Structure-activity relationships of 1,2,4-benzotriazine 1,4-dioxides as hypoxia-selective analogues of tirapazamine

Article abstract of DOI:10.1021/jm020367+

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxic cytotoxin currently in Phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. As part of a program to develop TPZ analogues with improved solubility/potency and therapeutic indices, we synthesized 34 1,2,4-benzotriazin-3-amine 1,4-dioxides (BTO) to examine structure-activity relationships (SAR) for ring substitution. The electronic, hydrophobic, and steric parameters of substituents at the 5-, 6-, 7-, and 8-positions were systematically varied, and the aqueous solubility and one-electron reduction potentials [E(1)] of the analogues were determined. For each compound, we determined cell killing of mouse SCCVII tumor cells in vitro under aerobic and hypoxic conditions by clonogenic survival and determined their relative hypoxic toxicity (RHT; relative to TPZ) and hypoxic cytotoxicity ratio (HCR). A subset of compounds was independently evaluated using a 96-well SRB proliferation assay, the data from which correlated well with that derived by the clonogenic endpoint. Most substituents, except 5- and 8-dimethylamino and 8-diethylamino, gave analogues less soluble than TPZ. E(1) values ranged from -240 mV through -670 mV (with TPZ having a value of -456 mV) and correlated well with the electronic parameter σ for substituents at the 5-, 6-, 7-, and 8-positions. Aerobic cytotoxic potency showed a strong positive correlation with E(1) (i.e., electron-withdrawing substituents increased aerobic toxicity). Hypoxic cytotoxicity also generally increased with increasing E(1), with a maximum (RHT up to 3.9-fold) seen in halo- and trifluoromethyl-substituted BTO derivatives having E(1) between ca. -370 to -400 mV. Analogues with high HCRs (>50) all had E(1)s in the range -450 to -510 mV (weakly electron-donating substituents) with the exception of the 8-CF₃ analogue, which had an HCR of 112 against SCCVII despite a high E(1) of -372 mV). The results suggest that ring-A substituents in BTO analogues can be used to predictably vary one-electron reduction potentials and also provide a much better definition than previously of the optimum range of these reduction potentials for a desirable biological activity profile (high HCR, RHT, and solubility).

Full text of DOI:10.1021/jm020367+

J . Med. Chem. 2003, 46, 169-182
169
Str u ctu r e-Activity Rela tion sh ip s of 1,2,4-Ben zotr ia zin e 1,4-Dioxid es a s
Hyp oxia -Selective An a logu es of Tir a p a za m in e
Michael P. Hay,*,† Swarna A. Gamage,† Mary S. Kovacs,‡ Frederik B. Pruijn,† Robert F. Anderson,†
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
Received August 26, 2002
Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxic cytotoxin
currently in Phase II/III clinical trials in combination with radiotherapy and with cisplatin-
based chemotherapy. As part of a program to develop TPZ analogues with improved solubility/
potency and therapeutic indices, we synthesized 34 1,2,4-benzotriazin-3-amine 1,4-dioxides
(BTO) to examine structure-activity relationships (SAR) for ring substitution. The electronic,
hydrophobic, and steric parameters of substituents at the 5-, 6-, 7-, and 8-positions were
systematically varied, and the aqueous solubility and one-electron reduction potentials [E(1)]
of the analogues were determined. For each compound, we determined cell killing of mouse
SCCVII tumor cells in vitro under aerobic and hypoxic conditions by clonogenic survival and
determined their relative hypoxic toxicity (RHT; relative to TPZ) and hypoxic cytotoxicity ratio
(HCR). A subset of compounds was independently evaluated using a 96-well SRB proliferation
assay, the data from which correlated well with that derived by the clonogenic endpoint. Most
substituents, except 5- and 8-dimethylamino and 8-diethylamino, gave analogues less soluble
than TPZ. E(1) values ranged from -240 mV through -670 mV (with TPZ having a value of
-456 mV) and correlated well with the electronic parameter σ for substituents at the 5-, 6-, 7-,
and 8-positions. Aerobic cytotoxic potency showed a strong positive correlation with E(1) (i.e.,
electron-withdrawing substituents increased aerobic toxicity). Hypoxic cytotoxicity also generally
increased with increasing E(1), with a maximum (RHT up to 3.9-fold) seen in halo- and
trifluoromethyl-substituted BTO derivatives having E(1) between ca. -370 to -400 mV.
Analogues with high HCRs (>50) all had E(1)s in the range -450 to -510 mV (weakly electron-
donating substituents) with the exception of the 8-CF3 analogue, which had an HCR of 112
against SCCVII despite a high E(1) of -372 mV). The results suggest that ring-A substituents
in BTO analogues can be used to predictably vary one-electron reduction potentials and also
provide a much better definition than previously of the optimum range of these reduction
potentials for a desirable biological activity profile (high HCR, RHT, and solubility).
In tr od u ction
ductases11-15 to form a radical anion, and this oxygen-
reversible process is the basis for its selective toxicity
to hypoxic cells.16 The radical anion is back-oxidized by
molecular oxygen under aerobic conditions, generating
superoxide radicals that mediate aerobic toxicity. It has
been proposed that under hypoxic conditions the pro-
tonated radical anion,17 or nitroxide11 undergoes frag-
mentation to produce the hydroxyl radical.18 A strong
oxidizing species must be formed as TPZ causes DNA
double-strand breaks under anoxic conditions, and these
correlate with cytotoxicity.19
It is now established that many human tumors
contain a significant fraction of hypoxic cells,1,2 due to
chaotic growth and an inefficient microvasculature
system within the tumor, leading to large intercapillary
distances and variable blood flow. Reduction in oxygen
tension within tumors leads to radioresistance, with up
to a 3-fold increase in radiation dose required to kill
severely hypoxic tumor cells. The presence of tumor
hypoxia and failure of local control by radiation therapy
have been linked.3 The phenomenon of tumor hypoxia
has been exploited in the development of a class of
anticancer agents termed ‘bioreductive drugs’,4-6 which
are selectively active against hypoxic cells in tumors.
Tirapazamine (TPZ, 1) is the leading bioreductive
agent,7-9 currently undergoing advanced clinical trials
for the potentiation of radiotherapy and cisplatin
chemotherapy.10 TPZ is reduced by one-electron re-
Although a Phase III trial has shown that TPZ in
combination with cisplatin significantly improves re-
sponse rate and median survival in non-small-cell lung
cancer,20 mixed results have been seen in earlier-stage
* To whom correspondence should be addressed. E-mail: m.hay@
auckland.ac.nz.
† The University of Auckland.
‡ Stanford University School of Medicine.
10.1021/jm020367+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/02/2002

170 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hay et al.
Sch em e 1a
trials of TPZ in combination with fractionated radiation
against head and neck carcinomas and glioblasto-
mas.21,22 Drug toxicity prevented TPZ being adminis-
tered with each fraction of radiation, which has been
shown theoretically to be the optimum administration
protocol.23
Previous structure-activity relationship (SAR) stud-
ies of A-ring-substituted 3-amino-1,2,4-benzotriazine
1,4-dioxides (BTO) have focused on a small set of 6- or
7-substituents [e.g., 6,7-dimethyl, 6-Cl, 7-Me, 7-alkoxy,
and mixtures of 6(7)-OMe and 6(7)-Cl].24 In this study,
hypoxic toxicity was shown to increase with increasing
E1/2 (the half wave potential of two-electron reduction,
measured polarographically). A more complex relation-
ship was observed between hypoxic cell selectivity
(HCR) and E1/2, with TPZ having the maximum HCR.
A subsequent paper examined the effect of a small set
of electron-withdrawing substituents (F, Cl, CF3, NO2,
Ph) in combination with a variety of 3-substituents.8
a
Reagents: Method A, cyanamide, HCl, then NaOH; Method
B, guanidine‚HCl, KOtBu, then NaOH; Method C, 30% H2O2,
HOAc; Method D, 70% H2O2, CF3CO2H; Method E, R2NH, MeCN.
Sch em e 2a
As part of a program to develop TPZ analogues with
improved solubility/potency and therapeutic indices, we
prepared a more diverse set of A-ring substituted BTOs
to explore the SAR for A-ring substitution more thor-
oughly, using substituents at all four ring positions and
without the confounding influence of substitution at the
3-position. We also measured one-electron reduction
potentials [E(1)] by pulse radiolysis rather than E1/2
values, as a more accurate determination of the one
electron reduction potential. We synthesized 34 3-amino-
BTOs, substituted at the 5-, 6-, 7-, and 8-positions with
groups spanning a wide range of electronic, hydrophobic,
and steric properties. The aqueous solubility, E(1)
values, and effect on the clonogenic survival of SCCVII
cells under aerobic and hypoxic conditions of the com-
pounds were determined, and hypoxic toxicity relative
to TPZ (RHT), and the hypoxic cytotoxicity ratio (HCR),
defined as the ratio of doses under aerobic and hypoxic
conditions producing equal toxicity, were calculated. For
a limited set of 12 compounds, including TPZ, a prolif-
eration assay (SRB) was used to determine IC50 values,
under oxic and anoxic conditions, against two cells lines
(murine SCCVII and human adenocarcinoma HT-29),
allowing a comparison between clonogenic survival (C10)
and proliferation (IC50) based assays.
a
Reagents: Method A, cyanamide, HCl, then NaOH; Method
B, guanidine‚HCl, KOtBu, then NaOH; Method C, 30% H2O2,
HOAc; Method D, 70% H2O2, CF3CO2H; Method E, R2NH, MeCN.
Sch em e 3a
Ch em istr y
a
Reagents: Method A, cyanamide, HCl, then NaOH; Method
For the substituted 3-amino-BTOs, 11 substituents
with widely different electronic, lipophilic, and steric
substituent parameters were chosen (NEt2, NMe2, OMe,
Me, F, Cl, CF3, MeSO2, n-BuSO2, NO2, and OH). The
majority of the compounds were prepared in a two-step
process; condensation-cyclization to form the 1-oxides,
followed by oxidation of these to give the 1,4-dioxides.
Two main condensation-cyclization reactions were
used. One (Method A: Schemes 1-4)25,26 employed
2-nitroanilines with cyanamide, followed by cyclization
of the intermediate guanidine with NaOH. Generally,
lower yields were noted for the more lipophilic com-
pounds, reflecting poor solubility in the reaction me-
dium. In several instances (7-CF3, 7-NO2, 5-Cl), it was
necessary to use an alternative method (Method B:
Schemes 2 and 4),27 involving reaction of 2-chloroni-
trobenzenes (7g, 7j, and 14e, respectively) with guani-
dine, followed by cyclization under basic conditions. This
B, guanidine‚HCl, KOtBu, then NaOH; Method C, 30% H2O2,
HOAc; Method D, 70% H2O2, CF3CO2H; Method E, R2NH, MeCN.
gave 1-oxides in modest yield (15-45%). Similarly,
reaction of 2,6-difluoronitrobenzene 3e with guanidine
followed by cyclization under basic conditions gave
1-oxide 4e in moderate yield (Scheme 1).
Sulfides 2h and 2i were prepared by nucleophilic
displacement of 3-chloro-2-nitroaniline (2f) with the
appropriate alkylthiolate salt (Scheme 5). Similarly,
displacement of 5-chloro-2-nitroaniline (10f) with BuSLi
gave 10i. Subsequent studies showed that displace-
ments of 8-chloro- and 6-chloro-1-oxides 4f and 11f with
nBuSLi gave the corresponding sulfides 4i and 11i,
n
whereas reaction of the 7-chloro-1-oxide 8f with BuSLi
gave mixtures of starting material, product, and the
corresponding nor-N-oxides from which pure 8h could
not be obtained. Reaction of the corresponding 7-bro-

Hypoxia-Selective Analogues of Tirapazamine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 171
Sch em e 4a
(Method D; Schemes 1-4) gave the corresponding 1,4-
dioxides. The oxidations provided moderate yields of 6-,
7-, and 8-substituted 1,4-dioxides, with significant losses
in the workup and purification of the products from
reaction mixtures containing large amounts of perox-
ides. Treatment of the reaction mix with strong base
led to decomposition of the product 1,4-dioxides. Sulfide
groups were oxidized to sulfone groups simultaneously
with N-oxidation. Dimethylamino- and diethylamino-
BTO derivatives were prepared by displacement of the
corresponding fluoro-1,4-dioxides with the appropriate
amine (Method E: Schemes 1-4).
A number of the planned BTOs could not be prepared.
The condensation reactions (Method A or B) failed to
give 6-nitro- and 8-nitro-1-oxides. Oxidation of 5-sub-
stituted 1-oxides 15c-f proved extremely difficult, with
extensive decomposition in the reaction leading to low
yields, and 5-CF3, 5-SO2Me, 5-SO2Bu, and 5-NO2 BTOs
could not be prepared at all. Nucleophilic displacement
of the 5-Cl-BTO 16e with LiSMe gave only starting
material. Attempts to prepare the proposed hydroxy-
BTOs were not also successful. Demethylation of the 7-
or 8-OMe-BTO analogues (9c and 5c, respectively) with
BBr3 gave only complex mixtures, while reaction with
LiSEt gave mixtures with reduction of both N-oxides.
Reaction of the 7-OMe 1-oxide 8c with pyridine hydro-
chloride gave intractable mixtures. While it was possible
to prepare 7-hydroxy-1,2,4-benzotriazine 1-oxide, we
were unable to find a protecting group (apart from OMe)
capable of surviving the oxidation conditions required
for 1,4-dioxide formation.
a
Reagents: Method A, cyanamide, HCl, then NaOH; Method
B, guanidine‚HCl, KOtBu, then NaOH; Method C, 30% H2O2,
HOAc; Method D, 70% H2O2, CF3CO2H; Method E, R2NH, MeCN.
mide gave complete reaction, but was accompanied by
significant amounts of 1-oxide reduction. Attempts to
prepare 4-(methylsulfanyl)-2-nitroaniline (6h ) by nu-
cleophilic displacement of the 4-chloride 6f were not
n
successful. Reaction of 5-chloro-1-oxide 15e with BuSLi
gave only starting material. A circuitous approach
utilizing the Newman-Kwart rearrangement28 and
vicarious nucleophilic substitution (VNS)29,30 gave two
of the required methylsulfanyl isomers 6h and 13h in
sufficient purity (Scheme 5). Thus, isomerization of
O-thiocarbamate 17 gave S-thiocarbamate 18, which
was hydrolyzed, and the intermediate thiol was alky-
lated with MeI to give sulfide 19. VNS Reaction of 19
with NH2OMe‚HCl gave nitroanilines 6h and 13h . A
similar sequence from 18 gave butylsufanylnitroanilines
6i and 13i as well as the isomeric 21. Curtius rear-
rangement of 5-methoxy-2-nitrobenzoic acid (22) gave
nitroaniline 10c.
P h ysicoch em ica l Mea su r em en ts. The solubilities
of the BTOs in R-MEM with 5% added fetal calf serum
was determined by HPLC (Table 1). None of the
analogues proved more soluble than TPZ itself (although
increasing solubility was not a major goal of the current
Oxidation of the substituted 1-oxides with peracetic
acid (Method C: Schemes 1-4) or trifluoroperacetic acid
Sch em e 5a
a
Reagents: (a) MeSLi, DMF; (b) nBuSLi, DMF; (c) 220 °C; (d) KOH, MeOH; (e) Me2SO4, KOH, MeOH; (f) NH2OMe‚HCl, KOtBu,
CuCl, DMF; (g) nBuBr, K2CO3, DMF; (h) DPPA, Et3N, tBuOH; (i) HCl, MeOH.

