluoroacetic anhydride
(3.5 mL, 24.3 mmol) was added dropwise to a stirred solution
of crude amine in pyridine (50 mL) at 5 °C and the solution
was stirred at 20 °C for 16 h. The solvent was evaporated and
the residue purified by chromatography, eluting with a gradi-
ent (30-50%) of EtOAc/pet. ether, to give trifluoroacetamide
45 (0.51 g, 22%) as a yellow solid: mp (EtOAc/pet. ether) 89-
90 °C; 1H NMR δ 8.22-8.26 (m, 2 H, H-8, NH), 7.71 (br dd, J
) 8.4, 7.0 Hz, 1 H, H-6), 7.59 (d, J ) 8.4 Hz, 1 H, H-5), 7.29
(br dd, J ) 8.5, 7.0 Hz, 1 H, H-7), 5.45 (br s, 1 H, NH), 4.12
(br dd, J ) 6.6, 6.5 Hz, 2 H, CH2N), 3.26-3.37 (m, 6 H, 3 ×
CH2N), 1.84-1.95 (m, 2 H, CH2), 1.71-1.77 (m, 2 H, CH2), 1.48
[s, 9 H, C(CH3)3]; 13C NMR δ 158.9, 157.3 (q, J ) 37 Hz), 156.8,
148.0, 135.6, 130.9, 126.5, 125.1, 120.4, 116 (q, J ) 288 Hz),
80.8, 44.5, 43.0, 38.8, 35.8, 29.7, 28.3 (3), 27.1; MS m/z 473
N-[3-({3-[(1,4-Dioxid o-1,2,4-b en zot r ia zin -3-yl)a m in o]-
p r op yl}a m in o)p r op yl]-4-a cr id in eca r boxa m id e (16). A so-
lution of 4-acridinecarboxylic acid 25 (846 mg, 4.35 mmol) and
CDI (846 mg, 5.21 mmol) in DMF (20 mL) was stirred at 50
°C for 1 h. The solvent was evaporated and the residue
recrystallized from DCM/pet. ether to give 4-(1H-imidazol-1-
ylcarbonyl)acridine (746 mg, 63%), which was used directly
without characterization. A solution of the amine 47 (320 mg,
0.82 mmol) in DCM (10 mL) was added dropwise to a stirred
solution of imidazolide (234 mg, 0.86 mmol) in THF (25 mL)
at 5 °C and the solution was stirred at 20 °C for 16 h. The
solvent was evaporated and the residue purified by chroma-
tography, eluting with a gradient (0-5%) of MeOH/DCM, to
give tert-butyl 3-[(4-acridinylcarbonyl)amino]propyl{3-[(1,4-
dioxido-1,2,4-benzotriazin-3-yl)amino]propyl}carbamate (330
mg, 67%) as a red gum: 1H NMR δ 11.92 (br s, 1 H, CONH),
8.98 (dd, J ) 7.2, 1.5 Hz, 1 H, H-3), 8.89 (s, 1 H, H-9), 8.26-
8.32 (m, 3 H, H-5, H-5′, H-8′), 8.16 (d, J ) 8.3 Hz, 1 H, H-1),
8.07 (d, J ) 8.8 Hz, 1 H, H-8), 7.82-7.89 (m, 3 H, H-3, H-6,
H-6′), 7.65-7.69 (m, 1 H, H-7′), 7.58-7.62 (m, 1 H, H-7), 7.48
(br s, 1 H, NH), 3.72 (dt, J ) 6.6, 6.0 Hz, 2 H, CH2N), 3.61 (dt,
J ) 6.6, 6.4 Hz, 2 H, CH2N), 3.38-3.50 (m, 4 H, 2 × CH2N),
2.04-2.08 (m, 2 H, CH2), 1.88-1.94 (m, 2 H, CH2), 1.40 [s, 9
H, C(CH3)3]; MS m/z 598 (MH+, 8%), 582 (6); HRMS calcd for
C
32H36N7O5 (MH+) m/z 598.2778, found 598.2772.
