Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB

Article abstract of DOI:10.1021/acsinfecdis.7b00112

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure-activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc₁ oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis.

Full text of DOI:10.1021/acsinfecdis.7b00112

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Article
Improved phenoxyalkylbenzimidazoles with activity
against Mycobacterium tuberculosis appear to target QcrB
N. Susantha Chandrasekara, Bryan J berube, Gauri Shetye, Somsundaram Chettiar,
Theresa O'Malley, Alyssa Manning, Lindsay Flint, Divya Awasthi, Thomas R Ioerger, James
C. Sacchettini, Thierry Masquelin, Philip A. Hipskind, Joshua Odingo, and Tanya Parish
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00112 • Publication Date (Web): 16 Oct 2017
Downloaded from http://pubs.acs.org on October 17, 2017
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Improved phenoxyalkylbenzimidazoles with activity against Mycobacterium tuberculosis appear
to target QcrB
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a
a
a
a
a
N. Susantha Chandrasekera, Bryan J. Berube, Gauri Shetye, Somsundaram Chettiar, Theresa
a
a
a
b
b
O’Malley, Alyssa Manning, Lindsay Flint, Divya Awasthi, Thomas R. Ioerger, James
c
c
a
a
Sacchettini, Thierry Masquelin, Philip A. Hipskind, Joshua Odingo, and Tanya Parish*
a
TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Avenue E,
Seattle, WA 98102, USA.
b
c
Departments of Computer Science and Engineering and Biochemistry and Biophysics, 301
Tarrow Street, Texas A&M University, College Station, TX 77843, USA.
c
Lilly Research Laboratories, 307 E Merrill St, Indianapolis, IN 46285, USA.
*
Corresponding author
TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Avenue E, Seattle,
WA 98102, USA.
Tel 206 858 6074
Fax 206 381 3678
Email tanya.parish@idri.org
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The phenoxy alkyl benzimidazoles (PABs) have good antiꢀtubercular activity. We expanded our
structureꢀactivity relationship studies to determine the core components of PABs required for
activity. The most potent compounds had minimum inhibitory concentrations against M.
tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells, as
well as activity against intracellular bacteria. We isolated resistant mutants against PAB
compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component
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of the cytochrome bc oxidase of the electron transport chain. QcrB mutant strains were resistant
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to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset,
suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for
the improved design of novel compounds to target intracellular M. tuberculosis.
Key Words:
Tuberculosis, benzimidazole, respiratory inhibitors, drugs
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Mycobacterium tuberculosis was a leading cause of death by infectious disease in the
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world in 2015 . Contributing to over 1.4 million deaths and infecting an estimated oneꢀthird of
the population, M. tuberculosis is a leading global health problem with major social and
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economic burdens . Over the last decade, there has also been an alarming increase in multidrugꢀ
resistant (MDRꢀTB) and extensively drugꢀresistant (XDRꢀTB) strains that are refractory to
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conventional therapeutics . Because of this, there is a dire need for novel drugs and
preventatives to combat this disease.
The last few years have seen promising leads for new antiꢀtuberculosis drugs with several
advancing to clinical trials. One series in the early stages of development, the phenoxy alkyl
benzimidazoles (PAB), has shown good promise with minimum inhibitory concentrations
(MICs) against M. tuberculosis in the nanomolar range, sterilizing activity under starvation
2
conditions, and low cytotoxicity against eukaryotic cells .
In this study, we further characterized the SAR for the PAB series and synthesized
compounds with antiꢀtubercular activity in the low nanomolar range, good cytotoxicity profiles,
and intracellular activity against M. tuberculosis. We also identified the probable target of PAB
compounds as QcrB – a component of the cytochrome bc oxidase of the electron transport
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chain. This knowledge will enable the elucidation of the mechanism of action for the strategic
design of new compounds and combination therapies to treat M. tuberculosis infection.
RESULTS AND DISCUSSION
PAB Structure-Activity Relationship
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The alkyl benzimidazoles were synthesized according to Scheme 1. Condensation of
appropriate 1,2ꢀdiaminobenzene derivative with propionic acid yielded the benzimidazole
intermediate (1) upon heating. Alkylation of the benzimidazole intermediate (1) was
accomplished by reacting with dibromoalkane to form Nꢀ(bromoalkyl)benzimidazole (2). This in
turn was treated with the corresponding anilines, thiophenols and phenols to yield the
corresponding benzimidazole alkylamine, alkyl thioether and alkyl ethers.
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Scheme 1: Synthesis of amine and thioether aryl, hetero aryl, cycloalkyl benzimidazoles.
In our previous study, we observed that compounds with heteroatoms (O, S, and N) as part of the
linker were tolerated, a linker length of three to four carbons was favored, and ethyl at the 2ꢀ
position and methyl at the 6ꢀposition on the benzimidazole were the most favorable substituents
2
on the core . We first investigated aryl and heteroaryl thioethers for their activity against M.
2
tuberculosis. Since compound 4 from our previous work showed good activity (Table 1) , we
made analogs (6 – 18) to explore the SAR around the phenyl group of 4. In our previous studies
on aryl ethers, we observed that the 4ꢀmethyl substitution improved both activity and selectivity.
In the case of the aryl thioether analogs, 4ꢀmethyl substitution (6) did not show any improvement
in activity, while 4ꢀmethoxy substitution (7) decreased activity by 4ꢀfold (MIC = 4.8 µM).
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Replacement of the phenyl with biaryls or a heteroaryl ring, such as naphthalene (8), quinoline
(9), quinazoline (10), benzothiazole (11 & 12) or benzimidazole (13 & 14) decreased activity
while replacement with thiazole (15 & 16) or tetrazole (17) resulted in no activity (MIC >20
µM). The 4ꢀphenylthiazole (18) was the most active thioether analog with an MIC of 0.73 µM.
However, it was fairly cytotoxic to eukaryotic cells (TC = 9.7 µM), despite the selectivity
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5
0
index (SI = 13) being similar to the earlier compounds.