172 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hay et al.
Ta ble 1. Physicochemical Properties of Ring-A-Substituted BTO Analogues
d
e
no.
R
synth method
solna (mM)
E(1)b (mV)
refc
logPmeas
logPtrain
1
5a
5b
5c
5d
5e
5f
5g
5h
5i
9a
9b
9c
9d
9e
9f
H
8.9
14.1
9.0
6.4
1.3
4.9
2.2
0.5
0.9
1.7
0.4
1.2
2.0
1.9
0.9
0.1
0.7
0.5
0.3
0.3
1.5
0.4
0.6
3.0
1.4
0.4
0.2
0.2
0.1
3.2
10.0
3.0
2.4
1.0
7.5
-456 ( 8
-554 ( 6
-545 ( 10
-503 ( 8
-510 ( 7
-400 ( 7
-388 ( 8
-372 ( 12
-309 ( 12
-314 ( 10
-
MV
TeQ
TQ
-0.34 ( 0.02
0.82 ( 0.01
-0.33
0.82
-0.24
-0.42
0.24
-0.28
0.17
0.48
-1.38
0.21
0.84
-0.23
-0.05
0.14
-0.28
0.44
0.81
-1.15
0.44
-0.60
0.84
-0.23
-0.05
0.08
-0.28
0.41
0.24
-1.27
0.32
0.84
8-NEt2
8-NMe2
8-OMe
8-Me
8-F
8-Cl
8-CF3
8-SO2Me
8-SO2Bu
7NEt2
7-NMe2
7-OMe
7-Me
E
E
A,C
A,C
B,D
A,C
A,C
A,D
A,D
E
MV
MV
MV
MV
MV
BV
DQ
-
0.24 ( 0.03
0.17 ( 0.005
0.53 ( 0.03
-1.38 ( 0.02
E
-525 ( 8
-494 ( 8
-474 ( 11
-400 ( 8
-397 ( 7
-345 ( 10
-297 ( 10
-296 ( 10
-260 ( 10
-671 ( 6
-668 ( 6
-558 ( 8
-493 ( 8
-443 ( 7
-391 ( 8
-335 ( 10
-258 ( 11
-240 ( 16
-489 ( 8
-481 ( 10
-427 ( 10
-
TQ
TQ
A,C
A,C
B,C
A,C
B,C
A,D
A,D
B,C
E
MV
MV
MV
DQ
DQ
DQ
DQ
TeQ
TeQ
TQ
0.13 ( 0.003
7-F
7-Cl
0.46 ( 0.04
0.81 ( 0.05
-1.15 ( 0.05
9g
9h
9i
9j
7-CF3
7-SO2Me
7-SO2Bu
7-NO2
6-NEt2
6-NMe2
6-OMe
6-Me
12a
12b
12c
12d
12e
12f
12g
12h
12i
16a
16b
16c
16d
16e
16f
E
A,D
A,C
A,C
A,C
A,C
A,D
A,D
E
MV
MV
MV
MV
DQ
DQ
MV
BV
BV
-
0.21 ( 0.03
6-F
6-Cl
6-CF3
6-SO2Me
6-SO2Bu
5-NEt2
5-NMe2
5-OMe
5-Me
E
-0.23
-0.42
0.13
0.10
-0.28
A,C
A,C
B,D
A,D
5-Cl
5-F
-401 ( 8
-394 ( 7
MV
MV
0.07 ( 0.01
a
b
Determined in FCS + 5% R-medium. One-electron potential in mV; determined by pulse radiolysis. c DQ; duroquinone (2,3,5,6-
tetramethylbenzoquinone) ) -260 ( 10 mV:44 BV; benzyl viologen (1,1′-dibenzyl-4,4′-bipyridinium) ) -375 ( 10 mV:45 MV; methyl viologen
(1,1′-dimethyl-4,4′-bipyridinium) ) -447 ( 7 mV:46 TQ; triquat (7,8-dihydrodipyrido-[1,2-a:2′,1′-c][1,4]diazepinediium) ) -548 ( 7 mV:46
TeQ, tetraquat (6,7,8,9-tetrahydrodipyrido[1,2-a:2′,1′-c][1,4]diazocinediium) ) -635 ( 5 mV.47 In n-octanol/water. Using the program
d
e
ACDLogP v. 4.5.
study). Octanol/water partition coefficients at pH 7.4
were determined by a low volume shake flask method,
with drug concentrations in both the octanol and buffer
phases analyzed by HPLC as previously described.31
These values were used to “train” ACD LogP prediction
software (v. 4.5, Advanced Chemistry Development Inc.,
Toronto, Canada) and measured and predicted values
are reported (Table 1).
Biologica l Assa ys
The efficacy of the compounds in killing aerobic and
hypoxic mouse SCCVII tumor cells in vitro was deter-
mined by clonogenic survival. Cytotoxicity was mea-
sured as the concentration required to reduce plating
efficiency to 10% of controls (C10), and the RHT was
determined as the intraexperimental ratio of hypoxic
TPZ C10 to hypoxic BTO C10 [RHT ) C10(TPZ)/C10-
(BTO)]. The intraexperimental differential between the
hypoxic and aerobic cytotoxicity for each compound was
calculated as the HCR [) C10(aerobic)/C10(hypoxic)]
(Table 2).
Pulse radiolysis experiments were performed on The
University of Auckland Dynaray 4 (4 MeV) linear
accelerator (200 ns pulse length with a custom-built
optical radical detection system). E(1) values for the
compounds were determined in anaerobic aqueous solu-
tions containing 2-propanol (0.1 M) buffered at pH 7.0
(10 mM phosphate) by measuring the equilibrium
constant32 for the electron transfer between the radical
anions of the compounds and the appropriate viologen
or quinone reference standard. Data were obtained at
three concentration ratios at room temperature (22 (
2 °C) and used to calculate the ∆E between the
compounds and references, allowing for ionic strength
effects on the equilibria.
Twelve compounds (Table 3) were evaluated using a
proliferation assay (Sulforhodamine B; SRB) to deter-
mine IC50 values under oxic and anoxic conditions. For
each experiment, all 12 compounds were simultaneously
tested under both conditions against both SCCVII and
HT-29 cell lines. Thus each set of IC50 determinations
is an intraexperiment comparison. Plates were stained
as described previously33 and IC50 values determined.
Results were averaged for three independent experi-
ments.

Hypoxia-Selective Analogues of Tirapazamine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 173
Ta ble 2. Clonogenic Assay Data for BTO Analogues in SCCVII
Cells
increased solubility compared to TPZ, while the 7-Cl 9f
was ca. 100-fold less soluble. Generally, the 5- and
8-substituted BTOs were more soluble than the corre-
sponding 6- and 7-substituted analogues, suggesting
that a peri interaction with the N-oxides may reduce
crystallinity via distortion of intermolecular hydrogen
bonding. Generally, simple substituents on ring-A low-
ered solubility, emphasizing the need for extended
substituents containing isolated polar groups to increase
aqueous solubility.
The hydrophobicity of TPZ and eleven other BTOs
were determined from HPLC analysis of drug distribu-
tion between octanol and aqueous buffer at pH 7.4
(Table 1). The measured value for TPZ (-0.34 ( 0.02)
was in good agreement with the literature value (-0.32)8
as was the value for the 7-Me-BTO 9d (measured 0.13
( 0.003; lit.24 0.2). The measured data, including
literature values, were used to train ACDLogP software,
which was then used to predict the values for the whole
set of BTOs. There was a strong correlation between
no.
R
C10(hyp)a(µM) C10(aer)b(µM) RHTc
HCRd
1
H
10.2 ( 0.5
(34)e
114
2644 ( 266
(9)e
1.0
258 ( 21
(9)e
16.7
5a 8-NEt2
5b 8-NMe2
5c 8-OMe
5d 8-Me
5e 8-F
5f 8-Cl
5g 8-CF3
5h 8-SO2Me
5i
9a 7NEt2
9b 7-NMe2
9c 7-OMe
9d 7-Me
9e 7-F
9f 7-Cl
9g 7-CF3
9h 7-SO2Me
9i
9j
12a 6-NEt2
12b 6-NMe2
12c 6-OMe
12d 6-Me
12e 6-F
12f 6-Cl
12g 6-CF3
12h 6-SO2Me
1904
2769
>135
2538
132
102
626
338
175
>725
3159
1934
2031
381
271
305
251
0.089
0.065
0.40
0.41
1.0
3.9
1.8
0.20
1.2
<0.14
0.043
0.53
0.63
1.9
1.1
1.1
0.32
1.3
1.9
157
17.6
25.4
24.7
10.2
2.58
5.56
50.8
8.31
>72.5
237
19.3
16.2
5.41
9.48
9.14
31.7
7.76
5.37
>50.8
>440
127
>5.3
102.9
13.0
39.4
112.5
6.7
8-SO2Bu
21.1
13.3
100.0
125.0
70.3
28.6
33.3
7.9
7-SO2Bu
7-NO2
>89.6
36.3
>1269
440
>11.5
6.8
the measured (logPmeas) and calculated logP (logPcalc
)
<0.20
<0.023
0.080
0.58
1.0
without training (eq 1a) but this improved after training
(eq 1b):
<1.0
ndf
2901
114
140
110
17.4
10.2
6.47
8.23
26.1
16.2
67.7
25.8
58.0
38.1
7.46
10.2
166.7
11.2
21.6
13.3
<10.5
<6.3
0.87
1.2
logPmeas ) 0.72 logPcalc + 0.15
1.6
1.2
n ) 12, r2 ) 0.903, F ) 93
(1a)
(1b)
<274
<102
58.7
30.5
47.9
<381
10.2
<76.1
0.39
0.63
0.15
0.39
0.18
0.27
1.4
12i
6-SO2Bu
logPmeas ) 1.01 logPcalc + 0.012
16a 5-NEt2
16b 5-NMe2
16c 5-OMe
16d 5-Me
16e 5-Cl
0.83
<10.0
1.4
n ) 12, r2 ) 0.997, F ) 2809
The main improvements came from including 5h , 9g,
and 9h in the training set. For the whole set of BTOs,
the trained logP was strongly correlated with the
substituent hydrophobicity parameter π (eq 2):
16f 5-F
1.0
<7.5
a
Concentration of drug for a one-log cell kill under hypoxia
b
normalized against TPZ (1) in the same experiment. Concentra-
tion of drug for
a one-log cell kill under aerobic conditions
normalized against TPZ (1) in the same experiment. c Intraex-
d
logPtrain ) 0.82π - 0.17
perimental Relative Hypoxic Toxicity; C10(TPZ)/C10(BTO). In-
traexperimental Hypoxic Cytotoxicity Ratio; C10(AER)/C10(HYPOX).
e Mean ( SEM, with number of measurements in brackets. f Not
determined due to poor solubility.
n ) 3, 5r2 ) 0.847, F ) 183
(2)
E(1) values were determined by pulse radiolysis for
TPZ and all but two of the series (Table 1). The
measured value for TPZ (-456 ( 8 mV) was in close
agreement with the value of -0.45 ( 0.01 V previously
reported.17 The values range widely, from -240 mV for
the 6-BuSO2-BTO (12i) to -671 mV for the 6-NEt2-BTO
(12a ). Regression analysis of substituents for each of
the four positions (including TPZ; 1 in each case) gave
Resu lts a n d Discu ssion
P h ysicoch em ica l Mea su r em en ts. The solubility of
TPZ in medium containing 5% fetal calf serum was
determined as 8.9 mM. This is in reasonable agreement
with the solubility (13.5 mM) previously determined in
0.9% NaCl solution.24 Three compounds (8-NEt2-BTO
5a , 8-NMe2-BTO 5b, and 5-NMe2-BTO 16b) showed
Ta ble 3. IC50 Assay Data for BTO Compounds in SCCVII and HT-29 Cells
IC50aSCCVII
IC50aHT-29
hypoxic
no.
name
hypoxic
oxic
RHTb
HCRc
oxic
RHTb
HCRc
1
5c
5e
5f
5h
9e
9f
9g
9j
12d
12f
16b
TPZ
8-OMe-BTO
8-F-BTO
8-Cl-BTO
8-MeSO2-BTO
7-F-BTO
2.4 ( 0.3
13.5 ( 2.9
1.2 ( 0.3
71.7 ( 8.7
288 ( 14
23.7 ( 4.6
8.2 ( 0.7
15.5 ( 2.1
17.6 ( 1.4
12.2 ( 1.2
23.9 ( 3.5
2.8 ( 0.2
217 ( 31
21.1 ( 3.2
2.1 ( 0.3
1.0
0.2
1.9
5.2
0.2
2.2
3.9
2.6
3.4
0.4
2.4
0.2
32.9 ( 7.2
25.7 ( 7.0
13.9 ( 3.6
15.9 ( 5.8
1.6 ( 0.3
19.3 ( 3.9
20.4 ( 2.7
27.7 ( 5.0
4.1 ( 0.3
5.3 ( 0.2
28.8 ( 4.4
2.1 ( 0.2
0.55 ( 0.25
15.1 ( 0.8
1.87 ( 0.25
0.73 ( 0.12
2.1 ( 0.9
0.79 ( 0.38
12.9 ( 0.8
1.3 ( 0.07
23.3 ( 0.7
196 ( 43
1.0
0.2
2.5
9.6
0.4
2.8
7.2
2.5
6.7
0.4
4.1
0.2
37.0 ( 7.5
21.9 ( 4.8
4.1 ( 0.4
624 ( 151
8.7 ( 1.5
0.45 ( 0.14
9.7 ( 0.5
7.7 ( 4.0
13.6 ( 1.3
4.5 ( 0.8
69.8 ( 14.9
47.8 ( 9.1
19.8 ( 4.4
79.1 ( 15.2
14.1 ( 4.0
469 ( 84
1.1 ( 0.3
21.7 ( 2.8
28.5 ( 8.4
35.7 ( 6.1
14.8 ( 0.1
35.8 ( 4.5
33.0 ( 7.7
0.18 ( 0.05
7-Cl-BTO
0.61 ( 0.07
0.92 ( 0.15
0.70 ( 0.09
5.6 ( 0.7
7-CF3-BTO
7-NO2-BTO
6-Me-BTO
6-Cl-BTO
42.6 ( 10.4
22.5 ( 4.0
0.18 ( 0.03
0.98 ( 0.17
12.2 ( 1.5
41.2 ( 8.6
4.2 ( 1.2
5-NMe2-BTO
a
b
IC50 values in µM (mean ( SEM, n g 3). Interexperimental relative hypoxic toxicity; C10(TPZ)/C10(BTO). c Interexperimental hypoxic
cytotoxicity ratio; C10(AER)/C10(HYPOX).