HCl-saturated MeOH (30 mL) was added to a solution of
the carbamate (328 mg, 0.55 mmol) in MeOH (30 mL) and the
solution stirred at 20 °C for 16 h. The solution was evaporated,
the residue dissolved in water (20 mL), and the solution
neutralized with KHCO3 and extracted with CHCl3 (5 × 50
mL). The combined organic fraction was dried and the solvent

486 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Hay et al.
evaporated to give compound 16 (247 mg, 90%) as a red solid:
1H NMR [(CD3)2SO] δ 11.38 (t, J ) 5.5 Hz, 1 H, CONH), 10.50
(br s, 1 H, NH), 9.28 (s, 1 H, H-9), 8.71 (dd, J ) 7.1, 1.5 Hz, 1
H, H-3), 8.35 (dd, J ) 8.4, 1.5 Hz, 1 H, H-1), 8.24 (d, J ) 8.7
Hz, 1 H, H-5), 8.19 (d, J ) 8.3 Hz, 1 H, H-8), 8.14 (d, J ) 8.5
Hz, 1 H, H-8′), 8.03 (d, J ) 8.5 Hz, 1 H, H-5′), 7.92-7.96 (m,
1 H, H-6), 7.83-7.88 (m, 1 H, H-6′), 7.75 (dd, J ) 8.3, 7.1 Hz,
1 H, H-2), 7.65-7.68 (m, 1 H, H-7), 7.48-7.54 (m, 1 H, H-7′),
7.38 (s, 1 H, NH), 3.64 (dt, J ) 6.9, 5.9 Hz, 2 H, CH2N), 3.46
(t, J ) 6.7 Hz, 2 H, CH2N), 2.79 (t, J ) 6.9 Hz, 2 H, CH2N),
2.70 (t, J ) 6.5 Hz, 2 H, CH2N), 1.88-1.94 (m, 2 H, CH2), 1.76-
1.82 (m, 2 H, CH2); 13C NMR [(CD3)2SO] δ 164.7, 149.6, 147.0,
145.4, 138.5, 138.0, 135.3, 134.4, 132.6, 131.8, 129.7, 128.5,
128.4, 128.3, 126.7, 126.4, 126.3, 125.5, 125.2, 121.0, 116.7,
47.1, 46.9, 39.6, 37.2, 29.3, 28.2; MS m/z 498 (MH+, 15%), 482
(5); HRMS calcd for C27H28N7O3 (MH+) m/z 498.2254, found
498.2258. Anal. (C27H27N7O3‚2H2O) C, H; N, calcd 18.4, found
17.1%.
Ack n ow led gm en t. The authors thank Dr. Maruta
Boyd, Sisira Kumara, Anna Chappell, and Karin Tan,
for technical assistance. The authors thank Degussa
Peroxide Ltd, Morrinsville, New Zealand, for the gener-
ous gift of 70% hydrogen peroxide. This work was
supported by the US National Cancer Institute under
Grant CA82566 (M.P.H., F.B.P., S.A.G., H.D.S.L.,
M.S.K.), the Health Research Council of New Zealand
(A.V.P., W.R.W.), and the Auckland Division of the
Cancer Society of New Zealand (W.A.D.).
Refer en ces
(1) Brown, J . M. SR 4233 (tirapazamine): A new anticancer drug
exploiting hypoxia in solid tumors. Br. J . Cancer 1993, 67, 1163-
1170.
(2) Denny, W. A.; Wilson, W. R. Tirapazamine: A bioreductive
anticancer drug that exploits tumour hypoxia. Exp. Opin. Invest.
Drugs 2000, 9, 2889-2901.
(3) Nordsmark, M.; Overgaard, M.; Overgaard, J . Pretreatment
oxygenation predicts radiation response in advanced squamous
cell carcinoma of the head and neck. Radiother. Oncol. 1996,
41, 31-40.
(4) Brizel, D. M.; Sibley, G. S.; Prosnitz, L. R.; Scher, R. L.; Dewhirst,
M. W. Tumor hypoxia adversely affects the prognosis of carci-
noma of the head and neck. Int. J . Radiat. Oncol. Biol. Phys.
1997, 38, 285-289.
(5) Wang, J .; Biedermann, K. A.; Brown, J . M. Repair of DNA and
chromosome breaks in cells exposed to SR 4233 under hypoxia
or to ionizing radiation. Cancer Res. 1992, 52, 4473-4477.
(6) Daniels, J . S.; Gates, K. S.; Tronche, C.; Greenberg, M. M. Direct
evidence for bimodal DNA damage induced by tirapazamine.
Chem. Res. Toxicol. 1998, 11, 1254-1257.