Table 1: Effect of phenyl replacements on biological activity
`
N
N
N
N
R
SR
3-5
6-18
MIC99a
b
TC50
c
Compound
Name
R-group
SI
(µM)
(µM)
d
3
IDRꢀ0333819
IDRꢀ0341914
IDRꢀ0351544
IDRꢀ0357446
IDRꢀ0458791
IDRꢀ0357447
IDRꢀ0357452
IDRꢀ0357451
Phenoxy
Phenylthio
Anilinyl
1.1 ± 0.4
1.4 ± 0.3
21 ± 7.1
31 ± 8.3
19
22
89
d
4
d
5
0.47 ± 0.09 42 ± 7.8
6
7
4ꢀMethylphenyl
1.4 ± 0.56 19 ± 0.71 14
4ꢀMethoxyphenyl
2ꢀNaphthyl
4.8 ± 0.1
4.1 ± 1.3
18 ± 2.6
10 ± 0
10 ± 2.1
6.0 ± 4.4
>20
34 ± 12.8
10 ± 0
7
2
8
9
2ꢀQuinolinyl
7 ± 0
0.4
1
10
2ꢀQuinoxalinyl
13 ± 0
1
1
IDRꢀ0351547 6ꢀEthoxyꢀ2ꢀbenzothiazolyl
IDRꢀ0351549 6ꢀChloroꢀ2ꢀbenzothiazolyl
12 ± 0.7
18 ± 2.2
1
12
3
1
1
1
1
3
4
5
6
IDRꢀ0351556
2ꢀBenzimidazolyl
18 ± 2.8 NC
16 ± 2.1 NC
IDRꢀ0351555 Nꢀmethylꢀ2ꢀbenzimidazolyl
>20
IDRꢀ0351552
IDRꢀ0351550
IDRꢀ0351557
IDRꢀ0351553
2ꢀThiazolyl
>20
>50
>50
>50
9.7
NC
NC
NC
13
5ꢀMethylꢀ2ꢀthiazolyl
NꢀMethylꢀ5ꢀtetrazolyl
4ꢀPhenylꢀ2ꢀthiazolyl
>20
17
>20
1
8
0.73
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a
MIC99 is the minimum concentration required to completely inhibit growth of M. tuberculosis in liquid culture.
b
MICs of active compounds are the average of two independent experiments ± standard deviation. TC50 is the
concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments
c
d
±
standard deviation. Selectivity index (SI) is TC /MIC. NC ꢀ not calculated. Data for compounds 3, 4, 5 are from
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compounds 6, 53, 54, respectively in .
In our previous SAR study, we observed that replacing the phenyl ether in compound 3 with 5ꢀ
2
(4ꢀchlorophenyl)oxadiazolꢀ2ꢀyl thioether as in 19 improved activity . We attempted to improve
this further by modifying the linker length (Table 2). Reduction of the linker length from four
carbons to three (20) or two (21) was detrimental to the antiꢀtubercular activity; in the case of the
propyl (20) linker, we observed a 39ꢀfold reduction in activity and further reduction of the linker
to an ethyl (21) resulted in complete loss of activity. The addition of a weaker electronꢀreleasing
group such as the methyl group on the 6ꢀposition of the benzimidazole core (22) retained good
potency (MIC = 0.085 µM). The combination of a 6ꢀmethyl on the benzimidazole core and a
propyl linker (23) was slightly less active in comparison (MIC =0.34 µM). Interestingly,
replacing the 6ꢀmethyl with 6ꢀmethoxy, a strong electronꢀdonating group, in compound 24,
reduced activity by almost 10ꢀfold (MIC = 2.5 µM).
Table 2: Effect of N-alkyl chain length and benzo substitution on biological activity.
MIC99a
TC50b
c
Compound
Name
Structure
SI
(µM)
(µM)
d
1
9
IDRꢀ0351551
0.10 ± 0.07
22 ± 9.2 220
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IDRꢀ0390229
IDRꢀ0351587
3.9 ± 0.21
>20
21 ± 2.1
5
35 ± 2.8 NC
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22
IDRꢀ0390228
IDRꢀ0390226
0.085 ± 0.007
0.34 ± 0.19
18 ± 0
212
2
2
3
4
21 ± 2.8 62
49 ± 2.6 20
IDRꢀ0458783
2.5 ± 0.2
a
MIC99 is the minimum concentration required to completely inhibit growth of M. tuberculosis in liquid culture.
b
MICs of active compounds are the average of two independent experiments ± standard deviation. TC50 is the
concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments
c
d
±
standard deviation. Selectivity index (SI) is TC /MIC. NC ꢀ not calculated. Data for compound 19 is from
50
2
compound 68 in .
We next explored the influence of nitrogen as the heteroatom in the linker (Table 3). We
synthesized compounds (25 ꢀ 35) to investigate the electronic and steric effects of different
aromatic substituents on the terminal aniline. The addition of the electron donating groups
methyl (25) or methoxy (26), made no improvement in activity, although the methyl analog was
slightly more active. A trifluoromethyl substituent at the meta position (27) had a comparable
MIC to compound 5. However, the cytotoxicity was significantly worse than that of 5 and the
selectivity was 8ꢀfold lower. The 3,5ꢀdimethoxy (28) lost activity, whereas the 3ꢀhalo and 4ꢀ
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methyl substituted anilines (29,30) showed comparable activity to that of 5. The substituents 3ꢀ
benzyloxy (31) and 4ꢀpyridyl (32) showed similar activity to that of 5, but the 3ꢀpyridyl (33) was
much less active (MIC = 3.7 µM). Moving from the aniline (5) to benzylamine (34) was
detrimental to the antiꢀtubercular activity. Replacing the terminal aniline with a morpholino
group also resulted in loss of activity (35).
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0
Table 3: Effect of anilinyl substitution on the biological activity.
