Benzoheterocyclic Oxime Carbamates Active against Mycobacterium tuberculosis: Synthesis, Structure-Activity Relationship, Metabolism, and Biology Triaging

Article abstract of DOI:10.1021/acs.jmedchem.1c00707

Screening of a library of small polar molecules against Mycobacterium tuberculosis (Mtb) led to the identification of a potent benzoheterocyclic oxime carbamate hit series. This series was subjected to medicinal chemistry progression underpinned by structure-activity relationship studies toward identifying a compound for proof-of-concept studies and defining a lead optimization strategy. Carbamate and free oxime frontrunner compounds with good stability in liver microsomes and no hERG channel inhibition liability were identified and evaluated in vivo for pharmacokinetic properties. Mtb-mediated permeation and metabolism studies revealed that the carbamates were acting as prodrugs. Toward mechanism of action elucidation, selected compounds were tested in biology triage assays to assess their activity against known promiscuous targets. Taken together, these data suggest a novel yet unknown mode of action for these antitubercular hits.

Full text of DOI:10.1021/acs.jmedchem.1c00707

pubs.acs.org/jmc
Article
Benzoheterocyclic Oxime Carbamates Active against Mycobacterium
tuberculosis: Synthesis, Structure−Activity Relationship, Metabolism,
and Biology Triaging
Renier van der Westhuyzen, Amanda Mabhula, Paul M. Njaria, Rudolf Muller, Denis Ngumbu Muhunga,
̈
Dale Taylor, Nina Lawrence, Mathew Njoroge, Christel Brunschwig, Atica Moosa, Vinayak Singh,
Srinivasa P.S. Rao, Ujjini H. Manjunatha, Paul W. Smith, Digby F. Warner, Leslie J. Street,
and Kelly Chibale*
Cite This: J. Med. Chem. 2021, 64, 9444−9457
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Screening of a library of small polar molecules
against Mycobacterium tuberculosis (Mtb) led to the identification of
a potent benzoheterocyclic oxime carbamate hit series. This series
was subjected to medicinal chemistry progression underpinned by
structure−activity relationship studies toward identifying a
compound for proof-of-concept studies and defining a lead
optimization strategy. Carbamate and free oxime frontrunner
compounds with good stability in liver microsomes and no hERG
channel inhibition liability were identified and evaluated in vivo for
pharmacokinetic properties. Mtb-mediated permeation and metab-
olism studies revealed that the carbamates were acting as prodrugs. Toward mechanism of action elucidation, selected compounds
were tested in biology triage assays to assess their activity against known promiscuous targets. Taken together, these data suggest a
novel yet unknown mode of action for these antitubercular hits.
small polar molecules (Mw 150−350 Da and clog P −1 to 3.5)
against Mtb. One of the hit series identified from this effort was
a novel pyrrolo[3,4-c]pyridine-1,3(2H)-dione series, which we
previously reported.9
INTRODUCTION
■
Tuberculosis (TB), caused by a single infectious pathogen
Mycobacterium tuberculosis (Mtb), represents a serious global
public health concern. The current treatment for drug-sensitive
TB, also called “first-line” TB treatment, was developed over
40 years ago and requires multiple drugs to be taken, often
daily, for 6 to 9 months.1 This drug treatment can cure active
drug-sensitive TB if treatment is completed properly with no
interruptions. However, the emergence of multidrug-resistant
(MDR), extensively drug-resistant (XDR), and so-called totally
drug-resistant (TDR) Mtb strains complicates TB treatment
severely.2−4 New drugs, novel targets, and treatment strategies
are critical to control the TB epidemic.
Among the existing clinically used TB drugs, which occupy a
broad chemical space, are small polar molecules exemplified by
isoniazid (INH) and pyrazinamide (PZA).5 These hydrophilic
molecules occupy a unique chemical space with respect to
molecular weight (<250 Da) and lipophilicity (clog P < 2.5)
and are able to cross the otherwise highly lipophilic
mycobacterial cell-wall to reach their site of action.6,7 This
permeation is apparently facilitated by the presence of
hydrophilic channels that allow access to polar nutrients.8
These observations encouraged researchers at the Novartis
Institute for Tropical Diseases (NITD) to conduct a high-
throughput phenotypic screen of an ∼6000-member library of
In continuation of our efforts to exploit chemical matter
arising from the screen, we now report the exploration of
another hit series containing oxime carbamates (Figure 1).
Gratifyingly, these compounds were also potently active
against a panel of five drug-susceptible clinical Mtb isolates.9
The hits were seemingly selective toward mycobacteria as they
were inactive (MIC > 125 μM) against a panel of 6 ESKAPE
bacterial pathogens (Table S1). These compounds exhibited
good selectivity versus a mammalian cell line as they were not
cytotoxic to Chinese hamster ovary (CHO) cells at
concentrations of 50 μM. This selective antimycobacterial
activity encouraged further progression of these compounds. In
this paper, we report the strategies that were adopted to
explore the in vitro anti-Mtb structure−activity relationship
Received: April 19, 2021
Published: June 17, 2021
https://doi.org/10.1021/acs.jmedchem.1c00707
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 9444−9457
9444

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
glacial acetic acid (AcOH) was slowly treated with sodium
nitrite. The 6-nitro intermediate was converted to the aromatic
amine by reduction under a hydrogen atmosphere followed by
acylation with acetyl chloride under basic conditions to give 4.
Finally, the oxime carbamate was introduced in the presence of
NEt3 by reaction of oximes with dimethylcarbamoyl chloride
to give a single geometric isomer, as revealed by 1H NMR. The
sulfamoyl analogue 8 could be prepared under similar
conditions from 5 and N,N-dimethylsulfamoyl chloride.
We initially set out to explore the SAR of this series (Table
1). The oxime carbamate moiety was required for anti-Mtb
Figure 1. HTS hit compounds 1 and 2 from screening a focused
library. cLog P and TPSA calculated by the ACD Percepta module.
ESKAPE bacterial strains: Enterobacter cloacae (ATCC 700323),
Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC BAA-
1705), Acinetobacter baumannii (ATCC 19606), Pseudomonas
aeruginosa (ATCC 27853), and Staphylococcus aureus (ATCC 25923).
Table 1. Optimization of the Benzoxazole Core
MIC
CHO IC
b50
a
compound
R1
R2
(μM)
(μM)
(SAR), cytotoxicity, solubility, and ADME/pharmacokinetic
(PK) profiles of the series toward identifying a compound for
proof-of-concept studies.
1
5
6
7
8
9
10
11
12
13
14
15
17
18
19
20
CONMe2
H
CONMe2
H
6-Me
6-Me
H
<0.16
>160
0.30
>160
5.0
<0.24
<0.24
<0.24
<0.24
<0.24
<0.24
<0.24
<0.24
<0.24
<0.24
<0.24
5.20
>100
>100
>100
n.d.
H
RESULTS AND DISCUSSIONS
■
SO2NMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
6-Me
4-Me
5-Me
5-Br
6-Br
6-CO2Me
6-CF3
6-Cl
5-Cl
6-F
5-F
6-NO2
6-NHCOMe
n.d.
Chemistry. We attempted to synthesize the 2-benzoxazolyl
acetonitrile intermediates by reacting 2-aminophenol with
malononitrile, as reported by Das and colleagues.10 However,
the yields obtained were in the region of 20%. After several
attempts with different reagents and conditions, a different
protocol was adapted, which increased the yield of 2-
benzoxazolyl acetonitrile to about 69%.11 The starting point
was the activation of malononitrile with absolute ethanol in the
presence of chlorotrimethylsilane (TMSCl) to give a more
reactive 2-cyanoacetimidic acid ethyl ester hydrochloride
intermediate 3 in high yields (Scheme 1). Appropriately
substituted 2-aminophenols were then reacted with 3 under
reflux in dichloromethane (DCM) for about 20 h to give the
corresponding 2-benzoxazolyl acetonitrile-based intermediates
in moderate to high yields. Oxime formation at the methylene
carbon was achieved via a nitrosation reaction, following a
literature protocol.12 In this case, a frozen solution of the
appropriate 2-benzoxazolyl acetonitrile-based intermediate in
>100
>100
n.d.
>100
7.05
46.3
>100
>100
>100
n.d.
46.8
>100
n.d.
21
Rifampicin
INH
0.009
0.14
n.d.
a
14 day Alamar Blue readout against Mtb H37RvMa in GAST/Fe
medium. Cytotoxicity against CHO cells. n.d., not determined.
b
a
Scheme 1. Synthesis of Benzoxazole analogues
a
Reagents and conditions: (a) EtOH, TMSCl, 0 °C, 17 h; (b) DCM, appropriately substituted 2-aminophenol, 25 °C, 10 min then reflux, 20 h; (c)
AcOH, NaNO2, 0−25 °C, 12 h; (d) dimethylcarbamoyl chloride or N,N-dimethylsulfamoyl chloride, triethanolamine, DCM, reflux, 15 h; (e)
dimethylcarbamoyl chloride, MeCN, K2CO3, reflux, 1 h; (f) H2, Pd/C, 15 h; and (g) MeCN, K2CO3, acetyl chloride, 40 °C, 0.5 h.
9445
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. Proposed prodrug-based activity of carbamate-functionalized oxime compounds.
Figure 3. Mtb-mediated in vitro metabolism of benzoxazole-based carbamates 6 and 15. Compounds were incubated in 7H9/OADC (culture
medium) and added to log-phase Mtb H37RvMa culture (live or heat-killed). PARs were calculated by dividing the peak area of the analyte by the
peak area of the internal standard, and all values were calculated as the percentage of the average PAR at time-point zero. Statistical significance was
assessed using one-way ANOVA with Dunnett’s multiple comparison test, comparing against the culture media incubation control at each time
point. *P < 0.05, **P < 0.01, and ***P < 0.001.
activity as the corresponding free oxime 5 resulted in complete
loss of activity. The carbonyl of the carbamate group could be
replaced with a sulfone in compound 8, resulting in a drop in
potency (MIC = 5 μM). The 6-Me group could be removed,
while activity was retained (compound 6 MIC of 0.3 μM).
Compounds incorporating small electron-withdrawing groups
were equipotent to 1. The 4-, 5-, and 6-positions around the
benzoxazole core were tolerant of various electron-withdrawing
groups (F, Cl, Br, ester, and nitro). However, the presence of
an ester group in the 6-position for 13 increased cytotoxicity
(IC50 = 7.05 μM) compared to 1. The single example of an
electron-donating group, such as an amide group 21, caused a
loss in potency.
