Discovery and optimization of benzotriazine Di-N-oxides targeting replicating and nonreplicating mycobacterium tuberculosis

Article abstract of DOI:10.1021/jm300123s

Compounds bactericidal against both replicating and nonreplicating Mtb may shorten the length of TB treatment regimens by eliminating infections more rapidly. Screening of a panel of antimicrobial and anticancer drug classes that are bioreduced into cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs) are potently bactericidal against replicating and nonreplicating Mtb. Medicinal chemistry optimization, guided by semiempirical molecular orbital calculations, identified a new lead compound (20q) from this series with an MIC of 0.31 μg/mL against H37Rv and a cytotoxicity (CC ₅₀) against Vero cells of 25 μg/mL. 20q also had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that BTOs have the potential to be developed into a new class of antitubercular drugs.

Full text of DOI:10.1021/jm300123s

Article
pubs.acs.org/jmc
Discovery and Optimization of Benzotriazine Di-N-Oxides Targeting
Replicating and Nonreplicating Mycobacterium tuberculosis
Sidharth Chopra,†,§ Gary A. Koolpe,† Arlyn A. Tambo-ong,† Karen N. Matsuyama,† Kenneth J. Ryan,†
Tran B. Tran,† Rupa S. Doppalapudi,† Edward S. Riccio,† Lalitha V. Iyer,† Carol E. Green,† Baojie Wan,‡
Scott G. Franzblau,‡ and Peter B. Madrid*,†
†Center for Infectious Disease and Biodefense Research, Bioscience Division, SRI International, 333 Ravenswood Avenue, Menlo
Park, California 94025-3493, United States
‡Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois
60612-7231, United States
S
* Supporting Information
ABSTRACT: Compounds bactericidal against both replicating and
nonreplicating Mtb may shorten the length of TB treatment regimens
by eliminating infections more rapidly. Screening of a panel of
antimicrobial and anticancer drug classes that are bioreduced into
cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs)
are potently bactericidal against replicating and nonreplicating Mtb.
Medicinal chemistry optimization, guided by semiempirical molecular orbital calculations, identified a new lead compound (20q)
from this series with an MIC of 0.31 μg/mL against H37Rv and a cytotoxicity (CC50) against Vero cells of 25 μg/mL. 20q also
had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a
panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic
toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that
BTOs have the potential to be developed into a new class of antitubercular drugs.
for shortening the length of treatment regimens. Metronida-
zole, a 5-nitroimidazole antibiotic primarily used for the
treatment of anaerobic bacterial infections, was one of the
first compounds shown to be bactericidal against NRP Mtb.2
Two newer 5-nitroimidazoles, PA-8243 and OPC-67683,4 have
greater potencies against Mtb and are being evaluated clinically
in combinations with standard TB drugs. So far, both of these
5-nitroimidazoles have been shown to have early bactericidal
activity in patients.5,6 Preclinical studies in murine models of
TB indicate that including bactericidal drugs with activity
against NRP Mtb in a combination regimen shortens the time
required to cure mice of the infection,7 but it remains to be
determined whether these findings will translate to the ability to
INTRODUCTION
■
The current R&D pipeline for new tuberculosis (TB) drugs is
inadequate to address emerging drug-resistant strains. Accord-
ingly, filling the early drug development pipeline with novel
therapies likely to slow additional drug resistance is urgent.
Combination chemotherapy has been a standard of care for TB
since the 1950s, when it was shown that combining drugs
slowed the development of drug resistance, particularly for
bactericidal compounds.1 The next major breakthrough in TB
treatment was the inclusion of rifampin and pyrazinamide in
the multidrug cocktail because those drugs significantly
accelerated the clearance of the infection, presumably through
their bactericidal activity against the populations of bacterial
persisters. The currently recommended direct observed therapy
short-course (DOTS) regimen has been shown to be highly
effective in treating drug-sensitive TB but requires at least six
months of treatment. The next major breakthrough in the
treatment of TB is likely to be the development of an improved
regimen that requires less treatment time, thus decreasing cost,
increasing patient compliance, and slowing the emergence of
multidrug-resistant TB (MDR-TB) strains.
shorten the length of clinical treatment regimens.
To develop new drugs that shorten TB treatment, we
evaluated additional classes of bioreductively activated com-
pounds with previously reported antimicrobial activities for
their activities against Mtb H37Rv. In the current study, we
identified BTOs as a new class of antitubercular compounds
with many unique properties that make them well-suited for
development as anti-TB drugs with the potential to shorten the
length of treatment.
Mycobacterium tuberculosis (Mtb) survival in stages of
nonreplicating persistence (NRP) that are tolerant to many
drugs provides a possible explanation for the long treatment
regimens required to eliminate an infection.2 The development
of new combination therapy regimens, including drugs that are
bactericidal against these NRP stages of Mtb, has the potential
Received: January 27, 2012
Published: June 12, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of bioreductively activated compounds with known antimicrobial activities.
a
Table 1. Antitubercular Activity of Different Scaffolds of Bioreductively-Activated Antimicrobial Compounds
LUMO
(eV)
TB MIC−SRI
(μg/mL)
TB MIC−ITR
(μg/mL)
LORA MIC−ITR
(μg/mL)
Vero Tox CC50
SI
classification
isoniazid
compd
INH
(μg/mL)
(CC50/MIC)
0.060
0.003
>10
>10
10
0.091
0.041
>32
>32
29
>20
2.0
11
>50
>50
>16
>16
16
>830
>15000
NA
rifampin
RIF
nitroimidazole
metronidazole
−1.303
−1.644
−1.802
−1.801
−1.652
−1.660
−1.452
etanidazole
26
NA
1
3.3
32
1.6
b
nitrofurans
nitrofurantoin
16
28
23
1.1
b
b
furazolidone
31
30
6.5
3.7
9.4
19
0.60
>50
2
3
0.98
10
3.8
>50
>16
quinoxaline-1,4-di-N-
oxides
28
>1.6
4
−2.436
−1.919
−1.279
−1.622
>20
10
>32
27
>32
>32
>32
7.3
>16
>16
>16
8.0
NA
>1.6
NA
1.6
5
6
>10
5.0
>32
3.7
benzotriazine-1,4-di-N-
oxides
TPZ
7
−1.703
−1.442
5.0
1.2
3.7
0.97
38
17
7.6
17
8a
0.57
0.37
b
a
NA = not available; ITR = Institute for Tuberculosis Research, University of Illinois at Chicago. Calculation performed for Z stereochemistry
around double bond.
MIC values against NRP Mtb in the low-oxygen recovery assay
(LORA)11 (Table 1). The primary screening was performed in
two independent laboratories with different sources of the
H37Rv strain to validate that the activities were not only
specific for a given laboratory strain, because wide variations in
lab strains of H37Rv have been reported.12 Additionally, we
tested all of the compounds for cytotoxicity against Vero cells
to give a selectivity index (SI) describing the relative
cytotoxicities of compounds for Mtb over a mammalian cell
line.
The data from the survey of bioreductively activated scaffolds
showed that the BTOs tested had consistent activities against
both actively replicating and nonreplicating Mtb. The MIC
values ranged from 0.57−5 μg/mL against the actively
RESULTS
■
Survey of Bioreductively Activated Antimicrobial
Scaffolds. Four classes of bioreductively-activated compounds
(i.e., compounds having cellular activities requiring the
enzymatic transfer of electrons from reductase enzymes) were
selected for screening against Mtb H37Rv. The compounds
included 2- and 5-nitroimidazoles, 2-nitrofurans, quinoxaline-
1,4-di-N-oxides, and BTOs (Figure 1). These particular classes
of compounds were selected based on prior reports of known
antimicrobial activities and based on availability through
commercial sources or the National Cancer Institute (NCI)
Developmental Therapeutics Program compound reposi-
tory.8−10 We screened the compounds for their MIC values
against Mtb H37Rv strains from two sources, as well as for their
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replicating H37Rv strains and from 0.37−7.3 μg/mL in the
LORA model of NRP. All of the compounds had some
cytotoxicity against the Vero cells with moderate SI values.
Using the results of the initial survey, we selected the anti-TB
activities of the BTOs, particularly analogues of 8a, for SAR
development and optimization studies.
Synthesis and Activity of N-Substituted BTOs. The first
set of new BTOs synthesized contained analogues with diverse
substitutions at the 3-amine position of the ring (Scheme 1).
pounds should dictate the selectivity of the compounds for
cytotoxicity to Mtb over mammalian cells. Variations to
substitutions on the heterocyclic ring should significantly alter
the electron reduction potentials and affect the selectivity index
of the compounds.
The synthetic route for making the ring-modified BTOs
began with either a substituted aniline starting material (11) or
a substituted 2-nitroaniline (12) when available. (Scheme 2)
The anilines were acylated, nitrated with fuming nitric acid, and
then deprotected by refluxing in hydrochloric acid to give a set
of 2-nitroanilines (12).13 The 2-nitroanilines were then
condensed with cyanamide in concentrated hydrochloric acid
followed by treatment with base to form the 1,2,4-
benzotriazine-(1N)-oxide ring. The 3-aminobenzotriazines
were then converted to 3-hydroxybenzotriazines by reaction
with either sodium nitrite or tert-butyl nitrite to form a
diazonium intermediate, which was hydrolyzed with acid. The
intermediates were chlorinated by refluxing in phosphorus
oxychloride to make the 3-chlorobenzotriazine-(1N)-oxide
intermediates (14a−g). Intermediates 14a−g were then reacted
with a subset of the primary amine side chains (Figure 2; side
chains a, b, d, e, f, i, and m). Side chains a, b, d, and f were
chosen because these low molecular weight side chains yielded
potent compounds without adding unnecessary additional
lipophilicity.14 Side chain i was chosen to sample a polar
group at this position, and m was chosen because it yielded one
of the most potent compounds from the series of side-chain-
substituted analogues. This set of ring-substituted BTOs was
then oxidized as described in Scheme 2 to give the compounds
listed in Table 3.
All of the ring-substituted BTOs were tested for
antitubercular activity and cytotoxicity (Table 3). The
modifications to the ring substitutions resulted in a wide
range of potencies (0.15−5 μg/mL) and cytotoxicities (<2.5−
100 μg/mL). Most importantly, several of the ring substitutions
led to a significant increase in SI values. The general trend
observed was that electron-donating substitutions led to
reduced cytotoxicities against mammalian cells. That trend
fits with the hypothesis that lowering the one-electron
reduction potential (E1/2) of the compounds may be effective
in increasing SI values.15 The best compounds from this series
were those with small alkyl substitutions to the benzotriazine
ring, such as the 5-methyl and 6,7-cyclopentyl substitutions.
These ring systems yielded compounds with SIs near 50.
Synthesis and Activity of Di-N-Alkyl BTOs. The SAR
data from the side chain and ring-substituted compounds
suggested that increasing the electron-donating groups around
the benzotriazine ring increased the selectivity index and that
modifying the alkylamine side chains increased potency but did
not considerably affect the SI. From these observations, we
Scheme 1. Synthesis of N-Alkyl 1,2,4-Benzotriazine-di-N-
a
oxides
a
Reagents and conditions: (a) RNH2 (2 equiv), TEA (2 equiv), DCM,
18 h, rt; (b) TFAA, H2O2, THF, 4 d.
