Synthesis and antimicrobial potential of nitrofuran-triazole congeners

Article abstract of DOI:10.1039/c5ob01353d

A series of 5-nitrofuran-triazole congeners were designed and synthesized by carrying out suitable structural modifications of the previously reported counterparts and were evaluated for their antimicrobial potential against both Gram-positive and Gram-ne

Full text of DOI:10.1039/c5ob01353d

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Synthesis and antimicrobial potential of nitrofuran-triazole
congeners
Ahmed Kamal,a,b,* S. M. Ali Hussaini,a M. Lakshmi Sucharitha,b Y. Poornachandra,a Faria Sultana,a
C. Ganesh Kumara
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A series of 5-nitrofuran-triazole congeners were designed and synthesized by carrying out suitable structural modifications
of the previously reported counterparts and were evaluated for their antimicrobial potential against both Gram-positive
and Gram-negative bacterial strains. The compounds exhibited promising inhibition towards different Gram-positive
pathogenic strains, while mild inhibitory effects were observed towards Gram-negative bacterial strains. Some of the
compounds 9f, 9g, 9l and 9m were most active among the series exhibiting MIC value of 1.9 µg/mL against different
bacterial strains. The bactericidal activity was found to be in coherence with the bacterial growth inhibition data. The
compounds were tested against fourteen different fungal strains and were found to possess excellent antifungal activities.
Interestingly, all the compounds were equipotent to miconazole against one or more of the tested fungal strains and
showed good activity against the other counterparts. Similar trend was observed in case of their minimum fungicidal
concentration values. Moreover, compound 9f exhibited two fold superior antifungal activity (MIC = 3.9 µg/mL) than the
standard miconazole (MIC = 7.8 µg/mL) against C. albicans and C. parapsilosis. These compounds also effectively inhibited
biofilm formation and compound 9f exhibited excellent anti-biofilm activity demonstrating biofilm inhibitory concentration
(BIC) as low as 0.8 µg/ml. A brief mechanistic study carried out on the most effective conjugate 9f indicated that it inhibits
the ergosterol biosynthesis thereby producing antifungal effects. Molecular modelling studies carried out to study the
binding modes of 9f that correlates well with the antifungal activity and supported by ergosterol biosynthesis inhibition
assay data. Most of these compounds exhibited ten times lower cytotoxicity to the normal cells compared to the
antimicrobial activity.
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common reason for bloodstream infections.5
Introduction
There are various classes of chemical substances such as
There has been a continuous battle between human beings and
disease causing microorganisms with history of millennia.
Infections caused by several microbes are posing great threat for
human survival.1 Worsening the situation, the emergence of
drug resistance is creating hurdles in the treatment.2 Fungal
infections such as candidiasis are accountable for causing
perilous diseases both in humans as well as in plants.3 Candida
species is involved in 75% of the fungal infections with Candida
albicans being an opportunistic pathogen mainly responsible for
45% of the total cases especially in immune compromised
patients.4 In addition, other Candida species such as C. glabrata
and C. krusei are emerging as serious hazards to the patients. In
the past two decades the growth of fungal infections has
increased by more than 200% and thus is the third most
imidazoles, pyrimidines, benzofurans, thiocarbamates, triazoles,
etc that are reported to possess antifungal activity and most of
these compounds exert their effect by targeting ergosterol
biosynthesis pathway.6 Ergosterol is particularly found in fungi
and is crucial for growth and normal membrane functioning of
fungal cells. In addition, it is also involved in regulating
membrane fluidity, integrity and asymmetry and has a role in
proper functioning of membrane-bound enzymes. Azoles such
as fluconazole (1), miconazole (2), etc are widely used antifungal
agents that exert their effect by inhibiting the enzyme lanosterol
14α-demethylase involved in ergosterol biosynthesis.7
Unfortunately, several fungal pathogens including C. glabrata
has developed resistance towards fluconazole which is one of
the clinically significant antifungal agents, while C. krusei is
insensitive to it.8 Therefore, the present situation demands
effective therapies for the treatment of infections caused by
several microorganisms including fungal pathogens.
Address here.
Address here.
Address here.
Furan derivatives especially nitrofurans are well known for their
antimicrobial potential.9 Some of the nitrofurans such as
nitrofurantoin (3) and nifuroxazide are agents of choice to treat
urinary tract and gastrointestinal infections. These compounds
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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are metabolically reduced by
a
class of enzymes called
and azide components were reacted following click chemistry
protocol18 to obtain the final compounds 9a-n in good yields (79-
92%).
nitroreductases to highly reactive intermediates which affect
various processes crucial for the survival of bacterial cells.10 It is
because of this intriguing mode of action, these compounds
tend to develop lower resistance as compared to other
antibiotics. Similarly, triazole scaffold is found in various
compounds with antibacterial,11 anticancer,12 antifungal13 and
antitubercular14 activities and tazobactam (4) is one of the
widely used antibiotic that contain this ring system (Figure 1). In
addition, the biofilm inhibitory potential of 1,2,3-triazoles is well
documented in the literature.15
H
N
a
OH
O2
N
O2N
O
O
O
O
5
6
R
N
N
N
c
H
N
O2
N
O
O
9a-n
9h
NH2
N3
9a
9b
: R=4-F
: R=2-Cl
: R=2-NO2
b
9i: R=4-NO2
9c: R=3,5-diOCH3
9d: R=4-OCH3
9e
: R=3Cl,4F
9f: R=2,3-DiCl
9g: R=3,4-diOCH3
9j: R=2-OCH3,4-NO2
9k
9l: R=H
9m:R=2-NO2,4-F
9n
: R=3-OH
R
R
Cl
: R=4-OH
N
N
N
7a-n
8a-n
N
N
O
O
H
N
O
N
N
N
S
N
N
O
Cl
OH
F
NH
N
O2N
O
N
Cl
O
O
N
OH
F
O
Reagents and Conditions: a) propargyl amine, EDC, HOBt,
CH2Cl2, rt, 8 h; b) NaNO2 in HCl, NaN3, 0 ˚C, 0.5-1 h; c)
CuSO4.5H2O, sodium ascorbate, t-BuOH/H2O (1:2), rt, 8 h.
Cl
4
1
2
3
Fig 1: Antimicrobial agents in market
Scheme 1: Synthesis of 5-nitrofuran-triazole conjugates.
In search for potent antimicrobial agents, we have recently
reported synthesis, antibacterial and anti-mycobacterial
activities of nitrofuramide linked triazole hybrids.16 Most of
these compounds demonstrated excellent antibacterial, anti-
mycobacterial and anti-biofilm activities. This propelled us to
study the effect of structural modification on the antimicrobial
activities of these compounds. In this regard, one end of the
molecule has been rigidified by removing the CH2 arm through
which the molecule is expected to rotate freely (Figure 2). We
herein report the synthesis, antimicrobial and anti-biofilm
potential of these nitrofuran-triazole congeners and their
mechanism of action.
Antimicrobial activity
The synthesized final nitrofuran-triazole congeners (9a-n) were
screened for their antimicrobial activity against both Gram-
positive as well as Gram-negative bacterial strains along with a
fungal strain, Candida albicans. The results shown in Tables 1 &
2
indicate that these compounds exhibited similar profile
towards various bacterial strains as previously reported analogs.