Mtb-Mediated Permeation and Metabolism. Mtb is
known to possess enzymes that can mediate biochemical
transformations on specific chemical groups of drug molecules
such as ester hydrolysis, addition of alkyl groups, and
reduction−oxidation reactions to name a few, which can
activate prodrugs or be detrimental if the drug molecules are
deactivated.13 Small polar anti-TB drugs, such as INH and
PZA, are prodrugs that are specifically activated within the
mycobacterial cell.14−16 In this regard, we hypothesized that
the carbamate group in our compounds masks the oxime to
improve its Mtb cell-membrane permeability in an analogous
fashion, as described in Figure 2. Once the carbamate crosses
the Mtb cell membrane and is within the bacilli, it can then be
9446
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 4. Carbamate metabolism by Mtb yields the free oxime, while the oxime remains stable. (a) Compound 15 undergoes rapid Mtb-mediated
metabolism, resulting in the accumulation of the free oxime metabolite 16, (b). (c) 16 does not undergo metabolism or spontaneous degradation.
(d) Mtb hydrolase activity hydrolyzes the carbamate to form the oxime. Compounds were incubated in 7H9/OADC growth medium (culture
media) and added to log-phase Mtb H37RvMa culture (live). PAR was calculated by dividing the peak area of the analyte by the peak area of the
internal standard and values calculated as the percentage of the average PAR at time-point zero.
Figure 5. Effect of carbamates and their corresponding free oximes on intracellular Mtb growth. (a) In vitro infected THP-1 macrophages treated
with selected compounds at 10 μM (showing how both oximes and the carbamates fail to control Mtb growth at high concentrations). (b)
Carbamates reduce intracellular growth in comparison to the untreated control. Carbamates (6, 15, and 1) are shown in dark green, maroon, and
dark blue, respectively; corresponding oximes (7, 16, and 5) are shown in light green, red, and light blue, respectively. Statistical significance was
assessed using one-way ANOVA with Dunnett’s multiple comparison test, comparing against the untreated control day 6 post treatment. ns not
significant; *P < 0.05, **P < 0.01, and ***P < 0.001.
enzymatically cleaved through esterase activity to form the free
oxime which then in turn binds to the target protein. This
hypothesis is in part supported by the observation that the free
oxime 7 is completely inactive at the highest concentration
tested, 160 μM.
To determine the potential metabolism of carbamates by
Mtb, we prepared three samples for representative compounds
6 and 15 as follows: (1) Mtb H37RvMa cultures incubated
with the compound of interest at sub-MIC concentration (0.1
μM), over 96 h, (2) similarly cultured, heat-killed Mtb control,
and (3) an equal volume of sterile growth media as a control
incubated with the same compound’s final concentration.
Control incubations were performed using heat-killed cells to
distinguish between enzymatic metabolism and aqueous
degradation. Samples were analyzed using liquid chromatog-
raphy coupled with mass spectrometry (LC−MS/MS), and the
results are represented as the percentage remaining by
comparing the analyte/internal standard peak area ratios
(PARs) at each time point with those at the beginning of
the incubation.
We found that, over time, the relative levels of carbamates
reduced gradually following incubation in culture media,
suggesting that the compounds were susceptible to sponta-
neous aqueous degradation (Figure 3). However, the relative
9447
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
levels reduced much more rapidly in incubations with Mtb live
cultures, with complete loss of compounds by 3 h (the earliest
time-point sampled). In contrast, no significant differences
were detected between culture media and heat-killed cultures.
To further test our prodrug hypothesis, we sought to
determine if the free oxime formed from the metabolism of
carbamates. We incubated compound 15 and its corresponding
oxime 16 in culture media alone and in the presence of live
Mtb (Figure 4). Carbamate hydrolysis was accompanied by the
formation of 16 as the only metabolite, with the same MS
transitions and retention time as a solvent-spiked oxime
compound. The free oxime compound 16 remained stable
when incubated with Mtb. These data confirm our hypothesis
that the carbamates permeated the Mtb cell membrane;
moreover, it seemed that hydrolysis occurs rapidly to form the
oxime which is then presumably the compound species that
reaches the molecular target.
Activity against Intracellular Mtb. We next assessed the
uptake of compounds 1, 6, and 15 and their oximes in Mtb-
infected THP-1 macrophages using a method reported by
Prideaux et al. 2015 and the associated impact on intracellular
bacillary survival.17 Owing to natural compound degradation in
growth media, macrophages were exposed to drug media,
replenished every 24 h to increase the chances of drug cellular
uptake. Analysis of macrophage cell cultures exposed to
compounds at high concentration, greater than 10× MIC for
all carbamates (10 μM), revealed significantly lower analyte
signals compared to media spiked with similar amounts of the
drug (Figure S2). To confirm whether or not this macrophage
uptake translated into effective intracellular killing, we treated
infected macrophages at the same concentration over a 6 day
period. We found that both the carbamates and their free
oxime derivatives failed to control intracellular bacterial
growth, presumably due to poor host cell penetration for the
highly active carbamates (Figure 5a). Even though bacteria
continued to grow, the carbamates did reduce the intracellular
bacterial counts in comparison to the untreated control
(Figure 5b).
Figure 6. Effect of increasing concentration on intracellular Mtb
growth. Increasing extracellular compound concentration up to 80
μM reduced intracellular bacterial growth by approximately 2-log in
comparison to the untreated control. Statistical significance was
assessed using one-way ANOVA with Dunnett’s multiple comparison
test, comparing against the untreated control day 6 post treatment. ns
not significant; *P < 0.05, **P < 0.01, and ***P < 0.001.
with a metabolically more stable group and/or modify the
physicochemical properties of the free oxime to the extent that
it may cross the cell membrane (Figure 2). We endeavored to
find a stable replacement for the oxime group. The oxime was
introduced into the benzothiazole core by reacting with
sodium nitrite using the same procedure as for the benzoxazole
(Scheme 2). Oxime carbamates and sulfamoyl compounds
a
Scheme 2. Synthesis of Benzothiazole analogues
An increase in compound concentrations for the selected
representative compound 15 and its free oxime revealed that
the carbamates could penetrate the host macrophage cells and
reduce intracellular bacterial burden in a dose-dependent
manner with approximately a 2-log reduction at 80 μM in
comparison to untreated cells (Figure 6).
Microsomal Metabolic Stability and Metabolite
Identification. The unusual oxime carbamate moiety
presented us with a significant optimization challenge as it
was anticipated that it would be labile under biological
conditions. Indeed, it was found that the hit compounds
displayed low metabolic stability in microsomal and S9
fractions. Metabolite identification studies showed that 2 was
metabolized extensively in liver microsomes and plasma by
hydrolysis of the carbamate to yield the corresponding oxime
22. This was confirmed using a sample of oxime 22 which had
the same retention time and fragmentation pattern as the
identified metabolite (Figure S1). No other metabolites were
detected. This metabolic reaction occurs even in the absence of
NADPH, indicating contribution by non-CYP450 enzymes.
The metabolic stability of 8 could not be determined due to
low solubility, while a carbonate (R1 = CO2Me) was found to
be chemically unstable.
a
Reagents and conditions: (a) NaNO2, AcOH, 0−25 °C, 12 h; (b)
dimethylcarbamoyl chloride, NEt3, DCM, reflux, 15 h (c) MsCl or
N,N-dimethylsulfamoyl chloride, NEt3, DCM, reflux, 15 h; and (d)
alkyl halide, NEt3, DCM, reflux, rt.
were prepared using the same protocol to give hit compound 2
and sulfamoyl 23. The oxime 22 was reacted with mesyl
chloride to give 24 under the same conditions. Alkyl ethers of
the oxime (25−32) were synthesized by alkylation of the
oxime with various alkyl halides.
Similar to the benzoxazole core, removal of the carbamate
group resulted in complete loss of activity for compound 22
(Table 2). Acyl sulfonamide and sulfone oximes maintained
The poor microsomal stability of the oxime carbamate
prompted a two-fold strategy to replace the carbamate moiety
9448
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 2. Optimization of Acyl Oxime Functionalities
Scheme 3. Synthesis of Benzothiazole analogues
a
Reagents and conditions: (a) NH2OH, NaHCO3, IPA, 65 °C; (b)
NH2OH, Na2CO3, water, EtOH, 75 °C;(c) (1) NH2OH, EtOH,
reflux, (2) Ac2O, PhMe, 120 °C; (d) NaNO2, AcOH, water, rt; (e)
hydrazine hydrate, EtOH, rt; (f) Ac2O or TFA2O Pyr, PhMe, 100 °C;
(g) POCl3, 80 °C; and (h) Lawesson’s reagent, dioxane, 100 °C.
a
14 day Alamar Blue readout against H37RvMa in GAST/Fe
b
DMAP.19 The formed ketones were converted to oximes 52
and 54-60 by reaction with hydroxylamine or O-methylhy-
droxylamine.
medium. Cytotoxicity against CHO cells. n.d., not determined.
some activity, indicating that other potentially labile groups are
also tolerated albeit would likely suffer the same fate as the
oxime carbamates. Alkylation of the oxime with a variety of
alkyl groups led to loss in potency in most cases. Benzyl 29 and
4-picolyl 31 ethers maintained some activity but were
significantly less active compared to 2.
Replacement of the nitrile group with H and Me
substituents resulted in no improvement in activity when
compared to free oxime 22 (Table 3). However, the CF3
replacement did contribute to the observed weak activity.
Replacement of the nitrile group with five-membered
heterocyclic isosteres was next investigated. While oxazole 52
and methyloxadiazoles 40 and 48 were inactive, thiadiazole 49
showed moderate activity. This activity was maintained with
methylthiazole 51. Activity of 49 could be improved to sub-
micromolar levels by replacement of the methyl with a CF3
group in compound 50. Interestingly, a benzothiazole dimer 54
also displayed good activity, while the methyl ether 55 lost all
activity, which was consistent with what was observed with the
oxime carbamates. A six-membered heterocyclic replacement
56 in the form of a pyrazine also maintained some potency, but
unfortunately cytotoxicity had eroded. Six-membered carbo-
cyclic 58 and cyclic alkyl groups 59-60 resulted in complete
loss of activity.
Alternative cores were synthesized similar to earlier
compounds by preparing the oxime from commercially
available nitriles followed by installation of the carbamate
(Scheme 5). 2-Benzothiazoleacetonitrile was transformed to 4-
amino-1,2,5-oxadiazole 67 in a one-pot two-step procedure, as
described in Scheme 5. Furoxane 68 was formed via
cycloaddition of the corresponding nitrile-oxide after chlori-
nation of oxime 34.
With the knowledge that activity could not be maintained
when replacing the acyl group with more metabolically stable
groups, we next investigated whether permeation of the free
oxime could be improved by modification of the nitrile
functionality. Free oximes with the nitrile replaced by H and
Me were prepared by reaction of the commercially available
aldehyde 33 or ketone 35 with hydroxylamine under basic
conditions (Scheme 3) to give a mixture of E/Z isomers.
Similarly, the hydrate 3718 could be converted to the oxime 38
to afford a CF3 replacement of the nitrile as a single isomer.
