Biochemical and microbiological evaluation of: N-aryl urea derivatives against mycobacteria and mycobacterial hydrolases

Article abstract of DOI:10.1039/c9md00122k

A focused library of 24 N-aryl urea derivatives was prepared and evaluated against serine esterases of Mycobacterium tuberculosis (Mtb) Rv3802c and Mtb Ag85C. The members of the library were evaluated for both selectivity and mode of inhibition. Furan-bas

Full text of DOI:10.1039/c9md00122k

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Med. Chem. Commun., 2019, DOI: 10.1039/C9MD00122K.
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DOI: 10.1039/C9MD00122K
ARTICLE
Biochemical and microbiological evaluation of N-aryl urea
derivatives against Mycobacteria and Mycobacterial hydrolases
Received 00th January 20xx,
Accepted 00th January 20xx
b†
c
a
Abhishek Vartak,a Christopher Goins, Vinicius Calado Nogueira de Moura, Celine M. Schreidah,
†
Alexander D. Landgraf,a Boren Lin,d Jianyang Du,d Mary Jackson,c Donald R. Ronninga* and Steven J.
Sucheck a*
DOI: 10.1039/x0xx00000x
A focused library of 24 N-aryl urea derivatives was prepared and evaluated against serine esterases of Mycobacterium
tuberculosis (Mtb) Rv3802c and Mtb Ag85C. The members of the library were evaluated for both selectivity and mode of
inhibition. Furan-based urea derivative 6c was found to be the most potent non-covalent inhibitor of Rv3802c with a Ki
value of 5.2 ± 0.7 µM. On the other hand, triazole-based ureas 10a and 10b selectively inhibited Ag85C irreversibly with a
kinact /Ki value of 2.3 ± 0.3 and 5.5 ± 0.4 × 10-3 µM-1min-1, respectively. The library was also evaluated for minimum
inhibitory concentration (MIC) against two strains of Mtb, Mycobacterium smegmatis, and Mycobacterium abscessus.
Compounds 4a and 4c were active against Mtb H37Rv mc26206 with MIC values of 3.12 and 1.5 µM, respectively. Closely
related 4e showed similar activity against Mtb H37Rv mc26206 but also possessed activity against Mtb H37Ra,
Mycobacterium smegmatis and Mycobacterium abscessus. Compounds 4a, 4c, and 4e all contained a common 1-
(cyclohexylmethyl)-3-phenylurea motif. In summary, we identified a selective non-covalent inhibitor of Rv3802c and
covalently irreversible inhibitors of Ag85C as well as the 1-(cyclohexylmethyl)-3-phenylurea motif which showed activity
against a variety of mycobacteria.
pathogen.2,3 The Mtb cell wall biosynthesis pathways have
been under investigation as potential drug targets for
decades.4,5 Specifically, mycolic acids are key components of
the mycobacterial outer membrane/cell wall superstructure
(mycolylarabinogalactan peptidoglycan), and the biosynthesis
of these unique fatty acids has been targeted for drug
discovery since researchers identified mycolic acids as a major
biological barrier for drug entry and determined that mycolic
acid biosynthesis is the target of the first-line TB drug
isoniazid.6,7
The enzyme Rv3802c has gained attention as a potential
drug target.8-10 It is one of the enzymes encoded by an
invariant mycobacterial gene cluster that encompasses the
genes Rv3799c-Rv3809c. Each of these genes is responsible for
various aspects of mycolic acid synthesis and the transfer of
mycolic acids to trehalose or arabinogalactan.10 The location of
the Rv3802c gene within the mycolic acid synthesis gene
cluster suggests that the enzyme is involved in the cell wall
biosynthesis.10 The enzyme is named cutinase-like because of
sequence similarities with the cutinase family of enzymes and
is retained in the periplasm following secretion because it
possesses an N-terminal transmembrane helix that remains
embedded in the plasma membrane.11 Early Rv3802c studies
1. Introduction
The World Health Organization (WHO) declared tuberculosis
(TB) a global health emergency in 1993.1 The epidemic is the
leading cause of death from a single infectious agent with an
estimated 1.3 million deaths in 2017.1 The emergence of multi-
drug (MDR) and extensively drug resistant (XDR) TB strains has
exacerbated the crisis. In 2017, half a million people developed
TB that was resistant to the most effective first line drug,
rifampicin.1 The meeting held by the United Nations (UN) in
September 2018 titled "United to End TB: An Urgent Global
Response to a Global Epidemic" underlines the need for
improved treatment against TB. The current standard TB
treatment includes 2-6 months of first line and second line
drugs in different phases. The overwhelming global burden of
the disease makes identifying new drug targets a high priority.
