Synthesis of amino acid appended indoles: Appreciable anti-fungal activity and inhibition of ergosterol biosynthesis as their probable mode of action

Article abstract of DOI:10.1016/j.ejmech.2014.04.063

Rationally designed compounds consisting of mono- and di-peptide appendages on bis-indole template were synthesized in appreciable yield. Some of these compounds exhibited significant antifungal activities against Candida albicans with their MIC₈₀ in μg/ml range. However, when used in combination with azoles, the antifungal activities of the azoles were considerably enhanced. The growth inhibition appeared to be specific to the fungal cells and mammalian cells were not affected by these compounds. It is shown that these compounds lower ergosterol levels in the fungal cells and probably act by targeting lanosterol 14α-demethylase, a key enzyme in the sterol biosynthetic pathway of C. albicans. The compounds do not appear to directly act on the fungal cell wall. Hence, the sensitivity of the fungal cells to these compounds cannot be attributed to cell wall damage and consequent accumulation of the compounds in the cell, though defects in cell wall due to defective sterol biosynthesis cannot be completely ruled out.

Full text of DOI:10.1016/j.ejmech.2014.04.063

European Journal of Medicinal Chemistry 80 (2014) 325e339
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of amino acid appended indoles: Appreciable anti-fungal
activity and inhibition of ergosterol biosynthesis as their probable
mode of action
,
b
Pooja a, Parteek Prasher a, Palwinder Singh a , Kalpana Pawar ,
*
Kunwar Somesh Vikramdeo b, Neelima Mondal b, Sneha Sudha Komath b
,
*
a Department of Chemistry, Guru Nanak Dev University, Amritsar, Punjab 143005, India
b School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Rationally designed compounds consisting of mono- and di-peptide appendages on bis-indole template
were synthesized in appreciable yield. Some of these compounds exhibited significant antifungal ac-
tivities against Candida albicans with their MIC80 in mg/ml range. However, when used in combination
with azoles, the antifungal activities of the azoles were considerably enhanced. The growth inhibition
appeared to be specific to the fungal cells and mammalian cells were not affected by these compounds. It
is shown that these compounds lower ergosterol levels in the fungal cells and probably act by targeting
Received 31 January 2014
Received in revised form
14 March 2014
Accepted 22 April 2014
Available online 24 April 2014
lanosterol 14a-demethylase, a key enzyme in the sterol biosynthetic pathway of C. albicans. The com-
Keywords:
Indole
Amino acid
Anti-fungal
pounds do not appear to directly act on the fungal cell wall. Hence, the sensitivity of the fungal cells to
these compounds cannot be attributed to cell wall damage and consequent accumulation of the com-
pounds in the cell, though defects in cell wall due to defective sterol biosynthesis cannot be completely
ruled out.
Lanosterol 14a-demethylase
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
and miconazole, all inhibitors of lanosterol 14a-demethylase
(Erg11); terbinafine, an allylamine inhibitor of Erg1, the sqaulene
epoxidase that functions upstream of Erg11; morpholines like
amorolfine which inhibit Erg24 and Erg2 that function downstream
of Erg11 in the same pathway; amphotericin B and nystatin, both
polyenes that bind to membrane ergosterol and cause cytotoxic
pores in the fungal cells [1,4]. More recently, echinocandins like
Fungal infections and their treatment continue to be major
challenges in the medical world today, not least due to the drug
resistance that these organisms are known to exhibit upon pro-
longed treatment with antifungals [1,2]. Because of the eukaryotic
nature of fungal cells and hence similarity of many of the essential
pathways to mammalian cell, the identification of appropriate drug
targets within these pathogens is highly complicated. However,
fungi and yeast, unlike mammals, depend predominantly on
ergosterol as the major membrane sterol while mammalian cells
require cholesterol for their function. Additionally, fungal cells have
a cell wall and the cell wall biosynthetic pathway is completely
absent in mammalian cells. Thus, most of the drugs currently
available for treatment of fungal infections either target membrane
ergosterol or the ergosterol biosynthetic pathway or target the
fungal cell wall [1]. Amongst the drugs of choice for treatment of
Candida infections are the azoles such as ketoconazole, fluconazole
caspofungin or micafungin have been used to inhibit
b-glucan
synthesis and thereby target the fungal cell wall [1,4,5] while nik-
komycin Z is being viewed as a potent antifungal drug due to its
ability to inhibit chitin synthases [4]. However, the many mecha-
nisms that pathogenic fungi adapt for developing resistance to
antifungals include overexpression of Erg11, a key enzyme of the
sterol biosynthetic pathway, accumulation of point mutations in
Erg11, overexpression of multidrug efflux pumps and reduction in
drug import into resistant cells [1]. Of these, a very common re-
ported mechanism in clinical drug resistant Candida albicans
strains, is the overexpression of Erg11 or point mutations in it to
counter the inhibition by azoles [1,3].
In a recent report, we showed that indole-based compounds can
act as good antifungals [6]. It was also predicted therein, using
homology and docking studies that these compounds may act by
* Corresponding authors.
E-mail addresses: palwinder_singh_2000@yahoo.com (P. Singh), sskomath@
yahoo.com (S.S. Komath).
http://dx.doi.org/10.1016/j.ejmech.2014.04.063
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

326
Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
interacting at the catalytic site of lanosterol 14
a
-demethylase
with 1,4-bis(bromomethyl)benzene and 1,3-dibromopropane,
respectively in the presence of KOH using DMSO as a solvent. Indole
2 and 9 were reacted with oxalyl chloride in dry ether to get cor-
responding oxoacetyl chlorides 3 and 10 (Schemes 1 and 2).
(molecular docking studies). Extending this work, another series of
indole based compounds is explored to study their effect on growth
of C. albicans and their probable mode of action. It is shown that a
number of these compounds could work very well in combination
therapies with azole drugs.
Treatment of compound 3 with
ride/ -alanine methyl ester hydrochloride/
hydrochloride/ -tryptophan methyl ester hydrochloride/
L
-valine methyl ester hydrochlo-
-proline methyl ester
-tyrosine
L
L
L
L
methyl ester hydrochloride in acetonitrile in presence of K2CO3
provided amino acid appended indoles 4ae8a. Hydrolysis of 4ae8a
with NaOH in acetoneewater gave compounds 4be8b in 70e80%
yields (Scheme 1).
2. Results and discussion
2.1. Chemistry
Through a similar sequence of reactions as depicted in Scheme 1,
Since antibiotics should be non-toxic to the host, killing only the
pathogen; the biological acceptability of indole and amino acids
was taken as the safe guard for non-toxicity of new compounds to
human system. To induce selectivity in the molecules, chiral amino
acids were suitably introduced on the indole to develop a library of
compounds. Selection of linker group between two indole moieties
was based on previous results where compounds with three carbon
and xylidene spacers exhibited significant antimicrobial activities
[6]. Compounds 2 and 9 were obtained by the reaction of indole
compound 10 on reaction with methyl ester hydrochlorides of
L-
valine, -alanine, -proline, -tryptophan and -tyrosine gave com-
L
L
L
L
pounds 11a,be15a,b (Scheme 2). Presence of amino acid residues in
compounds 4a,be8a,b and 11a,be15a,b has considerably increased
the solubility of these compounds in aqueous medium (observed
during biological studies) as compared to those of previously re-
ported compounds [6]. Further, compounds 4b, 6b, 11b and 13b
carrying valine and proline were coupled with other amino acids.
N
N
i)
N
H
2
(H3C)2HC
HN
1
O
HO
O
NH
O
ii)
OR
O
O
O
OR
N
O
O
Cl
Cl
N
N
O
O
N
iii)
viii)
vii)
viii)
3
N
OR
O
N
O
O
RO
O
O
HO
N
H
iv)
viii)
v)
viii)
viii)
vi)
O
N
H
CH(CH3)2
O
8a, 8b
4a, 4b
OR
OR
O
H3C
HN
O
HN
N
OR
NH
N
N
N
O
O
O
O
O
O
HN
RO
O
O
N
O
N
O
N
OR
O
O
CH3
N
N
N
H
H
O
5a, 5b
Reaction conditions:
i) KOH, BrCH2(C6H4)CH2Br, DMSO;
iii) L-valine methyl ester HCl, K2CO3, dry CH3CN;
v) L-proline methyl ester HCl, K2CO3, dry CH3CN;
vii) L-tyrosine methyl ester HCl, K2CO3, dry CH3CN;
RO
O
O
7a, 7b
6a, 6b
ii) (COCl)2, dry ether
iv) L-alanine methyl ester HCl, K2CO3, dry CH3CN
vi) L-tryptophan methyl ester HCl, K2CO3, dry CH3CN
viii) for a (R=CH3) to b (R=H): NaOH (3 eq), acetone - water (2:1)
Scheme 1. Synthesis of compounds 4e8.

Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
327
N
N
i)
O
N
RO
H
1
NH
O
9
HO
CH(CH3)2
O
O
ii)
HN
O
N
RO
O
O
Cl
O
Cl
vii)
viii)
N
iii)
N
O
N
O
viii)
N
10
O
N
HO
O
NH
viii)
O
vi)
RO
iv)
RO
viii)
O
15a, 15b
O
viii)
N
v)
O
HN
RO
HN
CH(CH3)2
CH3
11a, 11b
O
O
NH
HN
O
O
O
O
O
RO
O
RO
O
N
N
N
N
N
N
O
RO
RO
O
O
NH
O
O
HN
O
N
O
CH3
N
O
H
O
RO
14a, 14b
12a, 12b
13a, 13b
Reaction conditions:
i) KOH, BrCH2CH2CH2Br, DMSO;
iii) L-valine methyl ester HCl, K2CO3, dry CH3CN;
v) L-proline methyl ester HCl, K2CO3, dry CH3CN;
vii) L-tyrosine methyl ester HCl, K2CO3, dry CH3CN;
ii) (COCl)2, dry ether
iv) L-alanine methyl ester HCl, K2CO3, dry CH3CN
vi) L-tryptophan methyl ester HCl, K2CO3, dry CH3CN
viii) for a (R=CH3) to b (R=H): NaOH (3 eq), acetone - water (2:1)
Scheme 2. Synthesis of compounds 11e15.
Ethyl chloroformate, triethyl amine mediated coupling of 4b with
L
-
spacer between the two central indoles, was clearly the best in-
hibitor of fungal growth. It has nearly seven-fold better inhibition
than the ester form of the same compound (18a) and a three-fold or
better inhibition as compared to other compounds with the xyli-
dene spacer and carrying esterified di-peptide substituents of Pro
with amino acids other than Trp (17a, 17b; 19a, 19b). The presence
of dipeptide ProeTrp on 18b appeared to be slightly better for in-
hibition than a substituent containing Trp alone (7b) in our assay. In
general, it appears that compounds with single amino acid sub-
stituents were better inhibitors of growth of C. albicans than those
with dipeptide substituents. Additionally, the identity of the sub-
stituent amino acids did not appear to be significantly varying the
anti-fungal activities, although they improved the solubility of the
compounds (as observed during antifungal studies). In general,
amongst the compounds with single amino acids as substituents, a
flexible three-carbon spacer between the two central indoles
appeared to be better suited for fungal growth inhibition than the
more rigid xylidene spacer. For the more bulky Trp or Val substit-
uent, the choice of spacers did not appear to be as significant.
Therefore, a new category of compounds is identified with appre-
ciable MIC80 against C. albicans and they are worth to be pursued for
further studies.
alanine methyl ester hydrochloride and 6b with ester hydrochlo-
rides of -cysteine, -tryptophan and -tyrosine gave compounds 16
and 17e19, respectively (Scheme 3). Similarly, coupling of com-
pounds 11b and 13b with -alanine methyl ester hydrochloride and
ester hydrochlorides of -cysteine, -tryptophan and -tyrosine gave
L
L
L
L
L
L
L
compounds 20 and 21e23, respectively (Scheme 4). Hence, a library
of compounds with desirable substituents on indole moiety having
2/4 stereogenic centers were procured through simple synthetic
methodology. All the compounds were well characterized by NMR,
IR, HRMS spectral techniques.
2.2. Antifungal activity
2.2.1. MIC80
Inhibition in the growth of C. albicans in presence of test com-
pounds at ten different concentrations was determined in duplicate
and minimum concentration of the compounds causing 80% inhi-
bition in growth was calculated. As it is evident from the data given
in Table 1, the acid forms of the compounds have better antifungal
inhibitory potential than the corresponding esters. Compound 18b,
with ProeTrp substituent on both C-3 substituents and a xylidene

