Synthesis and evaluation of indole-based new scaffolds for antimicrobial activities - Identification of promising candidates

Article abstract of DOI:10.1016/j.bmcl.2011.04.001

Search for new antimicrobial agents led to the synthesis of series of N-1, C-3 and C-5 substituted bis-indoles. Their evaluation for antifungal and antibacterial activities resulted in the optimization of pyrrolidine/morpholine/ N-benzyl moiety at the C-3 end and propane/butane/xylidine groups as linkers between two indoles for significant inhibition of microbial growth. Preliminary investigations have identified three highly potent antimicrobial agents. Dockings of these molecules in the active sites of lanosterol demethylase, dihydrofolate reductase and topoisomerase II indicate their strong interactions with these enzymes.

Full text of DOI:10.1016/j.bmcl.2011.04.001

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3367–3372
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of indole-based new scaffolds for antimicrobial
activities—Identification of promising candidates
Palwinder Singh a, , Puja Verma a, Bhawna Yadav b, Sneha S. Komath b
⇑
a Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005, India
b School of Life Sciences, Jawaharlal Nehru University, New Delhi 160 067, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Search for new antimicrobial agents led to the synthesis of series of N-1, C-3 and C-5 substituted bis-
indoles. Their evaluation for antifungal and antibacterial activities resulted in the optimization of pyrrol-
idine/morpholine/N-benzyl moiety at the C-3 end and propane/butane/xylidine groups as linkers
between two indoles for significant inhibition of microbial growth. Preliminary investigations have iden-
tified three highly potent antimicrobial agents. Dockings of these molecules in the active sites of lanos-
terol demethylase, dihydrofolate reductase and topoisomerase II indicate their strong interactions with
these enzymes.
Received 14 February 2011
Revised 26 March 2011
Accepted 1 April 2011
Available online 7 April 2011
Keywords:
Indole derivatives
Antifungal
Ó 2011 Elsevier Ltd. All rights reserved.
Antibacterial
Docking
The increased emergence of microbial infections has led to mas-
sive increase in the rate of mortality, especially in the immuno-
compromised individuals, those suffering from tuberculosis,
cancer or AIDS. Fungal and bacterial infections constitute large pro-
portion of the infectious diseases resulting in 13 million deaths
each year worldwide.1 Although a number of antimicrobial drugs
are available, the smartness of microbes in developing mutant
strains, emergence of drug resistance,2–6 lack of specificity and nar-
row spectrum are major obstacles in current microbial therapy.
Hence the development of more efficacious, safe and target specific
new antimicrobial agents is an issue of current medicinal impor-
tance. As per the present status; most of the currently used antimi-
crobial drugs (Chart 1) target the microbe either at plasma/cell
membrane or ribosomes or DNA. Amphotericin B,7 naftifine,8,9 ter-
binafine,9,10 miconazole,11 fluconazole12 target the plasma mem-
brane; caspofungin,13 ampicillin,14 cefepime15 disturb cell wall
biosynthesis; clarithromycin,16 linezolid17 bind to ribosomes and
hinder protein biosynthesis while flucytosine,18 norfloxacin,19 tri-
methoprim20 block DNA replication. Further exploration of these
drugs revealed that: (1) antifungal drugs of azole class interact
with iron(II) of protoporphyrin IX in the active site of lanosterol
demethylase21, (2) trimethoprim and its analogues show H-bond
interactions with nitrogens of arginine in the active site of dihydro-
folate reducatse22 and (3) quinolone class of antibacterial drugs
interact at carbonyl oxygen and nitrogen of serine in the active site
of topoisomerase II.23 Despite a number of antimicrobial agents24–27
being reported, new compounds are required to meet increasing
demands for managing infections in the complex patient
population.
Since selective toxicity is a fundamental law for the develop-
ment of anti-infective agents- destroying only one form of life (mi-
crobe) without harming the host, it was kept in mind to develop
new molecules with non-toxicity to host. As indole carries a unique
place in the biological systems playing crucial roles in the form of
amino acid, growth hormone and alkaloids and its hydrophobic
nature28 makes the tryptophan to be present at or near the cata-
lytic/molecular recognition sites of enzymes; a library of N-1, C-3
and C-5 substituted bis-indoles was procured. Some bis- and tris-
indole alkaloids isolated from marine organisms, with significant
biological activities have been reported.29,30 2-(1H-Indol/5-bro-
mo-1H-indol-3-yl)-2-oxoacetyl chloride (2)31,32 was obtained from
the reaction of 1H-indole/5-bromo-1H-indole (1) with oxalyl chlo-
ride at 0–5 °C using dry ether as the reaction medium. Treatment
of 2 with various amines in presence of K2CO3 in acetonitrile gave
compounds 3a–g in 53–89% yield which on further reaction with
0.5 equiv of alkane dibromides in presence of NaH in dry acetoni-
trile at 0‘–5 °C gave target compounds 4a–o in 16–30% yields
(Scheme 1) along with the formation of compounds 5 (2–5%) and
6 (4–8%). Unreacted 3 (15–20%) was also recovered from these
reactions. Percentage yields of compounds 4 did not increase on
increasing the equivalence of alkyl dibromides to 1 w.r.t. 3.