The data illustrate that these compounds showed variable
inhibitory and bactericidal effects with minimum inhibitory
concentration (MIC) and minimum bactericidal concentration
(MBC) values ranging from 1.9-31.2 µg/mL. In analogy with
previous compounds, these compounds also showed selectivity
towards Gram-positive pathogenic strains like Staphylococcus
aureus (MLS-16) and Bacillus subtilis with MIC and MBC values
of <16 µg/mL except for the compound 9m (MBC value of 31.2
µg/mL against B. subtilis). In contrast, these compounds did not
inhibit growth of Micrococcus luteus (except 9f, 9g, 9j and 9n)
even at the maximum concentration used (125 µg/mL).
Compounds 9f, 9g, 9h and 9n displayed broad spectrum of
antimicrobial activity exhibiting excellent inhibitory as well as
bactericidal effects on Gram-positive bacterial strains and
moderate activity against Gram-negative bacterial strains.
Compounds 9f and 9g showed excellent inhibitory activity (MIC
= 1.9 µg/mL) against S. aureus MTCC 96, S. aureus MLS-16 MTCC
2940 and B. subtilis MTCC 121, while compounds 9l and 9m
exhibited identical activity only against the former two strains. In
addition, compounds 9b, 9c, 9h, 9i, 9j and 9k caused good
growth inhibition of these strains with MIC values 3.9 and/or 7.8
µg/mL. However, many of these compounds did not show
pronounced activity against Gram negative bacterial strains.
R
N
N
N
N
N
N
H
H
N
N
R
O2
N
O2
N
O
O
O
O
Fig 2: Strategy of present work
Results and discussion
Chemistry
The synthesis of the target compounds 9a-n is achieved in a
convergent manner and the route is outlined in Scheme 1. As
shown, 5-nitro-2-furoic acid (5) was coupled with propargyl
amine using EDC and HOBt in CH2Cl2 to obtain the corresponding
amide (6) containing terminal alkyne functionality. On the other
hand, substituted phenyl azides (8a-n) were prepared by the
well known Sandmeyer reaction of corresponding anilines (7a-
n), i.e., diazotization using NaNO2 and HCl followed by the
reaction with NaN3 in a single reaction vessel.17 Here differently
substituted anilines with both electron donating as well as
electron withdrawing substituents were employed to study the
effect of substituents on the bioactivity. Later, both the alkyne
Table 1: Antimicrobial activity of 5-nitrofuran-triazole
conjugates (9a-n). MIC values expressed in µg/mL
Co
mp
S.aa B.sb
S.ac M.ld
K.pe
E.cf
P.ag C.ah
9a
7.8 7.8
7.8 >125 >125 >125 >125 15.6
2 | J. Name., 2012, 00, 1-3
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9b
9c
9d
9e
9f
9g
9h
9i
3.9
3.9
7.8
7.8
1.9
1.9
3.9
7.8
3.9
3.9
1.9
3.9
7.8
7.8
7.8
1.9
1.9
3.9
3.9
3.9
7.8
7.8
3.9 >125 >125 >125 >125 15.6
1.9 >125 >125 >125 >125 15.6
3.9 >125 >125 >125 >125 7.8
9j
9k
9l
9m
9n
Cip
7.8
7.8
3.9
3.9
7.8
1.9
3.9
7.8
15.6
31.2
15.6
0.9
3.9
7.8
>125 >125
31.2 15.6
15.6
>125
15.6 >125
3.9
3.9
7.8
0.9
>125 >125 >125 >125
>125 >125 >125 >125
15.6
1.9
7.8 >125 31.2 >125 15.6
7.8
3.9
3.9
1.9
1.9 31.2 >125 >125
3.9 >125 >125 7.8
3.9 >125 >125 >125 >125 15.6
3.9 3.9 >125 >125 7.8 15.6
7.8 >125 31.2 7.8 >125 7.8
1.9 >125 >125 >125 >125 15.6
7.8
>125 >125
7.8
>125
1.9
31.2
1.9
15.6
0.9
7.8
a- Staphylococcus aureus MTCC 96, b- Bacillus subtilis MTCC 121,
c- S. aureus MLS16 MTCC 2940, d- Micrococcus luteus MTCC
2470, e- Klebsiella planticola MTCC 530, f- Escherichia coli MTCC
739, g- Pseudomonas aeruginosa MTCC 2453, Cip- Ciprofloxacin
>125 7.8
9j
9k
9l
9m 1.9 15.6 1.9 >125 >125 >125 >125 31.2
9n
7.8
7.8
3.9
7.8
>125 15.6
7.8
7.8
7.8
-
Surprisingly, these compounds showed excellent to good growth
inhibition of the fungal strain Candida albicans MTCC 3017.
Compounds 9f and 9g (MIC = 3.9 µg/mL) were found to exhibit
two fold better activity than the standard miconazole (MIC = 7.8
µg/mL), while compounds 9d, 9e, 9h, 9k and 9n were found to
be equipotent to it. Further, other compounds such as 9a, 9b,
9c, 9j, 9i and 9l also exhibited good activity against these strains
with MIC = 15.6 µg/mL. This observation is completely in
contrast with the antifungal activity of previously reported
nitrofuran based triazole congeners from our laboratory.16
Mic
-
-
-
-
-
-
-
Cip 0.9
0.9
0.9
0.9
0.9
0.9
0.9
a- Staphylococcus aureus MTCC 96, b- Bacillus subtilis MTCC
121, c- S. aureus MLS16 MTCC 2940, d- Micrococcus luteus
MTCC 2470, e- Klebsiella planticola MTCC 530, f- Escherichia coli
MTCC 739, g- Pseudomonas aeruginosa MTCC 2453, h- Candida
albicans MTCC 3017; Mic-Miconazole; Cip- Ciprofloxacin
Notably, the bactericidal activity (Table 2) was found to be in
accordance with the inhibitory activity against the respective
bacterial strains. Similarly, compounds 9f and 9g showed
excellent bactericidal effects against B. subtilis MTCC 121, S.
aureus MTCC 96 and S. aureus MLS-16 MTCC 2940 exhibiting
MBC 1.9 and/or 3.9 µg/mL. Whereas some other compounds
such as 9l and 9m showed good bactericidal activity (MBC 3.9
µg/mL) against S. aureus MTCC 96 and S. aureus MLS-16 MTCC
2940. Compound 9j displayed identical activity to 9l and 9m
against B. subtilis MTCC 121 and S. aureus MLS-16 MTCC 2940.
Several other compounds showed considerable bactericidal
activity displaying MBC 7.8 and/or 15.6 µg/mL and follow similar
trend as that of MIC values against the respective strains.
