Design, synthesis and bio-evaluation of C-1 alkylated tetrahydro-β-carboline derivatives as novel antifungal lead compounds

Article abstract of DOI:10.1016/j.bmcl.2019.126869

The field of antifungal agent has become static and development of resistance by the pathogen as well as limited clinical efficacy of marketed drugs demand the constant development of new antifungals. The presence of hydrocarbon chain of specific length linked with various different heterocycles was found to be an important structural feature in various antifungal lead compounds. Based on the prominent antimicrobial activity of β-carboline derivatives, a set of C1 alkylated tetrahydro-β-carboline derivatives were proposed to be active against fungi. To validate and confirm the role of suitable alkyl chains linked to a β-carboline scaffold, few related analogues having C1 aryl substituents were also synthesized in one step via classic Pictet-Spengler reaction. The synthesized library was evaluated for its antifungal activity against C. albicans, C. krusei, C. parapsilosis, C. kefyr, C. glabrata, C. tropicalis and C. neoformans. One of the library members (compound 12c), with n-alkyl chain of eight carbons exhibited potent antifungal activity against C. glabrata and C. kefyr. The lead compound, being selectively toxic also demonstrated prominent synergy enhancing the potency of antifungal drugs up to 10-fold. The time kill kinetic studies confirmed the efficacy of compound 12c, where the results obtained were comparable to that of Amp B. FE-SEM analysis revealed the increased asymmetry, disintegration and roughness of cell surface which could be because of the possible interaction of compound 12c at membrane level or interference in cell wall structure. Apoptosis/necrosis detection assay confirmed the significant apoptotic activity in C. glabrata cells after 12c treatment which was responsible for the rapid killing of C. glabrata cells.

Full text of DOI:10.1016/j.bmcl.2019.126869

Journal Pre-proofs
Design, Synthesis and Bio-evaluation of C-1 Alkylated Tetrahydro-β-carboline
Derivatives as Novel Antifungal Lead Compounds
Rahul Singh, Aanchal Jaisingh, Indresh K. Maurya, Deepak B. Salunke
PII:
S0960-894X(19)30845-5
DOI:
https://doi.org/10.1016/j.bmcl.2019.126869
BMCL 126869
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
26 August 2019
27 October 2019
26 November 2019
Please cite this article as: Singh, R., Jaisingh, A., Maurya, I.K., Salunke, D.B., Design, Synthesis and Bio-evaluation
of C-1 Alkylated Tetrahydro-β-carboline Derivatives as Novel Antifungal Lead Compounds, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126869
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Design, Synthesis and Bio-evaluation of C-1 Alkylated Tetrahydro-β-
carboline Derivatives as Novel Antifungal Lead Compounds
Rahul Singh,a Aanchal Jaisingh,a Indresh K. Mauryab,* and Deepak B. Salunkea,c,*
aDepartment of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University,
Chandigarh 160 014, India.
bDepartment of Microbial Biotechnology, Panjab University, Chandigarh 160 014, India.
cNational Interdisciplinary Centre of Vaccine, Immunotherapeutics and Antimicrobials
(NICOVIA), Panjab University, Chandigarh 160 014, India.
*Corresponding authors: salunke@pu.ac.in; drindresh@pu.ac.in
1