328
Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
(H3C)2HC
O
HN
N
O
O
OH
O
RO
H3C
OR
CH3
O
NH
HN
CH(CH3)2
NH
(H3C)2HC
HN
O
O
O
O
a
O
N
N
O
N
HO
O
O
16a, 16b
N
CH(CH3)2
H
O
RO
4b
O
O
HS
RO
HS
OH
HO
NH
NH
O
O
O
O
O
O
N
O
b
N
N
N
O
N
O
N
N
O
O
N
O
6b
17a, 17b
c
d
HO
O
OH
NH
RO
OR
O
HN
NH
N
OR
O
HN
O
RO
O
NH
O
O
HN
O
O
O
N
N
O
O
N
O
N
N
O
O
N
N
O
18a, 18b
17a: R = C2H5, 16a, 18a, 19a: R = CH3
16b-19b: R=H
19a, 19b
Reaction conditions:
(a): i, Et3N, C2H5OCOCl, DCM; ii, L-alanine methyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
(b): i,Et3N, C2H5OCOCl, DCM; ii, L-cysteine ethyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
(c): i, Et3N, C2H5OCOCl, DCM; ii, L-tryptophan methyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
(d): i, Et3N, C2H5OCOCl, DCM; ii, L-tyrosine methyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
Scheme 3. Synthesis of compounds 16e19.
2.2.2. Spot assay
between solid and liquid media. Thus we had effective concentra-
tions of 200 g/ml for compounds 12b, 13b, 14b, 15b and 150 g/ml
for compounds 7b, 18b. Overnight grown culture of BWP17 was
diluted to five different concentrations and 5 L from each con-
centration was spotted on eight YEPD plates containing: 1) solvent,
2) compound dissolved in the same solvent, 3) fluconazole, 4)
While looking for the possible mode of fungal growth inhibition
by these compounds, based upon MIC80 values, compounds 7b,12b,
13b, 14b, 15b and 18b were subjected to spot assays, either alone or
in combination with some anti-fungal drugs. The effect of com-
pounds on fungal growth was investigated in combination with
m
m
m
fluconazole (4
m
g/ml), ketoconazole (0.04 mg/ml) and terbinafine
compound
compound
þ
fluconazole,
ketoconazole,
5)
ketoconazole,
terbinafine and
6)
8)
(3 g/ml). In the culture plates, the compounds were used at a
m
þ
7)
concentration slightly higher than their MIC80 values, to compen-
sate for the fact that fungal cells often show differences in growth
compound þ terbinafine. 5
mL of each concentration was also
spotted on a plate containing YEPD alone to serve as positive

Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
329
(H3C)2HC
O
RO
H3C
O
OR
O
HN
O
OH
O
NH
HN
CH3
CH(CH3)2
(H3C)2HC
HN
O
O
O
N
O
a
NH
N
N
O
O
N
HO
O
O
N
H
CH(CH3)2
20a, 20b
O
RO
11b
O
O
HS
RO
HS
OH
HO
O
NH
O
NH
O
O
N
O
O
O
b
N
O
N
N
O
N
N
O
N
N
O
O
13b
21a, 21b
c
d
HO
OH
RO
OR
O
HN
O
OR
O
NH
O
O
NH
RO
HN
NH
O
HN
N
O
O
O
O
N
O
O
N
N
O
O
N
N
O
N
N
O
22a, 22b
23a, 23b
Reaction conditions:
21a: R = C2H5, 20a, 22a, 23a: R = CH3
20b-23b: R=H
(a): i, Et3N, C2H5OCOCl, DCM; ii, L-alanine methyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
(b): i,Et3N, C2H5OCOCl, DCM; ii, L-cysteine ethyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
(c): i, Et3N, C2H5OCOCl, DCM; ii, L-tryptophan methyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
(d): i, Et3N, C2H5OCOCl, DCM; ii, L-tyrosine methyl ester hydrochloride, Et3N, DCM; iii, NaOH, Acetone + water
Scheme 4. Synthesis of compounds 20e23.
control for growth (details in experimental section). The data for
spot assays using compound 18b are shown in Fig. 1. Normal fungal
growth was observed in YEPD and solvent plates at the three time
points at which the photographs were taken (part a, Fig. 1). Thus,
the solvent did not significantly affect the fungal growth. At the
concentrations in present studies, the three proven antifungal
agents, fluconazole, ketoconazole and terbinafine too did not
significantly affect growth at the 48 h time point (part b, Fig. 1),
though they did show some effects at the early time point (24 h). In
enhanced potency in combination with terbinafine, suggesting that
its action may be independent of the step at which terbinafine acts.
Similar effects on growth of BWP17 were observed in presence of
compounds 7b, 12b, 13b, 14b and 15b. Here too the compounds
showed greater potency in combination with fluconazole or keto-
conazole in inhibiting fungal growth (Figure S1). It must be pointed
out that very similar results were obtained when these compounds
were tested on another C. albicans strain, CAI4 (Figs. S2, S3).
The greater potency in fungal growth inhibition shown by these
compounds in combination with fluconazole and ketoconazole
might be due to their mode of action being similar to that of flu-
conazole/ketoconazole, namely, inhibiting lanosterol demethyla-
tion, the rate determining step of sterol biosynthesis, resulting in
comparison to these three drugs, 18b at 150 mg/ml inhibits growth
of the Candida cells very significantly. Notably, in combination with
the antifungals, particularly the azoles, 18b was a very potent
antifungal agent (part c, Fig. 1). In contrast, 18b showed no

330
Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
Table 1
MIC80 values of compounds 4e8 and 11e23 for BWP17 strain of Candida albicans.
Compounds with single amino acid
Compounds with two amino acids
Compd
Spacer group
L
-Amino acid/ester
MIC80
412
226.05
460.74
310.2
458.03
235.81
553.88
105.2
474.97
353.63
486.8
238.23
470.53
148.57
299.66
154.06
454.22
139.71
475.13
142.38
(m
g/ml)
Compd
Spacer group
L
-Amino acid/ester
MIC80
(mg/ml)
4a
4b
5a
5b
6a
6b
7a
7b
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
Val methyl ester
Val
Ala methyl ester
Ala
Pro methyl ester
Pro
Trp methyl ester
Trp
Tyr methyl ester
Tyr
Val methyl ester
Val
Ala methyl ester
Ala
Pro methyl ester
Pro
16a
16b
17a
17b
18a
18b
19a
19b
20a
20b
21a
21b
22a
22b
23a
23b
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
10,40-xylidenyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
1,3-propyl
ValeAla methyl ester
ValeAla
ProeCys ethyl ester
ProeCys
ProeTrp methyl ester
ProeTrp
ProeTyr methyl ester
ProeTyr
ValeAla methyl ester
ValeAla
ProeCys ethyl ester
ProeCys
ProeTrp methyl ester
ProeTrp
ProeTyr methyl ester
ProeTyr
1048.4
480.97
716.36
475.8
575.1
78.5
468.08
290.3
8a
8b
949.76
457.81
624.87
336.75
519.6
306.15
549
306.91
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
1,3-propyl
1,3-propyl
1,3-propyl
Trp methyl ester
Trp
Tyr methyl ester
Tyr
1,3-propyl
impaired ergosterol levels and growth retardation. To test whether
the ergosterol levels were actually affected in cells treated with
these compounds, the levels of ergosterol in BWP17 cells treated
overnight with 18b were assessed using GCeMS. Indeed, cells
treated with 18b showed lesser ergosterol as compared to the wild
type cells (Fig. 2). Interestingly, the level of lanosterol was found to
increase in cells treated with 18b (Fig. 2), suggesting that the sub-
terbinafine, which works upstream of the Erg11 step. Fig. 3 shows
the effect of 18b on the growth of erg11/ERG11 cells in the absence
or presence of terbinafine or fluconazole. The concentration of
fluconazole/terbinafine chosen were such that some sensitivity
could be observed in the 24 h time point (Fig. 3, part b), but the
growth caught up by the 48 h time point. Moreover, the growth of
erg11/ERG11 cells in fluconazole/terbinafine at the 48 h time point
was comparable to its growth in media lacking any drug (YEPD
alone). It may be also pointed out that the solvent alone has no
effect on the growth of these cells (Fig. 3, part a). As was also
observed for BWP17 cells (Fig. 1), the erg11/ERG11 cells were sen-
sitive to 18b itself. In combination with fluconazole, 18b showed
considerable potency, significantly inhibiting growth of this Erg11
deficient strain (Fig. 3, part c). This observation clearly indicated
that compound 18b functions by inhibition of the same step of
ergosterol biosynthesis as the azoles. On the other hand, 18b
showed no greater effect in combination with terbinafine (Fig. 3,
part c), suggesting that the upstream step catalyzed by Erg1 may
strate of lanosterol 14
To further confirm that the effect was at the level of lanosterol
-demethylase (Erg11) and not due to a generic defect arising
a-demethylase accumulated in these cells.
14
a
from sterol depletion, the effect of these compounds were tested on
the C. albicans strain, erg11/ERG11, created in the BWP17 back-
ground, wherein one copy of the ERG11 gene has been disrupted [7].
It was shown previously that the erg11/ERG11 strain is deficient in
ERG11 transcript levels and is sensitive to azoles [7]. Accordingly, it
was hypothesized that the erg11/ERG11 mutant cells should be
sensitive to the compounds also and the compounds should be
more potent in combination with fluconazole but not with
Fig. 1. Spot assays for the effect of compound 18b alone and in combination with fluconazole (Flu), ketoconazole (Keto) or terbinafine (Terb) on BWP17 cells.

Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
331
18b-treated and untreated cells were spotted on two plates, one
without fluconazole and another containing 4 g/ml fluconazole
m
(see Experimental Section for more details). As expected, cells
untreated with 18b showed mild sensitivity to fluconazole at the
24 h time point, but their growth caught up by the 48 h time point
and looked nearly comparable to its growth on the control plate
(YEPD) (Fig. 4 a, b). However, cells pre-treated with 18b, showed
high sensitivity to the same concentrations of fluconazole, and did
not manage to compensate for their growth defects even at the 48 h
time point (Fig. 4 c, d). Thus, the results support hypothesis that
both 18b and the azoles primarily target the same step in ergosterol
biosynthesis in C. albicans. This experiment also shows that even at
low concentration (50
mg/ml), compound 18b acts as antifungal
agent in combination with fluconazole. Similar effect was observed
for compounds 7b, 12b, 13b, 14b and 15b (Fig. S7).
Fig. 2. Ergosterol biosynthesis is altered in Candida albicans on treatment with 18b.
Total ergosterol content was decreased upon treatment with 75 g/ml of 18b in the
cells as compared to untreated cells as estimated by GCeMS (details in text). However,
the level of lanosterol was actually higher in the cells treated with 18b as compared to
untreated cells. The bars represent averages of two independent assays and the error
bars represent standard deviations.
2.2.3. Cell wall defects
m
It is also possible that the compounds directly target the fungal
cell wall, weakening it and resulting in higher accumulation of the
compounds in the cells, as it was demonstrated previously with
some acridone derivatives [9,10]. To test whether the compounds
have a direct effect on the cell wall, two different sets of experi-
ments were carried out. In the first, it was assessed whether the
compounds acted in concert with known cell wall disrupting
agents and therefore directly weakened the cell wall of BWP17 cells
treated with these compounds. The rationale for the design of the
experiment was similar to that for identifying Erg11 as the target of
the compounds. Disc diffusion assays with the compounds both in
the absence as well as presence of known cell wall disrupting
agents were performed. For this purpose two known cell wall
disrupting agents were used: Congo Red (CR), a dye known to
not be affected by 18b. Similar growth inhibition of erg11/ERG11
cells was observed in presence of compounds 7b, 12b, 13b, 14b and
15b (Figure S4).
The effect of these compounds on Cagpi19/CaGPI19 strain, which
has deficiencies in GPI anchor production [7,8] was also tested. This
strain too is ergosterol deficient and is specifically affected in ERG11
[7]. As expected, the Cagpi19/CaGPI19 cells were sensitive to the
indole-based compounds tested here and the compounds in com-
bination with azoles were very potent in inhibiting fungal growth
(Figs. S5, S6).
To further confirm the enhanced potency of these compounds in
the presence of azoles, the following experiment was designed. The
cells were first grown in the presence of compound 18b. If the Erg11
activity in these cells was inhibited by the compound, the cells were
expected to become more sensitive to azoles than before. With this
rationale, two cultures of BWP17 were grown, one in the absence of
compound 18b (18b untreated culture e labeled ‘a’) and the second
inhibit b-glucan synthesis and Calcofluor white (CFW), a dye that
inhibits chitin polymerization [11]. Effect of sodium dodecyl sulfate
(SDS), sensitivity to which is normally an indication of impaired cell
wall integrity [12,13] was also tested. As before, control plates with
no drug treatment and plates carrying solvent alone were also done
to show that the effects, if any were specifically due to the com-
pounds and/or the cell wall disruptive agents. Compounds 4e8 and
11e23 were loaded on sterile discs at a concentration slightly
higher than their MIC80 values and the discs were placed on a lawn
of BWP17 cells. The radius of the zone of clearance is an indication
of the effectiveness of the compounds/drugs in inhibiting fungal
growth (Table S1, Table S2). Compounds 7b, 12b, 13b, 14b, 15b and
18b were all capable of inhibiting fungal growth as also previously
in the presence of 50 mg of compound 18b (18b treated culture e
labeled ‘b’). After growing secondary cultures of these cells with
18b for 5 h and diluting them to five different concentrations (105,
104, 103, 102, 10 cells), 5
mL of each concentration of both
Fig. 3. Spot assays for effect of compound 18b alone and in combination with fluconazole or terbinafine on Candida albicans strain erg11/ERG11.

332
Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
Fig. 4. Spot assay for effect of compound 18b in combination with fluconazole on BWP17 growth inhibition.
shown in the spot assays. However, no significant effect was
observed when these compounds were used in combination with
CR, CFW or SDS. Thus, a direct effect of these compounds on the cell
wall integrity was ruled out.
integrity of C. albicans [16]. While the reasons for this are as yet
unclear, it is possible to speculate that deficiencies in ergosterol
lead to defects in transport and/or localization of proteins that
depend on ergosterol for their transport. These include GPI
anchored proteins involved in cell wall integrity as well as the
chitin synthases that are important for cell wall biogenesis. Thus, it
is expected that drugs that target Erg11 could also in turn affect cell
wall integrity and/or biogenesis.
In second experiment, the cells were grown for different time
periods in the absence or presence of compound 18b. These cells
were then stained by CFW for 20 min to study whether the cell wall
was intact. CFW binds to chitin and is used to visualize fungal cell
walls [8]. Given the short duration of exposure, CFW binding per se
is not expected to cause any defects in chitin polymerization under
these conditions. It was hypothesized that if the cells grown in the
presence of 18b have impaired cell walls, then their ability to pick
up CFW would reduce and much lower fluorescence from the CFW
bound to such cells will be detected. Alternatively, if the cell wall
integrity pathway is triggered due to cell wall damage [14] and it
results in higher chitin biosynthesis, greater amount of CFW
staining in the cells treated with 18b as compared to untreated cells
is expected. However, average fluorescence intensity (from 30 cells
in each case) did not indicate any variation in CFW staining be-
tween untreated cells and those treated with compound 18b for
different intervals of time (Fig. 5).
These results indicate that treatment of Candida cells with the
compounds do not result in any major cell wall aberrations. How-
ever, more subtle defects in the wall of cells treated with the
compound cannot be ruled out. Ergosterol biosynthesis is inti-
mately connected to cell wall biogenesis as well [15]. It has been
recently shown that fluconazole not only targets Erg11 but also
alters the secretome, the wall proteome as well as the cell wall
2.2.4. Specificity of 18b for fungal cells
One of the major challenges in drug design is to be able to show
that the drug targets the pathogen but not the host. To ascertain
whether the compounds under investigation were specifically
inhibitory to the growth of fungal cells (as hypothesized during
design of these molecules), mammalian HeLa cells were treated
with 18b and analyzed the cell viability using the MTT assay at
different time points. As can be seen from Fig. 6, the growth of HeLa
cells were not affected by even 300 mg/ml of 18b.
2.2.5. Molecular docking of compound 18b in the active site of
lanosterol 14 -demethylase
Since experimental results are favoring lanosterol 14
a
a
-deme-
thylase as the probable cellular target for the antifungal activities of
the compounds under present investigation, it was worth to check
Fig. 6. Viability of HeLa cells were unaffected by treatment with 18b. The cells were
grown in the presence of two different concentrations (150 mg/ml and 300 mg/ml) of
18b over 3 different time periods as specified in the figure. Cell viability was assessed
using the MTT assay as described in the Experimental Section. Control refers to a
parallel assay in which only the solvent (DMSO) was used in place of the compound.
The bars represent averages of two independent assays and the error bars represent
standard deviations.
Fig. 5. CFW staining of BWP17 cells grown in the absence or presence of 18b. The bars
represent averages of duplicates and the error bars represent standard deviations.

Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
333
the compatibility and interactions of the molecule in the active site
of this enzyme. Thence, the molecular docking of compound 18b in
the active site of the enzyme was performed. Due to non-
availability of fungal lanosterol demethylase, the crystal co-
ordinates of human lanosterol 14a-demethylase in complexation
with econazole (pdb ID 3jus) [17] were downloaded from protein
data bank (www.rcsb.org) and the active site of the enzyme was
located. Energy minimized conformation of compound 18b was
docked in the active site of the enzyme using ArgusLab 4.0.1 [18]. As
shown in Fig. 7, compound 18b gets buried in the interior of the
enzyme and fits in the same pocket where econazole is present
(Fig. 8). Compound 18b exhibited H-bond interactions with T763,
S644, H759 and D676 (Fig. 9). It is pertinent to note that human
lanosterol demethylase was used for docking purpose only other-
wise specificity of compound 18b for fungal cells has also been
checked.
3. Conclusions and outlook
Fig. 8. CPK model of the active site of lanosterol 14
a
-demethylase with econazole
(green wire frame) and compound 18b (ball stick). (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)
From the medicinal chemistry point of view, an easy synthetic
methodology has been demonstrated for generating amino acid
appended bis-indoles with 2/4 stereogenic centers. A detailed
investigation of the antifungal properties of these compounds us-
ing four different strains of C. albicans led to the identification of a
set of compounds, 7b, 12b, 13b, 14b, 15b and 18b with appreciable
fungal growth inhibitory activities. Remarkably, these compounds
showed enhanced potency with the azole class of drugs which
the ergosterol biosynthesis cannot be ruled out. The compounds
appear to be specific for fungal cells and do not affect the viability of
mammalian host cells. Overall, a new class of antifungal com-
pounds is identified which show good promise, either alone or in
combination with azoles, as probable drug candidates for anti-
fungal therapy.
target lanosterol 14a-demethylase (Erg11) but not with terbinafine,
which works on Erg1, an enzyme functioning upstream of Erg11 in
the sterol biosynthetic pathway. In addition, cells treated with 18b
tended to accumulate lanosterol, the substrate of Erg11. Sterol
deficient strains, erg11/ERG11 as well as Cagpi19/CaGPI19, affected
specifically in Erg11 levels, were also sensitive to these compounds,
lending credence to the hypothesis that the compounds work by
targeting Erg11 and consequently inhibiting ergosterol biosyn-
thesis. No direct effect of the compounds on the fungal cell wall was
detected. Thus, no altered sensitivity was observed with cell wall
disruption agents such as calcofluor white or Congo Red. Nor was
there any altered sensitivity to SDS in cells treated with these
compounds, as is frequently observed in cell wall defective strains.
However, more subtle defects in the cell wall due to inhibition of
4. Experimental section
4.1. Chemistry
All reactions were performed in oven-dried glassware with
magnetic stirring. Diethyl ether was distilled over activated anhy-
drous calcium chloride and further dried with sodium wire.
Acetonitrile was dried by refluxing over anhydrous phosphorus
pentaoxide, then over anhydrous K2CO3 and stored over activated
ꢀ
4 A molecular sieves. Dichloromethane was dried by refluxing over
ꢀ
activated anhydrous calcium chloride and stored over activated 4 A
molecular sieves. Triethyl amine was dried by refluxing over
Fig. 7. Crystal coordinates of lanosterol 14a-demethylase in association with compound 18b (generated in pymol). Compound 18b is pink in color while econazole is given blue and
light blue color. Dotted lines indicate H-bonding. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