Antifungal activity
The excellent growth inhibitory effects of these compounds
against Candida albicans MTCC 3017 prompted us to further test
these compounds against thirteen more fungal strains and the
results are tabulated in Table 3. As evident from the results, the
compounds showed excellent to good antifungal activity with
MIC values ranging between 3.9-62.5 µg/mL. Compound 9f was
found to be the most effective conjugate from the series
demonstrating two fold better activity (MIC = 3.9 µg/mL) than
one of the widely used antifungal agents, miconazole (MIC = 7.8
µg/mL) against C. albicans MTCC 183 and C. parapsilosis MTCC
1744. Additionally, this compound displayed equipotent activity
to miconazole in inhibiting the growth of C. albicans MTCC 227,
C. albicans MTCC 854, C. albicans MTCC 1637, C. albicans MTCC
7315, C. glabrata MTCC 3019, C. krusei MTCC 3020 and
Issatchenkia hanoiensis MTCC 4755. Also, compound 9g
displayed fungal growth inhibitory activity identical to
miconazole against all the strains tested except against C.
albicans MTCC 854, C. albicans MTCC 3958, C. albicans MTCC
4748. It is noteworthy that all the compounds demonstrated
equipotency to miconazole against one or more of the tested
fungal strains and good activity against the other counterparts.
Table 2: Minimum bactericidal concentration (MBC) of 5-
nitrofuran-triazole conjugates (9a-n). MBC expressed in µg/mL
Comp
9a
9b
9c
9d
9e
9f
9g
9h
9i
S.aa
B.sb
S.ac
M.ld
K.pe
E.cf
P.ag
15.6 15.6 15.6 >125 >125 >125 >125
7.8
7.8
15.6 15.6
15.6
3.9
3.9
7.8
15.6
3.9
15.6
7.8
3.9
3.9
>125 >125 >125 >125
>125 >125 >125 >125
>125 >125 >125 >125
7.8
1.9
3.9
7.8
7.8
15.6 >125
31.2
>125 >125
>125 >125
>125
31.2
15.6
15.6
>125
1.9
3.9
3.9
7.8
15.6
31.2
>125 >125
15.6
>125 >125 >125 >125
Table 3: Antifungal activity of 5-nitrofuran-triazole conjugates (9a-n). MIC values express in µg/mL
Compound
C.aa
C.ab
15.6
7.8
C.ac
31.2
15.6
31.2
15.6
C.ad
C.ae
15.6
31.2
31.2
15.6
C.af
C.ag
C.ah
C.pi
15.6
15.6
7.8
C.aj
31.2
7.8
C.gk
15.6
7.8
C.kl
15.6
7.8
I.hi
15.6
7.8
7.8
7.8
7.8
7.8
7.8
9a
9b
9c
9d
15.6
7.8
15.6
15.6
15.6
31.2
31.2
7.8
31.2
15.6
7.8
15.6
15.6
7.8
7.8
7.8
7.8
15.6
7.8
15.6
7.8
15.6
15.6
15.6
15.6
7.8
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15.6
3.9
7.8
7.8
7.8
7.8
7.8
7.8
15.6
15.6
7.8
15.6
15.6
15.6
7.8
7.8
15.6
15.6
7.8
7.8
7.8
7.8
3.9
15.6
15.6
7.8
15.6
7.8
15.6
7.8
7.8
7.8
9e
9f
7.8
7.8
15.6
7.8
7.8
7.8
7.8
7.8
7.8
7.8
9g
9h
9i
15.6
15.6
7.8
15.6
7.8
7.8
15.6
15.6
15.6
7.8
15.6
7.8
7.8
15.6
7.8
7.8
7.8
31.2
7.8
15.6
31.2
7.8
7.8
7.8
15.6
31.2
7.8
15.6
31.2
15.6
31.2
31.2
31.2
7.8
31.2
31.2
7.8
31.2
62.5
7.8
31.2
15.6
31.2
31.2
7.8
31.2
7.8
15.6
15.6
15.6
15.6
15.6
7.8
62.5
7.8
62.5
15.6
31.2
15.6
62.5
7.8
15.6
15.6
15.6
15.6
31.2
7.8
9j
15.6
15.6
31.2
15.6
7.8
9k
9l
31.2
62.5
7.8
15.6
62.5
7.8
62.5
31.2
7.8
31.2
31.2
62.5
7.8
7.8
15.6
7.8
15.6
7.8
15.6
62.5
7.8
9m
15.6
7.8
31.2
7.8
9n
Miconazole
7.8
7.8
7.8
7.8
a- C. albicans MTCC 183, b- C. albicans MTCC 227, c- C. albicans MTCC 854, d- C. albicans MTCC 1637, e- C. albicans MTCC 3018, f- C. albicans
MTCC 3958, g- C. albicans MTCC 4748, h- C. albicans MTCC 7315, i- C. parapsilosis MTCC 1744, j- C. aaseri MTCC 1962, k- C. glabrata MTCC
3019, l- C. krusei MTCC 3020, m- Issatchenkia hanoiensis MTCC 4755
Furthermore, these compounds were tested against the same
fourteen fungal strains to evaluate their minimum fungicidal
concentration (MFC) and the results are summarized in Table 4.
Similarly, the results correlated well with the antifungal activity
data and most of the compounds displayed MFC values identical
to the standard miconazole. In particular, compound 9f showed
MFC = 3.9 µg/mL, compared to the standard (MFC = 7.8 µg/mL)
against C. parapsilosis MTCC 1744 and displayed same efficacy
as miconazole against several other tested fungal strains. On the
whole, the results indicate that the compounds possess
excellent antifungal potential, in some cases better than and/or
equipotent to miconazole.
Table 4: Minimum fungicidal concentration (MFC) of 5-nitrofuran-triazole conjugates (9a-n). MFC expressed in µg/mL
Compound
C.aa
C.ab
C.ac
C.ad
C.ae
C.af
C.ag
C.ah
C.pi
C.aj
C.gk
C.kl
I.hi
9a
9b
9c
9d
9e
9f
15.6
15.6
15.6
31.2
31.2
7.8
31.2
15.6
15.6
31.2
15.6
15.6
15.6
31.2
15.6
62.5
31.2
62.5
62.5
15.6
15.6
62.5
31.2
62.5
31.2
15.6
15.6
31.2
15.6
31.2
62.5
15.6
62.5
62.5
15.6
15.6
15.6
31.2
31.2
31.2
15.6
15.6
15.6
15.6
7.8
31.2
31.2
31.2
15.6
15.6
7.8
31.2
62.5
62.5
31.2
31.2
15.6
15.6
31.2
31.2
31.2
15.6
62.5
62.5
15.6
7.8
15.6
31.2
31.2
15.6
31.2
31.2
31.2
15.6
15.6
31.2
31.2
31.2
31.2
31.2
15.6
15.6
62.5
31.2
15.6
15.6
31.2
15.6
15.6
31.2
62.5
15.6
62.5
62.5
62.5
7.8
15.6
31.2
31.2
15.6
15.6
15.6
15.6
31.2
15.6
62.5
15.6
15.6
31.2
62.5
7.8
15.6
31.2
15.6
31.2
15.6
3.9
31.2
15.6
15.6
31.2
15.6
31.2
15.6
31.2
15.6
62.5
31.2
62.5
31.2
62.5
7.8
31.2
15.6
15.6
15.6
31.2
15.6
15.6
15.6
62.5
62.5
15.6
31.2
15.6
31.2
15.6
31.2
15.6
31.2
15.6
31.2
7.8
9g
9h
9i
15.6
31.2
31.2
15.6
31.2
31.2
62.5
31.2
7.8
7.8
15.6
15.6
31.2
62.5
31.2
62.5
62.5
62.5
7.8
15.6
15.6
62.5
62.5
15.6
31.2
15.6
62.5
7.8
15.6
31.2
31.2
15.6
31.2
62.5
15.6
7.8
9j
62.5
15.6
31.2
62.5
15.6
15.6
9k
9l
9m
9n
Miconazole
a- C. albicans MTCC 183, b- C. albicans MTCC 227, c- C. albicans MTCC 854, d- C. albicans MTCC 1637, e- C. albicans MTCC 3018, f- C. albicans
MTCC 3958, g- C. albicans MTCC 4748, h- C. albicans MTCC 7315, i- C. parapsilosis MTCC 1744, j- C. aaseri MTCC 1962, k- C. glabrata MTCC
3019, l- C. krusei MTCC 3020, m- Issatchenkia hanoiensis MTCC 4755
The structure-activity relationships deduced from the
antimicrobial activity data reveal that the compounds possessing
ring activitating substituents at adjacent positions such as 3,4-
dimethoxy on the phenyl ring displayed excellent antibacterial
4 | J. Name., 2012, 00, 1-3
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Cip
0.6 ± 0.07
0.5 ± 0.08
0.4 ± 0.06
activity. This was also observed in case of weak ring deactivating
substituents such as 2,3-dichloro on the phenyl ring. Any type of
substituent is tolerable at ortho position and electron
withdrawing substituents are favourable at para position of the
phenyl ring. This is evident from the similar antibacterial
activities demonstrated by compounds 9b, 9h, 9i and 9j.
