Full text of DOI:10.1039/d0md00390e

Volume 12
Number 1
January 2021
Pages 1–146
RSC
Medicinal Chemistry
rsc.li/medchem
ISSN 2632-8682
RESEARCH ARTICLE
Marvin J. Miller et al.
Hydride-induced Meisenheimer complex formation reflects
activity of nitro aromatic anti-tuberculosis compounds

RSC
Medicinal Chemistry
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RESEARCH ARTICLE
Hydride-induced Meisenheimer complex
formation reflects activity of nitro aromatic anti-
tuberculosis compounds†
Cite this: RSC Med. Chem., 2021, 12,
62
Rui Liu,a Lowell Markley,a Patricia A. Miller,a Scott Franzblau,b Gauri Shetye,b
Rui Ma,b Karin Savková,c Katarína Mikušová, c Bei Shi Lee, d Kevin Pethe,de
a
Garrett C. Moraskif and Marvin J. Miller
*
The formation efficiency of hydride-induced Meisenheimer complexes of nitroaromatic compounds is
consistent with their anti-TB activities exemplied by MDL860 and benzothiazol N-oxide (BTO) analogs.
Herein we report that nitro cyano phenoxybenzenes (MDL860 and analogs) reacted slowly and
incompletely which reflected their moderate anti-TB activity, in contrast to the instantaneous reaction of
BTO derivatives to quantitatively generate Meisenheimer complexes which corresponded to their enhanced
anti-TB activity. These results were corroborated by mycobacterial and radiolabelling studies that
confirmed inhibition of the DprE1 enzyme by BTO derivatives but not MDL860 analogs.
Received 16th November 2020,
Accepted 13th December 2020
DOI: 10.1039/d0md00390e
rsc.li/medchem
hydride from a deazaflavin cofactor attacks the carbon
adjacent to the nitro group of PA824 to eventually trigger
Introduction
Nitroaromatic compounds have a rich history chemically,
biologically and microbiologically.1 While concerns are often
raised about potential toxicity of nitroaromatics, a number
are important therapeutic agents.2 Antibiotics like
metronidazole (1), nitrofurantoin (2) and chloramphenicol (3)
have been listed as essential medicines for treatment of
bacterial and fungal infections despite some complications.
Others, including nitro furans,3 and nitroimidazoles, like
PA824 (4)4 have anti-tuberculosis activity. In fact, PA824, now
called pretomanid, has recently been approved by the FDA for
use in combination with bedaquiline and linezolid for
treatment of select patients with extensively drug resistant
tuberculosis.5 Nitrobenzothiazinones like BTZ043 (5) and
pBTZ169 (6) have outstanding anti-tuberculosis activity and
are currently in advanced clinical trials.6 In both PA824 and
the BTZs, the nitro group plays an essential role. Biological
release of nitric oxide (NO) as the active agent.7 The
nitrobenzothiazinones (BTZ043 and pBTZ169) also are
prodrugs that are reductively activated to give a nitroso
moiety (7) that forms a covalent adduct (8) with an essential
cysteine of decaprenylphosphoryl-β-D-ribose 2′oxidase (DprE1)
that is essential for cell wall biosynthesis in Mycobacterium
tuberculosis (Mtb), the causative agent for tuberculosis.8–10
A
number of related electron deficient nitro aromatic
compounds have recently been shown to have a similar mode
of anti-TB activity.11–15
Detailed mechanistic understanding is still needed about
exactly how the nitro groups of the nitrobenzothiazinones
and related nitro aromatic compounds are reduced to the
important nitroso moieties. It is well known that the
essential DprE1 enzyme system is coupled to NADH/NAD
redox chemistry,8–10 but specifically how that important
biological chemistry proceeds remains of great interest. We
have previously shown that reactions of BTZ043 (5) with
nucleophiles like thiolates, cyanide and hydride proceeds
rapidly by cine attack ortho and para to the nitro group to
generate intermediate Meisenheimer complexes (9a, 9b eqn
(1)).16 The fate of the intermediate depends on the
nucleophile. Most relevant to anti-tuberculosis activity was
the observation that reactions with chemical hydride (NaBH4)
produced isolable reduction product 9b that is the same as
that obtained metabolically from BTZ043 (5).17 This is
reminiscent of the first step in the reductive activation of
PA824 through Michael addition of hydride at the carbon
adjacent to the nitro group (cine reaction). These
a Department of Chemistry and Biochemistry, University of Notre Dame, Notre
Dame, IN 46556, USA. E-mail: mmiller1@nd.edu
b Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at
Chicago, 833 South Wood Street, Chicago, Il 60612, USA
c Department of Biochemistry, Faculty of Natural Sciences, Comenius University in
Bratislava, Ilkovičova 6, 84215 Bratislava, Slovakia
d School of Biological Sciences, Nanyang Technological University, Singapore
637551
e Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
636921
f Department of Chemistry and Biochemistry, Montana State University, Bozeman,
MT 59717, USA
† Electronic supplementary information (ESI) available: NMR spectra of newly
synthesized compounds. See DOI: 10.1039/d0md00390e
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Meisenheimer complexes (9a and 9b) require only loss of
water to generate the corresponding, putatively active, nitroso
moiety 7. Reaction of BTZ043 with NaBD4 generates the
corresponding deuterated Meisenheimer complexes and
reoxidation results in net deuteration of the aromatic core.18
These results indicate that enzymatically induced biological
hydride reactions might proceed in a similar fashion to
generate the ultimate nitroso warhead. Thus, consideration
of appropriate combination of molecular recognition
elements and ability to be chemically susceptible to reductive
Meisenheimer complex formation, may be useful for
identification and evaluation of important and targeted
inhibitors (Fig. 1).
Interestingly, reduction of the N-oxide results in compounds
(10, n = 0) that are less toxic (IC50 > 80 μM) and retain good
(MICs as low as 0.1 μg mL−1), but not as potent activity.10,19
Nitro group containing MDL860 (13)20 and analogs also
have been found to react with an essential cysteine residue
that results in antiviral activity.21 MDL860 reportedly reacts
enzymatically with a cysteine thiol resulting in covalent
modification
(14)
from
a
nucleophilic
aromatic
substitution.22 We were intrigued by the similarity of the
aromatic core to that of the nitrobenzothiazinones (BTZ-like),
BTO and related anti-TB nitroaromatics. Herein, we report
that, like BTZ043, MDL860 and representative benzothiazol
N-oxide (BTO) derivatives undergo reversible reaction with
hydride and deuteride to form intermediate Meisenheimer
complexes (i.e. 16a and 16b). The results indicate that while
nitro aromatics based on a benzothiazol N-oxide (BTO)
scaffold (10) and nitro cyano phenoxybenzenes, (MDL860
analogs) reversibly form Meisenheimer complexes upon
reaction with hydride similar to BTZ043, they differ
considerably in their anti-TB activity (Schemes 1 and 2).
