Thiolates chemically induce redox activation of BTZ043 and related potent nitroaromatic anti-tuberculosis agents

Article abstract of DOI:10.1021/ja311058q

The development of multidrug resistant (MDR) and extensively drug resistant (XDR) forms of tuberculosis (TB) has stimulated research efforts globally to expand the new drug pipeline. Nitroaromatic compounds, including 1,3-benzothiazin-4-ones (BTZs) and related agents, are a promising new class for the treatment of TB. Research has shown that the nitroso intermediates of BTZs that are generated in vivo cause suicide inhibition of decaprenylphosphoryl- β-d-ribose 2′ oxidase (DprE1), which is responsible for cell wall arabinogalactan biosynthesis. We have designed and synthesized novel anti-TB agents inspired from BTZs and other nitroaromatic compounds. Computational studies indicated that the unsubstituted aromatic carbons of BTZ043 and related nitroaromatic compounds are the most electron-deficient and might be prone to nucleophilic attack. Our chemical studies on BTZ043 and the additional nitroaromatic compounds synthesized by us and others confirmed the postulated reactivity. The results indicate that nucleophiles such as thiolates, cyanide, and hydride induce nonenzymatic reduction of the nitro groups present in these compounds to the corresponding nitroso intermediates by addition at the unsubstituted electron-deficient aromatic carbon present in these compounds. Furthermore, we demonstrate here that these compounds are good candidates for the classical von Richter reaction. These chemical studies offer an alternate hypothesis for the mechanism of action of nitroaromatic anti-TB agents, in that the cysteine thiol(ate) or a hydride source at the active site of DprE1 may trigger the reduction of the nitro groups in a manner similar to the von Richter reaction to the nitroso intermediates, to initiate the inhibition of DprE1.

Full text of DOI:10.1021/ja311058q

Article
pubs.acs.org/JACS
Thiolates Chemically Induce Redox Activation of BTZ043 and Related
Potent Nitroaromatic Anti-Tuberculosis Agents
Rohit Tiwari, Garrett C. Moraski, Viktor Krchnak, Patricia A. Miller, Mariangelli Colon-Martinez,
̌
́
Eliza Herrero, Allen G. Oliver, and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
S
* Supporting Information
ABSTRACT: The development of multidrug resistant
(MDR) and extensively drug resistant (XDR) forms of
tuberculosis (TB) has stimulated research efforts globally to
expand the new drug pipeline. Nitroaromatic compounds,
including 1,3-benzothiazin-4-ones (BTZs) and related agents,
are a promising new class for the treatment of TB. Research
has shown that the nitroso intermediates of BTZs that are generated in vivo cause suicide inhibition of decaprenylphosphoryl-β-
D-ribose 2′ oxidase (DprE1), which is responsible for cell wall arabinogalactan biosynthesis. We have designed and synthesized
novel anti-TB agents inspired from BTZs and other nitroaromatic compounds. Computational studies indicated that the
unsubstituted aromatic carbons of BTZ043 and related nitroaromatic compounds are the most electron-deficient and might be
prone to nucleophilic attack. Our chemical studies on BTZ043 and the additional nitroaromatic compounds synthesized by us
and others confirmed the postulated reactivity. The results indicate that nucleophiles such as thiolates, cyanide, and hydride
induce nonenzymatic reduction of the nitro groups present in these compounds to the corresponding nitroso intermediates by
addition at the unsubstituted electron-deficient aromatic carbon present in these compounds. Furthermore, we demonstrate here
that these compounds are good candidates for the classical von Richter reaction. These chemical studies offer an alternate
hypothesis for the mechanism of action of nitroaromatic anti-TB agents, in that the cysteine thiol(ate) or a hydride source at the
active site of DprE1 may trigger the reduction of the nitro groups in a manner similar to the von Richter reaction to the nitroso
intermediates, to initiate the inhibition of DprE1.