ABSTRACT
The field of antifungal agent has become static and development of resistance by the
pathogen as well as limited clinical efficacy of marketed drugs demand the constant
development of new antifungals. The presence of hydrocarbon chain of specific length linked
with various different heterocycles was found to be an important structural feature in various
antifungal lead compounds. Based on the prominent antimicrobial activity of β-carboline
derivatives, a set of C1 alkylated tetrahydro-β-carboline derivatives were proposed to be
active against fungi. To validate and confirm the role of suitable alkyl chains linked to a β-
carboline scaffold, few related analogues having C1 aryl substituents were also synthesized in
one step via classic Pictet-Spengler reaction. The synthesized library was evaluated for its
antifungal activity against C. albicans, C. krusei, C. parapsilosis, C. kefyr, C. glabrata, C.
tropicalis and C. neoformans. One of the library members (compound 12c), with n-alkyl
chain of eight carbons exhibited potent antifungal activity against C. glabrata and C. kefyr.
The lead compound, being selectively toxic also demonstrated prominent synergy enhancing
the potency of antifungal drugs up to 10-fold. The time kill kinetic studies confirmed the
efficacy of compound 12c, where the results obtained were comparable to that of Amp B. FE-
SEM analysis revealed the increased asymmetry, disintegration and roughness of cell surface
which could be because of the possible interaction of compound 12c at membrane level or
interference in cell wall structure. Apoptosis/necrosis detection assay confirmed the
significant apoptotic activity in C. glabrata cells after 12c treatment which was responsible
for the rapid killing of C. glabrata cells.
Key words: Antifungals, Pictet-Spengler, -carboline, Candida glabrata, Synergistic effect,
SAR, Apoptosis
2

In recent years, fungal infections have emerged as a major cause of death in
immunocompromised patients and cause ~ 1.4 million death globally per years [1]. Most
common human fungal pathogens include Candida albicans and non-albicans species
namely, Candida krusei (C. krusei), Candida parapsilosis (C. parapsilosis), Candida kefyr
(C. kefyr), Candida tropicalis (C. tropicalis), Candida glabrata (C. glabrata) and
Cryptococcus neoformans (C. neoformnas) representing 4th leading cause of nosocomial
disease and accounting for ~75% of all fungal infections [2]. Although a large number of
antifungal agents are discovered, the pathogenic fungi are constantly developing resistance to
these drugs [3]. Overall, the field of antifungal agent has become static and there are only
four major groups of antifungals currently available in the market namely; azoles, polyenes,
allylamines and echinocandins [4]. Many of these antifungal agents are associated with
drawbacks such as development of resistance, side effects, limited clinical efficacy and poor
bioavailability [5] which demand the constant development of new antifungals. Recently
well-known clinical antifungals such as amphotericin B and fluconazole were also found less
potent due to the development of resistance against the most common fungal pathogens like
C. albicans, C. glabrata, and C. neoformans. [6].
During our recent investigation towards the synthesis of tetrahydro--carboline (THβC)
derivatives as potent indoleamine 2,3-dioxygenase (IDO1) inhibitors [7-8], it was observed
that few IDO1 inhibitors demonstrated promising antifungal activities. In particular, it was
interesting to observe potent antifungal activity of novel alkylated azole 1 which
demonstrated the inhibition of 14α-demethylase enzyme involved in ergosterol biosynthesis.
[9]. The presence of such hydrocarbon chain of specific length linked with various different
heterocycles was found to be an important structural feature in various other antifungal lead
compounds. These structures were proposed to be active using structure-based de novo
3

design based on the structure of lanosterol 14α-demethylase (CYP51) of fungi. Most active
analogues in this series include 7-hydroxy-2-heptyl-4-oximinochromane 2 [10], 2-decyl-
tetrahydroisoquinoline-6,7-diol 3 [11] and 5-methoxy-N-nonyl-tetrahydronaphthalen-2-amine
4 [12] (Figure 1).
N
HO
O
C6 chain
Cl
OH
N
N
C8 chain
N
1
2
OH
alkylated Fluconazole
derivative
C2-heptyl-4-oximinochromane
OCH3
Cl
HO
HO
C10 chain
C9 chain
N
3
N
H
3
4
C2-nonylamino-tetrahydronaphthalen
N2-decyl-tetrahydroisoquinoline
Figure 1. Alkylated heterocycles showing antifungal activities.
It was also observed that, various β-carboline derivatives are also known for their antifungal
activities. Specifically, the 9-(4-chlorophenoxy)propyl)-6-fluoro-tetrahydro-β-carboline 5
(Figure 2) showed promising in vitro antifungal activity and also exhibited good fungicidal
activity against both fluconazole sensitive and resistant C. albicans and demonstrated potent
inhibition against C. albicans hyphal growth and biofilm formation [13].
F
H
NH
O
N
H
O
N
Cl
C22
N
O
N
N
H
H
H
5
6
N
OH
Harmine
HN
N
Br
N
N
9
N
A
B
Manadomnzamine : 22-H
Manadomnzamine : 22-H
N
N
H
O
7
8
Xesdomanzamine A
Figure 2. β-Carboline derivatives showing antifungal activities.
4