Results and discussion
(1)
Reaction of MDL860, 13,23 with NaBD4 generated a red/
orange solution characteristic of Meisenheimer complex
formation. However, compared to the same reaction with
BTZ043 (5), the reaction was visibly slower and the color less
intense. Furthermore, monitoring the reductions by LC/MS
revealed that while the reduction of BTZ043 was essentially
instantaneous with complete conversion of starting material
to the Meisenheimer complex, extended reaction of MDL860
with 5, 10 or even 15 equivalents of NaBD4 gave incomplete
conversion to the Meisenheimer complex. As with the
Recently, compounds based on a benzothiazol N-oxide
(BTO) carboxamide scaffold with the meta nitro and CF3 groups
(10, n = 1, R = amine), also present and important in BTZ043,
are impressively potent anti-TB agents that, again, like BTZ043,
target the essential DprE1 enzyme.19 Detailed studies
demonstrated that just like BTZ043, the nitro group serves as a
prodrug and is first reduced to the corresponding nitroso
moiety 11 that then reacts with the key DprE1 cysteine thiolate
to generate a semimercaptal (12) that results in covalent
inhibition. Although these compounds have remarkable anti-
TB activity (MICs as low as 0.02 μg mL−1), the example
Meisenheimer
complexes
generated
from
BTZ043,
reoxidation of the Meisenheimer complexes (16a, b) from the
mixture with starting MDL860 regenerated the starting
MDL860, but also with deuterium incorporation (17a, b). In
contrast to the reported enzymatic reaction, did not result in
displacement of the dichlorophenol 15. Proton NMR analysis
after reduction with 5 equivalents of NaBD4 followed by
reoxidation revealed only 13% deuterium incorporation at
the two carbons ortho to the nitro group. In contrast, our
previous studies on complete deuteride reduction followed by
reoxidation of BTZ043 resulted in 57% and 24% deuterium
incorporation ortho and para, respectively. Repetition of the
reduction–reoxidation procedure five times on the same
samples of MDL860 and BTZ043 enhanced the amount of
deuterium content to 57% and 53% at the two positions ortho
described had moderate cytotoxicity (IC50
=
24 μM).19
Scheme 1 Nitro substituted benzothiazol-N-oxide reaction with the
Fig. 1 Nitro aromatic antibiotics and anti-tuberculosis agents.
DprE1 enzyme.
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scaffold, and the chemical shifts of the aromatic protons (δ
8.82 and 8.84) are essentially identical to those of BTZ043, it
reacted completely and visibly more quickly with NaBD4 than
did MDL860 to provide BTZ043 comparable vivid orange/red
Meisenheimer complexes. Oxidation of the complexes under
the same conditions reported previously for BTZ043,
regenerated the starting compounds and with deuterium
incorporation (24) of 48% and 61% ortho and para,
respectively, after one reduction – reoxidation cycle and 78%
and 89% after repeating the process five times. These results
were thus more similar to that observed with BTZ043 rather
than with MDL860.
This BTZ043-like hydride induced reactivity and
Meisenheimer complex formation prompted us to synthesize
additional analogs of MDL860, BTOs and the corresponding
BT (nitrone reduced) compounds and determine if they have
anti-TB activity that was reflected by their reactivity. As shown
by the representative example with the piperidine spiro ketal
component (19) of BTZ043 (eqn (2)), a set of MDL860 analogs
(26–36) was synthesized by direct nucleophilic aromatic
substitution reactions of commercially available 4-chloro-3-
cyano nitrobenzene (25) with various amines (Table 1).
Scheme 2 Enzymatic reaction and chemical reduction of MDL860 (13).
to the nitro group in MDL860 and 94% (ortho) and 57%
(para) in BTZ. It should be noted that the chemical shifts of
the aromatic protons of MDL860 and BTZ043 that are
exchanged with deuterium differ. In MDL860, the chemical
shifts of the two protons ortho to the nitro group are δ 8.36–
8.38 and 8.59–8.60 whereas in BTZ043 they are δ 8.78 (para)
and
δ 8.84 (ortho) suggesting differences in electron
deficiency of the corresponding aromatic carbons. Thus, it
became of interest to determine if the inherent difference in
reactivity and deuterium incorporation of BTZ043 and
analogs of MDL860, especially those with similar peripheral
substitution (i.e., compound 26), would have similar or
different anti-TB activity.
(2)
Reagent and conditions: a. N-methylmorpholine, DIPEA,
80 °C; or b. NaOH, DMF, 100 °C.
Based on Scheme 3, we also prepared a small additional
set of BTOs (Scheme 4, 39–41). Subsequent phosphite-
mediated reduction gave representative BT compounds (42
and 43, Scheme 4, Table 2).
Based on literature precedent,24,25 we also synthesized
BTO analogs, including 23 (Scheme 3) which incorporates the
same piperidine acetal component of BTZ043. Again, the key
step was nucleophilic aromatic substitution of 22 with
mercaptoacetamide (21) followed by nitro aldol like
cyclization to give the N-oxide (BTO, 23). As expected, since
the aromatic core of 23 more closely resembles the BTZ043
As noted, the hydride reactions with representatives of the
different classes of nitro aromatic compounds described above
indicated that the BTO/BT compounds visually reacted more
similarly to BTZ043 than did MDL860. This observation
corresponded to LC/MS analyses of the reduction reaction
mixtures. MDL860 (13, Table 1) itself and the separately
synthesized24 BTO ester (45) and unsubstituted amide (46,
Scheme 5) had no to low activity against TB (45, MIC > 5 μM in
7H12 and 4.97 μM in GAS and 46, 2.19 μM in 7H12 and 1.2 μM
in GAS). This was surprising because amide 46 had
demonstrated anticancer activity by inhibition of polo kinase
(PLK1) by reversible addition of an essential cysteine (Cys67) in
the ATP binding site to give a Meisenheimer complex (47).25
Therefore, it was anticipated that incorporation of substituents
to promote molecular recognition by the presumed DprE1
target would provide compounds with activity reflective of their
ease of formation of hydride induced Meisenheimer complex
formation. The anti-TB assay results shown in Tables 1 and 2
are consistent with these expectations.
Indeed, some substituted MDL860 analogs and all BTO
and BT derivatives had moderate to outstanding anti-TB
Scheme 3 Synthesis and reduction reoxidation chemistry of a BTO
with the BTZ043 side chain.
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Table 1 Structures of MDL860 analogs, anti-TB (H37Rv strain) activity and cytotoxicity (Vero cell)
MIC (μM)
IC50 (μM)
Cpd No.