the benzothiazinones (BTZ), as represented by benzothiazi-
none 043 (BTZ043, 3, Figure 1).11,12 The mode of action and
detailed structure−activity relationships of 2 have been
intensively studied in a number of laboratories around the
world, and it is now accepted that 2 is really a prodrug that
delivers nitric oxide (NO) as the active agent from the essential
nitro group.10 The chemistry is initiated through conjugate
addition with a hydride from a deazaflavin cofactor F420
present in deazaflavin-dependent nitroreductase (Ddn) to the
electron-deficient aromatic imidazole that induces a reductive
process (see Figure 2).13,14
BTZ043 has been shown to exhibit exceptional anti-TB
activity in vitro and in vivo.11 It is also a prodrug, which, upon
activation, results in the suicide inhibition of decaprenylphos-
phoryl-β-D-ribose 2′ oxidase (DprE1) of Mtb.15,16 Additional
studies substantiated the importance of the nitro group by
demonstrating that nitroreductase-producing mycobacteria
were resistant to 3.17,18 Chemical reduction of the nitro
group to the hydroxylamine or completely to the corresponding
amine also resulted in drastic loss of activity.11,15,16 Recent
enzymatic studies with DprE1 obtained from Mycobacterium
smegmatis (DprE1SM) with the BODIPY-X-labeled analogue of
3 (compound 4, Figure 3) suggest that the presence of the
INTRODUCTION
■
Tuberculosis (TB) has plagued the human race throughout
history.1 In 1882, when Robert Koch published his landmark
finding that TB was caused by Mycobacterium tuberculosis2
(Mtb), he also noted that 1/7 of all humans died of TB! Today,
1
approximately /3 of all people are infected with Mtb, making
TB persistently prevalent.3 Each year, about 8 million more are
infected and 2−3 million people die of TB. Heroic efforts
during the golden age of antibiotics in the 20th century led to
the discovery of several anti-TB agents that temporarily
stemmed the tide of lethal TB infections. While no single
drug has emerged that is completely effective against Mtb, when
given under carefully monitored conditions, combinations of
antibiotics have saved countless lives. Still, the number of
people being infected by Mtb grows every year as the increase
in global population far outweighs the slow reduction in its
incidence.4 The situation has been exacerbated by the
emergence of multidrug resistant (MDR) and extensively
drug resistant (XDR) strains.5 Thus, while the need for effective
treatment of TB is dire, no new anti-TB drug has been
marketed in decades and new TB drug discovery remains a
formidable task.6,7
Among the very potent and most intriguing leads related to
the discovery of new anti-TB agents are new nitroaromatic
compounds, including a series of nitrofurans 1;8 nitro-
imidazoles, such as the extensively studied PA824 (2);9,10 and
Received: November 14, 2012
Published: February 13, 2013
© 2013 American Chemical Society
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Figure 1. Representative structure of nitro aromatics as a class of anti-TB agents.
Crystal structures of DprE1 with BTZ043 and related
analogues were recently reported.20,21 The study reported by
Neres et al.21 substantiated the formation of the “semimercaptal
adduct” at the active site of DprE1 from reaction of the active-
site cysteine with the nitroso intermediate derived from 3 and
concomitant oxidation of FADH2 to FAD. However, the
mechanistic details related to the reduction of the nitro group
of BTZ043 to the nitroso intermediate are yet to be fully
elucidated. Specifically, how does FADH2, produced from FAD
in DprE1, which is an oxidase (or dehydrogenase), initiate such
reduction? Or if a reactive nitroso agent is generated by
exogenous reductases, would it survive long enough to react
with DprE1? Studies to address these questions will facilitate
the rational design, syntheses, and biological evaluations of new
anti-TB agents. Herein, we report that nitroaromatic
compounds, the design of which are based on BTZ043 and
other related nitroaromatic compounds, react directly with
thiolates and similarly basic and nucleophilic cyanide as well as
hydrides to initiate redox chemistry that results in reduction of
aromatic nitro groups to nitroso intermediates. The studies
described here suggest that a cysteine thiolate or a hydride at
the active site of DprE1 might induce the reduction of the nitro
group of BTZ043 to the nitroso intermediate.
Figure 2. Mechanism of antitubercular action of PA824.
RESULTS AND DISCUSSION
■
Intuitively, the structure of 3 suggests that it could be a
substrate for classical nucleophilic aromatic substitution
(Scheme 1) with the essential cysteine of DprE1. Therefore,
3 might simply be thought of as a thiol trap similar to the
Sanger reagent, 2,4-dinitrofluorobenzene, or 2,4-dinitrobenze-
nesulfonamides that react with thiols at the carbon bearing the
sulfonyl group and eventually release SO2 (see Supporting
Information, Scheme S1). The latter process has evolved into
the eventual use of related sulfonamides as amine-protecting
groups22 and most recently as a prodrug strategy for
development of new anti-TB agents that release SO2 as the
active agent.23
Figure 3. Proposed mechanism of DprE1 inhibition by BTZ-BODIPY-
X.16 (where BODIPY-X is X-substituted boron dipyrromethene).
DprE1SM substrate, decaprenylphosphoryl-β-D-ribosefuranose
(DPR), at the active site is important for the reduction of the
nitro group of 4 into the nitroso derivative. The authors
hypothesize that the reduced flavin adenine dinucleotide
(FADH2) generated during the oxidation of DPR to
decaprenylphosphoryl-D-2′-keto-erythro-pentofuranose (DPX)
is responsible for the nitro reduction of 4 into the nitroso
derivative (5), which, in turn, reacts with the essential cysteine
of DprE1SM to form a covalent semimercaptal adduct (6).16
DprE1 is an important enzyme required for arabinan
biosynthesis, an essential process for the cell wall assembly of
mycobacteria.19
However, several separate observations were inconsistent
with the standard nucleophilic aromatic substitution mecha-
nism as the mode of action for 3 at DprE1. First, the
compounds are remarkably selective and thus not general
arylating agents. Second, no such direct nucleophilic aromatic
Scheme 1. BTZ043 Possibly Acting As a Thiol Trap at the DprE1 Active Site
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substitution products of 3 or its analogues have been
reported.11,15,16 Prior oxidation of the sulfur of BTZ043 to
the sulfoxide or sulfone, followed by nucleophilic substitution at
the sulfur-bearing carbon, has been ruled out by isolation of the
previously mentioned semimercaptal.21 Additionally, Mulliken
charges calculated by the AM1 method for 3 indicate that the
unsubstituted aromatic carbons are electron-deficient, whereas
the aromatic carbon adjacent to sulfur is electron-rich. As a
result, nucleophilic attack at this position is unlikely (Figure 4;
see Supporting Information for the Mulliken charges for all the
atoms).