The complex cell wall structure of Mycobacterium
tuberculosis (Mtb) plays a crucial role in the survival of the
a. Department of Chemistry and Biochemistry, University of Toledo, 2801 West
Bancroft Street, Toledo, Ohio 43606, USA.
b. Center for Therapeutic Discovery, Lerner Research Institute, Cleveland Clinic
Foundation, Cleveland, OH, 44195, USA
c. Mycobacteria Research Laboratories, Department of Microbiology, Immunology
and Pathology, Colorado State University, Fort Collins, USA
d.Department of Biological Sciences, University of Toledo, 2801 West Bancroft
Street, Toledo, Ohio 43606, USA.
indicated
that
the
enzyme
possesses
thioesterase/phospholipase A activity.10 The essential role of
Corynebacterium glutamicum ortholog NCgl2775 in regulation
of cell wall lipid composition in response to stress was
observed with elevated mycolate content.12 Conditional
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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disruption of the M. smegmatisis ortholog MSMEG_6394 has consisted of 1 μM enzyme and were initiated upon the
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shown the gene to be essential for mycobacterial viability.13 titration of 100 μM (Km of RfB = 57.4 ± D1O5.I5: 10μ.M103).9/BCo9tMhDA00g18252CK
The structure of the MSMEG_6394 ortholog has revealed an and Rv3802c assays were performed in triplicate. All assays
α/β hydrolase fold with 68.8% sequence identity to the Mtb were performed with respective compounds (500 µM) in a
encoded Rv3802c. The more recent determination of the Mtb sodium phosphate buffer at pH 7.5. Determination of covalent
Rv3802c crystal structure identified the lipid binding site and or non-covalent inhibition was assessed via reaction progress
showed that structural changes in the enzyme are required for curves and time dependent inhibition (SI).
substrate recognition and hydrolysis of the fatty acyl chains of
phosphatidylinositol, which is the anchoring lipid for Scheme 1. Synthesis of N-phenyl ureas 2a-g
mycobacterial lipoarabinomannin.14 Combined with the study
from Meniche et al.,12 it seems clear that Rv3802c is an
essential enzyme that hydrolyses phosphatidylinositol and
plays a role in regulating mycomembrane lipid content.
Another α/β hydrolase encoded by a gene in the invariant
Rv3799c-Rv3809c gene cluster is Antigen 85A, which is one of
the three highly-similar homologs composing what is termed
as the Antigen 85 (Ag85) complex.15 The three enzymes of the
Ag85 complex: Ag85A, Ag85B and Ag85C, each possess a Ser-
His-Glu catalytic triad essential for catalysing mycolyl transfer
to components of the mycobacterila cell wall.16 A variety of
Ag85 inhibitors have been reported. Substrate analogs such as
trehalose and AG derivatives17-21 along with compounds that
mimic the tetrahedral intermediate such as phosphonates and
sulfonates have been synthesized and screened against these
enzymes.22,23
A variety of heterocyclic urea derivatives have been
synthesized over the past few years against many hydrolases.
Proschak et al. reported a library of substituted diaryl ureas
against soluble epoxide hydrolase (sEH).24 Cravatt et al. have
also reported the use of 1,2,3-triazole ureas as a chemotype
for serine hydrolase inhibitors.25,26 We report herein an
evaluation of N-aryl urea derivatives for their ability to inhibit
the serine hydrolases Mtb Ag85C and Rv3802c. In addition, the
library was screened for minimal inhibitory concentration
(MIC) activity against a variety of mycobacterial species and
strains. To our surprise, the 1-(cyclohexylmethyl)-3-phenylurea
motif emerged as an antimycobacterial motif.
Enzyme
activity of
Rv3802c at
500 µM (%)
Yield (%)
Compounds
2a
2b
92
77
43.0 ± 1.9
53.0 ± 10.7
2c
2d
2e
92
96
95
38.2 ± 8.5
50.5 ± 5.7
73.1 ± 2.8
2. Results and discussion
2f
93
73
84.3 ± 17.3
56.2 ± 7.7
2.1 Synthesis and Evaluation
We synthesized a small library of N-phenyl ureas 2a-g
(Scheme 1) in order to evaluate their potential for serine
hydrolase inhibition. Phenyl isocyanate was added to a
solution of each respective amine in anhydrous toluene and
refluxed. The desired N-phenyl urea derivatives were
recrystallized after 1 hr with excellent yields (Scheme 1).