174 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hay et al.
25-fold) more cytotoxic than predicted by E(1) alone,
possibly due to accelerated metabolic activation. It
excludes the 6-NMe2-BTO (12b), which was measured
at its solubility limit and had a very low E(1) value.
Addition of various hydrophobicity parameters (π for the
substituents, or logP values) to eq 4a gave small but
significant improvements (with π being best, eq 4b),
showing that, for this set of analogues, aerobic activity
was not very dependent on hydrophobicity.
log(aerobicC10) ) -0.12 - 6.81((0.90)E(1) ×
10-3 - 1.36((0.18)D - 0.23((0.11)π (4b)
n ) 25, r2 ) 0.83, F ) 33
Equations 4a and 4b show that (aerobic) potency in
the clonogenic assay is favored by higher reduction
potential. This is reasonable, since higher E(1)s will lead
to more rapid (aerobic) metabolism. Previous work34
F igu r e 1. Cytotoxicity of BTO analogues against SCCVII cells
(clonogenic assay) versus one electron reduction potential:
aerobic, 0; hypoxic, b; solid line eq 4a, dashed line eq 5a.
demonstrated a linear correlation (slope 10.4 V-1
)
the following equations:
between the (logarithm of) oxygen consumption rate in
CHO cells and the E1/2 for sixteen BTOs, based on other
data.24 The regression coefficient in eq 4 (5.9 V-1) is in
reasonable agreement with that, suggesting that under
aerobic conditions reactive oxygen species, formed by
redox-cycling of the BTO radical anion with oxygen, are
mainly responsible for toxicity.
Hypoxic C10 values could be determined for all but
three of the BTO analogues studied, and E(1) values
were measured for all but one of these (the 5-Me-BTO
16d , which could be prepared only in very low yield).
The hypoxic C10 values of the remaining thirty-one
compounds in SCCVII cells were less well predicted by
a linear relationship with E(1) (equation 5a and Figure
1):
5-sub: E(1) /mV ) -453 + 161σm
n ) 7, r2 ) 0.976, F ) 160
(3a)
(3b)
(3c)
(3d)
6-sub: E(1) /mV ) -454 + 282σp
n ) 10, r2 ) 0.987, F ) 596
7-sub: E(1) /mV ) -424 + 171σp
n ) 10, r2 ) 0.933, F ) 111
8-sub: E(1) /mV ) -492 + 287σm
n ) 10, r2 ) 0.946, F ) 106
log(hypoxicC10) ) 0.07 - 2.88((0.08)E(1) × 10-3
In all cases, the E(1) values were predicted very well
by the σ values of the substituents, with no significant
steric effects, even for 5- and 8-substituents. In both the
5- and 8-substituted series, the correlation fitted best
to σm, with the 8-substituents showing the stronger
dependence. In the 6- and 7-substituted series, the data
were fitted better by the σp parameter, with a stronger
dependence for the 6-substituents, which also spanned
the largest E(1) range of more than 400 mV. These well-
defined relationships make it possible to adjust E(1)
quite precisely by appropriate substitution of the 3-ami-
no-BTO nucleus.
(5a)
n ) 31, r2 ) 0.30, F ) 12
The significance of the QSAR was slightly improved
by the addition of hydrophobicity parameters (logP, π,
MR), with the best being π:
log(hypoxicC10) ) -0.47 - 4.32((0.05)E(1) ×
10-3 - 0.37((0.11)π (5b)
n ) 31, r2 ) 0.51, F ) 15
Clon ogen ic Assa y. Twenty-five of the thirty-four
BTO analogues were sufficiently soluble to obtain
aerobic cytotoxicity data in SCCVII cells, using a
clonogenic assay (concentration of drug for a one-log cell
kill; C10 in µM) (Figure 1). The aerobic logC10 values
showed a linear dependence on E(1), as shown by eq
4a:
Unlike the aerobic toxicity, there appeared to be a loss
of hypoxic potency at both low (< ca. -500 mV) and high
(>-370 mV) ends of the E(1) range (Figure 1). This is
reflected by the less strong linear correlation between
E(1) and hypoxic C10. The most potent compounds were
halo-substituted analogues; 8-Cl-BTO (5f), 7-F-BTO
(9e), 6-Cl-BTO (12f), 5-Cl-BTO (16e), and 8-CF3-BTO
(5g). BTO analogues with a dimethylamino or diethy-
lamino substituent at any position were considerably
less potent than TPZ under hypoxia, also resulting in
very low RHT values. With the exception of 5-Cl-BTO
(16e), analogues with a 5-substituent were less potent
than TPZ under hypoxia, and all 5-substituted BTO
analogues were considerably more potent than TPZ
under aerobic conditions. This resulted in low RHT
log(aerobicC10) ) -0.14 - 5.90((0.90)E(1) ×
10-3 - 1.41((0.19)D (4a)
n ) 25, r2 ) 0.79, F ) 42
This analysis includes a dummy variable (D), to
account for the fact that the four sterically strained
5-substituted analogues (16a -c, 16e) are much (about

Hypoxia-Selective Analogues of Tirapazamine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 175
F igu r e 3. Comparison of clonogenic assay (C10 values) with
proliferation assay (IC50 values, SRB) for SCCVII cells.
F igu r e 2. Hypoxic selectivity of BTO analogues against
SCCVII cells (clonogenic assay) versus one electron reduction
potential.
Thus another strategy to improve therapeutic indices
is to target the BTO analogue to the nucleus via a DNA
binding moiety. Indeed, preliminary studies show that
DNA-targeting does increase the potency of BTO ana-
logues.38
values and no hypoxic selectivity (Figures 1 and Table
2). Analogues contributing to the falloff in potency at
high reduction potential include methyl sulfones 5h , 9h ,
12h , and butyl sulfones 9i and 12i. The low logP values
for the methyl sulfones (-1.38, -1.15, and -1.27,
respectively) may contribute to poor uptake by cells
resulting in reduced potency. Another potential factor
contributing to reduced hypoxic potency for analogues
with high reduction potentials may be an increased rate
of metabolic depletion. It is notable that none of the BTO
analogues gave an overall increase in hypoxic potency
× solubility compared to TPZ (1).
Com p a r ison of C10 a n d IC50 Va lu es in SCCVII
Cells. Clonogenic survival (C10) assays have the advan-
tage of being a direct measure of cell sterilization, but
are time-consuming. Proliferation assays, such as the
SRB assay, enable higher throughput screening but are
potentially subject to artifacts related to different rates
of cell death or cell cycle arrest for different treatments.
To assess the suitability of rapid IC50 assays for further
development of TPZ analogues, we utilized a subset of
BTO compounds with a range of potencies and dif-
ferential toxicities, as determined by clonogenic sur-
vival, and compared IC50 values using the SRB assay
(Table 3). Despite the differences in exposure time (1 h
vs 4 h), endpoint (C10 vs IC50) and method for achieving
hypoxia, the SAR remained consistent, with the data
for all three parameters derived from both assays
showing good agreement (oxic cytotoxic potency, hypoxic
cytotoxic potency and HCR; R ) 0.93, 0.90, and 0.80,
respectively; P < 0.002). log-log regression analysis of
the data suggests the SRB assay is an adequate sur-
rogate for clonogenic survival, at least in the SCCVII
cell line (Figure 3). TPZ is more toxic under aerobic
conditions in the IC50 assay than would be predicted by
the C10 assay. This anomaly is not observed under
hypoxic conditions resulting in a higher hypoxic selec-
tivity being displayed by TPZ in the clonogenic assay
(258-fold; Table 2) than in the IC50 assay (33-fold; Table
3). However, for the other compounds in the study HCR
values for the two assays were in closer agreement
(Figure 3).
There was no clear relationship between HCR and
E(1) in the clonogenic assay, (Figure 2). None of the BTO
analogues displayed hypoxic selectivity greater than
TPZ. However, a cluster of compounds with good hy-
poxic selectivity (HCR g 50) [8-Me-BTO (5d ), 7-OMe-
BTO (9c), 7-Me-BTO (9d ) and 6-Me-BTO (12d )] had
weakly electron-donating substituents, giving E(1) val-
ues in the range of -510 to -450 mV. Other compounds
with E(1) values within this range that did not show
good selectivity were 5-NEt2-BTO (16a ) and 5-NMe2-
BTO (16b), further emphasizing that 5-substituted BTO
analogues are poor hypoxia-selective cytotoxins. Two
other analogues 8-CF3-BTO (5g) and 7-F-BTO (9e)
displayed good selectivity suggesting hypoxic selectivity
is influenced by a variety of factors as well as E(1).
The most common side effects of TPZ observed in
clinical trial21,22,35 were neutropenia, fatigue, muscle
cramps, and nausea and vomiting. The mechanisms of
these toxicities are poorly understood, especially whether
they arise from metabolism to the TPZ radical in normal
tissues under physiological hypoxia, aerobic redox cy-
cling leading to reactive oxygen species, or mitochon-
drial TPZ metabolism. One possible strategy to reduce
normal tissue toxicity is to increase the hypoxic selectiv-
ity of BTO analogues. The clonogenic assay data identi-
fies a range of E(1) values (-450 to -510 mV) where
maximal HCR was observed. It is expected that com-
pounds with lower E(1) values will have decreased rates
of metabolism under aerobic conditions, and the higher
selectivity may translate into improved therapeutic
indices. Much of the metabolism of TPZ occurs outside
the nucleus36 and does not result in DNA double strand
breaks, and mitochondrial toxicity induced by TPZ37 is
probably involved in the pathogenesis of muscle cramps.
Com p a r ison of SCCVII a n d H T-29 Cell Lin e
Scr een s. To evaluate potential intercell line variations
in BTO analogue SAR, the SRB assay was also used to
compare the murine cell line SCCVII with the human
adenocarcinoma cell line HT-29. Highly significant log-
log correlations between the two cell lines were seen for
all measured parameters (oxic cytotoxic potency, hypoxic
cytotoxic potency, and HCR; R ) 0.90, 0.99, and 0.92,
respectively; P < 0.001). This is illustrated for the
relationship between hypoxic IC50 in the two lines in
Figure 4. Thus the SAR for both the aerobic and anoxic