However, ring activitating and weak ring deactivating
substituents such as methoxy, hydroxyl, fluoro, etc. at para
position conferred comparatively lower antibacterial activity. On
the other hand, compounds exhibited excellent antifungal
activity against all the tested fungal strains indicating no
significant effect of substituents on the activity. Nevertheless,
compound 9f with 2,3-dichloro substituent exhibited enhanced
activity than miconazole against one of the fungal strains (C.
parapsilosis MTCC 1744) but was found to be equipotent against
other strains, analogous to several other derivatives.
Ergosterol biosynthesis inhibition
The excellent antifungal activity of these nitrofuran-triazole
conjugates propelled us to evaluate their mechanism of action.
It is earlier reported that most of the clinically significant
antifungal agents affect ergosterol biosynthesis pathway.
Therefore, the most effective conjugate 9f was investigated for
its effect on ergosterol biosynthesis. The experiment was carried
out using various concentrations (2, 4, 8, 16 µg/mL) of 9f and
miconazole, a known inhibitor of ergosterol biosynthesis. As
evident from Fig 3, the percentage of ergosterol formation in
absence of 9f and miconazole is 100% which was found to
decrease with the increase in the concentrations for both these
compounds. Interestingly, the amount of inhibition caused by 9f
is higher than that caused by the standard miconazole at all the
concentrations tested. This trend is in accordance with the
antifungal activity and thus supports the preliminary data. These
observations indicate that compound 9f exerts antifungal effects
by inhibiting ergosterol biosynthesis.
Anti-biofilm activity
Biofilms are surface-adhered bacterial communities embedded
in an extracellular matrix responsible for the growing resistance
in bacteria towards antibiotics.19 Several clinically significant
pathogens producing biofilms are implicated in more than 80%
of the bacterial infections.20 Therefore, developing newer
molecules having ability to inhibit biofilm formation could be
one of the solutions for the growing antibiotic resistance. In this
regard, the synthesized nitrofuran-triazole conjugates were
evaluated for anti-biofilm activity against three bacterial strains,
namely Staphylococcus aureus MTCC 96, Staphylococcus aureus
MLS-16 MTCC 2940 and Bacillus subtilis MTCC 530. The results
tabulated in Table 5 showed that these compounds exhibited
excellent to good anti-biofilm activity against all the three
strains tested. Particularly, compounds 9f, 9g, 9c and 9m
demonstrated potent efficacy in inhibiting biofilm formation
with IC50 values of 0.8, 1.1, 0.9 and 0.9 µg/ mL, respectively,
against Staphylococcus aureus MLS-16 MTCC 2940. It is
important to note that the ability to inhibit biofilm formation of
these compounds to a maximum extent is in coherence with the
antimicrobial activity against the respective strain.
120
9f
Miconazole
100
80
60
40
20
0
0
2
4
8
16
Concentration (µg/mL)
Fig 3: Effect of compound 9f and miconazole at various
concentrations on inhibition of ergosterol biosynthesis in
Candida parapsilosis MTCC 1744
Table 5: Anti-biofilm activity of 5-nitrofuran-triazole conjugates
(9a-n); IC50 values (μg/mL)
Cytotoxic activity
Compound
Staphylococcus
aureus
Bacillus
subtilis
S. aureus
MLS16
The therapeutic potential of these conjugates has been
determined by testing their cytotoxic activity against normal
human lung cell line (MRC5) and the results are represented in
Table 6. From the data it is evident that majority of the
compounds are not cytotoxic upto 100 µM concentration
whereas the antimicrobial activity of many of these conjugates is
below 10 µg/mL. These results suggest that the compounds
exhibit ten times lower cytotoxicity to the normal cells
compared to the antimicrobial activity and could be considered
to possess immense therapeutic potential.
MTCC 96
4.6 ± 0.22
2.9 ± 0.11
2.4 ± 0.23
4.2 ± 0.18
5.1 ± 0.36
1.1 ± 0.11
1.2 ± 0.12
2.6 ± 0.22
4.4 ± 0.18
3.0 ± 0.21
2.5 ± 0.35
1.1 ± 0.12
1.2 ± 0.09
3.3 ± 0.22
MTCC 121
5.2 ± 0.42
2.5 ± 0.26
3.8 ± 0.22
4.1 ± 0.28
4.7 ± 0.36
1.2 ± 0.18
1.1 ± 0.21
3.1 ± 0.18
2.7 ± 0.22
3.4 ± 0.36
4.7 ± 0.22
3.4 ± 0.25
8.8 ± 0.44
4.6 ± 0.31
MTCC 2940
4.8 ± 0.28
3.1 ± 0.15
0.9 ± 0.08
2.8 ± 0.12
4.5 ± 0.24
0.8 ± 0.22
1.1 ± 0.08
2.9 ± 0.22
2.9 ± 0.18
3.1 ± 0.21
3.8 ± 0.16
1.2 ± 0.16
0.9 ± 0.07
2.5 ± 0.15
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
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Table 6: Cytotoxic activity of 5-nitrofuran-triazole conjugates
(9a-n); IC50 values (µM)
& 4d). On the whole, 9f efficiently interacts with lanosterol 14α-
demethylase compared to miconazole and this is supported by
the antifungal activity as well as ergosterol biosynthesis
inhibition assay data.
Compound
MRC5
9a
9b
9c
9d
9e
9f
102.6 ± 0.36
98.9 ± 0.42
112.8 ± 0.28
99.5 ± 0.22
122.4 ± 0.18
142.8 ± 0.26
89.6 ± 0.24
88.2 ± 0.32
126.8 ± 0.18
107.3 ± 0.16
126.6 ± 0.28
108.8 ± 0.22
121.4 ± 0.24
151.8 ± 0.38
183.9 ± 0.42
9g
9h
9i
9j
9k
9l
9m
9n
Cip
Fig. 4: a) Binding pose of compound 9f in the binding site of
lanosterol 14α-demethylase; b) binding pose of miconazole; c)
binding pose of 9a; d) binding pose of 9d. Amino acid residues
that make interactions with the compounds are indicated in
violet color.