2g
The N-phenyl ureas 2a-g were screened for inhibitory
activity against Mtb Ag85C and Rv3802c. Mtb Ag85C and
Rv3802c were recombinantly expressed and purified as
previously reported.22 Activity of Ag85C was assessed using a
previously established assay.22 Briefly, reaction components
consisted of 500 nM enzyme and 4 mM trehalose, reactions
were initiated upon the titration of 100 μM resorufin butyrate
(RfB, Sigma Aldrich, 10 mM DMSO stock). Rv3802c activity was
initially assessed by monitoring the hydrolysis of RfB; reactions
Compounds 2a-g were found to selectively inhibit Rv3802c
while no inhibition of Ag85C was observed up to 500 µM
(Scheme 1). Linear progress curves indicated inhibition to be
non-covalent in nature. We suspect that the lack of irreversible
interaction is due to an inadequate leaving group ability of the
N-phenyl moiety and decided to include electron withdrawing
substituents on the phenyl isocyanate (Scheme 2). Only furan-
2-ylmethanamine and cyclohexanemethylamine were used for
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the next inhibitor series since these two amine derivatives Again, a moderate level of Rv3802c inhibition was observed
View Article Online
DOI: 10.1039/C9MD00122K
exhibited the best inhibitory activity in round I screening.
with the exception of 6c (Scheme 2). Compound 6c showed
88% inhibition of enzyme Rv3802c at 500 µM concentration
with a Ki = 5.2 ± 0.7 µM (Figure SI1). All progress curves were
linear, indicating equilibrium-based, non-covalent inhibition
except for compound 4a which was found to be highly reactive
to any free hydroxylate. No noticeable difference in inhibition
levels were observed if compounds, except 4a, were allowed
to incubate for 1 hour with respective enzyme prior to kinetic
reads, suggesting no evidence of slow covalent inhibition.
To investigate the structure activity relationship (SAR) of
Scheme 2. Synthesis of N-phenyl ureas 4a-e and 6a-c
6c, we incorporated imidazole and benzimidazole moieties (8c
and 8d, respectively) while retaining the N-3,5-difluorophenyl
moiety. We also synthesized secondary amines using reductive
amination of furfuraldehyde and 4-fluorobenzaldehyde in
combination with furfurylamine.27,28 The respective secondary
amines were reacted with 3,5-difluorophenyl isocyanates to
afford 8a-b (Scheme 3).
Enzyme
activity of
Rv3802c at
500 µM (%)
Yield (%)
Compounds
Scheme 3. Synthesis of N-3,5-difluorophenyl ureas 8a-d
4a
4b
4c
64
79
78
Too reactive
100
Enzyme
83.6 ±10.9
Yield
(%)
activity of
Rv3802c at
200 µM (%)
Compounds
4d
4e
74
81
66.0 ± 0.2
72.5 ± 3.5
8a
8b
70
81
100
100
8c
58
54
20.0 ± 1.2
100
6a
6b
6c
89
87
90
82.0 ± 3.8
54.9 ± 2.9
13.0 ±1.6
8d
Unfortunately, we did not observe any improvement in
activity compared to the initial hit 6c. Use of secondary amine
derivatives, 8a and 8b also eliminated the prior inhibitory
activity. The loss of inhibition may be attributed to the
increased steric demands of these derivatives.
Based on the reported success of triazole-ureas against
some lipases,25,26 we decided to synthesize triazole-urea
A
second
library
was
synthesized
using
cyclohexylmethylamine and furfurylamine combined with derivatives of cyclohexylmethylamine and furfurylamine as
various isocyanates (Scheme 2). The new series of compounds
(4a-e, 6a-c) was screened against Mtb Rv3802c and Ag85C.