Structure
MDL860
GASc
7H12d
Vero cell
13
45
2
>50
2.4
>50
>100
26a
27a
5
>5
>100
28a
29b
>5
>5
>5
>100
5
29
30b
>5
>5
>100
31b
32a
33a
>5
>5
5
>5
>5
>5
>100
>100
>100
34b
2
>5
>100
35b
36b
3
>5
>5
>100
>100
>5
4
PA-824
0.2
0.08
>100
a
b
c
Reaction condition: N-methylmorpholine, DIPEA, 80 °C. Reaction condition: NaOH, DMF, 100 °C. GAS: glycerol–alanine salts medium.
d
7H12: 7H9 medium + casitone, palmitic acid, albumin, and catalase.
activity. All MDL860 compounds (Table 1) were less active
than either the BTO or BT compounds (Table 2) and, as
might be expected based on their enhanced electrophilic
character, the BTOs were more active than the BT
compounds. Still, among the MDL860 compounds,
compound 26 with the same piperidine spiro ketal as in
BTZ043, was among the most active. Some of the other
compounds (27, 29, 33, 34, 35) with hydrophobic substituents
akin to pBTZ169 (6) also showed moderate activity. Removal
of the Boc protecting group from 35 to give the more
hydrophilic free amine 36, also reduced activity.
Incorporation of more elaborate amide substituents provided
compounds with outstanding anti-TB activity relative to the
ester (45) and simple amide (46). Addition of the BTZ043 like
piperidine spiro ketal (23, 0.24 μM) or more hydrophobic
moieties as in 39 (0.01 μM) and 40 (0.01 μM) or even the
large substituent in 41 (0.03 μM), which is the same as in the
potent anti-TB agent Q203 (44) resulted in dramatic
improvement in activity. Q203 exerts its anti-TB activity as a
Scheme 4 Syntheses of BTO and BT compounds.
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Table 2 Structures of BTO and BT analogs, anti-TB (H37RV strain),
M. vaccae activity and cytotoxicity (Vero cell)
permeability as well as binding. Reduction of the N-oxides to
give 42 and 43, resulted in more than ten-fold reduction of
anti-TB activity, consistent with reduced warhead
electrophilicity. Previous studies of BTO and BT type
compounds are consistent with these results.13 Those same
studies also indicated that while BTO compounds were
generally more active than the corresponding BT analogs, the
BTOs studied were notably cytotoxic. These results also
appear to correlate with the reduction potential of the nitro
aromatic compounds studied. This is also consistent with the
VERO cell studies also shown in Table 2 (compare 39 with 42
and 41 with 43). However, the reduced VERO cell toxicity of
BTO 42 merits further consideration.
While the anti-TB data shown in Tables 1 and 2, seems to
reflect the importance of incorporation of both a strongly
electron deficient warhead and hydrophobic binding/
molecular recognition components to enhance activity, we
also considered it important to determine if the compounds
actually did target and inhibit the DprE1 in mycobacteria.
First we tested the activity of BTZ043 (5), BTO 41, BT 43 and
Q203 (44) against additional strains of mycobacteria since it
is well known that in order for BTZ type compounds to be
active, the target mycobacteria must contain an essential
cysteine in the DprE1 active site. As shown in Table 2, not
only were all four of these compounds very active against
Mtb, but also M. vaccae, which is known to be susceptible to
BTZ043. However, Mycobacterium aurum, which is missing
the essential DprE1 cysteine, was resistant to all compounds
in Table 2 including BTZ043, BT 41 and BTO 43 (MIC = >1
μM, data not shown). This is consistent with all compounds
targeting DprE1 in the cells.
MIC (μM)
IC50 (μM)
Mv
MHc
Mtb
Mtb
7H12a
GASb
Vero cell
Cpd No. Structure
23
0.24
0.23
0.015 79.6
39
40
41
0.01
0.01
<0.001 76.0
0.01
<0.002 <0.001 >100
0.025
0.07
0.02
81.0
42
43
0.7
0.14
0.13
0.008 >100
0.125 >100
0.24
44
0.03
0.007
0.004 >100
To further corroborate this assumption we evaluated the
effects of the selected compounds by metabolic labeling of
Mtb H37Rv. Analysis of the radiolabeled lipids reveals DprE1
related activity as accumulation of trehalose dimycolates and
trehalose monomycolates due to lack of arabinan chains
serving as attachment sites for mycolates in the
mycobacterial cell wall12 (Fig. 2).
5
BTZ043
<0.004 <0.004 <0.001 >100
a
Mv = M. vaccae IMET 10670. 7H12: 7H9 medium + casitone,
b
The anti-TB data in Tables 1 and 2 along with the
radiolabeling studies clearly indicate, that as expected, the
BTO compounds (Table 2) are the most active against Mtb
H37Rv and, like BTZ043 and pBTZ169, inhibit the putative
target DprE1 enzyme in mycobacteria (Fig. 2). Interestingly,
BTO 23, with the BTZ043 piperidine spiro ketal moiety is less
active than the BTO derivatives with peripheral aromatic
substituents. As also reported for related BTO and BT
compounds, reduction of the N-oxide to give representative
BT compounds (42–43) considerably diminished activity but
with retention of DprE1 reactivity (Fig. 2). Although some of
the MDL860 analogs have moderate activity, which is
consistent with their less electrophilic warhead and visibly
slower formation of hydride induced Meisenheimer
complexes, they do not show DprE1 activity (Fig. 2). Despite
the reduced activity, further studies on the potential mode of
activity of the MDL860 analogs is merited, but beyond the
scope of this report.
palmitic acid, albumin, and catalase. GAS: glycerol–alanine salts
medium. c MH: Mueller–Hinton broth.
QcrB inhibitor, not by inhibition of DprE1, but also seems to
require significantly hydrophobic substituents, perhaps for
Scheme
5 Syntheses of BTO ester and unsubstituted amide and
reported reversible formation of Meisenheimer complex with cysteine
67 of oncology target polokinase 1 (PLK1).
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Conclusion
Overall, these results further suggest that design of potent
nitro aromatic anti-TB agents that target the essential DprE1
enzyme requires incorporation of substituents that promote
molecular recognition by the target enzyme as well as
strongly electron deficient warheads capable of rapid reaction
with hydride to form Meisenheimer complexes. These unique
intermediates may serve as precursors to the essential nitroso
moiety that inhibits DprE1 by forming covalent adducts.
Experimental
General methods
Fig. 2 Evaluation of the effects of the selected compounds on Mtb
H37Rv by metabolic radiolabeling. TLC analysis of the lipids from
radiolabeled Mtb H37Rv. Mycobacteria were incubated with [14C]-
acetate and BTZ043 (5) or target compounds at 100 × MIC for 24 h.
TMM, trehalose monomycolates; TDM, trehalose dimycolates; PE –
phosphatidylethanolamine; CL – cardiolipin; Con – control (containing
DMSO).
All solvents and reagents were obtained from commercial
sources and used without further purification unless
otherwise stated. Silica gel (230–400 mesh) was purchased
from Silicycle, Quebec City, Canada. All compounds are
>98% pure by HPLC analysis. All compounds were analyzed
for purity by HPLC and characterized by 1H and 13C NMR
using Bruker 400 MHz, 500 MHz NMR spectrometer.