176 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hay et al.
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 hertz, respec-
tively. Assignments were determined using COSY, HSQC, and
HMBC two-dimensional experiments. Mass spectra were
determined on a VG-70SE mass spectrometer using an ionizing
potential of 70 eV at a nominal resolution of 1000. High-
resolution spectra were obtained at nominal resolutions of
3000, 5000, or 10000 as appropriate. All spectra were obtained
as electron impact (EI) using PFK as the reference unless
otherwise stated. Solutions in organic solvents were dried with
anhydrous Na2SO4. Solvents were evaporated under reduced
pressure on a rotary evaporator. Thin-layer chromatography
was carried out on aluminum-backed silica gel plates (Merck
60 F254) 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 biological testing were analyzed at >99% purity by reverse
phase HPLC using a Philips PU4100 liquid chromatograph, a
Phenomenex BondClone 10-C18 stainless steel column (300
mm × 3.9 mm i.d.) and a Philips PU4120 diode array detector.
Chromatograms were run using various gradients of aqueous
(1 M NaH2PO4, 0.75 M heptanesulfonic acid, 0.5 M dibuty-
lammonium phosphate, and MilliQ water in a 1:1:1:97 ratio)
and organic (80% MeOH/MilliQ water) phases. DCM refers to
dichloromethane, DME refers to dimethoxyethane, DMF refers
to dry dimethylformamide, ether refers to diethyl ether, EtOAc
refers to ethyl acetate, EtOH refers to ethanol, MeOH refers
to methanol, and pet. ether refers to petroleum ether, boiling
range 40-60 °C; THF refers to tetrahydrofuran dried over
sodium benzophenone ketyl. All solvents were freshly distilled.
Nitroanilines 2d , 2f, 2g, 6c, 6d , 6e, 6f, 10d , 10e, 10f, 10g,
13d , 13f, chloronitrobenzenes 7g, 7j, 13e, and 2,6-difluoroni-
trobenzene 3e, were commercially available. Nitroanilines 2c,39
10h ,29 and 13c29 were prepared as previously described. The
synthesis of nitroanilines 2h , 2i, 6h , 6i, 10c, 10i, 13g, and
13h is described herein.
F igu r e 4. Comparison of Hypoxic IC50 values: HT-29 cells
versus SCCVII cells.
toxicity of BTO analogues is very similar in both cell
lines evaluated.
Con clu sion s
The use of substituted BTO analogues with a wide
range of electronic and lipophilic properties allowed a
better definition than previously of the influences of
these properties on cytotoxicity. One-electron reduction
potentials were highly dependent on substituent elec-
tronic properties, and aerobic C10s in the clonogenic
assay were highly dependent on E(1). However, all
5-substituted analogues were exceptionally toxic under
aerobic conditions, which lowered their HCR ratios.
Hypoxic C10s were less well predicted, but again reduc-
tion potential was the most important parameter. In
each case, potency was slightly increased with increas-
ing lipophilicity.
Overall, the results suggest that ring-A substituents
in BTO analogues can be used to predictably vary one-
electron reduction potentials, and that high hypoxic
cytotoxicity ratios (HCRs > 50) are best achieved by
weakly electron-donating substituents that result in
E(1) values between -450 to -510 mV. This defines a
narrow range of substituent electronic properties re-
quired to optimize the 1,2,4-benzotriazine-3-amine 1,4-
dioxide nucleus as a hypoxic cell cytotoxin and allows
the rational design of analogues that have improved
pharmacological properties (e.g., solubility and distribu-
tion) while retaining high HCR and RHT values. Our
efforts to improve the therapeutic indices of BTO
analogues by targeting compounds to the nucleus and
reducing the amount of unproductive metabolism will
be the subject of pending publication.
3-(Meth ylsu lfa n yl)-2-n itr oa n ilin e (2h ). A solution of
LiSMe (1.19 g, 22.0 mmol) in DMF (20 mL) was added
dropwise to a stirred solution of 3-chloro-2-nitroaniline (2f)
(3.17 g, 18.4 mmol) in DMF (80 mL) at 20 °C and the mixture
stirred for 2 h. The mixture was poured into water (300 mL)
and extracted with EtOAc (2 × 150 mL). The combined organic
fraction was washed with water (2 × 100 mL) and brine (50
mL) and dried and the solvent evaporated. The residue was
chromatographed, eluting with 20% EtOAc/pet. ether, to give
(i) starting material (0.51 g, 16%) and (ii) sulfide (2h ) (2.36 g,
70%) as red crystals, mp (EtOAc/pet. ether) 70-72 °C; 1H NMR
δ 7.21 (t, J ) 8.2 Hz, 1 H, H-5), 6.55 (d, J ) 8.2 Hz, 2 H, H-4,
H-6), 5.92 (br s, 2 H, NH2), 2.42 (s, 3 H, SCH3); 13C NMR δ
146.0, 141.6, 133.3, 131.0, 113.9 (2), 17.0; Anal. (C7H8N2O2S)
C, H, N.
3-(Bu tylsu lfa n yl)-2-n itr oa n ilin e (2i). A solution of LiSBu
(1.39 g, 14.5 mmol) in DMF (10 mL) was added dropwise to a
stirred solution of 3-chloro-2-nitroaniline (2f) (2.08 g, 12.05
mmol) in DMF (50 mL) at 20 °C and the mixture stirred for 2
h. The mixture was poured into water (300 mL) and extracted
with EtOAc (2 × 150 mL). The combined organic fraction was
washed with water (2 × 100 mL) and brine (50 mL) and dried
and the solvent evaporated. The residue was chromatographed,
eluting with 20% EtOAc/pet. ether, to give sulfide 2i (2.47 g,
91%) as a red oil, 1H NMR δ 7.17 (dd, J ) 8.2, 8.0 Hz, 1 H,
H-5), 6.62 (d, J ) 8.0 Hz, 1 H, H-4), 6.54 (dd, J ) 8.2, 1.0 Hz,
1 H, H-6), 5.74 (br s, 2 H, NH2), 2.87 (t, J ) 7.4 Hz, 2 H, CH2S),
1.64-1.72 (m, 2 H, CH2), 1.45-1.53 (m, 2 H, CH2), 0.95 (s, 3
H, CH3); 13C NMR δ 145.3, 140.1, 133.0, 132.4, 115.0, 113.9,
33.0, 29.8, 22.2, 13.6; MS (EI) m/z 226 (M+, 35%), 106 (100);
HRMS (EI) calcd for C10H14N2O2S (M+) m/z 226.0776, found
226.0773.
In addition, the excellent agreement between clono-
genic and proliferation assays for both hypoxic and
aerobic cytotoxic potency demonstrate the utility of the
IC50 assays for the higher throughput screening of
hypoxic cytotoxins, especially for SAR comparisons
within related series of analogues, where the influences
of other mechanisms of action are likely to be similar.
Exp er im en ta l Section
Analyses were carried out in the Microchemical Laboratory,
University of Otago, Dunedin, NZ. Melting points were
determined on an Electrothermal 2300 Melting Point Ap-
paratus. NMR spectra were obtained on a Bruker AM-400
4-(Meth ylsu lfa n yl)-2-n itr oa n ilin e (6h ) a n d 6-(Meth yl-
su lfa n yl)-2-n itr oa n ilin e (13h ). O-(3-Nitrophenyl) dimeth-
ylthiocarbamate28 (17) (14.05 g, 62.1 mmol) was heated at
1
spectrometer at 400 MHz for H and 100 MHz for 13C spectra.

Hypoxia-Selective Analogues of Tirapazamine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 177
235-240 °C for 3 h under N2, cooled to 20 °C, to give crude
S-(3-nitrophenyl) dimethylthiocarbamate (18) which was heated
at reflux temperature with KOH solution (7.5 M, 410 mL, 3.1
mol) and MeOH (200 mL) for 2 h. The mixture was cooled to
20 °C, Me2SO4 (59 mL, 0.62 mol) added dropwise, and the
mixture stirred at 20 °C for 16 h. The mixture was partitioned
between EtOAc (300 mL) and water (300 mL), the organic
fraction washed with water (3 × 100 mL) and brine (100 mL)
and dried, and the solvent evaporated. The residue was
chromatographed, eluting with 10% EtOAc/pet. ether, to give
1-(methylsulfanyl)-3-nitrobenzene (19) (9.6 g, 91%) as a soft
13C NMR δ 145.8, 142.5, 132.5, 127.0, 122.7, 115.6, 35.0, 31.6,
21.7, 13.6; MS (EI) m/z 226 (M+,100%), 209 (15), 192 (25);
HRMS (EI) calcd for C10H14N2O2S (M+) m/z 226.0776, found
226.0770; (ii) 4-(butylsulfanyl)-2-nitroaniline (6i) (1.12 g, 19%)
as a red oil, 1H NMR δ 8.16 (d, J ) 2.2 Hz, 1 H, H-3), 7.39 (dd,
J ) 8.7, 2.2 Hz, 1 H, H-5), 6.76 (d, J ) 8.7 Hz, 1 H, H-6), 6.06
(br s, 2 H, NH2), 2.83 (dd, J ) 7.3, 7.2 Hz, 2 H, CH3S), 1.56-
1.62 (m, 2 H, CH2), 1.38-1.48 (m, 2 H, CH2), 0.91 (dd, J )
7.4, 7.3 Hz, 3 H, CH3); 13C NMR δ 143.5, 139.1, 132.2, 128.3,
123.9, 119.3, 35.3, 31.2, 21.8, 13.6; MS (EI) m/z 226 (M+,100%),
170 (80); HRMS (EI) calcd for C10H14N2O2S (M+) m/z 226.0776,
found 226.0772; and (iii) 2-(butylsulfanyl)-4-nitroaniline (21)
1
solid, H NMR δ 8.05 (dd, J ) 2.0, 1.9 Hz, 1 H, H-2), 7.96 (dd,
1
(0.76 g, 13%) as a red oil, H NMR δ 8.16 (d, J ) 2.6 Hz, 1 H,
J ) 8.2, 2.0, 0.9 Hz, 1 H, H-4), 7.53 (ddd, J ) 7.9, 1.9, 1.0 Hz,
1 H, H-6), 7.43 (dd, J ) 8.2, 7.9 Hz, 1 H, H-5), 2.56 (s, 3 H,
SCH3); 13C NMR δ 148.6, 141.6, 131.9, 129.4, 120.3, 119.6, 15.4.
H-3), 8.00 (dd, J ) 9.1, 2.6 Hz, 1 H, H-5), 6.68 (d, J ) 9.1 Hz,
1 H, H-6), 5.07 (br s, 2 H, NH2), 2.79 (dd, J ) 7.2, 6.6 Hz, 2 H,
CH2S), 1.52-1.60 (m, 2 H, CH2), 1.38-1.47 (m, 2 H CH2), 0.91
(t, J ) 7.3 Hz, 3 H, CH3); 13C NMR δ 153.5, 138.7, 131.4, 125.7,
118.1, 113.0, 34.7, 31.5, 21.7, 13.6.
A solution of NH2OMe.HCl (2.83 g, 34.0 mmol) and ni-
trobenzene 19 (4.79 g, 28.3 mmol) in DMF (100 mL) was added
dropwise to a stirred mixture of KOtBu (13.0 g, 116.6 mmol)
and CuCl (0.28 g, 2.83 mmol) in DMF (50 mL) at 5 °C. The
mixture was stirred at 20 °C for 3 h and quenched with
saturated aqueous NH4Cl solution (100 mL). The mixture was
extracted with EtOAc (3 × 100 mL), the combined organic
fraction dried, and the solvent evaporated. The residue was
chromatographed, eluting with 10% EtOAc/pet. ether, to give
(i) 6-(methylsulfanyl)-2-nitroaniline (13h ) (2.11 g, 40%) as a
red oil, 1H NMR δ 8.11 (dd, J ) 8.7, 1.5 Hz, 1 H, H-3), 7.66
(dd, J ) 7.4, 1.5 Hz, 1 H, H-5), 6.92 (br s, 2 H, NH2), 6.66 (dd,
J ) 8.7, 7.4 Hz, 1 H, H-4), 2.38 (s, 3 H, SCH3); 13C NMR δ
145.0, 140.7, 132.5, 126.6, 124.5, 115.9, 18.2; MS (EI) m/z 184
(100, M+), 169 (10), 150 (30); HRMS calcd for C7H8N2O2S (M+)
m/z 184.0307, found 184.0304; (ii) 4-(methylsulfanyl)-2-nitroa-
niline (6h ) (0.8 g, 15%) as a red oil, 1H NMR δ 8.05 (d, J ) 2.3
Hz, 1 H, H-3), 7.34 (dd, J ) 8.7, 2.3 Hz, 1 H, H-5), 6.76 (d, J
) 8.7 Hz, 1 H, H-6), 6.05 (br s, 2 H, NH2), 2.46 (s, 3 H, SCH3);
13C NMR δ 143.1, 136.8, 132.2, 125.8, 125.1, 119.5, 18.0.
5-Meth oxy-2-n itr oa n ilin e (10c). A mixture of 5-methoxy-
2-nitrobenzoic acid (22) (10 g, 50.7 mmol), diphenylphosphoryl
azide (DPPA) (11.5 mL, 53.3 mmol), and Et3N (7.4 mL, 53.3
mmol) in t-BuOH (200 mL) was heated at reflux temperature
for 16 h. The solution was cooled to 20 °C and the solvent
evaporated. The residue was dissolved in DCM (300 mL) and
washed with water (2 × 100 mL), saturated aqueous KHCO3
(100 mL), and brine (50 mL), dried and the solvent evaporated.
The residue was suspended in MeOH (250 mL), concd HCl (50
mL) added, and the mixture stirred at 20 °C for 96 h. The
solvent was evaporated and the residue suspended in satu-
rated aqueous KHCO3 (400 mL) and stirred for 30 min. The
suspension was filtered and the solid washed with water (20
mL) and dried at 80 °C under reduced pressure. The solid was
chromatographed, eluting with a gradient (20-30%) of EtOAc/
pet. ether, to give 10c (8.26 g, 98%), as a yellow solid, mp 128-
130 °C (lit.29 mp 130-132 °C); 1H NMR δ 8.07 (d, J ) 9.5 Hz,
1 H, H-3), 6.28 (dd, J ) 9.5, 2.6 Hz, 1 H, H-4), 6.21 (br s, 2 H,
NH2), 6.15 (d, J ) 2.6 Hz, 1 H, H-6), 3.83 (s, 3 H, OCH3); 13C
NMR δ 165.4, 147.1, 128.5, 126.9, 106.7, 99.4, 55.7.
5-(Bu tylsu lfa n yl)-2-n itr oa n ilin e (10i). A solution of LiS-
nBu (3.34 g, 34.8 mmol) in DMF (30 mL) was added dropwise
to a stirred solution of 5-chloro-2-nitroaniline (10f) (5.0 g, 30.0
mmol) in DMF (50 mL) at 20 °C and the mixture stirred for 2
h. The mixture was poured into water (300 mL) and extracted
with EtOAc (2 × 150 mL). The combined organic fraction was
washed with water (2 × 100 mL) and brine (50 mL) and dried
and the solvent evaporated. The residue was chromatographed,
eluting with 20% EtOAc/pet. ether, to give 10i (6.12 g, 90%)
as a red solid, mp (EtOAc/pet. ether) 91-93 °C; 1H NMR δ
8.00 (d, J ) 7.5 Hz, 1 H, H-3), 6.52-6.56 (m, 2 H, H-4, H-6),
6.11 (br s, 2 H, NH2), 2.96 (dd, J ) 7.4, 7.3 Hz, 2 H, CH2S),
1.66-1.73 (m, 2 H, CH2), 1.44-1.53 (m, 2 H, CH2), 0.96 (s, 3
H, CH3); 13C NMR δ 149.0, 144.8, 129.6, 126.4, 115.2, 113.4,
31.3, 30.6, 22.0, 13.6; Anal. (C10H14N2O2S) C, H, N.
4-(Bu tylsu lfa n yl)-2-n itr oa n ilin e (6i) a n d 6-(Bu tylsu l-
fa n yl)-2-n itr oa n ilin e (13i). A mixture of crude 18 (7.0 g, 31
mmol) and KOH (7.5 M, 41 mL, 310 mmol) in MeOH (200 mL)
was heated a reflux temperature for 2 h. The mixture was
cooled to 5 °C and the pH adjusted to 2 with concd HCl. The
precipitate was collected, washed with water (20 mL), dis-
solved in EtOAc (200 mL), and dried, and the solvent was
evaporated. The residue was dissolved in DMF (100 mL), K2-
CO3 (5.15 g, 37.3 mmol) added, and the mixture stirred at 20
°C for 30 min. n-Butyl bromide (4.0 mL, 37.3 mmol) was added
and the mixture stirred at 80 °C for16 h. The mixture was
cooled and the solvent evaporated. The residue was partitioned
between EtOAc (300 mL) and water (300 mL), the organic
fraction washed with water (3 × 100 mL) and brine (100 mL)
and dried, and the solvent evaporated. The residue was
chromatographed, eluting with 10% EtOAc/pet. ether, to give
4-(butylsulfanyl)-2-nitrobenzene (20) (5.53 g, 84%) as a yellow
1
oil, H NMR δ 8.10 (dd, J ) 2.0, 2.0 Hz, 1 H, H-2), 7.97 (ddd,
J ) 8.1, 2.0, 0.9 Hz, 1 H, H-4), 7.57 (ddd, J ) 8.1, 2.0, 0.9 Hz,
1 H, H-6), 7.42 (dd, J ) 8.1, 8.1 Hz, 1 H, H-5), 3.01 (dd, J )
7.4, 7.3 Hz, 2 H, CH2S), 1.64-1.71 (m, 2 H, CH2), 1.43-1.53
(m, 2 H, CH2), 0.95 (t, J ) 7.3 Hz, 3 H, CH3); 13C NMR δ 148.6,
140.5, 133.5, 129.4, 121.9, 120.1, 32.6, 30.7, 21.9, 13.6; MS (EI)
m/z 211 (M+, 60%), 155 (100); HRMS (EI) calcd for C10H13NO2S
(M+) m/z 211.0667, found 211.0661.
A solution of NH2OMe‚HCl (2.61 g, 31.2 mmol) and 20 (5.5
g, 26.0 mmol) in DMF (40 mL) was added dropwise to a stirred
mixture of KOtBu (12.0 g, 106.7 mmol) and CuCl (0.26 g, 2.6
mmol) in DMF (50 mL) at 5 °C. The mixture was stirred at 20
°C for 6 h and quenched with saturated aqueous NH4Cl
solution (300 mL). The mixture was extracted with EtOAc (3
× 100 mL), the combined organic fraction dried, and the
solvent evaporated. The residue was chromatographed, eluting
with 5% EtOAc/pet. ether, to give (i) 6-(butylsulfanyl)-2-
nitroaniline (13i) (2.34 g, 40%) as a red oil, 1H NMR δ 8.12
(dd, J ) 8.7, 1.5 Hz, 1 H, H-3), 7.66 (dd, J ) 7.3, 1.5 Hz, 1 H,
H-5), 7.00 (br s, 2 H, NH2), 6.64 (dd, J ) 8.7, 7.3 Hz, 1 H,
H-4), 2.75 (dd, J ) 7.5, 7.2 Hz, 2 H, CH2S), 1.51-1.58 (m, 2 H,
CH2), 1.37-1.46 (m, 2 H, CH2), 0.90 (dd, 7.4,7.2 Hz, 3 H, CH3);
Meth od A: Con d en sa tion of 2-Nitr oa n ilin es w ith Cy-
a n a m id e. 2-Nitroaniline (4.3 mmol) and cyanamide (22 mmol)
were melted together at 100 °C cooled to ca. 50 °C and concd
HCl (5 mL) added carefully. The mixture was stirred until the
exotherm subsided then stirred at 100 °C for 2 h. If necessary,
more cyanamide (22 mmol) was added and the mixture stirred
at 100 °C for 4 h. The mixture was cooled to 20 °C and made
strongly basic with 7.5 M NaOH solution (ca. 50 mL) and the
mixture heated at 100 °C for 1 h then cooled to 20 °C and
diluted with water (100 mL). The precipitate was filtered,
washed with water (2 × 10 mL) and ether (2 × 10 mL) and
dried. If necessary, the solid was chromatographed, eluting
with a gradient (2-5%) of MeOH/CHCl3, to give the corre-
sponding 1,2,4-benzotriazin-3-amine 1-oxide.
Meth od B: Con d en sa tion of 2-Nitr oh a loben zen es w ith
Gu a n id in e. Guanidine hydrochloride (104 mmol) was added
to a stirred solution of KOtBu (104 mmol) in absol EtOH (80
mL) and the mixture stirred at 20 °C for 1 h. The mixture
was filtered and the filtrate added slowly to a stirred solution
of 2-nitrohalobenzene (26 mmol) in absol EtOH (50 mL). The
mixture was heated at reflux temperature for 72 h then cooled,