334
Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
dried over anhydrous sodium sulfate and solvent was removed by
distillation. The crude product was purified by column chroma-
tography using hexane and ethyl acetate as eluents.
4.1.2. General procedure for synthesis of compounds 4ae8a and
11ae15a (Procedure B)
Bis-indole 2/9 (1 mmol) was taken in dry ether (5 ml) followed
by addition of oxalyl chloride (2.4 mmol) to get corresponding
oxoacetyl chlorides 3 and 10, which were separated by filtration.
The solid obtained was added to dry acetonitrile containing po-
tassium carbonate (4 mmol) at 0e5 ꢀC, followed by addition of
amino acid ester hydrochloride (3 mmol). After 15 min stirring, the
reaction was quenched by addition of cold water. The crude product
was extracted with ethyl acetate (5 ꢂ 25 ml). The organic layer was
dried over anhydrous sodium sulfate and distilled under vacuum.
The pure product was obtained by column chromatography using
hexane and ethyl acetate as eluents.
4.1.3. General procedure for synthesis of compounds 4be8b and
11be15b (Procedure C)
Fig. 9. Compound 18b docked in the active site of lanosterol 14
a
-demethylase. Hs’ are
ꢀ
omitted for clarity. Yellow lines indicate in H-bonds in A. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
The ester derivative 4ae8a and 11ae15a (1 mmol) was dis-
solved in acetoneewater (2:1) followed by addition of sodium hy-
droxide (3 mmol). After the completion of reaction (TLC
monitoring), acetone was distilled off and the residue was acidified
with 1 N HCl and extracted with ethyl acetate (5 ꢂ 25 ml). Ethyl
acetate was passed through anhydrous sodium sulfate and distilled
under vacuum. The pure product was obtained by column chro-
matography using ethyl acetate and methanol as eluents.
ꢀ
sodium hydroxide and stored over activated 4 A molecular sieves.
Reactions conducted below room temperature were performed in
an ice bath and reactions performed above room temperature uti-
lized an oil bath preheated to the stated temperature. Thin-layer
chromatography was performed on silica gel GF254 plates
(0.25 mm); visualization of the developed chromatogram was
performed by UV and staining with iodine. All reagents were pur-
chased from commercial sources and used without further purifi-
cation unless otherwise noted. Organic solutions were
concentrated by rotary evaporation below 40 ꢀC at ca. 25 mmHg.
Column chromatography was performed with silica gel of 100e200
mesh using ethyl acetate, hexane and methanol as eluents. 1H and
13C NMR spectroscopy was performed on a JEOL NMR at 300 (1H) or
75 (13C) MHz using CDCl3, TFA, CD3OD and DMSO-d6 as solvents
and TMS as internal standard. Chemical shifts are reported in ppm
relative to tetramethylsilane or residual protiated solvent in parts
4.1.3.1. Compound 4b. Compound 4b was prepared from com-
pound 4a (400 mg, 0.5 mmol) following General Procedure C. The
crude residue was purified by silica gel chromatography (EtOAc:
MeOH 90:10) to yield 4b as light brown solid (320 mg, 83%),
ꢀ
25
[a
]
¼ ꢁ46.6 (c 1.2 in CH3OH); mp 246 C; IR (KBr, cmꢁ1): 3383,
D
2960, 1734, 1718, 1662, 1627, 1502, 1464, 1386, 1217, 1165, 742; dH
(300 MHz, CDCl3 þ TFA) 9.01 (s, 2H, ArH), 8.30e8.35 (m, 4H, 2H of
2 ꢂ NH, 2H of ArH), 7.35e7.40 (m, 6H, ArH), 7.18 (s, 4H, ArH), 5.40 (s,
4H, 2 ꢂ CH2), 4.54e4.59 (m, 2H, 2 ꢂ NCH), 2.39e2.46 (m, 2H,
2 ꢂ CH), 1.08 (d, 12H, J ¼ 6.6 Hz, 4 ꢂ CH3); dC (normal/DEPT-135)
(75 MHz, CDCl3 þ DMSO-d6) 179.19 (absent, C]O), 172.31 (absent,
C]O), 161.63 (absent, C]O), 140.91 (þve, ArCH), 135.79 (absent,
ArC), 135.14 (absent, ArC), 127.16 (absent, ArC), 127.05 (þve, ArCH),
123.53 (þve, ArCH), 122.93 (þve, ArCH), 121.96 (þve, ArCH), 111.70
(absent, ArC), 110.17 (þve, ArCH), 56.63 (þve, NCH), 50.07 (ꢁve,
CH2), 30.68 (þve, CH), 18.69 (þve, CH3), 17.14 (þve, CH3); HRMS
(ESI, m/z) calculated for C38H38N4O8Na [M þ Na]þ: 701.2587, found:
701.2555.
per million (
d
) and the coupling constants (J) were given in Hz. Data
for 1H are reported as chemical shift
(
d
ppm), multiplicity
(s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet),
coupling constant (J in Hz), integration. Data for 13C and DEPT-135
NMR spectra are reported in terms of chemical shift; þve signals
correspond to CH3 and CH carbons, ꢁve signals correspond to CH2
carbons while absent means a quaternary carbon. Infrared spec-
troscopic data was recorded on KBr plates as thin films on a Shi-
madzu FT-IR spectrometer. Optical rotations were measured on a
Jasco DIP-360 digital polarimeter using 1 cm glass cells with a so-
dium 589 nm filter. HRMS were acquired using Bruker MicroTOF QII
and Bruker autoflex TOF/TOF using electrospray ionization (ESI)
and MALDI mode, respectively. CHN analyses were recorded on
Thermoelectron FLASH EA1112 CHN analyzer.
4.1.3.2. Compound 5b. Compound 5b was prepared from 5a
(200 mg, 0.3 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
90:10) to yield 5b as lightꢀbrown solid (140 mg, 74%), [
a
]
¼ ꢁ18.6
D
(c 1.5 in CH3OH); mp 154 C; IR (KBr, cmꢁ1): 3365, 2937, 1734, 1674,
1624, 1506, 1464, 1386, 1209, 1172, 1064, 746; dH (300 MHz,
CDCl3 þ TFA) 8.96 (s, 2H, ArH), 8.55 (s, 2H, ArH), 8.29 (d, 2H,
J ¼ 7.5 Hz, NH), 7.33e7.41 (m, 6H, ArH), 7.20 (s, 4H, ArH), 5.42 (s, 4H,
2 ꢂ CH2), 4.70e4.74 (m, 2H, 2 ꢂ NCH), 1.67 (d, 6H, J ¼ 6.6 Hz,
2 ꢂ CH3); dC (normal/DEPT-135) (75 MHz, CDCl3 þ DMSO-d6)
179.43 (absent, C]O), 173.50 (absent, C]O), 161.51 (absent, C]O),
140.88 (þve, ArCH), 135.85 (absent, ArC), 135.28 (absent, ArC),
127.14 (þve, ArCH), 123.53 (þve, ArCH), 122.88 (þve, ArCH), 121.96
(þve, ArCH), 111.68 (absent, ArC), 110.28 (þve, ArCH), 50.04 (ꢁve,
CH2), 47.51 (þve, NCH),17.53 (þve, CH3); HRMS (ESI, m/z) calculated
for C34H30N4O8Na [M þ Na]þ: 645.1961, found: 645.1964.
4.1.1. General procedure for synthesis of compounds 2 and 9
(Procedure A)
A mixture of indole (1 mmol) and KOH (4 mmol) in DMSO (5 ml)
was stirred under nitrogen atmosphere. After stirring for 15 min,
1,3-dibromopropane/1,4-bis(bromomethyl)benzene (0.5 mmol)
was added to the reaction mass and stirred till disappearance of
indole (TLC monitoring). Reaction was quenched by adding 10 ml
cold water and extracted with ethyl acetate (5 ꢂ 25 ml). The
combined organic layer was washed with water and brine solution,

Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
335
4.1.3.3. Compound 6b. Compound 6b was prepared from 6a (1 g,
1.4 mmol) following General Procedure C. The crude residue was
ArH), 8.21 (d, 2H, J ¼ 6.9 Hz, ArH), 7.94 (d, 2H, J ¼ 8.7 Hz, 2 ꢂ NH),
7.09e7.21 (m, 6H, ArH), 4.46e4.50 (m, 2H, 2 ꢂ NCH), 4.13 (br s, 4H,
2 ꢂ CH2), 2.37e2.46 (m, 2H, 2 ꢂ CH), 2.27e2.35 (m, 2H, CH2), 1.01
(d, 12H, J ¼ 5.4 Hz, 4 ꢂ CH3); dC (normal/DEPT-135) (75 MHz, CDCl3)
179.02 (absent, C]O), 174.71 (absent, C]O), 162.87 (absent, C]O),
141.02 (þve, ArCH), 135.99 (absent, ArC), 127.54 (absent, ArC),
124.08 (þve, ArCH), 123.63 (þve, ArCH), 122.70 (þve, ArCH), 112.10
(absent, ArC), 109.77 (þve, ArCH), 57.52 (þve, NCH), 44.38 (ꢁve,
CH2), 30.71 (þve, CH), 29.19 (ꢁve, CH2),19.19 (þve, CH3), 17.72 (þve,
CH3); HRMS (ESI, m/z) calculated for C33H36N4O8Na [M þ Na]þ:
639.2431, found: 639.2421.
purified by silica gel chromatography (EtOAc: MeOH 85:15) to yield
25
6b as light brown solid (820 mg, 85%), [
a]
¼ ꢁ43.3ꢀ (c 1.5 in
D
CH3OH); mp 220 ꢀC; IR (KBr, cmꢁ1): 3460, 2955, 1723, 1635, 1521,
1452,1379,1222,1174,1070, 748; dH (300 MHz, CDCl3 þ TFA) 8.96 (s,
2H, ArH), 8.56 (s, 2H, ArH), 8.26 (s, 2H, ArH), 7.38e7.40 (m, 2H, ArH),
7.04 (d, 2H, J ¼ 5.7 Hz, ArH), 6.74 (s, 4H, ArH), 5.28 (s, 4H, 2 ꢂ CH2),
4.66e4.68 (m, 2H, 2 ꢂ NCH), 3.73e3.82 (m, 2H, NCH2), 3.60e3.61
(m, 2H, NCH2), 2.26e2.29 (m, 2H, CH2), 2.15e2.23 (m, 2H, CH2),
1.97e2.01 (m, 4H, 2 ꢂ CH2); dC (normal/DEPT-135) (75 MHz,
CDCl3 þ DMSO-d6) 184.76 (absent, C]O), 172.80 (absent, C]O),
164.97 (absent, C]O), 139.66 (þve, ArCH), 136.42 (absent, ArC),
135.31 (absent, ArC), 127.08 (þve, ArCH), 125.83 (absent, ArC),
123.55 (þve, ArCH), 122.70 (þve, ArCH), 121.67 (þve, ArCH), 112.72
(absent, ArC), 110.27 (þve, ArCH), 58.14 (þve, NCH), 50.00 (ꢁve,
CH2), 47.16 (ꢁve, NCH2), 28.49 (ꢁve, CH2), 21.67 (ꢁve, CH2); HRMS
(ESI, m/z) calculated for C38H34N4O8Na [M þ Na]þ: 697.2274, found:
697.2262.
4.1.3.7. Compound 12b. Compound 12b was prepared from 12a
(150 mg, 0.25 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
95:5) to yield 12b as light pink solid (120 mg, 84%), [
a
]
¼ ꢁ36.4 (c
D
1.4 in CH3OH), mp 144 ꢀC; IR (KBr, cmꢁ1): 3429, 1737, 1662, 1622,
1506, 1456, 1386, 1224, 1168, 746; dH (300 MHz, CDCl3 þ CD3OD)
8.79 (s, 2H, ArH), 8.31 (d, 2H, J ¼ 7.2 Hz, ArH), 7.15e7.28 (m, 6H,
ArH), 4.47e4.54 (m, 2H, 2 ꢂ NCH), 4.22 (t, 4H, J ¼ 6.75 Hz, 2 ꢂ CH2),
2.47e2.52 (m, 2H, CH2), 1.49 (d, 6H, J ¼ 7.2 Hz, 2 ꢂ CH3); dC (normal/
DEPT-135) (75 MHz, CDCl3 þ CD3OD) 181.02 (absent, C]O), 175.48
(absent, C]O), 163.64 (absent, C]O), 141.89 (þve, ArCH), 137.42
(absent, ArC),128.89 (absent, ArC), 125.49 (þve, ArCH),124.90 (þve,
ArCH), 123.99 (þve, ArCH), 113.57 (absent, ArC), 111.24 (þve, ArCH),
49.87 (þve, NCH), 45.67 (ꢁve, CH2), 30.72 (ꢁve, CH2), 19.05 (þve,
CH3); HRMS (ESI, m/z): calculated for C29H28N4O8Na [M þ Na]þ:
583.1799, found: 583.1786.
4.1.3.4. Compound 7b. Compound 7b was prepared from 7a
(170 mg, 0.2 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
80:20) to yield 7b as light brown solid (120 mg, 73%), [
a
]
¼ ꢁ23.8
D
(c 1.3 in DMSO); mp 180 ꢀC.); IR (KBr, cmꢁ1): 3381, 2929, 1734, 1674,
1624, 1500, 1464, 1384, 1230, 1170, 1057, 744; dH (300 MHz,
CDCl3 þ DMSO-d6) 8.91 (s, 2H, ArH), 8.16e8.30 (m, 4H, 4 ꢂ NH),
7.52e7.67 (m, 6H, ArH), 7.27 (s, 8H, ArH), 7.01e7.13 (m, 8H, ArH),
5.36 (s, 4H, 2 ꢂ CH2), 4.83 (br s, 2H, 2 ꢂ NCH), 3.35 (br s, 4H,
2 ꢂ CH2); dC (normal/DEPT-135) (75 MHz, CDCl3 þ DMSO-d6)
180.21 (absent, C]O), 173.05 (absent, C]O), 162.23 (absent, C]O),
141.44 (þve, ArCH), 136.34 (absent, ArC), 135.94 (absent, ArC),
127.65 (þve, ArCH), 127.42 (absent, ArC), 123.88 (absent, ArC),
123.73 (þve, ArCH), 123.22 (þve, ArCH), 122.18 (þve, ArCH), 121.23
(þve, ArCH), 118.75 (þve, ArCH), 118.48 (þve, ArCH), 111.92 (absent,
ArC), 111.52 (þve, ArCH), 110.99 (þve, ArCH), 109.34 (absent, ArC),
52.92 (þve, NCH), 50.26 (ꢁve, CH2), 27.24 (ꢁve, CH2); HRMS
(MALDI, m/z) calculated for C50H40N6O8Na [M þ Na]þ: 875.2805,
found: 875.281.
4.1.3.8. Compound 13b. Compound 13b was prepared from 13a
(1.15 g, 1.7 mmol) following General Procedure C. The crude residue
was purified by silica gel chromatography (EtOAc: MeOH 95:5) to
25
yield 13b as brown solid (820 mg, 75%), [
a
]
¼ ꢁ22.0 (c 1.5 in
D
CHCl3); mp 172 ꢀC; IR (KBr, cmꢁ1): 3454, 2949, 1743, 1620, 1525,
1452, 1383, 1215, 1176, 1091, 750; dH (300 MHz, CDCl3) 8.81 (s, 2H,
ArH), 8.43 (s, 2H, ArH), 7.38 (s, 6H, ArH), 4.79 (d, 2H, J ¼ 8.1 Hz,
2 ꢂ NCH), 4.31e4.36 (m, 2H, CH2), 4.14e4.19 (m, 2H, CH2), 3.76e
3.85 (m, 2H, NCH2), 3.62e3.64 (m, 2H, CH2), 2.5 (br s, 2H, CH2),
2.18e2.32 (m, 4H, 2 ꢂ CH2), 1.99e2.04 (m, 4H, 2 ꢂ CH2); dC (normal/
DEPT-135) (75 MHz, CDCl3) 184.57 (absent, C]O), 172.99 (absent,
C]O), 167.13 (absent, C]O), 142.52 (þve, ArCH), 136.56 (absent,
ArC), 126.62 (absent, ArC), 124.13 (þve, ArCH), 123.59 (þve, ArCH),
122.78 (þve, ArCH), 112.64 (absent, ArC), 109.96 (þve, ArCH), 58.47
(þve, NCH), 47.96 (ꢁve, NCH2), 44.05 (ꢁve, CH2), 28.78 (ꢁve, CH2),
28.26 (ꢁve, CH2), 23.99 (ꢁve, CH2); HRMS (ESI, m/z) calculated for
4.1.3.5. Compound 8b. Compound 8b was prepared from 8a
(170 mg, 0.2 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
80:20) to yield 8b as white solid (130 mg, 80%), [
a]
¼ ꢁ35.0 (c 1.4
D
in DMSO), mp 178 ꢀC; IR (KBr, cmꢁ1): 3392, 2926, 1732, 1664, 1616,
1508, 1388, 1232, 1174, 746 cmꢁ1
;
dH (300 MHz, CDCl3 þ DMSO-d6)
C
33H32N4O8 [M þ H]þ: 613.2298, found: 613.2249.
8.86 (s, 2H, ArH), 8.25 (s, 4H, 2H of 2 ꢂ NH, 2H of ArH), 7.50 (s, 2H,
ArH), 7.22 (s, 8H, ArH), 6.99 (d, 4H, J ¼ 6.3 Hz, ArH), 6.61 (d, 4H,
J ¼ 6.9 Hz, ArH), 5.48 (s, 4H, 2 ꢂ CH2), 4.44 (s, 2H, 2 ꢂ NCH), 3.02e
4.1.3.9. Compound 14b. Compound 14b was prepared from 14a
(100 mg, 0.1 mmol) following General Procedure C. The crude
3.11 (m, 4H,
2
ꢂ
CH2); dC (normal/DEPT-135) (75 MHz,
residue was purified by silica gel chromatography (EtOAc: MeOH
25
CDCl3 þ DMSO-d6) 179.45 (absent, C]O), 171.02 (absent, C]O),
162.05 (absent, C]O), 156.11 (absent, ArC), 140.43 (þve, ArCH),
136.03 (absent, ArC), 135.31 (absent, ArC), 129.90 (þve, ArCH),
127.29 (þve, ArCH), 123.75 (þve, ArCH), 123.13 (þve, ArCH), 122.16
(þve, ArCH), 115.34 (þve, ArCH), 111.70 (absent, ArC), 110.24 (ab-
sent, ArC), 53.20 (þve, NCH), 50.30 (ꢁve, CH2), 36.47 (ꢁve, CH2);
HRMS (MALDI, m/z) Calcd for C46H38N4O10Na [M þ Na]þ: 829.2486,
found: 829.247.
80:20) to yield 14b as light brown solid (79 mg, 83%), [
a
]
¼ ꢁ49.2
D
(c 1.4 in DMSO), mp 232 ꢀC; IR (KBr, cmꢁ1): 3375, 2926, 1730, 1670,
1616, 1500, 1386, 1226, 1166, 744; dH (300 MHz, CDCl3 þ TFA) 8.56
(s, 2H, ArH), 8.31 (d, 2H, J ¼ 6.3 Hz, ArH), 8.10 (d, 2H, J ¼ 5.7 Hz,
2 ꢂ NH), 7.60 (d, 2H, J ¼ 7.2 Hz, 2 ꢂ NH), 7.34 (d, 2H, J ¼ 5.4 Hz, ArH),
7.11e7.26 (m, 14H, ArH), 4.93 (br s, 2H, 2 ꢂ NCH), 4.22 (br s, 4H,
2 ꢂ CH2), 3.49 (br s, 4H, 2 ꢂ CH2), 2.56 (br s, 2H, CH2); dC (normal/
DEPT-135) (75 MHz, CDCl3 þ DMSO-d6) 181.11 (absent, C]O),
175.13 (absent, C]O), 161.93 (absent, C]O), 140.85 (þve, ArCH),
135.78 (absent, ArC), 127.91 (absent, ArC), 126.91 (absent, ArC),
123.37 (þve, ArCH), 122.66 (þve, ArCH), 121.58 (þve, ArCH), 120.32
(þve, ArCH), 118.38 (þve, ArCH), 117.89 (þve, ArCH), 111.36 (absent,
ArC), 110.69 (þve, ArCH), 54.67 (þve, NCH), 43.86 (ꢁve, CH2), 29.42
(ꢁve, CH2), 27.05 (ꢁve, CH2); HRMS (MALDI, m/z) calculated for
4.1.3.6. Compound 11b. Compound 11b was prepared from 11a
(100 mg, 0.15 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
95:5) to yield 11b as light brown solid (70 mg, 74%), [
a
]
¼ ꢁ43.3 (c
D
1.5 in CHCl3), mp 150 ꢀC; IR (KBr, cmꢁ1): 3460, 2966, 1734, 1676,
1635, 1506, 1388, 1222, 1168, 746; dH (300 MHz, CDCl3) 8.83 (s, 2H,
C
45H38N6O8Na [M þ Na]þ: 813.2649, found: 813.263.

