Indole-based novel small molecules for the modulation of bacterial signalling pathways

Article abstract of DOI:10.1039/c4ob02096k

Gram-negative bacteria such as Pseudomonas aeruginosa use N-acylated l-homoserine lactones (AHLs) as autoinducers (AIs) for quorum sensing (QS), a major regulatory and cell-to-cell communication system for social adaptation, virulence factor production, biofilm formation and antibiotic resistance. Some bacteria use indole moieties for intercellular signaling and as regulators of various bacterial phenotypes important for evading the innate host immune response and antimicrobial resistance. A range of natural and synthetic indole derivatives have been found to act as inhibitors of QS-dependent bacterial phenotypes, complementing the bactericidal ability of traditional antibiotics. In this work, various indole-based AHL mimics were designed and synthesized via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N,N′-dicyclohexylcarbodiimide (DCC) mediated coupling reactions of a variety of substituted or unsubstituted aminoindoles with different alkanoic acids. All synthesized compounds were tested for QS inhibition using a P. aeruginosa QS reporter strain by measuring the amount of green fluorescent protein (GFP) production. Docking studies were performed to examine their potential to bind and therefore inhibit the target QS receptor protein. The most potent compounds 11a, 11d and 16a showed 44 to 65% inhibition of QS activity at 250 μM concentration, and represent promising drug leads for the further development of anti-QS antimicrobial compounds.

Full text of DOI:10.1039/c4ob02096k

Organic &
Biomolecular Chemistry
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PAPER
Indole-based novel small molecules for the
modulation of bacterial signalling pathways†
Nripendra Nath Biswas,a Samuel K. Kutty,a Nicolas Barraud,b George M. Iskander,a
Renate Griffith,c Scott A. Rice,b,e Mark Willcox,d David StC. Blacka and
Naresh Kumar*a
Cite this: Org. Biomol. Chem., 2015,
13, 925
Gram-negative bacteria such as Pseudomonas aeruginosa use N-acylated L-homoserine lactones (AHLs)
as autoinducers (AIs) for quorum sensing (QS), a major regulatory and cell-to-cell communication system
for social adaptation, virulence factor production, biofilm formation and antibiotic resistance. Some bac-
teria use indole moieties for intercellular signaling and as regulators of various bacterial phenotypes
important for evading the innate host immune response and antimicrobial resistance. A range of natural
and synthetic indole derivatives have been found to act as inhibitors of QS-dependent bacterial pheno-
types, complementing the bactericidal ability of traditional antibiotics. In this work, various indole-based
AHL mimics were designed and synthesized via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC·HCl) and N,N’-dicyclohexylcarbodiimide (DCC) mediated coupling reactions of a
variety of substituted or unsubstituted aminoindoles with different alkanoic acids. All synthesized com-
pounds were tested for QS inhibition using a P. aeruginosa QS reporter strain by measuring the amount
of green fluorescent protein (GFP) production. Docking studies were performed to examine their potential
to bind and therefore inhibit the target QS receptor protein. The most potent compounds 11a, 11d and
16a showed 44 to 65% inhibition of QS activity at 250 µM concentration, and represent promising drug
leads for the further development of anti-QS antimicrobial compounds.
Received 1st October 2014,
Accepted 11th November 2014
DOI: 10.1039/c4ob02096k
www.rsc.org/obc
modification of existing drugs such as antifungal azoles, anti-
Introduction
bacterial β-lactams and quinolones.5 However, this strategy
Bacterial infection is a leading cause of hospitalization and merely delays the development of bacterial resistance, without
death throughout the world, and has primarily been treated total eradication of the infectious species. Basically, traditional
with antibiotics over the past half-century.1 However, history antibiotics ultimately kill or inhibit the growth of the bacteria.
has shown that the introduction of any novel antibiotic is They may exert their antimicrobial action by the disruption of
quickly followed by the evolution of clinically-significant resist- cell walls, inhibition of DNA or RNA synthesis, inhibition of
ant bacterial strains within a few years of introduction.2,3 The protein synthesis, inhibition of folate synthesis, or depolariz-
rapid increase of multidrug resistant strains worldwide ation of membrane potential.6 Since traditional antibiotics
coupled with the slowing pace of discovery of new antibiotics cause death of bacterial cells, this exerts selective pressure on
represent an urgent need to develop new and effective anti- bacteria and encourages the development and spread of anti-
bacterial therapies.4 One approach has involved the structural biotic drug resistance via different defensive phenotypes such
as biofilm formation.7 In this context, it is increasingly impor-
tant to understand bacterial virulence and survival mecha-
nisms in order to identify new therapeutic approaches to
combat bacterial infection.
aSchool of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia.
E-mail: n.kumar@unsw.edu.au; Fax: +61 2 9385 6141; Tel: +61 2 9385 4698
bCentre for Marine Bio-Innovation, School of Biotechnology and Biomolecular
Bacteria use various autoinducer (AI) chemical signals such
Sciences, UNSW Australia, Sydney, NSW 2052, Australia
as N-acylated L-homoserine lactones (AHLs) 1, 2, 3 (Fig. 1) to
monitor and coordinate their genome expression in a cell
density-dependent manner, which is a mechanism known as
quorum sensing (QS).8 This population-dependent regulatory
system plays an important role in the expression of genes and
phenotypes responsible for overcoming the host immune
response and regulating pathogenicity. Some typical QS con-
cSchool of Medical Sciences, UNSW Australia, Sydney, NSW 2052, Australia
dSchool of Optometry and Vision Science, UNSW Australia, Sydney, NSW 2052,
Australia
eThe Singapore Centre on Environmental Life Sciences Engineering and the School of
Biological Sciences, Nanyang Technological University, Singapore
†Electronic supplementary information (ESI) available: Bacterial cell growth
inhibition data for tested compounds and NMR spectra of synthesized com-
pounds. See DOI: 10.1039/c4ob02096k
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Fig. 1 Different types of natural N-acylated L-homoserine lactones.
Fig. 2 Indole derivatives that interfere with quorum sensing (QS).
enterohemorrhagic Escherichia coli (EHEC) (Fig. 2).29 The syn-
thetic indole derivative 7-fluoroindole 6 has also been reported
to reduce biofilm formation by four-fold and hemolytic activity
by 14-fold at a concentration of 1 mM in P. aeruginosa, and
also suppressed swarming motility, protease activity, extra-
cellular polymeric matrix production and QS-regulated viru-
lence factors in this species.30 Recently, it has been reported
that the Gram-positive bacteria Staphylococcus intermedius and
Staphylococcus delphini excreted an indole-based molecule
N-[2-(1H-indol-3-yl)ethyl]-urea 7, termed yayurea A, that inter-
fered with the QS system of a range of Gram-negative
bacteria.31
Inspired by these recent studies, we sought to examine the
LAS-dependent QS modulatory activity of novel indole-based
small molecules. We describe herein the design and synthesis
of AHL mimics containing indole-based, non-lactone head
groups and substituted or unsubstituted aliphatic tail groups.
The synthesized compounds were tested for QS inhibitory
activity in the P. aeruginosa MH602 lasB reporter strain.