Scheme 2. Design and Synthesis of Nitroaromatic Model
Compounds Based on BTZ043 and Other Nitrobenzamides
Figure 4. Key Mulliken charges for 3.
Other nitroaromatic compounds without the sulfur, and
especially meta-trisubstituted nitrobenzamides, were found to
have significant anti-TB activity.24−26 This was initially thought
to be surprising as they were structurally different from BTZs,
yet they were subsequently found to inhibit the same molecular
target, DprE1.17,25 The combination of these studies and
observations suggests that the molecular mode of action of 3
and simpler nitrobenzamides do not involve the simple
nucleophilic aromatic substitution, such as the one shown in
Scheme 1, at the active site of DprE1 with the thiol of the
essential cysteine.
In order to explore reactions of these nitroaromatic anti-TB
agents with thiols, we designed and synthesized simplified
nitrobenzamides 11 and 12 that can be considered to be
hybrids of 3 and the nitrobenzamides (7),17,24−26 as the latter
were found to have the same target as 3.17,25 Briefly, the
synthesis involved the 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDC) -mediated coupling of nitrobenzoic
acids, 8 and 9 with (S)-2-methyl-1,4-dioxa-8-azaspiro[4.5]-
decane (10) to obtain 11 and 12, respectively (Scheme 2). For
additional comparative studies, we also synthesized simple
nitrobenzamides 14 and 15 (Scheme 2). Compound 15 was
recently discovered to have anti-TB activity via high-throughput
screening.15
The similarity among the model and the prototype
nitroaromatics, such as 3 and 7, is emphasized in Scheme 2,
and we anticipated that studies of these model compounds,
which do not have a built-in leaving group for classical
nucleophilic aromatic substitution, would help determine if
they are reactive with thiols.
Before studying the reaction with thiols, we evaluated the
synthesized compounds (11, 12, 14, and 15) for their
antimycobacterial activity in vitro (see Supporting Information,
Table S1). As anticipated, dinitro compounds 11 and 14 were
more active than the mononitro analogues 12 and 15, against
Mycobacterium vaccae and MtbH37Rv [minimum inhibitory
concentration (MIC) = 0.8 μM for 11 and 0.25 μM for 14 vs
MIC = > 50 μM for 12 and 0.95 μM for 15 in 7H12 medium).
Although the purpose of this study was to determine inherent
chemical reactivity of nitroaromatic compounds to help discern
possible modes of biological activity, it was heartening to see
that none of these compounds was found to be toxic as judged
by VERO toxicity assay (see Supporting Information Table S1
for VERO toxicity data). Nitroaromatic compounds are often
avoided in medicinal chemistry because of potential toxicity
concerns; however, studies of 2 and 3 indicated that these
compounds have remarkably clean profiles, yet they are very
potent and selective anti-TB agents. Similarly, the biological
assays in H37Rv coupled with VERO toxicity data obtained for
11, 12, 14, and 15 indicated that these compounds have good
safety profiles and are potential leads for development of anti-
TB agents. When 3, 11, and 12 were exposed to cell cultures of
M. smegmatis for 24 h, 3 and 12 were found to be completely
reduced to the corresponding amines 16 and 18, respectively,
whereas 11 was found to be monoreduced to 17 (Scheme 3).
None of the above compounds was reduced in the non-
mycobacterial control experiments in cell culture medium.
These experiments indicate that the nitro groups present in 3,
11, and 12 undergo similar metabolic transformations in M.
smegmatis cell cultures and thus may have common target and
inactivating enzymes.
We then investigated the reaction between the hybrid
compounds and a series of thiols in both organic and mixed
aqueous solvents under neutral conditions. Interestingly,
prolonged treatment of 11, 12, 14, or 15 with methanethiol
and glutathione induced no reaction. However, upon addition
of sodium methanethiolate (NaSMe) to separate solutions of
11 and 15, we observed an immediate color change to a dark
red solution, reminiscent of the formation of a Meisenheimer
complex during classical nucleophilic aromatic substitution
reactions, even though neither 11 nor 15 contains a typical
leaving group.
In order to investigate the chemistry further, we carried out
liquid chromatography coupled to mass spectrometry (LC/
MS) studies of the reaction between 11 and NaSMe (Scheme
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corresponding to starting material, partially reduced compound
(compound 17), and possibly adducts with methanethiol
(compounds 20−23) and the corresponding dimeric forms of
the nitroso intermediate (azoxy analogue, compound 19)
generated in situ from 11 (see Supporting Information for the
LC/MS spectra).
