Synthesis and antitubercular activity of monocyclic nitroimidazoles: Insights from econazole

Article abstract of DOI:10.1016/j.bmcl.2010.12.128

We have designed and synthesized econazole-derived nitroimidazoles to investigate the antitubercular activity of the nitroimidazole compounds. The introduction of a nitro group at the 4-position of the imidazole on econazole abolished the antitubercular a

Full text of DOI:10.1016/j.bmcl.2010.12.128

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1515–1518
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitubercular activity of monocyclic nitroimidazoles:
Insights from econazole
Sang-Ho Lee a, Suhyun Kim a,c, Min-Han Yun a, Yong Sup Lee c, Sang-Nae Cho b, Taegwon Oh b, Pilho Kim a,
⇑
a Cancer & Infectious Diseases Research Center, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea
b Department of Microbiology and the Brain Korea 21 Project for the Medical Sciences, Yonsei University College of Medicine, Seoul, Republic of Korea
c Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed and synthesized econazole-derived nitroimidazoles to investigate the antitubercular
activity of the nitroimidazole compounds. The introduction of a nitro group at the 4-position of the imid-
azole on econazole abolished the antitubercular activity. However, alcoholic nitroimidazoles 4 and 6
compounds were active against Mycobacterium tuberculosis (Mtb). While the MIC value of econazole
Received 6 October 2010
Revised 22 December 2010
Accepted 25 December 2010
Available online 5 January 2011
was 16 lg/mL, the MIC of 6a and 6f turned out to be 0.5 lg/mL. In particular, the activity of 6f against
non-replicating Mtb was as good as PA-824, which is currently in clinical phase II studies as an antituber-
cular agent. Overall, alcohol compounds 4 and 6 tend to be more active than ether compounds 5 and 7.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Tuberculosis
Econazole
Anaerobic activity
Nitroimidazole
O2N
Tuberculosis (TB) still kills about 2 million people annually
worldwide.1 There have been no novel TB drugs developed for
the past four decades. Recently, nitroimidazole series compounds
such as PA-8242 and OPC-676833 (Fig. 1) entered into clinical stud-
ies and they are now in clinical phase II. Since then nitroimidazoles
have been explored as a promising scaffold for the TB drug devel-
opment.4–8 Since nitroimidazole compounds are active against
both replicating and non-replicating Mycobacterium tuberculosis
(Mtb), in the field of TB drug discovery this scaffold has been
thought to contribute to shortening the current lengthy TB drug
regimen.4 While PA-824 and OPC-67683 are bicyclic compounds
it has been reported that monocyclic nitroimidazoles also have
antitubercular activities such as metronidazole.4
Antifungal azole agents are known to have antimycobacterial
activity.9,10 Econazole, one of the antifungal agents, has been
known to be active against both replicating and non-replicating
Mtb,11 multi-drug resistant TB (MDR-TB),12 and even in mouse
TB model.11 The potential use of econazole as TB chemotherapeu-
tics was also investigated by using drug delivery systems.13,14
In an effort to discover novel antitubercular compounds, we ex-
plored the antitubercular activity of nitroimidazoles derived from
econazole. In terms of structure, econazole has an imidazole ring
moiety. In particular, we investigated the antitubercular activity
effect of the nitro functionality introduction on the imidazole of
econazole. Since both PA-824 and OPC-67683 have 4-nitroimidaz-
N
N
O
O2N
N
O
N
O
O
N
O
OCF3
OCF3
PA-824
Figure 1. PA-824 and OPC-67683.
OPC-67683
ole structural motif, our interest was to investigate the antituber-
cular activity of 4-nitroimidazoles derived from econazole (5 in
Fig. 2). It has been reported that it is advantageous to have an oxy-
R2
N
N
O2N
R1
N
N
O
O
R3
Cl
Cl
econazole (1)
Figure 2. Design of econazole-derived nitroimidazoles.
Cl
5
⇑
Corresponding author. Tel.: +82 42 860 7541; fax: +82 42 860 7348.