Additionally, docking studies were performed to examine their
binding potential with the target receptor.
trolled behaviors include the control of biofilm formation,
production of virulence and defense factors, conjugation, spor-
ulation and swarming motility.8,9 The QS system of Pseudomo-
nas aeruginosa, a causative agent of nosocomial infections and
infections of cystic fibrosis patients, has been widely studied
and it is clear that QS controls the expression of a broad range
of virulence determinants such as exotoxin, pyocyanin, pyover-
din etc.10 Importantly, QS and the phenotypes controlled by
this pathway are not essential genes and thus the inhibition of
QS is not lethal. Hence, blocking or interfering with the com-
munication between bacteria to prevent the production of viru-
lence factors or biofilm formation represents an attractive
alternative strategy to combat bacterial infections while redu-
cing the likelihood of engendering bacterial resistance.11,12
Numerous attempts have been directed towards producing
synthetic analogues of QS signaling molecules as antagonists
of bacterial communication systems. In P. aeruginosa, synthetic
AHLs, which mimic the structures of natural AHL molecules
can compete with their binding to the LasR receptor of the
LAS QS system, thereby inhibiting signal transduction and
gene transcription processes required for biofilm formation
and virulence expression.13 Research groups have previously
studied the effect of various structural modifications of natural
AHLs on receptor binding and AHL inhibitory activity, includ-
ing variations in the length and substitution pattern of the
acyl chain, as well as in the nature of the substituents on the
lactone ring.14–21 However, homoserine lactones are prone to
non-enzymatic hydrolysis at physiological pH and are also
readily degraded by mammalian lactonases, producing ring-
opened products that are inactive against QS.22 While various
small heterocycles and natural products have also been investi-
gated for QS inhibitory activity over the past few decades, the
application of indole heterocycles for this purpose has been
comparatively less explored.
Results
Synthetic schemes
The synthesis of the indole-based AHL mimics involved the
coupling of a variety of substituted or unsubstituted amino-
indoles with different alkanoic acids. The indole amines chosen
in the study were 3-amino, 5-amino and 7-amino indole deriva-
tives. Additionally, various alkyl chains ranging from 6 to 12
carbon atoms in length and mimicking natural autoinducer
chains, such as decyl, hexyl or phenylbutyl were explored.
To synthesize AHL mimics based on 3-amino-2-phenyl-
indole, the commercially available 2-phenylindole 8 was first
converted to 3-nitroso-2-phenylindole 9 by nitrosation, fol-
lowed by reduction to 3-amino-2-phenylindole 10 using hydra-
Indole is an intercellular signaling molecule employed by
diverse bacteria that regulates various bacterial behaviors in a
QS-dependent fashion, including antibiotic resistance, viru-
lence and biofilm formation.23–27 Further, bacterial oxygenases
can readily oxidize indole into various metabolites, such as
isatin and isoindigo, that have also been shown to play a role
in the control of biofilm formation via QS mechanisms.28
Consequently, small molecules based on the indole scaffold
could potentially be developed as inhibitors of bacterial QS
systems. Indole-derived plant secondary metabolites such as
indol-3-yl-acetonitrile (IAN) 4 and indole-3-carboxaldehyde
(I3CA) 5 were found to be active against biofilm formation in
zine with
a
palladium catalyst.32,33 EDC·HCl mediated
coupling of indole amine 10 with carboxylic acids at room
temperature yielded the targeted amides 11a–e in 25–82%
yield.34 Meanwhile, for the synthesis of the 3-oxo-substituted
amides 14a–c, the desired 2,2-dimethyl-1,3-dioxane-4,6-dione
(Meldrum’s acid) conjugate 13 was obtained by the coupling
reaction between Meldrum’s acid 12 and alkanoic acids using
DCC. Compound 13 was directly used for the next step and
was reacted with 3-amino-2-phenylindole 10 in DMF to give
the 3-oxo-substituted amides 14a–c in 33–39% (Scheme 1).34
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Scheme 2 Synthesis of AHL mimics based on 5-amino-2-methylindole.
Scheme 1 Synthesis of AHL mimics based on 3-amino-2-phenylindole.
For the synthesis of AHL mimics based on 5-amino-2-
methylindole, the commercially available 5-amino-2-methylin-
dole 15 was used as the starting material. Using the conditions
described above, the targeted amides 16a–e and 3-oxo-substi-
tuted amides 17a–c were synthesized in 46–63% and 20–30%
yields, respectively (Scheme 2).
For the synthesis of AHL mimics bearing the 7-aminoindole
scaffold, the commercially available 7-nitroindole 18 was first
reduced to 7-aminoindole 19 using hydrazine with a palladium
catalyst.33 Using similar amide coupling reactions, the targeted
amide compounds 20a–c and 3-oxo substituted amides 21a–c
were synthesized in 39–92% and 19–22% yields, respectively
(Scheme 3).
Scheme 3 Synthesis of AHL mimics based on 7-aminoindole.
Quorum sensing inhibition assay
In this assay, P. aeruginosa MH602 cultures were incubated
with various concentrations of the synthesized compounds at
37 °C. The fluorescence of GFP at λ = 535 nm and the optical
density at 600 nm (OD) of the cultures were recorded every
30 min. The percentage QS inhibition of the compounds was
calculated as the percentage difference of GFP intensity
between the sample and the control at the time point when
the fluorescence reached its maximum value in the control.
The assay was performed using the P. aeruginosa MH602 lasB
reporter strain (PlasB::gfp(ASV)) following the protocol develo-
ped by Hentzer et al.35 The production of AHL signals by this
reporter strain leads to an increase in unstable green fluo-
rescent protein (GFP-ASV) production as a function of an
active QS system. Compounds that inhibit bacterial QS systems
would be expected to reduce the expression of GFP in the
reporter strain.
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Table 1 Percentage inhibition of QS activity by the indole-based AHL
mimics as determined by the reduction of GFP fluorescence at λ =
535 nm in the P. aeruginosa MH602 lasB reporter strain (PlasB::gfp(ASV))
Concentrations (µM)
Fig. 3 Furanone 30 (control).
Entry
250
125
62.5
31.3
11a
11b
11c
11d
11e
14a
14b
14c
16a
16b
16c
16d
16e
17a
17b
17c
20a
20b
20c
21a
21b
21c
22
44.1 3.9a
37.9 4.3a
31.2 3.7a
65.0 3.9a
40.5 0.5a
7.5 0.8a
22.2 1.7a
9.9 3.5a
45.0 2.5a
31.8 2.7a
20.5 5.1a
20.9 3.2a
12.8 0.5a
14.2 2.1a
9.1 3.5a
30.0 3.5a
NA
31.8 4.7
25.7 2.5
22.2 2.2
54.2 2.9
16.6 5.1
NA
18.4 4.4
18.5 3.4
14.2 4.9
39.3 6.5
14.3 3.5
NA
NA
NA
19.8 4.0
NA
NA
NA
NA
NA
NA
9.0 7.6
NA
NA
NA
NA
5.5 7.3
4.3 1.9
NA
NA
2.6 0.6
NA
NA
NA
7.6 1.3
NA
NA
NA
NA
NA
NA
5.6 2.5
NA
NA
NA
NA
4.1 1.8
NA
28.9 4.4
17.3 1.9
10.0 2.5
9.2 5.1
1.4 0.2
NA
NA
14.0 6.4
NA
NA
NA
NA
NA
Fig. 4 QS inhibition assay for compound 11d in the P. aeruginosa
MH602 lasB reporter strain (PlasB::gfp(ASV)). (a) GFP fluorescence as a
function of time. (b) OD as a function of time.