Scheme 3. Cell Culture Experiments of Compounds 3, 11
and 12 with M. smegmatis
The 1H NMR of the crude reaction mixture after
neutralization and normal extractive workup indicated proton
signals corresponding to compound 19, which was later
1
confirmed by its isolation and characterization. The H NMR
spectra also indicated the presence of compound(s) with
substitution at the aromatic carbon between the two nitro
groups, indicating that some of the product(s) formed may be
similar to the thiol adducts shown in Scheme 4. Subsequent
chromatography of the crude material did not allow isolation of
pure samples of any of the compounds with a molecular weight
corresponding the thiol adducts, such as compounds 20−23.
As mentioned above, azoxy compound 19 was isolated and
fully characterized. Azoxy compounds like 19 are well-known to
be formed by dimerization of aryl nitroso species followed by
loss of a single oxygen. In fact, many electron-deficient aryl
nitroso compounds are known to exist in equilibrium with their
dimers,27 but subsequent loss of a single oxygen prohibits re-
formation of the monomeric nitroso agent. This process has
plagued applications of nitroso cycloaddition and ene
chemistry.28,29 It can be concluded from this study that
NaSMe induced the redox chemistry of electron-deficient
nitroaromatic compound as verified by the formation of
compound 19. This induction of the redox chemistry by
thiolates might mimic the reaction of nitroaromatic compounds
such as 3 with DprE1, provided that cysteine at the active site is
appropriately reactive (ionized).
4). The LC/MS analysis revealed generation of a complex
mixture containing compounds with molecular weights
Scheme 4. Probable Structures of Molecular Ions Found in
LC/MS Analysis of the Reaction between 11 and Sodium
a
Methanethiolate
The pKa of alkylthiols is typically in the range 10.5−11 and
that of the thiol of cysteine is 10.8, but as expected, substituents
influence thiol acidity.30 Incorporation of a β-hydroxy group on
ethanethiol to give mercaptoethanol reduces the pKa value to
9.5, and in cysteine ethyl ester, the thiol pKa is 9.05. The
biochemical reactivity of many functional groups in enzymes
depends dramatically on their states of protonation. Ionization
depends on the inherent pKa of the groups and the
microenvironment created by the enzyme.30 The pKa values
of functional groups found in the active sites of many enzymes
often are significantly affected, with changes of frequently >2
pKa units.30 Thus, it was exciting to consider that the thiol of
the essential cysteine residue of DprE1 enzyme may be
significantly ionized and/or nucleophilic in its microenviron-
ment to initiate redox chemistry on 3 similar to that observed
for the reaction of 11 with methanethiolate.
There are some indications in the literature regarding the
possible role of thiolate or thiols in the reduction of nitro
groups to the corresponding nitroso intermediates. Leblanc et
al.31 in their studies on the action of thiolate anion on
nitrofluoraromatics observed that the reaction of m-fluoroni-
trobenzene with methanethiolate resulted only in the formation
of the 3,3′-difluoroazoxybenzene, possibly via reduction of the
nitro to nitroso intermediate. Sodium dithionate,32 sodium
trimethylsilanethiolate,33 chlorotrimethylsilane in combination
with sodium sulfide,34 dihydrolipoamide in combination with
ferrous ion,35 and thioacetate36 and ethanethiol27 have all been
shown to reduce nitroaromatics to either aniline or amides
(reductive amidation), presumably via the formation of nitroso
and/or hydroxylamine intermediates.33−36 Sulfides such as
hydrogen sulfide, ammonium sulfide, sodium hydrosulfide, and
a
See Supporting Information for LC/MS spectra.
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Scheme 5. Reaction of 11 with Boc-Cysteamine
Scheme 6. Trapping of Nitroso Intermediate Generated in the Reaction of 11 and 3 with Boc-Cysteamine
disulfide ion have been used in the Zinin reduction strategy in
the past to achieve nitroaromatic reduction to anilines and
benzene hydroxylamines.37−41 However, except for a study with
ethanethiol in the presence of sodium hydroxide at 85 °C in
water,27 attempts were not made to either detect or isolate the
nitroso intermediates. The reaction conditions described in our
work are milder and can essentially mimic the enzyme
microenvironment. Thus, it was intuitive to hypothesize that
the cysteine thiol at the active site of DprE1 may initiate the
redox chemistry on 3 to convert it into the nitroso
intermediate, which would then be subsequently attacked by
the same cysteine thiolate to form the covalent semimercaptal
adduct. This hypothesis explains only the change in the
oxidation state of nitro group of 3 to the corresponding nitroso
intermediate. It does not, however, explain the corresponding
change in oxidation state of cysteine (thiol), a process that is
addressed later in this report. Nevertheless, this thiolate-
induced reactivity opens up new avenues for the exploration of
molecular mechanism of 3 and other anti-TB nitroaromatics.
While we encourage studies by those who have access to the
enzyme, we extended the fundamental studies with additional
thiolates such as Boc-cysteine, Boc-cysteamine, ethanethiol, and
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thiophenol (see Supporting Information for LC/MS of the
reactions of these thiols with 11). Indeed, simple changes from
the reactions of the same set of previously used thiols under
neutral conditions to basic conditions in mixed organic−
aqueous solvents (apparent pH 9.5−11.5) induced rapid
reaction with model nitroaromatic compounds such as 11
and BTZ043 (3).