P a r tition Coefficien ts. The octanol-water partition coef-
ficient (P) was measured using the shake flask method,56 using
GPR-grade octanol (BDH Laboratory Supplies). Briefly, lipo-
philic drugs were dissolved directly in octanol-saturated PBS
(137 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, 8.10 mM
Na2HPO4, pH 7.4) and hydrophilic drugs in PBS-saturated
octanol, to 25-100 µM. Equal volumes of PBS and octanol were
mixed on a Bellco roller drum (Bellco Glass, Inc., New J ersey)
at 20 rpm for 3 h at ambient temperature. The two solvent
layers were separated after a brief spin and analyzed by HPLC
directly (aqueous layer), or after addition of 4 volumes of
methanol (organic layer).
DNA Bin d in g Assa y. DNA binding was measured by
equilibrium dialysis using a Dianorm dialyzer (Diachema AG,
Ru¨schlikon-Zu¨rich, Switzerland) with 20 two-cell dialysis
chambers (1 mL each) as described previously.56 Solutions of
BTO (15 µM) with calf thymus DNA (Sigma type I) (25-1000
µM in base pairs) in KHE buffer (10 mM KCl, 2 mM
NaHEPES, 10 µM Na2EDTA, pH 7.0) were dialyzed (2 h at 37
°C) against free BTO (15 µM) in KHE buffer using a Cupro-
phan dialysis membrane (Medicell International Ltd., London,
UK) with a MW cutoff of 10 000 Da. After equilibrium was
reached, the contents of the dialysis chambers with KHE buffer
were collected and analyzed directly by HPLC. To the samples
containing DNA, 9 volumes of ice-cold ethanol were added and
the mixture was kept on ice for 1 h followed by a brief spin to
precipitate the DNA. Subsequently, the volume of the aqueous
ethanol was reduced using a Speed-Vac concentrator and
reconstituted with mobile phase followed by injection onto an
HPLC. BTO concentrations were calculated using TPZ as
internal standard, and the equilibrium association constants
(KDNA) and standard errors were obtained from the best fit
obtained by nonlinear regression analysis of direct plots of the
ratio of drug bound/base pair versus free drug concentra-
tion47,59 using the neighboring site exclusion model.65
(7) Anderson, R. F.; Shinde, S. S.; Hay, M. P.; Gamage, S. A.; Denny,
W. A. Activation of 3-amino-1,2,4-benzotriazine 1,4-dioxide
antitumor agents to oxidizing species following their one-electron
reduction. J . Am. Chem. Soc. 2003, 125, 748-756.
(8) Baker, M. A.; Zeman, E. M.; Hirst, V. K.; Brown, J . M.
Metabolism of SR 4233 by Chinese hamster ovary cells: Basis
of selective hypoxic cytotoxicity. Cancer Res. 1988, 48, 5947-
5952.
(9) Wang, J .; Biedermann, K. A.; Wolf, C. R.; Brown, J . M.
Metabolism of the bioreductive cytotoxin SR 4233 by tumour
cells: Enzymatic studies. Brit. J . Cancer 1993, 67, 321-325.
(10) Patterson, A. V.; Saunders, M. P.; Chinje, E. C.; Patterson, L.
H.; Stratford, I. J . Enzymology of tirapazamine metabolism: A
review. Anti-Cancer Drug Des. 1998, 13, 541-573.
(11) Delahoussaye, Y. M.; Evans, J . W.; Brown, J . M. Metabolism of
tirapazamine by multiple reductases in the nucleus. Biochem.
Phamacol. 2001, 62, 1201-1209.
(12) Evans, J . W.; Yudoh, K.; Delahoussaye, Y. M.; Brown, J . M.
Tirapazamine is metabolized to its DNA damaging radical by
intranuclear enzymes. Cancer Res. 1998, 58, 2098-2101.
(13) Peters, L. J .; Rischin, D.; Hicks, R. J .; Hughes, P. G.; Sizeland,
A. M. Extraordinary tumor control in phase I trial of concurrent
tirapazamine, cisplatin, and radiotherapy in patients with
advanced head and neck cancer. Int. J . Radiat. Oncol. Biol. Phys.
1999, 45, 148-149.