NA
NA
NA
The optical density (OD) of the cultures was utilized as a
measure of bacterial cell growth. A well-known QS inhibitor,
furanone 30, 22 (Fig. 3) was used as a positive control to vali-
date the assay protocol. Data for the most potent compound
11d are presented in Fig. 4.
The results showed that the synthesized compounds exhibi-
ted promising QS inhibitory activity as measured by the
reduction in GFP fluorescence, with the most potent com-
pound 11d inhibiting QS activity by 65% at 250 µM (Table 1).
The GFP inhibitory activities of the tested compounds were
found to be concentration-dependent. Additionally, the posi-
tive control compound furanone 30 reduced bacterial cell
growth by 32% at 250 µM concentration, whereas the syn-
thesized compounds showed less than 15% inhibition of bac-
terial cell growth at the same concentration (ESI†).
31.3 4.3a
12.5 3.6a
89.7 4.6b
NA
NA
41.4 2.7
NA
NA
26.0 5.2
9.0 1.6
64.2 3.5
a Growth inhibition less than 15%. b Growth inhibition greater than
30%; standard deviation of the mean from at least two independent
experiments. In each independent experiment, compounds were tested
in duplicate. NA = no activity.
Cys79 respectively played an important role in the binding of
the compounds to the LasR receptor. The indole rings and
phenyl-substituted tails of the tested compounds were able to
form multiple hydrophobic interactions that led to high
docking scores, which did not necessarily relate to activity.
Therefore, prudent analysis of docking scores and interactions
was required to understand the docking results with respect to
activity. It was observed that the orientation of the docked
ligand is crucial for inhibitory activity and an orientation
similar to the natural ligand OdDHL in the receptor pocket
was observed for synthesized compounds with high activity.
The amino acid sequence of LasR ligand binding region
(36–129), the docking poses of OdDHL and of the most potent
compound 11d are shown in Fig. 5(a–c) respectively.
Docking studies
In order to understand the nature of the interaction between
the novel indole-based AHL mimics and the QS receptor,
docking studies were performed. The crystal structure of the
QS signal receptor protein LasR with the agonist N-(3-oxodo-
decanoyl) L-homoserine lactone (OdDHL, 3c) was used for this
study (PDB code, 2UV0, resolution 1.8 Å).36 The GOLD algor-
ithm was used via the Accelrys Discovery Studio interface to
examine the ability of the compounds to bind to the receptor
protein LasR. The receptor agonist OdDHL was docked back
Discussion
into the protein as a control in order to determine the Bacteria communicate with each other using different autoin-
reliability of the docking method. The docking runs were ana- ducer molecules, such as AHLs, to regulate virulence factor
lyzed for the predicted binding interactions, including hydro- production, biofilm formation and antibiotic resistance in a
gen bonding, electrostatic and hydrophobic interactions, population-dependent manner. Many bacteria such as E. coli
between the compounds and the LasR receptor in the best use indole as an intercellular signaling molecule for QS-depen-
scoring pose and are presented in Table 2. The docking results dent phenotypes. Thus, AHL mimics bearing an indole moiety
suggested that hydrogen bonding and hydrophobic inter- as the head group and an acyl moiety as a tail have the poten-
actions to amino acid residues such as Thr75, Tyr56, Ser129, tial to inhibit QS. These synthetic AHL compounds with
Asp73, Val76, Gly126, Ala50, Leu40, Tyr47, Leu36, Ala127, and similar structures to natural AHL molecules can compete with
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Table 2 Docking of the indole-based AHL mimics to the LasR receptor protein
Docking
score
Unfavorable
interactions
Entry
H bonds
Π and hydrophobic interactions
11a
11b
52.20
50.85
Arg61, Thr75, Ser129
Tyr64
Tyr64, Tyr56, Val76, Cys79, Leu125, Ala127
Tyr47, Ala105, Trp88, Tyr93, Phe102, Val76, Cys79, Leu125,
Ala127, Ala50, Ile52
Thr75, Tyr56
—
11c
11d
11e
14a
55.43
61.58
60.26
38.55
—
Ile52, Ala70, Ala50, Val76, Cys79, Ala127
—
—
—
—
Thr75, Tyr56, Ser129
—
Tyr56, Ser129,
Val76, Gly126, Ala50, Leu40, Tyr47, Leu36, Ala127, Cys79
Phe101, Tyr56, Val76, Cys79, Leu125, Ala127, Ala50, Ile52
Try47, Trp88, Tyr93, Phe102, Val76, Ala127, Ala50, Ile52,
Ala70
14b
14c
60.25
58.93
Cys79, Ser129, Asp73
Tyr64
Tyr64, Trp88, Leu40, Leu125, Leu110, Leu36, Ala70
Trp88, Tyr47, Phe101, Ile52, Ala70, Ala50, Val76, Cys79,
Leu125, Ala127, Ala105, Leu110
—
Asp73, Thr75
16a
16b
16c
16d
16e
17a
17b
17c
20a
20b
20c
21a
21b
21c
OdDHL (3c)
60.97
68.13
71.02
60.87
73.26
70.03
67.86
74.63
58.48
52.59
62.48
68.34
62.25
71.40
61.70
Tyr56, Ser129, Leu110, Asp73
Arg61, Tyr56, Ser129, Trp88
Tyr56, Trp88
Tyr56, Trp88
Tyr56
Arg61, Tyr56, Trp88
—
Arg61, Tyr56, Trp88
Tyr56, Ser129, Asp73
—
Tyr56
Tyr56, Asp73
Trp88, Phe101, Ala127, Cys79, Leu125, Ala105, Leu110
Trp88, Phe101, Ala127, Val76, Cys79, Leu125, Leu36, Leu110
Tyr56, Ala105, Leu110, Leu40, Ala50, Ala127
Tyr56, Ala50, Leu40, Leu36, Leu110
Tyr56, Phe101, Leu36, Ala50, Ala70, Val76, Leu40, Leu125
Tyr56, Val76, Cys79, Leu125, Ala105, Leu110
Tyr56, Ala127, Leu125, Leu36
Phe101, Ala105, Leu110, Val76, Cys79, Leu125, Ala127
Trp88, Phe101, Leu40, Leu125, Ala105, Leu110
Leu110, Tyr56, Trp88, Ala50, Val76, Cys79, Ala127
Tyr56, Ala105, Leu110, Leu40, Ala50, Ala127
Trp88, Phe101, Val76, Cys79, Leu125, Ala105, Leu110
Ala105, Leu110, Trp88, Leu40, Ala50, Val76, Cys79, Ala127
Tyr47, Trp88, Phe101, Ala105, Leu110, Leu40, Ala50, Val76
Trp88
Tyr56
—
—
—
Arg 61
—
Arg61
—
Tyr64
Thr75
—
Asp73
—
Tyr64
Tyr64
Thr115, Ser129
Tyr56, Ser129, Asp73
Asp73, Tyr56, Trp60,
Arg61, Ser129, Tyr93
virulence expression and biofilm formation. In this work, 22
indole-based AHL mimics were synthesized using facile coup-
ling reactions as shown in Schemes 1–3. The amide series
compounds generally resulted in good yields and the 3-oxo
analogues generally gave moderate yields. The synthesized
compounds were analyzed by docking studies for their inter-
action with the LasR protein and further tested for QS inhibi-
tory activity against the P. aeruginosa MH602 lasB reporter
strain.