E-mail address: pkim@krict.re.kr (P. Kim).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.128

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S.-H. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1515–1518
O2N
N
R2
R2
R2
N
H
N
N
Br
R3
Cl
O2N
R1
N
O2N
R1
N
3
OH
R1
a
b
O
R3
OH
2
4
5
R1
=
R3
=
4-F, 4-Cl, 4-NO2, 4-Ph
2,4-dichloro, 2,4-difluoro
R2
=
2-F, 4-F, 4-Cl, 4-OCF3
2,4-difluoro, 2,5-difluoro, 3,5-difluoro
H, Me, Br
Scheme 1. Reagents and conditions: (a) 3, TBAI, K2CO3, MeOH or EtOH, reflux, 12 h, 38–50%; (b) benzyl bromides, NaH, TBAI, DMF, À78 °C to rt, 2.5 h, 90–94%.
gen or electron donating groups at the 2-position of nitroimidaz-
oles in order to have antitubercular activity.7,8 Thus, we also stud-
ied the effect of the 2-position substituents of the nitroimidazoles
for the activity.
ether 7 with various benzyl bromides. Ether 7 was also prepared
from bromoether 5 under the same reaction conditions as above.
The antitubercular activity of the synthesized compounds
against Mtb H37Rv was determined by the microdilution Alamar
blue assay.16 The MIC against non-replicating Mtb was determined
by using the green fluorescent protein (GFP) expressing Mtb
strain17 in the Wayne hypoxia model (anaerobic conditions).18
We found out that 4-nitro-econazole (5a) was not active, while
All of the compounds were prepared as racemic. At first, we pre-
pared 4-nitro-econazole (5a) as a control (Scheme 1). The synthesis
of 5a started from 2-chloro-1-(2,4-dichlorophenyl)ethanol (2a;
R1 = 2,4-dichloro) via introduction of 4-nitroimidazole (3a;
R2 = H) and subsequent benzylation. Compound 2a was refluxed
with 4-nitroimidazole (3a), TBAI, and K2CO3 in EtOH to obtain 4-
nitroimidazole 4a as a major product and corresponding 5-nitro-
imidazole compound (not shown) as a minor product.7 Benzylation
of 4a was accomplished with 4-chlorobenzyl bromide under NaH
and TBAI in DMF at À78 °C to rt to facilitate 5a (R3 = Cl) in good
yield.7 With this chemistry in hand, the two benzene rings of 5a
were derivatized with various substituents by using several differ-
ent chlorohydrins (2) and benzyl bromides. In case of benzyl bro-
mides, 4-trifluoromethoxy group was chosen because of the
presence of this substituent in both PA-824 and OPC-67683. Other
benzyl bromides were selected based on the structure of known
antifungal azole compounds. The 2-position of the nitroimidazole
was also varied with either 2-methyl-4-nitroimidazole (3b) or 2-
bromo-4-nitroimidazole15 (3c).
the MIC of econazole was 16 lg/mL, suggesting that the nitro
group introduction on econazole was ineffective to maintain the
antitubercular activity of econazole. In addition, all of the benzyl
ether series compounds 5 turned out to be inactive (Table 1). These
discouraging results led us to investigate the antitubercular activ-
ity of alcohol compounds 4, which are the precursors of 5. In the
case of alcohol series compounds 4, most of them were not active
except 4c (MIC 2
lg/mL), while compounds 4a and 4j were moder-
ately active (MIC 32
l
g/mL). Under these circumstances, our inter-
est was to introduce electron donating groups at the 2-position of
the imidazoles to enhance the activity. Overall, when the 2-posi-
tion bromine of the nitroimidazole was substituted with a meth-
oxy group, the activity was increased. In case of 6a, 6d, 6e, and
6h, the activity was increased 4-fold, compared with 4c, 4g, 4h,
and 4j, respectively. While 6c and 6l were 8-fold more active than
4f and 4k, respectively, 6b was 16-fold more active than 4e. In par-
A recent report suggested that electron donating groups at the
2-position of the nitroimidazole play a significant role in antituber-
cular activity.7 Thus, the 2-position bromine of nitroimidazole 4
was transformed to give rise to 6 in good yields with either meth-
oxy or thiomethoxy group (Scheme 2). Benzylation of 6 facilitated
ticular, the MIC values of both 6a and 6f were 0.5 lg/mL. However,
when the 2-position of the nitroimidazole was substituted with a
thiomethoxy group, the activity was decreased by 8-fold
(4C?6g). This data suggest that the oxygen atom at the 2-position
XMe
N
Br
N
O2N
O2N
N
N
a
OH
OH
R1
R1
(X = O, S)
6
4
b
Br
N
XMe
N
O2N
R1
O2N
R1
N
N
a
O
O
R3
R3
7
(X = O, S)
5
Scheme 2. Reagents and conditions: (a) NaOMe or NaSMe, MeOH, rt, 12 h, 60–93%; (b) benzyl bromides, NaH, TBAI, DMF, À78 °C to rt, 2.5 h, 90–94%.