well. Both amines were converted into isocyanates using
triphosgene followed by the coupling with commercially
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DOI: 10.1039/C9MD00122K
Scheme 4. Synthesis of triazole ureas 10a-c and 12a-b
Compounds
% yield
Inhibition against Rv3802c
Inhibition against Ag85C
Type of
inhibition
Enzyme
activity of
Rv3802c at
200 µM (%)
Kinact/Ki
Type of
inhibition
Enzyme
activity of
Ag85C at
Kinact/Ki
(x10-3 μM-
(x10-3 μM-
1Min-1)
1Min-1)
200 µM (%)
78
66
70
75
82
non-
covalent
43.0 ± 7.3
40.0 ± 4.1
-
-
covalent
covalent
covalent
-
2.3 ± 0.3
10a
10b
10c
12a
12b
non-
covalent
-
-
5.5 ± 0.4
covalent
1.6 ± 0.1
-
0.9 ± 0.1
non-
covalent
44.0 ± 6.6
72.5 ± 3.5
-
-
non-
covalent
80.0 ± 2.3
14.0 ± 2.9
-
-
non-
covalent
non-
covalent
titration of 4MH and obtained using λex = 360 nm and λem at
available triazoles to afford compounds 10a-c and 12a-b 450 nm on a Synergy H4 plate reader (Biotek). The 4MH assay
(Scheme 4).
provides superior signal to noise required to accurately
These compounds (10a-c and 12a-b) were initially screened determine kinact/Ki values.
at 200 μM (20 mM DMSO stock) with no pre-incubation
An interesting trend of inhibition was observed when the
period. Percent enzymatic activity was determined for non- N-3,5-difluorophenyl moiety was replaced with a series of
covalent inhibitors as previously described. Compounds that triazoles (Scheme 4). With the sterically hindered triazoles and
displayed progress curves that plateaued over time, indicating structurally rigid component such as a furan ring, non-covalent
covalent inhibition were serial diluted with DMSO and inhibition was observed against Rv3802c. But with the smaller
screened in triplicate at 150, 75, 37.5, and 18.75 μM with no triazole ring on one side and methylcyclohexanes on the other,
pre-incubation period. Following background subtraction, kobs compound 10c inhibited Rv3802c irreversibly with
a
were determined by fitting the reaction progress curves with a determined kinact/Ki value of 1.6 ± 0.1 x10-3 μM-1min-1.
one-phase association equation in PRISM 7. The kinact/Ki was Additionally, the inhibitory activity against Ag85C was
calculated by plotting the kobs as a function of inhibitor recovered with the triazole series of compounds. With the
푘푖푛푎푐푡
(
= 1 + (퐾i/[I])) in
methylfuran substituent, non-covalent inhibition was observed
concentration and fitting the data with kobs
against
Ag85C
while
derivatives
containing
PRISM 7 (Figure SI2 and SI3). Triazole compounds were
screened against Rv3802c using a different fluorometric
substrate than the one used for the Ag85C RfB assay. Briefly,
the previously published14 alternative assay uses 75 μM 4-
methylumbelliferyl heptanoate (4MH, Sigma Aldrich)(7.5 mM
DMSO stock) and 20 nM Rv3802c in 50 mM sodium phosphate
buffer pH 7.5 at 37°C. Kinetic reads were initiated upon
methylcyclohexanes ring showed irreversible inhibition (Figure
S2). The covalent inhibition of Ag85C with sterically hindered
triazoles 10a and 10b can be justified with the presence of a
larger active site in Ag85C compared to Rv3802c. The kinact/Ki
values of 10a and 10b are 2.3 ± 0.3 and 5.5 ± 0.4 × 10-3 µM-
1min-1 respectively.
2.2 MIC determination
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With a few active compounds in hand from enzyme
ARTICLE
chromatography (silica gel 60, f254) and spots were visualized
View Article Online
DOI: 10.1039/C9MD00122K
inhibition studies, we decided to evaluate MIC values of the
compound library against M. abscessus ATCC19977, Mtb
H37Rv mc26206, Mtb H37Ra ATCC25177, and M. smegmatis
mc2155 (Table SI1-SI3). A colorimetric resazurin microtiter
assay was used for Mtb and M. smegmatis while the M.
abscessus 96 well-plate was visually scanned for cell growth
after incubation.29 Surprisingly, compounds 4a, 4c and 4e had
better MIC values compared to the hits from enzyme inhibition
studies. The MIC values for each hit compounds are shown in
Scheme 1. The lack of activity of triazole-based covalent
inhibitors (10a-c) of serine hydrolases in whole cell assays may
suggest their inability to access the target enzyme. On the
other hand, the activity of hit compounds (Table 1) must be a
result of interactions with some other essential targets apart
from or in addition to Ag85C and Rv3802c.
