Proton-pumping-ATPase-targeted antifungal activity of cinnamaldehyde based sulfonyl tetrazoles

Article abstract of DOI:10.1016/j.ejmech.2011.12.007

Azoles are generally fungistatic, and resistance to fluconazole is emerging in several fungal pathogens. We designed a series of cinnamaldehyde based sulfonyl tetrazole derivatives. To further explore the antifungal activity, in vitro studies were conduct

Full text of DOI:10.1016/j.ejmech.2011.12.007

European Journal of Medicinal Chemistry 48 (2012) 363e370
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Proton-pumping-ATPase-targeted antifungal activity of cinnamaldehyde based
sulfonyl tetrazoles
,
,
Sheikh Shreaz a 1, Mohmmad Younus Wani b 1, Sheikh Rayees Ahmad d, Sheikh Imran Ahmad c,
Rimple Bhatia a, Fareeda Athar b, Manzoor Nikhat a, Luqman A. Khan a
,
*
a Enzyme Kinetics Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India
b Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India
c Department of Applied Sciences & Humanities, Jamia Millia Islamia, New Delhi 110025, India
d Department of Chemistry, Indian Institute of Technology, New Delhi 110016, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Azoles are generally fungistatic, and resistance to fluconazole is emerging in several fungal pathogens.
We designed a series of cinnamaldehyde based sulfonyl tetrazole derivatives. To further explore the
antifungal activity, in vitro studies were conducted against 60 clinical isolates and 6 standard laboratory
strains of Candida. The rapid irreversible action of these compounds on fungal cells suggested
a membrane-located target for their action. Results obtained indicate plasma membrane Hþ-ATPase as
site of action of the synthesized compounds. Inhibition of Hþ-ATPase leads to intracellular acidification
and cell death. Presence of chloro and nitro groups on the sulfonyl pendant has been demonstrated to be
a key structural element of antifungal potency. SEM micrographs of treated Candida cells showed severe
cell breakage and alterations in morphology.
Received 6 April 2011
Received in revised form
30 November 2011
Accepted 4 December 2011
Available online 9 December 2011
Keywords:
Antifungal activity
Candida
Ó 2011 Elsevier Masson SAS. All rights reserved.
Cinnamaldehyde
Tetrazole
Plasma membrane Hþ-ATPase
1. Introduction
[13]. However, a concrete mechanism for their antimicrobial action
has yet to be explained.
Candida albicans is an emerging cause of infection in both adults
and infants, and its resistance to azole antifungals is an increasing
problem [1,2]. Amphotericin B and azoles, basically fluconazole are
the currently available treatment options, which suffer from many
serious drawbacks [3e5]. Such serious global health problem
demands a renewed effort seeking the development of new anti-
microbial agents effective against pathogenic microorganisms
resistant to currently available treatments. To overcome this
problem we designed a series of spice oil cinnamaldehyde based
sulfonyl tetrazole derivatives. Sulfonamide derivatives possess
significant antimicrobial activity [6,7]. Some of them are HIV
protease inhibitors [8], carbonic anhydrase inhibitors [9], and
anticonvulsant agents [10,11]. Tetrazoles on the other extreme are
regarded as biologically equivalent to the carboxylic acid group
with strong growth inhibitory activity against different Candida
species [12]. It is reported that tetrazole derivatives were the most
active against C. albicans in comparison to standard antifungal drug
The yeast plasma membrane Hþ ATPase is a promising new
antifungal target [14,15]. This enzyme plays a crucial role in fungal
cell physiology, intracellular pH regulation, cell growth and has
been implicated in the pathogenicity of fungi through its effects on
dimorphism, nutrient uptake and medium acidification [14,16].
Previously we have reported proton translocating ATPase mediated
fungicidal activity of various essential oil components [17e19]. The
objective of this study was to further elucidate the antimicrobial
mechanism of action of newly synthesized cinnamaldehyde based
compounds by determining their effect on the activity of Hþ ATPase
located in the membranes of pathogenic Candida species. Impor-
tantly, we also illustrate that these synthesized compounds are
effective in vitro against Candida species which were demonstrated
to be largely insensitive to azoles.
2. Results and discussion
2.1. Chemistry
* Corresponding author. Tel.: þ91 (11) 26981717x3410.
E-mail address: profkhanluqman@gmail.com (L.A. Khan).
Authors contributed equally.
Present study was undertaken to synthesize some novel sulfonyl
tetrazole derivatives and study their probable antifungal behaviour.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.007

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S. Shreaz et al. / European Journal of Medicinal Chemistry 48 (2012) 363e370
Table 1
Target compounds were obtained in a two step reaction procedure
as outlined in Scheme 1. First of all, 5-[(E)-2-phenylethenyl]-2H-
tetrazole (1) was synthesized from cinnamaldehyde by following
a reported procedure [20]. 5-[(E)-2-phenylethenyl]-2H-tetrazole
was treated with different arylsulfonylchlorides (substituted/
unsubstituted), in presence of triethylamine using dry CH2Cl2 as
a solvent to get the target compounds. All the synthesized
compounds were characterized by elemental analysis, IR, 1H, 13C
NMR and ESI-MS studies and their data is presented in experi-
mental section.
Isolates used in the present study.