1,2,4-Oxadiazole 40 was prepared by reaction of 2-
benzothiazoleacetonitrile with hydroxylamine followed by
cyclization with acetic anhydride. Intermediates 45-47 were
synthesized by reacting ester 41 with hydrazine. The hydrazide
was then acylated and converted to oxadiazole and thiadiazoles
by reaction with POCl3 and Lawesson’s reagent, respectively.
These benzothiazole methylene compounds were then
converted to oximes using NaNO2 in acetic acid, which
yielded a mixture of E/Z isomers in most cases. Benzothiazole
was transformed to the required ketones by reacting with acid
chlorides or Weinreb amides (Scheme 4). Benzothiazole
ketone dimer 53 was formed by the reaction of triphosgene
with benzothiazole in the presence of triethylamine and
Replacing benzoxazole or benzothiazole cores of the hit
compounds with a benzimidazole resulted in loss of
9449
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 4. Synthesis of Benzothiazole analogues
a
Reagents and conditions: (a) (1) SOCl2, CHCl3, cat. DMF, (2) benzothiazole, DMAP, NEt3, MeCN, 80 °C; (b) NH2OH, K2CO3, EtOH, water,
50 °C; (c) benzothiazole, triphosgene, DMAP, MeCN, NEt3, 80 °C; (d) NH2OH·HCl, NaHCO3, EtOH, aniline, THF, 50 °C; (e) O-
methylhydroxylamine, cat. aniline, NaHCO3, MeOH, THF, 50 °C; (f) (1) benzothiazole, n-BuLi, −78 °C, THF, (2) Weinreb amide, THF, rt; (g)
NH2OH·HCl, NaHCO3, cat. aniline, MeOH, THF, 60 °C; (h) benzothiazole, DMAP, NEt3, MeCN, 80 °C; (i) (1) benzothiazole,
isoprpylmagnesium chloride, −15 °C, THF, (2) Weinreb amide, THF, rt; and (j) NH2OH·HCl, NaOAc, MeOH, reflux.
antitubercular activity, suggesting that hydrogen-bond donors
were less favored compared to hydrogen-bond acceptors or
that the additional benzimidazole NH impacts permeability
(Table 4). Replacement of the core with a phenyl and 2-
pyridyl led to the same reduction in potency, while removal of
the carbamate resulted in the loss of the marginal activity.
Replacement of the oxime with a trifluoromethyl ketone
hydrate 37 was not tolerated. Cyclization of the nitrile and
oxime in the form of an oxadiazole 67 or furoxanes 68 was not
tolerated.
We further profiled frontrunners from the oxime carbamate
and free oxime series with the aim of identifying metabolically
stable compounds that could potentially be evaluated in vivo.
Compound 6 retained the potency of the hit compounds and
displayed improved microsomal metabolic stability despite the
presence of the oxime carbamate (Table 5). Solubility was low,
human S9 fraction stability was moderate, and no human
ether-a-go-go-related gene (hERG) liability was found.
Dosing of 6 in mice showed clearance higher than mouse
liver blood flow notwithstanding the moderate to good in vitro
stability in microsomes, and the compound was not detected in
blood following oral administration at 20 mg/kg (Table 6).
The free oxime compound 50 had moderate potency but
improved solubility and microsomal metabolic stability
compared to the hit compounds. With the labile carbamate
group now removed and a better balance of activity, solubility,
and microsomal stability properties observed, the compound
was assessed in a mouse PK study (Table 6). The compound
was rapidly distributed into tissue, with a high volume of
distribution and rapidly cleared following IV administration in
the mouse, resulting in a short elimination half-life. Oral PK
showed that 50 was only 3% orally bioavailable. Taken
together, the PK exposure of 6 and 50 were not sufficient for
evaluation of in vivo efficacy in a mouse model of tuberculosis.
modulation in iniB expression if the test compound damages
the Mtb cell-wall and PrecA-LUX which detects modulation in
recA expressionan indicator of compounds inducing DNA
damage. Several attempts of generating spontaneous resistant
mutants failed where a range of inoculum (10−8 to 10−10 CFU)
was used. Nonetheless, based on this initial triage process, the
compounds were identified as novel hits involving a potential
novel mode of action in Mtb.
CONCLUSIONS
■
Phenotypic whole-cell screening of a hydrophilic small
molecule library yielded NCEs with excellent antimycobacte-
rial activity against both laboratory and clinical Mtb strains.
While the parent carbamates were highly potent, the
corresponding free oximes were inactive, due to their inability
to permeate the Mtb cell-wall. Attempts at replacing the
carbamate moiety with more metabolically stable groups
remained a challenge. On the other hand, modifying the
physicochemical properties of the free oxime proved successful
in delivering potent free oxime compounds exemplified by 50.
These active free oxime compounds can potentially benefit
from further optimization of in vivo intrinsic clearance. This
work also demonstrated that these compounds do not hit
promiscuous cell-wall biosynthesis and respiratory targets that
are so often confirmed as targets of whole-cell phenotypic
screening hits. Coupled with the potential of these compounds
to act via a novel mode of action in Mtb, this further justifies
continued future work to optimize this chemical series and/or
develop formulation strategies to improve permeation across
the Mtb cell-wall and identification of the target.
EXPERIMENTAL SECTION
■
General Methods. Purification of compounds were carried out by
column chromatography on silica gel 60 (Fluka), particle size 0.063−
0.2 mm (70−230 mesh), as the stationary phase. All target
BIOLOGY TRIAGING TOWARD MECHANISM OF
ACTION DETERMINATION
1
■
compounds and intermediates were characterized by H NMR and
LC−MS. NMR spectra were recorded on either a Varian Mercury-300
(1H 300.1 MHz, 13C 75.5 MHz) or Bruker-400 (1H 400.2 MHz, 13C
100.6 MHz) instrument using CDCl3, CD3OD, and DMSO-d6 as
solvents. LC−MS analysis was performed using an Agilent 1260
Infinity Binary Pump, Agilent 1260 Infinity Diode Array Detector
(DAD), Agilent 1290 Infinity Column Compartment, Agilent 1260
Infinity Standard Autosampler, and a Agilent 6120 Quadrupole
(Single) mass spectrometer, equipped with APCI and ESI multimode
ionization sources. Purities were determined by Agilent LC−MS using
a Kinetex Core C18 2.6 μm column (50 × 3 mm); Mobile Phase B:
0.4% acetic acid and 10 mM ammonium acetate in a 9:1 ratio of
To explore the mechanism of action, selected compounds were
tested in biology triage assays to assess their activity against
promiscuous targets. The tested compounds 1, 2, 6, 8, 49, and
50 retained activity against a QcrB mutant (A396T) and did
not show MIC modulation against a cytochrome-bd oxidase
knockout mutant strain (cydKO), thereby eliminating the
respiratory system of Mtb as a potential mechanism of action.20
The compounds did not show a positive signal in two standard
bioluminescence reporter assays: PiniB-LUX which detects
9450
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 3. Optimization of Nitrile Functionality
Scheme 5. Synthesis of Alternative cores
a
Reagents and conditions: (a) NaNO2, AcOH, 0−25 °C, 12 h; (b)
dimethylcarbamoyl chloride or N,N-dimethylsulfamoyl chloride,
triethanolamine, DCM, reflux, 15 h; (c) NaNO2, aq. HCl, MeOH,
rt; (d) aq. NaOH, NH2OH, reflux; (e) NCS, HCl gas, DMF, 18 h;
and (f) NEt3, EtOAc, 2 days.
Table 4. SAR of Alternative Core Structures
a
14 day Alamar Blue readout against H37RvMa in GAST/Fe
b
c
medium. Cytotoxicity against CHO cells. Cytotoxicity against
kidney epithelial cells extracted from an African green monkey
(VERO). n.d., not determined.
HPLC-grade methanol and type 1 water, Mobile Phase A: 0.4% acetic
acid in 10 mM ammonium acetate in HPLC-grade (type 1) water,
with flow rate = 0.9 mL/min; detector: diode array (DAD) and all
compounds were confirmed to have ≥95% purity. Compound 51 was
a generous gift from Pfizer. 2-Benzothiazoleacetonitrile was acquired
form Sigma-Aldrich.
General Procedure for Synthesis of Benzoxazole Oxime
Carbamates 1, 6, and 9−20. Triethylamine (5.0 equiv) and
dimethylcarbamoyl chloride (3.0 equiv) were successively added to
a suspension of the appropriate oxime intermediate (1.0 equiv) in
anhydrous DCM at room temperature. This solution was purged with
nitrogen gas for 10 min, heated to reflux for 15 h, and then cooled to
a
14 day Alamar Blue readout against H37RvMa in GAST/Fe
b
medium. Kinetic solubility assay at pH 6.5.
room temperature, and water (20 mL) was added. The mixture was
extracted with DCM (3 × 20 mL), and the combined organic phases
were rinsed with brine (2 × 50 mL), dried over anhydrous MgSO4,
9451
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
7.46 (t, J = 7.7 Hz, 1H), 3.17 (s, 3H), 3.11 (s, 3H). 13C NMR (151
MHz, CDCl3): δ 153.49, 151.38, 151.13, 141.16, 128.56, 127.00,
126.10, 121.83, 111.76, 107.14, 37.67, 36.33. LC−MS (ESI): m/z
304.1 [M + 46]+. HPLC purity >99%.
Table 5. Further Profiling of Leading Compounds
N-((Dimethylcarbamoyl)oxy)-4-methylbenzo[d]oxazole-2-carbi-
midoyl Cyanide (9). Yield (0.233 g, 69%). 1H NMR (400 MHz,
CDCl3): δ 7.46 (d, J = 7.7 Hz, 1H), 7.44−7.38 (m, 1H), 7.26 (dt, J =
7.2, 1.0 Hz, 1H), 3.19 (s, 3H), 3.14 (s, 3H), 2.70 (s, 3H). 13C NMR
(101 MHz, CDCl3): δ 152.51, 151.34, 150.86, 140.64, 132.68, 128.12,
127.05, 126.23, 108.72, 107.08, 37.51, 36.19, 16.40. LC−MS (ESI):
m/z 318.1 [M + 46]+. HPLC purity >99%.
compound
6
50
a
N-((Dimethylcarbamoyl)oxy)-5-methylbenzo[d]oxazole-2-carbi-
Mtb MIC (μM) GAST/Fe
0.30
0.60
>125
18
0.78
1.2
1
midoyl Cyanide (10). Yield (0.288 g, 71%). H NMR (400 MHz,
b
Mtb MIC (μM) 7H9
CDCl3): δ 7.68 (dt, J = 1.6, 0.8 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H),
7.35 (ddd, J = 8.5, 1.7, 0.6 Hz, 1H), 3.19 (s, 3H), 3.14 (s, 3H), 2.53
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 153.35, 151.29, 149.32,
141.29, 136.10, 129.77, 126.97, 121.30, 110.94, 107.05, 37.52, 36.19,
21.48. LC−MS (ESI): m/z 318.1 [M + 46]+. HPLC purity >99%.
5-Bromo-N-((dimethylcarbamoyl)oxy)benzo[d]oxazole-2-carbi-
c
ESKAPE bacteria
solubility (μM)
>125
67
>99/86/71
n.d.
d
e
H/R/MLM % remaining
>99/98/>99
60
f
human S9 % remaining
g
CHO IC50 (μM)
>100
>33
15.6
n.d.