S.-H. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1515–1518
1517
Table 1
Antitubercular activity and cytotoxicity of nitroimidazoles
R2
R2
N
O2N
N
XMe
XMe
N
N
N
O2N
O2N
R1
N
N
O2N
R1
N
O
O
OH
R1
R3
R3
OH
R1
4a-k
5a-q
R2
6a-i
7a-k (X = O, S)
(X = O, S)
b
Compound number
R1
R3
X
MICa
(l
g/mL)
IC50 (lg/mL)
4a
4b
4c
4d
4e
4f
4g
4h
4i
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Difluoro
2,4-Difluoro
4-F
4-Cl
4-NO2
4-Phenyl
H
2,4-Dimethyl
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Difluoro
2,4-Difluoro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Difluoro
2,4-Difluoro
2,4-Difluoro
2,4-Dichloro
2,4-Difluoro
4-F
4-Cl
4-NO2
4-Phenyl
2,4-Dichloro
H
2,4-Dimethyl
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Difluoro
2,4-Difluoro
2,4-Difluoro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Dichloro
2,4-Difluoro
H
32
>250
>250
100
CH3
Br
H
>256
2 (8)c
256
292
Br
Br
Br
Br
Br
Br
Br
H
CH3
Br
H
Br
H
64
128
64
64
>256
32
128
>200
>100
>100
>100
>100
>100
22
4j
4k
5a
5b
5c
5d
5e
5f
5g
5h
5i
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
2-F
4-F
2-F
4-F
2,4-Difluoro
2,5-Difluoro
4-OCF3
3,5-Difluoro
3,5-Difluoro
4-F
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>16
>31
>62.5
>25
>25
>63
>16
>63
>16
>25
>25
>25
>25
>6
>50
>50
>50
100
>100
>100
>100
>100
>100
59
H
Br
Br
H
H
H
H
Br
H
H
H
5j
5k
5l
5m
5n
5o
5p
5q
6a
6b
6c
6d
6e
6f
6g
6h
6i
7a
7b
7c
7d
7e
7f
7g
7h
7i
4-OCF3
3,5-Difluoro
O
O
O
O
O
O
S
O
O
O
S
O
O
O
O
O
O
S
0.5 (4)c
4 (4)c
16
16
16
0.5 (1)c
16
8
16
>256
>256
>256
>16
>100
84
>8
4-Cl
4-F
4-OCF3
4-Cl
4-F
4-OCF3
3,5-Difluoro
4-F
4-Cl
3,5-Difluoro
4-Cl
>13
>13
>13
>25
>25
>50
>13
>13
>135
>13
>32
>256
>256
>256
>256
>256
>256
16
7j
S
S
7k
Econazole
PA-824
0.13 (2)c
a
b
c
MIC against H37Rv.
IC50 against VERO cells.
Values in parenthesis represent MIC under anaerobic conditions.
of the nitroimidazole plays a significant role in the antitubercular
activity. In the case of ether series compounds 7, all of them were
inactive, suggesting that introducing lipophilic side chains on the
benzylic alcohol group in 6 abolishes the activity. The compounds
obic assays was just 2-fold, while PA-824 has more than 15-fold
difference, suggesting that this monocyclic nitroimidazole could
be optimized further to enhance anaerobic activity.