Moreover, amount of 5 and 6 builds up if more time was
given to the reaction. To see the effect of decreased flexibility
between the two indole rings on their biological profile, they
were linked through xylyl group. Reaction of compounds 3 with
⇑
Corresponding author. Tel.: +91 183 2258802 09x3495; fax: +91 183 2258819.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.001

3368
P. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3367–3372
H
OH
O
H
Cl
O
OH
OH
F
N
Cl
O
N
OH
O
N
O
N
O
N
F
Cl
N
HO
N
Cl
O
O
N
HO
OH
Fluconazole
Miconazole
Amphotericin B
NH2
NH2
F
N
CH3
CH3
N
O
N
H
CH3
CH3
H3C
Flucytosine
Naftifine
Terbinafine
OCH3
NH2
N
H
N
H
H
O
O
H
S
CH3
S
N
F
S
H
OH
N
O
N
H
CH3
OH
O
H2N
O
N
N+
N
N
O
O
COO-
HN
Ampicillin
Cefepime
Norfloxacin
O
H3C
HO
CH3
OCH3
O
NH2
N
OH
H3C
CH3
N
F
H3C
C2H5
CH3
CH3
O
O
O
N
HO
N
O
O
O
O
H2N
O
O
OCH3
CH3
CH3
O
CH3
NH
Trimethoprim
OH
CH3
O
Linezolid
CH3
O
Clarithromycin
Chart 1. Antimicrobial drugs.
a
,
a0-dibromo-p-xylene using NaH as base provided compounds
7a–f (35–55%) (Scheme 1). It was observed that reactions of 3 with
a0-dibromo-p-xylene were faster than the reactions of 3 with
wall disruptants. There was no noticeable zone of inhibition in case
of compounds 4e, 4h, 4k and 7c. Therefore, amongst a series of in-
dole derivatives, compounds 4b, 4d, 4f, 4j and 7d were found to in-
hibit the growth of C. albicans either alone or in combination with
cell wall/plasma membrane disrupting agents. It was observed that
compounds with piperidine moiety at the end of C-3 substituent
do not exhibit much antifungal activities. Better antifungal activity
of compound 4f than that of 4b, which differ only in the length of
spacer group between two indoles, indicates the role of spacer
group as well. However, further increase in the chain length of
the linker in case of compounds 4i and 4l did not improved their
antifungal activities. Replacement of pyrrolidine moieties of 4b
with benzylamine improved the antifungal activity of compound
4d while its further modification resulted in much better anti-
fungal profile of compound 7d. Hence, a proper combination of
the amine at C-3 with linkage group between two indoles is desir-
able for an appreciable fungal growth inhibition. Remarkably, fun-
gal growth inhibition zone diameter of compound 7d is
comparable to that of miconazole. Compounds 4b, 4d, 4f, 4j and
a,
alkyl dibromides. All the compounds were characterized by NMR
spectra, mass spectra and CHN analysis.33
Antifungal activities of these compounds were determined on
Candida albicans in solid as well as liquid phase using disc diffusion
and 96 well plate assays, respectively. Disc diffusion assay was per-
formed in YEPD medium either alone or in presence of cell wall
disrupting agents (SDS, CR, CFW).34,35 A specific number of cells
of C. albicans was spread over the medium and discs of 0.5 cm
diameter were placed over it. Solution of test compound and refer-
ence solvent were spotted over the discs. After incubation at 30 °C,
the diameter of inhibition zone was measured (Table 1, Fig. S1). We
also tested whether the compounds would enhance the toxic effect
of Rhodamine 6G (R6G), an antifungal drug and specific substrate
of two ATP-dependent drug pumps of C. albicans, Cdr1p and Cdr2p.