under UV light. Flash column chromatography was performed
on
1-(cyclohexylmethyl)-3-(4-fluorophenyl)urea (4d) Yield: 74%
(136 mg);1H NMR (600 MHz, DMSO): δ 7.37 (m, 2H, H-4,6),
7.04 (t, J = 8.9 Hz, 2H, H-1,3), 2.92 (t, J = 6.3 Hz, 2H, H-11), 1.6-
1.70 (m, 5H, H-12,13,17), 0.84-1.38 (m, 6H, H-14, 15, 16). 13
C
NMR (600 MHz, DMSO): δ 157.56, 155.99, 155.30, 136.99,
119.00, 115.15, 45.29, 37.99, 30.34, 26.10, 25.43. Melting
point: 149-151 °C. HRMS data: [M + H] m/z: calcd for
C14H20FN2O, 251.1560; found, 251.1566.
1-(cyclohexylmethyl)-3-(3,5-difluorophenyl)urea (4e) Yield: 81%
(186 mg);1H NMR (600 MHz, CDCl3): δ 6.91 (dd, J = 9.1, 2.2 Hz,
2H, H-4, 6), 6.45 (m, 1H, H-2), 3.08 (m, 2H, H-11), 1.61-1.72 (m,
5H, H-12,13,17), 0.84-1.45 (m, 6H, H-14, 15, 16). 13C NMR (600
MHz, CDCl3): δ 164.39, 162.77, 155.38, 141.56, 102.38, 98.19,
46.86, 38.49, 31.00, 26.55, 25.98. Melting point: 120-122 °C.
HRMS data: [M + H] m/z: calcd for C14H19F2N2O, 269.1465;
found, 269.1471.
The cytotoxic effect of the active compound on L929
murine fibroblast cells was investigated by a MTT cell viability
assays and are represented by the given IC50 value.30 The
results indicated that all compounds tested in this study
exhibited an IC50 (against L929 indicator cells)close to 20 times
higher than the MIC (against TB) suggesting a reasonable
selectivity index as a starting point for further investigation
(Table 1).
1-(3,5-difluorophenyl)-3-(furan-2-ylmethyl)urea (6c) Yield: 90%
(186 mg);1H NMR (600 MHz, DMSO): δ 7.60 (dd, J = 1.7, 0.7 Hz,
1H, H-17), 7.14 (dd, J = 10.1, 2.2 Hz, 1H, H-2), 6.79 (m, 2H, H-4,
6), 6.41 (dd, J = 3.1, 1.7 Hz, 1H, H-16), 6.27 (dd, J = 3.1, 0.7 Hz,
1H, H-15), 4.30 (d, J = 5.7 Hz, 2H, H-11). 13C NMR (600 MHz,
DMSO): δ 163.46, 161.86, 154.50, 152.79, 143.09, 142.13,
110.49, 106.64, 100.48, 96.1, 36.1. Melting point: 123-125 °C.
HRMS data: [M + H] m/z: calcd for C12H11F2N2O2, 253.0789;
found, 253.0778.
Table 1. MIC values (µM) of hits against Mycobacterium sp.
Mtb
H37Rv
IC50
mc26206
against
L929
cells
Mabs
ATCC
19977
Msmeg
mc2155
Compounds
3-(3,5-difluorophenyl)-1,1-bis(furan-2-ylmethyl)urea (8a) Yield:
70% (68 mg);1H NMR (600 MHz, CDCl3): δ 7.43 (dd, J = 1.8, 0.7
Hz, 2H, H-23, 16), 7.00 (dd, J = 9.2, 2.2 Hz, 2 H, H-4, 6), 6.47 (m,
1H, H-2), 6.38 (dd, J = 3.2, 1.8 Hz, 2H, H-15, 22), 6.27 (dd, J =
3.1, 0.7 Hz, 2H, H-14,21), 4.53 (s, 4H, H-11, 19). 13C NMR (600
MHz, CDCl3): δ 164.25, 164.15, 162.62, 162.52, 154.87, 150.60,
142.97, 110.91, 109.03, 102.61, 102.41, 98.24, 43.48. Melting
point: 98-99 °C. HRMS data: [M + H] m/z: calcd for
C17H15F2N2O3, 333.1051; found, 333.1049.