Since all of the nitroimidazole compounds have been derived
from the econazole scaffold, the synthesized compounds were
tested against Candida albicans ATCC90027 for their antifungal
activity. The broth microdilution method was used to determine
MIC of the compounds against Candida albicans.19 The yeast was
exposed to the compounds of which concentrations ranged from
of which MICs are lower than 8 lg/mL were tested against non-
replicating Mtb (4c, 6a, 6b, and 6f). All four of them were active
against non-replicating Mtb. In particular, the activity of 6f against
non-replicating Mtb was as good as PA-824. It is noteworthy that
in case of 6f the MIC value difference between aerobic and anaer-

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S.-H. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1515–1518
256 to 0.5
l
g/mL for 48 h, and then its growth was visualized using
Optimization efforts and pharmacokinetic profile studies are
underway and will be reported in due course.
Alamar blue dye.16 All of the nitroimidazole compounds were inac-
tive in the antifungal assays performed. While the MIC of econa-
zole was 16
l
g/mL against Candida albicans, the MICs of all of the
g/mL. Based on
Acknowledgments
nitroimidazoles in Table 1 were higher than 256
l
this dramatic difference on the antifungal activity between econa-
zole and the nitroimidazoles synthesized, we could speculate that
introduction of nitro functionality on econazole scaffold led to a
different mode of action, compared with that of econazole.
This research was supported in part by Korea Research Institute
of Chemical Technology (KRICT) and by the International Research
& Development Program of the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science and
Technology (MEST) of Korea (Grant No.: K20501000001-
09E0100-00110, FY 2009).
The cytotoxic effect of the compounds was tested on Vero cells
by MTT assay (Promega, USA) in accordance to the manufacturer’s
instruction. Initial compound solution was prepared in DMSO, and
2-fold serial dilutions were made in RPMI 1640 medium. Due to
limited solubility, the highest concentration at which each com-
pound was tested varied. The cell suspension which was in early
log phase was exposed to serially diluted compounds solution.
The cytotoxic effect was quantified by measuring the amount of
formazan that was generated in each well. Most of the nitroimidaz-
ole compounds have encouraging cytotoxicity profiles. For exam-
ple, the IC50 values of active compounds, such as 4c, 6a, 6b, and
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.128.
References and notes
1. http://www.who.int/mediacentre/factsheets/fs104/en/index.html.
2. http://clinicaltrials.gov/ct2/show/NCT00567840.
3. http://clinicaltrials.gov/ct2/show/NCT00685360.
6f, were either 100 lg/mL or above.
In conclusion, starting from 4-nitro-econazole (5a), various
monocyclic nitroimidazole compounds were designed and synthe-
sized. 4-Nitro-econazole (5a) turned out to be inactive against both
Mtb and Candida albicans, suggesting that the introduction of nitro
functionality on econazole led to the loss of both antitubercular
and antifungal activities. However, in lieu of ether functionality
in 5 or 7, having an alcohol group such as 4 and 6 was advanta-
geous for the antitubercular activity. In both 4 and 6 series com-
pounds, the 2-position oxygen of the nitroimidazole turned out
to be important for the activity. The alcohol compounds 4 and 6
tend to be more active than ether compounds 5 and 7. In particular,
4. Barry, C. E., 3rd; Boshoff, H. I.; Dowd, C. S. Curr. Pharm. Des. 2004, 10, 3239.
5. Sutherland, H. S.; Blaser, A.; Kmentova, I.; Franzblau, S. G.; Wan, B.; Wang, Y.;
Ma, Z.; Palmer, B. D.; Denny, W. A.; Thompson, A. M. J. Med. Chem. 2010, 53,
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6. Palmer, B. D.; Thompson, A. M.; Sutherland, H. S.; Blaser, A.; Kmentova, I.;
Franzblau, S. G.; Wan, B.; Wang, Y.; Ma, Z.; Denny, W. A. J. Med. Chem. 2010, 53,
282.