Classification of isolates
Type of isolate
Sensitive (Standard, n ¼ 6)
ATCC 10261
ATCC 44829
ATCC 90028
ATCC 750
C. albicans
C. albicans
C. albicans
C. tropicalis
C. glabrata
C. krusei
ATCC 90030
ATCC 6258
Sensitive (Clinical, n ¼ 40)
Invasive (n ¼ 8)
C. albicans(3), C. tropicalis(1),
C. glabrata(3), C. krusei (1)
C. albicans(6), C. glabrata(3), C. krusei (1)
2.2. Pharmacology
Cutaneous (n ¼ 10)
Respiratory:
2.2.1. Minimal inhibitory concentration
Bronchioalveolar (n ¼ 7)
C. albicans (3), C. glabrata(2), C. krusei (2)
C. albicans(3), C. tropicalis(1), C. glabrata(3),
C. guilliermondii(1), C. krusei (1)
Oropharyngeal (n ¼ 9)
Table 2 summarizes the in vitro susceptibilities of 46 Candida
isolates of fluconazole-susceptible and 20 Candida isolates of
fluconazole-resistant category (Table 1). The data are reported as
MICs required to inhibit 90% growth of the Candida cell population.
Resistanta (Clinical, n ¼ 20)
Invasive (n ¼ 7)
C. albicans(4), C. tropicalis(1),
C. glabrata(1), C. krusei (1)
C. tropicalis(2), C. glabrata(2),
C. guilliermondii(2), C. krusei (1)
C. albicans(3), C. tropicalis(2), C. glabrata(1)
Species exhibiting fluconazole MIC ꢀ 64
mg/ml were considered as
Cutaneous (n ¼ 7)
resistant.
Respiratory (n ¼ 6)
2.2.2. Disc diffusion
a
Fluconazole MIC ꢀ 64
mg/ml considered as resistant.
The results summarized in Table 3 give the sensitivity assay,
using standard discs of compound (1a), compound (1b), compound
(1c), compound (1d), compound (1e) and fluconazole (100 mg/ml).
Both the standard and clinical types of Candida isolates showed
high degree of sensitivity as is evident from the zone of clearance in
each case. Index of sensitivity defined as
effect on the strains tested in the present study. Our results show
that the growth was inhibited by synthesized compounds, and the
halo was completely clear. Fig. S1 in the supplementary information
indicates potential fungicidal activity in fluconazole-sensitive
strains, whereas in case of the resistant strains fluconazole
showed a turbid halo and in most of the cases the zone was absent
an indication of its fungistatic nature.
X
Zone diameterðmmÞ=concentrationðmgÞ
¼ clearingðmmÞ=mg
2.2.3. Growth studies (Turbidometric measurement)
is greater (3.47 ꢁ 0.53) for resistant isolates when treated with
compound (1d) and least (0.73 ꢁ 0.15) for clinical isolates when
treated with compound (1a). Results obtained demonstrated that
the ability to kill Candida species is dependent on the concentration
of the test compound. The discs impregnated with DMSO (negative
control) showed no zone of inhibition and hence 10% DMSO had no
Growth pattern of Candida species was investigated at different
concentrations of compound (1a), compound (1b), compound (1c),
compound (1d) and compound (1e). Fig. 1 (A)e(C) shows the effect
of different concentrations of compound 1c, compound 1d and
compound 1e on growth pattern of C. albicans ATCC 10261.
Significant and pronounced effect is observed for other tested
species (data not shown). Control cells showed a normal pattern of
growth with lag phase of 4 h, active exponential phase of 8e10 h
before attaining stationary phase. Increase in concentration of test
agents leads to significant decrease in growth with suppressed and
delayed exponential phases with respect to control. At MIC90 values
almost complete cessation of growth was observed for all the yeast
species. Effectiveness of growth inhibition was measured by
comparing optical densities on full growth (24 h) at half MIC of
respective compounds. Average decrease in optical densities of four
Candida isolates of fluconazole-susceptible and three Candida
isolates of fluconazole-resistant category with respect to control
was 60.64% for compound (1a), 71.06% for compound (1b), 82.92%
for compound (1c), 91.53% for compound (1d) and 77.31% for
compound (1e) respectively.
Table 2
Minimum inhibitory concentrations (90%) of the synthesized compounds against
Candida isolates.
Bioactive
Mean MIC90 (mg/ml)
compound
Fluconazole-sensitive
Fluconazole-resistant
(n ¼ 46)
(n ¼ 20)
Compound (1a)
Compound (1b)
Compound (1c)
Compound (1d)
Compound (1e)
250e450
150e250
80e110
60e90
200e300
150e250
70e90
50e70
100e200
Scheme 1. Synthesis of 1-(phenyl/substituted phenyl) sulfonyl]-5-[(E)-2-phenyl
ethenyl]-1H-tetrazoles.
150e200

S. Shreaz et al. / European Journal of Medicinal Chemistry 48 (2012) 363e370
365
Table 3
In vitro sensitivity of compound (1a), compound (1b), compound (1c), compound (1d) and compound (1e) against Candida isolates as determined by disc diffusion assay.