For example, reaction of 11 with Boc-cysteamine in
acetonitrile/water at an apparent pH of 11.4 resulted in the
formation of the azoxy product, thus indicating the transient
generation of its predecessor nitroso species. It should be noted
that only two products, 24, which was formed by the oxidation
of the thiol, and the azoxy intermediate 19, were isolated from
the reaction mixture after workup (Scheme 5). Attempts to
isolate and characterize the corresponding semimercaptal
(Scheme 5, compound 25) or other thiol adducts similar to
the ones shown in Scheme 4 were not fruitful because of their
instability.
The reaction of 11 with Boc-cysteine at pH 10−11
essentially duplicated the observations above and only the
Boc-cystine (oxidized cysteine) and nitroso-derived azoxy
intermediates were detected by LC/MS analysis (see
Supporting Information). Incorporation of a smaller group at
the aromatic carbon between the two nitro substituents, such as
a 4-methyl derivative of 11 (compound 39, see Supporting
Information), did not significantly alter the outcome of this
redox chemistry. Thus, reaction of 39 with NaSMe resulted in
formation of a mononitro reduced product and a corresponding
azoxy dimer as judged by LC/MS analyses.
To confirm the generation of the nitroso intermediate in
these reactions, we repeated the reaction of 11 with Boc-
cysteamine under basic conditions (apparent pH 10−12) as a
representative thiolate anion equivalent and with NaSMe in the
presence of α-terpinene and 1,3-cyclohexadiene to trap any
nitroso intermediate as a hetero-Diels−Alder cycloadduct
before it could be converted into the azoxy dimer (Scheme 6,
see Supporting Information for additional reactions of 11 and
Boc-cysteamine in the presence of 1,3-cyclohexadiene). The
LC/MS analyses of these reactions indicated the formation of
the corresponding hetero-Diels−Alder cycloadducts. When
BTZ043 was subjected to similar reaction conditions with
NaSMe in the presence of α-terpinene, formation of the hetero-
Diels−Alder cycloadduct was not detected. Only the reduced
amine 16 and the azo product 27 were detected by LC/MS
analyses, and after 24 h, 3 was completely consumed to afford
16 and 27 (Scheme 6). The formation of the reduced amine
and the azo intermediate is still consistent with the in situ
formation of the nitroso intermediate in the reaction42,27 and
suggests that such reactions proceed by reduction of the nitro
group to the nitroso intermediate prior to formation of the
amine, azoxy, or azo intermediate. It should be noted that the
outcome of the reaction of 11 with NaSMe in presence of 1,3-
cyclohexadiene remained unaltered under deoxygenated
conditions (see Supporting Information for experimental
conditions and LC/MS spectra). Similar results were obtained
when 3 was subjected to reaction with glutathione under basic
conditions in the presence of 1,3-cyclohexadiene (see
Supporting Information for LC/MS spectra). These experi-
ments clearly indicate that thiolates induce redox chemistry of 3
and 11, a process that appears to mimic the redox activation of
BTZ analogues at DprE1 (see Figure 2).
formation of the hetero-Diels−Alder cycloadduct (26)
generated in the reaction of 11 with Boc-cysteamine and verify
that 3 indeed undergoes nitro reduction to the nitroso
intermediate prior to subsequent reduction to amine, we
decided to subject 3 to a reaction with stannous chloride
(SnCl2·2H2O) in the presence of 1,3-cyclohexadiene. The
above reaction produced the expected nitroso cycloadduct 40
(see Supporting Information), which was isolated, purified, and
fully characterized.
While redox chemistry was shown to be induced by reaction
with thiolates, attempts to isolate and individually characterize
the thiol adducts such as the semimercaptal were unsuccessful,
perhaps because of the well-documented instability of the latter
in aqueous solution and under basic conditions.43−45
Several interesting questions still remain, most notably: What
is the fate of the thiol(ate) after the nitro reduction, and what is
the detailed mechanism of the redox chemistry? As mentioned
earlier, direct LC/MS studies of the model reactions indicated
the putative formation of the thiol adducts and the partially
reduced form of the nitro substrates (nitroso equivalents), all of
which were unstable and could not be isolated (Scheme 4). In
the model experiments with Boc-cysteamine and Boc-cysteine,
the corresponding disulfides were detected. While redox
chemistry of thiols in solution would likely induce disulfide
formation, the enzyme and model chemistries can proceed
differently once the redox chemistry is initiated. The enzyme
DprE1 apparently contains a single cysteine thiol at the active
site,11,15 so it is unlikely that a disulfide could form. There is
only one other cysteine, located at ∼15 Å from the cysteine
present at the active site as observed in the X-ray crystal
structure of DprE1, and only a conformational change could
bring these two cysteines in close proximity to form a disulfide
bond. However, oxidized forms such as a sulfenic acid or
semimercaptal would cause the enzyme to be inactivated. To
test for possible formation of sulfenic acid, the reaction between
11 and Boc-cysteamine was carried out with two different
sulfenic acid trapping agents (see Supporting Information),
namely, dimedone46 and 4-chloro-7-nitrobenzofurazan (NBD-
Cl).47 However, reactions of 11 with dimedone were not
conclusive and did not suggest any adduct formed with sulfenic
acid under the reaction conditions, whereas the reaction of 11
with NBD-Cl, as expected, resulted in nucleophilic displace-
ment of the chlorine by Boc-cysteamine (compound 41, see
Supporting Information).