Molecular modelling studies
The ergosterol biosynthesis inhibition assay confirmed that 9f
inhibits ergosterol biosynthesis which prompted us to study the
binding modes of this compound with the enzyme lanosterol
14α-demethylase (CYP 51) using computational techniques. The
protein structure of CYP51 was downloaded from protein data
bank (PDB code: 3GW9)21a and the docking studies were
performed using the software Autodock 4.2.21b The results
indicate that 9f binds efficiently into the binding pocket of the
enzyme with a binding energy of -8.70 kcal/mol whereas
miconazole interacts with a binding energy of -8.13 kcal/mol. As
shown in Fig 4a, the nitro group of nitrofuran moiety of 9f
establishes a couple of hydrogen bonding interactions with NH
of Leu 357 (1.9 Å) and Met 358 (2.1 Å). The amino acid residues
in the proximity of 9f include Tyr 103, Phe 105, Phe 290, Ala 291,
Thr 295, Pro 355, Met 460, Val 461 and Heme 480. A series of
hydrophobic interactions have been observed between most of
these amino acid residues and the aromatic ring of 9f. On the
other hand, miconazole establishes only one hydrogen bond
between its oxygen atom and OH of Tyr 103 (1.7 Å) which is
represented in Fig 4b. This could be responsible for its lower
binding energy compared to 9f. Interestingly, the molecular
modelling studies comply with the antifungal activities of the
compounds. As evident from the binding studies there is no
significant interaction of the aromatic substituent with the
enzyme active site and this is in accordance with the antifungal
activity data. However, 9f with 2,3-dichloro substituent on the
aromatic ring conferred comparatively better activity which
could be attributed to the polar interactions of chlorine atom
with the Tyr 103 as represented in Fig 4a. This supposition is
supported by studying the binding modes of 9a and 9d, wherein
the fluoro and methoxy substituents have not shown any
interactions with the amino acid residues in the proximity (Fig 4c
Conclusion
In conclusion, we have synthesized a series of nitrofuran-triazole
congeners carrying out suitable structural modifications on the
previously reported counterparts. The target compounds were
conveniently synthesized employing click chemistry protocol and
were evaluated for both antibacterial and antifungal activities.
All the compounds showed very good to moderate inhibitory
and bactericidal effects against most of the Gram-positive and
Gram-negative bacterial strains, respectively. Further, the
compounds displayed excellent antifungal activity against
fourteen different Candida strains and compound 9f was found
to be the most active compound against C. parapsilosis MTCC
1744. These compounds were also found to be effective
inhibitors of biofilm formation which may contribute to the
development of antibiotic resistance in bacteria. Furthermore,
ergosterol biosynthesis inhibition assay indicated that
compound 9f inhibited ergosterol biosynthesis thereby inducing
antifungal effects. Molecular modelling studies are carried out
to study the binding modes of 9f that correlates with the
antifungal activity as well as ergosterol biosynthesis inhibition
assay data. These compounds exhibited ten times lower
cytotoxicity to the normal human cells compared to the
antimicrobial activity and could be considered to possess
immense therapeutic potential. Therefore, a small structural
modification could be responsible for a big change in the
biological properties of
a compound which indicates that
libraries of compounds with diverse biological properties could
be generated after suitable structural modifications of the
known scaffold. The work reported herein is anticipated to
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provide an insight in the development of newer antibacterial
and antifungal agents. Moreover, compound 9f could be
considered as a promising candidate for further investigation.
General procedure for the synthesis of compounds (9a-n)
To a solution of tert-butanol (2 ml) and water (4 ml), 5-
nitro-N-(prop-2-ynyl)furan-2-carboxamide (6, 1 mmol) was
Acknowledgements
added while stirring. After thorough mixing substituted
phenyl azides (8a-n, 1 mmol), CuSO4.5H2O (0.25 mmol) and
sodium ascorbate (0.5 mmol) were added and the reaction
was stirred for 8hrs. On completion of the reaction water (5
ml) and chloroform (20 ml) were added and the aqueous
layer was extracted with chloroform (2x25 ml). The
combined organic layer was dried over anhydrous Na2SO4
and concentrated by rotary evaporation to afford crude
product which was further purified by column
chromatography using ethyl acetate and hexane as solvent
system.
The authors Y.P and F.S acknowledge CSIR, New Delhi and UGC,
New Delhi for the award of fellowships and Open Source Drug
Discovery (OSDD), CSIR, New Delhi for financial assistance.
Experimental Section
Chemistry
All chemicals and reagents were obtained from Aldrich (Sigma–
Aldrich, St. Louis, MO, USA) or Alfa Aesar (Johnson Matthey
Company, Ward Hill, MA, USA). Reactions were monitored by
TLC, performed on silica gel glass plates containing 60 F-254, and
visualization on TLC was achieved by UV light or iodine indicator.
Column chromatography was performed with Merck 60–120
mesh silica gel. 1H and 13C NMR spectra were recorded on
Gemini Varian-VXR-unity (200, 400, 500 MHz) or Bruker
UXNMR/XWIN-NMR (300 MHz) instruments. Chemical shifts (δ)
are expressed in ppm relative to internal standard TMS.
Multiplicities of NMR signals are represented as singlet (s),
doublet (d), doublet of doublet (dd), triplet (t), quartet (q) and
multiplet (m). ESI spectra were recorded on Micro mass, Quattro
LC using ESI+ software with capillary voltage 3.98 kV and ESI
mode positive ion trap detector.
N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-
nitrofuran-2-carboxamide (9a)
1
Yield: 86%; M.p: 192-194 °C; H NMR (500 MHz, CDCl3): δ
8.02 (s, 1H), 7.70 (dd, J = 9.0, 4.4 Hz, 2H), 7.35 (d, J = 3.8 Hz,
1H), 7.31 (s, 1H), 7.28 (d, J = 3.8 Hz, 1H), 7.23 (d, J = 8.8 Hz,
2H), 4.81 (d, J
=
5.5 Hz, 2H); 13C NMR (75 MHz,
CDCl3+DMSO-d6): δ 162.3, 158.9, 155.2, 150.1, 146.9, 131.8,
121.0, 120.2, 115.4, 115.1, 114.4, 111.5, 33.3; ESI-MS: 332
[M+H]+, HRMS Calcd for C14H11O4N5F [M+H]+ 332.07896
Found: 332.07829.
N-((1-(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-
nitrofuran-2-carboxamide (9b)
Synthesis of 5-nitro-N-(prop-2-ynyl)furan-2-carboxamide
Yield: 88%; M.p: 161-163 °C; 1H NMR (500 MHz,
CDCl3+CD3OD): δ 8.12 (s, 1H), 7.62-7.56 (m, 2H), 7.52-7.45
(m, 2H), 7.39 (d, J = 3.8 Hz, 1H), 7.34 (s, 1H), 7.29 (d, J = 3.8
Hz, 1H), 4.77 (s, 2H); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ
156.0, 150.8, 147.8, 143.7, 134.1, 130.3, 127.9, 127.1,
124.3, 115.2, 111.9, 34.2; ESI-MS: 348 [M+H]+, HRMS Calcd
for C14H11O4N5Cl [M+H]+ 348.04941 Found: 348.04886.