The substitutions at this position were designed to determine
how steric and electronic variations at this position affected the
potency and selectivity of the compounds. The side chain
modified analogues were synthesized by reacting the primary
amine starting materials (Figure 2) with 3-chloro-benzotriazine-
1-N-oxide (9). The amine adducts were then oxidized using
pertrifluoroacetic acid, prepared in situ by adding trifluoroacetic
anhydride (TFAA) to a stirring solution of the compound in
excess hydrogen peroxide. The complete oxidation took 3−4 d
with additional equivalents of TFAA added until the oxidation
was complete by TLC.
Results revealed that acyclic and cyclic alkyl groups of various
sizes were tolerated at the 3-amine position but that none of the
modifications significantly increased the compounds’ SIs.
(Table 2) All of the compounds were active (MIC ≤10 μg/
mL); the most active were those with very hydrophobic side
chains (8j and 8m). Incorporation of a polar ether group (8i)
into the side chain resulted in a significant loss of activity but
with a comparable reduction in cytotoxicity. The overall results
from this set indicated that both cyclic and acyclic substituents
at this position were tolerated with the more hydrophobic
groups resulting in the most potent compounds. None of the
modifications in this set significantly improve the compounds’
SIs, which ranged from 2.9−8.3.
Synthesis and Activity of Ring-Substituted BTOs. The
hypothesized mechanism of action for these BTOs is the
bioreductive activation of the ring into a cytotoxic radical
species that causes irreparable DNA damage to Mtb. Given this
mechanism, the electron reduction potentials of the com-
Figure 2. Amine diversity elements used for side chains on benzotriazines.
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Table 2. Antitubercular Activity of Side-Chain-Substituted BTOs
compd
LUMO (eV)
TB MIC−SRI (μg/mL)
TB MIC−ITR (μg/mL)
LORA MIC−ITR (μg/mL)
Vero Tox CC50 (μg/mL)
SI (CC50/MIC)
8b
8c
8d
8e
8f
−1.652
−1.414
−1.589
−1.555
−1.536
−1.514
−1.530
−1.412
−1.523
−1.495
−1.532
−1.553
−1.501
2.5
1.2
1.8
1.8
0.82
0.84
0.68
0.75
0.50
0.99
4.9
21
8.0
8.9
10
8.3
6.4
7.1
4.2
3.6
3.2
5.3
>5.0
3.7
7.1
3.3
5.5
2.9
1.9
1.2
0.94
3.6
2.5
2.5
3.8
8.9
7.9
26
8g
8h
8i
2.5
1.6
5.0
1.9
10.0
0.62
1.2
6.6
>50
2.3
8.9
4.1
3.4
7.3
8j
0.22
0.92
0.52
0.60
1.9
<0.12
0.36
0.38
0.42
2.3
8k
8l
1.2
8m
8n
0.62
2.5
a
Scheme 2. Synthesis of Ring Substituted 1,2,4-Benzotriazine-di-N-oxides
a
Reagents and conditions: (a) Ac2O, TEA, DCM; (b) fHNO3, Ac2O, DCM; (c) HCl, reflux; (d) NCNH2, concd HCl; (e) NaNO2, HCl; (f)
tBuONO, tBuOH, H2O; (g) POCl3, reflux; (h) RNH2 (2 equiv), TEA (2 equiv), DCM, 18 h, rt; (i) TFAA, H2O2, THF, 4 d.
concluded it was desirable to synthesize new compounds with
more electron-donating groups on the alkylamine side chain. In
previous attempts, compounds with trisubstituted amine side
chains could not be synthesized because the final oxidation
reaction conditions did not produce the desired di-N-oxide
product. To make these compounds, we thus explored several
alternative oxidizing agents to pertrifluoroacetic acid, identify-
ing HOF·CH3CN as a sufficiently strong reagent to carry out
the desired transformation without producing significant
overoxidation side products.16,17 This new route allowed
synthesis of a series of di-N-alky BTOs (Scheme 3). For this
route, it proved preferable to oxidize the 3-chlorobenzotriazine
intermediates so that the amine diversification step was the last
step in the library synthesis from common intermediates 16
and 19.
When tested for antitubercular activity and cytotoxicity, the
new di-N-alkyl BTOs were found to have increased SIs (Table
4). The antitubercular activities of the compounds were
generally comparable to those of the ring-substituted analogues,
but cytotoxicities against Vero cells were significantly reduced.
From this set of compounds, we identified several new
compounds with SIs >50. From the set of secondary amine
starting materials used for this set of compounds (Scheme 3),
small alkyl groups were found to have the greatest activities.
The addition of polar or hydrophilic groups designed to
increase solubility resulted in a significant loss of anti-TB
activity.
determine whether or not the compounds had cross-resistance
with existing TB drugs and maintained their potencies against
diverse strains of Mtb. The resulting data indicated that no
cross-resistance occurred and that the compounds had either
equal or increased potencies against the drug-resistant strains of
Mtb.
Antimicrobial Spectrum of Activity for BTOs. Given the
long treatment times and complex drug combination regimens,
antitubercular drugs should have a narrow spectrum of
antimicrobial activity. Two BTOs, 8a and 20q, were profiled
for their antimicrobial activities against a diverse panel of Gram-
positive and Gram-negative bacterial pathogens (Supporting
Information Table 1). Remarkably, the BTOs were highly
selective in their activities for mycobacteria, with potent activity
against Mtb and weaker activity against Mycobacterium
smegmatis. Mycobacterium abscessus, which is naturally resistant
to many classes of antibiotics, was resistant to both 8a and
20q.18
Mutagenic Potential of BTOs. A liability in developing
any bioreductively activated antimicrobial drug is the potential
for mutagenicity. Because the basis for activity of these
compounds is the generation of intermediate radical species,
undesirable mutagenicity poses a risk, as has been documented
for many classes of bioreductive drugs, including the BTO
tirapazamine (TPZ).19,20 The most convenient methodology
for assessing the potential to induce genetic damage is to use
the plate incorporation method with Salmonella typhimurium
strains TA98 and TA100, commonly known as the Ames test.
The Salmonella tester strains have mutations in the histidine
operon, a mutation that leads to defective lipopolysaccharide
Activity Against Drug-Resistant Mtb Strains. We also
tested a set of the most potent and selective BTOs against a
panel of single-drug resistant Mtb strains (Table 5) to
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Table 3. Antitubercular Activity of Ring-Substituted BTOs
compd
LUMO (eV)
TB MIC−SRI (μg/mL)
TB MIC−ITR (μg/mL) LORA MIC−ITR (μg/mL)
Vero Tox CC50 (μg/mL)
SI (CC50/MIC)
15aa
15ab
15ad
15ae
15af
−1.704
−1.918
−1.857
−1.820
−1.696
−1.716
−1.808
−1.666
−1.870
−1.812
−1.655
−1.675
−1.759
−1.652
−1.793
−1.757
−1.749
−1.541
−1.66
−1.629
−1.409
−1.567
−1.524
−1.254
−1.352
−1.244
−1.195
−1.324
−1.284
0.62
0.31
0.31
0.31
0.62
5.0
0.35
0.93
0.40
0.33
0.68
2.0
0.75
3.5
4.6
1.3
1.6
2.3
<2.5
6.3
<2.5
2.6
1.3
1.6
2.6
2.6
1.1
4.2
3.8
18
7.3
2.2
10
0.88
0.34
0.47
1.9
7.3
<4.0
1.3
<4.0
2.1
2.2
10
15ai
15am
15ba
15bb
15bd
15bf
15bi
0.31
1.2
<0.12
<0.12
0.21
<0.12
0.94
1.4
0.21
0.41
0.22
<0.12
0.63
0.78
0.23
0.46
0.41
0.46
0.41
16
0.62
0.15
0.62
1.2
4.2
2.1
7.3
6.7
24
15bm
15ca
15cd
15ce
15cm
15da
15dd
15dm
15ea
15ed
15ee
15fa
0.15
0.62
0.15
0.15
0.31
2.5
0.22
0.49
0.46
0.35
0.36
15
110
2.6
6.3
16
0.80
16
0.62
0.62
0.62
0.31
0.31
1.2
0.96
0.97
0.51
0.47
0.43
0.95
0.96
0.93
3.5
0.90
1.6
9.8
>50
22
>80
36
0.48
0.43
0.27
0.83
0.44
0.36
2.5
22
70
3.7
36
12
29
15fd
15fm
15ga
15gd
15gm
0.62
1.2
34
54
>50
>100
>50
>50
>41
>40
>20
>10
2.5
2.5
5.1
2.9
5.0
5.6
3.5
(rfa), and a deletion that covers genes involved in the synthesis
of biotin (bio) and in the repair of ultraviolet-induced DNA
damage (uvrB).21,22 These mutations make the strains more
permeable to many molecules and increase their susceptibility
to the mutagenic effects of these molecules. Both strains
contain plasmid pKM101 that causes an increase in error-prone
DNA repair. Given that the hypothesized mechanism of the
BTO compounds is to selectively form cytotoxic radical species
in bacteria over mammalian cells, a microbial mutagenicity
assay is not ideal but was used to benchmark their activities in
this assay. Two BTOs, 15fa and 20q, with good potency and
selectivity profiles, were evaluated in the Ames assay in both the
presence and absence of an Aroclor 1254-induced rat-liver
metabolic activation system containing 10% S9 (MA).
Individual plate counts and their means and standard
deviations, along with the condition of the background lawn,
are presented in Supporting InformationI Table 2. There were
dose-related increases in the number of revertant colonies for
both 15fa and 20q with both strains TA98 and TA100 in the
presence and absence of metabolic activation. To assess their
mutagenic potential in a nonmicrobial system, 15fa and 20q
were also evaluated in the mouse lymphoma cell tk+/−→tk−/−
gene mutation (MOLY) assaya routine genetic toxicology
assay used to assess the mutagenic potential of compounds in
mammalian cells (Table 6).23,24 In the presence or absence of
metabolic activation (S9), 15fa, at noncytotoxic dose levels of
10, 25, 50, and 100 μg/mL, showed a significant increase in
mutation frequency (MF). For 20q, both in the presence or
absence of S9, mutation frequency did not increase at
noncytotoxic dose levels up to 100 μg/mL for 4 h exposure
and up to 10 μg/mL for 24 h exposure in the absence of S9. In
conclusion, 20q gave negative response for mutation frequency
in the presence or absence of S9.
Scheme 3. Synthesis of Di-N-alkyl 1,2,4-Benzotriazine-di-N-
a
oxides
a
Reagents and conditions: (a) F2 (g), H2O, MeCN; (b) RNH2 (2
equiv), TEA (2 equiv), DCM, 1 h, rt.