MIC99a
TC50b
c
Compound
Name
R
SI
(µM)
(µM)
d
5
IDRꢀ0351544
IDRꢀ0357431
IDRꢀ0458786
IDRꢀ0351602
IDRꢀ0351605
Anilinyl
0.47 ± 0.09
0.50 ± 0.1
1.4 ± 1.1
1.1 ± 0.9
4.4 ± 1.5
0.45 ± 0.07
0.35 ± 0.07
0.60 ± 0
0.80 ± 0.7
3.7 ± 0.8
>20
42 ± 7.8
39 ± 1.4
>100
89
78
>71
11
5
2
2
2
5
6
7
4ꢀMethylanilinyl
4ꢀMethoxyanilinyl
3ꢀTrifluoromethylanilinyl
3,5ꢀDimethoxyanilinyl
12 ± 2.6
21 ± 4.6
17 ± 0
28
2
3
9
0
IDRꢀ0351599 3ꢀBromoꢀ4ꢀmethylanilinyl
IDRꢀ0458790 3ꢀChloroꢀ4ꢀmethylanilinyl
38
151
23
28
5
53 ± 27.0
14 ± 3.8
22 ± 2.1
19 ± 3.8
>50
31
IDRꢀ0351600
IDRꢀ0351601
IDRꢀ0351606
IDRꢀ0351603
IDRꢀ0357457
3ꢀBenzyloxyanilinyl
4ꢀ(2ꢀPyridyl)anilinyl
3ꢀ(2ꢀPyridyl)anilinyl
NꢀBenzylamino
3
3
3
3
2
3
4
5
NC
NC
NꢀMorpholinyl
>20
>100
a
MIC99 is the minimum concentration required to completely inhibit growth of M. tuberculosis in liquid culture.
b
MICs of active compounds are the average of two independent experiments ± standard deviation. TC50 is the
concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments
c
d
± standard deviation. Selectivity index (SI) is TC /MIC. NC ꢀ not calculated. Data for compound 5 is from
50
2
compound 54 in .
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ꢀBromoꢀNꢀ(3ꢀ(2ꢀethylꢀ6ꢀmethylꢀ1Hꢀbenzo[d]imidazolꢀ1ꢀyl)propyl)ꢀ3ꢀmethylaniline, compound
2
2
62 from Chandrasekera et al. was the most potent and selective in our previous study . This
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compound contained only three carbons in the lipophilic linker. We prepared additional analogs
36 - 43 to understand the SAR surrounding the phenyl region of threeꢀcarbon tethered
benzimidazole molecules. The phenyl ether analog (36) had good activity (MIC = 5.2 µM) but
the corresponding aniline analog (37) was 10ꢀfold more active. Electron withdrawing 4ꢀmethyl
substitution on phenyl ether (38) or thioether (39) analogs improved activity. The aniline analog
with a 4ꢀmethyl group (40) also showed good activity (MIC = 0.12 µM) and selectivity (SI =
258). In comparison, double substitution to the more electronꢀrich 3,4ꢀdimethylaniline analog
(41) slightly reduced activity but a combination of electronꢀwithdrawing group at 3ꢀposition and
methyl at 4ꢀposition (42) resulted in the most potent of all the alkyl benzimidazoles (Table 4)
with a MIC of 0.040 µM and good selectivity (SI = 275). Interestingly, a 3ꢀchlorine substitution
instead of the 3ꢀbromine substitution was ~10ꢀfold less active (MIC = 0.55 µM).
Table 4: Effect of phenyl ring substitutions on biological activity.
MIC99a
b
TC50
(µM)
c
Compound
Name
R
SI
(µM)
d
3
6
IDRꢀ0257287
IDRꢀ0357449
IDRꢀ0357444
IDRꢀ0357448
Phenoxy
Anilinyl
5.2 ± 1.9
0.45 ± 0.2
1.6 ± 0.2
4.5 ± 0.8
>50
>10
64
9
3
7
8
29 ± 4.9
14 ± 0.7
24 ± 2.8
3
4ꢀMethylphenoxy
4ꢀMethylphenylthio
39
5
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0
IDRꢀ0357450
IDRꢀ0357442
IDRꢀ0357458
4ꢀMethylanilinyl
3,4ꢀDimethylanilinyl
3ꢀBromoꢀ4ꢀmethylanilinyl
0.12 ± 0.07
31 ± 3.5
>100
258
>154
275
29
41
0.65 ± 0.4
0.04 ± 0
4
4
2
3
11 ± 0.7
16 ± 0
IDRꢀ0357443 3ꢀChloroꢀ4ꢀmethylphenoxy 0.55 ± 0.35
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
MIC99 is the minimum concentration required to completely inhibit growth of M. tuberculosis in liquid culture.
b
MICs of active compounds are the average of two independent experiments ± standard deviation. TC50 is the
concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments
c
d
2
±
standard deviation. Selectivity index (SI) is TC /MIC. Data for compound 36 is from compound 5 in .
50
We then investigated the effect of 6ꢀmethyl substitution on the benzimidazole core bearing a 4ꢀ
carbon tether to various aromatics (Table 5). The aniline (44), phenyl thioether (45) and phenyl
ether (46) had comparable activities. 4ꢀmethoxy substitution on phenyl ether (47) or aniline (48)
had minimal effect on activity, but this substitution resulted in 10ꢀfold shift in activity of a
corresponding phenyl thioether (49). Both 3ꢀchloroaniline (50) and 4ꢀchlorophenyl thioether (53)
were less active but a 3ꢀtrifluoromethyl substitution on aniline (51) retained all activity of parent
compound 44. We also tested 3ꢀbenzyloxy aniline (52) with good activity.
Table 5: Effect of phenyl SAR on biological activity.
MIC99a
b
TC50
(µM)
c
Compound
Name
R
SI
(µM)
d
4
4
IDRꢀ0357433
IDRꢀ0341934
Anilinyl
0.32 ± 0.09
0.22 ± 0.02
22 ± 0.7
9.5 ± 0.7
69
43
4
5
Phenylthio
1
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d
4
6
IDRꢀ0341930
IDRꢀ0458750
IDRꢀ0458784
IDRꢀ0458789
IDRꢀ0357439
Phenoxy
0.27 ± 0.09
18 ± 2.5
27 ± 6.4
14 ± 2.1
20 ± 2.1
16 ± 9.0
67
68
22
9
4
7
4ꢀMethoxyphenoxy
4ꢀMethoxyanilinyl
4ꢀMethoxyphenylthio
3ꢀChloroanilinyl
0.4 ± 0.1
0.65 ± 0.07
2.3 ± 0.4
48
4
5
9
0
2.1 ± 0.9
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
5
1
2
IDRꢀ0357436
3ꢀTrifluoromethylanilinyl
0.55 ± 0.4
5.7 ± 1.5
10
IDRꢀ0357435
IDRꢀ0357445
IDRꢀ0578347
IDRꢀ0390234
IDRꢀ0458788
IDRꢀ0390235
IDRꢀ0458745
3ꢀBenzyloxyanilinyl
4ꢀChlorophenylthio
Anilinyl
1.8 ± 0.76
2.6 ± 0.6
18 ± 0.7
16 ± 1.4
> 50
10
6
53
5
5
4
5
0.056 ± 0.020
0.066 ± 0.018
0.35 ± 0.2
>893
470
260
200
38
4ꢀMethylanilinyl
4ꢀMethoxyanilinyl
2,4ꢀDimethylanilinyl
4ꢀMethoxyphenylthio
31 ± 0.7
91 ± 12.3
14 ± 0.7
33 ± 11
56
5
5
7
8
0.070 ± 0.03
0.88 ± 0.2
a
MIC99 is the minimum concentration required to completely inhibit growth of M. tuberculosis in liquid culture.
b
MICs of active compounds are the average of two independent experiments ± standard deviation. TC50 is the
concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments
c
d
± standard deviation. Selectivity index (SI) is TC /MIC. Data for compounds 44 and 46 are from compound 55
50
2
and 34, respectively in .