Detailed mechanistic studies are merited, and are ongoing, to
explore this redox chemistry initiated by thiol(ate)s. Thiols and
aminothiols are known to have a dramatic effect on redox
behavior of other nitroaromatic drugs such as metronidazole48
and even influence the enzymatic reduction of nitroimida-
zoles,49 but detailed mechanisms remain elusive. Thiols are
known to react with aromatic nitroso agents to give unstable
thionitroxide radicals [ARN(O•)SR]50 and, as mentioned
earlier, form semimercaptals.44,51,52 Thiols, including gluta-
thione, also react with one-electron reduction products of
nitroaromatic drugs,53 but in some cases the thiol acts as a
reducing agent only after alternative formation of the nitro
radical anion.48
So far, the accumulated data present a strong case for the
redox chemistry between 3, 11, or an analogue of 11 with
thiol(ate)s. To unambiguously synthesize a thiol adduct
corresponding to addition to 11, and to demonstrate that a
related appropriately substituted electron-deficient aromatic
compound can undergo classical nucleophilic aromatic
We have extensively studied nitroso cycloaddition and ene
reactions and their utility in syntheses.28 To substantiate the
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Scheme 7. Reaction of 11 with KCN in CH3CN/H2O
substitution, a 4-chloro derivative of 11 (compound 42, see
Supporting Information) was synthesized and subjected to
reaction with Boc-cysteamine in the presence of an organic
base. As expected, the reaction with Boc-cysteamine generated
only the products of the nucleophilic aromatic substitution
(compound 43, see Supporting Information). No such simple
substitution products were detected by direct LC/MS analyses
of reactions of 11 (which does not contain a classical leaving
group) with either NaSMe or Boc-cysteamine.
Recalling that the Mulliken charge calculations indicated that
the unsubstituted aromatic carbons of 3 are most electrophilic,
we considered an alternative mechanism for generation of
nitroso intermediates based on the well-known von Richter
reaction, which is initiated by cine addition to electron-deficient
aromatic compounds, especially nitroaromatics.54,55 Cine
addition to aromatic nitro compounds that initiates reduction
of the nitro group to a transient, reactive nitroso intermediate
has ample precedent (see the reaction described under ref 54),
which illustrates the mechanism of the classic von Richter
reaction for conversion of p-nitrotoluene I to m-toluic acid III
via formation of 5-methyl-2-nitrosobenzamide II).54,55 Also,
though it has never been referenced as such, the first step of the
prodrug chemistry of PA824 (2) is, in fact, a cine addition. In
the von Richter reaction, the nucleophilic cyanide carbon is
incorporated into the product. Mulliken charges calculated by
the AM1 method for 3 and 11 (Figure 4 and see Supporting
Information) indicated that the unsubstituted aromatic carbons,
which are ortho to the nitro substituents, are electron-deficient.
The electronic character of the nitroaromatic portion of 3 and
model hybrids reported herein appear to make them ideally
suited for cine addition chemistry.
Although 2 and 3 have different targets and different modes
of action, we hypothesize that the molecular mode of action of
the latter also might be initiated by a cine addition of the
essential cysteine sulfhydryl group either ortho or para to its
essential nitro group. Mechanisms can be written to
accommodate this hypothesis and also incorporate generation
of a nitroso intermediate that could lead to covalent
modification of the enzyme target, including generation of
the corresponding semimercaptal (see Supporting Information,
Scheme S3).
To further demonstrate the propensity of the nitroaromatic
compounds toward cine addition chemistry, we explored the
possibility of 11 undergoing a von Richter reaction. Thus, it was
subjected to a reaction with potassium cyanide (KCN) in
acetonitrile/water at room temperature, essentially the same
conditions described for the reactions with thiol(ates). LC/MS
of the reaction mixture indicated the formation of at least three
identifiable reaction products. One of the reaction products
corresponded to a single nucleophilic addition of a cyanide,
whereas the mass of the other two reaction products was
related to double addition of cyanide and hence were isomeric
(Scheme 7). The product corresponding to single cyanide
addition was characterized by X-ray crystallography (see
Supporting Information) and it turned out to be compound
31, whereas only one of the isomeric products related to double
cyanide addition was characterized by X-ray and it turned out
to be compound 32. The formation of 31 and 32 can only be
explained via the formation of the nitroso intermediate, 29,
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which is formed first after the attack of cyanide at the position
between both of the nitro groups (Scheme 7). Nucleophilic
substitutions of hydrogen in electron-deficient nitroarenes have
been reviewed.56,57 Electron-deficient arenes have been shown
to regenerate the aromaticity by loss of hydride after the initial
attack by cyanide.58−60 (see conversion of 11 into 28). Such
loss of hydride may help explain the reduction of 29 to 30,
which eventually led to the formation of 31. When the reaction
was repeated in the presence of α-terpinene, LC/MS of the
crude reaction mixture indicated the product correspondrd to
the trapped hetero-Diels−Alder cycloadduct of the nitroso
intermediate with a cyanide addition (see Supporting
Information). All of the above observations unequivocally
substantiate the fact that cine addition of 11 results in the
generation of nitroso intermediate such as 29. This reaction still
differs from the classical von Richter reaction in that one of the
nitro groups becomes a part of the newly formed isooxazole
ring system and the reaction was carried out at room
temperature.