1
h
midoyl Cyanide (11). Yield (0.105 g, 56%). H NMR (400 MHz,
hERG IC50 (μM)
CDCl3): δ 8.09 (d, J = 2.0 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.67
(dd, J = 8.5, 2.1 Hz, 1H), 3.13 (s, 3H), 3.09 (s, 3H). 13C NMR (101
MHz, CDCl3): δ 157.21, 152.77, 141.98, 130.12, 126.53, 122.86,
122.71, 117.74, 112.87, 107.65, 37.41, 36.28. LC−MS (ESI): m/z
382.0, 384.0 (1:1) [M + 46]+. HPLC purity >99%.
PPB (fu)
n.d.
1.53
91.7
0.002
2.64
128
i
clog P
i
TPSA
a
14 day Alamar Blue readout against H37RvMa in GAST/Fe
b
medium. 14 day Alamar Blue readout against H37RvMa in
6-Bromo-N-((dimethylcarbamoyl)oxy)benzo[d]oxazole-2-carbi-
c
1
Middlebrook 7H9 medium. ESKAPE bacteria strains: Enterobacter
midoyl Cyanide (12). Yield (0.187 g, 49%). H NMR (400 MHz,
cloacae (ATCC 700323), Escherichia coli (ATCC 25922), Klebsiella
pneumoniae (ATCC BAA-1705), Acinetobacter baumannii (ATCC
19606), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus
CDCl3): δ 7.84 (d, J = 1.9 Hz, 1H), 7.77 (d, J = 8.5, 1H), 7.63 (dd, J
= 8.5, 1.9 Hz, 1H), 3.19 (s, 3H), 3.14 (s, 3H). 13C NMR (101 MHz,
CDCl3): δ 153.75, 151.33, 151.13, 140.15, 129.73, 126.54, 122.58,
121.89, 115.15, 106.82, 37.57, 36.21. LC−MS (ESI): m/z 382.0,
384.0 (1:1) [M + 46]+. HPLC purity >99%.
d
aureus (ATCC 25923). Kinetic solubility assay at pH 6.5.
e
Percentage remaining at 30 min assessed in human, rat, and mouse
f
Methyl 2-(cyano(((dimethylcarbamoyl)oxy)imino)methyl)benzo-
liver microsomes. Percentage remaining at 40 min assessed in the
1
g
h
[d]oxazole-6-carboxylate (13). Yield (0.282 g, 73%). H NMR (400
human S9 fraction. Cytotoxicity against CHO cells. Human ether-a-
go-go related gene (hERG) K+ channel inhibition determined with
MHz, CDCl3): δ 8.33 (d, J = 1.1 Hz, 1H), 8.20 (dd, J = 8.5, 1.4 Hz,
1H), 7.95 (d, J = 8.4 Hz, 1H), 4.00 (s, 3H), 3.20 (s, 3H), 3.15 (s,
3H). 13C NMR (101 MHz, CDCl3): δ 165.83, 155.52, 151.05, 150.65,
144.32, 130.28, 127.39, 126.56, 121.38, 113.24, 106.79, 52.66, 37.60,
36.23. LC−MS (ESI): m/z 362.1 [M + 46]+. HPLC purity >99%.
N-((Dimethylcarbamoyl)oxy)-6-(trifluoromethyl)benzo[d]-
oxazole-2-carbimidoyl Cyanide (14). Yield (0.105 g, 59%). 1H NMR
(400 MHz, CDCl3): δ 8.03 (d, J = 8.5, 1H), 7.95 (d, J = 2.0, 1H),
7.79 (dd, J = 8.5, 2.0 Hz, 1H), 3.20 (s, 3H), 3.15 (s, 3H). 13C NMR
(101 MHz, CDCl3): δ 155.48, 150.99, 150.36, 143.38, 130.39 (q),
126.37, 124.89, 123.19, 122.31, 109.49, 106.72, 37.61, 36.22. LC−MS
(ESI): m/z 372.1 [M + 46]+. HPLC purity >99%.
i
electrophysiology-based hERG assay using IonWorks HT. Calculated
using the ACD Percepta module. PPB = plasma protein binding; n.d.,
not determined.
Table 6. PK Parameters of Compound 6 and 50 in C57/BL6
a
Mice Following Intravenous and Oral Administration
6
50
parameters
IV
PO
IV
5.0
5.4
PO
nominal dose (mg/kg)
Cmax (μM)
5.0
3.9
20
n.d.
20.0
0.13
6-Chloro-N-((dimethylcarbamoyl)oxy)benzo[d]oxazole-2-carbi-
1
midoyl Cyanide (15). Yield (0.160 g, 81%). H NMR (400 MHz,
Tmax (h)
0.17
1.8
CDCl3): δ 7.83 (dd, J = 8.6, 0.5 Hz, 1H), 7.67 (dd, J = 1.9, 0.5 Hz,
1H), 7.47 (dd, J = 8.6, 1.9 Hz, 1H), 3.19 (s, 3H), 3.14 (s, 3H). 13C
NMR (101 MHz, CDCl3): δ 153.92, 151.11 (2C), 139.74, 134.43,
126.98, 126.51, 122.20, 112.17, 106.81, 37.56, 36.20. LC−MS (ESI):
m/z 338.1 [M + 46]+. HPLC purity >99%.
t1/2 terminal (h)
CLb (mL/min/kg)
Vd (L/kg)
AUC0−∞ (min. μmol/L)
F (%)
0.13
n.d.
2.6
157
34.7
98
165
1.82
118
n.d.
<1
12
3
5-Chloro-N-((dimethylcarbamoyl)oxy)benzo[d]oxazole-2-carbi-
1
a
midoyl Cyanide (17). Yield (0.207 g, 72%). H NMR (400 MHz,
Mean values from three animals. n.d., not determined due to low oral
CDCl3): δ 7.90 (dd, J = 2.1, 0.5 Hz, 1H), 7.60 (dd, J = 8.8, 0.5 Hz,
1H), 7.52 (dd, J = 8.8, 2.0 Hz, 1H), 3.19 (s, 3H), 3.14 (s, 3H). 13C
NMR (101 MHz, CDCl3): δ 154.49, 151.08, 149.52, 141.95, 131.71,
128.82, 126.51, 121.49, 112.41, 106.79, 37.57, 36.21. LC−MS (ESI):
m/z 338.1 [M + 46]+. HPLC purity >99%.
exposure. Dash indicates that the value was not measured or was not
relevant.
and concentrated to give a residue, which was column-chromato-
graphed (EtOAc/hexane) to give the desired product.
N-((Dimethylcarbamoyl)oxy)-6-fluorobenzo[d]oxazole-2-carbi-
1
midoyl Cyanide (18). Yield (0.225 g, 67%). H NMR (400 MHz,
N-((Dimethylcarbamoyl)oxy)-6-methylbenzo[d]oxazole-2-carbi-
midoyl Cyanide (1). Yield (0.312 g, 77%). 1H NMR (300 MHz,
CDCl3): δ 7.77 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 0.7 Hz, 1H), 7.29
(dd, J = 8.3, 1.0 Hz, 1H), 3.18 (s, 3H), 3.13 (s, 3H), 2.55 (s, 3H). 13C
NMR (101 MHz, CDCl3): δ 153.55, 152.89, 151.38, 139.61, 139.04,
127.47, 127.00, 121.01, 111.51, 107.07, 37.51, 36.19, 22.06. LC−MS
(ESI): m/z 318.1 [M + 46]+. HPLC purity >99%.
N-((Dimethylcarbamoyl)oxy)benzo[d]oxazole-2-carbimidoyl Cy-
anide (6). Yield (0.252 g, 79%). 1H NMR (600 MHz, CDCl3): δ 7.89
(d, J = 7.9 Hz, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H),
CDCl3): δ 7.87 (dd, J = 8.8, 5.0 Hz, 1H), 7.83 (dd, J = 8.5, 2.3 Hz,
1H), 7.35 (ddd, J = 9.8, 8.8, 2.3 Hz, 1H), 3.19 (s, 3H), 3.14 (s, 3H).
13C NMR (101 MHz, CDCl3): δ 163.77, 161.28, 153.97, 151.09,
137.47, 126.50, 122.44, 114.77, 106.85, 99.70, 37.55, 36.19. LC−MS
(ESI): m/z 322.1 [M + 46]+. HPLC purity >99%.
N-((Dimethylcarbamoyl)oxy)-5-fluorobenzo[d]oxazole-2-carbi-
1
midoyl Cyanide (19). Yield (0.167 g, 62%). H NMR (400 MHz,
DMSO-d6): δ 7.83 (dd, J = 8.9, 4.0 Hz, 1H), 7.78 (dd, J = 8.5, 2.6 Hz,
1H), 7.40 (ddd, J = 9.6, 8.9, 2.7 Hz, 1H), 3.20 (s, 3H), 3.16 (s, 3H).
9452
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
13C NMR (101 MHz, DMSO-d6): δ 164.08, 160.23, 155.98, 148.31,
CDCl3): δ 8.19 (ddd, J = 8.2, 1.3, 0.7 Hz, 1H), 7.89 (ddd, J = 7.8, 1.4,
0.7 Hz, 1H), 7.65−7.36 (m, 2H), 5.00 (s, 2H), 4.30 (q, J = 7.1 Hz,
2H), 1.32 (t, J = 7.1 Hz, 3H). LC−MS (ESI): m/z 290.0 [M + 1]+.
HPLC purity 94%.
142.20, 126.57, 122.91, 114.57, 112.81, 107.49, 37.53, 36.21. LC−MS
(ESI): m/z 322.1 [M + 46]+. HPLC purity >99%.
N-((Dimethylcarbamoyl)oxy)-6-nitrobenzo[d]oxazole-2-carbimi-
doyl Cyanide (20). Yield (0.114 g, 55%). 1H NMR (400 MHz,
DMSO-d6): δ 8.57 (d, J = 2.0 Hz, 1H), 8.45 (dd, J = 8.8, 2.0 Hz, 1H),
8.05 (d, J = 8.8 Hz, 1H), 3.21 (s, 3H), 3.16 (s, 3H). 13C NMR (101
MHz, DMSO-d6): δ 157.18, 150.82, 150.13, 147.29, 145.41, 126.05,
121.96, 121.74, 108.31, 106.54, 37.66, 36.25. LC−MS (ESI): m/z
349.1 [M + 46]+. HPLC purity 96%.