We finally probed the effect of combination of the optimized residues, namely the threeꢀcarbon
linker and a 6ꢀmethyl substitution on the benzimidazole core (54 – 58). Four compounds had a
selectivity index of > 200 (54, 55, 56 & 57) (Table 5). The aniline analogs 54, 55 and 57 had the
best activities with comparable MICs of 0.061 µM, 0.067 µM and 0.070 µM, respectively. While
5
4 had no cytotoxicity at 50 µM, the addition of one (55) or two (56) methyl groups on the
phenyl ring caused a stepꢀwise increase in cytotoxicity towards eukaryotic cells, thus reducing
the selectivity index from > 819 (54) to 470 and 200 for 55 and 57, respectively (Table 5). The
methoxy analog (56) was 5ꢀfold less active (Table 5).
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Several phenoxy alkyl imidazoles 59 – 68 were evaluated to study the importance of the
benzimidazole core for the antiꢀtubercular activity of PAB (Table 6). Many of the compounds
lost activity (59, 60, 64 & 67), while others maintained only moderate activity compared to
benzimidazole variants (61, 62, 63, 65, 66 & 68). These data show the benzene of the
benzimidazole core is not strictly required; several imidazole variants maintain moderate activity
against M. tuberculosis. However, the benzene ring does appear to confer optimal antiꢀ
tubercular activity to PAB compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6: SAR of phenoxy alkyl imidazole analogs with varying linker length.
MIC99a
b
TC50
c
Compound
Name
SI
(µM)
(µM)
5
9
IDRꢀ0122293
IDRꢀ0124213
IDRꢀ0124480
IDRꢀ0257359
IDRꢀ0262047
IDRꢀ0262049
> 20
> 20
20
> 50
NC
NC
0.8
3
6
6
0
1
ND
16 ± 0
35 ± 11
21
62
11 ± 2.3
20
6
6
3
4
1
> 20
ND
NC
65
66
IDRꢀ0262053
IDRꢀ0155469
20
40
2
2
9.9 ± 1.7
19 ± 9.3
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6
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IDRꢀ0123367
IDRꢀ0168354
> 20
89 ± 2.5
15 ± 0.3
NC
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
68
8.6 ± 0.9
a
MIC99 is the minimum concentration required to completely inhibit growth of M. tuberculosis in liquid culture.
b
MICs of active compounds are the average of two independent experiments ± standard deviation. TC50 is the
concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments
c
± standard deviation. Selectivity index (SI) is TC /MIC. ND – not determined. NC ꢀ not calculated.
50
PAB compounds target QcrB
To identify the target of PAB compounds, we isolated resistant mutants against three different
PABs. Of the seven confirmed mutants, five contained mutations in qcrB, the cytochrome b
subunit of the cytochrome bc oxidase (Rv2196), while two of the three mutants raised against
1
compound 4 contained mutations in Rv1339, a gene of unknown function (Table 7).
Table 7: Resistant mutant strains isolated against PAB compounds.
Compound
3
Name
Structure
Gene
Mutation
N
N
IDRꢀ0333819
qcrB
A179P
N
N
O
S
Rv1339
qcrB
V90M
A179P
Y94C
4
IDRꢀ0341914
IDRꢀ0351551
Rv1339
qcrB
M342T
W312C
W312G
1
9
qcrB
qcrB
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We confirmed targeting of QcrB by PABs by testing the MIC of 30 active compounds against
LPꢀ0351551ꢀRM1 (QcrBM342T), one of the isolated resistant mutants. All compounds tested
showed a minimum 5ꢀfold shift in MIC compared to the parental strain with 78% of the
compounds having at least a 10ꢀfold higher MIC (Table 8). Most compounds had similar MICs
against LPꢀ0341914ꢀRM5 (Rv1339V90M) as against the wildꢀtype strain (Table 9). Six
compounds had 5ꢀfold shifts in MIC against the Rv1339V90M mutant, but in all cases the
observed resistance shift was significantly smaller than those observed when the same
compounds were tested against the QcrBM342T mutant (Tables 8 + 9). Together, these data
indicate the Rv1339 gene product is unlikely to play a major role in resistance to the PAB series
and implicates QcrB as the target of PAB compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 8: Cross-resistance of QcrBM342T mutant strain to PAB compounds.
WT
MIC90 (µM)
QcrB
MIC90 (µM)
Fold-
change
M342T
a
Compound
Name
a
3
4
6
IDRꢀ0333819
IDRꢀ0341914
IDRꢀ0357446
IDRꢀ0351553
IDRꢀ0351551
IDRꢀ0390229
IDRꢀ0390228
IDRꢀ0458786
IDRꢀ0351605
IDRꢀ0458790
IDRꢀ0351601
IDRꢀ0357449
IDRꢀ0357444
IDRꢀ0357442
16
20
>100
>100
>100
>100
47
>6.3
>5.0
>12
>18
313
>13
>17
>15
5.0
8.3
5.7
0.15
8
1
8
19
2
2
0
2
>100
>100
>100
5
6
26
6.9
1
2
3
8
0
1.6
0.55
2.7
7.6
3.1
93
58
32
22
40
3
3
7
8
>100
>100
35
>37
>13
11
41
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4
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4
IDRꢀ0357443
IDRꢀ0357433
IDRꢀ0341930
IDRꢀ0458789
IDRꢀ0357435
IDRꢀ0357445
IDRꢀ0578347
IDRꢀ0390235
IDRꢀ0458745
IDRꢀ0341919
IDRꢀ0341920
IDRꢀ0341931
IDRꢀ0341936
IDRꢀ0341926
IDRꢀ0341921
IDRꢀ0357430
IDRꢀ0390233
3.8
0.21
1.3
9.4
6.7
16
44
8.6
12
41
46
73
56
4
5
9
2
98
10
70
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
53
>100
4.1
>6.3
41
5
5
4
7
0.10
1
6.2
6.2
21
58
4.6
0.92
5
98
b
6
7
9
0
16
17
b
b
b
b
b
b
b
>100
87
>20
11
71
7.7
14
7
7
2
3
>100
31
>7.1
8.9
>7.7
55
3.5
13
74
>100
48
7
7
5
6
0.88
0.66
>100
>152
a
MIC is the concentration required to inhibit growth of M. tuberculosis by 90%. LPꢀ0351551ꢀ
9
0
b
RM1 is QcrBM342T
.