to induce rearomatization with simultaneous formation of the
disulfide. The aromatized product 36 is at the nitroso oxidation
state and can thus generate nitroso intermediate 37 by
dehydration. In the absence of other reagents, nitroso moieties
are well-known to rearrange to azoxy (38) or azo compounds
(27). The cofactor FADH2 present near the active site of the
enzyme may help regenerate the thiol functionality by acting as
a hydride donor (see Scheme 9 for the regeneration of thiol
from disulfide).
This chemical scheme accounts for the redox chemistry but
not the formation of isolable adducts such as the reported
semimercaptal. The model experiments reported by Makarov
and co-workers11,15 and others illustrated that separately
generated nitroso agents do react with thiols to produce
adducts, as reported by many others in the literature. Recent
additional crystallographic studies with DprE1SM with 3 are
consistent with formation of the semimercaptal adduct.20,21
Our model von Richter reactions on 3 and 11 with KCN
substantiated our proposed mechanism for the conversion of
nitro groups present in these compounds to the putative
nitroso moiety, as demonstrated by the observed products
(Schemes 7 and 8). Specifically, two products from the reaction
of 11 with KCN were also analyzed by X-ray crystallography to
unequivocally support the proposed structures and thereby the
hypothesis behind the mechanism of their formation. Again,
while the chemical mechanism in Scheme 9 is consistent with
the model reactions, it remains to be seen if the only other
cysteine thiolate in the enzyme is available for formation of the
disulfide in a manner similar to that suspected for the chemical
model reactions. However, related redox chemistry, especially
any oxidation of the enzyme’s essential cysteine, would
inactivate the enzyme. Mechanisms can be written that again
start with the cine addition of the active-site cysteine to 3,
followed by oxidative elimination to generate a sulfenic acid
with concomitant formation of the nitroso intermediate of 3
(see Supporting Information, Scheme S3). While attempts to
trap related sulfenic acids with dimedone (which is well-known
to react with sulfenic acids to form characterizable adducts)
failed in the chemical model studies, their formation might have
been circumvented by the rapid formation of disulfide.
Recently, use of labeled dimedone derivatives to detect
generation of trace amounts of sulfenic acids from cysteine
residues in enzymatic reactions has been reported.61−63 This
would be an appropriate study for those with access to the
enzyme system.
Alternatively, the attack of the hydride from FADH2 in a
manner similar to the attack of the thiolates and cyanide
described herein to generate the nitroso intermediate cannot be
ruled out. To explore the possibility of such hydride reduction
from FADH2 of the nitro group to the nitroso intermediate by
cine addition mechanism, we subjected 11 to reaction with
sodium triaceotxyborohydride [NaBH(OAc)3] in the presence
of 1,3-cyclohexadiene in tetrahydrofuran (THF). LC/MS
analysis of the crude reaction mixture indicated the formation
of the corresponding hetero-Diels−Alder cycloadduct similar to
compound 26 described in Scheme 6 (see Supporting
Information for LC/MS). We anticipated that reduction of
the nitro group to the nitroso intermediate prior to formation
of the hetero-Diels−Alder cycloadduct occurred by cine
addition of the hydride and not by direct reduction of the
nitro group by NaBH(OAc)3, as the later reagent is not known
to cause a direct nitro reduction.64 Such reduction of the
nitroaromatic compounds by nucleophilic attack of the hydride
As a further indication of the susceptibility of these types of
compounds to the von Richter reaction, we treated BTZ043
with K13CN under conditions similar to those described for 11
(Scheme 8). As shown in Scheme 8, two major products, 33
a
Scheme 8. Reaction of 3 with K13CN in CH3CN/H2O
a
Asterisk indicates 99% C-13 enrichment.
and 34, were isolated, corresponding to the generation of single
and double cyanide additions. The isolated compounds were
assigned structures on the basis of our previous cyanide
substitution experiments with 11 and 1H and 13C NMR studies
(see Supporting Information).
All of the studies reported herein clearly indicate that the
reaction with thiolates induces redox chemistry in which the
nitro group is unquestionably converted to the nitroso
intermediate, which then undergoes further reactions, including
classical dimerization. Trapping with dienes verified the
generation of the nitroso moiety. Further chemistry of the
nitroso and thiolate is possible. LC/MS and NMR studies of
the reactions of thiolates with nitroaromatic compounds
indicate at least transient formation of adducts corresponding
to the mass of the thiolate combined with the nitroso moiety,
but they seem to be short-lived and not yet amenable to
isolation and full characterization.