Candida isolates
Sensitivity index (mm/mg)
Compound (1a)
Compound (1b)
Compound (1c)
Compound (1d)
Compound (1e)
Standard (n ¼ 6)
Clinical (n ¼ 10)
Resistant (n ¼ 10)
0.77 ꢁ 0.18
0.73 ꢁ 0.15
0.98 ꢁ 0.21
0.81 ꢁ 0.41
1.05 ꢁ 0.09
1.27 ꢁ 0.23
2.77 ꢁ 0.33
2.31 ꢁ 0.51
3.29 ꢁ 0.47
3.16 ꢁ 0.41
2.71 ꢁ 0.37
3.47 ꢁ 0.53
1.42 ꢁ 0.60
1.18 ꢁ 0.19
1.71 ꢁ 0.28
Fluconazole (100
m
g/ml) showed inhibitory zone (mm) of 14.33 (ꢁ2.58), 15.8 (ꢁ1.61) and 02 (ꢁ1.24) for standard, clinical and resistant isolates. Sensitivity index is expressed
as mean ꢁ SD and was calculated as diameter of zone of inhibition (mm)/concentration of drug (mg/ml). n is number of isolates. Each isolate was tested in duplicate.
2.2.4. Proton efflux measurements
2.2.5. Measurement of intracellular pH
Hþ-efflux is an immediate event associated with Hþ ATPase
activity. Proton-pumping ability of fungi mediated by the Hþ
ATPase at the expense of energy is crucial for the regulation of
internal pH and growth regulation of fungal cell. Fungal cells
depleted of carbon-source when exposed to glucose, rapidly acidify
medium to generate proton motive force for nutrient uptake.
Candida cells susceptible to the synthesized compounds were
examined for the ability to pump intracellular protons to the
external medium (as measured by the alteration of pH of the
external medium) in the presence of compound (1a), compound
(1b), compound (1c), compound (1d) and compound (1e). Table 4
gives relative rates of Hþ-efflux by Candida sp. in presence of
Intracellular pH and the Hþ pump are thought to play an
important role in yeast growth. Many cellular processes are regu-
lated by the internal pH, and many transport processes depend on
the Hþ cycle. Generally internal pH of yeast cells is maintained
between 6.0 and 7.5 by Hþ ATPase activity. We tried to investigate
whether cells with normal Hþ ATPase activity maintain the
constant internal pH as compared to cells with low activity. Fig. 2
shows changes in the pattern of pHi with control and treated
cells. Only yeast control cells with normal Hþ ATPase activity
maintain the pHi (6.52), while the treated cells show increase in
internal acidification. The decrease in pHi was more in cells
exposed to compound (1d) than rest of the four compounds.
synthesized compounds and fluconazole (5 m
g/ml). Hþ-extrusion
inhibition in standard isolates was 32.37%, 50.22%, 89.90%, 96.05%
and 62.88% when treated with compound (1a), compound (1b),
compound (1c), compound (1d) and compound (1e), respectively.
Hþ-extrusion rate was also decreased to 21.00%, 38.91%, 80.75%,
88.89% and 56.82% for compound (1a), compound (1b), compound
(1c), compound (1d) and compound (1e), in clinical isolates. In case
of resistant isolates the decrease was 36.81%, 54.54%, 93.56%,
97.21% and 66.35% when cells were treated with compound (1a),
compound (1b), compound (1c), compound (1d) and compound
(1e), respectively. Glucose (5 mM) stimulated Hþ-efflux in all the
strains by 4e5 folds. Glucose-stimulated Hþ-efflux was also
inhibited by the synthesized compounds. Glucose-stimulated Hþ-
efflux rates in standard, resistant and clinical isolates with respect
to control, were 16.68%, 16.81% and 11.17% in the presence of
compound (1a), 36.85%, 36.37% and 26.29% in the presence of
compound (1b), 60.53%, 61.90% and 49.57% in presence of
compound (1c), 64.50%, 69.86% and 59.42% in presence of
compound (1d) and 45.35%, 45.91% and 38.57% in presence of
compound (1e), respectively. Detailed studies of test compounds on
the same can give us more insight into the possible mechanisms of
action.
2.2.6. Scanning electron microscopy
Finally SEM observations were used to study the anticandidal
activity of the synthesized compound. Interaction between
compound (1d) and Candida cells was demonstrated by SEM after
a 14 h exposure. Fig. 3 results clearly showed differences in
morphology between untreated and compound 1d treated Candida
cells. On the basis of present microscopic analyses of the Candida
cells, it can be concluded that compound (1d) causes irreparable
damage to Candida cells. The SEM micrographs for untreated
C. albicans Seq No 1138 shows well defined shape with normal
smooth surfaces Fig. 3 (A). There was complete damage to the cell of
compound 1d treated fungi, indicated by black arrow in Fig. 3 (B). In
addition to this multiple breaks were also noticed in the membrane
of the treated cell which is indicated by white arrow in Fig. 3 (B).
Interesting finding is that compound 1d causes complete cellular
damage at MIC90 concentrations.
2.2.7. Hemolytic activity
To evaluate the toxicity of synthesized compounds, they were
tested against human red blood cells. Effects of test agents on
human red blood cells are shown in Fig. 4. Compound (1a),
A
B
C
2.5
2
2
2.5
2
1.8
1.6
1.4
1.2
1
1.5
1
1.5
1
0.8
0.6
0.4
0.2
0
0.5
0
0.5
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hrs)
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hrs)
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hrs)
Control
15µg/ml
30µg/ml
60µg/ml
Control
37.5µg/ml
75µg/ml
150µg/ml
Control
25µg/ml
50µg/ml
100µg/ml
Fig. 1. Effect of synthesized compounds on growth of C. albicans ATCC 10261. Growth curve plotted against absorbance at 595 nm and time (h) shows complete inhibition of growth
at MIC90 values. (A) In presence of compound 1c. (B) In presence of compound 1d. (C) In presence of compound 1e.