Compound 4f showed excellent fungal growth inhibitory pro-
file, having higher zone of inhibition alone as well as in combina-
tion with CFW, CR, SDS than the solvent control besides
enhancing the toxic effects of R6G. Compound 7d showed most
promising inhibition of fungal growth (12.5 mm zone of inhibition)
with 4 mm higher inhibition zone in comparison to the solvent
(8.5 mm). Other compounds contributing to the toxicity of R6G
were 4a, 4b, 4c, 4d, 4g, 4i, 4j, 4o and 7b. Compounds 4b, 4c, 4d,
4j, 4l, 7b and 7d proved to be antifungal candidates with their
own inhibitory profiles as well as in combination with one or
two of the cell wall disruptants while compounds 4a, 4i, 4n, 4o
and 7a inhibited the fungal growth only in combination with cell
7d exhibit IC50 465
lM, 121 lM, 78 lM, 340 lM and 20 lM,
respectively (Table 1).36
Antibacterial activities of compounds under present investiga-
tion were tested using Escherichia coli strain, having an ampicillin
resistance gene encoding plasmid. The results in terms of inhibi-
tion zone diameter (mm) are given in Table 2 and the pictures of
inhibition zones in comparison with the solvent system are shown
in Figure S2.
It was observed that presence of compounds 4a, 4b, 4d, 4k and
4m in the culture medium increased the zone inhibition diameter

P. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3367–3372
3369
O
O
NR1R2
O
Cl
O
R
R
R
i)
ii)
N
H
N
N
3a: R=H, NR1R2=piperidinyl
3b: R=H, NR1R2=pyrrolidinyl
3c: R=H, NR1R2=morpholinyl
3d: R=H, NR1R2=benzylamine
3e: R=Br, NR1R2=piperidinyl
3f: R=Br, NR1R2=pyrrolidinyl
3g: R=Br, NR1R2=morpholinyl
H
H
1
2
3
iii)
iv)
O
O
NR1R2
NR1R2
O
O
R
R
NR1R2
O
O
R
NR1R2
N
N
R
O
N
O
( )n
+
+
N
( )n
( )n
N
O
Br
5a-o
R
6a-o
NR1R2
N
O
4a-o
O
R
NR1R2
4a, 5a, 6a: R=H, NR1R2=piperidinyl, n=1
O
7a-f
4b, 5b, 6b: R=H, NR1R2=pyrrolidinyl, n=1
4c, 5c, 6c: R=H, NR1R2=morpholinyl, n=1
4d, 5d, 6d: R=H, NR1R2=benzylamine, n=1
4e, 5e, 6e: R=H, NR1R2=piperidinyl, n=2
4f, 5f, 6f: R=H, NR1R2=pyrrolidinyl, n=2
4g, 5g, 6g: R=H, NR1R2=morpholinyl, n=2
4h, 5h, 6h: R=H, NR1R2=piperidinyl, n=3
4i, 5i, 6i: R=H, NR1R2=pyrrolidinyl, n=3
4j, 5j, 6j: R=H, NR1R2=morpholinyl, n=3
4k, 5k, 6k: R=H, NR1R2=piperidinyl, n=4
4l, 5l, 6l: R=H, NR1R2=pyrrolidinyl, n=4
4m, 5m, 6m: R=H, NR1R2=morpholinyl, n=4
4n, 5n, 6n: R=Br, NR1R2=piperidinyl, n=1
4o, 5o, 6o: R=Br, NR1R2=pyrrolidinyl, n=1
7a: R=H, NR1R2=piperidinyl
7b: R=H, NR1R2=pyrrolidinyl
7c: R=H, NR1R2=morpholinyl
7d: R=H, NR1R2=benzylamine
7e: R=Br, NR1R2=piperidinyl
7f: R=Br, NR1R2=morpholinyl
Reaction conditions:
i) dry ether, 0-5 oC, (COCl)2
ii) dry ACN, K2CO3, NHR1R2
iii) NaH, dry ACN, BrCH2(CH2)nCH2Br
iv) NaH, dry ACN, BrCH2(C6H4)CH2Br
Scheme 1.