178 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hay et al.
acidified with concd HCl and the solvent evaporated. The
residue was suspended in 0.5 M HCl and the precipitate was
filtered. The aqueous fraction was washed with CHCl3, basified
with aq NH3, and extracted into EtOAc. The organic fraction
was dried and the solvent evaporated. The residue was
suspended in 10% aq NaOH and heated at 100 °C for 2 h. The
precipitate was filtered, washed with water (2 × 10 mL), ether
(2 × 10 mL) and dried. If necessary, the solid was chromato-
graphed, eluting with a gradient (2-5%) of MeOH/CHCl3, to
give the corresponding 1,2,4-benzotriazin-3-amine 1-oxide.
7-F lu or o-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e (8e).
Method A using 6e gave 8e (78%) as a yellow powder, mp
(DMF) 280-290 °C (dec) [lit.40 mp 290 °C (dec)]; 1H NMR
[(CD3)2SO] δ 7.89 (dd, J ) 8.6, 2.9 Hz, 1 H, H-8), 7.76 (ddd, J
) 9.3, 8.8, 2.9 Hz, 1 H, H-6), 7.62 (dd, J ) 9.3, 5.2 Hz, 1 H,
H-5), 7.35 (br s, 2 H, NH2); Anal. (C7H5FN4O) C, H, N, F.
7-Ch lor o-1,2,4-ben zotr azin -3-am in e 1-Oxide (8f). Method
A using 6f gave 8f (39%) as a yellow powder, mp (DCM/pet.
ether) 309 °C (dec) [lit.27 (HOAc) 306-308 °C]; 1H NMR [(CD3)2-
SO] δ 8.14 (d, J ) 1.7 Hz, 1 H, H-8), 7.80 (dd, J ) 8.8, 1.9 Hz,
1 H, H-6), 7.56 (d, J ) 9.0 Hz, 1 H, H-5), 7.48 (br s, 2 H, NH2).
7-Tr iflu or om eth yl-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(8 g). Method B using 7g gave 8g (30%) as a yellow powder,
mp (DCM/pet. ether) 290 °C (dec) [lit.40 (acetone/toluene) 301-
8-Met h oxy-1,2,4-b en zot r ia zin -3-a m in e 1-Oxid e (4c).
Method A using 2c gave 4c (51%) as a yellow powder, mp
1
(H2O) 235-239 °C; H NMR [(CD3)2SO] δ 7.62 (dd, J ) 8.3,
8.0 Hz, 1 H, H-6), 7.15 (br s, 2 H, NH2), 7.02 (d, J ) 8.3 Hz, 1
H, H-7), 6.80 (d, J ) 8.0 Hz, 1 H, H-5), 3.83 (s, 3 H, OCH3);
13C NMR [(CD3)2SO] δ 160.0, 153.3, 151.4, 135.4, 122.6, 117.0,
105.1, 56.4; Anal. (C8H8N4O2) C, H, N.
1
302 °C]; H NMR [(CD3)2SO] δ 8.38 (br s, 1 H, H-8), 8.01 (dd,
J ) 8.9, 2.0 Hz, 1 H, H-6), 7.72 (br s, 2 H, NH2), 7.68 (d, J )
8.9 Hz, 1 H, H-5); 13C NMR [(CD3)2SO] δ 161.1, 150.6, 130.7
(J ) 2.9 Hz), 129.3, 127.5, 123.4 (q, J ) 272.0 Hz), 123.6 (q, J
) 32.1 Hz), 118.0 (q, J ) 10.9 Hz).
8-Meth yl-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e (4d ).
Method A using 2d gave 4d (100%) as a yellow powder, mp
1
7-(Meth ylsu lfa n yl)-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(8h ). Method A using 6h gave 8h (56%) as a red solid, mp
(DMF) 265 °C (dec); H NMR [(CD3)2SO] δ 7.59 (dd, J ) 8.3,
7.3 Hz, 1 H, H-6), 7.35 (d, J ) 8.0 Hz, 1 H, H-5), 7.18 (s, 2 H,
NH2), 7.10 (dd, J ) 7.2, 0.8 Hz, 1 H, H-7), 2.79 (s, 3 H, CH3);
Anal. (C8H8N4O) C, H, N.
1
(MeOH/CHCl3) 245-247 °C; H NMR [(CD3)2SO] δ 7.79 (d, J
) 2.1 Hz, 1 H, H-8), 7.67 (dd, J ) 8.9, 2.1 Hz, 1 H, H-6), 7.47
(d, J ) 8.9 Hz, 1 H, H-5), 7.28 (s, 2 H, NH2), 2.58 (s, 3 H, SCH3);
13C NMR [(CD3)2SO] δ 159.9, 147.0, 135.7, 134.6, 130.1, 126.5,
113.4, 14.6; Anal. (C8H8N4OS) C, H, N.
8-F lu or o-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e (4e).
Method B using 3e gave 4e (49%) as a yellow powder, mp
1
DCM/pet. ether) 270-278 °C (dec) (lit.40 mp 271 °C (dec); H
7-(Bu tylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(8i). Method A using 6i gave 8i (68%) as a red solid, mp
NMR [(CD3)2SO] δ 7.69 (ddd, J ) 10.9, 8.3, 5.2 Hz, 1 H, H-6),
7.45 (br s, 2 H, NH2), 7.31 (dd, J ) 9.6, 1.0 Hz, 1 H, H-5), 7.09
(ddd, J ) 12.0, 8.0, 1.0 Hz, 1 H, H-7); 13C NMR [(CD3)2SO] δ
160.2 (d, J ) 5.3 Hz), 153.5 (d, J ) 264.3 Hz), 151.1 (d, J )
3.2 Hz), 135.2 (d, J ) 4.4 Hz), 121.7 (d, J ) 4.5 Hz), 121.1,
110.0 (d, J ) 20.8 Hz); HRMS (EI+) calcd for C7H5FN4O (M+)
m/z 180.0360, found 180.0441.
1
(MeOH/CHCl3) 215-217 °C; H NMR [(CD3)2SO] δ 7.87 (d, J
) 2.1 Hz, 1 H, H-8), 7.68 (dd, J ) 8.9, 2.1 Hz, 1 H, H-6), 7.46
(d, J ) 8.9 Hz, 1 H, H-5), 7.07 (br s, 2 H, NH2), 3.04 (dd, J )
7.3, 7.2 Hz, 2 H, CH2S), 1.55-1.63 (m, 2 H, CH2), 1.37-1.45
(m, 2 H, CH2), 0.88 (t, J ) 7.3 Hz, 3 H, CH3); 13C NMR [(CD3)2-
SO] δ 160.0, 147.3, 135.9, 133.9, 130.0, 126.4, 115.8, 31.6, 30.1,
21.2, 13.4; Anal. (C11H14N4OS) C, H, N.
8-Ch lor o-1,2,4-ben zotr iazin -3-am in e 1-oxide (4f). Method
A using 2f gave 4f (30%) as a yellow powder, mp (DMF) 280-
7-Nitr o-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e (8j). Method
B using 7j gave 8j (15%) as a yellow powder, mp (DMF) 269-
272 °C [lit.27 mp (pyridine/EtOH) 290 °C]; 1H NMR [(CD3)2-
SO] δ 8.82 (d, J ) 2.6 Hz, 1 H, H-8), 8.44 (dd, J ) 9.4, 2.6 Hz,
1 H, H-6), 8.03 (br s, 2 H, NH2), 7.64(d, J ) 9.3 Hz, 1 H, H-5).
6-Meth oxy-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e (11c).
Method A using 10c gave 11c (63%) as a yellow powder, mp
1
290 °C (dec); H NMR [(CD3)2SO] δ 7.63 (dd, J ) 8.4, 7.8 Hz,
1 H, H-6), 7.45 (dd, J ) 8.6, 1.0 Hz, 1 H, H-7), 7.42 (br s, 2 H,
NH2), 7.36 (dd, J ) 7.6, 1.1 Hz, 1 H, H-5); HRMS (EI) calcd
for C7H5N4O35Cl (M+) m/z 196.0152, found 196.0152; calc for
C7H5N4O37Cl (M+) m/z 198.0122, found 198.0124.
8-Tr iflu or om eth yl-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(4 g). Method A using 2g gave 4g (75%) as a yellow powder,
mp (DCM/pet. ether) 280-286 °C (dec); 1H NMR [(CD3)2SO] δ
7.78-7.87 (m, 3 H, H-5, H-6, H-7), 7.55 (br s, 2 H, NH2); Anal.
(C8H5F3N4O) C, H, N, F.
8-(Meth ylsu lfa n yl)-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(4h ). Method A using 2h gave 4h (68%) as a yellow powder,
mp (H2O) 271-275 °C; 1H NMR [(CD3)2SO] δ 7.63 (dd, J )
8.3, 8.0 Hz, 1 H, H-6), 7.28 (s, 2 H, NH2), 7.17 (d, J ) 8.3 Hz,
1 H, H-5), 6.98 (d, J ) 8.0 Hz, 1 H, H-7), 2.39 (s, 3 H, SCH3);
13C NMR [(CD3)2SO] δ 159.9, 151.1, 137.1, 134.7, 127.9, 120.1,
118.7, 15.7; Anal. (C8H8N4OS) C, H, N.
8-(Bu tylsu lfa n yl)-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(4i). Method A using 2i gave 4i (26%) as a red/brown solid,
mp (MeOH/CHCl3) 233-236 °C; 1H NMR [(CD3)2SO] δ 7.61
(dd, J ) 8.2, 7.4 Hz, 1 H, H-6), 7.27 (s, 2 H, NH2), 7.16 (dd, J
) 8.2, 0.8 Hz, 1 H, H-5), 7.04 (dd, J ) 7.4, 0.8 Hz, 1 H, H-7),
2.86 (t, J ) 7.3 Hz, 2 H, CH2S), 1.62-1.70 (m, 2 H, CH2), 1.44-
1.52 (m, 2 H, CH2), 0.93 (s, 3 H, CH3); 13C NMR [(CD3)2SO] δ
159.8, 151.1, 136.4, 134.7, 128.0, 120.1, 119.1, 31.0, 28.8, 21.7,
13.5; Anal. (C11H14N4OS) C, H, N.
7-Met h oxy-1,2,4-b en zot r ia zin -3-a m in e 1-Oxid e (8c).
Method A using 6c gave 8c (93%) as a yellow powder, mp
(HOAc) 269-271 °C [lit.26 mp (HOAc) 271 °C]; 1H NMR [(CD3)2-
SO] δ 7.48-7.53 (m, 3 H, H-5, H-6, H-8), 7.10 (br s, 2 H, NH2),
3.88 (s, 3 H, OCH3); 13C NMR [(CD3)2SO] δ 159.3, 156.3, 144.9,
129.7, 128.3, 127.3, 97.9, 55.8.
1
(CHCl3) 265-270 °C; H NMR [(CD3)2SO] δ 8.04 (d, J ) 9.5
Hz, 1 H, H-8), 7.24 (br s, 2 H, NH2), 6.95 (dd, J ) 9.5, 2.6 Hz,
1 H, H-7), 6.86 (d, J ) 2.6 Hz, 1 H, H-5), 3.91 (s, 3 H, OCH3);
13C NMR [(CD3)2SO] δ 164.7, 160.7, 151.3, 125.0, 121.5, 117.0,
103.8, 56.0; MS (EI+) m/z 192 (M+, 100%), 176 (5); HRMS calcd
for C8H8N4O2 (M+) m/z 192.0647, found 192.0653; Anal.
(C8H8N4O2) C, H, N.
6-Met h yl-1,2,4-b en zot r ia zin -3-a m in e 1-Oxid e (11d ).
Method A using 10d gave 11d (87%) as a yellow powder, mp
1
(DMF) 263 °C (dec) [lit.41 mp (HOAc) 284-286 °C]; H NMR
[(CD3)2SO] δ 8.02 (d, J ) 8.8 Hz, 1 H, H-8), 7.33 (s, 1 H, H-5),
7.27(br s, 2 H, NH2), 7.18 (dd, J ) 8.8, 1.7 Hz, 1 H, H-7), 2.42
(s, 3 H, CH3); Anal. (C8H8N4O) C, H, N.
6-F lu or o-1,2,4-b en zot r ia zin -3-a m in e 1-Oxid e (11e).
Method A using 10e gave 11e (61%) as a yellow powder, mp
(DCM/pet. ether) 276-280 °C (lit.40 mp 268 °C); 1H NMR
[(CD3)2SO] δ 8.21 (dd, J ) 9.5, 5.9 Hz, 1 H, H-8), 7.50 (br s, 2
H, NH2), 7.30 (dd, J ) 10.0, 2.6 Hz, 1 H, H-5), 7.21 (ddd, J )
8.8, 7.5, 2.6 Hz, 1 H, H-7); 13C NMR [(CD3)2SO] δ 164.5 (d, J
) 254.6 Hz), 160.8, 150.6 (d, J ) 16.1 Hz), 127.4, 123.4 (d, J
) 11.1 Hz), 114.1 (d, J ) 26.2 Hz), 109.5 (d, J ) 23.1 Hz);
Anal. (C7H5FN4O) C, H, N, F.
6-Ch lor o-1,2,4-ben zotr a zin -3-a m in e 1-Oxid e (11f).
Method A using 10f gave 11f (53%) as a yellow solid, mp
(DCM/pet. ether) >320 °C [lit.41 mp (HOAc) >300 °C]; 1H NMR
[(CD3)2SO] δ 8.13 (d, J ) 9.2 Hz, 1 H, H-8), 7.60 (d, J ) 2.11
Hz, 1 H, H-5), 7.53 (br s, 2 H, NH2), 7.33 (dd, J ) 9.2, 2.1 Hz,
1 H, H-7).
7-Meth yl-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e (8d ).
Method A using 6d gave 8d (74%) as a yellow powder, mp
(DMF) 270 °C (dec) [lit.27 mp (Methylcellosolve) 282 °C]; 1H
NMR [(CD3)2SO] δ 7.94 (s, 1 H, H-8); 7.64 (dd, J ) 8.7, 1.9
Hz, 1 H, H-6), 7.46 (d, J ) 8.6 Hz, 1 H, H-5), 7.21 (s, 2 H,
NH2), 2.42 (s, 3 H, CH3); Anal. (C8H8N4O) C, H, N.
6-Tr iflu or om eth yl-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(11 g). Method A using 10g gave 11g (29%) as a yellow solid,
mp (DCM/pet. ether) 280-284 °C (dec); 1H NMR [(CD3)2SO] δ
8.31 (d, J ) 8.9 Hz, 1 H, H-8), 7.85 (br s, 1 H, H-5), 7.65 (br s,