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Table 4. Antitubercular Activity of Di-N-alkyl BTOs
compd
LUMO (eV)
TB MIC−SRI (μg/mL)
TB MIC−ITR (μg/mL)
LORA MIC−ITR (μg/mL)
Vero Tox CC50 (μg/mL)
SI (CC50/MIC)
17o
17p
17q
20q
20r
20s
20t
−1.483
−1.624
−1.286
−1.196
−1.311
−1.335
−1.344
−1.289
−1.751
−1.293
−1.258
0.31
2.5
0.46
3.7
1.3
19
12
5.4
17
40
2.2
56
0.31
0.31
0.62
0.62
0.31
0.31
5.0
NT
0.46
0.40
0.30
0.15
0.24
15
NT
0.99
0.49
1.5
25
80
21
13
24
13
0.41
0.44
30
17
53
20u
20v
20w
20x
40
130
3.4
>40
>10
17
1.2
0.66
13
0.47
15
>50
>50
5.0
Table 5. MIC of BTOs against Single Drug Resistant Mtb Strains
MIC values (μg/mL) of Mtb strains resistant to
streptomycin
ATCC 35820
p-amino-salicyclic acid
isoniazid
ATCC 35822
kanamycin
ATCC 35827
ethionamide
ATCC 35830
ethambutol
ATCC 35837
compd
H37Rv
ATCC 35821
8a
1.2
1.2
1.2
1.2
1.2
2.5
1.2
1.2
1.2
8c
2.5
1.2
2.5
1.2
2.5
15ea
15fa
15fd
15ga
17q
20q
20r
20s
20t
0.62
1.2
0.16
0.63
0.31
1.2
0.31
1.2
0.62
2.5
0.62
1.2
0.31
1.2
0.62
1.2
0.62
1.2
0.62
1.2
1.2
1.2
0.62
1.2
0.62
1.2
1.2
1.2
0.31
0.62
0.62
0.62
0.31
0.31
1.2
0.02
0.16
0.15
<0.07
<0.07
<0.07
0.31
0.62
0.31
0.31
0.15
0.07
0.15
0.62
0.31
0.31
0.15
<0.07
<0.07
0.07
0.31
0.31
0.63
0.31
0.31
0.15
0.15
0.62
0.31
0.62
0.31
0.15
0.07
0.15
0.31
0.31
0.62
0.31
0.31
0.15
0.31
0.62
20u
20w
compounds is still in the proof-of-concept stage, a single dose
(100 mg/kg) of 8a and 20q was administered to mice in order
to profile the pharmacokinetics for the BTOs (Table 7). Our
Table 6. Mutagenic Potential of BTOs in MOLY Assay (S9 =
Rat Metabolic Activation; Positive = A Significant Induction
of Mutation Frequency; Negative = No Significant Induction
of Mutation Frequency; NT = Not Tested)
Table 7. Pharmacokinetic Parameters for 8a and 20q after
Oral Administration to Female Mice
dose
(μg/
mL)
+S9, 4 h
exposure
(repeat)
−S9, 4 h
−S9, 24 h
+S9, 4 h
exposure
compd
exposure
exposure
dose
compd (mg/kg) (h)
Tmax
(h)
Cmax
AUClast
AUCinf
(h·ng/mL)
t1/2
15fa
10
25
positive
positive
positive
positive
negative
negative
negative
negative
NT
positive
positive
positive
positive
negative
positive
positive
positive
positive
negative
negative
negative
negative
(ng/mL) (h·ng/mL)
NT
8a
100
100
1.5
1.9
0.08
0.25
9540
9767
6578
6937
50
NT
20q
11219
11248
100
10
NT
20q
negative
negative
NT
targeted drug candidate profile is an orally administered
therapeutic that requires a maximum of once-a-day dosing
and preferable 1−3 times a week dosing in order to be
compatible with current first line TB drugs in a fixed dose
combination tablet.25 To achieve this profile, a mouse
elimination half-life between 4−12 h is being targeted in our
development program. The elimination half-life for each
compound was shorter than our ideal target values, but the
Tmax values indicate that they are rapidly absorbed after oral
administration (Figure 3). Both compounds resulted in good
exposure based on Cmax and AUC values, indicating these
compounds show promise for further development as
antitubercular drugs.
a
25
negative
50
negative
negative
100
NT
a
Induction of mutation frequency but slightly higher than 40 × 10−6
net over mean solvent control, biologically considered negative.
Physiochemical Properties and Mouse Pharmacoki-
netics. To assess the potential of BTOs to be developed into
orally bioavailable therapeutics, some of the physiochemical
properties and ADME properties were profiled for 8a and 20q.
The solubility of these two compounds was measured in
triplicate using the shake-flask method in a 0.9% saline solution
at pH 7.4. Both 8a and 20q had acceptable solubilities of 1.35
and 0.28 mg/mL, respectively.
DISCUSSION
■
To validate BTOs as a new lead series for the treatment of
TB, it was also important to evaluate their systemic exposure in
the mouse after oral administration. Because this series of
Bioreductively activated antimicrobials have proven to be
effective drugs for a number of different types of infections and
have also been evaluated for the treatment of cancer as hypoxic
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The quinoxaline oxides have also been evaluated as potential
antitubercular agents with potent activity against replicating and
nonreplicating Mtb.34−39 Members of this class have been
developed that have excellent selectivities for Mtb and
demonstrated efficacy in low-dose aerosol infection models in
mice.40 The quinoxaline oxides tested in our panel of
bioreductively activated compounds were originally synthesized
as part of a hypoxic cytotoxin program for cancer, and the
antitubercular activity they exhibited was limited. Optimizing
this class of compounds for antitubercular activity has led to the
development of a new class of lead compounds with potent in
vitro activities and clear in vivo efficacy.40
Our initial survey identified BTOs as a new class of
bioreductively activated antitubercular agents with good
selectivity against Mtb. BTOs are well-studied and have been
developed into antitumor agents, as exemplified by the drug
TPZ.41,42 Because hypoxic areas in solid tumors resist standard
chemotherapy, bioreductive hypoxic-specific cytotoxins such as
TPZ have been included in combination therapies to
circumvent tumor resistance and improve the effectiveness of
chemotherapy.43,44 The treatment of a TB granuloma presents
a similar challenge: many of the metabolic subpopulations of
Mtb found in lung granulomas resist antitubercular drugs used
in conventional treatment regimens. The development of new
antitubercular agents targeting the subpopulations of Mtb
resistant to other drugs may increase the overall efficacy of
treatment regimens.
TPZ’s selective activity toward hypoxic cells is attributed to
increased activation by cytosolic and nuclear reductases that
convert the drug to radical metabolites and to reduced back-
oxidation to the parent compound under hypoxic condi-
tions.45−48 These metabolites have been shown to directly or
indirectly cause double-strand DNA breaks in hypoxic cells.49
The antitubercular activity of BTOs differs from antitumor
activity in that most of the compounds tested are equally active
against both actively replicating Mtb and hypoxia-induced NRP
Mtb. This finding suggests that the reductases in Mtb
responsible for activating the BTOs are expressed during
both of the replicating and nonreplicating metabolic states and
that back-oxidation to the parent compound under normoxic
conditions does not significantly affect antitubercular activity. A
detailed investigation into the molecular mechanism for the
antitubercular activity of BTOs is under investigation.
Given knowledge about TPZ and the hypothesized
mechanism of action for BTOs, multiple Mtb reductases are
likely to be capable of activating the parent compounds. This
aspect of their activity made it difficult to rely on analysis of
SAR data for the optimization of the cellular activities of the
BTOs. Instead, we focused on the physiochemical properties of
the compounds, such as the electron reduction potentials, to
guide the design of new compounds. On the basis of our
hypothesized mechanism of action, the electron reduction
potentials of the BTOs are fundamentally related to their
activity. The total cellular activity of compounds depends on
many factors, including the ability to bind to the reductases
responsible for bioactivation and enter the bacterial cytosol.
The potential involvement of multiple activating reductase
enzymes makes it less likely that pharmacophore-based
approaches will be useful for modeling activities and guiding
optimization efforts.
Figure 3. Plasma drug concentrations of 8a and 20q in female Balb/c
mice (n = 3 mice per time point) after po administration of 100 mg/kg
of each compound. Blood was collected at time points through 24 h
postdose, processed to plasma, and analyzed for each of the test
compounds. The concentrations at the 24 h time point for both
compounds was lower than the limit of quantitation.
cytotoxins and radiation sensitizers. These highly oxidized
compounds can be selectively reduced through the transfer of
electrons from reductase enzymes. The single- or double-
electron reduction produces reactive intermediate species that
cause irreversible cellular damage, leading to cell death. The
mechanisms of bioreductive compounds allow them to inflict
bactericidal activity against Mtb under various growth
conditions and metabolic states, provided the activating
reductase enzyme is present. Thus, the compounds may have
bactericidal activity against specific metabolic subpopulations of
Mtb bacilli that are commonly found in lung granulomas and
are resistant to other standard therapeutics. The primary
motivation for evaluating bioreductively activated compounds
as antitubercular drugs is based on the hypothesis that
bactericidal compounds active against multiple subpopulations
of Mtb bacilli will shorten the length of drug treatment required
to eliminate an infection.
Among the panel of bioreductively activated antimicrobial
scaffolds tested against Mtb, 5-nitroimidazoles have been
thoroughly explored for their antitubercular activity:26−30 2-
nitroimidazoles (e.g., etanidazole), on the other hand, have
quite different properties relating to their electron reduction
potentials and have been primarily used as tumor hypoxia
imaging agents.31 In our initial screening, the 2-nitroimidazoles
did not show potent activity against actively replicating Mtb but
did demonstrate some selective activity against the hypoxia-
induced nonreplicating Mtb. This trend mimics the activity
observed in mammalian cells that 2-nitroimidazoles are
selectively reduced in hypoxic, but not in normoxic, cancer
cells.
The nitrofurans have been used for more than 50 years to
treat bacterial and parasitic diseases. They have also been
reported to have potent antitubercular activity.32,33 Of the three
nitrofurans we tested in our screen, compound 2 was the only
one identified with potent activity against both replicating and
nonreplicating Mtb as well as good selectivity over a
mammalian cell line. Compound 2 was originally developed
as a candidate antibiotic for Staphylococcus aureus infections but
was not advanced to development because of genetic toxicity.
Despite the interesting in vitro activity profile of compound 2,
its genetic toxicity made it unsuitable for development as an
antitubercular drug.