The results from the QS inhibition assay (Table 1) indicated
that for the 3-amino-2-phenylindole-based compounds, the
amide derivatives 11a–e were more active compared to the
3-oxo-amide compounds 14a–c. Furthermore, compound 11d
bearing a six-carbon acyl chain was the most active compound
in the series. In contrast, AHL mimics (11a–b and 14a–c) con-
taining longer hydrophobic chains showed lower activity. This
is consistent with the results of the molecular docking study,
which showed compound 11d having the highest docking
score of 61.58 out of all the 3-amino-2-phenyl-based com-
pounds (Table 2). Moreover, compound 11d was predicted to
form both hydrogen bonding and hydrophobic interactions
with LasR at the binding site. Additionally, the orientation of
the docked molecule appears to be crucial for the activity of
compounds 11a–e, as only 11a and 11d docked in a similar
head to tail orientation compared to the natural agonist
OdDHL, and also showed better QS inhibitory activity in the
QS assay. Even though the compounds with opposite orien-
Fig. 5 (a) Amino acid sequence for the LasR ligand binding region
(amino acids color coded according to the nature of interaction).
(b) Schematic representation of the natural ligand OdDHL (3c).
(c) Docked poses of natural ligand OdDHL (left) and Compound 11d
(right) with LasR. Selected interactions shown as dashed lines (green: H
bonds; pink: hydrophobic interactions; orange and cream: interaction
involving π systems).
their binding to the LasR protein. Binding of these artificial tation may show higher docking scores, this orientation gener-
signaling molecules to the LuxR receptor prevents signal trans- ally does not appear to facilitate QS inhibitory activity against
duction from leading to gene transcription, thereby inhibiting P. aeuroginosa in the series.
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For the 5-amino-2-methylindole-based compounds, the reported as QS inhibitors38 (one representative and the most
amide analogues 16a–e again showed better activity compared potent QS inhibitory compound is 24 (Fig. 6). However, to
to their 3-oxo amide 17a–c counterparts. However, in contrast date, this study is the first attempt to make AHL mimics
to the previous results, 5-amino-2-methylindole-based com- bearing indole as a head group as QS inhibitors. The current
pounds 16a and 16b containing longer acyl chains were the work may provide an opportunity to develop new QS based
most active, suggesting that more hydrophobic acyl chains antimicrobial therapeutics without affecting normal bacterial
could be beneficial for indole mimics lacking a bulky substitu- growth and thus avoiding resistance to antibiotics.
ent at the 2-position. In the docked poses, the orientation of
the head and tail of 16 and 17 in a similar fashion to OdDHL,
as well as the orientation of the amide bond between indole
and the linker and its H-bonding propensity increases the
Conclusion
docking score and supports the observed activity. In the case In this work, twenty two different AHL mimics based on
of 7-aminoindole, a drastic loss in activity was observed for the indoles have been designed, synthesized, biologically evalu-
amide analogues 20a–c, while only minimal activity was seen ated, and docked into the LasR receptor to study the possible
for the 3-oxo amide analogues 21a–c. Although the docking interactions such as hydrogen bonding, electrostatic and
scores were higher for these series of compounds, it was hydrophobic interactions. Biological assays (QS inhibition)
observed that the proximity of the two ‘NH’ groups the indole were performed using a P. aeruginosa MH602 reporter strain.
‘NH’ and the 7-substituted ‘NH’, led to unfavorable clashes N-(2-Phenyl-1H-indol-3-yl)hexanamide 11d showed 65% QS
with the receptor protein. These indicate that 7-amino substi- inhibitory activity at 250 µM concentration, which makes it the
tution is not ideal for QS inhibitory activity.
most potent compound studied. This is also supported by the
Comparing across the three series of compounds, AHL results of the molecular docking study, where compound 11d
amide mimics based on the 3-amino-2-phenylindole scaffold showed the highest docking score of the 3-amino-2-phenyl-
were more active than those based on 5-amino-2-methyl or based compounds (Table 2), and was predicted to form both
7-amino indoles. A trend between activity and docking score hydrogen bonding and hydrophobic interactions with LasR at
could be observed for the 3-amino-2-phenyl compounds, but the binding site. An optimized chain length for AHLs bearing
not for the other two series. For the 3-oxo mimics, the indole as a head group was found to be 6 carbons. All syn-
5-amino-2-methyl compounds 17a–c and the 7-aminoindole thesized compounds were found to be non-toxic to bacterial
compound 21b were more active than the 3-amino-2-phenyl- cells (≤15% cell death) which may overcome the main draw-
compounds 14a–c. Thus, in the case of 3-oxo compounds, less backs of traditional antibiotics (antibiotic resistance). This
bulky or no substitution was preferable for activity. Overall, study could further assist in the development of novel indole-
AHL mimics based on 3-amino-2-phenylindole and 5-amino-2- based antibacterials (QS inhibitors) without killing bacteria
methylindole bearing different acyl or aryl tail groups were and thereby not enhancing resistance.
found to be moderate QS inhibitors.
The Blackwell group synthesized the AHL mimic 23 (Fig. 6)
with an indole tail and in qualitative experiments on engin-
Materials and methods
General chemistry details
eered P. aeruginosa at 50 µM concentration found significant
GFP inhibition followed by biofilm reduction.14,37 A recently
isolated indole based natural compound N-[2-(1H-indol-3-yl)- All chemical reagents were purchased from commercial
ethyl]-urea 7, or yayurea A from the Gram-positive bacteria sources (Alfa-Aesar and Sigma Aldrich) and used without
Staphylococcus intermedius and Staphylococcus delphini also further purification. Solvents were sourced from commercial
interferes with the QS mediated pyocyanin production in sources and used as obtained. Reactions were performed using
P. aeruginosa, but this effect was found to be associated with oven-dried glassware under an atmosphere of nitrogen and in
about 50% bacterial cell killing compared to the QS inhibitory anhydrous conditions (as required). Room temperature refers
effect.31 Some attempts to modify indoles have been made by to the ambient temperature (22–24 °C). Yields refer to
substitution at different positions of the indole ring and chromatographically and spectroscopically pure compounds
unless otherwise stated. Reactions were monitored by thin
layer chromatography (TLC) precoated with Merck silica gel 60
F254. Visualization was performed by the quenching of short
or long wavelength UV fluorescence or by staining with potass-
ium permanganate or ninhydrin solution. Flash chromato-
graphy was carried out using Grace Davison LC60A 6–35 µm
silica gel. Preparative thin layer chromatography was carried
out on 3 × 200 × 200 mm glass plates coated with Merck
60GF254 silica gel. Infrared spectra were recorded using a Cary
630 FTIR spectrophotometer. Ultraviolet spectra were
Fig. 6 Indole-based representative QS inhibitors.