Chemical shifts are reported in ppm (δ) relative to the
residual solvent peak in the corresponding spectra, and
coupling constants (J) are reported in hertz (Hz). The liquid
chromatography mass spectrum (LC/MS) analyses were
The structural similarity of BTO 41 and BT 43 to Q203 (44)
also prompted us to determine if they might be dual DprE1/
QcrB inhibitors. Thus, BTO (41) and BT (43) were subjected
to an ATP depletion assay in M. bovis BCG wild type and BCG
ΔcydAB as described before.26 In this assay, a QcrB inhibitor
such as Q203 depleted ATP levels much more in the BCG
ΔcydAB background relative to the parental strain (see Fig. 3),
while bedaquiline is equally potent in both strains due to its
action of the FOF1 ATP synthase (Fig. 3). Conversely, both
compounds 41 and 43 deregulated ATP levels at higher doses,
carried out on
a Waters ZQ instrument consisting of
chromatography module Alliance HT, photodiode array
detector 2996, and mass spectrometer Micromass ZQ, using a
3 × 50 mm Pro C18 YMC reverse phase column. Mobile
phases: 10 mM ammonium acetate in HPLC grade water (A)
and HPLC grade acetonitrile (B). A gradient was formed from
which is
a
characteristic of
a
drug targeting cell-wall
10% to 80% of B in 10 min at 0.7 mL min−1
.
synthesis. Thus, despite their structural similarity to Q203,
neither of the two compounds (41 nor 43) appear to target
QcrB, or any other components of the oxidative
phosphorylation pathway. Thus, consistent with previous
reports, the activity of the new BTO and BT compounds is
due to inhibition of DprE1.
Deuterated MDL860 (17a, b) MDL860 (13). Deuterated
MDL860 (17a, b) MDL860 (13), prepared according to the
literature23 was dissolved in 1 mL of THF and diluted with 5
mL of MeOH. To the solution was added 5 equiv. of NaBD4
and the reaction was monitored by LC/MS and TLC. When
MDL860 was totally converted to the regioisomeric mixture of
Meisenheimer complexes,
1
equiv. of diisopropyl
azodicarboxylate (DIAD) was added to the reaction solution.
LC/MS and TLC indicated that the reduction products had re-
oxidized to give partially deuterated MDL860 as a major
product. To this reaction mixture was added 5 equiv. of
NaBD4 (2×) further reduce the partially deuterated MDL860.
Next, 1 equiv. of DIAD was added again to regenerate
MDL860 but with enhanced deuteration. The reduction and
the re-oxidation sequences was repeated
5 times. The
reaction was quenched by addition of acetic acid and
concentrated under reduced pressure. The residue was
purified by silica gel column chromatography eluting with
hexanes/EtOAc to give deuterated MDL860 as a white solid in
40% overall yield for the 5 step process. Deuteration after a
Fig. 3 Evaluation of compound activity against QcrB. The effect of the
test compounds on intracellular ATP levels was tested in M. bovis BCG
1
single redox cycle: H-NMR (CDCl3, 500 MHz) δ 6.95–6.97 (m,
1H), 7.04 (dd, 1H, J1 = J2 = 5 Hz), 7.31 (d, 1H, J = 5 Hz), 7.57
(d, 1H, J = 10 Hz), 8.36–8.38 (m, 0.87 H), 8.59–8.60 (m, 0.87
H). Deuteration after five redox cycles: 1H-NMR (CDCl3, 500
parental strain (a) and
a genetic knockout of cydAB (b). Various
concentrations of 41 are shown in filled circles; 43 shown in crosses;
Q203 shown in hollow triangles; and bedaquiline (BDQ) shown in asterisks.
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MHz) δ 6.95–6.97 (m, 1H), 7.04 (dd, 1H, J1 = J2 = 5 Hz), 7.31
(d, 1H, J = 5 Hz), 7.57 (d, 1H, J = 10 Hz), 8.36–8.38 (m, 0.43
H), 8.59–8.60 (m, 0.47 H).
45.48–45.57, 71.03, 72.54, 106.91, 121.40, 122.40–122.50,
123.57, 127.57, 131.14–131.41, 142.87, 144.39, 145.36, 155.79.
HRMS calculated for C17H17F3N3O6S (M + H+) 448.0761;
found 448.0785.
(S)-1-(2-Methyl-1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-2-
(tritylthio)ethan-1-one (20). To a solution of mercaptoacetic
acid (920 mg, 10 mmol) and triphenylmethanol (2.6 g, 10
mmol) in chloroform (15 mL) was added TFA (0.99 mL, 13
mmol). After stirring at room temperature for 2 h, the
mixture was concentrated, and the residue was purified by
column chromatograph on silica gel eluting with hexanes/
EtOAc (5/1, v/v) to give the trityl protected thioacetic acid as a
colorless oil (2.3 g, 70% yield) that was directly used for
Deuterated BTO (24). Deuterated 24 was synthesized
according to the same method as used for the synthesis of
deuterated MDL860 (17a, b). The product was obtained as a
yellow solid. Deuteration after a single redox cycle: 1H-NMR
(CDCl3, 500 MHz) δ 1.31 (t, 3H, J = 5.0 Hz), 1.87–1.96 (m, 4H),
3.49–3.52 (m, 1H), 3.58–3.62 (m, 2H), 3.87–3.94 (m, 2H),
4.10–4.13 (m, 1H), 4.25–4.32 (m, 1H), 8.82 (s, 0.52H), 8.84 (s,
0.39H). Deuteration after five redox cycles: 1H-NMR (CDCl3,
500 MHz) δ 1.31 (t, 3H, J = 5.0 Hz), 1.86–1.93 (m, 4H), 3.46–
3.51 (m, 1H), 3.58–3.60 (m, 2H), 3.86–3.93 (m, 2H), 4.09–4.11
(m, 1H), 4.25–4.29 (m, 1H), 8.80 (s, 0.22H), 8.83 (s, 0.11H).
1
formation of the corresponding amides. H-NMR (CDCl3, 500
MHz) δ 3.04 (s, 2H), 7.21–7.25 (m, 3H), 7.28–7.32 (m, 6 H),
7.41–7.43 (m, 6H).
To a mixture of the trityl acid (1 mmol, 334 mg) and (S)-2-
methyl-1,4-dioxa-8-azaspiro[4.5]decane (19, 1 mmol, 157 mg)
in 8 mL of DMF was added HATU (1.5 mmol, 570 mg) and
DIPEA (1.5 mmol, 276 μL). The solution was stirred at room
temperature overnight. The reaction was quenched with H2O
and extracted with EtOAc. The EtOAc layer was washed with
brine, dried with Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by column
chromatography on silica gel eluting with hexanes/EtOAc (5/1
General synthetic method to synthesize MDL860 analogs (26–36)
Method A. 2-Chloro-5-nitrobenzonitrile (25, 0.4 mmol, 74
mg) and amines (0.5 mmol) were dissolved in
N-methylmorpholine (4 mL). To the mixture was added
DIPEA (1 mL) and the reaction was stirred at 80 °C. When
the reaction was completed as monitored by TLC, water was
added to quench the reaction, and extracted with DCM. The
DCM layer was collected and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography eluting with DCM/MeOH.