For many classes of bioreductively activated classes of
compounds such as the BTOs, E1/2 has been shown to be a
major determinant of the potency and selectivity for
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bioreductive cytotoxic activity, and a linear relationship has
been shown to exist between the energy of the LUMO and the
E1/2 value.50 Given this relationship, we used the semiempirical
calculation of LUMO energies as part of our design strategy to
identify compounds with increased selectivity for Mtb over
mammalian cells. Mtb uses the F420 deazaflavin cofactor, which
has a much lower reduction potential than the classical
hydrogen carrier NAD(P)+ (−380 vs −320 mV).51 The use
of the lower reduction potential F420-requiring reductases may
provide a basis for the antimicrobial selectivity for many classes
of bioreductive drugs, as has been proposed for the 5-
nitroimidazole PA-824.3,15 Analysis of the calculated LUMO
energies for the BTOs revealed that compounds with higher
LUMO energies were generally less toxic to the mammalian
Vero cell line, whereas antitubercular activities had no
correlation with LUMO energies. When the CC50 toxicity
values were plotted against the calculated LUMO energies, only
a weak correlation existed (R2 value of 0.56), which was to be
expected because the overall activity was multifactorial and not
based on a single molecular property. Despite this limited
correlation, the calculation of LUMO energies proved a
valuable predictive tool in designing new BTO compounds
with less toxicity to mammalian cells and with higher SIs. The
calculations indicated that LUMO energies, as expected from
introducing electron-donating groups into the aromatic ring
system, generally increased.
elimination of genetic toxicity and mutagenicity. These major
developmental liabilities pose mechanism-based risks for any
bioreductively activated drug. Because the MOLY assay
indicated that TPZ, the most advanced clinical BTO under
evaluation for its anticancer activity, is mutagenic, eliminating
its mutagenic activity was essential for developing it as an
antitubercular drug candidate. To mitigate that risk, we
increased the selectivity of the compounds sufficiently so that
they did not produce the reactive radical compounds in
mammalian cells that give rise to mutagenicity. Surprisingly,
both of the compounds tested in the MOLY assay (15fa and
20q) had similar selectivities and cytotoxicities against Vero
cells but had dramatically different mutagenic potentials. That
discrepancy demonstrates the difficulty of rational approaches
for reducing genetic toxicity. Fortunately, our lead compound,
20q, had no mutagenic activity at concentrations up to 100 μg/
mL.
Analysis of the physiochemical properties and mouse
pharmacokinetic data for 8a and 20q show that the class has
good drug-like properties. The solubilities of the compounds
are generally very good, most likely due to the polar nature of
the N-oxide groups on the benzotriazine rings. The initial
pharmacokinetics data for the series indicates that the lead
compounds are not yet optimal with respect to clearance and
overall exposure but are sufficient to warrant further medicinal
chemistry optimization.
A key lead optimization challenge for developing a
bioreductively activated antitubercular drug is achieving
sufficient potency and selectivity for Mtb. The SAR indicates
that hydrophobic side chains generally resulted in more potent
antitubercular compounds, which was expected because
compounds must pass through the hydrophobic mycobacterial
cell wall to exert their activity. Increasing hydrophobicity also
seemed to increase cytotoxicity to mammalian cells, presumably
through increased cell permeability. Modifications to the
substitutions on the BTO ring resulted in substantial changes
in the selectivity of the compounds. In general, introduction of
electron-withdrawing groups such as halogens resulted in more
potent compounds against Mtb (MICs of 0.15−0.31 μg/mL),
but those compounds were also more cytotoxic to Vero cells.
Electron-donating substitutions on the ring tended to slightly
decrease potency against Mtb (MICs of 0.31−1.2 μg/mL) but
substantially decreased toxicity, leading to compounds with
overall improved selectivity profiles. To balance these two
opposing SAR trends, we selected compounds with fused
tricyclic ring systems (15f), which had the most balanced
potency and selectivity profiles.
The development of the oxidation chemistry to produce the
di-N-alkyl BTO compounds allowed us to make compounds
with 2−4-fold increases in potency against Mtb, while either
maintaining or decreasing toxicity against Vero cells. All of the
compounds made in this series had very good potencies (MICs
of 0.31−0.62 μg/mL), except for compounds with more
hydrophilic side chains containing a morpholino (17p), a
sulfone (20v), or a sulfamide (20x); all those compounds
demonstrated significant reductions in antitubercular activities
(MICs of 2.5−5 μg/mL). Overall, the SAR trends suggested
that optimal compounds should have electron-rich ring systems
(lower E1/2 values; higher LUMO energies) with hydrophobic
side chains to afford compounds with maximal potencies and
selectivities.
CONCLUSION
■
In summary, the development of bioreductively activated anti-
infective drugs is a clinically proven strategy that has only
recently been applied to the development of therapeutics for
TB. The success of 5-nitroimidazoles such as PA-824 in
preclinical and clinical studies demonstrates that developing a
safe and effective therapeutic from this mechanistic class is
possible. BTOs have long been known for their anticancer
activity as radiation sensitizers and hypoxic cytotoxins, but the
present work demonstrates that newer members of this class
can be developed with potent and selective antitubercular
activity. As with any new therapeutic class, the evaluation of
BTOs in in vivo efficacy models will provide further insight into
the bactericidal activity of this class and their potential for
shortening the required length of therapy.
EXPERIMENTAL METHODS
General. All reagents and starting materials were purchased from
commercial sources and used without further purification. H NMR
were recorded on a Varian Utility 300 or 400 MHz spectrometer, in
deuterated chloroform (CDCl3), methanol (CD3OD), or dimethyl
sulfoxide (DMSO-d6) solvent. Chemical shifts were reported as ppm
■
1
1
downfield from an internal TMS standard (δ = 0.0 for H NMR) or
from solvent reference. Coupling constants (J values) were measured
in hertz. Low resolution electrospray mass spectra (ES-MS) were
collected on a Finnigan liquid chromatography quadrupole (LCQ)
Duo liquid chromatography tandem mass spectrometer (LC-MS-MS)
(Thermoquest). Crude products were purified by flash chromatog-
raphy using a Biotage Isolera flash purification system with SiliaSep-
HP high-performance flash cartridges (SiliCycle). All compounds were
1
purified to purities >95% as assessed by HPLC and H NMR. The
HPLC method used an Ace reversed-phase C18 column (100 mm ×
4.6 mm, 3 μm) running a binary gradient with water (with 0.1% TFA)
and acetonitrile (with 0.1% TFA). Purity was measured using the
wavelengthMAX reading from a photodiode array detector scanning
from 220−600 nm on a 12 min gradient running from 0% to 100%
acetonitrile/TFA at 1 mL/min on a Waters Alliance HPLC. All known
Another major challenge for the development of a safe and
effective bioreductively activating antitubercular drug is the
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compounds were referenced throughout this work and data for any
new compounds is given below.
Solvent was evaporated, and the product was purified by flash
chromatography using silica gel and a mobile phase gradient of 0−10%
EtOAc in CH2Cl2. Isolated yields were from 35−40%, with the
remainder of the mass being recovered starting material.
General Procedure for Amination/Oxidation. Method A. A
primary amine (2.2 mmol) starting material was added to a stirring
solution of 9 (200 mg, 1.1 mmol) dissolved in DCM (10 mL) with
triethylamine (5.5 mmol) at room temperature. The solution was
stirred for 24−48 h until all of the 9 starting material was consumed as
monitored by TLC (5% MeOH/DCM mobile phase). The bulky
amine side chains required heating to 45 °C for complete conversion.
The entire reaction was evaporated under reduced pressure to give the
crude products 10a−n, which were directly subjected to oxidation
conditions. The crude mono-N-oxides were dissolved in a mixture of
THF (8 mL) and 50% H2O2 (4 mL) and then slowly treated with
TFAA (3.3 mmol) while stirring at room temperature. The oxidation
was monitored by TLC and additional equivalents of TFAA (3.3
mmol) were added every 12 h until the reactions were >90% complete
or no additional product was being formed. Upon completion of the
reaction, the reaction was quenched with 10 mL of saturated sodium
bicarbonate and extracted three times with 10 mL of chloroform. The
combined organic layers were evaporated and purified by semi-
preparative reversed-phase HPLC.
General Procedure for 3-Aminobenzotriazine 1-Oxides.
Method B. In a 1 L three-neck flask with mechanical stirring and a
reflux condenser was placed a 2-nitroaniline (12a−h, 171 mmol),
cyanamide (1.37 mol, 8 equiv), and Et2O (30 mL). The mixture was
heated to 100 °C for 1 h and then cooled to 50−55 °C. Using an
addition funnel, concentrated HCl (72 mL) was added dropwise with
mechanical stirring (CAUTION: highly exothermic reaction). Once
the addition was complete and there was no further gas evolution, the
mixture was heated to 110 °C for 3 h. The reaction was then cooled to
50 °C, and a NaOH solution (7.5 M, 160 mL) was slowly added. This
was then reheated to 110 °C with an oil bath for 3 h, cooled, and
poured into 3 L of H2O. The resulting solid was filtered off, washed
with H2O and Et2O, and then dried under vacuum. If necessary, the
product was purified by flash chromatography using silica gel with a
mobile phase gradient of 0−10% MeOH in CH2Cl2.
General Procedure for 3-Chlorobenzotriazine 1-Oxides.
Method C. NaNO2 (1.5 g, 21 mmol) was added in small portions
to a stirring solution of a 3-aminobenzotriazine 1-oxide (13a−e, 7
mmol) in trifluoroacetic acid (15 mL) at room temperature. After 1 h,
50 mL of H2O was added and the solid was filtered off and dried under
vacuum. The solid was then suspended in 20 mL of POCl3 and heated
to reflux for 3 h. The solution was cooled, and the majority of the
excess POCl3 was removed by distillation and then was dissolved in
CH2Cl2 (40 mL). This solution was then washed with H2O and brine,
and the solvent was evaporated. The crude residue was purified by
flash chromatography using silica gel and a gradient of 0−10% EtOAc
in CH2Cl2.
General Procedure for 3-Chlorobenzotriazine 1-Oxides.
Method D. tBuONO (90%, 15 mL) and H2SO4 (1 mL) were slowly
added to a stirring solution of a 3-aminobenzotriazine 1-oxide (13f−g,
9 mmol) in tBuOH (50 mL) and H2O (5 mL). After heating to 60 °C
overnight, the mixture added to 100 g of ice and the resulting solid was
filtered off and dried under vacuum. The solid was then suspended in
20 mL of POCl3 and heated to reflux for 3 h. The solution was cooled,
and the majority of the excess POCl3 was removed by distillation and
then was dissolved in CH2Cl2 (40 mL). This solution was then washed
with H2O and brine, and the solvent was evaporated. The crude
residue was purified by flash chromatography using silica gel and a
gradient of 0−10% EtOAc in CH2Cl2.
General Procedure for Amination of 3-Chlorobenzotriazine-
1,4-Di-N-Oxides. Method F. To a stirring solution of the 3-
chlorobenzotriazine-1,4-di-N-oxide (2 mmol) in dimethoxyethane (12
mL) at 0 °C was added an amine (2.2 mmol) and triethylamine (2.4
mmol). The mixture was allowed to warm to room temperature and
was stirred for 16 h. After the reaction, solvent was evaporated and the
crude product was purified by flash chromatography using silica gel
and a mobile phase gradient of 0−10% MeOH in CH2Cl2. Typical
yields were from 80−90%.
3-(Ethylamino)benzo[e][1,2,4]triazine 1,4-Dioxide (8a). Syn-
1
thesized from 9 by method A. Red solid. H NMR (DMSO-d6, 400
MHz): δ 8.28 (br s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 8.5 Hz,
1H), 7.90 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 8.6 Hz, 1H), 3.43 (m, 2H),
1.18 (t, J = 7.0 Hz, 3H). MS (ESI+): m/z 207.0 ((M + H)+).