Harmine 6 was recommended as new antimicrobial biorational by Nenaah based on its potent
activity against Proteus vulgaris, Bacillus subtilis and C. albicans [14]. Recently, Zhou et al.
demonstrated 2-phenyl-3,4-dihydroisoquinolin-2-ium 7 as promising lead compound for the
development of new antifungal agents [15]. Xestomanzamine A (8) and two novel
manzamine type alkaloids, named Manadomanzamines A (9A) and B (9B), isolated from an
Indonesian sponge Acanthostrongylophora spp. (Haplosclerida: Petrosiidae) having β-
carboline skeleton also demonstrated potent antimicrobial activities [16]. Interestingly, the
C22β-H isomer 9A was found to be selectively active against C. albicans, whereas the C22α-
H isomer 9B was active against C. neoformans.
Considering the importance of β-carboline scaffold, specific stereochemistry and heterocycle
linked with appropriate alkyl chain for determining the antifungal activity, a set of C1
alkylated tetrahydro-β-carboline derivatives were proposed to be active against fungi. The
construction of tetrahydro-β-carboline chemotype from L-tryptophan also provide an
opportunity to synthesize cis- and trans diastereomers to understand the effect of specific
stereochemistry on the biological activity.
Tetrahydro-β-carboline (THβC) framework is observed in various indole based natural
products. Specifically, C-1 substituted optically active THβC derivatives have attracted
considerable interest of synthetic chemist owing to the presence of this scaffold in various
biologically active natural products and marketed drugs [17]. Pictet Spengler reaction
discovered in 1911 is one of the most efficient, reliable and shortest routes to synthesize
optically active C-1 substituted THβC framework [18]. This reaction proceeds through the
formation of imine intermediate between tryptamine and aldehyde followed by
intramolecular cyclisation through spiroindole which leads to the generation of THβC adduct
5

[19]. We planned to synthesize various C-1 substituted THβC derivatives wherein, L-
tryptophan methyl ester was used instead of Tryptamine, so as to obtain both cis and trans
THβCs to study the effect of specific stereochemistry at C1 and C3 position on the anti-
fungal activity.
O
O
O
O
O
OH
NH2
OCH3
NH3
OCH3
OCH3
+
R1
H
NH
NH
SOCl2, MeOH
70 oC, 24h
isopropanol
Cl
Reflux
8h
N
R1
Trans isomer
12a 12h
N
R1
Cis isomer
11a 11h
N
N
H
H
H
H
L-Tryptophan
10
-
-
a
b
c
d
e
f
g
h
O
O
O
O
O
O
H
S
O
H
O
H
H
H
H
H
H
10
3
5
7
N
O
Various aldehydes used for the library synthesis
O
Scheme 1. Synthesis of 11a-11h (cis) and 12a-12h (trans).
L-Tryptophan was refluxed with SOCl2 in methanol to provide L-tryptophan methyl ester
hydrochloride 10 in 95% yield (Scheme 1) [20]. Having the compound 10 in hand, next we
envisioned to synthesize both cis and trans isomers of THβC derivatives in one step with
high yield and approximate equal ratio (cis:trans ~ 50/50) for the ease of chromatographic
separation. In the Pictet-Spengler reaction of L-Tryptophan methyl ester with aldehydes or
ketones, the cis isomer is predominantly formed under kinetically controlled condition.
Whereas, the selectivity is transferred towards trans isomer under thermodynamically
controlled condition, which exclusively depend upon the nature of reagents, solvents, reaction
time and temperature [21]. We recently observed that the Pictet-Spengler reaction of L-
tryptophan methyl ester hydrochloride with various aldehydes in isopropanol in 8 hours of
reflux quantitatively resulted in the formation of both the isomers with low cis/trans
selectivity [20]. Following the optimized protocol, a library of THβC derivatives (11a – 11h
and 12a – 12h) was synthesized in moderate to good yield (50-70%) and with almost equal
6