General synthetic method B. 2-Chloro-5-nitrobenzonitrile
(0.4 mmol, 74 mg) and amines (0.5 mmol) were dissolved in
DMF (10 mL). Then NaOH (0.5 mmol, 20 mg) was added to
the solution and the mixture was stirred at 100 °C overnight.
The reaction was diluted with EtOAc and washed with H2O.
The organic layer was combined and concentrated under
reduced pressure. The residue was purified by silica gel
column chromatography eluting with DCM and MeOH.
(S)-2-(2-Methyl-1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-5-
1
to 3/1, v/v) to give 20 as a white solid in 76% yield. H-NMR
(CDCl3, 500 MHz) δ 1.25 (d, 3H, J = 5.0 Hz), 1.54–1.66 (m, 4H),
2.93 (s, 2H), 3.06–3.11 (m, 2H), 3.40–3.45 (m, 1H), 3.48–3.58
(m, 1H), 3.64–3.69 (m, 1H), 4.02–4.06 (m, 1H), 4.17–4.25 (m,
1H), 7.21–7.24 (m, 3H), 7.28–7.31 (m, 6 H), 7.45–7.47 (m, 6H).
(S)-2-Mercapto-1-(2-methyl-1,4-dioxa-8-azaspiro[4.5]decan-
8-yl)ethan-1-one (21). To a solution of 20 (100 mg, 0.2 mmol)
in anhydrous DCM (8 mL) was added TFA (0.2 mL), followed
by the addition of Et3SiH (0.22 mmol, 1.1 equiv., 27 μL) at
room temperature. The mixture was stirred for 30 min and
monitored by LC/MS. Then saturated sodium bicarbonate aq.
solution was added slowly. The organic layer was separated,
and the aqueous layer was extracted with DCM twice. The
combined organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure. Compound 21 was
obtained as an oil which was directly used for the next step
without purification.
nitrobenzonitrile (26). 1H-NMR (CDCl3, 400 MHz) δ 1.30 (d,
3H, J = 4.0 Hz), 1.89–1.95 (m, 4H), 3.50 (t, 1H, J = 8.0 Hz),
3.58–3.64 (m, 4H), 4.10 (q, 1H, J = 4.0 Hz), 4.26–4.29 (m, 1H),
6.96 (d, 1H, J = 8.0 Hz), 8.22 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz),
8.40 (d, 1H, J = 4.0 Hz). 13C-NMR (CDCl3, 400 MHz) δ 18.71,
35.47, 36.62, 49.23, 49.25, 70.98, 72.51, 101.92, 106.53, 117.27,
117.98, 129.13, 131.65, 139.44, 158.61. HRMS calculated for
C15H18N3O4 (M + H+) 304.1292; found 304.1313.
(S)-2-(2-Methyl-1,4-dioxa-8-azaspiro[4.5]decane-8-carbonyl)-
7-nitro-5-(trifluoromethyl)benzo[d]thiazole
3-oxide
(23).
Compound 21 (48 mg, 0.2 mmol) and 2-chloro-1,3-dinitro-5-
(trifluoromethyl)benzene (22, 54 mg, 0.2 mmol) were
dissolved in EtOH (10 mL) at room temperature. Then 10
equiv. of Et3N (2 mmol, 288 μL) was added, and the mixture
was stirred overnight. The color of the solution changed from
colorless to brown. The solution was concentrated to dryness
and the residue was purified by silica gel column
chromatography eluting with hexanes/EtOAc (4/1, v/v) to give
product 23 as a yellow solid in 58% yield. 1H-NMR (CDCl3,
500 MHz) δ 1.30 (t, 3H, J = 5.0 Hz), 1.86–1.96 (m, 4H), 3.47–
3.52 (m, 1H), 3.57–3.61 (m, 2H), 3.85–3.94 (m, 2H), 4.09–4.13
(m, 1H), 4.24–4.31 (m, 1H), 8.81 (s, 1H), 8.83 (s, 1H). 13C-
NMR (CDCl3, 500 MHz) δ 18.67, 34.86, 37.22, 41.47–41.55,
2-((3-Fluorobenzyl)amino)-5-nitrobenzonitrile (27). 1H-
NMR (CDCl3, 400 MHz) δ 4.56 (d, 2H, J = 4.0 Hz), 5.94 (t, 1H,
J = 4.0 Hz), 6.62 (d, 1H, J = 8.0 Hz), 7.01–7.05 (m, 2H), 7.10–
7.13 (m, 1H), 7.34–7.39 (m, 1H), 8.19 (1, dd, J1 = 4.0 Hz, J2 =
8.0 Hz), 8.37 (d, 1H, J = 4.0 Hz). 13C-NMR (CDCl3, 400 MHz) δ
47.22, 96.10, 110.76, 114.12, 114.34, 115.40, 115.61, 115.84,
122.74, 122.77, 130.00, 130.33, 131.08, 138.05, 138.71, 138.78,
153.70, 162.24, 164.70. HRMS calculated for C14H11FN3O2 (M
+ H+) 272.0830; found 272.0830.
2-((4-Methoxybenzyl)amino)-5-nitrobenzonitrile (28). 1H-
NMR (CDCl3, 400 MHz) δ 3.81 (s, 3H), 4.46 (d, 2H, J = 4.0
Hz), 5.77 (t, 1H, J = 4.0 Hz), 6.68 (d, 1H, J = 12.0 Hz), 6.89–
68 | RSC Med. Chem., 2021, 12, 62–72
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6.93 (m, 2H), 7.24–7.26 (m, 2H), 8.19 (1, dd, J1 = 4.0 Hz, J2 =
8.0 Hz), 8.34 (d, 1H, J = 4.0 Hz). 13C-NMR (CDCl3, 400 MHz) δ
47.40, 55.60, 95.78, 110.79, 114.81, 115.92, 127.89, 128.88,
130.02, 130.28, 137.66, 153.76, 159.83. HRMS calculated for
C15H13N3O3 (M + H+) 284.1030; found 284.1030.