3-(Cyclopropylamino)benzo[e][1,2,4]triazine 1,4-Dioxide
(8b). Synthesized from 9 by method A. Red solid. 1H NMR
(DMSO-d6, 400 MHz): δ 8.41 (d, J = 2.4 Hz, 1H), 8.20 (dd, J = 8.8,
0.8 Hz, 1H), 8.10 (dd, J = 8.8, 0.8 Hz, 1H), 7.92 (m, 1H), 7.56 (m,
1H), 2.76 (m, 1H), 0.74 (m, 4H). MS (ESI+): m/z 219.0 ((M + H)+).
3-(tert-Butylamino)benzo[e][1,2,4]triazine 1,4-Dioxide (8c).
Synthesized from 9 by method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.31 (d, J = 8.8 Hz, 1H), 8.26 (d, J = 8.8 Hz, 1H), 7.83 (t, J =
7.0 Hz, 1H), 7.45 (t, J = 7.0 Hz, 1H), 7.19 (s, 1H), 1.55 (s, 9H). MS
(ESI+): m/z 235.0 ((M + H)+).
3-(Cyclobutylamino)benzo[e][1,2,4]triazine 1,4-Dioxide (8d).
Synthesized from 9 by method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.44 (d, J = 8.0 Hz, 1H), 8.17 (dd, J = 8.8, 0.8 Hz, 1H), 8.10
(dd, J = 8.8, 0.8 Hz, 1H), 7.91 (m, 1H), 7.54 (m, 1H), 4.33 (m, 1H),
2.24 (m, 4H), 1.67 (m, 2H). MS (ESI+): m/z 233.0 ((M + H)+).
3-(Cyclopentylamino)benzo[e][1,2,4]triazine 1,4-Dioxide
(8e). Synthesized from 9 by method A. Red solid. 1H NMR
(DMSO-d6, 400 MHz): δ 8.18 (dt, J = 8.8, 0.8 Hz, 1H), 8.10−8.06
(m, 2H), 7.92 (m, 1H), 7.54 (m, 1H), 4.18 (m, 1H), 1.92 (m, 2H),
1.70 (m, 4H), 1.56 (m, 2H). MS (ESI+): m/z 247.0 ((M + H)+).
3-(Cyclohexylamino)benzo[e][1,2,4]triazine 1,4-Dioxide (8f).
Synthesized from 9 by method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.16 (dd, J = 8.8, 0.8 Hz, 1H), 8.08 (dt, J = 8.8, 0.8 Hz, 1H),
7.95 (d, J = 9.2 Hz, 1H), 7.89 (m, 1H), 7.52 (m, 1H), 3.71 (m, 1H),
1.85 (m, 2H), 1.72 (m, 2H), 1.58 (m, 1H), 1.51−1.42 (m, 2H), 1.35−
1.26 (m, 2H), 1.20−1.09 (m, 1H). MS (ESI+): m/z 261.0 ((M +
H)+).
3-(Cycloheptylamino)benzo[e][1,2,4]triazine 1,4-Dioxide
(8g). Synthesized from 9 by method A. Red solid. 1H NMR
(DMSO-d6, 400 MHz): δ 8.18 (dt, J = 8.8, 0.8 Hz, 1H), 8.09 (dt, J =
8.8, 0.8 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.91 (m, 1H), 7.53 (m, 1H),
3.92 (m, 1H), 1.89 (m, 2H), 1.76−1.42 (m, 10H). MS (ESI+): m/z
275.0 ((M + H)+).
3-(Pentan-2-ylamino)benzo[e][1,2,4]triazine 1,4-Dioxide
(8h). Synthesized from 9 by method A. Red solid. 1H NMR
(DMSO-d6, 400 MHz): δ 8.16 (dt, J = 8.8, 0.4 Hz, 1H), 8.07 (dt, J =
8.8, 0.4 Hz, 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.90 (m, 1H), 7.52 (m, 1H),
3.97 (m, 1H), 1.65 (m, 1H), 1.49 (m, 1H), 1.30 (m, 2H), 1.20 (d, J =
6.4 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H). MS (ESI+): m/z 249.0 ((M +
H)+).
3-((1-Methoxybutan-2-yl)amino)benzo[e][1,2,4]triazine 1,4-
Dioxide (8i). Synthesized from 9 by method A. Red solid. 1H
NMR (DMSO-d6, 400 MHz): δ 8.18 (dt, J = 8.8, 0.8 Hz, 1H), 8.09
(dt, J = 8.8, 0.8 Hz, 1H), 7.92 (m, 2H), 7.54 (m, 1H), 3.50 (dd, J = 10,
6.4 Hz, 1H), 3.40 (dd, J = 10, 5.2 Hz, 2H), 3.23 (s, 3H), 1.62 (m, 2H),
0.87 (m, 3H). MS (ESI+): m/z 265.0 ((M + H)+).
General Procedure for HOF·CH3CN Oxidation. Method E.
(CAUTION: F2 and HOF·CH3CN are extremely strong oxidants and
corrosive materials.) F2 (10%) with nitrogen was slowly bubbled into
100 mL of CH3CN with 10 mL of H2O for 1 h at −15 °C. The
resulting oxidant was added in one portion to a cooled (0 °C) solution
of the benzotriazine 1-N-oxide (12.6 mmol) dissolved in CH2Cl2 (80
mL). The mixture was stirred for 10 min and then quenched with
saturated NaHCO3 (40 mL). The biphasic mixture was then diluted
with H2O (40 mL) and extracted with CHCl3 (80 mL). The organic
layer was then washed with H2O, brine, and dried over MgSO4.
3-((4-(tert-Butyl)cyclohexyl)amino)benzo[e][1,2,4]triazine
1
1,4-Dioxide (8j). Synthesized from 9 by method A. Red solid. H
NMR (DMSO-d6, 400 MHz): δ 8.17 (td, J = 8.4, 0.8 Hz, 1H), 8.08
(m, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.91 (m, 1H), 7.57−7.50 (m, 1H),
4.06 (m, 1H), 1.94 (m, 2H), 1.75 (m, 1H), 1.58 (m, 2H), 1.14 (m,
4H), 0.83 (d, J = 3.2 Hz, 9H). MS (ESI+): m/z 317.1 ((M + H)+).
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(R)-3-((1-Cyclohexylethyl)amino)benzo[e][1,2,4]triazine 1,4-
7-Chloro-3-(ethylamino)benzo[e][1,2,4]triazine 1,4-Dioxide
(15ba). Synthesized from 4-chloro-2-nitroaniline using method C
and method A. Red solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.41 (t, J
= 6.0 Hz, 1H), 8.19 (d, J = 2.4 Hz, 1H), 8.10 (d, J = 9.2 Hz, 1H), 7.91
(dd, J = 9.2, 2.4 Hz, 1H), 3.41 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H). MS
(ESI+): m/z 241.0 ((M + H)+).
1
Dioxide (8k). Synthesized from 9 by method A. Red solid. H NMR
(DMSO-d6, 400 MHz): δ 8.16 (dd, J = 8.8, 0.8 Hz, 1H), 8.08 (dd, J =
8.8, 0.8 Hz, 1H), 7.89 (m, 2H), 7.51 (m, 1H), 3.75 (m, 1H), 1.72−
1.55 (m, 6H), 1.18 (d, J = 6.8 Hz, 3H), 1.12 (m, 3H), 0.92 (m, 2H).
MS (ESI+): m/z 289.0 ((M + H)+).
3-((5-Methylhexan-2-yl)amino)benzo[e][1,2,4]triazine 1,4-
Dioxide (8l). Synthesized from 9 by method A. Red solid. 1H
NMR (DMSO-d6, 400 MHz): δ 8.16 (dd, J = 8.8, 0.8 Hz, 1H), 8.09
(dd, J = 8.4, 0.4 Hz, 1H), 7.94 (d, J = 9.6 Hz, 1H), 7.89 (m, 1H), 7.52
(m, 1H), 3.91 (m, 1H), 1.66 (m, 1H), 1.50 (m, 2H), 1.20 (d, J = 6.8
Hz, 3H), 1.15 (m, 2H), 0.83 (dd, J = 6.4, 2.0 Hz, 6H). MS (ESI+): m/
z 277.0 ((M + H)+).
7-Chloro-3-(cyclopropylamino)benzo[e][1,2,4]triazine 1,4-
Dioxide (15bb). Synthesized from 4-chloro-2-nitroaniline using
method C and method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.47 (br s, 1H), 8.19 (d, J = 2.0 Hz, 1H), 8.09 (d, J = 9.2 Hz,
1H), 7.90 (dd, J = 9.2, 2.0 Hz, 1H), 2.73 (m, 1H), 0.77−0.67 (m, 4H).
MS (ESI+): m/z 253.0 ((M + H)+).
7-Chloro-3-(cyclobutylamino)benzo[e][1,2,4]triazine 1,4-Di-
oxide (15bd). Synthesized from 4-chloro-2-nitroaniline using method
3-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)benzo[e]-
[1,2,4]triazine 1,4-Dioxide (8m). Synthesized from 9 by method A.
Red solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.18 (dt, J = 8.8, 0.4 Hz,
1H), 8.09 (dd, J = 8.8, 0.8 Hz, 1H), 7.90 (m, 1H), 7.73 (d, J = 6.8 Hz,
1H), 7.53 (m, 1H), 3.65 (m, 1H), 2.26 (m, 2H), 1.69 (m, 2H), 1.50
(m, 3H), 1.14 (m, 3H). MS (ESI+): m/z 273.0 ((M + H)+).
3-((1R,3S,5r,7r)-Adamantan-2-ylamino)benzo[e][1,2,4]-
triazine 1,4-Dioxide (8n). Synthesized from 9 by method A. Red
1
C and method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.55
(d, J = 7.6 Hz, 1H), 8.16 (d, J = 1.6 Hz, 1H), 8.09 (d, J = 9.2 Hz, 1H),
7.89 (dd, J = 9.2, 2.0 Hz, 1H), 4.31 (m, 1H), 2.22 (m, 4H), 1.66 (m,
2H). MS (ESI+): m/z 267.0 ((M + H)+).
7-Chloro-3-((1-methoxybutan-2-yl)amino)benzo[e][1,2,4]-
triazine 1,4-Dioxide (15bg). Synthesized from 4-chloro-2-nitroani-
1
line using method C and method A. Red solid. H NMR (DMSO-d6,
1
solid. H NMR (DMSO-d6, 400 MHz): δ 8.18 (dd, J = 8.8, 0.8 Hz,
400 MHz): δ 8.18 (d, J = 2.4 Hz, 1H), 8.10 (d, J = 9.2 Hz, 1H), 7.96
(d, J = 8.8 Hz, 1H), 7.90 (dd, J = 9.2, 2.4 Hz, 1H), 3.97 (m, 1H), 3.49
(dd, J = 9.6, 6.4 Hz, 1H), 3.39 (m, 1H), 3.23 (m, 3H), 1.61 (m, 2H),
0.86 (t, J = 7.2 Hz, 3H). MS (ESI+): m/z 299.0 ((M + H)+).