(6)
To 5-nitro-2-furoic acid
(5, 1mmol) in CH2Cl2, HOBt
(1.2mmol), EDC (1.2mmol) and propargyl amine (1 mmol)
were added and the reaction was stirred for 12h. After
completion of the reaction, water was added. Subsequently
the reaction mixture was washed with saturated NaHCO3
solution followed by 2N HCl solution. The combined organic
layer was separated, dried over anh. Na2SO4 and
N-((1-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-
evaporated on rotavapor to afford
6
in good yields. 1H NMR
5-nitrofuran-2-carboxamide (9c)
(300 MHz, CDCl3+DMSO-d6): δ 7.36 (d, J = 3.5 Hz, 1H), 7.29
(d, J = 3.5 Hz, 1H), 6.76 (bs, 1H), 4.26-4.25 (m, 2H), 2.33 (t, J
= 2.6 Hz, 1H).
1
Yield: 81%; M.p: 128-130 °C; H NMR (400 MHz, CDCl3): δ
8.09 (s, 1H), 7.75 (s, 1H), 7.34 (d, J = 3.8 Hz, 1H), 7.28 (d, J =
3.8 Hz, 1H), 6.88 (d, J = 2.2 Hz, 2H), 6.52 (t, J = 2.2 Hz, 1H)
4.83 (d, J = 6.0 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 161.5,
156.5, 147.6, 144.1, 138.2, 121.1, 116.1, 112.1, 100.8, 99.0,
55.7, 34.5; ESI-MS: 374 [M+H]+; HRMS Calcd for C16H16O6N5
[M+H]+ 374.10951 Found: 374.10879.
General procedure for the synthesis of compounds (8a-n)
The azide derivatives were conveniently synthesized by well
known Sandmeyer protocol.17 Substituted anilines (8a-n
)
were dissolved in aq. HCl and cooled to 0 °C. Ice cooled
solution of NaNO2 (2 eq.) was added and mixture was
stirred for 5-10 minutes at 0-5 °C. Subsequently Sodium
azide (2 eq) was added and the reaction mixture was stirred
for 0.5-1 h. After completion of the reaction, the azide
derivatives were extracted using EtOAc. The organic layer
was dried over anh. Na2SO4 and concentrated under
reduced pressure. The crude product was directly used for
further reactions.
N-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-
nitrofuran-2-carboxamide (9d)
1
Yield: 87%; M.p: 177-179 °C; H NMR (500 MHz, CDCl3): δ
7.97 (s, 1H), 7.61 (d, J = 9.0 Hz, 2H), 7.54 (s, 1H), 7.35 (d, J =
3.7 Hz, 1H), 7.27 (d, J = 3.8 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H)
4.81 (d, J = 5.9 Hz, 2H); 13C NMR (75 MHz, CDCl3+ CD3OD): δ
160.8, 157.9, 152.3, 148.4, 145.2, 130.8, 122.8, 122.4,
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116.7, 115.4, 112.9, 55.9, 35.1; ESI-MS: 344 [M+H]+, HRMS
Calcd for C15H14O5N5 [M+H]+ 344.09895 Found: 344.09857.
d6): δ 157.6, 152.5, 148.8, 147.8, 141.9, 126.1, 122.3, 121.3,
116.5, 113.3, 35.1; ESI-MS: 359 [M+H]+; HRMS Calcd for
C14H10O6N6Na [M+Na]+ 381.05540 Found: 381.05476.
N-((1-(3-Chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-
yl)methyl)-5-nitrofuran-2-carboxamide (9e)
N-((1-(2-Methoxy-4-nitrophenyl)-1H-1,2,3-triazol-4-
yl)methyl)-5-nitrofuran-2-carboxamide (9j)
1
Yield: 81%; M.p: 167-169 °C; H NMR (300 MHz, CDCl3): δ
8.03 (s, 1H), 7.84 (dd, J = 6.2, 2.6 Hz, 1H), 7.66-7.59 (m, 1H),
7.38-7.33 (m, 2H), 7.30 (d, J = 8.5 Hz, 2H), 4.81 (d, J = 5.5 Hz,
2H); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 157.5, 155.2,
150.0, 146.8, 144.1, 132.2, 120.9, 120.2, 118.9, 116.6,
114.5, 111.6, 33.2; ESI-MS: 366 [M+H]+, HRMS Calcd for
C14H10O4N5ClF [M+H]+ 366.03999 Found: 366.03966
Yield: 72%; M.p: 192-194 °C; 1H NMR (300 MHz,
CDCl3+CD3OD): δ 8.45 (s, 1H), 8.05 (d, J = 4.3 Hz, 3H), 7.50
(s, 1H), 7.44 (d, J = 3.7 Hz, 1H), 7.31 (d, J = 3.7 Hz, 1H), 4.76
(s, 2H), 4.09 (s, 3H); 13C NMR (75 MHz, CDCl3+CD3OD+
DMSO-d6+CF3COOD): δ 159.3, 156.6, 153.1, 150.7, 147.9,
130.4, 124.8, 119.7, 115.9, 112.2, 108.4, 107.5, 56.2, 33.9;
ESI-MS: 389 [M+H]+, HRMS Calcd for C15H13O7N6 [M+H]+
389.08402 Found: 389.08409
N-((1-(2,3-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-
nitrofuran-2-carboxamide (9f)
N-((1-(4-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-
1
Yield: 85%; M.p: 127-128 °C; H NMR (500 MHz, CDCl3): δ
nitrofuran-2-carboxamide (9k)
8.04 (s, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H),
7.40 (dt, J = 7.5, 0.6 Hz, 1H), 7.37-7.35 (m, 1H), 7.29-7.27
(m, 1H), 4.85 (d, J = 5.8 Hz, 2H); 13C NMR (75 MHz,
CDCl3+DMSO-d6): δ 156.4, 151.2, 147.9, 143.9, 136.1, 134.4,
131.5, 127.8, 125.9, 124.7, 115.8, 112.2, 34.6; ESI-MS: 382
[M+H]+, HRMS Calcd for C14H10O4N5Cl2 [M+H]+ 382.01044
Found: 382.01016
Yield: 72%; M.p: 209-211 °C; 1H NMR (300 MHz,
CDCl3+CD3OD): δ 7.55 (d, J = 8.7 Hz, 2H), 7.47-7.40 (m, 3H),
7.31 (s, 1H), 6.95 (d, J = 8.1 Hz, 2H), 4.74 (s, 2H); ESI-MS:
330 [M+H]+, 13C NMR (75 MHz, CDCl3+CD3OD): δ 157.6,
156.8, 151.2, 147.5, 128.8, 122.1, 115.8, 112.0, 34.3; HRMS
Calcd for C14H12O5N5 [M+H]+ 330.08329 Found: 330.08330
N-((1-(3,4-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-
5-Nitro-N-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)furan-2-
5-nitrofuran-2-carboxamide (9g)
carboxamide (9l)