2-((1-(4-Chlorophenyl)ethyl)amino)-5-nitrobenzonitrile
(29). 1H-NMR (CDCl3, 400 MHz) δ 1.64 (d, 3H, J = 8.0 Hz),
4.63–4.69 (m, 1H), 5.61 (d, 1H, J = 8.0 Hz), 6.43 (d, 1H, J = 8.0
Hz), 7.23–7.27 (m, 2H), 7.32–7.35 (m, 2H), 8.07 (1, dd, J1 = 4.0
Hz, J2 = 8.0 Hz), 8.34 (d, 1H, J = 4.0 Hz). 13C-NMR (CDCl3, 400
MHz) δ 24.71, 53.59, 96.14, 111.59, 115.80, 127.14, 129.71,
129.88, 130.07, 134.02, 137.92, 140.67, 152.78. HRMS
calculated for C15H13ClN3O2 (M + H+) 302.0691; found
302.0691.
tert-Butyl
4-(4-((2-cyano-4-nitrophenyl)amino)phenoxy)
piperidine-1-carboxylate (35). 1H-NMR (CDCl3, 400 MHz) δ
1.47 (s, 9H), 1.73–1.81 (m, 2H), 1.92–1.97 (m, 2H), 3.32–3.39
(m, 2H), 3.68–3.74 (m, 2H), 4.46–4.52 (m, 1H), 6.84 (d, 1H, J =
12.0 Hz), 6.91–7.00 (m, 2H), 7.16–7.19 (m, 2H), 8.12 (dd, 1H,
J1 = 4.0 Hz, J2 = 8.0 Hz), 8.39 (d, 1H, J = 4.0 Hz). 13C-NMR
(CDCl3, 400 MHz) δ 28.67, 30.68, 40.70, 72.87, 96.19, 112.26,
115.86, 116.23, 117.56, 124.64, 127.20, 129.89, 130.07, 130.15,
138.46, 153.36, 155.05, 156.65. HRMS calculated for
C23H27N4O5 (M + H+) 439.1976; found 439.1976.
5-Nitro-2-((4-(piperidin-4-yloxy)phenyl)amino)benzonitrile
1
(36). H-NMR (DMSO-d6, 400 MHz) δ 1.76–1.85 (m, 2H), 2.05–
2.11 (m, 2H), 3.08 (brs, 2H), 3.25 (brs, 2H), 4.62–4.66 (m, 1H),
6.85 (d, 1H, J = 12.0 Hz), 7.05–7.07 (m, 2H), 7.22–7.24 (m, 2H),
8.14 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz), 8.50 (d, 1H, J = 4.0 Hz),
9.36 (s, 1H). 13C-NMR (DMSO-d6, 400 MHz) δ 27.98, 41.37,
70.04, 96.19, 114.25, 116.63, 117.46, 124.94, 127.02, 130.35,
131.92, 132.50, 137.84, 154.06, 155.35. HRMS calculated for
C18H19N4O3 (M + H+) 339.1452; found 339.1440.
Trityl amides 37. Trityl amides 37 were synthesized
according to the same method as used for the synthesis of 20.
Thioamides 38. Thioamides 38 were synthesized by the
same procedure used for 21. All the reactions were monitored
by LC/MS and the intermediates were directly used for the
next step without purification.
5-Nitro-2-((pyridin-2-ylmethyl)amino)benzonitrile (30). 1H-
NMR (CDCl3, 400 MHz) δ 4.59 (d, 2H, J = 4.0 Hz), 6.71 (d, 1H,
J = 8.0 Hz), 7.07 (brs, 1H), 7.24–7.27 (m, 1H), 7.29 (d, 1H, J =
8.0 Hz), 7.69–7.73 (m, 1H), 8.19 (dd, 1H, J1 = 4.0 Hz, J2 = 8.0
Hz), 8.31 (d, 1H, J = 4.0 Hz), 8.58 (d, 1H, J = 4.0 Hz). 13C-NMR
(CDCl3, 400 MHz) δ 47.80, 95.95, 110.89, 115.93, 121.87,
123.25, 130.14, 130.32, 137.35, 137.49, 149.59, 153.60, 154.34.
HRMS calculated for C13H11N4O2 (M + H+) 255.0877; found
255.0887.
2-(6-Amino-9H-purin-9-yl)-5-nitrobenzonitrile (31). 1H-
NMR (DMSO-d6, 400 MHz) δ 7.57 (s, 2H), 8.12 (d, 1H, J = 8.0
Hz), 8.18 (s, 1H), 8.60 (s, 1H), 8.71 (dd, 1H, J1 = 4.0 Hz, J2 =
8.0 Hz), 9.03 (d, 1H, J = 4.0 Hz). 13C-NMR (DMSO-d6, 400
MHz) δ 111.27, 115.38, 119.12, 129.98, 130.11, 130.23, 140.44,
141.90, 147.57, 150.50, 154.30, 157.13. HRMS calculated for
C12H8N7O2 (M + H+) 282.0734; found 282.0734.
N-Oxides (BTOs) 39–41. N-Oxides (BTOs) 39–41 were
synthesized by the same procedure used for 23.
2-((4-Methoxybenzyl)carbamoyl)-7-nitro-5-(trifluoromethyl)
benzo[d]thiazole 3-oxide (39). Yellow solid in 83% yield. 1H-
NMR (CDCl3, 400 MHz) δ 3.81 (s, 3H), 4.68 (d, 2H, J = 4.0
Hz), 6.89 (d, 2H, J = 8.0 Hz), 7.32 (d, 2H, J = 8.0 Hz), 8.80–
8.81 (m, 1H), 8.84–8.85 (m, 1H), 10.41 (s, 1H). 13C-NMR
(CDCl3, 500 MHz) δ 43.36, 55.55, 114.49, 122.18–122.22,
122.81–122.84, 123.47, 126.87, 129.09, 129.52, 131.35, 131.63,
143.05, 146.13–146.23, 156.46, 159.52. HRMS calculated for
C17H13F3N3O5S (M + H+) 428.0552; found 428.0523.
2-(((2,3-Dihydrobenzofuran-5-yl)methyl)carbamoyl)-7-nitro-
5-(trifluoromethyl)benzo[d]thiazole 3-oxide (40). Yellow solid
in 60% yield. 1H-NMR (CDCl3, 500 MHz) δ 3.20 (d, 2H, J = 10.0
Hz), 4.57 (d, 2H, J = 10.0 Hz), 4.65 (d, 2H, J = 5.0 Hz), 6.74 (d,
1H, J = 10.0 Hz), 7.12–7.14 (m, 1H), 7.24–7.25 (m, 1H), 8.80–
8.81 (m, 1H), 8.84 (s, 1H), 10.39 (t, 1H, J = 5.0 Hz). 13C-NMR
(CDCl3, 500 MHz) δ 29.85, 43.65, 71.64, 109.64, 121.30, 122.17–
122.20, 122.81–122.83, 125.12, 126.86, 128.03, 128.26, 129.03,
131.35, 143.05, 146.17, 146.23, 156.41, 160.12. HRMS calculated
for C18H12F3N3O5S (M + Na+) 462.0368; found 462.0342.
7-Nitro-2-((4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)
benzyl)carbamoyl)-5-(trifluoromethyl)benzo[d]thiazole 3-oxide
2-(Cyclopropylamino)-5-nitrobenzonitrile (32). 1H-NMR
(DMSO-d6, 400 MHz) δ 0.60–0.64 (m, 2H), 0.79–0.84 (m, 2H),
2.55–2.60 (m, 1H), 7.11 (d, 1H, J = 12.0 Hz), 7.86 (s, 1H), 8.21
(dd, 1H, J1 = 4.0 Hz, J2 = 8.0 Hz), 8.37 (d, 1H, J = 4.0 Hz). 13C-
NMR (DMSO-d6, 400 MHz) δ 7.99, 25.79, 94.54, 113.32,
116.69, 130.22, 131.43, 137.00, 156.00. HRMS calculated for
C10H10N3O2 (M + H+) 204.0768; found 204.0768.