930 | Org. Biomol. Chem., 2015, 13, 925–937
measured using a Cary 100 Bio UV-visible spectrophotometer
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in the designated solvents and data reported as wavelength (λ) dichloromethane (15 ml) was added dodecanoic acid (0.15 g,
in nm and absorption coefficient (ε) in M−1 cm−1. High-resolu- 0.75 mmol) and EDC·HCl (0.23 g, 1.20 mmol). The reaction
tion mass spectrometry was performed by the Bioanalytical mixture was stirred at room temperature for 30 h and then
Mass Spectrometry facility, UNSW. Melting points were further diluted with dichloromethane (20 ml). The mixture
obtained using Mel-Temp melting point apparatus and are was washed with water (40 ml × 3), saturated potassium bi-
uncorrected. Proton and Carbon NMR was recorded in desig- sulfate solution, sodium bicarbonate solution and then brine,
nated solvents using Bruker DPX 300 or a Bruker Avance 400 dried over sodium sulfate and the solvent was evaporated
spectrometer as designated. Chemical shifts (δ) are quoted in in vacuo. Purification of the residue by silica gel chromato-
parts per million (ppm), to the nearest 0.01 ppm and intern- graphy afforded the title compound as green solid (0.10 g,
1
ally referenced relative to the solvent nuclei. H NMR spectral 37%); mp 92–94 °C; UV (MeOH): λmax 305 nm (ε 20 231 M−1
data are reported as follows: [chemical shift in ppm; multi- cm−1), 236 (21 910), 205 (29 213); IR (neat): νmax 3315, 3215,
plicity in br, broad; s, singlet; d, doublet; t, triplet; q, quartet; 3054, 2917, 2847, 2338, 2106, 1881, 1729, 1641, 1456, 1258,
quint, quintet; sext, sextet; sept, septet; m, multiplet; or as a 1070, 1020, 798 cm−1
;
1H NMR (300 MHz, DMSO-d6):
combination of these (e.g. dd, dt etc.)]; coupling constant (J) in δ 0.84–0.88 (m, 3H, CH3), 1.27–1.33 (m, 16H, CH2), 1.65 (t, J =
hertz, integration, proton count and assignment. 6.9 Hz, 2H, CH2), 2.37 (t, J = 7.1 Hz, 2H, CH2), 6.97–7.79 (m,
3-Nitroso-2-phenyl-1H-indole (9). A solution of 2-phenyl-1- 9H, ArH), 9.36 (s, 1H, NH), 11.36 (s, 1H, indole NH); 13C NMR
H-indole (5.00 g, 25.87 mmol) in acetic acid (50 ml) was cooled (75 MHz, DMSO-d6): δ 14.4 (CH3), 22.6 (CH2), 26.0 (CH2), 29.2
to 18 °C and a solution of sodium nitrite (1.60 g, 23.19 mmol) (2 × CH2), 29.3 (2 × CH2), 29.5 (2 × CH2), 31.8 (CH2), 36.0
in water (3 ml) was added drop-wise, while keeping the temp- (CH2), 111.3 (ArC), 111.8 (2 × ArCH), 119.5 (ArCH), 122.4
erature of the reaction mixture at 20 °C. The resulting reaction (ArCH), 126.7 (ArC), 127.2 (ArCH), 127.2 (ArCH), 127.8 (ArCH),
mixture was stirred at room temperature for 30 min and then 129.0 (ArCH), 129.0 (ArCH), 131.8 (ArC), 132.1 (ArC), 135.0
diluted with ice water (250 ml). The precipitate was filtered (ArC), 173.1 (CO); HRMS (+ESI): Found m/z 413.2563 [M + Na]+,
and the resulting yellow solid was washed with water and then C26H34N2ONa requires 413.2569.
with methanol to yield the desired product as yellow solid
N-(2-Phenyl-1H-indol-3-yl)decanamide (11b). To a solution
(4.50 g, 78%); mp 263–265 °C; UV (MeOH): λmax 254 nm of 2-phenyl-1H-indol-3-amine (0.22 g, 1.04 mmol) in dry
(ε 25 231 M−1 cm−1), 210 (29 910); IR (neat): νmax 3400, 2845, dichloromethane (15 ml) was added decanoic acid (0.15 g,
1
1610, 1548, 1435 cm−1; H NMR (300 MHz, DMSO-d6): δ 7.33 0.80 mmol) and EDC·HCl (0.21 g, 1.12 mmol). The reaction
(t, J = 7.4 Hz, 1H, ArH), 7.45–7.57 (m, 5H, ArH), 8.12 (d, J = 7.4, mixture was stirred at room temperature for 23 h and then
1H, ArH), 8.25 (dd, J = 7.4, 1.7 Hz, 2H, ArH), 13.79 (s, 1H, NH); further diluted with dichloromethane (20 ml). The mixture
13C NMR (75 MHz, DMSO-d6): δ 127.7 (3 × ArCH), 129.0 (3 × was washed with water (40 ml × 3), saturated potassium bisul-
ArC), 129.9 (3 × ArCH), 131.4 (3 × ArCH); HRMS (+ESI): Found fate solution, sodium bicarbonate solution and then brine,
m/z 223.0862, [M + H]+, C14H11N2O requires 223.0863.
dried over sodium sulfate and the solvent was evaporated
2-Phenyl-1H-indol-3-amine (10). To a boiling solution of in vacuo. Purification of the residue by silica gel chromato-
3-nitroso-2-phenyl-1H-indole (1.00 g, 4.50 mmol) in absolute graphy afforded the title compound as green solid (0.11 g,
ethanol (50 ml) was added 10% Pd/C (0.10 g), followed by the 29%); mp 64 °C; UV (MeOH): λmax 305 nm (ε 24 179 M−1 cm−1),
addition of hydrazine hydrate (2.25 g, 45.00 mmol) drop-wise 236 (35 707); IR (neat): νmax 3244, 3054, 2918, 2849,2339, 2100,
over a period of 15 min. The mixture was heated under reflux 1874, 1636, 1487, 1453, 1337, 1238, 1182, 1106, 918, 737,
1
for further 2 h, followed by filtration through a filter column of 690 cm−1; H NMR (300 MHz, DMSO-d6): δ 0.85–0.89 (m, 3H,
silica. The ethanol was removed by rotary evaporation and the CH3), 1.27–1.33 (m, 12H, CH2), 1.65 (t, J = 7.2 Hz, 2H, CH2),
residue was dissolved in dichloromethane (50 ml). The organic 2.36 (t, J = 7.3 Hz, 2H, CH2), 6.97–7.78 (m, 9H, ArH), 9.35 (s,
layer was then washed with water (50 ml × 3) and then with 1H, NH), 11.35 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO-
brine, dried over sodium sulfate and evaporated in vacuo to d6): δ 14.4 (CH3), 22.6 (CH2), 26.0 (CH2), 29.2 (CH2), 29.2 (CH2),
yield the title compound an white solid (0.65 g, 69%); mp 29.3 (CH2), 29.5 (CH2), 31.8 (CH2), 36.0 (CH2), 111.3 (ArC),
119 °C; UV (MeOH): λmax 254 nm (ε 25 231 M−1 cm−1), 205 111.8 (ArCH), 118.8 (ArCH), 119.5 (ArCH), 122.4 (ArCH), 126.7
(27 310); IR (neat): νmax 3400, 2850, 1610 cm−1 1H NMR (ArC), 127.2 (ArCH), 127.2 (ArCH), 127.8 (ArCH), 129.0 (ArCH),
;
(300 MHz, DMSO-d6): δ 4.46 (s, NH2), 6.90 (t, J = 7.5 Hz, 2H, 129.0 (ArCH), 131.8 (ArC), 132.1 (ArC), 135.0 (ArC), 173.1 (CO);
ArH), 7.04 (t, J = 7.5 Hz, 2H, ArH), 7.16–7.25 (m, 2H, ArH), 7.43 HRMS (+ESI): Found m/z 385.2249 [M + Na]+, C24H30N2ONa
(t, J = 7.4 Hz, 2H, ArH), 7.65 (d, J = 7.6 Hz, 2H, ArH), 7.79 (d, requires 385.2250.