7-Chloro-3-(cyclohexylamino)benzo[e][1,2,4]triazine 1,4-Di-
oxide (15bj). Synthesized from 4-chloro-2-nitroaniline using method
1H), 8.11 (dd, J = 8.8, 1.2 Hz, 1H), 7.93 (m, 1H), 7.55 (m, 1H), 7.39
(d, J = 8.4 Hz, 1H), 4.00 (m, 1H), 2.04 (m, 2H), 1.88 (m, 8H), 1.72
(br s, 2H), 1.61 (m, 2H). MS (ESI+): m/z 313.1 ((M + H)+).
3-(Ethylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-Dioxide
(15aa). Synthesized from 4-fluoro-2-nitroaniline using method C
and method A. Red solid. 1H NMR (CDCl3, 400 MHz): δ 8.26 (dd, J
= 4.9, 9.6, 1H), 7.92 (dd, J = 1.7, 8.0, 1H), 7.57 (m, 1H), 6.94 (br-t,
1H), 3.57 (p, J = 7.3, 2H), 1.29 (t, J = 7.3, 3H). MS (ESI+): m/z 224.9
((M + H)+).
3-(Cyclopropylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-
Dioxide (15ab). Synthesized from 4-fluoro-2-nitroaniline using
method C and method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.37 (br s, 1H), 8.15 (dd, J = 9.6, 5.2 Hz, 1H), 7.97 (dd, J =
8.4, 2.8 Hz, 1H), 7.84 (m, 1H), 2.72 (m, 1H), 0.72 (m, 4H). MS (ESI
+): m/z 236.9 ((M + H)+).
1
C and method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.17
(d, J = 2.4 Hz, 1H), 8.08 (d, J = 9.2 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H),
7.89 (dd, J = 9.2, 2.4 Hz, 1H), 3.70 (m, 1H), 1.82 (m, 2H), 1.70 (m,
2H), 1.57 (m, 1H), 1.46 (m, 2H), 1.31 (m, 2H), 1.12 (m, 1H). MS
(ESI+): m/z 295.1 ((M + H)+).
3-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-7-
chlorobenzo[e][1,2,4]triazine 1,4-Dioxide (15bn). Synthesized
from 4-chloro-2-nitroaniline using method C and method A. Red solid.
1H NMR (DMSO-d6, 400 MHz): δ 8.18 (dd, J = 2.4, 0.4 Hz, 1H), 8.08
(dd, J = 9.6, 0.8 Hz, 1H), 7.90 (dd, J = 9.2, 2.4 Hz, 1H), 7.85 (m, 1H),
3.62 (m, 1H), 2.28 (d, J = 4.0 Hz, 1H), 2.22 (br s, 1H), 1.69 (m, 2H),
1.56 (m, 1H), 1.47 (m, 2H), 1.14 (m, 3H). MS (ESI+): m/z 307.1
((M + H)+).
7-Bromo-3-(ethylamino)benzo[e][1,2,4]triazine 1,4-Dioxide
(15ca). Synthesized from 4-bromo-2-nitroaniline using method C
and method A. Red solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.41 (t, J
= 6.4 Hz, 1H), 8.32 (m, 1H), 8.01 (m, 2H), 3.40 (m, 2H), 1.17 (t, J =
7.2 Hz, 3H). MS (ESI+): m/z 286.9 ((M + H)+).
7-Bromo-3-(cyclobutylamino)benzo[e][1,2,4]triazine 1,4-Di-
oxide (15 cd). Synthesized from 4-bromo-2-nitroaniline using
method C and method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.58 (m, 1H), 8.31 (m, 1H), 8.02 (m, 2H), 4.32 (m, 1H),
2.32 (m, 4H), 1.67 (m, 2H). MS (ESI+): m/z 312.9 ((M + H)+).
7-Bromo-3-(cyclopentylamino)benzo[e][1,2,4]triazine 1,4-
Dioxide (15ce). Synthesized from 4-bromo-2-nitroaniline using
method C and method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.32 (t, J = 1.2 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.01 (m,
2H), 4.16 (m, 1H), 1.92 (m, 2H), 1.70 (m, 4H), 1.56 (m, 2H). MS
(ESI+): m/z 326.9 ((M + H)+).
3-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-7-
bromobenzo[e][1,2,4]triazine 1,4-Dioxide (15cn). Synthesized
from 4-bromo-2-nitroaniline using method C and method A. Red
solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.33 (t, J = 1.2 Hz, 1H), 8.02
(m, 2H), 7.89 (m, 1H), 3.62 (m, 1H), 2.30 (m, 1H), 2.23 (m, 1H),
1.72 (m, 2H), 1.59−1.40 (m, 3H), 1.23−1.01 (m, 3H). MS (ESI+):
m/z 352.9 ((M + H)+).
3-(Cyclobutylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-Di-
oxide (15ad). Synthesized from 4-fluoro-2-nitroaniline using method
1
C and method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.43
(d, J = 8.0 Hz, 1H), 8.15 (dd, J = 9.6, 5.2 Hz, 1H), 7.93 (dd, J = 8.8,
2.8 Hz, 1H), 7.83 (m, 1H), 4.30 (m, 1H), 2.23 (m, 4H), 1.67 (m, 2H).
MS (ESI+): m/z 250.9 ((M + H)+).
3-(Cyclopentylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-Di-
oxide (15ae). Synthesized from 4-fluoro-2-nitroaniline using method
1
C and method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.14
(dd, J = 9.6, 5.2 Hz, 1H), 7.97 (m, 1H), 7.94 (dd, J = 8.4, 2.8 Hz, 1H),
7.83 (m, 1H), 4.13 (m, 1H), 1.91 (m, 2H), 1.67(m, 4H), 1.55 (m,
2H). MS (ESI+): m/z 265.0 ((M + H)+).
7-Fluoro-3-((1-methoxybutan-2-yl)amino)benzo[e][1,2,4]-
triazine 1,4-Dioxide (15ag). Synthesized from 4-fluoro-2-nitroani-
1
line using method C and method A. Red solid. H NMR (DMSO-d6,
400 MHz): δ 8.16 (dd, J = 9.6, 5.2 Hz, 1H), 7.95 (dd, J = 9.2, 2.8 Hz,
1H), 7.85 (m, 2H), 3.95 (m, 1H), 3.49 (dd, J = 10, 6.4 Hz, 1H), 3.39
(dd, J = 9.6, 5.2 Hz, 1H), 3.23 (s, 3H), 1.61 (m, 2H), 0.86 (t, J = 7.2
Hz, 3H). MS (ESI+): m/z 283.0 ((M + H)+).
3-(Cyclohexylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-Di-
oxide (15aj). Synthesized from 4-fluoro-2-nitroaniline using method
1
C and method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.14
(dd, J = 9.2, 5.2 Hz, 1H), 7.95 (dd, J = 8.8, 2.8 Hz, 1H), 7.91 (m, 1H),
7.83 (m, 1H), 3.69 (m, 1H), 1.84 (m, 2H), 1.71 (m, 2H), 1.58 (m,
1H), 1.51−1.41 (m, 2H), 1.35−1.25 (m, 2H), 1.12 (m, 1H). MS (ESI
+): m/z 279.0 ((M + H)+).
3-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-7-
fluorobenzo[e][1,2,4]triazine 1,4-Dioxide (15an). Synthesized
from 4-fluoro-2-nitroaniline using method C and method A. Red
3-(Ethylamino)-7-methoxybenzo[e][1,2,4]triazine 1,4-Diox-
ide (15da). Synthesized from 4-methoxy-2-nitroaniline using method
1
C and method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.08
1
solid. H NMR (DMSO-d6, 400 MHz): δ 8.15 (dd, J = 9.6, 4.8 Hz,
(t, J = 6.0 Hz, 1H), 8.04 (d, J = 9.6 Hz, 1H), 7.57 (dd, J = 9.6, 2.8 Hz,
1H), 7.46 (d, J = 2.4 Hz, 1H), 3.90 (s, 3H), 3.38 (m, 2H), 1.17 (t, J =
7.2 Hz, 3H). MS (ESI+): m/z 237.0 ((M + H)+).
3-(Cyclobutylamino)-7-methoxybenzo[e][1,2,4]triazine 1,4-
Dioxide (15dd). Synthesized from 4-methoxy-2-nitroaniline using
1H), 7.95 (dd, J = 9.2, 2.8 Hz, 1H), 7.84 (m, 1H), 7.74 (d, J = 7.2 Hz,
1H), 3.61 (m, 1H), 2.28 (d, J = 4.0 Hz, 1H), 2.22 (br s, 1H), 1.69 (m,
2H), 1.56 (m, 1H), 1.45 (m, 2H), 1.12 (m, 3H). MS (ESI+): m/z
291.0 ((M + H)+).
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method C and method A. Red solid. 1H NMR (DMSO-d6, 400 MHz):
δ 8.25 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 9.6 Hz, 1H), 7.57 (dd, J = 9.2,
2.8 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 4.30 (m, 1H), 3.90 (s, 3H), 2.22
(m, 4H), 1.67 (m, 2H). MS (ESI+): m/z 263.0 ((M + H)+).
3-Chlorobenzo[e][1,2,4]triazine 1,4-Dioxide (16). Synthesized
from 9 by method E. Yellow solid. H NMR (CDCl3, 300 MHz): δ
1
8.55 (d, J = 8.5, 1H), 8.48 (d, J = 8.6, 1H), 8.07 (t, J = 8.5 Hz, 1H),
7.90 (t, J = 8.6 Hz, 1H). MS (ESI+): m/z 198.0 ((M + H)+).
3-Chloro-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-Di-
oxide (19). Synthesized from 18 by method E. Yellow solid. 1H NMR
(CDCl3, 300 MHz): δ 8.34 (s, 1H), 8.26 (s, 1H), 3.17 (q, J = 7.8 Hz,
4H), 2.27 (quin, J = 7.8, 2H). MS (ESI+): m/z 238.0 ((M + H)+).
3-(Diethylamino)benzo[e][1,2,4]triazine 1,4-Dioxide (17o).
3-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-7-
methoxybenzo[e][1,2,4]triazine 1,4-Dioxide (15dn). Synthesized
from 4-methoxy-2-nitroaniline using method C and method A. Red
1
solid. H NMR (DMSO-d6, 400 MHz): δ 8.04 (d, J = 9.6 Hz, 1H),
7.56 (m, 2H), 7.46 (d, J = 2.4 Hz, 1H), 3.90 (s, 3H), 3.61 (m, 1H),
2.30 (d, J = 4.0 Hz, 1H), 2.23 (br s, 1H), 1.69 (m, 2H), 1.57−1.35 (m,
3H), 1.23−1.09 (m, 3H). MS (ESI+): m/z 303.0 ((M + H)+).
Ethyl-(5-methyl-1,4-dioxy-benzo[1,2,4]triazin-3-yl)-amine
(15ea). Synthesized from 6-methyl-2-nitroaniline using method C and
1
Synthesized from 16 using method F. Red solid. H NMR (CDCl3,
300 MHz): δ 8.36 (d, J = 7.4 Hz, 1H), 8.31 (d, J = 7.4 Hz, 1H), 7.85
(t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H), 3.81 (q, J = 6.9 Hz, 4H),
1.32 (t, J = 7.0 Hz, 6H). MS (ESI+): m/z 235.0 ((M + H)+).