propositions. The diastereomers were purified by column chromatography using 230 - 400
1
mesh silica gel and the formation of desired product was confirmed by IR, H-NMR,13C-
NMR and mass spectrometry. The purity of the synthesized library was confirmed by HPLC.
To check the validity of our hypothesis, C1-substituted THβC derivatives 11a-h and 12a-h
were tested for their in vitro antifungal activity against seven fungal strains i.e C. albicans, C.
krusei, C. parapsilosis, C. kefyr, C. glabrata, C. tropicalis, C. neoformans using
amphotericin B (Amp B) as a positive control (Table 1). The preliminary in vitro antifungal
activity confirmed that the trans-isomer 12c bearing an eight-carbon linear aliphatic chain at
C1 position exhibited significant antifungal activity. The respective cis-isomer (11c) was also
found to be active but was active against only C. neoformans having MIC value 39.02 µM.
Compound 12c exhibited multi-spectrum antifungal activity against seven fungal strains and
showed promising activity against C. kefyr and C. glabrata with MIC values of 9.70 µM for
both. This observation confirmed the effect of specific stereochemistry at C1 and C3 position
on the observed biological activity. The antifungal activity of other C1 aliphatic chain bearing
compounds 11a, 12a, 11b, 12b (with C1 substituent chain length of less than eight carbons)
and 11d, 12d (with C1 substituent chain length of more than eight carbons) was much lower
than 11c and 12c suggesting the importance of substituent with appropriate chain length at C1
position. Haitao et al. [22] previously described the presence of narrow hydrophobic cleft at
S3 subunit of CYP51, making stronger non-covalent interaction with lipophobic n-alkyl
chains. Our observation suggested the possibility of interaction of compound 12c at the S3
subunit of CYP51. No antifungal activity was observed for compounds bearing C1 aromatic
and heteroaromatic substituents (11e-h and 12e-h) confirming the significance of C1
aliphatic substituent for the antifungal activity.
7