Compounds 69ꢀ76 are compounds 13, 15, 22, 23, 29, 30, 56, 62,
2
respectively in .
Table 9: Cross-resistance of M. tuberculosis Rv1339V90M mutant strain to PAB compounds.
WT
Rv1339
MIC90 (µM)
Fold-
change
V90M
Compound
3
Name
a
a
MIC90 (µM)
IDRꢀ0333819
IDRꢀ0351553
IDRꢀ0351551
IDRꢀ0390229
IDRꢀ0390228
IDRꢀ0458786
IDRꢀ0458790
IDRꢀ0351601
IDRꢀ0357449
IDRꢀ0357444
IDRꢀ0341930
16
5.7
0.15
8
6.2
9.6
1.5
10
0.4
1.7
10
1
1
2
2
2
3
3
3
3
8
9
0
2
6
0
2
7
8
1.3
0.5
0.4
0.5
12
6
2.9
3
6.9
1.6
0.55
2.7
7.6
1.3
0.77
6.3
1.8
6.9
1.7
0.7
0.9
1.3
46
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9
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
5
9
2
IDRꢀ0458789
IDRꢀ0357435
IDRꢀ0357445
IDRꢀ0458745
IDRꢀ0341919
IDRꢀ0341920
IDRꢀ0341931
IDRꢀ0341936
IDRꢀ0341926
IDRꢀ0341921
IDRꢀ0357430
IDRꢀ0390233
9.4
6.7
16
10
3.5
12
1.1
0.5
0.8
1.5
6.7
0.5
0.8
0.9
0.6
0.6
0.8
0.9
53
5
8
b
4.6
0.92
5
6.8
6.2
2.3
5.9
13
6
9
b
b
b
b
b
b
b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
70
7
7
1
2
7.7
14
73
3.5
13
2.1
7.8
0.74
0.62
7
7
4
5
0.88
0.66
76
a
b
MIC90 is the concentration required to inhibit growth of M. tuberculosis by 90%. Compounds 69, 70, 71, 72, 73,
2
7
4, 75, 76 are compounds 13, 15, 22, 23, 29, 30, 56, 62, respectively from .
Structurally, the benzene of the benzimidazole core of the PAB series is not required for antiꢀ
tuberculosis activity, as some compounds with just an imidazole ring (PABꢀII) still have
moderate activity against M. tuberculosis (Table 6). However, the PABꢀII compounds appear to
have a different target or mode of action, since the QcrBM342T mutant strain was not resistant to
this set (Table 10). This argues that while the benzene ring of the core may not be strictly
required for wholeꢀcell activity, this ring may be responsible for targeting of QcrB specifically. It
would be interesting to elucidate the target of PABꢀII compounds to determine their mechanism
of action.
Table 10: Cross-resistance of M. tuberculosis QcrBM342T mutant strain to PAB-II
compounds.
WT
MIC90
QcrB
MIC90 (µM)
Fold-
change
M342T
a
Compound
Name
a
1
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9
(
µM)
63
IDRꢀ0262047
IDRꢀ0155469
IDRꢀ0168354
34
8.1
9.4
47
8.7
10
1.4
1.1
1.1
6
6
6
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
MIC90 is the concentration required to inhibit growth of M. tuberculosis by 90%.
As a key component of the mycobacterial electronꢀtransport chain, QcrB receives electrons from
menaquinol and serves as a pump shuttling protons into the periplasmic space, thereby
3
establishing the proton gradient required for ATP production . Over the last few years, the
cytochrome bc oxidase has been discovered to be the target of novel drugs against numerous
1
infectious diseases including malaria and Leishmania parasites, as well as multiple classes of
compounds active against M. tuberculosis, including the drug Q203, an imidazopyridine amide
4
ꢀ8
currently in clinical trials . Two of the resistance mutations found in our study cause amino
acid changes at position 312 of QcrB, which sits in the same pocket as the resistant mutant at
8
position 313 found for Q203 . This suggests the PAB compounds may be binding in the same
location and have a similar mechanism of action as Q203. To test this, we examined crossꢀ
resistance of PAB compounds to a strain of M. tuberculosis containing the same mutation found
in Q203ꢀresistant isolates (QcrBT313I). PAB compounds were highly resistant to the QcrBT313I
variant; the MIC for PAB compounds ranged from 13ꢀfold up to 200ꢀfold higher compared to
9
0
the wildꢀtype strain (Table 11). The foldꢀchange in MIC against QcrBT313I was larger than that
for QcrBM342T for every compound tested except IDRꢀ0351551, the compound against which
QcrBM342T was raised. When Q203 was tested against QcrBW312C, the MIC was greater than 100
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nM compared to just 2.8 nM for H37RvꢀLP. Together, these data suggest an overlapping binding
site between Q203 and PAB compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 11: Cross-resistance of QcrBT313I mutant strain to PAB compounds.
WT
QcrB
MIC90 (µM)
Fold-
change
a
T313I
Compound
19
Name
MIC90
a
(µM)
IDRꢀ0351551
IDRꢀ0351605
IDRꢀ0351601
IDRꢀ0357433
IDRꢀ0357435
IDRꢀ0578347
IDRꢀ0341919
IDRꢀ0341931
Q203
0.39
2.4
63
> 100
> 100
89
161
> 41
> 52
198
> 45
87
2
3
8
2
1.9
44
0.45
2.2
5
5
2
4
> 100
8.7
0.10
0.87
7.7
b
b
b
69
61
70
7
7
1
7
> 100
1.7
> 13
193
0.0088
a
b
MIC is the concentration required to inhibit growth of M. tuberculosis by 90%. Compounds
9
0
2
8
6
9 and 71 are compounds 13 and 22, respectively in . Compound 77 is from .