5-Nitro-2-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)
1
benzonitrile (33). H-NMR (CDCl3, 400 MHz) δ 3.52–3.54 (m,
4H), 3.68–3.70 (m, 4H), 6.96 (d, 2H, J = 8.0 Hz), 7.03 (d, 1H, J
= 8.0 Hz), 7.52 (d, 2H, J = 8.0 Hz), 8.31 (dd, 1H, J1 = 4.0 Hz, J2
= 8.0 Hz), 8.48 (d, 1H, J = 4.0 Hz). 13C-NMR (DMSO-d6, 400
MHz) δ 47.06, 49.96, 100.42, 114.63, 118.00, 118.84, 126.86,
126.90, 126.93, 129.68, 132.08, 139.38, 153.27, 158.20. HRMS
calculated for C18H16F3N4O2 (M
377.1217.
+
H+) 377.1220; found
2-(4-Benzylpiperidin-1-yl)-5-nitrobenzonitrile (34). 1H-NMR
(CDCl3, 400 MHz) δ 1.44–1.54 (m, 2H), 1.78–1.87 (m, 3H),
2.61 (d, 2H, J = 8.0 Hz), 2.99–3.05 (m, 2H), 3.94–3.97 (m, 2H),
6.93 (d, 1H, J = 8.0 Hz), 7.16 (s, 1H), 7.17–7.18 (m, 1H), 7.19–
7.24 (m, 1H), 7.29–7.32 (m, 2H), 8.19 (dd, 1H, J1 = 4.0 Hz, J2 =
8.0 Hz), 8.38 (d, 1H, J = 4.0 Hz). 13C-NMR (CDCl3, 400 MHz) δ
32.22, 37.84, 43.09, 51.52, 101.44, 117.49, 117.78, 126.41,
128.62, 129.11, 129.35, 131.79, 139.06, 140.03, 158.81. HRMS
1
(41). Orange solid in 44% yield. H-NMR (CDCl3, 500 MHz) δ
1.82–1.90 (m, 2H), 1.93–1.96 (m, 2H), 2.64–2.71 (m, 1H),
2.80–2.85 (m, 2H), 3.80–3.82 (m, 2H), 4.67 (d, 2H, J = 5.0 Hz),
6.94–6.97 (m, 2H), 7.15–7.17 (m, 2H), 7.25–7.28 (m, 2H),
7.29–7.31 (m, 2H), 8.81 (s, 1H), 8.84 (s, 1H), 10.38 (t, 1H, J =
5.0 Hz). 13C-NMR (CDCl3, 500 MHz) δ 33.36, 42.10, 43.43,
50.45, 116.93, 121.27, 122.16–122.19, 122.78, 126.88, 127.60,
128.29, 129.22, 131.32, 131.61, 143.05, 144.84, 146.25, 147.81,
calculated for C19H20N3O2 (M
322.1554.
+
H+) 322.1550; found
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151.50, 156.42. HRMS calculated for C28H23F6N4O5S (M + H+)
641.1294; found 641.1288.
compounds at 100× of the highest desired final concentration
were prepared in dimethyl sulfoxide (DMSO). A 2 μl aliquot
of DSMO stock solution was added to the assay plate
containing either the 7H12 medium (4.7 g 7H9 broth, 1 g
casitone (Bacto), 5 g bovine serum albumin (BSA), 4 mg
catalase and 5.6 mg palmitic acid for 1 L media) or GAS
medium (0.3 g bacto casitone, 0.05 g ferric ammonium
citrate, 4 g K2HPO4, 2 g citric acid anhydrous, 1 g L-alanine,
1.2 g MgCl2 6H2O, 0.6 g K2SO4, 2 g NH4Cl, 10 mL glycerol for
1 L of media). Compounds were then twofold serially diluted
nine times. M. tuberculosis strain H37Rv (ATCC 27294) stock
was thawed, sonicated and diluted in either MABA or GAS
media to get the final inoculum of 3 to 5 × 105 cells per mL.
Bacterial culture was added to the plates and then incubated
for 7 days at 37 °C. Wells that contained only drug and no
bacterial inoculum were used to quantify auto fluorescence
of the compounds. In addition to drug only controls, controls
with bacteria only and medium only were also included per
microtiter plate. After the 7 day incubation at 37 °C, 32.5 μl
of resazurin dye/tween 80 mixture (0.6 mM resazurin dye and
12 μl of 20% Tween 80) was added to each well, and the
plates were incubated for an additional 18 to 24 h at 37 °C.
Fluorescence was measured at 530 nm excitation and 590 nm
emission using a CLARIOstar (BMG LABTECH, Ortenberg,
Germany) plate reader. The MIC was defined as the
minimum concentration required to achieve a 90% reduction
in fluorescence relative to untreated bacterial controls.
Cytotoxicity in mammalian cells. Cytotoxicity was tested
using the Vero cell (ATCC CRL-81) line as previously
described.28 Briefly, eagle's minimum essential medium
(MEM) containing 10% fetal bovine serum (FBS) plus
penicillin and streptomycin was used for culturing Vero cells.
Vero cells were washed with 0.25% trypsin–EDTA 1× solution
in Hanks' balanced salt solution (HBSS; pH 7.4), and
adjusted to the density of 3 to 5 × 105 cells per ml in MEM.
Test compounds were added to the 100 μl of the cell
suspension in a 96 well plate and then incubated at 37 °C for
72 h; visual inspection was performed after 24 h. Then 20 μl
of 0.6 mM resazurin was added into each well and incubated
for 4 h. The fluorescence was measured at excitation/
emission wavelengths of 530/590 nm. The concentration of
test compound effecting a reduction in fluorescence of 50%
relative to untreated cells (IC50) was calculated.
N-(4-Methoxybenzyl)-7-nitro-5-(trifluoromethyl)benzo[d]
thiazole-2-carboxamide (42). Triethyl phosphite (0.12 mmol,
20 μL) was added dropwise to a solution of 39 (0.1 mmol, 42
mg) in EtOH (15 mL). The mixture was heated to reflux for 1
h and monitored by TLC. Upon completion, the solution was
concentrated and the residue was purified by silica gel
column chromatography eluting with hexanes/EtOAc (4/1, v/v)
to give 42 as a yellow solid in 86% yield (35 mg). 1H-NMR
(CDCl3, 500 MHz) δ 3.81 (s, 3H), 4.65 (d, 2H, J = 5.0 Hz),
6.90–6.92 (m, 2H), 7.31–7.34 (m, 2H), 7.66 (t, 1H, J = 5.0 Hz),
8.60–8.61 (m, 1H), 8.73–8.74 (m, 1H). 13C-NMR (CDCl3, 500
MHz) δ 43.83, 55.56, 114.54, 119.79–119.87, 127.58–127.67,
129.04, 129.71, 130.15, 130.43, 135.43, 143.31, 154.89, 158.77,
159.67, 170.63. HRMS calculated for C17H13F3N3O4S (M + H+)
412.0602; found 412.0573.