J = 7.4 Hz, 2H, ArH), 10.47 (s, NH); 13C NMR (75 MHz, DMSO-
4-Phenyl-N-(2-phenyl-1H-indol-3-yl)butanamide (11c). To a
d6): δ 111.2 (ArCH), 117.8 (ArCH), 118.7 (ArC), 119.3 (ArCH), solution of 2-phenyl-1H-indol-3-amine (0.23 g, 1.10 mmol) in
122.4 (ArCH), 123.4 (ArCH), 123.4 (ArCH), 125.4 (ArCH), 125.5 dry dichloromethane (15 ml) was added 4-phenyl butyric acid
(2 × ArC), 125.5, 129.1 (2 × ArCH), 134.0 (ArC), 135.4 (ArC); (0.20 g, 1.20 mmol) and EDC·HCl (0.34 g, 1.76 mmol). The
HRMS (+ESI): Found m/z 209.1069 [M + H]+, C14H13N2 requires reaction mixture was stirred at room temperature for 24 h and
209.1070.
then further diluted with dichloromethane (20 ml). The
N-(2-Phenyl-1H-indol-3-yl)dodecanamide (11a). To a solu- mixture was washed with water (40 ml × 3), saturated potass-
tion of 2-phenyl-1H-indol-3-amine (0.17 g, 0.83 mmol) in dry ium bisulfate solution, sodium bicarbonate solution and then
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1
brine, dried over sodium sulfate and the solvent was evapor- 685 cm−1; H NMR (300 MHz, DMSO-d6): δ 2.78 (t, J = 7.4 Hz,
ated in vacuo. Purification of the residue by silica gel chrom- 2H, CH2), 3.09 (t, J = 7.4 Hz, 2H, CH2), 6.96–7.71 (m, 14H,
atography afforded the title compound as a greenish solid ArH), 9.41 (s, 1H, NH), 10.84 (s, 1H, indole NH), 11.33 (s, 1H,
(0.10 g, 25%); mp 139 °C; UV (MeOH): λmax 305 nm (ε 16 014 indole NH); 13C NMR (75 MHz, DMSO-d6): δ 21.6 (CH2), 36.7
M−1 cm−1), 236 (17 334), 205 (28 717); IR (neat): νmax 3251, (CH2), 111.3 (ArC), 111.8 (2 × ArCH), 114.3 (ArC), 118.7 (ArCH),
3023, 2921, 2340, 2110, 1880, 1637, 1490, 1451, 1336, 1239, 118.9 (ArCH), 119.0 (ArCH), 119.4 (ArCH), 121.4 (ArCH), 122.3
1143, 1027, 910, 824, 737, 692 cm−1 1H NMR (300 MHz, (ArCH), 122.8 (ArCH), 126.6 (ArC), 127.1 (2 × ArCH), 127.6
;
DMSO-d6): δ 1.90–2.00 (m, 2H, CH2), 2.40 (t, J = 7.9 Hz, 2H, (ArC), 127.8 (ArCH), 129.1 (2 × ArCH), 131.6 (ArC), 132.1 (ArC),
CH2), 2.68 (t, J = 7.8 Hz, 2H, CH2), 6.97–7.79 (m, 14H, ArH), 135.0 (ArC), 136.8 (2 × ArC), 172.8 (CO); HRMS (+ESI): Found
9.40 (s, 1H, NH), 11.36 (s, 1H, indole NH); 13C NMR (75 MHz, m/z 402.1576 [M + Na]+, C25H21N3ONa requires 402.1579.
DMSO-d6): δ 27.8 (CH2), 35.2 (CH2), 35.4 (CH2), 111.2 (ArC),
3-Oxo-N-(2-phenyl-1H-indol-3-yl)dodecanamide (14a). To a
111.8 (ArCH), 118.8 (ArCH), 119.5 (ArCH), 122.4 (ArCH), 126.3 solution of decanoic acid (0.25 g, 1.44 mmol) in dry dichloro-
(ArCH), 126.7 (ArC), 127.2 (ArCH), 127.2 (ArCH) 127.9 (ArCH), methane (20 ml) was added DCC (0.34 g, 1.63 mmol),
128.5 (ArCH), 128.8 (ArCH), 128.8 (ArCH), 128.8 (ArCH), 129.1 4-dimethylaminopyridine (DMAP) (0.19 g, 1.54 mmol) and
(ArCH), 129.1 (ArCH), 131.9 (ArC), 132.2 (ArC), 135.0 (ArC), Meldrum’s acid (0.21 g, 1.44 mmol) under a N2 atmosphere.
142.3 (ArC), 172.8 (CO); HRMS (+ESI): Found m/z 377.1625 The reaction mixture was stirred at room temperature for 24 h
[M + Na]+, C24H22N2ONa requires 377.1629.