6-Acetamido-N-((dimethylcarbamoyl)oxy)benzo[d]oxazole-2-
carbimidoyl Cyanide (21). Yield (0.071 g, 47%). 1H NMR (400
MHz, DMSO-d6): δ 10.41 (br s, 1H), 8.27 (d, J = 2.1 Hz, 1H), 7.86
(d, J = 8.9 Hz, 1H), 7.55 (dd, J = 8.9, 2.1 Hz, 1H), 3.09 (s, 3H), 3.05
(s, 3H), 2.12 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 169.13,
153.97, 151.59, 151.42, 140.03, 136.04, 127.62, 121.59, 118.41,
108.41, 101.59, 37.62, 36.03, 24.76. LC−MS (ESI): m/z 361.1 [M +
46]+. HPLC purity >99%.
(E)-N-(2-(Dimethylamino)-2-Oxoethoxy)benzo[d]thiazole-2-car-
1
bimidoyl Cyanide (26). Yield (135 mg, 48%). H NMR (400 MHz,
CDCl3): δ 8.17 (ddd, J = 8.1, 1.4, 0.7 Hz, 1H), 7.96−7.79 (m, 1H),
7.64−7.43 (m, 2H), 5.13 (s, 2H), 3.07 (s, 3H), 3.01 (s, 3H). LC−MS
(ESI): m/z 289.1[M + 1]+. HPLC purity 99%.
(E)-N-Propoxybenzo[d]thiazole-2-carbimidoyl Cyanide (28).
1
Yield (51 mg, 21%). H NMR (400 MHz, CDCl3): δ 8.09 (ddd, J
= 8.2, 1.3, 0.7 Hz, 1H), 7.82 (ddd, J = 7.8, 1.4, 0.7 Hz, 1H), 7.50−
7.37 (m, 2H), 4.41 (t, J = 6.7 Hz, 2H), 1.81 (h, J = 7.4 Hz, 2H), 0.98
(t, J = 7.4 Hz, 3H). LC−MS (ESI): m/z 246.1 [M + 1]+. HPLC
purity >99%.
(E)-N-(Benzyloxy)benzo[d]thiazole-2-carbimidoyl Cyanide (29).
Yield (130 mg, 44%). 1H NMR (400 MHz, CDCl3): δ 8.08 (ddd, J =
8.1, 1.4, 0.7 Hz, 1H), 7.80 (ddd, J = 7.8, 1.4, 0.6 Hz, 1H), 7.55−7.26
(m, 7H), 5.43 (s, 2H). LC−MS (ESI): m/z 294.0 [M + 1]+. HPLC
purity >99%.
(E)-N-(Pyridin-3-ylmethoxy)benzo[d]thiazole-2-carbimidoyl Cya-
nide (30). Yield (100 mg, 68%). 1H NMR (300 MHz, CDCl3): δ 8.80
(s, 1H), 8.71 (d, J = 4.5 Hz, 1H), 8.22−8.11 (m, 1H), 8.03 (d, J = 8.1
Hz, 1H), 7.94−7.79 (m, 1H), 7.63−7.44 (m, 3H), 5.57 (s, 2H). LC−
MS (ESI): m/z 295.1 [M + 1]+. HPLC purity 97%.
(E)-N-((Dimethylcarbamoyl)oxy)benzo[d]thiazole-2-carbimidoyl
Cyanide (2). Triethylamine (0.700 mL, 5.00 mmol) and dimethyl-
carbamoyl chloride (0.276 mL, 3.00 mmol) were successively added
to a suspension of oxime 22 (0.200 g, 0.984 mmol) in anhydrous
DCM (10 mL) at 0 °C. This solution was stirred at 0 °C for 30 min
and left at rt o/n. Water (10 mL) was added, the mixture was
extracted with DCM (3 × 20 mL), and the combined organic phases
were rinsed with brine (2 × 50 mL), dried over anhydrous MgSO4,
and concentrated to give a residue, which was purified by flash
column chromatography with EtOAc/hexane (3/7) to give 2 (108
(E)-N-(Pyridin-4-ylmethoxy)benzo[d]thiazole-2-carbimidoyl Cya-
1
nide (31). Yield (46 mg, 31%). H NMR (300 MHz, DMSO-d6): δ
8.70−8.54 (m, 2H), 8.26−8.08 (m, 2H), 7.70−7.52 (m, 2H), 7.51−
7.40 (m, 2H), 5.66 (s, 2H). LC−MS (ESI): m/z 295.0 [M + 1]+.
HPLC purity 99%.
(E)-N-(Pyridin-2-ylmethoxy)benzo[d]thiazole-2-carbimidoyl Cya-
nide (32). Yield (116 mg, 39%). 1H NMR (300 MHz, CDCl3): δ 8.64
(dd, J = 4.6, 2.2 Hz, 1H), 8.21−8.11 (m, 1H), 7.92−7.71 (m, 2H),
7.63−7.43 (m, 3H), 7.38−7.28 (m, 1H), 5.68 (s, 2H). LC−MS
(ESI): m/z 295.0 [M + 1]+. HPLC purity 98%.
1
mg, 40%) as a brown solid. H NMR (400 MHz, CDCl3): δ 8.23−
8.18 (m, 1H), 7.97−7.90 (m, 1H), 7.62−7.49 (m, 2H), 3.17 (s, 3H),
3.10 (s, 3H). LC−MS (ESI): m/z 275.0 [M + 1]+. HPLC purity 94%.
(E)-N-Hydroxybenzo[d]thiazole-2-carbimidoyl Cyanide (22). The
synthesis of compound 22 was performed according to the literature
procedure of Ilkun et al.12
N-((N,N-Dimethylsulfamoyl)oxy)benzo[d]thiazole-2-carbimidoyl
Cyanide (23). Triethylamine (0.700 mL, 5.00 mmol) and N,N-
dimethylsulfamoyl chloride (0.322 mL, 3.00 mmol) were successively
added to a suspension of oxime 22 (0.200 g, 0.984 mmol) in
anhydrous DCM (10 mL) at 0 °C. This solution was stirred at 0 °C
for 30 min and left at rt o/n. Water (10 mL) was added, the mixture
was extracted with DCM (3 × 20 mL), and the combined organic
phases were rinsed with brine (2 × 50 mL), dried over anhydrous
MgSO4, and concentrated to give a residue, which was purified by
flash column chromatography with EtOAc/hexane (10/90 to 20/80)
to give 23 (55 mg, 18%). 1H NMR (300 MHz, CDCl3): δ 8.20−8.11
(m, 1H), 7.86 (ddd, J = 7.1, 2.0, 0.7 Hz, 1H), 7.62−7.44 (m, 2H),
3.08 (s, 6H). LC−MS (ESI): m/z 311.0 [M + 1]+. HPLC purity 99%.
N-((Methylsulfonyl)oxy)benzo[d]thiazole-2-carbimidoyl Cyanide
(24). Triethylamine (0.700 mL, 5.00 mmol) and MsCl (0.232 mL,
3.00 mmol) were successively added to a suspension of oxime 22
(0.200 g, 0.984 mmol) in anhydrous DCM (10 mL) at 0 °C. This
solution was stirred at 0 °C for 30 min and left at rt o/n. Water (10
mL) was added, the mixture was extracted with DCM (3 × 20 mL),
and the combined organic phases were rinsed with brine (2 × 50 mL),
dried over anhydrous MgSO4, and concentrated to give a residue,
which was purified by flash column chromatography with EtOAc/
(E)-N-Methoxybenzo[d]thiazole-2-carbimidoyl Cyanide (27). To
a solution of 22 (200 mg, 0.984 mmol) in DCM (10 mL) was added
triethylamine (0.411 mL, 2.95 mmol) followed by methyl iodide
(0.123 mL, 1.968 mmol) under a nitrogen atmosphere. The mixture
was stirred for 3 h at rt, diluted with DCM, and washed with water.
After drying over MgSO4, filtration, and concentration, the crude
product was purified by flash column chromatography using EtOAc/
1
hexane (10/90 to 30/70) to yield 27 (44 mg, 21%). H NMR (300
MHz, CDCl3): δ 8.21−8.09 (m, 1H), 7.92−7.84 (m, 1H), 7.58−7.44
(m, 2H), 4.33 (s, 3H). LC−MS (ESI): m/z 218.0 [M + 1]+. HPLC
purity 95%.
N-[(1,3-Benzothiazol-2-yl)methylidene]oxime (34). Benzothia-
zole-2-carboxaldehyde (200 mg, 1.22 mmol) was dissolved in
methanol (3 mL) and tetrahydrofuran (THF) (3 mL) and placed
in a pressure tube. To this mixture was added a solution of
hydroxylamine hydrochloride (500 mg, 7.20 mmol) in saturated
NaHCO3 (1 mL) solution, and the mixture was stirred at 60 °C for 18
h. The cooled mixture was diluted with 60 mL of water and then
extracted with EtOAc (2 × 80 mL). The organic phase was dried over
Na2SO4 and evaporated to give 34 (197 mg, 90%) as a light-yellow
solid. 1H NMR (300 MHz, CDCl3): δ [isomer ratio 2:1] 8.52 & 8.15
(s & s 1H), 8.21−8.07 (m, 1H), 8.04−7.88 (m, 2H), 7.63−7.44 (m,
2H). LC−MS (ESI): m/z 179.0 [M + 1]+. HPLC purity 98%.
N-[-1-(1,3-Benzothiazol-2-yl)ethylidene]hydroxylamine (36). 2-
Acetylbenzothiazole (80 mg, 0.45 mmol) was dissolved in isopropanol
(2 mL); then, a solution of hydroxylamine hydrochloride (500 mg,
7.20 mmol) in NaHCO3 (2 mL) solution was added, and the mixture
was heated to 65 °C overnight. The mixture was diluted with 50 mL
of NaHCO3 solution and extracted with EtOAc (2 × 50 mL). The
organic phase was dried over Na2SO4 and evaporated. Flash
chromatography with hexane/DCM/EtOAc (70/20/10 to 50/30/
20) and crystallization from EtOAc yielded 36 (62 mg, 71%) in the
form of white crystals. 1H NMR (600 MHz, MeOD): δ [isomer ratio
1:1] 8.20−8.13 & 8.13−8.06 (m & m, 2H), 7.67−7.61 & 7.61−7.55
1
hexane (20/80 to 50/50) to give 24 (71 mg, 26%). H NMR (300
MHz, CDCl3): δ 8.30−8.21 (m, 1H), 8.00−7.92 (m, 1H), 7.68−7.55
(m, 2H), 3.37 (s, 3H). LC−MS (ESI): m/z 282.0 [M + 1]+. HPLC
purity 95%.
General Procedure for Synthesis of 25, 26, 28-32. To a solution
of 22 (1 equiv) in DCM (0.1−0.2 M) was added triethylamine (5
equiv) followed by the appropriate bromide or chloride (3 equiv)
under a nitrogen atmosphere. The mixture was stirred o/n at rt or
until the starting material was consumed, diluted with DCM, and
washed with water. After drying over MgSO4, filtration, and
concentration, the crude product was purified by flash column
chromatography using EtOAc/hexane to yield the desired product.