Table 1
Candida albicans growth inhibition
Compd (concn
lg/ml)/solvent
YEPD
YEPD+CFW(100
l
g/ml)
YEPD+CR (150
lg/ml)
YEPD+SDS (0.01%)
YEPD+R6G(20
lg/ml)
IC50 (mM)
4a (374)
Solventa
4b (428)
Solventb
4c (375)
Solventa
4d (250)
Solventa
4e (150)
Solventa
4f (375)
Solventa
4g (250)
Solventa
4h (450)
Solventa
4i (375)
Solventa
4j (375)
Solventa
4k (238)
Solventb
4l (545)
Solventc
4m (357)
—
—
6.5
7.0
5.5
7.0
8.0
7.0
8.0
9.0
6.0
7.5
8.0
7.0
5.0
7.5
7.0
7.5
8.0
7.0
7.0
7.5
6.0
7.5
10.5
12.5
6.5
7.5
7.0
6.5
6.0
8.5
7.0
8.5
7.5
6.5
6.5
8.0
7.0
7.0
6.5
6.5
6.5
6.0
8.0
7.0
6.5
6.5
7.0
15.5
15
6.5
6.0
6.5
6.0
7.5
6.0
7.0
6.0
—
7.0
7.5
6.0
7.5
7.0
6.5
7.0
6.5
6.0
8.0
7.0
5.5
6.0
12.5
14.5
—
—
6.5
7.5
6.5
7.0
6.5
9.0
8.0
6.0
6.5
7.5
6.5
6.0
6.5
6.0
6.5
5.5
6.5
7.5
6.5
6.5
7.5
14.5
13.5
5.5
7.0
6.5
6.0
6.5
7.0
7.0
9.0
6.0
6.5
7.5
7.0
7.5
6.5
6.5
6.5
5.5
7.0
7.5
6.5
—
0.465
561.6
0.121
566
0.078
365
874.7
627.1
0.340
163.7
—
7.0
11.0
15.0
6.5
—
553.3
(continued on next page)

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P. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3367–3372
Table 1 (continued)
Compd (concn
lg/ml)/solvent
YEPD
YEPD+CFW(100
lg/ml)
YEPD+CR (150
lg/ml)
YEPD+SDS (0.01%)
YEPD+R6G(20
lg/ml)
IC50 (mM)
Solventb
4n (500)
Solventb
4o (257)
Solventb
7a (300)
Solventb
7b (187)
Solventa
7c (250)
Solventa
7d (600)
Solventb
6.0
6.0
7.5
11.5
11.5
—
6.0
6.5
7.0
11.5
12.5
—
6.0
6.5
7.0
6.0
6.5
11.5
11.0
—
6.5
9.5
7.0
11.5
11.5
7.5
5.0
7.0
7.0
6.0
7.5
10.5
13.0
6.0
8.0
7.5
11.5
11.5
7.5
8.0
8.0
7.0
6.5
6.5
12.5
12.0
6.5
7.5
9.5
12.0
11.0
6.0
6.5
7.0
6.0
6.0
7.0
11.0
11.0
401
29.2
407
8.0
7.5
6.5
6.5
6.5
12.5
8.5
12.0
7.0
363.3
376
0.02
Miconazole (300)
Solventd
—
Fluconazole (300)
Solventd
15.0
7.0
—
—
Diameter (mm) of zone of inhibition in disc diffusion assay and IC50 of compounds 4 and 7.
a
DMSO.
DMSO+acetone.
Acetone+CHCl3.
CH3OH.
b
c
d
Table 2
Anti-bacterial activity
Comp (concn
g/ml)/solvent
Inhibition zone
diameter (mm)
Comp (concn
g/ml)/solvent
Inhibition zone
diameter (mm)
l
l
4a (250)
Solventa
4b (428)
Solventb
4c (500)
Solventa
4d (250)
Solventa
4e (300)
Solventa
4f (500)
Solventa
4g (333)
Solventa
4h (450)
Solventa
4i (500)
Solventa
4j (375)
Solventa
9.5
8.5
8.0
7.0
4k (238)
Solventb
4l (545)
Solventc
4m (357)
Solventb
4n (500)
Solventb
4o (257)
Solventb
7a (300)
Solventb
7b (187)
Solventa
7c (250)
Solventa
7d (600)
Solventb
7.5
6.5
11.5
13.0
7.5
6.5
9.0
11.0
13.5
9.5
9.0
8.5
7.0
8.5
10.5
10.0
9.0
6.0
10.0
12.0
10.0
10.5
10.0
10.5
10.0
10.0
9.5
8.5
10.0
8.0
10.0
8.5
9.5
10.0
Diameter (mm) of zone of inhibition of compounds 4 and 7 on LB-Amp agar plate.
a
DMSO.
DMSO+acetone.