To provide further evidence supporting QcrB as the target of PAB compounds, we performed
initial mechanistic studies surrounding QcrB. Inhibition of QcrB leads to reduced proton
pumping across the bacterial membrane. An inability to pump protons disrupts ATP production
and increases the concentration of protons within the cell, thus lowering intrabacterial pH
(pHIB). As would be expected following QcrB inhibition, PAB compounds caused ATP
depletion over a 24 hr timeꢀperiod (Fig. 1A), and significantly decreased pHIB; all PAB
compounds caused a reduction in pHIB to at least 6.7 with 8/16 compounds dropping the pHIB
below 6.5, similar to that seen with the ionophore monensin (Figure 1B). The one PABꢀII
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compound tested showed no activity in this assay, consistent with the conclusion QcrB is not its
target. Together with the resistant mutant isolation, these data strongly support QcrB as the target
for PAB compounds and suggest potential mechanisms for compound activity.
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Previous studies identified PAB compounds as bacteriostatic against actively growing M.
2
tuberculosis, while bactericidal against nutrientꢀstarved bacteria . We tested this for two
compounds, including one of our most potent compounds, IDRꢀ0578347 (54). Both compounds
had similar activity; when tested at 10X their respective MICs, the compounds inhibited growth
of M. tuberculosis grown aerobically, but displayed bactericidal activity only in the context of
nutrient starvation (Fig. 2A and 2B). One possible explanation for the differential activity stems
from the elucidation of QcrB as the target. Under replicating conditions individual bacteria
contain large amounts of ATP necessary to support growth and division of bacterial cells. Under
starvation conditions ATP is still required to maintain basal cellular processes, but the amount of
9
ATP/cell is much reduced . It is possible ATP depletion mediated by PAB compounds during
replicative growth is only enough to inhibit growth and force the bacteria into a nonꢀreplicative
state, while under starvation conditions any reduction from the basal amount of ATP being
produced is enough to cause bacterial cell death. This hypothesis still needs to be tested
experimentally.
The number of series of compounds that target QcrB has led to an intriguing debate within the
field. On one hand, the number of times this target has been identified highlights the importance
3
of the cytochrome bc complex and ETC to mycobacterial survival . However, there has been
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concern as to the promiscuity of the target, the similarity to the human electron transport chain,
and the ability of M. tuberculosis to adapt by using the bd oxidase as an alternative electron
10ꢀ11
acceptor under oxygenꢀlimited conditions
. Recent work used the adaptability of M.
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tuberculosis against itself by using combination therapy targeting the ETC . Based on our
results, it is possible the PAB series could similarly be used in combination with other electronꢀ
transport inhibitors. These studies are currently underway.
Table 12: Activity of PAB compounds against intracellular M. tuberculosis.
Intracellular
IC50 (µM)
Intracellular
IC90 (µM)
c
d
Compound
18
ID
TC50 (µM)
SI
a
b
IDRꢀ0351553
IDRꢀ0357433
IDRꢀ0341930
IDRꢀ0578347
IDRꢀ0341926
IDRꢀ0341939
0.27 ± 0.05
0.040 ± 0.002
0.070 ± 0.006
0.86 ± 0.14
0.15 ± 0.04
0.29 ± 0.06
17 ± 3.5
49 ± 14.9
28 ± 0.5
>50
19
325
95
4
4
4
6
54
0.0073 ± 0.0009 0.028 ± 0.001
>1785
85
7
3
0.093 ± 0.01
0.48 ± 0.03
0.30 ± 0.05
1.3 ± 0.16
26 ± 3.2
38 ± 5.6
e
7
7
29
a
b
c
Intracellular IC50 is the concentration required to inhibit growth of M. tuberculosis inside macrophages by 50%.
Intracellular IC90 is the concentration required to inhibit growth of M. tuberculosis inside macrophages by 90%.
TC50 is the concentration required to inhibit growth of RAW cells by 50%. Data are the average of four experiments
d
e
2
± standard deviation. Selectivity index (SI) = TC /IC . Compound 77 is compound 67 from .
50
90
As for the potential consequence of targeting a component of the electron transport chain, our
previously published data suggests the inhibitors described herein are specific to M. tuberculosis,
2
as PAB compounds do not inhibit growth of several other bacteria, including M. smegmatis . In
fact, when tested against M. tuberculosis in a highꢀcontent analysis of M. tuberculosis
macrophage infection, PAB compounds showed exquisite sensitivity towards bacterial cells with
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MICs against M. tuberculosis inside macrophages similar to those found in liquid broth and
selectivity indexes ranging from 19 to nearly 2000 (Table 12). Several compounds even appeared
to be slightly more active inside macrophages than in liquid culture. Together, these data show
the penetrance of these compounds into eukaryotic cells, while highlighting the safety of the
PAB compounds, offering a promising therapeutic window that would allow for treatment of
infection while minimizing host cell toxicity.
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CONCLUSION
This work investigated a range of possible amine and thioether linkages as a strategy to identify
improved alkyl benzimidazole molecules. Several compounds showed good potency and
selectivity, as well as activity against intracellular bacteria. The key determinants for optimal
antimycobacterial activity of the compounds are methyl groups in both the Cꢀ6 position of the
benzimidazole and the para position of the terminal phenyl ring, a three or four carbon atom
linker, a nitrogen as the hetero atom on the alkyl chain, and a benzimidazole as the core moiety.
The PAB compounds appear to target QcrB, a component of the M. tuberculosis electron
transport chain, suggesting they could be useful in combination therapy with other electronꢀ
transport chain inhibitors.
METHODS
Bacterial strains and growth conditions
1
3
M. tuberculosis H37Rv (London Pride) (ATCC 25618) was used for all work. M. tuberculosis
strains were grown in Middlebrook 7H9 medium containing 10% v/v OADC (oleic acid,
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albumin, dextrose, catalase) supplement (Becton Dickinson) and 0.05% w/v Tween 80 (7H9ꢀTwꢀ
OADC) under aerobic conditions.