(14) Craighead, P. S.; Pearcey, R.; Stuart, G. A phase I/II evaluation
of tirapazamine administered intravenously concurrent with
cisplatin and radiotherapy in women with locally advanced
cervical cancer. Int. J . Radiat. Oncol. Biol. Phys. 2000, 48, 791-
795.
(15) von Pawel, J .; von Roemeling, R.; Gatzemeier, U.; Boyer, M.;
Elisson, L. O.; Clark, P.; Talbot, D.; Rey, A.; Butler, T. W.; Hirsh,
V.; Olver, I.; Bergman, B.; Ayoub, J .; Richardson, G.; Dunlop,
D.; Arcenas, A.; Vescio, R.; Viallet, J .; Treat, J . Tirapazamine
plus cisplatin versus cisplatin in advanced nonsmall-cell lung
cancer: A report of the international CATAPULT I study group.
Cisplatin and tirapazamine in subjects with advanced previously
untreated nonsmall-cell lung tumors. J . Clin. Oncol. 2000, 18,
1351-1359.
(16) Del Rowe, J .; Scott, C.; Werner-Wasik, M.; Bahary, J . P.; Curran,
W. J .; Urtasun, R. C.; Fisher, B. Single-arm, open-label phase
II study of intravenously administered tirapazamine and radia-
tion therapy for glioblastoma multiforme. J . Clin. Oncol. 2000,
18, 1254-1259.
(17) Rishin, D.; Peters, L.; Hicks, R.; Hughes, P.; Fisher, R.; Hart,
R.; Sexton, M.; D’Costa, I.; von Roemeling, R. Phase I trial of
concurrent tirapazamine, cisplatin, and radiotherapy in patients
with advanced head and neck cancer. J . Clin. Oncol. 2001, 19,
535-542.
(18) Rauth, A. M.; Melo, T.; Misra, V. Bioreductive therapies: An
overview of drugs and their mechanisms of action. Int. J . Radiat.
Oncol. Biol. Phys. 1998, 42, 755-762.
Clon ogen ic Assa ys. Clonogenic survival of mouse SCCVII
tumor cells in vitro was determined for 1 h drug exposure of
cells under aerobic and hypoxic conditions as previously
described.57 Hypoxic C10 values are corrected for interexperi-
mental variation in hypoxia by using TPZ as an internal
standard: C10(BTO,calc) ) C10(BTO,meas) × [C10(TPZ,mean)/
C
10(TPZ,meas)].
IC50 Assa ys. IC50 assays were determined for BTOs under
aerobic and hypoxic conditions as previously described.57 For
each experiment, compounds were simultaneously tested
under both oxic and hypoxic conditions against the HT-29 cell
line and included TPZ as an independent internal control at
the front and back of the assay (n ) 2). Final data were pooled
from a series of seven independent experiments and are cal-
culated using interexperimental means. In all cases, 8-methyl-
5-nitroquinoline was used as a second internal control to
confirm that strict hypoxia was present during the experi-
ment.66 Plates were stained as described previously67 and IC50
values determined. Results were averaged for two to six
independent experiments for each compound.

DNA-Targeted 1,2,4-Benzotriazine 1,4-Dioxides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 487
(19) Skov, K. A. Workshop report: DNA targeted hypoxic cytotoxins
and radiosensitizers. Int. J . Radiat. Biol. 1989, 56, 387-393.
(20) Denny, W. A.; Roberts, P. B.; Anderson, R. F.; Brown, J . M.;
Wilson, W. R. NLA-1: A 2-nitroimidazole radiosensitizer tar-
geted to DNA by intercalation. Int. J . Radiat. Oncol. Biol. Phys.
1992, 22, 553-556.
(21) Cowan, D. S. M.; Matejovic, J . F.; McClelland, R. A.; Rauth, A.
M. DNA-targeted 2-nitroimidazoles: In vitro and in vivo studies.
Br. J . Cancer 1994, 70, 1067-1074.
(22) Papadopoulou, M. V.; J i, M.; Rao, M. K.; Bloomer, W. D. 9-[3-
(2-Nitro-1-imidazolyl)propylamino]cyclopenteno[b]quinoline hy-
drochloride (NLCPQ-1): A novel DNA-affinic bioreductive agent
as chemosensitizer I. Oncol. Res. 1997, 9, 249-257.
(23) Papadopoulou, M. V.; J i, M.; Rao, M. K.; Bloomer, W. D. 4-[3-
(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochlo-
ride (NLCQ-1), a novel bioreductive compound as a hypoxia-
selective cytotoxin. Oncol. Res. 2001, 12, 185-192.