3-Morpholinobenzo[e][1,2,4]triazine 1,4-Dioxide (17p). Syn-
1
method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.34 (t, J =
1
thesized from 16 using method F. Red solid. H NMR (CDCl3, 300
6.4 Hz, 1H), 8.05 (dq, J = 8.8, 0.8 Hz, 1H), 7.60 (m, 1H), 7.38 (m,
1H), 3.40 (p, J = 6.8 Hz, 2H), 2.95 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H).
MS (ESI+): m/z 221.0 ((M + H)+).
MHz): δ 8.39 (d, J = 8.7 Hz, 1H), 8.35 (d, J = 9.6 Hz, 1H), 7.90 (t, J =
7.0 Hz, 1H), 7.65 (t, J = 7.0, 1H), 3.91 (m, 4H), 3.87 (m, 4H). MS
(ESI+): m/z 249.1 ((M + H)+).
3-(Cyclobutylamino)-5-methylbenzo[e][1,2,4]triazine 1,4-Di-
oxide (15ed). Synthesized from 6-methyl-2-nitroaniline using
method C and method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.43 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.59 (dt, J
= 7.2, 1.2 Hz, 1H), 7.38 (m, 1H), 4.31 (m, 1H), 2.96 (s, 3H), 2.27−
2.17 (m, 4H), 1.68 (m, 2H). MS (ESI+): m/z 247.0 ((M + H)+).
3-(Cyclopentylamino)-5-methylbenzo[e][1,2,4]triazine 1,4-
Dioxide (15ee). Synthesized from 6-methyl-2-nitroaniline using
method C and method A. Red solid. 1H NMR (DMSO-d6, 400
MHz): δ 8.05 (m, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.59 (dt, J = 7.2, 1.2
Hz, 1H), 7.38 (dd, J = 8.8, 7.2 Hz, 1H), 4.15 (m, 1H), 2.96 (s, 3H),
1.94 (m, 2H), 1.68 (m, 4H), 1.57 (m, 2H). MS (ESI+): m/z 261.0
((M + H)+).
3-(Pyrrolidin-1-yl)benzo[e][1,2,4]triazine 1,4-Dioxide (17q).
1
Synthesized from 16 using method F. Red solid. H NMR (CDCl3,
300 MHz): δ 8.31 (d, J = 5.8 Hz, 2H), 7.84 (t, J = 5.8, 1H), 7.49 (t, J =
5.8 Hz, 1H), 4.04 (m, 4H), 1.98 (m, 4H). MS (ESI+): m/z 233.0 ((M
+ H)+).
3-(Pyrrolidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]-
triazine 1,4-Dioxide (20q). Synthesized from 19 using method F.
Red solid. 1H NMR (CDCl3, 300 MHz): δ 8.11 (s, 1H), 8.10 (s, 1H),
4.03 (t, J = 6.6, 4H), 3.07 (t, J = 7.3 Hz, 2H), 3.04 (t, J = 7.5 Hz, 2H),
2.19 (quin, J = 7.4 Hz, 2H), 1.98 (m, 4H). MS (ESI+): m/z 273.0 ((M
+ H)+).
3-(Piperidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]-
triazine 1,4-Dioxide (20r). Synthesized from 19 using method F.
Red solid. 1H NMR (CDCl3, 300 MHz): δ 8.15 (s, 1H), 8.11 (s, 1H),
3.76 (m, 4H), 3.05 (m, 4H), 2.18 (quintet, J = 7.5 Hz, 2H), 1.69 (m,
6H). MS (ESI+): m/z 287.0 ((M + H)+).
3-(Dimethylamino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]-
triazine 1,4-Dioxide (20s). Synthesized from 19 using method F.
Red solid. 1H NMR (CDCl3, 300 MHz): δ 8.15 (s, 1H), 8.10 (s, 1H),
3.31 (s, 6H), 3.05 (quint, J = 6.9 Hz, 4H), 2.15 (quint, J = 7.3 Hz, 2H).
MS (ESI+): m/z 247.0 ((M + H)+).
3-(Ethylamino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine
1,4-Dioxide (15fa). Synthesized from 6-nitro-2,3-dihydro-1H-inden-
5-amine using method D and method A. Red solid. 1H NMR (CDCl3,
400 MHz): δ 8.11 (s, 1H), 8.07 (s, 1H), 6.94 (br-t, 1H), 3.59 (p, J =
7.2, 2H), 3.09 (t, J = 6.7, 2H), 3.03 (t, J = 7.7, 2H), 2.20 (p, J = 7.5,
2H), 1.33 (t, J = 7.3, 3H). MS (ESI+): m/z 247.0 ((M + H)+).
Cyclobutyl-(5,8-dioxy-2,3-dihydro-1H-5,6,8-triaza-
cyclopenta[b]naphthalen-7-yl)-amine (15fd). Synthesized from
6-nitro-2,3-dihydro-1H-inden-5-amine using method D and method A.
3-(Benzyl(methyl)amino)-7,8-dihydro-6H-indeno[5,6-e]-
1
Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.32 (d, J = 8.0 Hz,
[1,2,4]triazine 1,4-Dioxide (20t). Synthesized from 19 using
1H), 7.99 (br s, 1H), 7.95 (br s, 1H), 4.32 (m, 1H), 3.07−2.97 (m,
4H), 2.22 (m, 4H), 2.07 (m, 2H), 1.67 (m, 2H). MS (ESI+): m/z
273.0 ((M + H)+).
1
method F. Red solid. H NMR (CDCl3, 300 MHz): δ 8.24 (s, 1H),
8.16 (s, 1H), 7.25−7.35 (m, 5H), 5.1 (s, 2H), 3.17 (s, 3H), 3.12
(quint, J = 7.6 Hz, 4H), 2.21 (quint, J = 7.6 Hz, 2H). MS (ESI+): m/z
323.0 ((M + H)+).
3-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-7,8-dihydro-
6H-indeno[5,6-e][1,2,4]triazine 1,4-Dioxide (15fn). Synthesized
from 6-nitro-2,3-dihydro-1H-inden-5-amine using method D and
3-(Methyl(phenethyl)amino)-7,8-dihydro-6H-indeno[5,6-e]-
[1,2,4]triazine 1,4-Dioxide (20u). Synthesized from 19 using
1
method A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.00 (br
1
method F. Red solid. H NMR (CDCl3, 300 MHz): δ 8.09 (s, 1H),
s, 1H), 7.94 (br s, 1H), 7.63 (d, J = 6.8 Hz, 1H), 3.63 (m, 1H), 3.07−
2.97 (m, 4H), 2.30 (d, J = 4.0 Hz, 1H), 2.24 (br s, 1H), 2.07 (m, 2H),
1.70 (m, 2H), 1.57 (m, 1H), 1.49 (m, 2H), 1.15 (m, 3H). MS (ESI+):
m/z 313.0 ((M + H)+).
8.05 (s, 1H), 7.21 (d, J = 7.4 Hz, 2H), 7.11 (t, J = 5.2 Hz, 2H), 7.00 (t,
J = 7.4 Hz, 1H), 4.16 (t, J = 7.6 Hz, 2H), 3.24 (s, 3H), 2.97−3.11 (m,
6H), 2.18 (quint, J = 7.6 Hz, 2H). MS (ESI+): m/z 337.0 ((M + H)+).
3-(Thiomorpholinosulfone-1-yl)-7,8-dihydro-6H-indeno[5,6-
e][1,2,4]triazine 1,4-Dioxide (20v). Synthesized from 19 using
3-(Ethylamino)-6-methoxy-7-methylbenzo[e][1,2,4]triazine
1
1,4-Dioxide (15ga). Synthesized from 5-methoxy-4-methyl-2-nitro-
method F. Red solid. H NMR (CDCl3, 300 MHz): δ 8.17 (s, 1H),
1
aniline using method D and method A. Red solid. H NMR (DMSO-
8.14 (s, 1H), 4.35 (m, 2H), 3.26 (m, 2H), 3.12 (m, 2H), 2.23 (quint, J
= 7.4 Hz, 2H). MS (ESI+): m/z 337.0 ((M + H)+).
d6, 400 MHz): δ 8.17 (t, J = 6.0 Hz, 1H), 7.99 (d, J = 1.2 Hz, 1H), 7.33
(s, 1H), 4.00 (s, 3H), 3.42−3.36 (m, 2H), 2.26 (d, J = 0.8 Hz, 3H),
1.17 (t, J = 7.2 Hz, 3H). MS (ESI+): m/z 251.0 ((M + H)+).
3-(Cyclobutylamino)-6-methoxy-7-methylbenzo[e][1,2,4]-
triazine 1,4-Dioxide (15gd). Synthesized from 5-methoxy-4-methyl-
3-(2-Methylpiperidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e]-
[1,2,4]triazine 1,4-Dioxide (20w). Synthesized from 19 using
1
method F. Red solid. H NMR (CDCl3, 300 MHz): δ 8.16 (s, 1H),
8.12 (s, 1H), 4.18 (t, J = 7.0 Hz, 1H), 4.18 (d, J = 13.2 Hz, 1H), 3.32
(m, 1H), 3.03 (m, 4H), 2.16 (quint, J = 7.3 Hz, 2H), 1.92 (m, 1H),
1.62 (m, 6H), 1.33 (d, J = 7.0 Hz, 3H). MS (ESI+): m/z 301.0 ((M +
H)+).
1
2-nitroaniline using method D and method A. Red solid. H NMR
(DMSO-d6, 400 MHz): δ 8.31 (d, J = 8.4 Hz, 1H), 7.98 (m, 1H), 7.33
(s, 1H), 4.31 (m, 1H), 4.00 (s, 3H), 2.26 (d, J = 0.8 Hz, 3H), 2.22 (m,
4H), 1.67 (m, 2H). MS (ESI+): m/z 277.0 ((M + H)+).
3-(4-(Methylsulfonyl)piperazin-1-yl)-7,8-dihydro-6H-indeno-
3-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-6-methoxy-7-
methylbenzo[e][1,2,4]triazine 1,4-Dioxide (15gn). Synthesized
from 5-methoxy-4-methyl-2-nitroaniline using method D and method
[5,6-e][1,2,4]triazine 1,4-Dioxide (20x). Synthesized from 19 using
1
method F. Red solid. H NMR (CDCl3, 300 MHz): δ 8.15 (s, 1H),
8.12 (s, 1H), 3.95 (m, 4H), 3.42 (m, 4H), 3.07 (quint, J = 5.9 Hz, 4H),
2.79 (s, 3H), 2.17 (quint, J = 7.7 Hz, 2H). MS (ESI+): m/z 366.0 ((M
+ H)+).
Calculation of LUMO Energies. Molecular orbital energy
calculations were performed using the semiempirical quantum
1
A. Red solid. H NMR (DMSO-d6, 400 MHz): δ 8.01 (d, J = 1.2 Hz,
1H), 7.60 (d, J = 6.8 Hz, 1H), 7.32 (s, 1H), 4.00 (s, 3H), 3.63 (m,
1H), 2.33 (m, 1H), 2.27 (s, 3H), 2.24 (br s, 1H), 1.69 (m, 2H), 1.49
(m, 3H), 1.16 (m, 3H). MS (ESI+): m/z 317.0 ((M + H)+).