A consistent mechanism (Scheme 9) for generation of the
nitroso intermediate invokes initial attack of thiolate in a cine
addition to give 35. In the model chemical reaction, an
additional thiolate can directly attack the thioether sulfur of 35
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Scheme 9. Potential Mechanism for the Reaction of Thiolates with BTZ043 to Initiate Redox Chemistry That Mimics the
Essential Cysteine-Containing Enzymatic Process
has been studied by deuterium labeling experiments in the past
and is known to involve the hydride attack on a carbon atom β
to the nitro group (cine addition).65
work offers an alternate hypotheses for the activation of these
compounds, in that the cysteine thiol(ate) at the active site of
DprE1 may induce nitro reduction to nitroso intermediates,
which subsequently cause inhibition of DprE1. Nevertheless,
FADH2 may also be responsible for this particular reduction, as
demonstrated by our preliminary studies with hydride that are
consistent with the cine addition chemistry described herein.
CONCLUSION
■
The current anti-TB treatment regimen is 40 years old and
suffers from poor patient compliance due to long-term drug
administration, side effects, and treatment costs that led to the
emergence of MDR and XDR strains. There is a growing
demand for new agents effective against TB.66 BTZ043 (3) and
PA 824 (2) have been shown to kill M. tuberculosis in vitro, ex
vivo, and in mouse models of TB. The simple compounds
designed, synthesized, and described here have promising anti-
TB activity and are nontoxic, and our work shows that these
may share a similar mode of action with BTZ043. Our chemical
studies described herein indicate that the unsubstituted
aromatic carbon between the electron-withdrawing groups of
11 and BTZ043 is susceptible to addition of nucleophiles such
as thiolates, cyanide, and even hydrides. Thiolates as such can
be responsible for reduction of the nitro group present in
hitherto-mentioned compounds to the corresponding nitroso
intermediates. The formation of the nitroso intermediates
under such reaction conditions was verified either by isolation
of the hetero-Diels−Alder cycloadducts or by isolation of the
azoxy or azo intermediates. Additionally, reactions of KCN with
11 and BTZ043 gave products whose formation could only be
explained by in situ generation of the nitroso intermediates,
thus verifying that structures such as 11 and BTZ043 are
potential substrates for cine addition chemistry. Therefore, our
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional text, six schemes, and one table with complete
experimental section along with details of all biological assays
and cell culture experiments, Mulliken charges for 3 and 11,
LC/MS spectra and experimental procedures related to nitroso
trapping experiments, details related to sulfenic acid trapping
experiments, and characterization of compounds (PDF);
crystallographic information files (CIF) for compounds 31
and 32. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
mmiller1@nd.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported in part by Grant 2R01AI054193
from the National Institutes of Health (NIH). The same grant
■
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(17) Ribeiro, A. L.; Degiacomi, G.; Ewann, F.; Buroni, S.; Incandela,
M. L.; Chiarelli, L. R.; Mori, G.; Kim, J.; Contreras-Dominguez, M.;
Park, Y. S.; Han, S. J.; Brodin, P.; Valentini, G.; Rizzi, M.; Riccardi, G.;
Pasca, M. R. PLoS One 2011, 6, No. e26675.
supported the antituberculosis assays, which were performed at
the Institute for Tuberculosis Research, College of Pharmacy,
University of Illinois at Chicago, under the direction of
Professor Scott G. Franzblau. We thank Dr. Albert Hinnen of
Alere Technologies GmbH and Alere Inc. for providing
(18) Manina, G.; Bellinzoni, M.; Pasca, M. R.; Neres, J.; Milano, A.;
̌
De Jesus Lopes Ribeiro, A. L.; Buroni, S.; Skovierova,
́ ̌ ́
H.; Dianiskova,
BTZ043 and partial financial support, Dr. Ute Mollmann at
̈
P.; Mikusova, K.; Marak, J.; Makarov, V.; Giganti, D.; Haouz, A.;
̌
́
́
the Leibniz Institute for Natural Product Research and
Lucarelli, A. P.; Degiacomi, G.; Piazza, A.; Chiarelli, L. R.; De Rossi, E.;
Salina, E.; Cole, S. T.; Alzari, P. M.; Riccardi, G. Mol. Microbiol. 2010,
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Infection Biology, Hans Knoll Institute, Beutenbergstrasse
̈
11a, D-07745 Jena, Germany for helpful discussions, and Dr.
Anthony Serianni, University of Notre Dame, for providing
K13CN. M.C.-M. thanks CBBI for financial support. We thank
the Office of the Vice President of Research at the University of
Notre Dame for financial support for the purchase of the
copper microfocus source used in this research. We thank the
University of Notre Dame, especially the Mass Spectrometry
and Proteomics Facility (Bill Boggess, Michelle Joyce, and
Nonka Sevova), which is supported by Grant CHE-0741793
from the National Science Foundation (NSF).
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