Determination of minimum inhibitory concentration (MIC)
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MICs were performed as previously described ; briefly MIC s were determined against M.
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tuberculosis H37RvꢀLP constitutively expressing codonꢀoptimized mCherry grown in 7H9ꢀTwꢀ
OADC under aerobic conditions. Bacterial growth was measured by OD590 after 5 days of
incubation at 37 °C. MIC was defined as the minimum concentration required for >99% growth
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inhibition using the Gompertz model. MIC s were determined against M. tuberculosis H37Rvꢀ
9
0
LP or indicated mutants under the same growth conditions. MIC is defined as the concentration
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0
of compound required to inhibit growth of M. tuberculosis by 90% and was determined from the
1
5
LevenbergꢀMarquardt leastꢀsquares plot .
Cytotoxicity assay
Cytotoxicity was determined against the Vero African green monkey kidney cell line (ATCC
CCLꢀ81). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), High Glucose,
GlutaMAX™ (Invitrogen), 10% v/v FBS (Fetal Bovine Serum), and 1x of Penicillinꢀ
Streptomycin Solution (100 units/mL of penicillin, 100 µg/mL of streptomycin). Cells were
plated into 96ꢀwell plates and allowed to adhere for 24 hours before exposure to compounds for
48 h. CellTiterꢀGlo® Reagent (Promega) was added to 96ꢀwell plates and relative luminescent
units (RLU) were measured. Inhibition curves were fitted using the Levenberg–Marquardt
algorithm. Toxic concentration (TC ) was defined as the concentration of compound that gave
5
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50% inhibition of growth.
Resistant mutant isolation
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M. tuberculosis H37RvꢀLP was plated on 7H10ꢀOADC plates containing 5X or 10X MIC of
2
three PAB compounds #3, 4, and 19 (Compounds 6, 53, and 68 from ). Resistant clones raised
against compounds 4 and 19 were isolated, confirmed and whole genomeꢀsequenced as
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16
previously described . The resistant clone raised against compound 3 was isolated, and
confirmed by sequencing.
ATP depletion assay
M. tuberculosis H37RvꢀLP was grown in duplicate at 37 °C for 24 hrs in the presence of 3ꢀfold
dilutions of PAB compounds starting at 200 µM. ATP levels were measured using the BacTiterꢀ
Glo assay kit (Promega) according to the manufacturer’s instructions. OD590 of the duplicate
plate was measured using a Synergy H4 plate reader (BioTek). ATP/OD was calculated by
dividing relative fluorescent units from the BacTiterꢀGlo plate by OD590 readings.
pH homeostasis assay
1
7
M. tuberculosis H37RvꢀLP containing a pHꢀsensitive ratiometric GFP reporter was added to
compounds at 100 µM final concentration in phosphocitrate buffer at pH 4.5. After 48 hrs
fluorescence was read and intrabacterial pH was determined based on the ratio between
Ex396/Em510 and Ex475/Em510.
Determination of compound kill kinetics
5
M. tuberculosis H37RvꢀLP was inoculated into 7H9ꢀTwꢀOADC at 1 x 10 CFU/mL in the
presence of 10X the respective MIC of either compound 46 or 54 or a DMSO control (final
DMSO concentration of 2%). For starvation experiments, H37RvꢀLP was washed and
5
resuspended in phosphateꢀbuffered saline (PBS) + 0.05% Tyloxapol at 1 x 10 CFU/mL for 2
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weeks prior to compound addition. Serial dilutions were performed at the indicated times and
bacteria were plated on 7H10ꢀOADC agar plates for CFU enumeration.
Intracellular activity
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18
Activity against intracellular bacteria was tested as described . In brief, RAW 264.7 cells were
batchꢀinfected overnight in RPMIꢀ1640, 10% v/v FBS, 2 mM Glutagro, 1 mM sodium pyruvate
1
9
medium with M. tuberculosis DREAM8 (an H37Rv variant constitutively expressing dsRED)
at a multiplicity of infection of 1. Cells were harvested, washed twice and seeded into 384ꢀwell
3
assay plates at 3.3 x 10 cells per well. Infected cells were exposed to compounds for 72 hours,
Macrophage nuclei were stained with SYBR Green I dye and imaged on an ImageXpress Micro
with a 4X objective using a FITC filter set. M. tuberculosis were imaged using a Texas Red filter
set. Images were analyzed by setting a lower bound for pixel intensity for each channel to define
nuclei and bacteria above background. The integrated intensity of each channel above this
threshold was determined. Raw data were normalized to the average integrated control wells.
Growth inhibition curves were fitted using the Levenberg–Marquardt algorithm. The IC and
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IC90 were defined as the compound concentration that produced 50% or 90% of the growth
inhibitory response, respectively, for each readout.
COMPOUND SYNTHESIS
Compounds 59ꢀ68 were purchased from Chembridge.
General Methods
1
H NMR (300MHz) spectra were recorded on a Bruker Biospin NMR spectrometer. Reactions
were monitored using thinꢀlayer chromatography (TLC) using Whatman silica gel 60 Å plates
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with florescent indicator and visualized using a UV lamp (254 nm). Flash chromatography was
performed on Grace with GraceResolv Normal Phase disposable silica columns. High
performance liquid chromatography (HPLC) was performed on a Gilson 322 HPLC pump with a
Gilson UV/VISꢀ155 detector and a Phenomenex Gemini C18 column (10 µm, 250 mm x 10
mm). The purity of all final products was >95% as determined by HPLC analysis conducted on
an Agilent 1100 HPLCꢀMS system (Phenomenex Gemini C18 column, 5 ꢁm, 3 x 50 mm, 0.45
mL/min, UV 254 nm, room temperature) with gradient elution (5ꢀ95% acetonitrile in water over
8 min with all solvents containing 0.05% formic acid). Liquid chromatography electrospray
ionization mass spectroscopy (LCꢀMS/ESIꢀMS) were acquired on an Agilent LC/MSDꢀSL with
a 1100 HPLC and G1956B mass spectrometer with a Phenomenex Gemini 5 ꢁm C18 110Å 50x3
mm column. Highꢀresolution mass spectra (HRMSꢀESI) were acquired by the Mass
Spectrometry Laboratory at the University of Michigan on an Agilent QꢀTOF HPLCꢀMS.