Ethyl (E)-2-(((Benzo[d]thiazol-2-yl(cyano)methylene)amino)-
oxy)acetate (25). Yield (55 mg, 63%). 1H NMR (400 MHz,
9453
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(m & m, 2H), 2.81 & 2.78 (s & s, 3H). LC−MS (ESI): m/z 193.1 [M
+ 1]+. HPLC purity >99%.
pressure tube to 50 °C for 5 h. The mixture was cooled and
evaporated onto silica gel. Flash chromatography with hexane/
1
N-[1-(1,3-Benzothiazol-2-yl)-2,2,2-trifluoroethylidene]-
hydroxylamine (38). 37 (700 mg, 2.80 mmol) was dissolved in water
(3 mL) and ethanol (5 mL). Hydroxylamine hydrochloride (800 mL,
11.5 mmol) and Na2CO3 (1.0 g, 9.43 mmol) were added, and the
mixture was heated to 75 °C for 24 h. The reaction mixture was
evaporated, and the remaining material was diluted with a NaHCO3
(50 mL) solution and extracted with EtOAc (2 × 70 mL). The
organic phase was dried over Na2SO4 and evaporated. Flash
chromatography with hexane/EtOAc (90/10 to 70/30) yielded 38
EtOAc/DCM (90/10/0 to 40/10/50) yielded 55 (35 mg, 46%). H
NMR (300 MHz, CDCl3): δ 8.30−8.19 (m, 1H), 8.11−7.91 (m, 1H),
7.67−7.50 (m, 2H), 4.41 (s, 3H). LC−MS (ESI): m/z 326.0 [M +
1]+. HPLC purity >99%.
N-[(1,3-Benzothiazol-2-yl) (pyrazin-2-yl)methylidene]-
hydroxylamine (56). Benzothiazol-2-yl(pyrazin-2-yl)methanone
(100 mg, 0.414 mmol) was dissolved in THF (3 mL) and MeOH
(3 mL). A solution of hydroxylamine hydrochloride (500 mg, 7.19
mmol) in NaHCO3 (1 mL) solution was added, and the mixture was
heated in a pressure tube with 1 drop of aniline to 60 °C for 18 h. The
cooled mixture was treated with NaHCO3 (50 mL) solution and
extracted with EtOAc (50 mL). The organic phase was dried over
NaSO4 and evaporated. Flash chromatography with EtOAc/DCM
(40/60) yielded 56 (80 mg, 76%) as a white solid after crystallization
from EtOAc. 1H NMR (300 MHz, CDCl3): δ (isomer ratio 4:1) 9.95
& 9.51 (s & s, 1H), 8.85−8.68 (m, 2H), 8.26−7.94 (m, 2H), 7.69−
7.52 (m, 2H). HPLC purity 97%.
Benzothiazol-2-yl(benzothiazol-6-yl)methanone Oxime (57).
Benzothiazol-2-yl(benzothiazol-6-yl)methanone (100 mg, 0.337
mmol) and 3 drops of aniline were dissolved in THF (3 mL) and
MeOH (3 mL). A solution of hydroxylamine hydrochloride (600 mg,
8.63 mmol) was added in NaHCO3 (1 mL) solution. The mixture was
heated in a sealed pressure tube to 50 °C for 16 h, and 50 mL of
NaHCO3 solution was added to the cooled mixture and extracted
with EtOAc (2 × 50 mL), then dried over Na2SO4, and evaporated.
Flash chromatography with hexane/EtOAc/DCM (50/30/20) and
crystallization from the almost evaporated product fractions yielded a
mixture of isomers 57 (86 mg, 82%) as a yellow solid. 1H NMR (300
MHz, DMSO-d6): δ [isomer ratio 1:1] 13.33 & 12.70 (bs & s, 1H),
9.48 & 9.45 (s & s, 1H), 8.44 & 8.35 (d & d, J = 3.0 & 3.0 Hz, 1H),
8.27−7.88 (m, 3H), 7.84 & 7.70 (dd & dd, J = 6.0 & 3.0 Hz & J = 6.0
& 3.0 Hz, 1H), 7.61−7.44 (m, 2H). LC−MS (ESI): m/z 312.0 [M +
1]+. HPLC purity 97%.
1
(250 mg, 36%). H NMR (300 MHz, CDCl3): δ 14.25 (br s, 1H),
8.12−8.08 (m, 1H), 7.98−7.94 (m, 1H), 7.62−7.49 (m, 2H). LC−
MS (ESI): m/z 247.0 [M + 1]+. HPLC purity 99%.
General Procedure for Synthesis of 40, 48, 49 and 50. To a
solution of either 39, 45, 46, or 47 (1 equiv) in acetic acid (7.5 mL)
and water (0.75 mL) was added sodium nitrite (1 equiv). After
stirring for 2 h at rt, the reaction mixture was diluted with water (40
mL) and the desired product was isolated by filtration of the
precipitate that had formed.
Benzo[d]thiazol-2-yl(5-methyl-1,2,4-oxadiazol-3-yl)methanone
1
Oxime (40). Yield (109 mg, 49%). H NMR (400 MHz, DMSO-d6):
δ 10.64 (s, 1H), 8.29 (dddd, J = 11.9, 8.1, 1.4, 0.7 Hz, 2H), 7.80−7.55
(m, 2H), 2.28 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 169.18,
153.13, 152.71, 149.43, 144.98, 134.74, 127.76, 124.45, 123.26, 23.74.
LC−MS (ESI): m/z 261.0 [M + 1]+. HPLC purity >99%.
Benzo[d]thiazol-2-yl(5-methyl-1,3,4-oxadiazol-2-yl)methanone
1
Oxime (48). H NMR (300 MHz, DMSO-d6): δ [isomer ratio 5:1]
8.29−8.22 & 8.18−8.14 (m & m, 1H), 8.13−8.09 & 8.03−8.00 (m &
m, 1H), 7.65−7.58 & 7.56−7.52 (m & m, 2H), 2.65 & 2.63 (s & s,
3H). LC−MS (ESI): m/z 261.0 [M + 1]+. HPLC purity 99%.
Benzo[d]thiazol-2-yl(5-methyl-1,3,4-thiadiazol-2-yl)methanone
Oxime (49). 1H NMR (300 MHz, CDCl3): δ [isomer ratio 1:1]
8.23−8.07 & 8.07−8.03 (m & m, 1H), 7.71−7.40 (m, 2H), 2.93 (s,
1H), 2.85 (s, 1H). LC−MS (ESI): m/z 277.0 [M + 1]+. HPLC purity
96%.
Benzo[d]thiazol-2-yl(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)-
methanone Oxime (50). Yield (200 mg, 46%). 1H NMR (600 MHz,
DMSO-d6): δ [isomer ratio 2:3] 8.26 & 8.23 (d, J = 8.0 Hz & d, J =
8.1 Hz, 1H), 8.18 & 8.10 (d, J = 7.8 Hz, & d, J = 8.0 Hz, 1H), 7.65 &
7.63−7.58 (d, J = 7.7 Hz & m, 1H), 7.58−7.51 (m, 1H). LC−MS
(ESI): m/z 331.2 [M + 1]+. HPLC purity 97%.
(1,3-Benzothiazol-2-yl)(phenyl)methanone Oxime (58). A sol-
ution of (1,3-benzothiazol-2-yl)(phenyl)methanone (200 mg, 0.83
mmol) in THF (15 mL) and EtOH (20 mL) was treated with a 50%
solution of NH2OH (1.2 mL, 18.2 mmol) in water and stirred for 3
days at 50 °C. The organic solvents were evaporated, and the
remaining mixture was extracted with EtOAc (2 × 50 mL), dried over
Na2SO4, and evaporated. Purification by flash chromatography with
1
(1,3-Benzothiazol-2-yl)(1,3-oxazol-5-yl)methanone Oxime (52).
To a solution of (1,3-benzothiazol-2-yl)(1,3-oxazol-5-yl)methanone
(100 mg, 0.43 mmol) in EtOH (10 mL) was added a solution of
hydroxylamine hydrochloride (650 mg, 9.34 mmol) in water (5 mL)
and NaHCO3 (2 mL) solution and K2CO3 (250 mg). This mixture
was heated to 50 °C for 3 days. The organics were evaporated, the
remaining mixture was extracted with EtOAc (2 × 50 mL), and the
organic layer was dried over Na2SO4 and evaporated. Flash
chromatography with EtOAc/hexane (1/3) yielded 52 (51 mg,
hexane/EtOAc (90/10) yielded 58 (80 mg, 38%). H NMR (300
MHz, CDCl3): δ 8.11−7.95 (m, 1H), 7.90−7.80 (m, 1H), 7.70−7.30
(m, 7H), 7.20−7.14 (m, 1H). LC−MS (ESI): m/z 255.0 [M + 1]+.
HPLC purity 99%.
Benzothiazol-2-yl(cyclopentyl)methanone Oxime (59). A mixture
of benzo[d]thiazol-2-yl(cyclopentyl)methanone (282 mg, 1.22
mmol), hydroxylamine hydrochloride (93 mg, 1.34 mmol), and
sodium acetate (110 mg, 1.34 mmol) in methanol was heated to
reflux for 2 h under a nitrogen atmosphere. The reaction mixture was
cooled, and the methanol was removed under reduced pressure. The
residue was diluted with EtOAc (30 mL) and extracted with 2 N
NaOH (15 mL). The organic layer was washed with water (15 mL)
and concentrated under reduced pressure. Flash chromatography with
hexane/EtOAc (70/30) gave 59 (230 mg, 77%) as a mixture of two
1
48%). H NMR (300 MHz, CDCl3): δ 8.79 (s, 1H), 8.21 (s, 1H),
8.12−8.02 (m, 2H), 7.63−7.50 (m, 2H). LC−MS (ESI): m/z
246.1[M + 1]+. HPLC purity 95%.
Bis(benzo[d]thiazol-2-yl)methanone Oxime (54). 53 (200 mg,
0.67 mmol) was dissolved in ethanol (20 mL) and THF (20 mL). A
solution of hydroxylamine hydrochloride (800 mg, 11.5 mmol) in sat.
NaHCO3 (2 mL) solution was added, and the mixture was heated
with aniline (0.2 mL, 2.15 mmol) to 50 °C for 16 h. The cooled
mixture was diluted with a 50 mL NaHCO3 solution and extracted
with EtOAc (2 × 50 mL). The organic phase was dried over Na2SO4
and evaporated. Flash chromatography with hexane/EtOAc/CHCl3
(50/15/35 to 40/25/35 to 0/70/30) and crystallization from EtOAc
yielded 54 (58 mg, 28%). 1H NMR (300 MHz, CDCl3): δ 8.23−8.09
(m, 3H), 7.99−7.96 (m, 1H), 7.69−7.48 (m, 4H). LC−MS (ESI): m/
z 312.0 [M + 1]+. HPLC purity >99%.