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S. Shreaz et al. / European Journal of Medicinal Chemistry 48 (2012) 363e370
Table 4
Effect of compound (1a), compound (1b), compound (1c), compound (1d) and compound (1e) on the rate of Hþ-efflux by various Candida isolates at pH 7. Cells were sus-
pended in 0.1 mM CaCl2 and 0.1 M KCl at 25 ꢂC.
Incubation with
Range of relative Hþ-efflux rate
Standard
Clinical
Resistant
Control
1
1*
1**
Compound 1a (MIC90
Compound 1b (MIC90
)
)
0.67 ꢁ 0.02 (32.37)
0.49 ꢁ 0.03 (50.22)
0.09 ꢁ 0.01 (89.90)
0.03 ꢁ 0.02 (96.05)
0.36 ꢁ 0.01 (62.88)
4.19
0.78 ꢁ 0.03 (21.00)
0.60 ꢁ 0.04 (38.99)
0.18 ꢁ 0.03 (80.75)
0.10 ꢁ 0.02 (88.89)
0.42 ꢁ 0.02 (56.82)
3.80
0.62 ꢁ 0.02 (36.81)
0.44 ꢁ 0.04 (54.45)
0.05 ꢁ 0.02 (93.56)
0.02 ꢁ 0.01 (97.21)
0.33 ꢁ 0.01 (66.35)
3.71
Compound 1c (MIC90
)
Compound 1d (MIC90
)
Compound 1e (MIC90
)
Glucose (5 mM)
Glucose þ Compound 1a
Glucose þ Compound 1b
Glucose þ Compound 1c
Glucose þ Compound 1d
Glucose þ Compound 1e
3.48 ꢁ 0.51 (16.68)
2.65 ꢁ 0.60 (36.85)
1.64 ꢁ 0.63 (60.53)
1.48 ꢁ 0.63 (64.50)
2.29 ꢁ 0.63 (45.35)
3.37 ꢁ 0.48 (11.17)
2.78 ꢁ 0.40 (26.29)
1.90 ꢁ 0.26 (49.57)
1.53 ꢁ 0.22 (59.42)
2.32 ꢁ 0.33 (38.57)
3.09 ꢁ 0.24 (16.81)
2.35 ꢁ 0.26 (36.70)
1.41 ꢁ 0.17 (61.90)
1.12 ꢁ 0.18 (69.86)
1.99 ꢁ 0.25 (45.91)
Control had an average (of 4 independent recordings) Hþ-efflux rate of 5.58 nmol/min/mg cells in standard isolates (1); 5.51 nmol/min/mg yeast cells in clinical isolates (1*)
and 5.73 nmol/min/mg yeast cells in resistant isolates (1**). Values in parentheses give the %-age inhibition of Hþ-efflux w.r.t. control.
compound (1b), compound (1c), compound (1d), compound (1e)
and the reference compound fluconazole showed a viability of 98%,
yeast physiology may not be better equipped to counteract the
antifungal properties of these synthesized compounds.
93%, 91%, 97%, 90%, 89% at the concentration range of 10
50 g/ml and 100 g/ml the viability was 95%, 90%, 84%, 89%, 85%,
71% and 93%, 87%, 79%, 86%, 78%, 45%. At much higher concentra-
tions 300 g/ml and 600 g/ml compound (1a), compound (1b),
compound (1c), compound (1d), compound (1e) showed 10%, 17%,
24%, 16%, 26% and 11%, 20%, 25%, 18%, 28% hemolysis while as
conventional antifungal therapeutic agent showed 79% and 82%
hemolysis. At the MIC90 concentration of synthesized compounds,
which had profound effect on growth, Hþ ATPase activity and
ultrastructure of Candida, the hemolysis observed was very little.
This indicates that the synthesized compounds have low cytotoxic
activity.
m
g/ml. At
In liquid medium on full growth (24 h) at half MIC, decrease in
optical densities of 7 randomly selected Candida isolates with
respect to control was 60.64% for compound 1a, 71.06% for
compound 1b, 82.92% for compound 1c, 91.53% for compound 1d
and 77.31% for compound 1e respectively. Control cells showed
normal growth pattern. At lower concentration of the synthesized
compounds slight decline in curve was observed as compared to
control, whereas at MIC90 values of the test agents, normal S shaped
curve declined to flat line showing almost complete arrest of cell
growth (Fig. 1). Anticandidal activity order of the compounds on
solid medium leads to similar conclusion, compound
m
m
m
m
(1d)
> compound (1c) > compound (1e) > compound
The fungal cell wall may be considered to be a prime target for
selectively toxic antifungal agents because of its chitin structure,
absent from human cells. In the present study we demonstrated
that the synthesized compounds at MIC90 values exhibit fungicidal
and not fungistatic activity, by halo assay and growth curve studies
against all the Candida isolates in vitro. In addition to our six
laboratory wild-type Candida strains, forty (susceptible) and
twenty (resistant), recently obtained clinical isolates were also
tested for evidence of antifungal activity of these compounds.