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General procedure for synthesis of 1H-benzo[d]imidazole intermediates (1):
2
1Hꢀbenzo[d]imidazole intermediates were synthesized according to reported procedure.
General procedure for synthesis of 1-(2-bromoalkyl)-1H-benzo[d]imidazole intermediates
2):
(
1ꢀ(2ꢀbromoalkyl)ꢀ1Hꢀbenzo[d]imidazole intermediates were synthesized according to reported
2
procedure .
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For the synthesis of 1ꢀ(2ꢀbromoalkyl)ꢀ6ꢀmethylꢀ1Hꢀbenzo[d]imidazole intermediates, two
1
isomers were formed and separated chromatographically. Both isomers were characterized by H
2
NMR and the desired intermediate was carried forward .
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General procedure for preparation of compounds 6-18, 25-35, 37-43, 45 and 47-58:
To a solution of benzimidazole in dimethylformamide were added 5 molar equivalents of
potassium carbonate and 2 molar equivalents of the appropriate alcohol (or thiol). The reaction
was stirred overnight at room temperature or until disappearance of the starting material
monitored by TLC. Heating of the reaction to 50 °C was necessary to prepare the amine analogs
using the same protocol. The reaction mixture was washed with water and extracted with ethyl
acetate. The organics were dried with anhydrous sodium sulfate and concentrated in vacuo. The
crude mixture was purified by column chromatography.
General procedure for preparation of compounds 20-24:
To a solution of benzimidazole in dimethylformamide were added 5 molar equivalents of
potassium carbonate and 2 molar equivalents of 5ꢀ(4ꢀchlorophenyl)ꢀ1,3,4ꢀoxadiazoleꢀ2ꢀthiol.
The reaction was stirred overnight at room temperature or until all of the starting material
disappeared. The reaction mixture was washed with water and extracted with ethyl acetate. The
organics were dried with anhydrous sodium sulfate and concentrated in vacuo. The crude
mixture was purified by column chromatography.
2-ethyl-1-(4-(p-tolylthio)butyl)-1H-benzo[d]imidazole
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+
1
Compound 6: Yield = 80%. H NMR (300 MHz, Chloroformꢀd) δ 1.46 (t, J = 7.5 Hz, 3H), 1.55
1.78 (m, 2H), 1.78 – 2.05 (m, 2H), 2.32 (s, 3H), 2.69 – 2.99 (m, 4H), 4.07 (t, J = 7.4 Hz, 2H),
.06 – 7.73 (m, 8H). LCMS – ESI (M+H) : 325.2. HRMS (ESI): calcd for C H N S ,
25.1733; found, 325.1738.
–
7
3
+
+
2
0
25
2
2-ethyl-1-(4-((4-methoxyphenyl)thio)butyl)-1H-benzo[d]imidazole
1
Compound 7: Yield = 74%. H NMR (300 MHz, Chloroformꢀd) δ 1.48 (t, J = 7.5 Hz, 3H), 1.54
1.72 (m, 2H), 1.82 – 2.08 (m, 2H), 2.70 – 2.98 (m, 4H), 3.81 (s, 3H), 4.10 (t, J = 7.3 Hz, 2H),
.82 (d, J = 8.6 Hz, 2H), 7.17 – 7.37 (m, 5H), 7.56 – 7.87 (m, 1H). LCMS – ESI (M+H) : 341.0.
HRMS (ESI): calcd for C H N OS , 341.1682; found, 341.1681.
–
6
+
+
2
0
25
2
2-ethyl-1-(4-(naphthalen-2-ylthio)butyl)-1H-benzo[d]imidazole
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Compound 8: Yield = 66%. H NMR (300 MHz, Chloroformꢀd) δ 1.45 (t, J = 7.5 Hz, 3H), 1.63
–
1.82 (m, 2H), 1.84 – 2.15 (m, 2H), 2.84 (q, J = 7.5 Hz, 2H), 3.02 (t, J = 6.9 Hz, 2H), 4.08 (t, J
7.4 Hz, 2H), 7.09 – 7.32 (m, 3H), 7.32 – 7.56 (m, 3H), 7.65 – 7.86 (m, 5H). LCMS – ESI
M+H) : 361.2. HRMS (ESI): calcd for C H N S , 361.1733; found, 361.1738.
=
(
+
+
23 25 2
2
-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)quinolone
1
Compound 9: Yield = 66%. H NMR (300 MHz, Chloroformꢀd) δ 1.44 (t, J = 7.5 Hz, 3H), 1.73
–
2.21 (m, 4H), 2.68 – 3.04 (m, 2H), 3.24 – 3.53 (m, 2H), 4.01 – 4.28 (m, 2H), 7.10 – 7.51 (m,
5H), 7.55 – 7.96 (m, 5H). LCMS – ESI (M+H) : 362.2. HRMS (ESI): calcd for C H N S ,
+
+
2
2
24
3
362.1685; found, 362.1691.
2
-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)quinoxaline
2
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1
Compound 10: Yield = 78%. H NMR (300 MHz, Chloroformꢀd) δ 1.46 (t, J = 7.5 Hz, 3H), 1.77
–
3
3
2.13 (m, 4H), 2.75 – 3.05 (m, 2H), 3.24 – 3.49 (m, 2H), 4.03 – 4.38 (m, 2H), 7.01 – 7.41 (m,
H), 7.45 – 7.94 (m, 4H), 7.89 – 8.11 (m, 1H), 8.44 – 8.75 (m, 1H). LCMS – ESI (M+H) :
63.2. HRMS (ESI): calcd for C H N S , 363.1638; found, 363.1643.
+
+
21 23
4
6
-ethoxy-2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)benzo[d]thiazole
1
Compound 11: Yield = 60%. H NMR (300 MHz, Chloroformꢀd) δ 1.37 – 1.61 (m, 6H), 1.74 –
2
1
.31 (m, 4H), 2.63 – 3.02 (m, 2H), 3.19 – 3.47 (m, 2H), 3.95 – 4.29 (m, 4H), 6.84 – 7.12 (m,
H), 7.14 – 7.40 (m, 4H), 7.66 – 7.88 (m, 2H). LCMS – ESI (M+H) : 412.2. HRMS (ESI): calcd
for C H N OS , 412.1512; found, 412.1517.
+
+
2
22 26
3
6
-chloro-2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)benzo[d]thiazole
2
9
ACS Paragon Plus Environment