Hypoxia-Selective Analogues of Tirapazamine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 179
2 H, NH2), 7.56 (dd, J ) 8.9, 1.6 Hz, 1 H, H-7); 13C NMR
[(CD3)2SO] δ 160.8, 148.4, 134.8 (q, J ) 32.7 Hz), 131.5, 123.5
(q, J ) 4.0 Hz), 123.1 (q, J ) 273.1 Hz), 122.0, 119.3; Anal.
(C8H5F3N4O) C, H, N, F.
6-(Meth ylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(11h ). Method A using 10h gave 11h (55%) as a yellow powder,
mp (MeOH/CHCl3) 248-250 °C; 1H NMR [(CD3)2SO] δ 7.99
(d, J ) 8.3 Hz, 1 H, H-8), 7.30 (br s, 2 H, NH2), 7.16-7.19 (m,
2 H, H-5, H-7), 2.59 (s, 3 H, SCH3); 13C NMR [(CD3)2SO] δ
160.7, 149.2, 149.1, 127.3, 122.9, 119.8, 118.0, 14.0; Anal.
(C8H8N4OS) C, H, N.
evaporated and the residue chromatographed, eluting with a
gradient (0-3%) of MeOH/CHCl3 to give dialkylamino-1,2,4-
benzotriazin-3-amine 1,4-dioxide.
8-Dieth yla m in o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Diox-
id e (5a ). Method E using 5e gave 5a (87%) as a purple powder,
mp (DCM/pet. ether) 154-156 °C; 1H NMR [(CD3)2SO] δ 7.77
(br s, 2 H, NH2), 7.66 (t, J ) 8.2 Hz, 1 H, H-6), 7.56 (d, J ) 8.0
Hz, 1 H, H-5), 6.93 (d, J ) 7.5 Hz, 1 H, H-7), 3.24 (q, J ) 6.9
Hz, 4 H, 2 × CH2), 1.05 (t, J ) 6.9 Hz, 6 H, 2 × CH3); 13C
NMR [(CD3)2SO] δ 150.9, 145.9, 141.7, 135.2, 126.3, 115.6,
107.0, 46.6 (2), 12.3(2); Anal. (C11H15N5O2) C, H, N.
6-(Bu tylsu lfa n yl)-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e
(11i). Method A using 10i gave (i) starting material (10i) (60%)
and (ii) 11i (30%) as a red solid, mp (MeOH/CHCl3) 180-182
°C; 1H NMR [(CD3)2SO] δ 7.99 (d, J ) 9.1 Hz, 1 H, H-8), 7.31
(br s, 2 H, NH2), 7.21 (d, J ) 2.0 Hz, 1 H, H-5), 7.17 (dd, J )
9.1, 2.0 Hz, 1 H, H-7), 3.12 (dd, J ) 7.3, 7.2 Hz, 2 H, CH2S),
1.61-1.68 (m, 2 H, CH2), 1.41-1.49 (m, 2 H, CH2), 0.92 (s, 3
H, CH3); 13C NMR [(CD3)2SO] δ 160.6, 149.1, 147.9, 127.3,
123.4, 119.9, 118.8, 30.1, 29.9, 21.3, 13.4; Anal. (C11H14N4OS)
C, H, N.
5-Met h oxy-1,2,4-b en zot r ia zin -3-a m in e 1-Oxid e (15c).
Method A using 13c gave 15c (66%) as a yellow powder, mp
(HOAc) 267 °C (dec) [lit.41 mp (HOAc) >270 °C]; 1H NMR
[(CD3)2SO] δ 7.65-7.69 (m, 1 H, H-7), 7.39 (br s, 2 H, NH2),
7.23-7.27 (m, 2 H, H-6, H-8), 3.92 (s, 3 H, OCH3); 13C NMR
[(CD3)2SO] δ 159.7, 153.3, 141.5, 130.0, 123.9, 113.4, 110.7,
55.9; Anal. (C8H8N4O2) C, H, N.
8-Dim et h yla m in o-1,2,4-b en zot r ia zin -3-a m in e 1,4-Di-
oxid e (5b). Method E using 5e gave 5b (80%) as a purple
powder, mp (DCM/pet. ether) 170-172 °C; H NMR [(CD3)2-
1
SO] δ 7.77 (br s, 2 H, NH2), 7.65 (t, J ) 8.3 Hz, 1 H, H-6), 7.51
(dd, J ) 7.8, 0.8 Hz, 1 H, H-5), 6.87 (dd, J ) 8.1, 0.8 Hz, 1 H,
H-7), 2.87 [s, 6 H, N(CH3)2]; 13C NMR [(CD3)2SO] δ 150.4,
147.4, 140.9, 135.0, 124.4, 111.7, 105.3, 43.7 (2); Anal.
(C9H11N5O2) C, H; N: calc 31.7, found 31.0%.
8-Meth oxy-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (5c).
Method C using 4c gave 5c (54%) as an orange powder, mp
(H2O) 208-213 °C (dec); 1H NMR [(CD3)2SO] δ 7.90 (br s, 2 H,
NH2), 7.80 (dd, J ) 8.5, 8.0 Hz, 1 H, H-6), 7.68 (d, J ) 8.5 Hz,
1 H, H-5), 7.03 (dd, J ) 8.0 Hz, 1 H, H-7), 3.94 (s, 3 H, OCH3);
13C NMR [(CD3)2SO] δ 153.8, 150.7, 140.4, 135.7, 123.3, 107.9,
107.4, 56.8; Anal. (C8H8N4O3‚1/2H2O) C, H; N: calcd 25.8,
found 25.3%.
8-Meth yl-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (5d ).
Method C using 4d gave 5d (34%) as an orange powder, mp
5-Met h yl-1,2,4-b en zot r ia zin -3-a m in e 1-Oxid e (15d ).
Method A using gave 13d gave 15d (89%) as a yellow solid,
mp (DCM/pet. ether) 253-255 °C; 1H NMR [(CD3)2SO] δ 7.98
(d, J ) 8.6, Hz, 1 H, H-8), 7.64 (d, J ) 7.1 Hz, 1 H, H-6), 7.33
(br s, 2 H, NH2), 7.23 (dd, J ) 8.6, 7.1 Hz, 1 H, H-7), 2.49 (s,
3 H, CH3); 13C NMR [(CD3)2SO] δ 156.7, 148.0, 135.1, 134.3,
129.8, 124.0, 117.4, 16.8; Anal. (C8H8N4O) C, H, N.
5-Ch lor o-1,2,4-b en zot r ia zin -3-a m in e 1-Oxid e (15e).
Method B using 14e gave 15e (45%) as a yellow solid, mp
1
(DCM/pet. ether) 225 °C; H NMR [(CD3)2SO] δ 8.04 (d, J )
8.7 Hz, 1 H, H-5), 7.93 (br s, 2 H, NH2), 7.77 (dd, J ) 8.6, 7.3
Hz, 1 H, H-6), 7.35 (d, J ) 7.2 Hz, 1 H, H-7), 2.85 (s, 3 H,
CH3); 13C NMR [(CD3)2SO] δ 150.5, 139.8, 134.5, 134.3, 130.6,
129.1, 115.2, 23.1; Anal. (C8H8N4O2) C, H; N: calcd 29.15,
found 28.4.
8-F lu or o-1,2,4-ben zotr ia zin -3-a m in e-1,4-Dioxid e (5e).
Method D using 4e gave 5e (67%) as a red powder, mp (DCM/
pet. ether) 210-211 °C; 1H NMR [(CD3)2SO] δ 8.12 (br s, 2 H,
NH2), 7.94 (d, J ) 8.8 Hz, 1 H, H-5), 7.85 (ddd, J ) 9.0, 8.4,
4.6 Hz, 1 H, H-6), 7.35 (dd, J ) 12.2, 7.5 Hz, 1 H, H-7); 13C
NMR [(CD3)2SO] δ 153.6 (d, J ) 264.1 Hz) 151.3, 140.1, 135.3
(d, J ) 9.7 Hz), 122.4 (d, J ) 7.6 Hz), 113.1 (d, J ) 5.4 Hz),
112.4 (d, J ) 21.5 Hz); Anal. (C7H5FN4O2) C, H, F; N: calcd
28.6, found 28.1%.
1
(HOAc) 251-254 °C; H NMR [(CD3)2SO] δ 8.11 (dd, J ) 8.7,
1.0 Hz, 1 H, H-8), 7.95 (dd, J ) 7.6, 1.0 Hz, 1 H, H-6), 7.67 (br
s, 2 H, NH2), 7.29 (dd, J ) 8.7, 7.6 Hz, 1 H, H-7); 13C NMR
[(CD3)2SO] δ 160.1, 145.7, 135.1, 131.1, 128.6, 123.6, 119.1;
Anal. (C7H5ClN4O) C, H, N.
5-F lu or o-1,2,4-ben zotr ia zin -3-a m in e 1-Oxid e (15f).
Method A using 13f gave 15f (43%) as a yellow powder, mp
(DMF) 252-256 °C (lit.40 mp 278 °C) 1H NMR [(CD3)2SO] δ
7.96 (dd, J ) 8.7, 1.0 Hz, 1 H, H-8), 7.66 (ddd, J ) 10.3, 7.9,
1.2 Hz, 1 H, H-6), 7.61 (br s, 2 H, NH2), 7.28 (ddd, J ) 8.6,
8.0, 5.1 Hz, 1 H, H-7); 13C NMR [(CD3)2SO] δ 160.1 (d, J ) 5.6
Hz), 154.9 (d, J ) 253.1 Hz), 139.7 (d, J ) 16.1 Hz), 131.1 (d,
J ) 4.2 Hz), 122.8 (d, J ) 7.6 Hz), 119.4 (d, J ) 17.7 Hz),
115.8 (d, J ) 4.4 Hz); Anal. (C7H5FN4O) C, H, N, F.
8-Ch lor o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (5f).
Method C using 4f gave 5f (16%) as a red powder, mp (DMF)
1
231 °C; H NMR [(CD3)2SO] δ 8.13 (d, J ) 8.7 Hz, 1 H, H-5),
8.12 (br s, 2 H, NH2), 7.80 (t, J ) 8.2 Hz, 1 H, H-6), 7.62 (d, J
) 7.6 Hz, 1 H, H-7); 13C NMR [(CD3)2SO] δ 151.1, 140. 9, 134.4,
129.3, 128.1, 126.4, 116.7; Anal. (C7H5ClN4O2) C, H, N, Cl.
8-Tr iflu or om eth yl-1,2,4-ben zotr ia zin -3-a m in e 1,4-Di-
oxid e (5g). Method C using 4g gave 5g (27%) as a red powder,
mp (DCM/pet. ether) 265-275 °C (dec); 1H NMR [(CD3)2SO] δ
8.49 (d, J ) 8.1 Hz, 1 H, H-7), 8.20 (br s, 2 H, NH2), 7.97-8.04
(m, 2 H, H-5, H-6); 13C NMR [(CD3)2SO] δ 151.4, 140.4 (q, J )
9.0 Hz), 133.8, 127.6, 127.2 (q, J ) 7.9 Hz), 122.8, 122.5 (q, J
) 271.5 Hz), 121.7 (q, J ) 32.9 Hz); Anal. (C8H5F3N4O2) C, H,
N, F.
Meth od C: Oxid a tion of 1-Oxid es w ith 30% H2O2/
HOAc. Hydrogen peroxide (30%, 12 mL) was added dropwise
to a suspension of 1-oxide (2.60 mmol) in HOAc (25 mL) and
the suspension stirred at 50 °C for 4 h. The solution was
diluted with water (100 mL) and carefully neutralized with
solid NaHCO3. The solution was extracted with CHCl3 (5 ×
50 mL), the combined organic extracts were dried, and the
solvent was evaporated. The residue was chromatographed,
eluting with a gradient (0-5%) of MeOH/CHCl3, to give the
corresponding 1,2,4-benzotriazin-3-amine 1,4-dioxide.
Meth od D: Oxid a tion of 1-Oxid es w ith 70% H2O2/
CF 3CO2H. Hydrogen peroxide (70%, 4 mL) (CAUTION) was
added dropwise to a stirred suspension of 1-oxide (1.65 mmol)
in trifluoroacetic acid (5 mL) and the mixture stirred at 50 °C
for 7 days. The mixture was diluted with water (50 mL) and
carefully neutralized with solid NaHCO3. The precipitate was
filtered, washed with water (2 × 5 mL), and dried to give the
corresponding 1,2,4-benzotriazin-3-amine 1,4-dioxide.
Meth od E: Am in a tion of F lu or o-1,2,4-ben zotr ia zin -3-
a m in e 1,4-Oxid es. Fluoro-1,2,4-benzotriazin-3-amine 1,4-
dioxide (0.4 mmol) was dissolved in acetonitrile (8 mL), and a
solution of 40% aq dimethylamine (3 mL) or diethylamine (3
mL) was added and stirred at 20 °C for 24 h. The solvent was
8-(Meth ylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1,4-Di-
oxid e (5h ). Method D using 4h gave 5h (38%) as a red solid,
mp (H2O) 290-300 °C; 1H NMR [(CD3)2SO] δ 8.48 (d, J ) 8.6
Hz, 1 H, H-5), 8.32 (d, J ) 7.0 Hz, 1 H, H-7), 8.27 (br s, 2 H,
NH2), 8.03 (dd, J ) 8.6, 7.0 Hz, 1 H, H-6), 3.61 (s, 3 H, SO2-
CH3); 13C NMR [(CD3)2SO] δ 151.6, 140.3, 135.4, 133.9, 130.1,
127.5, 123.5, 45.1; MS (FAB+) m/z 257 (MH+, 5%), 242 (1);
HRMS (FAB+) calcd for C8H9N4O4S (MH+) m/z 257.0345, found
257.0345.
8-(Bu tylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1,4-Diox-
id e (5i). Method D using 4i gave 5i (21%) as a red solid, mp
1
(water) 220-230 °C; H NMR [(CD3)2SO] δ 8.49 (dd, J ) 8.6,
0.7 Hz, 1 H, H-5), 8.30 (dd, J ) 7.5, 0.6 Hz, 1 H, H-7), 8.26 (br
s, 2 H, NH2), 8.04 (dd, J ) 8.6, 7.5 Hz, 1 H, H-6), 3.83 (s, 2 H,
SO2CH2), 1.58-1.64 (m, 2 H, CH2), 1.32-1.41 (m, 2 H, CH2),
0.85 (t, J ) 7.3 Hz, 3 H, CH3); 13C NMR [(CD3)2SO] δ 151.6,