Synthesized derivatives exhibit varying degrees of antifungal
activity. Generally, all the fluconazole-susceptible as well as-
resistant Candida isolates investigated were found to be sensitive
to the synthesized compounds. The use of total mean MICs ob-
tained gave a good indication of the overall antimicrobial effec-
tiveness of each synthesized compound. This may indicate that the
(1b) > compound (1a). The conclusion that compound 1d has
higher antifungal activity than rest of the compounds is based on
the differences in compound concentrations needed to inhibit yeast
growth (Table 2). These results suggest that presence of nitro and
chloro groups in the structure plays a key role in the antifungal
activity. A total of two of the five compounds feature a nitro group
(compound 1d) and chloro group (compound 1c) in their structure,
these represented the most active antifungal compounds. However,
in this study, the less effective compounds were shown to be
compound (1a), compound (1b) and compound (1e), Compound 1a
bears an unsubstituted phenyl ring while as the phenyl ring is
substituted at para and meta positions by a methyl group in
compounds 1b and 1e respectively. Furthermore, it was also
observed that in less effective compounds the presence and posi-
tion of methyl group has a marked effect on the antifungal potential
of the compound. These findings indicate that the presence of
electron withdrawing groups on the phenyl ring of the sulfonyl
pendant increases the antifungal activity of the compounds under
investigation than the compounds bearing electron donating
groups. Based upon the results it will also be necessary to optimize
the led compound by substitution in C-4 position of phenyl ring of
the sulfonyl fragment by electron withdrawing groups, which seem
to be very important for antifungal effect, besides the position of
the substituents seems to be an important factor behind the anti-
fungal potential of the tested compounds.
6.6
6.5
6.4
6.3
6.2
6.1
The in vitro hemolytic assay is a feasible screening tool for
gauging in vivo toxicity to host cells [21,22]. The comparative study
of synthesized compounds indicates that the test compounds were
significantly less cytotoxic than conventional antifungals.
6
Control compound compound compound compound compound
(1a)
(1b)
(1c)
(1d)
(1e)
Low MIC values and low cytotoxicity obtained against Candida
with synthesized compounds encouraged us to study their mode of
action. Although tetrazole derivatives showed higher anticandidal
activity than that of comparable membrane-targeting antifungal
Fig. 2. Intracellular pH in presence of synthesized compounds in Candida cells. Mid
logarithmic cells were incubated with MIC90 of compound (1a), compound (1b),
compound (1c), compound (1d) and compound (1e). Remarkable decline in pH as
shown in figure is indicative of induced acidity.

S. Shreaz et al. / European Journal of Medicinal Chemistry 48 (2012) 363e370
367
Fig. 3. Scanning electron micrographs of C. albicans Seq No 1138 (A) Untreated control and (B) Treated with compound 1d. The black arrows indicate damage to the cell while as the
white arrows indicate breakage of the membrane.
drugs [13], however, specific mechanisms involved in the antimi-
crobial action of tetrazoles remain poorly characterised. The rapid
irreversible action of these compounds suggests that there may be
a cellular target(s) accessible to the compound externally. Candida
isolates showing susceptibility to the test compounds also showed
inhibition of Hþ-ATPase-mediated proton pumping suggesting that
the two events are linked. It is to be noted that the inhibition of Hþ-
ATPase function was achieved at the MIC90 concentrations of the
compounds. The decrease in Hþ-extrusion being less when cells
were exposed to the test compounds in presence of glucose. It is
well established that plasma membrane ATPase undergoes modi-
fication in glucose medium [23]. Glucose induced acidification of
the external medium by yeast cells are a convenient measure of Hþ-
ATPase-mediated proton pumping [24]. The enzyme may exist in
a different conformational state in the two situations. It is thus
possible that the synthesized compounds may be directly inter-
acting with the enzyme, which serves as the primary reason for
their antifungal activity. Thus, it would be useful to further inves-
tigate the interaction of these compounds with the purified PM-
ATPase enzyme and study its activity in both steady state and
pre-steady state. Regulation of pHi, appears to be a fundamental
prerequisite for growth of Candida and activation of plasma
membrane ATPase as it is involved in maintenance of pHi [16]. We
therefore studied the role of plasma membrane ATPase activation
in the regulation of pHi, in control as well as treated cells. The pHi
was near neutrality in absence of test compounds while as in
presence of the MIC90 of the synthesized compounds pHi declined
to 6.46 (compound 1a), 6.36 (compound 1b), 6.24 (compound 1c),
6.21 (compound 1d) and 6.31 (compound 1e) respectively. Surface
features of cells after 14 h treatment with compound 1d revealed
extensive cell damage and significant breakage of cell membrane of
compound 1d treated cells Fig. 3 (B). In addition to this significant
morphological change in cell shape was also observed. Taken
together our results emphasize that multiple drug susceptibility
assays therefore should be used for exact assessment. The excel-
lence of these compounds demand more insight studies into all of
the possible mechanisms of these compounds.