1
isomers as a green solid. H NMR (300 MHz, DMSO-d6) [isomer
ratio 3:1]: δ 12.66 & 12.27 (s & s, 1H), 8.20−7.98 (m, 2H), 7.62−
7.44 (m, 2H), 3.85−3.65 (m, 1H), 2.20−1.54 (m, 8H). LC−MS
(ESI): m/z 247.1 [M + 1]+. HPLC purity >99%.
Benzo[d]thiazol-2-yl(cyclohexyl)methanone Oxime (60). A mix-
ture of benzo[d]thiazol-2-yl(cyclohexyl)methanone (100 mg, 0.408
mmol), hydroxylamine hydrochloride (31.2 mg, 0.449 mmol), and
sodium acetate (36.8 mg, 0.449 mmol) in 5 mL of methanol was
heated to reflux for 2 h under a nitrogen atmosphere. The reaction
mixture was cooled, and the methanol was removed under reduced
pressure. The residue was diluted with EtOAc (20 mL), and 2 N
NaOH (7 mL) was added. The organic layer was washed with water
(7 mL) and concentrated under reduced pressure. Flash chromatog-
raphy with hexane/EtOAc (70/30) yielded 60 (81 mg, 76%) as a
Bis(benzothiazol-2-yl)methanone O-methyl Oxime (55). A
mixture of 53 (70 mg, 0.236 mmol), O-methylhydroxylamine (500
mg, 10.63 mmol, dissolved in 1 mL NaHCO3 solution), and 10 drops
of aniline in THF (2 mL) and MeOH (2 mL) was heated in a sealed
9454
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
mixture of two isomers in the form of a yellow solid. H NMR (300
MHz, DMSO-d6): δ [isomer ratio 7:3] 12.70 & 12.21 (s & s, 1H),
8.23−8.03 (m, 2H), 7.62−7.45 (m, 2H), 3.60−3.20 (m, 1H), 2.30−
1.20 (m, 10H). LC−MS (ESI): m/z 261.1 [M + 1]+. HPLC purity
>99%.
Synthetic procedures and procedures used for in vitro
Mtb studies and PK and metabolism studies (PDF)
SMILES file with Mtb MIC values (CSV)
AUTHOR INFORMATION
Corresponding Author
N-((Dimethylcarbamoyl)oxy)-1H-benzo[d]imidazole-2-carbimi-
doyl Cyanide (63). Triethylamine (0.700 mL, 5.00 mmol) and
dimethylcarbamoyl chloride (0.276 mL, 3.00 mmol) were successively
added to a suspension of oxime 61 (0.200 g, 1.07 mmol) in anhydrous
DCM (10 mL) at 0 °C. This solution was stirred at 0 °C for 30 min
and left at rt o/n. Water (10 mL) was added, the mixture was
extracted with DCM (3 × 20 mL), and the combined organic phases
were rinsed with brine (2 × 50 mL), dried over anhydrous MgSO4,
and concentrated to give a residue, which was purified by flash
column chromatography with EtOAc/hexane (5/5 to 7/3) to give 63
(157 mg, 57%). 1H NMR (300 MHz, CDCl3): δ 7.76−7.66 (m, 2H),
7.45−7.33 (m, 2H), 3.17 (s, 3H), 3.11 (s, 3H). LC−MS (ESI): m/z
258.1 [M + 1]+. HPLC purity 99%.
■
Kelly Chibale − Drug Discovery and Development Centre
(H3D), Department of Chemistry, South African Medical
Research Council Drug Discovery and Development Research
Unit, Department of Chemistry, and Institute of Infectious
Disease and Molecular Medicine, University of Cape Town,
Cape Town 7701, South Africa; orcid.org/0000-0002-
1327-4727; Phone: +27-21-6502553;
Email: Kelly.Chibale@uct.ac.za; Fax: +27-21-6505195
Authors
N-((Dimethylcarbamoyl)oxy)benzimidoyl Cyanide (65). Triethyl-
amine (0.953 mL, 6.84 mmol) and dimethylcarbamoyl chloride
(0.378 mL, 4.11 mmol) were successively added to a suspension of
oxime 62 (0.200 g, 1.37 mmol) in anhydrous DCM (10 mL) and
dimethylformamide (DMF) (2 mL) at 0 °C. This solution was stirred
at 0 °C for 30 min and left at rt o/n. Water (10 mL) was added, and
the solid that formed was purified by recrystallization (DCM/hexane)
to give 63 (163 mg, 55%). 1H NMR (300 MHz, CDCl3): δ 8.03−7.90
(m, 2H), 7.61−7.43 (m, 3H), 3.12 (s, 3H), 3.07 (s, 3H). LC−MS
(ESI): m/z 218.0 [M + 1]+. HPLC purity >99%.
N-((Dimethylcarbamoyl)oxy)picolinimidoyl Cyanide (66). Trie-
thylamine (0.953 mL, 6.84 mmol) and dimethylcarbamoyl chloride
(0.378 mL, 4.11 mmol) were successively added to a suspension of
oxime 64 (0.200 g, 1.07 mmol) in anhydrous DCM (10 mL) at 0 °C.
This solution was stirred at 0 °C for 30 min and left at rt o/n. Water
(10 mL) was added, the mixture was extracted with DCM (20 mL x
3), and the combined organic phases were rinsed with brine (2 × 50
mL), dried over anhydrous MgSO4, and concentrated to give a
residue, which was purified by flash column chromatography with
EtOAc/hexane (6/4) to give 66 (82 mg, 35%). 1H NMR (300 MHz,
CDCl3): δ 8.85−8.57 (m, 1H), 8.17 (dt, J = 8.0, 1.1 Hz, 1H), 7.81
(td, J = 7.8, 1.8 Hz, 1H), 7.45 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 3.16 (s,
3H), 3.09 (s, 3H). LC−MS (ESI): m/z 219.1 [M + 1]+. HPLC purity
>99%.
Renier van der Westhuyzen − Drug Discovery and
Development Centre (H3D), Department of Chemistry,
University of Cape Town, Cape Town 7701, South Africa
Amanda Mabhula − South African Medical Research Council
Drug Discovery and Development Research Unit, Department
of Chemistry, University of Cape Town, Cape Town 7701,
South Africa
Paul M. Njaria − South African Medical Research Council
Drug Discovery and Development Research Unit, Department
of Chemistry, University of Cape Town, Cape Town 7701,
South Africa
̈
Rudolf Muller − Drug Discovery and Development Centre
(H3D), Department of Chemistry, University of Cape Town,
Cape Town 7701, South Africa
Denis Ngumbu Muhunga − Drug Discovery and Development
Centre (H3D), Department of Chemistry, University of Cape
Town, Cape Town 7701, South Africa
Dale Taylor − Drug Discovery and Development Centre
(H3D), Division of Clinical Pharmacology, University of
Cape Town, Cape Town 7701, South Africa
Nina Lawrence − Drug Discovery and Development Centre
(H3D), Division of Clinical Pharmacology, University of
Cape Town, Cape Town 7701, South Africa
Mathew Njoroge − Drug Discovery and Development Centre
(H3D), Division of Clinical Pharmacology, University of
Cape Town, Cape Town 7701, South Africa; orcid.org/
0000-0003-1276-2224
Christel Brunschwig − Drug Discovery and Development
Centre (H3D), Division of Clinical Pharmacology, University
of Cape Town, Cape Town 7701, South Africa
Atica Moosa − SAMRC/NHLS/UCT Molecular
Mycobacteriology Research Unit, Department of Pathology,
University of Cape Town, Cape Town 7701, South Africa
Vinayak Singh − Drug Discovery and Development Centre
(H3D), Department of Chemistry and South African Medical
Research Council Drug Discovery and Development Research
Unit, Department of Chemistry, University of Cape Town,
Cape Town 7701, South Africa; orcid.org/0000-0001-
9002-2489
Srinivasa P.S. Rao − Novartis Institute for Tropical Diseases
(NITD), Singapore 138670, Singapore; Present
Address: Novartis Institute for Tropical Diseases (NITD),
5959 Horton Street, Emeryville, California 94608, United
States.; orcid.org/0000-0002-7156-5725
4-(Benzo[d]thiazol-2-yl)-1,2,5-oxadiazol-3-amine (67). The
oxime 22 (200 mg, 1.00 mmol) was dissolved in MeOH (3.33
mL), and the pH was adjusted to 12 with 50% aq. NaOH. 50%
aqueous NH2OH (2.73 mL) was added, and the mixture was stirred
for 2 h at 90 °C. The solid that formed was isolated by filtration to
1
give 67 (120 mg, 55%). H NMR (300 MHz, DMSO-d6): δ 8.47−
8.06 (m, 2H), 7.87−7.34 (m, 2H), 6.83 (s, 2H). LC−MS (ESI): m/z
219.0 [M + 1]+. HPLC purity >99%.
3,4-Bis(benzo[d]thiazol-2-yl)-1,2,5-oxadiazole 2-Oxide (68). N-
[(1,3-Benzothiazol-2-yl)methylidene]hydroxylamine (197 mg, 1.10
mmol) was dissolved in DMF (3 mL); then, NCS (195 mg, 1.46
mmol) was added, and ∼10 mL of HCl gas (use HCl gas over conc.
HCl) was filled into the flask and then closed with a plastic stopper.
The mixture was stirred at rt overnight. EtOAc (50 mL) was added,
and the solution was washed with water (2 × 50 mL) and dried over
Na2SO4. To this solution was added triethylamine (500 mg, 4.94
mmol), and the solution was stirred at rt for 2 days. This solution was
washed with 50 mL of water/1 mL of HCl, dried over Na2SO4, and
evaporated to ∼3 mL, and the product was allowed to crystallize to
1
give 68 (42 mg, 22%) in the form of an orange solid. H NMR (300
MHz, CDCl3): δ 8.30−8.03 (m, 4H), 7.67−7.52 (m, 4H). LC−MS
(ESI): m/z 352.9 [M + 1]+. HPLC purity 98%.
ASSOCIATED CONTENT
■
sı
* Supporting Information
Ujjini H. Manjunatha − Novartis Institute for Tropical
Diseases (NITD), Singapore 138670, Singapore; Present
Address: Novartis Institute for Tropical Diseases (NITD),
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00707.
9455
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
5959 Horton Street, Emeryville, California 94608, United
States.; orcid.org/0000-0002-7461-9303
Paul W. Smith − Novartis Institute for Tropical Diseases
(NITD), Singapore 138670, Singapore; orcid.org/0000-
0002-0146-1410
Digby F. Warner − SAMRC/NHLS/UCT Molecular
Mycobacteriology Research Unit, Department of Pathology
and Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Cape Town 7701, South Africa
Leslie J. Street − Drug Discovery and Development Centre
(H3D), Department of Chemistry, University of Cape Town,
Cape Town 7701, South Africa
REFERENCES
■
(1) World Health Organization. Guidelines for Treatment of Drug-
Susceptible Tuberculosis and Patient Care; World Health Organization:
Geneva, 2017.