180 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hay et al.
140.4, 133.8, 133.7, 130.0, 127.6, 123.6, 55.6, 24.6, 20.8, 13.3;
MS (EI+) m/z 298 (M+, 2%), 282 (12), 253 (10), 201 (30), 162
(100); HRMS (EI+) calcd for C11H14N4O4S (M+) m/z 298.0736,
found 298.0746.
7-Dieth yla m in o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Diox-
id e (9a ). Method E using 9e gave 9a (5%) as a purple powder,
mp (DCM/pet. ether) 323 °C (dec); 1H NMR [(CD3)2SO] δ 7.98
(d, J ) 9.7 Hz, 1 H, H-5), 7.60 (dd, J ) 9.7, 2.9 Hz, 1 H, H-6),
7.50 (br s, 2 H, NH2), 6.97 (d, J ) 2.7 Hz, 1 H, H-8), 3.47 (q,
J ) 7.0 Hz, 4 H, 2 × CH2), 1.15 (t, J ) 7.0 Hz, 6 H, 2 × CH3),
13C NMR [(CD3)2SO] δ 149.0, 148.9, 131.8, 131.2, 124.2, 118.1,
94.6, 44.1 (2), 12.0 (2); HRMS (EI) calcd for C11H15N5O2 (M+)
m/z 249.1226, found 249.1231.
7-Dim et h yla m in o-1,2,4-b en zot r ia zin -3-a m in e 1,4-Di-
oxid e (9b). Method E using 9e gave 9b (76%) as a purple
powder, mp (DCM/pet. ether) 205-215 °C (dec); 1H NMR
[(CD3)2SO] δ 7.99 (d, J ) 9.6 Hz, 1 H, H-5), 7.63 (dd, J ) 9.6,
2.7 Hz, 1 H, H-6), 7.55 (br s, 2 H, NH2), 6.96 (d, J ) 2.7 Hz,
1 H, H-8), 3.06 [s, 6 H, N(CH3)2], 13C NMR [(CD3)2SO] δ
149.2; 148.5, 131.5, 131.5, 124.4, 117.7, 95.6, 39.9 (2); Anal.
(C9H11N5O2‚1/2H2O) C, H, N.
7-Meth oxy-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (9c).
Method C using 8c gave 9c (30%) as a red powder, mp (HOAc)
210-214 °C [lit.42 mp (H2O) 213-214 °C]; 1H NMR [(CD3)2SO]
δ 8.07 (d, J ) 9.4 Hz, 1 H, H-5), 7.87 (br s, 2 H, NH2), 7.59
(dd, J ) 9.4, 2.6 Hz, 1 H, H-6), 7.47 (d, J ) 2.6 Hz, 1 H, H-8),
3.92 (s, 3 H, OCH3); 13C NMR [(CD3)2SO] δ 157.9, 150.5, 134.3,
131.0, 127.7, 118.7, 99.1, 56.1; Anal. (C8H8N4O3) C, H, N.
7-Meth yl-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (9d ).
Method C using 8d gave 9d (25%) as a red powder, mp (DMF)
215 °C [lit.26 mp (H2O) 220 °C]; 1H NMR [(CD3)2SO] δ 8.05 (d,
J ) 8.8 Hz, 1 H, H-5), 8.01 (s, 1 H, H-8), 7.93 (br s, 2 H, NH2),
7.79 (dd, J ) 8.8, 1.7 Hz, 1 H, H-6), 2.48 (s, 3 H, CH3); Anal.
(C8H8N4O2) C, H; N: calcd 29.2, found 28.4%
7-Nit r o-1,2,4-b en zot r ia zin -3-a m in e 1,4-Dioxid e (9j).
Method C using 8j gave 9j (22%) as a red powder, mp (DMF)
242-243 °C (dec) [lit.43 mp (DMF/acetone) 286-288 °C]; 1H
NMR [(CD3)2SO] δ 8.84 (d, J ) 2.4 Hz, 1 H, H-8), 8.56 (m, 3
H, H-6, NH2), 8.27 (d, J ) 9.5 Hz, 1 H, H-5); 13C NMR [(CD3)2-
SO] δ 153.1, 144.1, 140.5, 129.8, 128.3, 119.2, 118.1; Anal.
(C7H5N5O4) C, H, N.
6-Dieth yla m in o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Diox-
id e (12a ). Method E using 12e gave 12a (69%) an orange
1
powder, mp (DCM/pet. ether) 209 °C (dec); H NMR [(CD3)2-
SO] δ 7.96 (d, J ) 9.8 Hz, 1 H, H-8), 7.71 (br s, 2 H, NH2),
7.13 (dd, J ) 9.8, 2.0 Hz, 1 H, H-7), 6.87 (d, J ) 1.8 Hz, 1 H,
H-5), 3.55 (q, J ) 6.8 Hz, 4 H, 2 × CH2), 1.19 (t, J ) 6.9 Hz,
6 H, 2 × CH3); 13C NMR [(CD3)2SO] δ 152.0, 151.3, 139.8,
122.8, 122.4, 115.5, 90.2, 44.5, 12.1; Anal. (C11H15N5O2) C, H,
N.
6-Dim et h yla m in o-1,2,4-b en zot r ia zin -3-a m in e 1,4-Di-
oxid e (12b). Method E using 12e gave 12b (83%) as a red
1
powder, mp (DCM/pet. ether) 238 °C (dec); H NMR [(CD3)2-
SO] δ 7.98 (d, J ) 9.8 Hz, 1 H, H-8), 7.75 (br s, 2 H, NH2),
7.17 (dd, J ) 9.8, 2.7 Hz, 1 H, H-7), 6.85 (d, J ) 2.7 Hz, 1 H,
H-5), 3.16 [s, 6 H, N(CH3)2]; 13C NMR [(CD3)2SO] δ 154.0,
151.4, 139.5, 122.6, 122.4, 115.7, 90.8, 39.9; Anal. (C9H11N5O2)
C, H, N.
6-Meth oxy-1,2,4-ben zotr iazin -3-am in e 1,4-Dioxide (12c).
Method D using 11c gave 12c (30%) as a red powder, mp
1
(EtOAc/MeOH) 234-236 °C; H NMR [(CD3)2SO] δ 8.11 (d, J
) 9.6 Hz, 1 H, H-8), 8.02 (br s, 2 H, NH2), 7.41 (d, J ) 2.6 Hz,
1 H, H-5), 7.15 (dd, J ) 9.6, 2.6 Hz, 1 H, H-7), 3.99 (s, 3 H,
OCH3); 13C NMR [(CD3)2SO] δ 164.9, 151.8, 140.1, 125.7, 123.2,
119.1, 96.7, 56.6; Anal. (C8H8N4O3) C, H, N.
6-Meth yl-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (12d ).
Method C using 11d gave 12d (57%) as a red powder, mp
(DMF) 225-229 °C; 1H NMR [(CD3)2SO] δ 8.09 (d, J ) 8.9
Hz, 1 H, H-8), 8.00 (br s, 2 H, NH2), 7.95 (s, 1 H, H-5), 7.40
(dd, J ) 9.0, 3.1 Hz, 1 H, H-7), 2.51 (s, 3 H, CH3); 13C NMR
[(CD3)2SO] δ 151.4, 146.9, 138.2, 128.9, 128.8, 120.9, 115.6,
21.6; Anal. (C8H8N4O2) C, H, N.
6-F lu or o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (12e).
Method C using 11e gave 12e (40%) as an orange powder, mp
(DMF) 241-246 °C (dec) (lit.40 mp >300 °C); 1H NMR [(CD3)2-
SO] δ 8.30 (dd, J ) 9.5, 5.2 Hz, 1 H, H-8), 8.20 (br s, 2 H,
NH2), 7.83 (dd, J ) 9.2, 2.6 Hz, 1 H, H-5), 7.43-7.48 (m, 1 H,
H-7); 13C NMR [(CD3)2SO] δ 164.8 (d, J ) 256.3 Hz), 151.8,
139.8 (d, J ) 12.1 Hz), 128.1, 125.2 (d, J ) 11.1 Hz), 116.4 (d,
J ) 26.7 Hz), 101.9 (d, J ) 28.5 Hz); Anal.(C7H5FN4O2) C, H,
N, F.
6-Ch lor o-1,2,4-ben zotr a zin -3-a m in e 1,4-Dioxid e (12f).
Method C using 11f gave 12f (20%) as a red powder, mp (DMF)
230-240 °C (dec); 1H NMR [(CD3)2SO] δ 8.21 (m, 3 H, H-8,
NH2), 8.11 (d, J ) 2.2 Hz, 1 H, H-5), 7.56 (dd, J ) 9.2, 2.4 Hz,
1 H, H-7); 13C NMR [(CD3)2SO] δ 150.9, 140.4, 138.7, 129.4,
127.0, 123.5, 115.9; HRMS (EI+) calcd for C7H535ClN4O2 (M+)
m/z 212.0101, found 212.0102; calcd for C7H537ClN4O2 (M+) m/z
214.0072, found 214.0073.
7-F lu or o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (9e).
Method C using 8e gave 9e (53%) as a red powder, mp (DCM/
pet. ether) 240-250 °C (dec) [lit.40 mp >300 °C]; 1H NMR
[(CD3)2SO] δ 8.20 (dd, J ) 9.5, 5.1 Hz, 1 H, H-8), 8.03 (br s, 2
H, NH2), 7.98 (dd, J ) 8.7, 2.7 Hz, 1 H, H-5), 7.88 (ddd, J )
10.2, 7.4, 2.0 Hz, 1 H, H-6); 13C NMR [(CD3)2SO] δ 105.9 (d, J
) 27.4), 120.2 (d, J ) 9.1 Hz), 125.1 (d, J ) 26.7 Hz), 130.7 (J
) 10.1 Hz), 136.0, 151.2 (d, J ) 5.0 Hz), 159.5 (d, J ) 249.2
Hz); Anal. (C7H5FN4O2) C, H, N, F.
7-Ch lor o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (9f).
Method C using 8f gave 9f (50%) as a red powder, mp (DMF)
1
248-250 °C (dec) [lit.26 (HOAc) 269 °C]; H NMR [(CD3)2SO]
δ 8.21 (d, J ) 2.1 Hz, 1 H, H-8), 8.14 (m, 3 H, H-5, NH2), 7.94
(dd, J ) 9.2, 2.2 Hz, 1 H, H-6); 13C NMR [(CD3)2SO] δ 151.5,
137.4, 135.5, 131.1, 130.8, 119.3, 119.4; Anal. (C7H5ClN4O2)
C, H, N, Cl.
7-Tr iflu or om eth yl-1,2,4-ben zotr ia zin -3-a m in e 1,4-Di-
oxid e (9g). Method C using 8g gave 9g (76%) as red crystals,
mp (DCM/pet. ether) 256-260 °C (lit.40 >300 °C); 1H NMR
[(CD3)2SO] δ 8.45 (br s, 1 H, H-8). 8.40 (br, 2 H, NH2), 8.30 (d,
J ) 8.9 Hz, 1 H, H-5), 8.18 (dd, J ) 9.1, 1.8 Hz, 1 H, H-6); 13
C
NMR [(CD3)2SO] δ 152.5, 139.8, 130.3 (q, J ) 2.8 Hz), 130.0,
126.0 (q, J ) 33.4 Hz), 123.1 (q, J ) 272.0 Hz), 119.4 (q, J )
4.4 Hz), 119.2; Anal. (C8H5F3N4O2) C, H, N, F.
6-Tr iflu or om eth yl-1,2,4-ben zotr ia zin -3-a m in e 1,4-Di-
oxid e (12 g). Method C using 11g gave 12g (24%) as a red
1
powder, mp (DCM/pet. ether) 238-242 °C; H NMR [(CD3)2-
SO] δ 8.40 (d, J ) 9.2 Hz, 1 H, H-8), 8.37 (br s, 1 H, H-5), 8.30
(br s, 2 H, NH2), 7.81 (dd, J ) 9.1, 1.8 Hz, 1 H, H-7); Anal.
(C8H5F3N4O2-1/2H2O) C, H, N, F.
7-(Meth ylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1,4-Di-
oxid e (9h ). Method D using 8h gave 9h (55%) as a red solid,
mp (water/EtOH) 306-310 °C; 1H NMR [(CD3)2SO] δ 8.61 (br
s, 1 H, H-8), 8.40-8.53 (m, 2 H, H-5, H-6), 8.31 (br s, 2 H,
NH2), 3.37 (s, 3 H, SO2CH3); 13C NMR [(CD3)2SO] δ 152.7,
140.0, 137.8, 131.6, 129.9, 121.8, 119.8, 43.1; Anal. (C8H8N4O4S‚
1/2EtOH) C, N; H: calcd 4.0, found 3.3
7-(Bu tylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1,4-Diox-
id e (9i). Method D using 8i gave 9i (22%) as a red solid, mp
(water) 255-262 °C; 1H NMR [(CD3)2SO] δ 8.55 (d, J ) 1.5
Hz, 1 H, H-8), 8.50 (br s, 2 H, NH2), 8.31 (d, J ) 9.0 Hz, 1 H,
H-5), 8.26 (dd, J ) 9.0, 1.5 Hz, 1 H, H-6), 3.46 (dd, J ) 7.9, 7.7
Hz, 2 H, SO2CH2), 1.49-1.57 (m, 2 H, CH2), 1.30-1.38 (m, 2
H, CH2), 0.83 (br s, 3 H, CH3); 13C NMR [(CD3)2SO] δ 152.7,
140.1, 136.1, 132.1, 130.1, 122.6, 119.1, 54.0, 24.1, 20.6, 13.4;
Anal. (C11H14N4O4S) C, H, N.
6-(Meth ylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1,4-Di-
oxid e (12h ). Method D using 11h gave 12h (67%) as a red
powder, mp (DMF/MeOH) 281 °C (dec); 1H NMR [(CD3)2SO] δ
8.57 (d, J ) 1.9 Hz, 1 H, H-5), 8.42 (d, J ) 9.1 Hz, 1 H, H-8),
8.34 (br s, 2 H, NH2), 7.96 (dd, J ) 9.1, 1.9 Hz, 1 H, H-7), 3.41
(s, 3 H, SO2CH3); 13C NMR [(CD3)2SO] δ 152.1, 146.0, 138.1,
132.2, 123.6, 123.0, 117.2, 42.8; Anal. (C8H8N4O4S) C, H; N,
calcd 21.9; found 21.2%.
6-(Bu tylsu lfon yl)-1,2,4-ben zotr ia zin -3-a m in e 1,4-Diox-
id e (12i). Method D using 11i gave 12i (41%) as a red solid,
mp (EtOH/H2O) 203-206 °C; 1H NMR [(CD3)2SO] δ 8.52 (s, 1
H, H-5), 8.42 (d, J ) 9.0 Hz, 1 H, H-7), 8.34 (br s, 2 H, NH2),
7.91 (d, J ) 9.0 Hz, 1 H, H-8), 3.51 (s, 2 H, SO2CH2), 1.50-