6057
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chemistry program MOPAC2009.52 The calculation was performed
with the neglect of diatomic differential overlap (NDDO) approx-
imation method with the PM6 parametrization. The geometries of all
compounds were optimized using the default eigenvector following
routine until a self-consistent field was achieved. The energies of the
LUMO for each molecule is approximated by the eigenvalues (in eV)
of the appropriate orbital generated using the EIGEN and VECTORS
keywords.
Bacterial Strains, Growth Conditions, And Chemicals. Mtb
strains were obtained from the American Type Culture Collection
(ATCC, VA) and Colorado State University (CSU) and cultured in
roller bottles (Corning Inc.) using Middlebrook 7H9 broth (Difco
Laboratories) supplemented with 0.2% glycerol, 0.05% Tween-80, and
10% albumin-dextrose-catalase (Difco Laboratories). Middlebrook
7H10 agar (Difco Laboratories), supplemented with 0.2% glycerol and
10% oleic acid-albumin-dextrose-catalase (Difco Laboratories), was
used to visualize colonies. Mtb H37Rv was grown in 7H9 broth at 37
°C to the midlog phase. The antibiotics and resazurin were purchased
from Sigma (St. Louis, MO) and resuspended according to the
manufacturer’s instructions. Experiments performed at the Institute for
Tuberculosis Research at the University of Illinois Chicago (TB MIC-
ITR) were performed according to published methods.11,53
Determination of Antibiotic Susceptibility. To determine the
MIC of compounds against Mtb, the resazurin-based microplate assay
was performed. Briefly, the compounds were resuspended in DMSO
and tested in a range from 10−0.08 mg/L following a 2-fold dilution
scheme. After addition of the bacterial cells ∼105 colony forming unit
(CFU)/mL, the 96-well plates were incubated at 37 °C for 5 d. The
addition of 0.05 mL of 0.1% resazurin followed, with additional
incubation for 2 d at 37 °C. Fluorescence was measured using a
Fluoroskan Ascent or Victor 3 microplate fluorimeter (Thermo
Scientific, USA) with an excitation of 530 nm and emission of 590 nm.
Wells containing compounds only were used to detect autofluor-
escence of compounds. The lowest drug concentration that inhibited
≥90% growth was considered to be the MIC. In addition to the
fluorescence readouts, all of the MIC values are also scored by visual
inspection for confirmation of the MIC value. A 2-fold variation in
MIC was considered to be within the error range of the assay. The
final concentration of DMSO in all wells was 0.625%. These data are
presented in Tables 1−5.
Antimicrobial Activity against NRP Mtb Cells. The antimicro-
bial activity of various compounds against NRP Mtb cells was
determined as described previously using LORA.7 Briefly, Mtb H37Rv
cells were suspended in Middlebrook 7H12 broth and sonicated for 15
s. Cultures were diluted to obtain an OD570 of 0.03−0.05 and 3000−
7000 relative light units (RLU) per 100 μL. Two-fold serial dilutions
of antimicrobial agents were prepared in a volume of 100 μL in black
96-well microtiter plates, and 100 μL of the cell suspension were
added. The microplate cultures were placed under anaerobic
conditions (O2 <0.16%) using an Anoxomat model WS-8080
(MART Microbiology) using three cycles of evacuation and filling
with a mixture of 10% H2, 5% CO2, and the balance N2. An anaerobic
indicator strip was placed inside the chamber to visually confirm the
removal of O2. Plates are incubated at 37 °C for 10 d and then
transferred to an ambient gaseous condition (5% CO2-enriched air)
incubator for a 28-h “recovery”. On day 11 (after the 28-h aerobic
recovery), 100 μL of culture were transferred to white 96-well
microtiter plates for determination of luminescence. The MIC was
defined as the lowest drug concentration effecting an inhibition of
∼90% relative to drug-free controls.
of the Cell Titer-Glo reagent followed, with mixing the contents for 2
min on an orbital shaker to induce cell lysis and further incubation at
room temperature for 10 min to stabilize the luminescent signal. The
plates were read and the luminescence was recorded. Each test plate
contained a set of controls including media only (for background
subtraction) and 0.625% DMSO controls (for calculating the percent
viability for all test wells). In addition, each test plate contained an
ATP standard curve, which was used to calculate the ATP units in each
test well. The toxicity was defined as the concentration at which host
cells are 50% viable (IC50).
Mouse Lymphoma Cell tk+/−→tk−/− Gene Mutation Assay.
Experiments were conducted using a standard procedure for evaluating
mutagenic potential.23,24 The details on cell line, experimental
procedures, and data interpretation have been previously published.54
The following end points were evaluated in cells following exposure to
15fa and 20q: cell growth during expression periods, relative
suspension growth, relative total growth, relative cloning efficiency,
mutation frequency, and numbers of small (≤0.6 mm in diameter)
colonies from trifluorothymidine-resistant (TFTr) cells.
Solubility Assays. Compounds were ground into a fine powders
using a mortar and pestle and added to a 25 mL glass Erlenmeyer flask
with 5 mL of a 0.9% saline solution at pH 7.4. Approximately 20 mg of
compound was added to each flask to ensure saturation. The flasks
were then vigorously stirred using a magnetic stir bar at 25 °C for 48 h.
The samples were then filtered through a Whatman PVDF membrane
(0.45 μm pore size) syringe filter. The concentration of compound in
the supernatant was then measured using HPLC quantitation at the
maximum absorption wavelength for each compound. The integration
was then fit to a standard curve generated for each compound (R2
values >0.99) to determine the equilibrium solubility for each
compound.
Pharmacokinetic Studies in Female CD1Mice. All pharmaco-
kinetic studies were performed in accordance with SRI International’s
animal care policies in an AAALAC and OLAW accredited facility. The
procedure for the pharmacokinetics studies followed a previously
described method.55 Briefly, the plasma pharmacokinetics of selected
BTO derivatives were determined in female CD1 mice after
administration of a single dose (100 mg/kg) by oral gavage. Blood
was collected from three mice per time point at 5, 15, 30, and 60 min
and 2, 4, 6, 8, and 24 h after dose administration. Means and standard
deviations were calculated for the plasma drug concentrations at each
time point. Pharmacokinetic analysis was performed using non
compartmental methods (WinNonlin Professional, version 5.2,
Pharsight Corp, Mountain View, CA). The following parameters
were calculated: time to maximum plasma concentration (Tmax),
maximum plasma concentration (Cmax), maximum plasma concen-
tration extrapoloated to time 0 (C0), and area under the plasma
concentration−time curve to the last time point (AUClast) and to
infinity (AUCinf).
ASSOCIATED CONTENT
■
S
* Supporting Information
Spectrum of antimicrobial activity for BTOs; experimental
method for the salmonella/microsome plate incorporation
assay (Ames screen); evaluation of 15fa and 20q in the
salmonella/microsome plate incorporation assay (Ames screen)
This material is available free of charge via the Internet at
http://pubs.acs.org.
Cytotoxicity against Vero Cells. The compounds were tested for
cytotoxicity against Vero cells using the Cell Titer-Glo luminescent cell
viability assay (Promega). The Vero cells (Vero ATCC CCL-81) were
grown and maintained in minimal essential medium (MEM) + 0.25%
fetal bovine serum (FBS). Compound dilutions were done in
accordance with Clinical and Laboratory Standards Institute (CLSI)
guidelines in 96-well plates. Each well contained 5 μL of compound
and 95 μL of host Vero cells at a concentration of ∼5 × 104 cells/mL.
Plates were incubated for 72 h at 37 °C + 5% CO2, followed by
equilibration of the plate to room temperature for ∼30 min. Addition
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-650-859-2253. Fax: 1-650-859-3153. E-mail: peter.
madrid@sri.com.
Present Address
§Scientist-Fellow, Department of Biotechnology, CSIR-Central
Institute for Medicinal and Aromatic Plants, P.O. CIMAP,
Lucknow-226015, India
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Article
(8) Compounds 1−6 were obtained through the National Institute of
Health, National Cancer Institute, Developmental Therapeutics
Program, 2003.
Notes
The authors declare no competing financial interest.
(9) Dirlam, J. P.; Presslitz, J. E.; Williams, B. J. Synthesis and
antibacterial activity of some 3-[(alkylthio)methyl]quinoxaline 1-oxide
derivatives. J. Med. Chem. 1983, 26, 1122−1126.
ACKNOWLEDGMENTS
■
The project described was supported by award R56AI090817
from the National Institute of Allergy and Infectious Diseases.
The content is the sole responsibility of the authors and does
not necessarily represent the official views of the National
Institute of Allergy and Infectious Diseases or the National
Institutes of Health. Some of the antimicrobial screening was
also supported by the National Institutes of Health and the
National Institute of Allergy and Infectious Diseases, contract
HHSN266200600011C (N01-AI-60011).
(10) 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 cytotoxins
with antitumor activity. Anti-Cancer Drug Des. 1998, 13, 575−592.
(11) Cho, S. H.; Warit, S.; Wan, B.; Hwang, C. H.; Pauli, G. F.;
Franzblau, S. G. Low-oxygen-recovery assay for high-throughput
screening of compounds against nonreplicating Mycobacterium tuber-
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(12) Ioerger, T. R.; Feng, Y.; Ganesula, K.; Chen, X.; Dobos, K. M.;
Fortune, S.; Jacobs, W. R., Jr.; Mizrahi, V.; Parish, T.; Rubin, E.;
Sassetti, C.; Sacchettini, J. C. Variation among genome sequences of
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ABBREVIATIONS USED
■
ATCC, American Type Culture Collection; BTO, 1,2,4-
benzotriazine di-N-oxides; CC50, cytotoxicity concentration;
CDCl3, deuterated chloroform; CD3OD, deuterated methanol;
CFU, colony forming unit; CLSI, Clinical and Laboratory
Standards Institute; CSU, Colorado State University; DOTS,
direct observed therapy short-course; E1/2, one electron
reduction potential; ES-MS, electrospray mass spectra; FBS,
fetal bovine serum; LC-MS-MS, liquid chromatograph tandem
mass spectrometer; LCQ, liquid chromatography quadrupole;
LORA, low-oxygen recovery assay; MDR-TB, multidrug-
resistant TB; MEM, minimal essential medium; MLM, mouse
liver microsome; MOLY, mouse lymphoma cell mutation assay;
Mtb, Mycobacterium tuberculosis; NRP, nonreplicating persis-
tence; RLU, relative light unit; SD, standard deviation; SI,
selectivity index; TB, tuberculosis; TFAA, trifluoroacetic
anhydride; TI, therapeutic index; TPZ, tirapazamine; XDR,
extensively drug-resistant
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Pruijn, F. B.; Anderson, R. F.; Shinde, S. S.; Wilson, W. R.; Denny, W.
A. Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia selective cytotoxins.
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Their Leads in Successful Drug Discovery Programs. J. Med. Chem.
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