336
Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
4.1.3.10. Compound 15b. Compound 15b was prepared from 15a
(100 mg, 0.12 mmol) following General Procedure C. The crude
3.66e3.80 (m, 4H, 2 ꢂ NCH2), 3.15e3.28 (m, 4H, 2 ꢂ SCH2), 2.28e
2.32 (m, 2H, CH2), 1.93e2.03 (m, 6H, 3 ꢂ CH2); 13C NMR (normal/
DEPT-135) (75 MHz, CDCl3 þ DMSO): dC 183.74 (absent, C]O),
173.27 (absent, C]O), 170.41 (absent, C]O), 165.27 (absent, C]O),
140.06 (þve, ArCH), 136.42 (absent, ArC), 135.30 (absent, ArC),
127.05 (absent, ArC), 123.56 (þve, ArCH), 122.70 (þve, ArCH),
122.06 (þve, ArCH), 112.85 (absent, ArC), 110.27 (þve, ArCH), 59.67
(þve, NCH), 52.10 (þve, NCH), 50.27 (ꢁve, CH2), 47.54 (ꢁve, NCH2),
32.00 (ꢁve, SCH2), 28.49 (ꢁve, CH2), 24.41 (ꢁve, CH2); HRMS
(MALDI, m/z) Calcd for C44H44N6O10S2Na [M þ Na]þ: 903.2458,
found: 903.243.
residue was purified by silica gel chromatography (EtOAc: MeOH
25
80:20) to yield 15b as light brown solid (75 mg, 78%), [
a
]
¼ ꢁ16.6
D
(c 1.5 in DMSO), mp > 250 ꢀC; IR (KBr, cmꢁ1): 3390, 2929, 1668,
1616, 1506, 1388, 1232, 1199, 1172, 748; dH (300 MHz, CDCl3 þ TFA)
8.67 (s, 2H, ArH), 8.39 (s, 2H, ArH), 8.19 (d, 2H, J ¼ 6.0 Hz, 2 ꢂ NH),
7.22e7.35 (m, 6H, ArH), 7.15 (d, 4H, J ¼ 6.6 Hz, ArH), 6.82 (d, 4H,
J ¼ 6.6 Hz, ArH), 4.89 (s, 2H, 2 ꢂ NCH), 4.32 (s, 4H, 2 ꢂ CH2), 3.33e
3.38 (m, 2H, CH2), 3.10e3.23 (m, 2H, CH2), 2.61 (s, 2H, CH2); dC
(normal/DEPT-135) (75 MHz, CDCl3 þ DMSO-d6) 181.72 (absent,
C]O), 170.81 (absent, C]O), 162.33 (absent, C]O), 156.02 (absent,
ArC), 141.45 (þve, ArCH), 136.34 (absent, ArC), 130.59 (þve, ArCH),
128.65 (absent, ArC), 127.23 (absent, ArC), 123.71 (þve, ArCH),
123.11 (þve, ArCH), 122.03 (þve, ArCH), 115.12 (þve, ArCH), 111.79
(absent, ArC), 111.11 (þve, ArCH), 55.43 (þve, NCH), 44.34 (ꢁve,
CH2), 36.60 (ꢁve, CH2), 29.81 (ꢁve, CH2); HRMS (MALDI, m/z)
calculated for C41H36N4O10Na [M þ Na]þ: 767.2329, found: 767.234.
4.1.5.3. Compound 18b. Compound 18b was prepared from 18a
(170 mg, 0.2 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
70:30) to yield 18b as light brown solid (120 mg 73%), [
a
]
¼ ꢁ41.4
D
(c 1.4 in DMSO); mp 193 ꢀC. IR (KBr, cmꢁ1): 3325, 2964, 1737, 1635,
1500, 1464, 1386, 1236, 1170, 748; dH (300 MHz, CDCl3 þ TFA): 8.71
(s, 2H, ArH), 8.45 (d, 2H, J ¼ 7.8 Hz, ArH), 8.14 (d, 2H, J ¼ 4.5 Hz,
2 ꢂ NH), 7.58 (d, 2H, J ¼ 7.5 Hz, 2 ꢂ NH), 7.34 (d, 2H, J ¼ 8.1 Hz, ArH),
7.11e7.27 (m, 14H, ArH), 7.11 (s, 4H, ArH), 5.33 (s, 4H, 2 ꢂ CH2),
4.90e4.96 (m, 2H, 2 ꢂ NCH), 4.60e4.64 (m, 2H, 2 ꢂ NCH), 3.70e
3.80 (m, 4H, 2 ꢂ NCH2), 3.42e3.49 (m, 4H, 2 ꢂ CH2), 2.26e2.33 (m,
2H, CH2), 1.94e2.06 (m, 6H, 3 ꢂ CH2); 13C NMR (normal/DEPT-135)
(75 MHz, CDCl3 þ DMSO): dC 178.80 (absent, C]O), 171.07 (absent,
C]O), 164.97 (absent, C]O), 161.53 (absent, C]O), 140.83 (þve,
ArCH), 135.81 (absent, ArC), 135.63 (absent, ArC), 135.02 (absent,
ArC), 127.18 (absent, ArC), 127.01 (þve, ArCH), 126.69 (absent, ArC),
123.59 (þve, ArCH), 123.06 (þve, ArCH), 122.46 (þve, ArCH), 122.18
(þve, ArCH), 121.73 (þve, ArCH), 119.14 (þve, ArCH), 118.02 (þve,
ArCH), 111.77 (absent, ArC), 110.78 (þve, ArCH), 109.99 (þve, ArCH),
109.15 (absent, ArC), 58.13 (þve, NCH), 52.15 (þve, NCH), 50.09
(ꢁve, CH2), 47.14 (ꢁve, NCH2), 28.49 (ꢁve, CH2), 27.34 (ꢁve, CH2),
21.64 (ꢁve, CH2); HRMS (MALDI, m/z) Calcd for C60H54N8O10Na
[M þ Na]þ: 1069.3861, found: 1069.387.
4.1.4. General procedure for synthesis of dipeptides 16ae23a
(Procedure D)
Compound 4b/6b/11b/13b (1 mmol) was dissolved in dry DCM
in the presence of triethyl amine (2 mmol) at 0 ꢀC followed by
addition of ethyl chloroformate (2 mmol). Solution of amino acid
ester hydrochloride (2 mmol) and triethyl amine (2 mmol) in DCM
was added to above mixture. The reaction mixture was stirred and
monitored by TLC. After 15 min stirring, the reaction mixture was
washed with 1 N HCl, 0.5 M sodium bicarbonate and again with
water. After drying over sodium sulfate and distillation the pure
product was obtained by column chromatography.
4.1.5. General procedure for synthesis of dipeptides 16be23b
The ester derivative 16ae23a (1 mmol) were hydrolyzed and
purified following General Procedure C to get compounds 16be
23b.
4.1.5.1. Compound 16b. Compound 16b was prepared from 16a
4.1.5.4. Compound 19b. Compound 19b was prepared from 19a
(75 mg, 0.08 mmol) following General Procedure C. The crude
(150 mg, 0.14 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
residue was purified by silica gel chromatography (EtOAc: MeOH
25
25
75:25) to yield 16b as light brown solid (60 mg, 83%), [
a]
¼ ꢁ30.0
75:25) to yield of 19b as brown solid (105 mg, 72%); [
a
]
¼ ꢁ16.2 (c
D
D
(c 1.5 in DMSO), mp 182 ꢀC, IR (KBr, cmꢁ1): 3383, 2966, 1734, 1624,
1506, 1464, 1386, 1172, 748 cmꢁ1; 1H NMR (300 MHz, CDCl3 þ TFA):
dH 8.96 (s, 2H, ArH), 8.55 (s, 2H, ArH), 8.30e8.32 (m, 4H, 4 ꢂ NH),
7.26e7.40 (m, 6H, ArH), 7.20 (s, 4H, ArH), 5.42 (s, 4H, 2 ꢂ CH2), 4.72
(t, 2H, J ¼ 6.9 Hz, 2 ꢂ NCH), 4.56 (t, 2H, J ¼ 7.05 Hz, 2 ꢂ NCH), 2.39e
2.46 (m, 2H, 2 ꢂ CH), 1.67 (d, 6H, J ¼ 6.6 Hz, 2 ꢂ CH3), 1.08 (d, 12H,
J ¼ 6.6 Hz, 4 ꢂ CH3); 13C NMR (normal/DEPT-135) (75 MHz,
CDCl3 þ DMSO-d6): dC 179.72 (absent, C]O), 173.79 (absent, C]O),
169.80 (absent, C]O), 161.81 (absent, C]O), 140.88 (þve, ArCH),
135.82 (absent, ArC), 135.33 (absent, ArC), 127.10 (þve, ArCH),
123.43 (þve, ArCH), 122.79 (þve, ArCH), 121.80 (þve, ArCH), 111.64
(absent, ArC), 110.35 (þve, ArCH), 57.54 (þve, NCH), 49.94 (ꢁve,
CH2), 47.37 (þve, NCH), 31.11 (þve, CH), 18.87 (þve, CH3), 17.50
(þve, CH3), 17.17 (þve, CH3); HRMS (MALDI, m/z) Calcd for
1.6 in DMSO); mp 195 ꢀC; IR (KBr, cmꢁ1): 3309, 2974, 1716, 1616,
1516, 1456, 1379, 1226, 1174, 750 cmꢁ1
;
dH (300 MHz, CDCl3 þ TFA):
8.42 (s, 2H, ArH), 8.27 (d, 2H, J ¼ 7.2 Hz, 2 ꢂ NH), 7.89 (d, 2H,
J ¼ 8.4 Hz, ArH), 7.36 (s, 2H, ArH), 7.17e7.20 (m, 4H, ArH), 7.04 (s, 4H,
ArH), 6.89 (d, 4H, J ¼ 8.1 Hz, ArH), 6.67 (d, 4H, J ¼ 8.1 Hz, ArH), 5.26
(s, 4H, 2 ꢂ CH2), 4.78e4.81 (m, 2H, 2 ꢂ NCH), 4.66e4.73 (m, 2H,
2 ꢂ NCH), 3.76e3.85 (m, 2H, NCH2), 3.62e3.64 (m, 2H, NCH2),
2.94e3.07 (m, 4H, 2 ꢂ CH2), 2.17e2.32 (m, 4H, 2 ꢂ CH2), 1.99e2.03
(m, 4H,
2
ꢂ
CH2); 13C NMR (normal/DEPT-135) (75 MHz,
CDCl3 þ DMSO-d6): dC 184.95 (absent, C]O), 172.82 (absent, C]O),
170.76 (absent, C]O), 165.11 (absent, C]O), 155.79 (absent, ArC),
140.55 (þve, ArCH), 136.48 (absent, ArC), 136.19 (absent, ArC),
135.76 (absent, ArC),130.01 (þve, ArCH), 127.50 (þve, ArCH),125.89
(absent, ArC), 123.51 (þve, ArCH), 122.72 (þve, ArCH), 121.49 (þve,
ArCH), 114.84 (þve, ArCH), 112.33 (absent, ArC), 110.90 (þve, ArCH),
59.30 (þve, NCH), 53.41 (þve, NCH), 49.93 (ꢁve, CH2), 47.51 (ꢁve,
NCH2), 36.05 (ꢁve, CH2), 28.72 (ꢁve, CH2), 24.27 (ꢁve, CH2); HRMS
(MALDI, m/z) Calcd for C56H52N6O12Na [M þ Na]þ: 1023.3541,
found: 1023.352.
C
44H48N6O10Na[M þ Na]þ: 843.3329, found: 843.332.
4.1.5.2. Compound 17b. Compound 17b was prepared from 17a
(100 mg, 0.1 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
80:20) to yield 17b as light brown solid (75 mg, 80%), [
a
]
D
¼ ꢁ29.1
(c 1.2 in DMSO), mp 192 ꢀC, IR (KBr, cmꢁ1): 3429, 2953, 2582, 1732,
4.1.5.5. Compound 20b. Compound 20b was prepared from 20a
(100 mg, 0.16 mmol) following General Procedure C. The crude
1624, 1521, 1456, 1379, 1224, 1176, 1068, 750 cmꢁ1 1H NMR
;
(300 MHz, CDCl3 þ TFA): dH 8.96 (s, 2H ArH), 8.56 (s, 2H, ArH), 8.26
(s, 2H, 2 ꢂ NH), 7.19e7.28 (m, 6H, ArH), 7.11 (s, 4H, ArH), 5.33 (s, 4H,
2 ꢂ CH2), 4.77e4.78 (m, 2H, 2 ꢂ NCH), 4.67e4.68 (m, 2H, 2 ꢂ NCH),
residue was purified by silica gel chromatography (EtOAc: MeOH
25
90:10) to yield 20b as light brown solid (95 mg, 75%), [
a
]
D
¼ ꢁ16.2
(c 1.6 in DMSO); mp > 250 ꢀC, IR (KBr, cmꢁ1): 3383, 2966, 1635,

Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
337
1506, 1456, 1392, 1226, 1168, 748 cmꢁ1
;
dH (300 MHz, CDCl3 þ TFA):
1519, 1456, 1384, 1220, 1174, 752; dH (300 MHz, CDCl3 þ TFA): 8.05e
8.26 (m, 4H, 2H of ArH, 2H of 2 ꢂ NH), 7.39 (d, 6H, J ¼ 9 Hz, ArH),
7.07 (d, 5H, J ¼ 5.4 Hz, ArH), 6.70e6.87 (m, 5H, ArH), 4.93 (s, 2H,
2 ꢂ NCH), 4.61e4.66 (m, 2H, 2 ꢂ NCH), 4.20e4.23 (m, 4H, 2 ꢂ CH2),
3.62e3.71 (m, 4H, 2 ꢂ NCH2), 3.23e3.27 (m, 2H, CH2), 3.06 (br s, 2H,
CH2), 2.55 (br s, 2H, CH2), 2.30e2.39 (m, 4H, 2 ꢂ CH2), 1.96 (br s, 4H,
2 ꢂ CH2); dC (normal/DEPT-135) (75 MHz, CDCl3): 179.79 (absent,
C]O), 170.83 (absent, C]O), 166.58 (absent, C]O), 161.78 (absent,
C]O), 155.86 (absent, ArC), 140.16 (þve, ArCH), 135.56 (absent,
ArC), 129.63 (þve, ArCH), 125.83 (absent, ArC), 123.46 (þve, ArCH),
122.84 (þve, ArCH), 121.88 (þve, ArCH), 115.07 (þve, ArCH), 111.43
(þve, ArCH), 109.75 (absent, ArC), 57.92 (þve, NCH), 52.99 (þve,
NCH), 47.42 (ꢁve, NCH2), 43.80 (ꢁve, CH2), 36.20 (ꢁve, CH2), 29.15
(ꢁve, CH2), 28.24 (ꢁve, CH2), 23.45 (ꢁve, CH2); HRMS (MALDI, m/z)
Calcd for C51H50N6O12Na [M þ Na]þ: 961.3384, found: 961.337.
8.83 (s, 2H, ArH), 8.29 (d, 2H, J ¼ 7.2 Hz, 2 ꢂ NH), 8.21 (d, 2H,
J ¼ 6.9 Hz, 2 ꢂ NH), 7.94 (d, 2H, J ¼ 8.7 Hz, ArH), 7.09e7.21 (m, 6H,
ArH), 4.46e4.50 (m, 4H, 4 ꢂ NCH), 4.13 (br s, 4H, 2 ꢂ CH2), 2.37e
2.44 (m, 2H, CH2), 2.27e2.35 (m, 2H, 2 ꢂ CH), 1.47 (d, 6H, J ¼ 7.2 Hz,
2 ꢂ CH3), 1.01 (d, 12H, J ¼ 5.4 Hz, 4 ꢂ CH3); dC (normal/DEPT-135)
(75 MHz, CDCl3 þ DMSO-d6): 178.46 (absent, C]O), 173.60 (absent,
C]O), 162.31 (absent, C]O), 156.54 (absent, C]O), 140.00 (þve,
ArCH), 135.57 (absent, ArC), 126.98 (absent, ArC), 123.61 (þve,
ArCH), 123.06 (þve, ArCH), 122.13 (þve, ArCH), 111.69 (absent, ArC),
109.36 (þve, ArCH), 56.96 (þve, NCH), 49.09 (þve, NCH), 43.81
(ꢁve, CH2), 30.15 (þve, CH), 28.85 (ꢁve, CH2),18.63 (þve, CH3),17.16
(þve, CH3); HRMS (MALDI, m/z) Calcd for C39H46N6O10Na
[M þ Na]þ: 781.3173, found: 781.322.
4.1.5.6. Compound 21b. Compound 21b was prepared from 21a
(80 mg, 0.09 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
4.2. Antifungal activities
4.2.1. Evaluation of MIC80
25
90:10) to yield 21b as brown solidꢁ(151 mg, 68%), [
a
]
¼ ꢁ50.6 (c 1.5
D
in DMSO), mp 240 ꢀC, IR (KBr, cm ): 3392, 2976, 2522, 1716, 1683,
100
added to each well in the 96-well flat bottom transparent cell
culture ELISA plate (200 L in column 11). Each compound was
screened in duplicates. A known amount of test compounds were
first dissolved in DMSO/THF and diluted to 100 L with YEPD.
100 L of compound solution was added to the first well of the row
making final concentration of the compound 5 mg/ml in 200
mL of yeast extract peptone dextrose medium (YEPD) was
1624, 1521, 1456, 1381, 1174, 752 cmꢁ1
;
dH (300 MHz, CDCl3 þ TFA)
8.41 (s, 2H, ArH), 8.32 (s, 2H, 2 ꢂ NH), 7.49 (s, 2H, ArH), 7.28 (s, 6H,
ArH), 4.82 (s, 2H, 2 ꢂ NCH), 4.73 (s, 2H, 2 ꢂ NCH), 4.25 (m, 4H,
2 ꢂ CH2), 3.79 (br s, 4H, 2 ꢂ NCH2), 3.50e3.54 (m, 2H, SCH2), 3.14e
3.34 (m, 2H, SCH2), 2.60 (br s, 2H, CH2), 2.30 (br s, 2H, CH2), 2.00 (br
m
m
m
s, 6H,
3
ꢂ
CH2); 13C NMR (normal/DEPT-135) (75 MHz,
m
L
CDCl3 þ DMSO-d6): dC 183.78 (absent, C]O), 170.57 (absent, C]O),
169.45 (absent, C]O), 165.22 (absent, C]O), 139.62 (þve, ArCH),
136.21 (absent, ArC), 126.01 (absent, ArC), 123.73 (þve, ArCH),
122.97 (þve, ArCH), 122.11 (þve, ArCH), 112.83 (absent, ArC), 109.44
(þve, ArCH), 59.64 (þve, NCH), 52.05 (þve, NCH), 47.53 (ꢁve,
NCH2), 43.70 (ꢁve, CH2), 31.99 (ꢁve, SCH2), 28.81 (ꢁve, CH2), 27.61
(ꢁve, CH2), 24.38 (ꢁve, CH2); HRMS (MALDI, m/z) Calcd for
(total volume in each well). A two-fold serial dilution of the com-
pound was done in the next 9 wells, making ten concentrations of
compound ranging from 5 mg/ml to 0.009 mg/ml. Corresponding
solvent control was also diluted in similar fashion. Candida cells
(BWP17) which were already grown in culture medium for 24 h, at
30 ꢀC (primary culture) were resuspended in 0.9% saline solution to
give an optical density of 0.1 at 600 nm (OD600) followed by dilution
C
39H42N6O10S2Na [M þ Na]þ: 841.2302, found: 841.231.
(ꢂ100) in YEPD medium. 100
mL of diluted cells was added to each
well except one, which was a control well to monitor microbial
contamination during the study. Another control well contained
the growth medium and the fungal cells only to serve as positive
control. The plate was incubated at 30 ꢀC for 48 h and OD600 was
monitored using a microplate reader. The percent fungal growth
inhibition for all the concentrations of compounds were deter-
mined with respect to positive control as:
4.1.5.7. Compound 22b. Compound 22b was prepared from 22a
(100 mg, 0.09 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
80:20) to yield 23b as light brown solid (95 mg, 97%), [
a
]
¼ ꢁ33.5
D
(c 1.4 in DMSO), mp > 250 ꢀC, IR (KBr, cmꢁ1): 3400, 2953, 1616,
1521, 1446, 1381, 1203, 1176, 1158, 744 cmꢁ1
;
dH (300 MHz,
CDCl3 þTFA): 8.81 (s, 2H, ArH), 8.43 (s, 2H, ArH), 8.31 (d, 2H,
J ¼ 6.3 Hz, 2 ꢂ NH), 8.10 (d, 2H, J ¼ 5.1 Hz, 2 ꢂ NH), 7.60 (d, 2H,
J ¼ 7.2 Hz, ArH), 7.38 (s, 6H, ArH), 7.16e7.18 (m, 8H, ArH), 4.93e4.95
(m, 2H, 2 ꢂ NCH), 4.79 (d, 2H, J ¼ 8.1 Hz, 2 ꢂ NCH), 4.17e4.22 (m,
4H, 2 ꢂ CH2), 3.80 (q, 2H, J ¼ 8.7 Hz, NCH2), 3.62e3.64 (m, 2H,
NCH2), 3.49 (br s, 4H, 2 ꢂ CH2), 2.55 (br s, 2H, CH2), 2.18e2.39 (m,
4H, 2 ꢂ CH2), 1.99e2.04 (m, 4H, 2 ꢂ CH2); dC (normal/DEPT-135)
(75 MHz, CDCl3 þ DMSO-d6): 179.60 (absent, C]O), 172.99 (absent,
C]O), 171.67 (absent, C]O), 162.29 (absent, C]O), 140.74 (þve,
ArCH), 136.47 (absent, ArC), 136.08 (absent, ArC), 127.43 (absent,
ArC), 127.27 (absent, ArC), 123.99 (þve, ArCH), 123.64 (þve, ArCH),
123.37 (þve, ArCH), 122.42 (þve, ArCH), 121.49 (þve, ArCH), 118.95
(þve, ArCH), 118.22 (þve, ArCH), 111.96 (absent, ArC), 111.62 (þve,
ArCH), 110.27 (þve, ArCH), 108.86 (absent, ArC), 58.47 (þve, NCH),
52.81 (þve, NCH), 47.97 (ꢁve, NCH2), 44.33 (ꢁve, CH2), 29.64 (ꢁve,
CH2), 28.78 (ꢁve, CH2), 27.59 (ꢁve, CH2), 23.99 (ꢁve, CH2); HRMS
(MALDI, m/z) Calcd for C55H52N8O10Na [M þ Na]þ: 1007.3704,
found: 1007.374.
% Inhibition ¼ ½f1 ꢁ ðOD600C= OD600PÞg
ꢁ f1 ꢁ ðOD600S=OD600PÞgꢃ ꢂ 100
Where, OD600C, OD600S and OD600P correspond to OD600 of com-
pound well, solvent well and positive control well, respectively. The
MIC80 values were determined by plotting graph of percent inhi-
bition versus concentration of the compound using Graphpad
Prism software.
4.2.2. Disc diffusion assay
BWP17 cells were grown overnight in YEPD medium at 30 ꢀC.
108 cells were spread on five 90 mm diameter YEPD-agar plates
containing: (i) YEPD only, (ii) calcofluor white (0.01%), (iii) congo
red (200 mg/mL) and (iv) sodium dodecylsulphate (100 mg/mL). The
plates were left to dry for about 10 min. Sterile filter paper discs
were placed on this plate with the help of sterile forceps. Solution of
appropriate concentrations of compound was spotted on these
paper discs. Paper discs spotted with respective solvents were
taken as controls. Plates were incubated at 30 ꢀC and the growth of
fungal cells was noted after 48 h. Average diameter of the circle
where no fungal growth was observed corresponded to inhibition
zone diameter.
4.1.5.8. Compound 23b. Compound 23b was prepared from 23a
(100 mg, 0.1 mmol) following General Procedure C. The crude
residue was purified by silica gel chromatography (EtOAc: MeOH
25
75:25) to yield 23b as light brown solid (81 mg, 82%), [
a
]
¼ ꢁ25.3
D
(c 1.5 in DMSO), mp 195 ꢀC, IR (KBr, cmꢁ1): 3354, 2953, 1732, 1651,