and then filtered through filter paper. The filtrate was evapor-
N-(2-Phenyl-1H-indol-3-yl)hexanamide (11d). To a solution ated by rotary evaporation and the residue was dissolved in di-
of 2-phenyl-1H-indol-3-amine (0.16 g, 0.77 mmol) in dry methylformamide (DMF) (7 ml). 2-Phenyl-1H-indol-3-amine
dichloromethane (15 ml) was added hexanoic acid (0.099 g, (0.20 g, 0.96 mmol) was added and the mixture was heated at
0.85 mmol) and EDC·HCl (0.24 g, 1.23 mmol). The reaction 80 °C for 15 h. After cooling to room temperature, water
mixture was stirred at room temperature for 21 h and then (20 ml) was added and the mixture was extracted with ethyl
further diluted with dichloromethane (20 ml). The mixture acetate (30 ml × 3), washed with brine, dried over sodium
was washed with water (40 ml × 3), saturated potassium bisul- sulfate and the solvent was evaporated in vacuo. Purification of
fate solution, sodium bicarbonate solution and then brine, the residue by silica gel chromatography afforded the title
dried over sodium sulfate and the solvent was evaporated compound as a green solid (0.15 g, 39%); mp 79–81 °C; UV
in vacuo. Purification of the residue by silica gel chromato- (MeOH): λmax 279 nm (ε 38 836 M−1 cm−1); IR (neat): νmax
graphy afforded the title compound as an off-white solid 3250, 3054, 2920, 2848, 2318, 2106, 1882, 1713, 1625, 1568,
(0.19 g, 82%); mp. 120–121 °C; UV (MeOH): λmax 305 nm 1522, 1452, 1310, 1239, 1084, 918, 840, 738, 690 cm−1
;
1H
(ε 25 186 M−1 cm−1), 236 (27 269), 205 (37 319); IR (neat): νmax NMR (300 MHz, DMSO-d6):
δ
0.83–0.88 (m, 3H, CH3),
3235, 3055, 2925, 2855, 2340, 2108, 1883, 1638, 1506, 1453, 1341, 1.25–1.73 (m, 16H, CH2), 2.59 (t, J = 7.4 Hz, 2H, CH2), 3.59 (s,
1
1240, 1189, 969, 916, 842, 738, 690 cm−1; H NMR (300 MHz, 2H, CH2), 7.01–7.82 (m, 9H, ArH), 9.61 (s, 1H, NH), 11.40 (s,
DMSO-d6): δ 0.88–0.93 (m, 3H, CH3), 1.32–1.38 (m, 2H, CH2), 1H, indole NH); 13C NMR (75 MHz, DMSO-d6): δ 14.4 (CH3),
1.65 (t, J = 7.1 Hz, 2H, CH2), 2.36 (t, J = 7.2 Hz, 2H, CH2), 23.5 (CH2), 24.9 (CH2), 25.8 (CH2), 29.0 (CH2), 29.2 (CH2), 29.4
6.97–7.78 (m, 9H, ArH), 9.35 (s, 1H, NH), 11.35 (s, 1H, indole (CH2), 33.8 (CH2), 51.2 (CH2), 110.8 (ArC), 111.9 (ArCH), 119.0
NH); 13C NMR (75 MHz, DMSO-d6): δ 14.4 (CH3), 22.4 (CH2), (ArCH), 119.5 (ArCH), 122.5 (ArCH), 126.4 (ArC), 127.2 (2 ×
25.6 (CH2), 31.5 (CH2), 36.0 (CH2), 111.3 (ArC), 111.8 (ArCH), ArCH), 128.0 (ArCH), 129.11 (2 × ArCH), 131.7 (ArC), 132.0
118.8 (ArCH), 119.5 (ArCH), 122.4 (ArCH), 126.7 (ArC), 127.2 (ArC), 134.5 (ArC), 166.9 (CO), 205.5 (CO); HRMS (+ESI): Found
(ArCH), 127.2 (ArCH), 127.9 (ArCH), 129.1 (ArCH), 129.1 (ArCH), m/z 427.2358 [M + Na]+, C26H32N2O2Na requires 427.2356.
131.8 (ArC), 132.1 (ArC), 135.0 (ArC), 173.1 (CO); HRMS (+ESI):
3-Oxo-N-(2-phenyl-1H-indol-3-yl)octanamide (14b). To
solution of hexanoic acid (0.17 g, 1.44 mmol) in dry dichloro-
a
Found m/z 329.1625 [M + Na]+, C20H22N2ONa requires 329.1629.
3-(1H-Indol-3-yl)-N-(2-phenyl-1H-indol-3-yl)propanamide (11e). methane (20 ml) was added DCC (0.34 g, 1.63 mmol), DMAP
To solution of 2-phenyl-1H-indol-3-amine (0.20 g, (0.19 g, 1.54 mmol) and Meldrum’s acid (0.21 g, 1.44 mmol)
a
0.96 mmol) in dry dichloromethane (15 ml) was added 3-(1H- under a N2 atmosphere. The reaction mixture was stirred at
indol-3-yl)propanoic acid (0.20 g, 1.06 mmol) and EDC·HCl room temperature for 24 h and then filtered through filter
(0.29 g, 1.54 mmol). The reaction mixture was stirred at room paper. The filtrate was evaporated by rotary evaporation and
temperature for 24 h and then further diluted with dichloro- the residue was dissolved in DMF (7 ml). 2-Phenyl-1H-indol-3-
methane (20 ml). The mixture was washed with water (40 ml × 3), amine (0.20 g, 0.96 mmol) was added and the mixture was
saturated potassium bisulfate solution, sodium bicarbonate heated at 60 °C for 24 h. After cooling to room temperature,
solution and then brine, dried over sodium sulfate and the water (20 ml) was added and the mixture was extracted with
solvent was evaporated in vacuo. Purification of the residue by ethyl acetate (30 ml × 3), washed with brine, dried over sodium
silica gel chromatography afforded the title compound as an sulfate and the solvent was evaporated in vacuo. Purification of
off-white solid (0.14 g, 37%); mp 187–188 °C; UV (MeOH): λmax the residue by silica gel chromatography afforded the title
304 nm (ε 28 686 M−1 cm−1), 222 (77 598), 204 (66 214); compound as an off-white powder (0.12 g, 36%); mp
IR (neat): νmax 3331, 3042, 2921, 2776, 2340, 2113, 1859, 1736, 145–147 °C; UV (MeOH): λmax 279 nm (ε 21 603 M−1 cm−1); IR
1648, 1482, 1453, 1331, 1248, 1157, 1096, 1004, 912, 828, 732, (neat): νmax 3337, 3188, 3048, 2928, 2850, 2112, 1718, 1646,
932 | Org. Biomol. Chem., 2015, 13, 925–937
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1
1541, 1448, 1325, 1196, 1085, 963, 891, 827, 737, 682 cm−1; H ArH), 7.07–7.16 (m, 2H, ArH), 7.70 (s, 1H, ArH), 9.55 (s, 1H,
NMR (300 MHz, DMSO-d6):
δ
0.84–0.89 (m, 3H, CH3), NH), 10.75 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO-d6):
1.24–1.59 (m, 6H, CH2), 2.58 (t, J = 7.3, 2H, CH2), 3.60 (s, 2H, δ 13.9 (CH3), 14.4 (CH3), 22.6 (CH2), 25.8 (CH2), 29.2 (2 × CH2),
CH2), 7.01–7.82 (m, 9H, ArH), 9.60 (s, 1H, NH), 11.39 (s, 1H, 29.3 (CH2), 29.4 (CH2), 29.5 (2 × CH2), 31.8 (CH2), 36.9 (CH2),
indole NH); 13C NMR (75 MHz, DMSO-d6): δ 14.3 (CH3), 23.1 99.6 (ArCH), 110.3 (ArCH), 110.5 (ArCH), 114.1 (ArCH), 128.9
(CH2), 24.9 (CH2), 33.8 (CH2), 42.7 (CH2), 51.2 (CH2), 110.8 (ArC), 131.6 (ArC), 133.2 (ArC), 136.5 (ArC), 170.9 (CO); HRMS
(ArC), 111.8 (ArCH), 119.0 (ArCH), 119.5 (ArCH), 122.5 (ArCH), (+ESI): Found m/z 351.2403 [M + Na]+, C21H32N2ONa requires
126.4 (ArC), 127.2 (2 × ArCH), 128.0 (ArCH), 129.1 (2 × ArCH), 351.2407.