1
Yield: 85%; M.p: 168-170 °C; H NMR (500 MHz, CDCl3): δ
Yield: 84%; M.p: 124-126 °C; 1H NMR (300 MHz, CDCl3+
CD3OD): δ 7.81 (s, 3H), 7.58-7.46 (m, 5H), 7.34 (s, 1H), 5.07
(s, 2H); 13C NMR (75 MHz, CDCl3+CD3OD): δ 156.7, 151.2,
147.7, 138.5, 129.9, 128.8, 120.8, 116.1, 112.2, 34.2; ESI-
MS: 314 [M+H]+, HRMS Calcd for C14H12N4O5 [M+H]+
314.0883 Found: 314.0879.
8.09 (s, 1H), 7.66 (s, 1H), 7.35 (d, J = 3.4 Hz, 1H) 7.33 (s, 1H),
7.29 (d, J = 3.4 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 6.95 (d, J =
8.5 Hz, 1H), 4.84 (s, 2H), 3.95 (d, J = 7.9 Hz, 6H); 13C NMR
(100 MHz, CDCl3): δ156.5, 151.4, 149.7, 147.6, 130.1, 116.1,
112.7, 112.2, 111.2, 104.9, 56.3, 56.2, 34.5; ESI-MS: 374
[M+H]+, HRMS Calcd for C16H16O6N5 [M+H]+ 374.10951
Found: 374.10937
N-((1-(4-Fluoro-2-nitrophenyl)-1H-1,2,3-triazol-4-
yl)methyl)-5-nitrofuran-2-carboxamide (9m)
5-Nitro-N-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-
Yield: 76%; M.p: 98-100 °C; 1H NMR (300 MHz, CDCl3+
CD3OD): δ 8.11 (s, 1H), 7.90 (dd, J = 7.4, 2.6 Hz, 1H), 7.72-
7.66 (m, 1H), 7.59 (dt, J = 7.2, 2.8 Hz, 2H), 7.42 (d, J = 3.6 Hz,
1H), 7.30 (d, J = 3.8 Hz, 1H), 4.76 (s, 2H); 13C NMR (75 MHz,
CDCl3+ CD3OD): δ 163.8, 160.4, 156.8, 147.5, 144.7, 129.7,
126.1, 124.9, 121.1, 115.8, 113.1, 112.0, 34.1; ESI-MS: 377
[M+H]+, HRMS Calcd for C14H10O6N6F [M+H]+ 377.06404
Found: 377.06398
yl)methyl)furan-2-carboxamide (9h)
Yield: 76%; M.p: 115-117 °C; 1H NMR (300 MHz,
CDCl3+DMSO-d6): δ 9.31 (t, J = 5.4 Hz, 1H), 8.07 (s, 1H), 8.00
(dd, J = 7.9, 1.1 Hz, 1H), 7.77 (dt, J = 7.5, 1.1 Hz, 1H), 7.67
(dt, J = 7.9, 1.1 Hz, 1H), 7.58 (dd, J = 7.7, 1.1 Hz, 1H), 7.38 (d,
J = 3.8 Hz, 1H), 7.28 (d, J = 3.8 Hz, 1H), 4.65 (d, J = 5.8 Hz,
2H); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 155.5, 147.4,
144.2, 132.8, 129.7, 128.7, 126.3, 124.3, 123.1, 114.6,
111.5, 33.6; ESI-MS: 359 [M+H]+, HRMS Calcd for
C14H11O6N6 [M+H]+ 359.07346 Found: 359.07331
N-((1-(3-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-
nitrofuran-2-carboxamide (9n)
Yield: 81%; M.p: 172-174 °C; 1H NMR (300 MHz,
CDCl3+CD3OD): δ 8.27 (s, 1H), 7.47-7.40 (m, 2H), 7.38-7.29
(m, 2H), 7.24-7.16 (m, 2H), 7.276.93 (dd, J = 8.3, 1.5 Hz, 1H),
4.73 (s, 2H); 13C NMR (75 MHz, CDCl3+CD3OD): δ 158.3,
156.8, 151.3, 147.5, 137.4, 130.3, 115.8, 112.0, 111.1,
107.5, 34.2; ESI-MS: 330 [M+H]+, HRMS Calcd for
C14H12O5N5 [M+H]+ 330.08329 Found: 330.08287
5-Nitro-N-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-
yl)methyl)furan-2-carboxamide (9i)
Yield: 78%; M.p: 193-195 °C; 1H NMR (300 MHz,
CD3OD+DMSO-d6): δ 8.73 (s, 1H), 8.45 (d, J = 9.0 Hz, 2H),
8.18 (d, J = 9.0 Hz, 2H), 7.62 (d, J = 3.9 Hz, 1H), 7.38 (d, J =
3.9 Hz, 1H), 4.71 (s, 2H); 13C NMR (75 MHz, CD3OD +DMSO-
8 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5OB01353D
Journal Name
ARTICLE
Minimum fungicidal concentration (MFC)
Biology
Antimicrobial activity
Fungicidal assay (NCCLS, 2000) was performed in sterile 2.0 ml
microfuge tubes against a panel of different pathogenic Candida
strains including Candida albicans MTCC 183, C. albicans MTCC
227, C. albicans MTCC 854, C. albicans MTCC 1637, C. albicans
MTCC 3017, C. albicans MTCC 3018, C. albicans MTCC 3958, C.
albicans MTCC 4748, C. albicans MTCC 7315, C. parapsilosis
MTCC 1744, C. aaseri MTCC 1962, C. glabrata MTCC 3019, C.
krusei MTCC 3020, Issatchenika hanoiensis MTCC 4755 which
were cultured overnight in Mueller Hinton broth. Serial dilutions
of test compounds were prepared in Mueller Hinton broth using
different concentrations ranging from 0 to 125 µg mL-1. To the
test compounds, 100 µl of overnight cultured bacterial
suspensions were added to reach a final concentration of 1.5 ×
108 cfu mL-1 (equal to 0.5 McFarland) and incubated at 37 ºC for
The antibacterial and antifungal activities of the nitrofuran-
triazole conjugates were determined using well diffusion
method22 (Amsterdam, 1996) against different pathogenic
reference strains such as Micrococcus luteus MTCC 2470,
Bacillus subtilis MTCC 121, Staphylococcus aureus MTCC 96,
Staphylococcus aureus MLS16 MTCC 2940, Klebsiella planticola
MTCC 530, Escherichia coli MTCC 739, Pseudomonas aeruginosa
MTCC 2453 and different Candida strains such as Candida
albicans MTCC 183, C. albicans MTCC 227, C. albicans MTCC 854,
C. albicans MTCC 1637, C. albicans MTCC 3017, C. albicans MTCC
3018, C. albicans MTCC 3958, C. albicans MTCC 4748, C. albicans
MTCC 7315, C. parapsilosis MTCC 1744, C. aaseri MTCC 1962, C.
glabrata MTCC 3019, C. krusei MTCC 3020, Issatchenika
hanoiensis MTCC 4755 procured from the Microbial Type
Culture Collection (MTCC), CSIR-Institute of Microbial
Technology, Chandigarh, India. The pathogenic reference strains
were seeded on the surface of the media Petri plates, containing
Muller-Hinton agar with 0.1 ml of previously prepared microbial
suspensions individually containing 1.5 × 108 cfu mL-1 (equal to
0.5 McFarland). Wells of 6.0 mm diameter were prepared in the
media plates using a cork borer and the synthesized nitrofuran-
triazole conjugates at a dose range of 125 - 0.9 µg well-1 were
added in each well under sterile conditions in a laminar air flow
chamber. Standard antibiotic solution of ciprofloxacin (bacterial
strains) and miconazole (Candida strains) at a dose range of 125
- 0.9 µg well-1 and the well containing methanol served as
positive and negative controls, respectively. The plates were
incubated for 24 h at 37 °C for bacterial strains and 30 °C for
different Candida strains and the well containing the least
concentration showing the inhibition zone was considered as
the minimum inhibitory concentration. All experiments were
carried out in duplicates and mean values are represented.