Table 1. In-vitro antifungal activity. MIC in µM (µg/mL) of 11a-11h (cis) and 12a-12h
(trans).
Candida
kefyr
>250
Candida
albicans
>250
Candida
krusei
>250
Candida
Tropicalis parapsilosis
>250
(>80.70)
>250
(>80.70)
>250
(>87.72)
>250
(>87.72)
>250
(>94.73)
19.57
(7.41)
>250
Candida
Candida Cryptococcus
Comp.
11a
12a
11b
12b
11c
glabrata
neoformans
>250
(>80.70)
156.25
(50.40)
>250
(>87.72)
>250
(>87.72)
>250
(>94.73)
>250
>250
(>80.70) (>80.70)
(>80.70) (>80.70)
(>80.70)
>250
(>80.70)
156.25
(54.82)
>250
>250
(>80.70)
>250
(>87.72) (>87.72)
>250 >250
(>87.72) (>87.72)
>250 >250
(>94.73) (>94.73)
156.25
(50.70)
>250
156.25
(50.44)
>250
(>87.72) (>87.72)
>250 >250
(>87.72) (>87.72)
62.5
(23.68)
9.70
>250
(>80.70)
>250
(>87.72)
62.5
(23.68)
9.70
39.06
(14.80)
19.57
39.06
(14.80)
>250
19.57
(7.41)
>250
39.06
(14.80)
>250
12c
(3.67)
>250
(3.67)
(7.41)
>250
>250
11d
12d
11e
(>101.74) (>101.74) (>101.74)
>250 >250 >250
(>101.74) (>101.74) (>101.74)
(>101.74) (>101.74) (>101.74)
>250 >250 >250
(>101.74) (>101.74) (>101.74)
(>101.74)
>250
(>101.74)
>250
>250
(>78.09) (>78.09)
>250 >250
(>78.09) (>78.09)
>250 >250
(>76.84) (>76.84)
>250 >250
(>76.84) (>76.84)
>250 >250
(>87.64) (>87.64)
>250 >250
(>87.64) (>87.64)
>250 >250
(>76.59) (>76.59)
>250 >250
(>76.59) (>76.59)
>250
>250
(>78.09)
>250
(78.09)
>250
(>76.84)
>250
(>76.84)
>250
(>87.64)
>250
(>87.64)
>250
(>76.59)
>250
(>76.59)
>250
(>78.09)
>250
(78.09)
>250
(>76.84)
>250
(>76.84)
>250
(>87.64)
>250
(>87.64)
>250
(>76.59)
>250
(>76.59)
>250
>250
(>78.09) (>78.09)
(>78.09)
>250
(78.09)
>250
>250
(78.09)
>250
>250
(78.09)
>250
12e
11f
(>76.84) (>76.84)
>250 >250
(>76.84) (>76.84)
>250 >250
(>87.64) (>87.64)
>250 >250
(>87.64) (>87.64)
>250 >250
(>76.59) (>76.59)
125 >250
(>76.59) (>76.59)
(>76.84)
>250
(>76.84)
>250
(>87.64)
>250
(>87.64)
>250
(>76.59)
>250
12f
11g
12g
11h
12h
Amp B
(>76.59)
0.78
1.56
(1.44)
0.78
(0.72)
0.78
(0.72)
1.56
(11.55)
12.50
(11.55)
0.39
(0.36)
(0.72)
The repeated treatment or long time use of antifungal drugs in clinic has led to the
development of resistance against various fungal strains. A combination therapy with
synergistic effect is an emerging approach to tackle this prevailing problem, in which potency
of a particular drug/compound can be altered under the influence of another compound. C.
glabrata have emerged as the second most common cause of candidacies after C. albicans.
8

Therefore, the synergistic activity of the lead compound (12c) was studied with known
antifungals amphotericin B (Amp B), fluconazole (FLC) and ketoconazole (KTZ) against C.
glabrata by checkerboard method. In vitro activity of 12c was evaluated with combination of
marketed antifungal drugs such as Amp B, KTZ and FLC against C. glabrata strain (Table
2). The effect is considered to be synergistic if fractional inhibitory concentration index [FICI
= (MIC of drug in combination/MIC of drug alone + MIC of tested compound in
combination/MIC of tested compound alone)] ≤ 0.5. Interestingly, approximately 8- and 11-
fold enhancement of antifungal activity was observed for 12c and drug Amp B against C.
glabrata, respectively with FICI value of 0.24, suggesting a strong synergy between both 12c
and Amp B. The result observed is quite interesting as very low dosage of highly toxic Amp
B is required in the combination. In case of Fluconazole (FLC), the MIC value of 12c and
FLC was lowered to ~4 fold, and FIC value of 0.48 also indicated the effective synergy
between FLC and compound 12c. In the combination of 12c and KTZ, the drug required to
inhibit the growth of C. glabrata decreased to ~9 fold and the amount of lead compound 12c
required was reduced to ~4 fold. The calculated fractional inhibitory concentration of 0.38 for
the combination of KTZ and 12c also confirmed the effective synergy among these drugs.
Table 2. Synergistic study of compound 12c with known antifungals (Amp B =
Amphotericin B, FLC = Fluconazole, KTZ = Ketoconazole) against C. glabrata
MIC (µM)
Amp B
0.78
MIC (µM)
12c
MIC (µM)
Amp B + 12c
0.07 + 1.21
FLC + 12c
3.26 + 2.42
KTZ + 12c
1.63 + 2.42
FIC index
0.24
Effect
Synergy
9.71
12c
9.71
12c
FLC
13.07
KTZ
15.06
0.48
0.35
Synergy
Synergy
9.71
Antimicrobial compounds are intended to have selective toxicity against the microbes under
investigation. Mammalian cell toxicity of lead compound 12c (up to 50 µM concentration)
9