(Mtb
H37Ra
ATCC
(µM)
25177)
H
H
N
N
O
O
N
O
3.12 (25)
>100
>100
15.6
57
56
4a
H
H
N
N
O
F3C
1.5
(12.5)
3-(3,5-difluorophenyl)-1-(4-fluorobenzyl)-1-(furan-2-
>100
4c
ylmethyl)urea (8b) Yield: 81% (72 mg); 1H NMR (600 MHz,
CDCl3): δ 7.44 (dd, J = 1.8, 0.7 Hz, 1H, H-16), 6.46-7.35 (m, 7H,
H-2, 4, 6, 21-25)6.37 (dd, J = 3.2, 1.8 Hz, 1H, H-15), 6.23 (dd, J =
H
H
F
N
N
3.2, 0.7 Hz, 1H, H-14), 4.57 (s, 2H, H-19), 4.44 (s, 2H, H-11). 13
C
O
3.12 (25)
25
62.5
70
F
NMR (600 MHz, CDCl3): δ 164.28, 163.45, 162.65, 162.55,
161.81, 155.14, 150.36, 143.16, 141.58, 129.59, 129.54,
116.15, 116.01, 110.98, 109.10, 102.65, 102.45, 98.36, 50.11,
43.74. Melting point: 142-143 °C. HRMS data: [M + H] m/z:
calcd for C19H16F3N2O2, 361.1164; found, 361.1157.
4e
3. Experimental
1-((1H-benzimidazol-2-yl)methyl)-3-(3,5-difluorophenyl)urea
(8c) Yield: 58% (355 mg); 1H NMR (600 MHz, DMSO): δ 7.76 (m,
2H, H-19, 22), 7.51 (dd, J = 9.2, 2.2 Hz, 2 H, H-4, 6), 7.30 (m, 1H,
H-2), 7.16 (m, 2H, H-20, 21), 4.79 (d, 5.52, 2H, H-11). 13C NMR
(600 MHz, DMSO): δ 163.48, 163.38, 161.88, 161.77, 155.17,
3.1 General Method
All fine chemicals were obtained from Acros Organics, Alfa
Aesar, Oakwood chemicals or Sigma-Aldrich. The solvents were
purified using a PureSolv MD5 Solvent Purification System
(SPS). Reactions were monitored using thin-layer
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153.23, 142.78, 131.40, 125.45, 114.03, 100.67, 100.47, 96.59, the hydrolysis of RfB; reactions consisted of 1 μM enzyme and
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DOI: 10.1039/C9MD00122K
79.49, 36.56. Melting point: 92-94 °C. HRMS data: [M + H] m/z: were initiated upon the titration of 100 μM (Km of RfB = 57.4 ±
calcd for C15H13F2N4O, 303.1057; found, 303.1055.
15.5 μM). Both Ag85C and Rv3802c assays were performed in
triplicate in 50 mM sodium phosphate buffer pH 7.5 at 37 °C.
(8d) Determination of covalent or non-covalent inhibition was
1-((1H-imidazol-2-yl)methyl)-3-(3,5-difluorophenyl)urea
Yield: 54% (280 mg); 1H NMR (600 MHz, DMSO): δ 7.13 (dd, J = assessed via reaction progress curves and time dependent
9.2, 2.2 Hz, 2 H, H-4, 6), 6.75 (m, 1H, H-2), 6.70 (m ,2H, H-15, inhibition.32
16), 4.29 (d, J = 5.7 Hz, 2H, H-11). 13C NMR (600 MHz, DMSO):
Compound series 2, 4, and 6 were dissolved in DMSO for a
δ 163.49, 161.89, 154.68, 145.10, 143.14, 100.43, 100.23, stock concentration of 50 mM for initial inhibition screening at
96.08, 37.13. Melting point: 204-206 °C. HRMS data: [M + H] 500 μM in triplicate. The tested compounds were incubated
m/z: calcd for C11H11F2N4O, 253.0901; found, 253.0898.