24 h. After 24
h of incubation, the minimum fungicidal
concentration (MFC) was determined by sampling 10 µl of
suspension from the tubes onto Mueller Hinton agar plates and
were incubated for 24 h at 37 ºC to observe the growth of test
organisms. MFC is the lowest concentration of compound
required to kill a particular bacterium. All the experiments were
carried in duplicates.
Biofilm inhibition crystal violet assay
The nitrofuran-triazole conjugates were screened in sterile 96
well polystyrene microtiter plates using the modified biofilm
inhibition assay24 (Kamal et al., 2015) against a panel of
pathogenic bacterial strains including Staphylococcus aureus
MTCC 96, Staphylococcus aureus MLS16 MTCC 2940 and Bacillus
subtilis MTCC 121, which were cultured overnight in tryptone
soy broth (supplemented with 0.5% glucose). The test
compounds of predetermined concentrations ranging from 0 to
125 µg/mL were mixed with the bacterial suspensions having an
initial inoculum concentration of 5 × 105 cfu mL-1. Aliquots of 100
µl were distributed in each well and then incubated at 37 ºC for
24 h under static conditions. The medium was then discarded
and washed with phosphate buffered saline to remove the non-
adherent bacteria. Each well of the microtiter plate was stained
with 100 µl of 0.1% crystal violet solution followed by 30 min
incubation at room temperature. Later the crystal violet solution
from the plates was discarded, thoroughly washed with distilled
water for 3 to 4 times and air dried at room temperature. The
crystal violet stained biofilm was solubilized in 95% ethanol (100
µL) and the absorbance was recorded at 540 nm using TRIAD
multimode reader (Dynex Technologies, Inc, Chantilly, VA, USA).
Blank wells were employed as background check. The inhibition
data were interpreted from the dose-response curves, where
IC50 value is defined as the concentration of inhibitor required to
inhibit 50% of biofilm formation under the above assay
conditions. All the experiments were carried out in triplicates
and the values are indicated as mean ± S.D.
Minimum bactericidal concentration (MBC)
Bactericidal assay (NCCLS, 2000) was performed in sterile 2.0 ml
microfuge tubes against
a panel of pathogenic bacterial
strains,23 including Micrococcus luteus MTCC 2470,
Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS-16
MTCC 2940, Bacillus subtilis MTCC 121, Escherichia coli MTCC
739, Pseudomonas aeruginosa MTCC 2453 and Klebsiella
planticola MTCC 530 which were cultured overnight in Mueller
Hinton broth. Serial dilutions of test compounds were prepared
in Mueller Hinton broth using different concentrations ranging
from 0 to 125 µg mL-1. To the test compounds, 100 µl of
overnight cultured bacterial suspensions were added to reach a
final concentration of 1.5 × 108 cfu mL-1 (equal to 0.5 McFarland)
and incubated at 37 ºC for 24 h. After 24 h of incubation, the
minimum bactericidal concentration (MBC) was determined by
sampling 10 µl of suspension from the tubes onto Mueller
Hinton agar plates and were incubated for 24 h at 37 ºC to
observe the growth of test organisms. MBC is the lowest
Quantification of ergosterol content
The total intracellular sterols from C. parapsilosis MTCC 1744
were extracted using the method of Breivik and Owades (1957)
with slight modifications.25 A single C. parapsilosis MTCC 1744
concentration of compound required to kill
a particular
bacterium. All the experiments were carried in duplicates.
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DOI: 10.1039/C5OB01353D
ARTICLE
Journal Name
7. C. Sheng, W. Wang, X. Che, G. Dong, S. Wang, H. Ji, Z. Miao,
J. Yao, W. Zhang, ChemMedChem, 2010, 5, 390.
colony cultured overnight in Sabouraud dextrose agar was
inoculated in 50 ml of Sabouraud dextrose broth containing
varying concentrations of the test compounds including 0, 2, 4, 8
and 16 µg mL-1. The culture was incubated at 30°C for 20 h with
continuous shaking. The stationary-phase cells were harvested
by centrifugation at 8,000 rpm for 5 min and washed with sterile
distilled water. The net wet weight of the cell pellet was
determined. Three millilitres of 25% alcoholic potassium
hydroxide solution was added to each pellet and vortex mixed
for 1 min. The cell suspensions were transferred to sterile glass
screw-cap tubes and were incubated in water bath at 85 oC for 1
h and then allowed to cool to room temperature. Sterols were
then extracted by addition of a mixture of sterile distilled water
and n-heptane (1:3) followed by vigorous vortex mixing for 3 to
4 min. The heptane layer was transferred to a clean glass tube
and stored at -20°C for duration of up to 24 h. An aliquot (20 µL)
of the sterol extract diluted fivefold in 100% ethanol was
scanned spectrophotometrically from 240 to 300 nm. The
presence of ergosterol and 24(28)dehydroergosterol
[24(28)DHE, a late sterol pathway intermediate] in the extracted
8. a) M. Irfan, B. Aneja, U. Yadava, S. I. Khan, N. Manzoor, C. G.
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sample exhibited
a characteristic four-peaked curve. The
absence of detectable ergosterol content in the extracts was
indicated by a flat line. A dose-dependent decrease in the height
of the absorbance peaks was evident which corresponded to the
decreased ergosterol concentration. Ergosterol content was
calculated as a percentage of the wet weight of the cell using
the following equations:
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% ergosterol + % 24(28) DHE = [(A281.5/ 290) × F]/pellet weight
% 24(28) DHE = [(A230/518) × F]/pellet weight, and
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approved standard fifth edition, NCCLS: Wayne, PA, 2000.
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%ergosterol = [% ergosterol + %24(28) DHE] - %24(28) DHE,
Where F is the factor for dilution in ethanol and 290 and 518 are
the E values (in percentages per centimetre) determined for
crystalline ergosterol and 24(28) DHE, respectively.
Molecular modeling procedure
All the geometries are optimized in Gaussian 09 using PM3 semi-
empirical method.26 Protein structure was downloaded from
Protein Data Bank (PDB ID: 3GW9). Docking studies were
performed using AutoDock 4.2 software. The analysis of
intermolecular interactions has been performed using Pymol, v.
0.99.27
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10 | J. Name., 2012, 00, 1-3
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Journal Name
View Article Online
DOI: 10.1039/C5OB01353D
ARTICLE
a Ahmed Kamal, S. M. Ali Hussaini, Y. Poornachandra, Faria Sultana, C.
Ganesh Kumar; Medicinal Chemistry & Pharmacology, CSIR-Indian
Institute of Chemical Technology, Hyderabad 500 007, India; Email:
ahmedkamal@iict.res.in
bAhmed Kamal, M. Lakshmi Sucharitha; Department of Medicinal
Chemistry, National Institute of Pharmaceutical Education and Research
(NIPER), Hyderabad-500 037, India
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