338
Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
4.2.3. Spot assay
glass beads (4 g) and methanol (10 ml). After vortexing each set 10
times (1 min each), the supernatant was decanted into fresh vials.
20 mL CHCl3 was added to each vial and kept on rocker for 2 h. The
Specified concentrations of compounds 7b, 12b, 13b, 14b, 15b
and 18b and solvent control (DMSO 30 L) were added to the YEPD-
m
agar medium and air-dried for 10 min. Similarly, drug plates (flu-
conazole, ketoconazole and terbinafine) and compoundedrug
combination plates were prepared. Plate with YEPD-agar only acted
as positive control. Primary cultures of C. albicans (BWP17, CAI4,
erg11/ERG11 and Cagpi19/CaGPI19) were grown overnight in YEPD
medium at 30 ꢀC (primary culture). 4% of each primary culture was
suspended in YEPD and incubated for 4 h to prepare secondary
cultures. The OD600 of the secondary culture was recorded and the
cell suspension diluted to OD600 of 0.1 by using 0.9% saline solution.
Five serial dilutions of this secondary culture (with OD600 nm of 0.1)
were performed with 0.9% saline solution in 5 vials each with OD600
0.1, 0.02, 0.004, 0.0008 and 0.00016 corresponding to fungal cell
suspension was filtered through Whatman filter paper. 600 mL of
0.9% saline solution was added to each vial and vortexed. The upper
layer was removed and the remaining solution was dried under
nitrogen gas. 400
m
L of CHCl3, 3 ml ethanol and 300 ꢀmL of alc. KOH
(33% w/v) was added to each vial and boiled at 60 C for 45 min,
cooled to room temperature and 5 ml hexane and 1 ml water was
added to each vial. After vortexing for 5 min, the organic layer was
separated. 100
internal standard, dried with nitrogen gas and resuspended in
100 L CHCl3. The contents of each vial were analyzed within 24 h
mL of 1 mg/ml cholesterol was added to each vial as
m
with GCeMS for ergosterol level in both the cultures (grown with
and without compound) as described previously [7]. Quantification
was done by normalizing the area for the ergosterol peak relative to
the internal standard (cholesterol) and the data were normalized to
cell weights.
count 105,104,103,102 and 10 cells/ml. 5
mL from each of the five cell
suspensions were spotted on the plates. The plates were incubated
at 30 ꢀC for 48 h and photographs were taken after 24 h, 36 h and
48 h.
4.2.7. Mammalian cell viability assays
4.2.4. Spot assay to show effect of the compounds in the presence of
azoles
Effect of the compound on cell proliferation was measured using
MTT assay. HeLa cells (4000 cells/well) were incubated in triplicate
in a 96-well plate in the presence of various concentrations of the
BWP17 cells were grown in two sets, one in YEPD medium only
(normal culture) and the other in YEPD medium containing com-
pound at a concentration nearly half of its MIC80 for 24 h at 30 ꢀC.
Culture grown in presence of compound was further incubated for
6 h to attain sufficient fungal growth corresponding to the normal
untreated culture (compared by OD600). Two sets of secondary
cultures containing 4% inoculums from respective primary cultures
were further grown in two media e one YEPD alone and one YEPD
with compound (at concentration nearly half of its MIC80) for 4 h.
Five serial dilutions of both the cell suspensions from secondary
culture were made with 0.9% saline solution to give OD600 0.1, 0.02,
compound in a final volume of 200 ml for different time lengths
(24 h, 48 h and 72 h) at 37 ꢀC in a humidified 5% CO2 chamber. Cells
treated with the vehicle alone (DMSO) served as a control. At the
end of each time point, 20 ml of MTT solution (5 mg/ml in PBS)
added to each well and incubated for another 4 h. After discarding
the supernatant at the end of the 4 h, the resultant formazan
crystals produced were dissolved in 200 mL of DMSO. The absor-
bance value (A) measured at 570 nm by a microplate reader. Per-
centage viability calculated by using the following formula:
Percentage of cell viabilityðCVÞ : CVð%Þ
¼ ðAbsorbance of the experimental samples=
Absorbance of the control sampleÞ ꢂ 100
0.004, 0.0008 and 0.00016. 5
culture were added to YEPD plate and plate containing 4
fluconazole. Similarly, 5 L from cell suspensions from culture
containing compound was added to both the plates. The plates
were incubated and snapshots were taken at time intervals of 24 h,
36 h and 48 h.
m
L cell suspensions from normal
m
g/ml
m
4.2.7.1. Docking procedure. Compounds were built using the
builder toolkit of the software package ArgusLab 4.0.1 and energy
minimized using semi-empirical quantum mechanical method
4.2.5. CFW staining
PM3. The crystal coordinates of lanosterol 14a-demethylase (pdb ID
Two cultures of BWP17, one in YEPD and the other in YEPD
3jus) carrying econazole as ligand in its binding site were down-
loaded from protein data bank. Compound 18b was pasted in the
work space carrying the structure of the enzyme. The docking
programme implements an efficient grid based docking algorithm
which approximates an exhaustive search within the free volume
of the binding site cavity. The conformational space was explored
by the geometry optimization of the flexible ligand (rings were
treated as rigid) in combination with the incremental construction
of the ligand torsions. Thus, docking occurs between the flexible
ligand parts of the compound and enzyme. The ligand orientation
was determined by a shape scoring function based on Ascore and
the final positions were ranked by lowest interaction energy values.
H-bond and hydrophobic interactions between the compound and
enzyme were explored.
medium containing 75
mg/ml of compound 18b were grown at
30 ꢀC for 24 h. Both the cell suspensions were diluted in PBS
(phosphate buffer saline) to give an OD600 0.2. Each of the two cell
suspensions were divided into four sets of 500
from cell suspensions of normal culture were stained with CFW
(100 g/ml) and the other two sets were left unstained. Similarly,
mL volume. Two sets
m
two sets from cell suspensions of culture grown in the presence of
compound 18b were also stained with CFW and two left unstained.
All the cell suspensions were kept on rocker for 1 h in dark. The cells
from each suspension were pelleted down by centrifugation at
5000 rpm for 5 min. Each pellet was washed with PBS twice fol-
lowed by addition of 20
prepared by spotting 5
with cover slips. The slides were observed under florescence mi-
mL of 50% glycerol solution. The slides were
mL from each glycerol stock, and covered
croscope to study the effect of CFW staining.
Conflict of interest
4.2.6. Sterol extraction and GCeMS analysis
A 10 ml primary culture of BWP17 was grown overnight. Taking
1% of the primary culture, two sets of secondary cultures (each
The authors declare they have no conflict of interests.
Acknowledgments
50 mL), one in presence of compound 18b (75
mg/mL) and the
second in absence of the compound, were grown overnight. Both
the cultures were pelleted down by centrifugation at 5000 rpm for
5 min. The pellet weights were equalized, followed by addition of
Financial support from Department of Science & Technology,
New Delhi (SR/S1/OC-82A/2010 dated 19.3.2012) to PS and SSK is

Pooja et al. / European Journal of Medicinal Chemistry 80 (2014) 325e339
339
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MIC80
YEPD
Minimum concentration needed for 80% inhibition of
fungal growth
Yeast extract peptone dextrose
GCeMS Gas chromatographyemass spectroscopy
CFW
CR
[10] P. Singh, J. Kaur, B. Yadav, S.S. Komath, Bioorganic & Medicinal Chemistry 18
(2010) 4212e4223.
Calcoflour white
Congo red
Sodium dodecyl sulfate.
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SDS
Appendix A. Supplementary data
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