was evaluated against normal human embryonic kidney cell lines (HEK-293) and cervical
cancer cell lines (HeLa) by performing MTT assay. At 10 µM, the cytotoxicity of compound
12c was observed to be 10% and 14% against normal HEK-293 and HeLa cell lines,
respectively (Figure 3). Even at higher concentration range of 12c (20µM-50µM), 70-80% of
HeLa and HEK-293 cells were viable. It is noteworthy to mention that in the combination
study, maximum 2.5 µM of 12c was used at which more than 95% HEK-293 cells were
viable, demonstrating selective toxicity of the lead compound against the fungal strains.
120
HEK-293
HeLa cells
100
80
60
40
20
0
0
3.12
6.25
12.5
25
50
Conc. of 12c (µM)
Figure 3. Cell toxicity study of 12c against HEK-293 and HeLa cell lines.
Time kill assay demonstrate the efficacy of drug/compound by determining the time required
to kill the targeted microorganism at MIC. Time kill kinetic study of 12c was carried against
C. glabrata at MIC = 9.20 µM (Figure 4). Compound 12c rapidly inhibited the growth of C.
glabrata and the results were comparable to that of Amp B. In particular, 12c took 12 hrs to
kill the complete colony of C. glabrata, on the other hand Amp B took about 4 hrs to achieve
the same.
10

16
14
12
10
8
Untreated C. glabrata cells
(12c) Treared C. glabrata cells
Amp B Treated C. glabrata cells
6
4
2
0
0
4
8
12
16
20
24
Time (hours)
Figure 4. Time kill kinetic study of 12c and Amp. B against C. glabrata
Many synthetic as well as natural compounds exhibited antifungal activity by inducing
apoptotic/necrotic activity in Candida spp. Further, the inhibition of Candida cells growth
and survival might result from induced apoptotic activity. Therefore, generation of apoptosis
by compound 12c was evaluated by the externalization of phosphatidylserine (PS, apoptosis
marker) from the inner leaflet of C. glabrata cell membrane. The C. glabrata cells treated
with compound 12c for 4 hrs. were compared by using annexin V-FITC and PI staining with
untreated cells (Figure 5). The annexinV-FITC specifically binds to externalized PS whereas
the PI binds to Candida cell membrane. Both unstained (Figure 5A) and stained (Figure 5B)
C. glabrata cells showed no significant necrosis and apoptosis compared to compound 12c
treated C. glabrata cells (Figure 5C). A significant apoptotic activity in C. glabrata cells
after 12c treatment was clearly observed leading to rapid killing of C. glabrata cells.
11

(A)
(B)
(C)
0.0%
0.0%
0.1%
3.0%
1.4%
87.7%
3.2%
5.6%
95.5%
100 %
0.0%
3.5%
FL-1 (FITC-annexin V)
FL-1 (FITC-annexin V)
FL-1 (FITC-annexin V)
Figure 5. Detection of necrosis/apoptosis marker in compound 12c treated C. glabrata by
removal of phosphatidylserine from cell membrane. (A) Unstained C. glabrata cells. (B) C.
glabrata cells + FITC-annexin V + PI and (C) C. glabrata cells + FITC-annexin V + PI +
compound 12c (at 09.71 µM). Both treated and untreated C. glabrata cells were incubated at
30ºC for 4hrs. in RPMI-1640 media.
Ergosterol, a component of fungal cell membranes, is essential for fungal cell growth. Most
of the antifungals either inhibit ergosterol biosynthesis by targeting CYP51 enzyme of the
fungi (such as azole drugs) or directly bind or interact with ergosterol (such as Amp B)
interfering the functioning and integrity of the fungal cell membrane leading to alterations of
cell morphology and subsequent death of fungal cells. The effect of compound 12c treatment
on the morphology of C. glabrata cells at MIC was observed using Field Emission Scanning
Electron Microscopy (FE-SEM) (Figure 6). Increased asymmetry, disintegration and
roughness of cell surface was clearly observed which could be because of the possible
interaction of compound 12c at membrane level or interference in cell wall structure.
12