with respective enzyme for 15 minutes at room temperature
prior to kinetic reads, which were initiated upon addition of
3.3 General procedure for synthesis of urea derivatives (10a- resorufin butyrate (RfB). Kinetic reads were obtained using λex
c, 12a-b) = 500 nm and λem at 590 nm on a Synergy H4 plate reader
Triphosgene (1.1 equiv.) was added to a solution of amine in (Biotek). Reaction rates were calculated using a linear fit
anhydrous toluene at 0 °C. The reaction was refluxed overnight between time points 2 to 6 minutes, with reads every 30
and appeared complete by TLC. The solvent was evaporated seconds. Following background subtraction, percent enzyme
and the residue was dissolved in EtOAc (20 mL). The reaction activity was determined by (Vo/Vo’)*100 where Vo is inhibited
mixture was then washed with H2O (×2, 15 mL) and organic reaction and Vo’ is uninhibited reaction. All progress curves
layer was collected, dried and used without further were linear, indicating equilibrium based, non-covalent
purification. Isocyanate intermediates were dissolved in dry inhibition except for compound 4a which was found to be
DCM (10 mL) followed by addition of the respective secondary highly reactive to any free hydroxylate. No noticeable
amines. The reactions were stirred under N2 at RT. The difference in inhibition levels were observed if compounds,
reactions were monitored by TLC and appeared complete after except 4a, were allowed to incubate for 1 hour with respective
15 h. The solvent was evaporated and the residue was enzyme prior to kinetic reads, suggesting no evidence of slow
subjected to flash column chromatography on silica gel covalent inhibition.
Compound 6C was serial diluted from 50 mM to 156 μM in
DMSO for KI determination. The respective concentrations of
6c were added to Rv3802c (1 % v/v final) in triplicate and
allowed to incubate for 15 minutes at RT prior to addition of
RfB and initiation of the kinetic read. The reaction rates were
determined as previously stated with the background
subtracted. The resulting rates were plotted as a function of
inhibitor concentration and the Ki determined by fitting the
data with the Morrison Ki equation in PRISM 7 (Figure S1).
The series 8 compounds were screened in triplicate at 200
μM (20 mM DMSO stock) and allowed to incubate with
enzyme 15 minutes at room temperature prior to initiation of
the reaction by adding RfB. Data were acquired and processed
in an identical fashion to that of compound series 2, 4, and 6.
All progress curves were linear, indicating equilibrium based,
non-covalent inhibition. No noticeable difference in inhibition
levels were observed if compounds were allowed to incubate
for 1 hour with respective enzyme prior to initiation of the
kinetic reads, suggesting no evidence of slow covalent
inhibition.
(hexanes:EtOAc) to afford the desired products.
N-(cyclohexylmethyl)-3-(methylthio)-4H-1,2,4-triazole-4-
carboxamide (10b) Yield: 66% (35 mg); 1H NMR (600 MHz,
CDCl3): δ 8.76 (s, 1H, H-10), 3.26 (t, J = 6.6 Hz, 2H, H-4), 2.63 (s,
3H, H-12), 1.61-1.72 (m, 5H, H-5,13,17), 0.84-1.45 (m, 6H, H-
14, 15, 16). 13C NMR (600 MHz, CDCl3): δ 164.28, 144.38,
46.76, 38.14, 30.85, 26.44, 25.90, 14.50. Melting point: 102-
104 °C. HRMS data: [M + Na] m/z: calcd for C11H18NaN4OS,
277.1099; found, 277.1090.
N-(cyclohexylmethyl)-1H-1,2,3-triazole-1-carboxamide
(10c)
Yield: 70% (42 mg); 1H NMR (600 MHz, CDCl3): δ 8.29 (d, J = 1.2
Hz, 1H, H-1), 7.77 (d, J = 1.2 Hz, 1H, H-2), 3.35 (t, J = 6.6 Hz, 2H,
H-9), 1.61-1.72 (m, 5H, H-5, 10, 11, 15), 0.84-1.45 (m, 6H, H-12,
13, 14). 13C NMR (600 MHz, CDCl3): δ 147.56, 134.58, 122.57,
47.09, 38.09, 30.84, 26.43, 25.88. Melting point: 115-117 °C.
HRMS data: [M + H] m/z: calcd for C10H17N4O, 209.1402; found,
209.1410.
3.3 In vitro Inhibition Assays
Mtb Ag85C and Rv3802c were recombinantly expressed
and purified as previously reported.31 Following purification,
both enzymes were dialyzed into a 50 mM sodium phosphate
pH 7.5 buffer for all inhibition assays. Activity of Ag85C was
Conclusions
We synthesized a library of N-aryl urea derivatives that was
evaluated against two mycobacterial hydrolases. The furan-
based urea derivative 6c inhibited Rv3802c selectively,
exhibiting a Ki value of 5.2 ± 0.7 µM in a non-covalent fashion.