3. Conclusion
Present study has achieved the excellent synthesis of cinna-
maldehyde based sulfonyl tetrazoles. The results of the study also
90
80
70
60
50
40
30
20
10
0
10
50
100
300
1e
600
Test agents (µg/ml)
1a
1b
1c
1d
Fluconazole
Fig. 4. Hemolysis caused by different agents: compound (1a), compound (1b),
compound (1c), compound (1d), compound (1e) and fluconazole. Hemolysis was
determined by an absorbance reading at 450 nm and compared to hemolysis achieved
with 1% Triton X-100 (reference for 100% hemolysis). The data are means of triplicate
experiments.

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S. Shreaz et al. / European Journal of Medicinal Chemistry 48 (2012) 363e370
present a concrete mechanism for their antimicrobial action. The
synthesized compounds show significant anticandidal activity both
in liquid and solid medium. These compounds have insignificant
toxicity at MIC90. Tentative mechanism of action appears to origi-
nate from inhibition of plasma membrane ATPase activity. These
synthesized compounds could be promising drugs after improved
formulations and also advocates the determination of optimal
concentrations for clinical applications.
4.2.2. 5-[(E)-2-Phenylethenyl]-1-(phenylsulfonyl)-1H-tetrazole (1a)
Yield 72%; White crystals; mp 182e185 ꢂC; Anal. Calc. for
C15H12N4O2S: C 57.68, H 3.87, N 17.94%, found: 57.70, H, 3.84, N,
18.05%; IR nmaxcmꢃ1: 3072 (AreH), 3045 (CeH), 1650 (C]N) 1150
(S]O); 1H NMR (DMSO-d6)
d(ppm): 7.80e7.72 (2H, m) 7.64 (2H, m),
7.53 (1H, d, J ¼ 15.2 Hz), 7.48e7.38 (3H, m), 7.33e7.22 (3H, m), 7.10
(1H, d, J ¼ 15.1); 13C NMR (DMSO-d6)
d(ppm): 167.5 (C]N), 145.6,
137.2, 123.4, 115.7, 113.0, (AreC), 128.5, 108.0; ESI-MS m/z: [Mþþ1]
313.
4. Experimental Protocol
4.2.3. 1-[(4-Methylphenyl)sulfonyl]-5-[(E)-2-phenylethenyl]-1H-
tetrazole (1b)
All the chemicals were purchased from Aldrich (USA) and Merck
(Germany). Precoated aluminum sheets (silica gel 60 F254), Merck
(Germany) were used for thin-layer chromatography (TLC) and
spots were visualized under UV light. Elemental analyses were
performed on Heraeus Vario EL III analyzer at Central Drug
Research Institute, Lucknow, India. The results were within ꢁ0.4%
of the theoretical values. IR spectra were recorded on PerkineElmer
model 1600 FT-IR RX1 spectrophotometer as KBr discs. 1H NMR and
13C NMR spectra were recorded on Bruker AVANCE 300 spec-
trometer using DMSO-d6 as solvent with TMS as internal standard.
Splitting patterns are designated as follows; s, singlet; d, doublet;
dd, doublet of doublets; t, triplet; m, multiplet. Chemical shift
values are given in ppm. ESI-MS was recorded on a MICROMASS
QUATTRO II triple quadrupole mass spectrometer.
Yield 68%; Yellow crystals; mp 175e178 ꢂC; Anal. Calc. for
C16H14N4O2S: C 58.88, H 4.32, N 17.17%, found: C 59.03, H 4.30, N
17.22%; IR nmaxcmꢃ1: 3068 (AreH), 3038 (CeH), 1660 (C]N) 1158
(S]O); 1H NMR (DMSO-d6)
d(ppm): 7.89e7.80 (2H, m) 7.75 (2H, m),
7.60 (1H, d, J ¼ 15.2 Hz), 7.55e7.48 (2H, m), 7.46e7.36 (3H, m), 7.33
(1H, d, J ¼ 15.1) 2.35 (3H, s, CH3); 13C NMR (DMSO-d6)
d(ppm): 168.0
(C]N), 147.5, 135.0, 125.4, 112.5, 113.0, (AreC), 132.0, 104.5, 28.0;
ESI-MS m/z: [Mþþ1] 327.
4.2.4. 1-[(4-Chlorophenyl)sulfonyl]-5-[(E)-2-phenylethenyl]-1H-
tetrazole (1c)
Yield 75%; White crystals; mp 170e173 ꢂC; Anal. Calc. for
C15H11ClN4O2S: C 51.95, H 3.20, N 16.16%, found: C 52.01, H 3.24, N
16.27%; IR nmaxcmꢃ1: 3080 (AreH), 3055 (CeH), 1650 (C]N) 1165
(S]O) 735 (CeCl stretch); 1H NMR (DMSO-d6)
d(ppm): 7.85e7.86
4.1. General procedure for the transformation of cinnamaldehyde
into 5-[(E)-2-phenylethenyl]-2H-tetrazole (1)
(2H, m) 7.70 (2H, m), 7.65 (1H, d, J ¼ 15.2 Hz), 7.52e7.46 (2H, m),
7.40e7.32 (3H, m), 7.26 (1H, d, J ¼ 15.1); 13C NMR (DMSO-d6)
d
(ppm): 165.5 (C]N), 148.0, 130.0, 125.5, 115.0, 110.4, (AreC),129.5,
110.5; ESI-MS m/z: [Mþþ1] 348.