(24) Wilson, W. R.; Denny, W. A.; Twigden, S. J .; Baguley, B. C.;
Probert, J . C. Selective cytotoxicity of nitracrine to hypoxic cells.
Br. J . Cancer 1984, 49, 215-223.
(25) Wilson, W. R.; Siim, B. G.; Denny, W. A.; van Zijl, P. L.; Taylor,
M. L.; Chambers, D. M.; Roberts, P. B. 5-Nitro-4-(N,N-dimethyl-
(aminopropyl)amino)quinoline (5-nitroquine), a new DNA affinic
hypoxic cell radiosensitizer and bioreductive agent: Comparison
with nitracrine. Radiat. Res. 1992, 131, 257-265.
(26) Denny, W. A.; Wilson, W. R. Considerations for the design of
nitrophenyl mustards as agents with selective toxicity for
hypoxic tumour cells. J . Med. Chem. 1986, 29, 879-887.
(27) Durand, R. E. Slow penetration of anthracyclines into spheroids
and tumors: A therapeutic advantage? Cancer Chemother.
Pharmacol. 1990, 26, 198-204.
(28) Wilson, W. R.; Denny, W. A. DNA-binding nitroheterocycles as
hypoxia-selective cytotoxins. In Radiation Research, a Twentieth-
Century Perspective; Dewey, W. C., Edington, M., Fry, R. J . M.,
Hall, E. J ., Whitmore, G. F., Eds; Academic Press: San Diego,
1992; Vol. 2, pp 796-801.
(40) Panicucci, R.; Heal, R.; Laderoute, K.; Cowan, D.; McClelland,
R. A.; Rauth, A. M. NLP-1: A DNA intercalating hypoxic cell
radiosensitizer and cytotoxin. Int. J . Radiat. Oncol. Biol. Phys.
1989, 16, 1039-1043.
(41) Cowan, D. S.; Panicucci, R.; McClelland, R. A.; Rauth, A. M.
Targeting radiosensitizers to DNA by attachment of an inter-
calating group: Nitroimidazole-linked phenanthridines. Radiat.
Res. 1991, 127, 81-89.
(42) Papadopoulou, M. V.; Epperly, M. W.; Shields, D. S.; Bloomer,
W. D. Radiosensitization and hypoxic cell toxicity of NLA-1 and
NLA-2, two bioreductive compounds. J pn. J . Cancer Res. 1992,
83, 410-414.
(43) Papadopoulou, M. V.; Rosenzweig, H. S.; Doddi, M.; Bloomer,
W. D. 9-[3-(2-Nitro-1-imidazolyl)propylamino]-1,2,3,4-tetra-
hydroacridine hydrochloride. A novel DNA-affinic hypoxic cell
cytotoxin and radiosensitizer. Comparison with NLA-1. Oncol.
Res. 1994, 6, 439-448.
(44) Hicks, K. O.; Pruijn, F. B.; Sturman, J . R.; Denny, W. A.; Wilson,
W. R. Multicellular resistance to tirapazamine is due to re-
stricted extravascular transport: A pharmacokinetic/pharma-
codynamic study using multicellular layer cultures. Cancer Res.
2003, 63, 5970-5977.
(45) J ain, R. K. The next frontier of molecular medicine: Delivery of
therapeutics. Nat. Med. 1998, 4, 655-657.
(46) Tannock, I. F. Tumor physiology and drug resistance. Cancer
Metastasis Rev. 2001, 20, 123-132.
(47) Delahoussaye, Y. M.; Hay, M. P.; Pruijn, F. B.; Denny, W. A.;
Brown, J . M. Improved potency of the hypoxic cytotoxin
tirapazamine by DNA-targeting. Biochem. Pharmacol. 2003, 65,
1807-1815.
(48) Robbins, R. F.; Schofield, K. Polyazabicyclic compounds. Part
II. Further derivatives of benzo-1,2,4-triazine. J . Chem. Soc.
1957, 3186-3194.
(49) Gamage, S. A.; Spicer, J . A.; Rewcastle, G. W.; Denny, W. A. A
new synthesis of substituted acridine-4-carboxylic acids and the
anticancer drug N-[2-(dimethylamino)ethyl]acridine-4-carbox-
amide (DACA). Tetrahedron Lett. 1997, 38, 699-702.