7-Nitro-N-(4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)
benzyl)-5-(trifluoromethyl)benzo[d]thiazole-2-carboxamide
(43). Compound 43 was synthesized according to the same
method as 42. 1H-NMR (CDCl3, 500 MHz) δ 1.82–1.91 (m, 2H),
1.94–1.98 (m, 2H), 2.65–2.71 (m, 1H), 2.81–2.86 (m, 2H), 3.81–
3.84 (m, 2H), 4.63 (d, 2H, J = 10.0 Hz), 6.97–6.98 (m, 2H),
7.16–7.17 (m, 2H), 7.25–7.31 (m, 2H), 7.67 (brs, 1H), 8.60 (s,
1H), 8.73 (s, 1H). 13C-NMR (CDCl3, 500 MHz) δ 33.36, 42.06,
43.94, 50.39, 116.91, 119.79–119.82, 121.28, 127.46, 127.58–
127.61, 128.28, 129.46, 130.13, 130.41, 135.44, 143.31, 144.79,
147.85, 151.65, 154.90, 158.73, 170.76. HRMS calculated for
C28H23F6N4O4S (M + H+) 625.1357; found 625.1339.
2-(Ethoxycarbonyl)-7-nitro-5-(trifluoromethyl)benzo[d]
thiazole 3-oxide (45). Compound 45 and 46 were synthesized
according to the literature method.25 To a solution of ethyl
2-mercaptoacetate (18, 1 mmol, 120 mg) and 2-chloro-1,3-
dinitro-5-(trifluoromethyl)benzene (22, 1 mmol, 270.55 mg) in
EtOH (6 mL) was added Et3N (1.1 mmol, 153 μL). The mixture
was stirred at room temperature for 3 h and monitored by LC/
MS. When the reaction was completed, the solution was
concentrated to dryness and the residue was purified by silica
gel column chromatography eluting with hexanes and EtOAc
1
(4/1, v/v). Compound 45 was a yellow solid in 45% yield. H-
NMR (CDCl3, 500 MHz) δ 1.49 (t, 3H, J = 10.0 Hz), 4.56 (q, 2H, J
= 10.0 Hz), 8.86 (m, 1H), 8.88 (m, 1H).
2-Carbamoyl-7-nitro-5-(trifluoromethyl)benzo[d]thiazole
3-oxide (46). Compound 45 (0.2 mmol, 67 mg) and
ammonium hydroxide (0.24 mmol, 48 μL) was suspended in
EtOH (8 mL) and stirred for 2 days. The solution was
concentrated, and the desired compound 46 was
M. vaccae and M. aurum susceptibility testing by the broth
microdilution method. Antimycobacterial activity of the
compounds was determined by measuring their minimum
inhibitory concentrations (MIC) using the broth microdilution
method. Each well of a 96-well microtiter plate was filled with
50 μL of sterile Mueller–Hinton (MH) broth. Each test
compound was dissolved in DMSO making a 2 mM solution,
then diluted with sterile MH broth. Exactly 50 μL of the
compound solution was added to the first well of the microtiter
plate and 2-fold serial dilutions were made down each row of
1
recrystallized with methanol. H-NMR (DMSO-d6, 500 MHz) δ
8.88 (m, 1H), 8.90 (brs, 1H), 8.97 (m, 1H), 9.36 (brs, 1H).
Biological assays
MIC versus M. tuberculosis. All experiments with M.
tuberculosis were conducted within a biosafety level 3 (BSL3)
laboratory using a previously described microplate Alamar
blue assay (MABA).27 Briefly, stock solutions of test
the plate.
A preculture of mycobacteria was grown in
mycobacteria preculture broth for 2 days. This was diluted to
McFarland standard 0.5 (1.5 × 108 CFU) with saline. 100 μL of
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the mycobacterial suspension was further diluted with 14.9 mL
of MH broth. Exactly 50 μL of mycobacterial inoculum (1 × 106
CFU mL−1 in MH media) was then added to each well giving a
total volume of 100 μL per well and 5 × 105 CFU mL−1. The plate
was incubated at 37 °C. After 42 h, each well was examined
visually for mycobacterial growth. The MIC was recorded as the
lowest compound concentration required to inhibit growth as
judged by turbidity relative to a row of wells diluted with the
solvent DMSO as a growth control. Ciprofloxacin was included
in a control row at a concentration gradient of 5–0.0025 μg
mL−1. If on visual examination of plate, the turbidity was too
low to be detected then resazurin was added to each well and
the plate was incubated for an additional 14–18 h. Wells were
then examined for color change.
and reported data. K. S. performed radiolabelling DprE1
studies and reported data. K. M. designed and supervised
radiolabelling DprE1 studies, analyzed, interpreted, reported
data and helped to draft the manuscript. B. S. L. performed
target selectivity studies to determine whether the
compounds were dual inhibitor and reported data. K. P.
designed target selectivity studies to determine whether the
compounds were dual inhibitor, analyzed, reported data and
also helped to draft the manuscript. G. M. synthesized
precursor components to molecules, organized data base for
all data and edited the manuscript. M. J. M. conceived of the
project, organized the project and oversaw all collaborative
interactions, guided the syntheses, interpreted all data and
was the primary author of the manuscript.
Conflicts of interest
Analysis of the effects of the studied compounds on lipids of
M. tuberculosis H37Rv
There are no conflicts to declare.
Mtb H37Rv were grown shaking at 37 °C in GAS medium
containing 0.05% tyloxapol until OD = 0.34. Aliquots of 95 μl
were transferred into Eppendorf tubes. Subsequently, the
drugs or DMSO (final conc. of the drugs was 100 × MIC and
DMSO was at 2%) and 0.1 μCi [14C]-acetate [specific activity:
110 mCi mmol−1, American Radiolabeled Chemicals, Inc.]
were added to the tubes. After 24 h the whole cultures were
transferred into 1.5 ml of chloroform : methanol (2 : 1), and
treated for 2 h at 65 °C. The samples were then subjected to
biphasic Folch wash, TLC analysis in CHCl3/CH3OH/H2O
(20 : 4 : 0.5, v/v), and autoradiography (Biomax MR-1 film,
Kodak, 5 days at −80 °C).
Acknowledgements
This work was supported in part by NIH grants R01 AI054193
and R37 AI054193 and the University of Notre Dame (George &
Winifred Clark professorship, M. J. M). K. M. acknowledges
support by Ministry of Education, Science, Research and Sport
of the Slovak Republic (contract 0395/2016 for Slovak/Russian
cooperation in science 2015-15075/33841:1-15E0) and the Slovak
Research and Development Agency (project APVV-15-0515).
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