Acetone+chloroform.
b
c
Figure 1. Compound 4f docked in the active site of lanosterol demethylase. H-bond
distances are given in Å. Hs’ are omitted for clarity.
by 1 mm, while that of compounds 4e, 4f, 4g, 7a and 7c increased
the zone inhibition diameter by 0.5 mm in comparison to the sol-
vent controls. Compound 4o, with an increase of 4 mm in inhibi-
tion zone diameter was found to be most promising candidate
for antibacterial activity. Comparison of antibacterial activity of
4b and 4o indicates that presence of 5-Br substituent in 4o might
be responsible for higher antibacterial activity of this compound.
No noticeable zone of inhibition was observed in case of other
compounds. Results showed that antibacterial properties of these
compounds are less sensitive to the changes in C-3 substituents
than the antifungal ones. It was observed that compounds 4e, 4f
and 4g with four carbon spacer between two indoles have equal
antibacterial activity while compounds 4h, 4i and 4j with five car-
bon spacer did not show antibacterial activity. Significant inhibi-
tion zone diameters of compounds 4d (12 mm) and 4o
(13.5 mm) which are comparable to the inhibition zone diameters
of clinically used antibacterial drugs apalcillin and piperacillin
(612 mm to 617 mm)37 indicates the suitability of these com-
pounds as antibacterial agents.
Although the cellular target is not defined in the experimental
antimicrobial investigations of these molecules, it was desirable
to investigate the interactions of the active compounds with the
enzymes targeted by clinically used antimicrobial drugs viz., squa-
lene epoxidase, b-1,3-D-glucan synthase, lanosterol demethylase,
dihydrofolate reductase, topoisomerase II, trans-peptidase and
DNA dependent polymerase. Search of Protein Data Bank showed
the availability of crystal structures of lanosterol demethylase,

P. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3367–3372
3371
Figure 4. Compound 4o docked in the active site of topoisomerase II. H-bond
distances are given in Å. Hs’ are omitted for clarity.
(Fig. 3). In the active site of TOPO II, compound 4o showed a num-
ber of H-bond interactions using all the carbonyl oxygens and pyr-
rolidinyl nitrogens with T430, H423, Y28 and Y547 amino acids
(Fig. 4). Docking results indicate the probability of these com-
pounds to interfere with sterol biosynthesis in C. albicans resulting
into fungal growth arrest and DNA replication leading to bacterial
death.
Figure 2. Compound 7d docked in the active site of lanosterol demethylase. H-
bond distances are given in Å. Hs’ are omitted for clarity.
In conclusion, amongst a series of N-1, C-3 and C-5 substituted
indoles, preliminary investigations have identified three highly po-
tent antifungal and antibacterial compounds. Compounds 4o and
7d with 4 mm higher zone of inhibition than the solvent control
and 4f, 7d with IC50 78 lM and 20 lM were found to be most
promising antibacterial and antifungal candidates. Work is under-
going to further improve the potencies of these compounds and ex-
plore their modes of action.
Acknowledgements
Financial assistance from DST, New Delhi and CSIR, New Delhi is
gratefully acknowledged. P.V. thanks CSIR for fellowship. CDRI,
Lukhnow is acknowledged for recording mass spectra.
Supplementary data
Supplementary data (experimental details, 1H NMR spectra of
compounds, bio-evaluation procedures, photographs of disc diffu-
sion assays and docking procedure) associated with this article
can be found, in the online version, at doi:10.1016/j.bmcl.2011.
04.001.
Figure 3. Compound 4o docked in the active site of dihydrofolate reductase. H-
bond distances are given in Å. Hs’ are omitted for clarity.
dihydrofolate reductase and topo II in combination with econazole,
trimethoprim analogue and dextrazoxazone, respectively. Dock-
ing38 of 4f, 7d in the active sites of lanosterol demethylase showed
that these compounds interact through H-bond formation between
their carbonyl oxygens and H314, Y145, T135 and I379, M378 ami-
no acids, respectively (Figs. 1 and 2). Compound 4f also showed H-
bond interactions through its pyrrolidinyl nitrogen with Y145 and
compound 7d through its amino nitrogen and indole nitrogen with
I379 and T135 amino acids, respectively.
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CH2), 27.18 (ꢀve, CH2), 45.89 (ꢀve, NCH2), 46.71 (ꢀve, NCH2), 47.38 (ꢀve,
NCH2), 109.84 (+ve, ArCH), 113.46 (absent, ArC), 122.71 (+ve, ArCH), 123.26
(+ve, ArCH), 123.96 (+ve, ArCH), 126.87 (absent, ArC), 136.57 (absent, ArC),
138.73 (+ve, ArCH), 164.72 (absent, C@O), 184.91 (absent, C@O); Anal. Calcd
for C32H34N4O4: C, 71.35; H, 6.36; N, 10.40. Found C, 71.12; H, 6.11; N, 10.00.