(50) Corey, E. J .; Nicolaou, K. C.; Balanson, R. D.; Machida, Y. A
useful method for the conversion of azides to amines. Synthesis
1975, 590-591.
(51) Rolla, F. Sodium borohydride reactions under phase transfer
conditions: Reductions of azides to amines. J . Org. Chem. 1982,
47, 4327-4329.
(29) Denny, W. A.; Atwell, G. J .; Rewcastle, G. W.; Baguley, B. C.
Potential antitumor agents. 49. 5-Substituted derivatives of N-[2-
(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide with in
vivo solid-tumor activity. J . Med. Chem. 1987, 30, 658-663.
(30) Atwell, G. J .; Rewcastle, G. W.; Baguley, B. C.; Denny, W. A.
Potential antitumor agents. 50. In vivo solid-tumor activity of
derivatives of N-[2-(dimethylamino)ethyl]acridine-4-carbox-
amide. J . Med. Chem. 1987, 30, 664-669.
(31) Denny, W. A.; Atwell, G. J .; Baguley, B. C. Minimal DNA-
intercalating agents as anti-tumour drugs: 2-Styrylquinoline
analogues of amsacrine. Anti-Cancer Drug Des. 1987, 2, 263-
270.
(52) Hassner, A.; Levy, L. A. Addition of iodine azide to olefins.
Stereospecific introduction of azide functions. J . Am. Chem. Soc.
1965, 87, 4203-4204.
(53) Koziara, A.; Zwierzak, A. Optimized procedures for one-pot
conversion of alkyl bromides into amines via the Staudinger
reaction. Synthesis 1992, 11, 1063-1065.
(54) Bayley H.; Standring, D. N.; Knowles, J . R. Propane-1,3-
dithiol: A selective reagent for the efficient reduction of alkyl
and aryl azides to amines. Tetrahedron Lett. 1978, 39, 3633-
3634.
(55) Huang, T. L.; Dredar, S. A.; Manneh V. A.; Blankenship, J . W.;
Fries, D. S. Inhibition of N8-acetylspermidine deacetylase by
active-site-directed metal coordinating inhibitors. J . Med. Chem.
1992, 35, 2414-2418.
(56) Siim, B. G.; Hicks, K. O.; Pullen, S. M.; van Zijl, P. L.; Denny,
W. A.; Wilson, W. R. Comparison of aromatic and tertiary amine
N-oxides of acridine DNA intercalators as bioreductive drugss
Cytotoxicity, DNA binding, cellular uptake, and metabolism.
Biochem. Pharmacol. 2000, 60, 969-978.
(57) Hay, M. P.; Gamage, S. A.; Kovacs, M. S.; Pruijn, F. B.; Anderson,
R. F.; Patterson, A. V.; Wilson, W. R.; Brown, J . M.; Denny, W.
A. Structure-activity relationships of 1,2,4-benzotriazine 1,4-
dioxides as hypoxia-selective analogues of tirapazamine. J . Med.
Chem. 2003, 46, 169-182.
(58) Kelson, A. B.; McNamara, J . P.; Pandey, A.; Ryan, K. J .; Dorie,
M. J .; McAfee, P. A.; Menke, D. R.; Brown, J . M.; Tracy, M. 1,2,4-
Benzotriazine 1,4-dioxides. An important class of hypoxic cyto-
toxins with antitumor activity. Anti-Cancer Drug Des. 1998, 13,
575-592.
(59) Crenshaw, J . M.; Graves, D. E.; Denny, W. A. Interactions of
acridine antitumor agents with DNA: Binding energies and
groove preferences. Biochem. 1995, 34, 13682-13687.
(60) Anderson, R. F.; Harris, T. A.; Hay, M. P.; Denny, W. A.
Enhanced Conversion of DNA Radical Damage to Double Strand
Breaks by 1,2,4-Benzotriazine 1,4-dioxides linked to a DNA
Binder compared to Tirapazamine. Chem. Res. Toxicol. 2003,
16, 1477-1483.
(61) Durand, R. E.; Olive, P. L. Evaluation of bioreductive drugs in
multicell spheroids. Int. J . Radiat. Oncol. Biol. Phys. 1992, 22,
689-692.