Hypoxia-Selective Analogues of Tirapazamine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 181
1.57 (m, 2 H, CH2), 1.31-1.40 (m, 2 H, CH2), 0.83 (t, J ) 7.3
Hz, 3 H, CH3); 13C NMR [(CD3)2SO] δ 152.1, 144.4, 138.2, 132.3,
123.6, 123.4, 118.0, 53.7, 24.1, 20.6, 13.3; Anal. (C11H14N4O4S‚
1/2EtOH) C, H, N.
survival in plastic Petri dishes. Ten to fourteen days later the
dishes were stained with crystal violet (0.25% in 95% ethanol),
and colonies containing more than 50 cells were counted.
IC50 Assa ys. Drug exposures were performed in 96-well
plates (Nunc) using either a 37 °C humidified incubator (20%
O2, 5% CO2) or in the incubator compartment (37 °C) of an
anaerobic chamber (Shell Lab) where palladium catalyst
scrubbed gas (90% N2, 5% H2, 5% CO2) ensures severe anoxia
(<0.001% O2). For each experiment, all 12 compounds were
simultaneously tested under both conditions against both
SCCVII and HT-29 cell lines. Thus each set of IC50 determina-
tions is an intraexperiment comparison. Cell cultures were
grown in RMEM (Gibco) containing 5% heat inactivated FCS
and maintained in exponential growth phase. For each indi-
vidual experiment an appropriate number of cells were seeded
(SCCVII ) 200; HT-29 ) 1000) into wells in RMEM + 10%
FCS + 10 mM added glucose + 100 µM 2′-deoxycytidine
(2′dCyd), and allowed to attach for 3 h. High glucose (final
concentration 17mM) and the presence of 2′-dCyd minimize
hypoxia-induced cell cycle arrest. Replicates were then exposed
to BTOs, using 2-fold serial dilutions in triplicate, for a further
4 h. Subsequently cells were washed free of compound using
complete media (without glucose/2′-dCyd) and allowed to grow
for 5 (oxic) or 6 (anoxic) days. Plates were stained as described
previously33 and IC50 values determined. Results were aver-
aged for three independent experiments.
5-Dieth yla m in o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Diox-
id e (16a ). Method E using 16f gave 16a (96%) as a deep red
1
gum, H NMR [(CD3)2SO] δ 7.86 (br s, 2 H, NH2), 7.76 (dd, J
) 8.7, 1.1 Hz, 1 H, H-8), 7.36 (br dd, J ) 8.7, 7.7 Hz, 1 H,
H-7), 7.22 (dd, J ) 7.7, 0.8 Hz, 1 H, H-6), 3.24 (q, J ) 7.0 Hz,
4 H, 2 × CH2), 1.00 (t, J ) 7.0 Hz, 6 H, 2 × CH3); 13C NMR
[(CD3)2SO] δ 152.1, 141.8, 132.9, 132.8, 126.6, 124.3, 113.2,
46.6 (2), 11.4 (2); HRMS (EI+) calcd for C11H15N5O2 (M+) m/ z
249.1226, found 249.1231.
5-Dim et h yla m in o-1,2,4-b en zot r ia zin -3-a m in e 1,4-Di-
oxid e (16b). Method E using 16f gave 16b (58%) as a purple
1
powder, mp (DCM/pet. ether) 143-146 °C; H NMR [(CD3)2-
SO] δ 7.87 (br s, 2 H, NH2), 7.71 (d, J ) 8.6 Hz, 1 H, H-8),
7.35 (t, J ) 8.2 Hz, 1 H, H-7), 7.18 (d, J ) 7.8 Hz, 1 H, H-6),
2.81 [s, 6 H, N(CH3)2]; 13C NMR [(CD3)2SO] δ 152.1, 141.8,
132.7, 126.9, 119.5, 114.7, 112.0, 44.9 (2); Anal. (C9H11N5O2)
C, H, N.
5-Meth oxy-1,2,4-ben zotr iazin -3-am in e 1,4-Dioxide (16c).
Method C using 15c gave 16c (5%) as an orange powder, mp
(H2O) 195-200 °C; 1H NMR [(CD3)2SO] δ 7.88 (br s, 2 H, NH2),
7.78 (dd, J ) 8.7, 1.2 Hz, 1 H, H-8), 7.43 (dd, J ) 8.7, 7.8 Hz,
1 H, H-7), 7.32 (d, J ) 7.8 Hz, 1 H, H-6), 3.90 (s, 3 H, OCH3);
13C NMR [(CD3)2SO] δ 152.3, 150.9, 132.2, 131.4, 128.6, 115.2,
112.6, 57.3; MS (EI) m/z 208 (M+, 5%), 192 (100); HRMS (EI)
calcd for C8H8N4O3 (M+) m/z 208.0596, found 208.0594.
5-Meth yl-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (16d ).
Method C using 15d gave 16d (4%) as a red powder, mp (H2O)
Ack n ow led gm en t. The authors thank Dr Maruta
Boyd, Sarath Liyanage, Li Fong Leong, and Karin Tan
for technical assistance. The authors thank Degussa
Peroxide Ltd, Morrinsville, NZ for the generous gift of
70% hydrogen peroxide. This work was supported by
the US National Cancer Institute under Program Project
Grant 1PO1-CA82566-01A1 (M.P.H., S.A.G., M.S.K.,
F.B.P., K.O.H.), the Health Research Council of New
Zealand (R.F.A., W.R.W., A.V.P.), the Auckland Division
of the Cancer Society of New Zealand (W.A.D.), and the
Australian Institute of Nuclear Sciences and Engineer-
ing (M.P.H.).
1
226-232 °C; H NMR [(CD3)2SO] δ 8.08 (d, J ) 8.7 Hz, 1 H,
H-8), 7.94 (br s, 2 H, NH2), 7.61 (d, J ) 7.2 Hz, 1 H, H-6), 7.41
(dd, J ) 8.6, 7.3 Hz, 1 H, H-7), 3.00 (s, 3 H, CH3); HRMS (EI)
calcd for C8H8N4O2 (M+) m/z 192.0647, found 192.0644.
5-Ch lor o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (16e).
Method D using 15e gave 16e (18%) as a red solid, mp (MeOH/
CHCl3) 219 °C (dec); 1H NMR [(CD3)2SO] δ 8.19 (d, J ) 8.8
Hz, 1 H, H-8), 8.14 (br s, 2 H, NH2), 7.90 (d, J ) 7.7 Hz, 1 H,
H-6), 7.45 (dd, J ) 8.8, 7.7 Hz, 1 H, H-7); 13C NMR [(CD3)2SO]
δ 152.6, 137.2, 135.2, 132.6, 126.4, 122.4, 121.0; MS (EI+) m/z
214 (M+, 4%), 212 (M+, 12), 198 (35), 196 (100); HRMS (EI+)
calcd for C7H535ClN4O2 (M+) m/z 212.0101, found 212.0093,
calcd for C7H537ClN4O2 (M+) m/z 214.0072, found 214.0065;
Anal. (C7H5ClN4O2) C, H, N.
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5-F lu or o-1,2,4-ben zotr ia zin -3-a m in e 1,4-Dioxid e (16f).
Method D using 15f gave 16f (50%)as a red powder, mp (DCM/
pet. ether) 236-239 °C; 1H NMR [(CD3)2SO] δ 8.11 (br s, 2 H,
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