MS (FAB) 539 (M++1).
Compound 7d: White solid, Yield: 49%; mp 184 °C. IR (KBr, cmꢀ1): 1628 (C@O),
1681 (C@O), 3123 (N–H), 3349 (N–H); UV (THF + HEPES buffer) kmax (e) 220
15. Grassi, G. G.; Grassi, C. J. Antimicrob. Chemother. 1993, 32, 87.
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21. Strushkevich, N.; Usanov, S. A.; Park, H.-W. J. Mol. Biol. 2010, 397, 1067.
22. Cody, V.; Pace, J. Acta Crystallogr., Sect. D 2011, 67, 1.
23. Laponogov, I.; Sohi, M. K.; Veselkov, D. A.; Pan, X.-S.; Sawhney, R.; Thompson,
A. W.; McAuley, K. E.; Fisher, L. M.; Sanderson, M. R. Nat. Struct. Mol. Biol. 2009,
16, 667.
(26,220), 259 (19,660), 330 (17,400); 1H NMR (300 MHz, CDCl3): d 4.54 (d, 4H,
J = 6 Hz, 2 ꢁ NCH2), 5.33 (s, 4H, 2 ꢁ NCH2), 7.11 (s, 4H, ArH), 7.26–7.27 (m, 4H,
ArH), 7.30–7.35 (m, 12H, ArH), 7.84 (br s, 2H, NH), 8.40 (d, 2H, J = 7.5 Hz, ArH),
9.07 (s, 2H, ArH); 13C NMR (normal/DEPT-135) (75 MHz, CDCl3): d 43.33 (ꢀve,
NCH2), 50.73 (ꢀve, NCH2), 110.49 (+ve, ArCH), 112.46 (absent, ArC), 122.75
(+ve, ArCH), 123.58 (+ve, ArCH), 124.11 (+ve, ArCH), 127.62 (+ve, ArCH), 127.67
(absent, ArC), 127.74 (+ve, ArCH), 128.79 (+ve, ArCH), 135.63 (absent, ArC),
136.38 (absent, ArC), 137.37 (absent, ArC), 141.54 (+ve, ArCH), 162.32 (absent,
C@O), 179.93 (absent, C@O); Anal. Calcd for C42H34N4O4: C, 76.58; H, 5.20; N,
8.51. Found C, 76.80; H, 5.38; N, 8.34. MS (FAB) 659 (M++1).
34. Roncero, C.; Duran, A. J. Bacteriol. 1985, 163, 1180.
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36. Evaluation of IC50: Ten serial dilutions of the compounds and the corresponding
solvent control were made with synthetic dextrose minimal medium (SDMM)
in duplicate in 96-well flat bottom transparent cell culture ELISA plate. Candida
cells which were already grown in culture medium for 24 h, at 30 °C were
resuspended in a 0.9% saline solution to give an optical density of 0.1 at 600 nm
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(OD600). The cells were then diluted 100 times in SDM medium. 100 ll of
diluted cells was added to each well except column 11, which was our media
control to monitor contamination during the study. Column 12 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 micro plate
reader. The percent fungal growth inhibition of the compounds were
determined with respect to positive control after subtracting the percent
inhibition by the solvent control. The IC50 values were determined by plotting
graph of percent inhibition versus concentration of the compound using
Graphpad Prism software.
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33. Experimental data for selected compounds: Compound 4f: Light brown solid,
Yield: 29%; mp 165 °C. IR (KBr, cmꢀ1): 1626 (C@O); UV (THF + HEPES buffer)
kmax (e
) 224 (17,320), 251 (22,620), 314 (18,160); 1H NMR (300 MHz, CDCl3): d
1.90 (m, 12H, 6 ꢁ CH2), 3.57–3.65 (m, 8H, 4 ꢁ CH2 of pyrrolidine), 4.11 (m, 4H,
2 ꢁ CH2), 7.24–7.33 (m, 6H, ArH), 8.08 (s, 2H, ArH), 8.35–8.39 (m, 2H, ArH); 13
C
37. Robert, J. F.; Valerie, L. H. Antimicrob. Agents Chemother. 1984, 26(5), 660.
38. ArgusLab, Thompson, M. A. Planaria Software LLC, Seattle, WA 98155.
NMR (normal/DEPT-135) (75 MHz, CDCl3): d 23.86 (ꢀve, CH2), 26.24 (ꢀve,