On the other hand the 1,2,3-triazole derivative 10c showed
irreversible inhibition of Ag85C with a kinact/Ki value of 1.6 ± 0.1
assessed using
a
previously established assay.31 Briefly,
reaction components consisted of 500 nM enzyme and 4 mM
trehalose, reactions were initiated upon the addition of 100
μM resorufin butyrate (RfB, Sigma Aldrich)(10 mM DMSO
stock). Rv3802c activity was initially assessed by monitoring
x10-3 μM-1min-1. The presence of
a 2-methylfuran or
6 | J. Name., 2012, 00, 1-3
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ARTICLE
methylcyclohexane ring on the triazole ureas affected the 8. Saravanan, P.; Avinash, H.; Dubey, V. K.; Patra, S., J. Mol.
View Article Online
DOI: 10.1039/C9MD00122K
mode of inhibition against of Ag85C, where the
Graph. Model. 2012, 38, 235-242.
methylcyclohexane-containing derivatives bind Ag85C 9. West, N. P.; Cergol, K. M.; Xue, M.; Randall, E. J.; Britton,
irreversibly. The MIC values of compounds 4a, 4c, and 4e W. J.; Payne, R. J.; Chem. Comm. 2011, 47, 5166-5168.
against Mtb H37Rv mc26206 are observed to be in the range of 10. Parker, S. K.; Barkley, R. M.; Rino, J. G.; Vasil, M. L. PloS
1.5-6.25 µM. The structures all share common 1- one 2009, 4, e4281.
(cyclohexylmethyl)-3-phenylurea motif which may represent 11. West, N. P.; Chow, F. M.; Randall, E. J.; Wu, J.; Chen, J.;
a
the active pharmacophore. However, the antibacterial activity
Ribeiro, J. M.; Britton, W. J. The FASEB J. 2009, 23,
of 4a, 4c and 4e appears to be largely independent of the
1694-1704.
Rv3802c and Ag85C enzyme inhibitory activity. This distinction 12. Meniche, X.; Labarre, C.; De Sousa-D'Auria, C.; Huc, E.;
can be indicative of a different target and mechanism of action
of these compounds. The hit compounds were evaluated for
Laval, F.; Tropis, M.; Bayan, N.; Portevin, D.; Guilhot, C.;
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the cytotoxicity against
a
mammalian cell line and 13. Crellin, P. K., Vivian, J. P., Scoble, J., Chow, F. M., West,
demonstrated good selectivity. These hits can potentially be
converted into probes for mycobacterial target identification
and used as tools in chemical genomics-based experiments.
N. P., Brammananth, R., Proellocks, N. I., Shahine, A., Le
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R. J. Biol. Chem. 2018, 293, 1363-1372.
Conflicts of interest
15. Ronning, D. R.; Klabunde, T.; Besra, G. S.; Vissa, V. D.;
There are no conflicts to declare.
Belisle,
J. T.; Sacchettini, J. C. Nat. Struct. Mol. Biol.
2000, 7, 141-146.
16. Thanna, S.; Sucheck, S. J. MedChemComm. 2016, 7, 69-85.
17. Belisle, J. T.; Vissa, V. D.; Sievert, T.; Takayama, K.;
Brennan, P. J.; Besra, G. S. Science 1997, 276, 1420-1422.
18. Rose, J. D.; Maddry, J. A.; Comber, R. N.; Suling, W. J.;
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19. Sanki, A. K.; Boucau, J.; Srivastava, P.; Adams, S. S.;
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Notes
AUTHOR INFORMATION
Corresponding Authors
*E-mail: donald.ronning@utoledo.edu;
steve.sucheck@utoledo.edu.
Author Contributions
SJS and AV designed and synthesized the compounds and wrote
that portion of the paper. CG, CMS, and DRR performed Ag85C and
Rv3802c isolation and enzyme inhibition studies and wrote that
portion of the paper. MJ, AV, and AL performed MIC studies and
edited key aspects of the paper. BL and JD performed the MTT
assay.
Bioorganic
Med.Chem. 2004, 12, 6397-6413.
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24. Buscató, E. l.; Blöcher, R.; Lamers, C.; Klingler, F.-M.;
Funding Sources
This work was supported by the National Institutes of Health Grant
AI105084 to SJS and DRR and Grant AI135313 to SJS.
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DOI: 10.1039/C9MD00122K
N-aryl urea derivatives were synthesized and some showed activity against mycobacterial hydrolases
while others showed antimicrobial activity against mycobacterial species