(2) Dheda, K.; Gumbo, T.; Maartens, G.; Dooley, K. E.; McNerney,
R.; Murray, M.; Furin, J.; Nardell, E. A.; London, L.; Lessem, E.;
Theron, G.; van Helden, P.; Niemann, S.; Merker, M.; Dowdy, D.;
Van Rie, A.; Siu, G. K. H.; Pasipanodya, J. G.; Rodrigues, C.; Clark, T.
G.; Sirgel, F. A.; Esmail, A.; Lin, H.-H.; Atre, S. R.; Schaaf, H. S.;
Chang, K. C.; Lange, C.; Nahid, P.; Udwadia, Z. F.; Horsburgh, C. R.;
Churchyard, G. J.; Menzies, D.; Hesseling, A. C.; Nuermberger, E.;
McIlleron, H.; Fennelly, K. P.; Goemaere, E.; Jaramillo, E.; Low, M.;
Jara, C. M.; Padayatchi, N.; Warren, R. M. The Epidemiology,
Pathogenesis, Transmission, Diagnosis, and Management of Multi-
drug-Resistant, Extensively Drug-Resistant, and Incurable Tuber-
culosis. Lancet Respir. Med. 2017, 5, 291−360.
(3) Tiberi, S.; du Plessis, N.; Walzl, G.; Vjecha, M. J.; Rao, M.;
Ntoumi, F.; Mfinanga, S.; Kapata, N.; Mwaba, P.; McHugh, T. D.;
Ippolito, G.; Migliori, G. B.; Maeurer, M. J.; Zumla, A. Tuberculosis:
Progress and Advances in Development of New Drugs, Treatment
Regimens, and Host-Directed Therapies. Lancet Infect. Dis. 2018, 18,
e183−e198.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00707
Funding
The project was funded through a Global Health grant
(number OPP1066878) received from the Bill and Melinda
Gates Foundation and the Strategic Health Innovation
Partnerships (SHIP) unit of the South African Medical
Research Council (SAMRC). The University of Cape Town,
SAMRC, and South African Research Chairs Initiative of the
Department of Science and Innovation, administered through
the South African National Research Foundation, are gratefully
acknowledged for support (K.C.). D.F.W. acknowledges the
financial support of the SAMRC-SHIP Unit with funds
received from the South African Department of Science and
Innovation.
(4) Parida, S. K.; Axelsson-Robertson, R.; Rao, M. V.; Singh, N.;
Master, I.; Lutckii, A.; Keshavjee, S.; Andersson, J.; Zumla, A.;
Maeurer, M. Totally Drug-Resistant Tuberculosis and Adjunct
Therapies. J. Intern. Med. 2015, 277, 388−405.
(5) Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. The
Challenge of New Drug Discovery for Tuberculosis. Nature 2011,
469, 483−490.
́
(6) Bardou, F.; Raynaud, C.; Ramos, C.; Lanéelle, M. A.; Lanrelle, G.
Mechanism of Isoniazid Uptake in Mycobacterium Tuberculosis.
Microbiology 1998, 144, 2539−2544.
Notes
The authors declare no competing financial interest.
(7) Raynaud, C.; Lanéelle, M.-A.; Senaratne, R. H.; Draper, P.;
Lanéelle, G.; Daffé, M. Mechanisms of Pyrazinamide Resistance in
Mycobacteria: Importance of Lack of Uptake in Addition to Lack of
Pyrazinamidase Activity. Microbiology 1999, 145, 1359−1367.
(8) Faller, M.; Niederweis, M.; Schulz, G. E. The Structure of a
Mycobacterial Outer-Membrane Channel. Science 2004, 303, 1189−
1192.
(9) van der Westhuyzen, R.; Winks, S.; Wilson, C. R.; Boyle, G. A.;
Gessner, R. K.; Soares De Melo, C.; Taylor, D.; De Kock, C.; Njoroge,
M.; Brunschwig, C.; Lawrence, N.; Rao, S. P. S.; Sirgel, F.; Van
Helden, P.; Seldon, R.; Moosa, A.; Warner, D. F.; Arista, L.;
Manjunatha, U. H.; Smith, P. W.; Street, L. J.; Chibale, K.
Pyrrolo[3,4-c]Pyridine-1,3(2H)-Diones: A Novel Antimycobacterial
Class Targeting Mycobacterial Respiration. J. Med. Chem. 2015, 58,
9371−9381.
(10) Das, J.; Rao, C. V. L.; Sastry, T. V. R. S.; Roshaiah, M.; Sankar,
P. G.; Khadeer, A.; Kumar, M. S.; Mallik, A.; Selvakumar, N.; Iqbal, J.;
Trehan, S. Effects of Positional and Geometrical Isomerism on the
Biological Activity of Some Novel Oxazolidinones. Bioorg. Med. Chem.
Lett. 2005, 15, 337−343.
(11) Ammar, H. B.; Kaddachi, M. T.; Kahn, P. H. Conversion of
Malononitrile into 2-Cyanomethyl Compounds. Phys. Chem. News
2003, 9, 137−139.
(12) Ilkun, O. T.; Archibald, S. J.; Barnes, C. L.; Gerasimchuk, N.;
Biagioni, R.; Silchenko, S.; Gerasimchuk, O. A.; Nemykin, V. N.
Benz(2-Heteroaryl)Cyanoximes and Their Tl(i) Complexes: New
Room Temperature Blue Emitters. Dalton Trans. 2008, 42, 5715−
5729.
(13) Awasthi, D.; Freundlich, J. S. Antimycobacterial Metabolism:
Illuminating Mycobacterium Tuberculosis Biology and Drug Discov-
ery. Trends Microbiol. 2017, 25, 756−767.
(14) Lei, B.; Wei, C. J.; Tu, S.-C. Action Mechanism of
Antitubercular Isoniazid: Activation by Mycobacterium Tuberculosis
KatG, Isolation, and Characterization of InhA inhibitor. J. Biol. Chem.
2000, 275, 2520−2526.
ACKNOWLEDGMENTS
■
We thank Carmen de Kock, Nesia Barnes, Duane Knowles,
Virgil Verhoog, Sumaya Salie, and Warren Olifant at in vitro
DMPK and parasitology divisions of H3D for excellent
technical assistance in generating all in vitro DMPK and
cytotoxicity data Marianna de Kock and Claudia Spies,
Frederick Sirgel, Paul van Helden, SAMRC, and Stellenbosch
University, South Africa, for excellent technical assistance in
generating MICs against clinical TB isolates. Ronnett Seldon is
acknowledged for conducting antimycobacterial assays with
H37RvMa.
ABBREVIATIONS
■
AcOH, acetic acid; ADME, absorption, distribution, metabo-
lism and excretion; CHO, Chinese hamster ovarian; DCM,
dichloromethane; DMAP, 4-(dimethylamino)pyridine; hERG,
human ether-a-go-go-related gene; HLM, human liver micro-
some; HTS, high-throughput screening; INH, isoniazid; LC−
MS/MS, liquid chromatography coupled with mass spectrom-
etry; MDR, multidrug-resistant; MIC, lowest concentration of
the drug that inhibits growth of more than 90% of the bacterial
population; MLM, mouse liver microsome; Mtb, Mycobacte-
rium tuberculosis; NCE, new chemical entity; NITD, Novartis
Institute for Tropical Diseases; NMR, nuclear magnetic
resonance; PAR, peak area ratio; PK, pharmacokinetics; PPB,
plasma protein binding; PZA, pyrazinamide; RLM, rat liver
microsome; SAR, structure−activity relationship; TB, tuber-
culosis; TDR, totally drug-resistant; TMSCl, chlorotrimethyl-
silane; TPSA, total polar surface area; VERO, kidney epithelial
cells extracted from an African green monkey; XDR,
extensively drug-resistant
9456
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(15) Konno, K.; Feldmann, F. M.; McDermott, W. Pyrazinamide
Susceptibility and Amidase Activity of Tubercle Bacilli. Am. Rev.
Respir. Dis. 1967, 95, 461−469.
(16) Scorpio, A.; Zhang, Y. Mutations in PncA, a Gene Encoding
Pyrazinamidase/Nicotinamidase, Cause Resistance to the Antituber-
culous Drug Pyrazinamide in Tubercle Bacillus. Nat. Med. 1996, 2,
662−667.
(17) Prideaux, B.; Via, L. E.; Zimmerman, M. D.; Eum, S.; Sarathy,
J.; O’Brien, P.; Chen, C.; Kaya, F.; Weiner, D. M.; Chen, P.-Y.; Song,
T.; Lee, M.; Shim, T. S.; Cho, J. S.; Kim, W.; Cho, S. N.; Olivier, K.
N.; Barry, C. E.; Dartois, V. The Association between Sterilizing
Activity and Drug Distribution into Tuberculosis Lesions. Nat. Med.
2015, 21, 1223−1227.
(18) Khodakovskiy, P. V.; Volochnyuk, D. M.; Panov, D. M.; Pervak,
I. I.; Zarudnitskii, E. V.; Shishkin, O. V.; Yurchenko, A. A.; Shivanyuk,
A.; Tolmachev, A. A. 2-(Trifluoroacetyl)Imidazoles, 2-Trifluoroacetyl-
1,3-Thi-azoles, and 2-Trifluoroacetyl-1,3-Oxazoles. Synthesis 2008,
2008, 948−956.
(19) Stavenger, R. A.; Cui, H.; Dowdell, S. E.; Franz, R. G.;
Gaitanopoulos, D. E.; Goodman, K. B.; Hilfiker, M. A.; Ivy, R. L.;
Leber, J. D.; Marino, J. P.; Oh, H.-J.; Viet, A. Q.; Xu, W.; Ye, G.;
Zhang, D.; Zhao, Y.; Jolivette, L. J.; Head, M. S.; Semus, S. F.; Elkins,
P. A.; Kirkpatrick, R. B.; Dul, E.; Khandekar, S. S.; Yi, T.; Jung, D. K.;
Wright, L. L.; Smith, G. K.; Behm, D. J.; Doe, C. P.; Bentley, R.;
Chen, Z. X.; Hu, E.; Lee, D. Discovery of Aminofurazan-
Azabenzimidazoles as Inhibitors of Rho-Kinase with High Kinase
Selectivity and Antihypertensive Activity. J. Med. Chem. 2007, 50, 2−
5.
(20) Moosa, A.; Lamprecht, D. A.; Arora, K.; Barry, C. E.; Boshoff,
H. I. M.; Ioerger, T. R.; Steyn, A. J. C.; Mizrahi, V.; Warner, D. F.
Susceptibility of Mycobacterium Tuberculosis Cytochrome bd Oxidase
Mutants to Compounds Targeting the Terminal Respiratory Oxidase,
Cytochrome c. Antimicrob. Agents Chemother. 2017, 61, No. e01338-
17.
9457
https://doi.org/10.1021/acs.jmedchem.1c00707
J. Med. Chem. 2021, 64, 9444−9457