Figure 6. FE-SEM images of control (I) and 12c treated (II) C. glabrata
Overall, a series of C1-alkyl/aryl THβC derivatives with both cis and trans stereochemistry
were synthesized using Pictet-Spengler reaction between L-trytophan and various aliphatic/
aromatic aldehydes in moderate to good yield. A mixture of nitromethane and toluene (1:1) at
reflux for 8 hrs was found to be the optimum condition for the desired PS cyclization. The
synthesized derivatives were screened for antifungal activity against seven fungal strains. As
envisioned, compound 12c having specific stereochemistry (1R, 3S) at C1, C3 position with
n-alkyl chain of eight carbons exhibited potent antifungal activity against all the fungal
strains. Predominant activity was observed against C. glabrata and C. kefyr with MIC of 9.20
µM. The in vitro antifungal study of 12c in combination with marketed drugs such as Amp B,
KTZ, FLC demonstrated prominent synergy enhancing the potency of both active compound
and antifungal drugs up to 10-fold. The FIC of 12c with Amp, FLC and KTZ was 0.24, 0.48
and 0.35, respectively, confirming the strong synergistic effect. The lead compound 12c also
exhibited selective toxicity against the fungal strains. The cytotoxicity against normal
mammalian cells (HEK-293) and cancer cell line (HeLa) was observed within the acceptable
range. The time kill kinetic studies confirmed the efficacy of compound 12c, which killed the
given colony of C. glabrata in 12 hours and the results were comparable to that of Amp B. A
significant apoptotic activity in C. glabrata cells after 12c treatment was clearly observed
13

leading to rapid killing of fungal cells. FE-SEM analysis revealed the disintegration and
roughness of the fungal cell surface. Further SAR investigation on this lead compound to
improve the activity is currently ongoing.
Acknowledgments
DBS is thankful to UGC New Delhi for start-up research grant and DBT New Delhi for the
award of Ramalingaswami Fellowship. The support from UGC-CAS, DST-PURSE-II, DST-
SAIF, and Panjab University Development fund is gratefully acknowledged. IKM is thankful
to DST, New Delhi for DST-Inspire Faculty research grant. RS is thankful to CSIR, New
Delhi, for the award of Research Fellowship.
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17

TOC GRAPHIC
 Apoptosis/Necrosis Evaluation
 Time Kill Kinetics
 Cell Surface Morphology
O
OCH3
16
14
12
10
8
87.7%
3.2%
NH
12c
N
H
Untreated C. glabrata cells
(12c) Treared C. glabrata cells
Amp B Treated C. glabrata cells
6
D
4
5.6%
3.5%
MIC:
2
• C. kefyr = 9.70 µM (3.67 μg/mL)
0
• C. glabrata = 9.70 µM (3.67 μg/mL)
0
4
8
12
16
20
24
Time (hours)
FIC Index (Synergy)
• 0.24 for 12c + Amp B
• 0.48 for 12c + FLC
after 12c treatment, increased
asymmetry and damage of C.
glabrata cells surface were observed.
Compound 12c rapidly inhibited
12c significantly induced apoptotic-like
programmed cell death in C. glabrata.
the growth of C. glabrata
• 0.35 for 12c + KTZ
18