(62) Hicks, K. O.; Fleming, Y.; Siim, B. G.; Koch, C. J .; Wilson, W.
R. Extravascular diffusion of tirapazamine: Effect of metabolic
consumption assessed using the multicellular layer model. Int.
J . Radiat. Oncol. Biol. Phys. 1998, 42, 641-649.
(32) Rewcastle, G. W.; Denny, W. A.; Baguley, B. C. Potential
antitumor agents. 51. Synthesis and antitumor activity of
substituted phenazine-1-carboxamides. J . Med. Chem. 1987, 30,
843-851.
(33) Palmer, B. D.; Rewcastle, G. W.; Atwell, G. J .; Baguley, B. C.;
Denny, W. A. Potential antitumor agents. 54. Chromophore
requirements for in vivo antitumor activity among the general
class of linear tricyclic carboxamides. J . Med. Chem. 1988, 31,
707-712.
(34) Atwell, G. J .; Bos, C. D.; Baguley, B. C.; Denny, W. A. Potential
antitumor agents. 56. “Minimal” DNA-intercalating ligands as
antitumor drugs: Phenylquinoline-8-carboxamides. J . Med.
Chem. 1988, 31, 1048-1052.
(35) Atwell, G. J .; Baguley, B. C.; Denny, W. A. Potential antitumor
agents. 57. 2-phenylquinoline-8-carboxamides as “minimal”
DNA-intercalating antitumor agents with in vivo solid tumor
activity. J . Med. Chem. 1989, 32, 396-401.
(36) Hicks, K. O.; Pruijn, F. B.; Baguley, B. C.; Wilson, W. R.
Extravascular transport of the DNA intercalator and topo-
isomerase poison N-[2-(dimethylamino)ethyl]acridine-4-carbox-
amide (DACA): Diffusion and metabolism in multicellular layers
of tumor cells. J . Pharmacol. Exp. Ther. 2001, 297, 1088-1098.
(37) McCrystal, M. R.; Evans, B. D.; Harvey, V. J .; Thompson, P. I.;
Porter, D. J .; Baguley, B. C. Phase I study of the cytotoxic
agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide. Can-
cer Chemother. Pharmacol. 1999, 44, 39-44.
(38) Twelves, C.; Campone, M.; Coudert, B.; Van den Bent, M.;
de J onge, M.; Dittrich, C.; Rampling, R.; Sorio, R.; Lacombe, D.;
de Balincourt, C.; Fumoleau, P. Phase II study of XR5000
(DACA) administered as a 120-h infusion in patients with
recurrent glioblastoma multiforme. Ann. Oncol. 2002, 13, 777-
780.
(39) Papadopoulou, M. V.; J i, M.; Rao, M. K.; Bloomer, W. D. 4-[3-
(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochlo-
ride (NLCQ-1), a novel bioreductive agent as radiosensitizer in
vitro and in vivo: Comparison with tirapazamine. Oncol. Res.
2000, 8, 325-333.

488 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Hay et al.
(63) Kyle, A. H.; Minchinton, A. I. Measurement of delivery and
metabolism of tirapazamine to tumour tissue using the multi-
layered cell culture model. Cancer Chemother. Pharmacol. 1999,
43, 213-220.
(64) Mason, J . C.; Tennant, G. Heterocyclic N-oxides. Part VI.
Synthesis and nuclear magnetic resonance spectra of 3-amino-
benzo-1,2,4-triazines and their mono- and di-N-oxides. J . Chem.
Soc. (B) 1970, 911-916.
(65) McGhee, J . D.; von Hippel, P. H. Theoretical aspects of DNA-
protein interactions: Co-operative and non-co-operative binding
of large ligands to a one-dimensional homogeneous lattice. J .
Mol. Biol. 1974, 86, 469-489.
(66) Siim, B. G.; Atwell, G. J .; Wilson, W. R. Oxygen dependence of
the cytotoxicity and metabolic activation of 4-alkylamino-5-
nitroquinoline bioreductive drugs. Br. J . Cancer 1994, 70, 596-
603.
(67) Wilson, W. R.; Thompson, L. H.; Anderson, R. F., Denny, W. A.
Hypoxia-selective antitumor agents. 2. Electronic effects of
4-substituents on the mechanisms of cytotoxicity and meta-
bolic stability of nitracrine analogues. J . Med. Chem. 1989, 32,
31-38.
J M030399C