A
solution of (2E)-3-phenylprop-2-enal (cinnamaldehyde)
(5 mmol) and iodine (5.5 mmol) in ammonia water (50 ml of 30%
solution) and THF (5 ml) was stirred at room temperature for 1 h.
The dark solution became colorless at the end of reaction. A
mixture of NaN3 (6.0 mmol) and ZnBr2 (7.5 mmol) was then added.
The reaction mixture was heated at reflux for 12e15 h with
vigorous stirring. HCl (40 ml of 3 M solution) and EtOAc (100 ml)
were added, and vigorous stirring was continued until no solid was
present and the aqueous layer had a pH of 1. The organic phase was
concentrated in vacuo, and the remaining solids were rinsed with
EtOAc (50 ml) to give a pure tetrazole product.
4.2.5. 1-[(4-Nitrophenyl)sulfonyl]-5-[(E)-2-phenylethenyl]-1H-
tetrazole (1d)
Yield 80%; White crystals; mp 165e168 ꢂC; Anal. Calc. for
C15H11N5O4S: C 50.42, H 3.10, N 19.60%, found: C 50.45, H 3.14, N
19.67%; IR nmaxcmꢃ1: 3067 (AreH), 3045 (CeH), 1660 (C]N), 1380
(NO2 stretch), 1152 (S]O stretch); 1H NMR (DMSO-d6)
d(ppm):
7.82e7.75 (2H, m) 7.73 (2H, m), 7.68 (1H, d, J ¼ 15.0 Hz), 7.50e7.43
(2H, m), 7.38e7.30 (3H, m), 7.25 (1H, d, J ¼ 14.3); 13C NMR (DMSO-
d6) d(ppm): 168.0 (C]N), 145.0, 133.5, 127.0, 113.7, 110.5, (AreC)
130.0, 110.5; ESI-MS m/z: [Mþþ1] 358.
4.2. General procedure for the synthesis of 5-[(E)-2-phenylethenyl]-
2-(phenyl/substituted phenyl sulfonyl)-2H-tetrazoles (1aee)
4.2.6. 1-[(3-Methylphenyl)sulfonyl]-5-[(E)-2-phenylethenyl]-1H-
tetrazole (1e)
To a solution of 5-[(E)-2-phenylethenyl]-2H-tetrazole 1 (1 eq.)
and triethylamine (3 eq.) in dry dichloromethane at 0 ꢂC was
added aryl sulfonyl chlorides (1.2 eq.). The reaction mixture was
stirred at 0 ꢂC for about 2 h and stirring was continued at room
temperature for about 4e5 h (completion of reaction was moni-
tored by TLC). After the completion of reaction the reaction mass
was quenched with distilled water and extracted with dichloro-
methane. Finally the combined organic layer was washed with
distilled water again and dried over anhydrous Na2SO4. After
removal of the solvent in vacuum, the residue was purified by
recrystallization.
Yield 70%; Light Yellow crystals; mp 165e168 ꢂC; Anal. Calc. for
C16H14N4O2S: C 58.86, H 4.33, N 17.17%, found: C 58.98, H 4.34, N
17.20%; IR nmaxcmꢃ1: 3074 (AreH), 3046 (CeH), 1648 (C]N) 1175
(S]O); 1H NMR (DMSO-d6)
d(ppm): 7.76e7.68 (2H, m) 7.75 (2H, m),
7.63 (1H, d, J ¼ 14.8 Hz), 7.50e7.42 (2H, m), 7.45e7.34 (3H, m), 7.28
(1H, d, J ¼ 15.1) 2.25 (3H, s, CH3); 13C NMR (DMSO-d6)
d(ppm):
168.0 (C]N), 148.9, 137.6, 126.8, 115.5, 110.5, (AreC), 127.8, 112.5,
28.0; ESI-MS m/z: [Mþþ1] 327.
4.3. Determination of MIC90
Microtiter assay: Cells were grown for 48 h at 30 ꢂC to obtain
single colonies, which were resuspended in a 0.9% normal saline
solution to give an optical density at 600 nm (OD600) of 0.1. The cells
were then diluted 100-fold in YNB medium containing 2% glucose.
The diluted cell suspensions were added to the wells of round-
4.2.1. 5-(2-Phenylethenyl)-2H-tetrazole (1)
Solid; yield: 84%; mp 152e155 ꢂC; Anal. Calc. for C9H8N4: C
62.78, H 4.68, N 32.54%, found: C 62.73, H 4.72, N 32.58%; IR
nmaxcmꢃ1: 3280 (NeH br stretch), 2864 (CeH), 1632 (C]N); 1H
NMR (DMSO-d6)
d
(ppm): 7.79 (2H, m), 7.66 (1H, d, J ¼ 15.2 Hz),
bottomed 96-well microtiter plates (100 ml/well) containing equal
7.46e7.36 (3H, m), 7.32 (1H, d, J ¼ 15.0 Hz), 7.09 (1H, NH); 13C NMR
volumes of medium and different concentrations of synthesized
compounds [25]. A drug-free control was also included. The plates
were incubated at 30 ꢂC for 24 h. The MIC test end point was
(DMSO-d6)
d(ppm): 147.50 (C]N), 132.12 (C]C); ESI-MS m/z:
[Mþþ1] 173.