7. Kim, P.; Zhang, L.; Manjunatha, U. H.; Singh, R.; Patel, S.; Jiricek, J.; Keller, T. H.;
Boshoff, H. I.; Barry, C. E., 3rd; Dowd, C. S. J. Med. Chem. 2009, 52, 1317.
8. Kim, P.; Kang, S.; Boshoff, H. I.; Jiricek, J.; Collins, M.; Singh, R.; Manjunatha, U.
H.; Niyomrattanakit, P.; Zhang, L.; Goodwin, M.; Dick, T.; Keller, T. H.; Dowd, C.
S., ; Barry, C. E., 3rd J. Med. Chem. 2009, 52, 1329.
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Gurcha, S. S.; Besra, G. S.; Munro, A. W. Microbiology 2002, 148, 2937.
10. Jackson, C. J.; Lamb, D. C.; Kelly, D. E.; Kelly, S. L. FEMS Microbiol. Lett. 2000, 192,
159.
11. Ahmad, Z.; Sharma, S.; Khuller, G. K. FEMS Microbiol. Lett. 2006, 261, 181.
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Antimicrob. Agents 2006, 28, 543.
13. Ahmad, Z.; Sharma, S.; Khuller, G. K. Nanomed. Nanotechnol. Biol. Med. 2007, 3,
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18. Wayne, L. G.; Hayes, L. G. Infect. Immun. 1996, 64, 2062.
The detailed experimental procedure for the MIC under anaerobic conditions is
as follows: Mtb H37Rv-GFP was grown in tight-sealed test tube with 0.5-head
space ratio for 14 days. Initial compound solution was prepared in DMSO, and
two fold serial dilutions were made in DMSO in a microplate. The serially-
diluted compound solution was dispensed in a microplate, and Mtb H37Rv-
GFP in non-replicating phase was dispensed in the plate in an anaerobic
chamber. The plates containing compounds dilutions and Mtb H37Rv-GFP
were incubated at 37 °C for 7 days in the anaerobic chamber. After exposure
Mtb H37Rv-GFP was diluted 10 fold in fresh media, and incubated for 2 days.
The fluorescence was measured in a Fluostar Optima microplate fluorometer
(BMG Labtech., Germany) in the bottom-reading mode with excitation at
485 nm and emission at 520 nm. The MIC against non-replicating Mtb was
defined as the lowest concentration of compounds that inhibited fluorescence
by 80%, compared to the fluorescence of bacteria only wells.
19. Tiballi, R. N.; He, X.; Zarins, L. T.; Revankar, S. G.; Kauffman, C. A. J. Clin.
Microbiol. 1995, 33, 915.
the MIC of both 6a and 6f turned out to be 0.5
lg/mL and the
anaerobic MIC of 6f was as good as that of PA-824.
To our knowledge, monocyclic nitroimidazoles active against
both aerobic and anaerobic Mtb have not been reported before.
Metronidazole, a class of monocyclic 5-nitroimidazole compound,
is known to have activities against not aerobic but anaerobic
Mtb.4 A recent study reported that some monocyclic nitroimidaz-
oles are active against not anaerobic but aerobic Mtb.7 Thus, in
our present study we have discovered monocyclic nitroimidazoles
active against both aerobic and anaerobic Mtb for the first time.
Although the mode of action of the nitroimidazole compounds
prepared has not been studied, based on the trend of antitubercu-
lar activities, we could speculate that the antitubercular activities
come from similar mode of action as that of PA-824. It is also clear
that an oxygen atom at the 2-position of nitroimidazoles helps to
increase the antitubercular activity. However, in terms of anti-
fungal activities, regardless of the presence of an oxygen atom at
the 2-position of nitroimidazoles, all of the nitroimidazoles pre-
pared did not have antifungal activities. Thus, we could also spec-
ulate that the mode of action of the nitroimidazoles prepared is
quite different from that of econazole.
Encouraging biological data including anaerobic activities sug-
gested that the monocyclic nitroimidazole compounds prepared
could be further pursued to optimize as antitubercular agents.