S. Shreaz et al. / European Journal of Medicinal Chemistry 48 (2012) 363e370
369
evaluated both visually and by observing the OD620 in a microplate
reader (BIO-RAD, iMark, US) and is defined as the lowest compound
concentration that gave ꢀ90% inhibition of growth compared to the
controls.
suspension was followed on pH meter with constant stirring. The
value of external pH at which nystatin permeabilization induced no
further shift was taken as an estimate of pHi.
4.8. Scanning electron microscopy (SEM)
4.4. Disc diffusion halo assays
Candida cell suspensions from overnight cultures were prepared
in YEPD medium. Test compound at equivalent to MIC90 concen-
tration was added to the cell (w1 ꢄ 106) and incubated for 14 h at
30 ꢂC and prepared for an electron microscopy. All Candida cells
were fixed with 2% glutaraldehyde in 0.1% phosphate buffer for 1 h
at room temperature (20 ꢂC) [27,28]. Washed with 0.1 M phosphate
buffer (pH 7.2) and post fixed 1% OsO4 in 0.1 M phosphate buffer for
1 h at 4 ꢂC. For SEM, samples were dehydrated in acetone and
dropped on round glass cover slip with HMDS and dried at room
temperature then sputter coating with gold and observed under
the SEM (Zeiss EV040).
Strains were inoculated into liquid YPD medium and grown
overnight at 37 ꢂC. The cells were then pelleted and washed three
times with distilled water. Approximately 105cells/ml were inocu-
lated in molten agar media at 40 ꢂC and poured into 100-mm-
diameter petri plates. The synthesized compounds initially dissolved
in 10% DMSO were further diluted in distilled water to concentration
ranges of 10 fold of their respective MICs. 4-mm-diameter sterile
filter discs were impregnated with the test compounds as described
earlier [18]. 10% DMSO (solvent) and 100 mg/ml of fluconazole were
also applied on the discs to serve as negative and positive controls,
respectively. The diameter of zone of inhibition was recorded in
millimeters after 48 h and was compared with that of control. This
experiment was performed on fluconazole-sensitive [standard
(n ¼ 6), clinical (n ¼ 10)], and fluconazole-resistant (n ¼ 10) isolates
selected randomly. Results are reported as mean ꢁ standard error of
mean (SEM) of all three respective categories.
4.9. Hemolytic activity
Human erythrocytes from healthy individuals were collected in
tubes containing EDTA as anti-coagulant. The erythrocytes were
harvested by centrifugation for 10 m at 2000 rpm at 20 ꢂC, and
washed three times in PBS. To the pellet, PBS was added to yield
a 10% (v/v) erythrocytes/PBS suspension. The 10% suspension was
4.5. Growth curve studies
then diluted 1:10 in PBS. From each suspension,100
ml was added in
For growth studies, 106 cells/ml (optical density A600 ¼ 0.1)
culture of Candida cells were inoculated and grown aerobically in
YEPD broth for control along with varied concentrations of the
synthesized compounds in individual flasks. Growth was recorded
turbidometrically at 595 nm using LaboMed Inc. Spectrophotom-
eter (USA) as described previously [18,19]. The growth rate study of
different Candida species in absence as well as in presence of
inhibitor was performed for each concentration in triplicate,
average of which was taken into consideration.
triplicate to 100 L of a different dilution series of synthesized
m
compounds (or fluconazole) in the same buffer in eppendorf tubes.
Total hemolysis was achieved with 1% Triton X-100. The tubes were
incubated for 1 h at 37 ꢂC and then centrifuged for 10 m at
2000 rpm at 20 ꢂC. From the supernatant fluid, 150
ml was trans-
ferred to a flat-bottomed microtiter plate (BIO-RAD, iMark, US), and
the absorbance was measured spectrophotometrically at 450 nm.
The hemolysis percentage was calculated by following equation:
% hemolysis ¼ [(A450 of test compound treated sample ꢃ A450 of
buffer treated sample)/(A450 of 1% Triton X ꢃ A450 of buffer treated
sample)] ꢄ 100%.
4.6. Proton efflux measurements
The proton pumping activity of Candida species was determined
by monitoring acidification of external medium by measuring the
pH as described previously [17,26]. Briefly, mid-log phase cells
harvested from YEPD medium were washed twice with distilled
water and routinely 0.1 g cells were suspended in 5 ml solution
containing 0.1 M KCl, 0.1 mM CaCl2 in distilled water. Suspension
was kept in a double-jacketed glass container with constant stir-
ring. The container was connected to a water circulator at 25 ꢂC.
Initial pH was adjusted to 7.0 using 0.01 M HCl/NaOH. Synthesized
compounds were added to achieve the desired concentrations
(MIC90) in 5 ml solution. For glucose stimulation experiments,
Acknowledgment
Sheikh Shreaz greatly acknowledges the financial support by
ICMR (India) grant 45/93/09-Pha/BMS, (S.R.F). Authors are thankful
to Dr. Sumathi Muralidhar from Sexually Transmitted Disease Lab,
Safdarjung Hospital, New Delhi, India for providing the Candida
strains. Authors are also thankful to AIRF, JNU for SEM analysis.
Appendix. Supplementary Data
100 ml of glucose was added to achieve a final concentration of
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.12.007.
5 mM in total volume of suspension. Hþ-extrusion rate was calcu-
lated from the volume of 0.01 N NaOH consumed.
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