131.7 (ArC), 132.0 (ArC), 134.5 (ArC), 166.9 (CO), 205.5 (CO);
HRMS (+ESI): Found m/z 371.1731 [M + Na]+, C22H24N2O2Na of 2-methyl-1H-indol-5-amine (0.18 g, 1.24 mmol) in dry
requires 371.1730. dichloromethane (15 ml) was added decanoic acid (0.23 g,
N-(2-Methyl-1H-indol-5-yl)decanamide (16b). To a solution
3-Oxo-6-phenyl-N-(2-phenyl-1H-indol-3-yl)hexanamide (14c). 1.36 mmol) and EDC·HCl (0.38 g, 1.98 mmol). The reaction
To a solution of 4-phenyl butyric acid (0.24 g, 1.44 mmol) in mixture was stirred at room temperature for 23 h and then
dry dichloromethane (20 ml) was added DCC (0.34 g, further diluted with dichloromethane (20 ml). The mixture
1.63 mmol), DMAP (0.19 g, 1.54 mmol) and Meldrum’s acid was washed with water (40 ml × 3), saturated potassium bi-
(0.21 g, 1.44 mmol) under a N2 atmosphere. The reaction sulfate solution, sodium bicarbonate solution and then brine,
mixture was stirred at room temperature for 24 h and then fil- dried over sodium sulfate and the solvent was evaporated
tered through filter paper. The filtrate was evaporated by rotary in vacuo. Purification of the residue by silica gel chromato-
evaporation and the residue was dissolved in DMF (7 ml). graphy afforded the title compound as an off-white solid
2-Phenyl-1H-indol-3-amine (0.20 g, 0.96 mmol) was added and (0.17 g, 46%); mp 98 °C; UV (MeOH): λmax 238 nm (ε 60 207
the mixture was heated at 60 °C for 24 h. After cooling to room M−1 cm−1); IR (neat): νmax 3405, 3223, 3044, 2912, 2846, 2340,
temperature, water (20 ml) was added and the mixture was 2108, 1638, 1538, 1470, 1303, 1258, 1028, 971, 882, 775,
1
extracted with ethyl acetate (30 ml × 3), washed with brine, 698 cm−1; H NMR (300 MHz, DMSO-d6): δ 0.85 (t, J = 6.9 Hz,
dried over sodium sulfate and the solvent was evaporated 3H, CH3), 1.25–1.29 (m, 12H, CH2), 1.59 (t, J = 7.0 Hz, 2H,
in vacuo. Purification of the residue by silica gel chromato- CH2), 2.26 (t, J = 7.2 Hz, 2H, CH2), 2.35 (s, 3H, indole ring CH3),
graphy afforded the title compound as an off-white powder 6.03 (s, 1H, ArCH), 7.07–7.16 (m, 2H, ArCH), 7.71 (s, 1H, ArCH),
(0.127 g, 33%); mp 118–120 °C; UV (MeOH): λmax 279 nm 9.55 (s, 1H, NH), 10.75 (s, 1H, indole NH); 13C NMR (75 MHz,
(ε 24 581 M−1 cm−1); IR (neat): νmax 3386, 3222, 3052, 2915, DMSO-d6): δ 13.9 (CH3), 14.4 (CH3), 22.6 (CH2), 25.8 (CH2), 29.2
2328, 117, 1874, 1713, 1636, 1452, 1307, 1258, 1100, 1006, 919, (2 × CH2), 29.3 (CH2), 29.4 (CH2), 31.8 (CH2), 36.9 (CH2), 99.6
1
843, 737, 687 cm−1; H NMR (300 MHz, DMSO-d6): δ 1.83–1.89 (ArCH), 110.3 (ArCH), 110.5 (ArCH), 114.1 (ArCH), 128.9 (ArC),
(m, 2H, CH2), 2.56–2.64 (m, 4H, CH2), 3.61 (s, 2H, CH2), 131.6 (ArC), 133.2 (ArC), 136.5 (ArC), 170.9 (CO); HRMS (+ESI):
6.99–7.82 (m, 14H, ArH), 9.61 (s, 1H, NH), 11.40 (s, 1H, indole Found m/z 323.2093 [M + Na]+, C19H28N2ONa requires 323.2094.
NH); 13C NMR (75 MHz, DMSO-d6): δ 25.4 (CH2), 34.8 (CH2),
N-(2-Methyl-1H-indol-5-yl)-4-phenylbutanamide (16c). To a
42.2 (CH2), 51.2 (CH2), 110.7 (ArC), 111.8 (ArCH), 119.1 solution of 2-methyl-1H-indol-5-amine (0.20 g, 1.38 mmol) in
(ArCH), 119.6 (ArCH), 122.5 (ArCH), 123.5 (ArCH), 126.3 dry dichloromethane (15 ml) was added 4-phenyl butyric acid
(ArCH), 126.4 (ArC), 127.2 (2 × ArCH), 128.0 (ArCH), 128.8 (3 × (0.25 g, 1.52 mmol) and EDC·HCl (0.42 g, 2.21 mmol). The
ArCH), 129.1 (2 × ArCH), 131.7 (ArC), 132.0 (ArC), 135.0 (ArC), reaction mixture was stirred at room temperature for 17 h and
142.2 (ArC), 166.9 (CO), 205.3 (CO); HRMS (+ESI): Found m/z then further diluted with dichloromethane (20 ml). The
419.1736 [M + Na]+, C26H24N2O2Na requires 419.1730.
mixture was washed with water (40 ml × 3), saturated potass-
N-(2-Methyl-1H-indol-5-yl)dodecanamide (16a). To a solu- ium bisulfate solution, sodium bicarbonate solution and then
tion of 2-methyl-1H-indol-5-amine (0.20 g, 1.38 mmol) in dry brine, dried over sodium sulfate and the solvent was evapor-
dichloromethane (15 ml) was added dodecanoic acid (0.30 g, ated in vacuo. Purification of the residue by silica gel chrom-
1.52 mmol) and EDC·HCl (0.42 g, 2.21 mmol). The reaction atography afforded the title compound as a white solid (0.25 g,
mixture was stirred at room temperature for 21 h and then 63%); mp 118–120 °C; UV (MeOH): λmax 239 nm (ε 59 156 M−1
further diluted with dichloromethane (20 ml). The mixture cm−1); IR (neat): νmax 3402, 3225, 3021, 2918, 2857, 2341, 2109,
was washed with water (40 ml × 3), saturated potassium bisul- 1857, 1632, 1534, 1477, 1372, 1273, 1247, 1205, 1132, 1030,
fate solution, sodium bicarbonate solution and then brine, 968, 883, 779, 739, 697 cm−1 1H NMR (300 MHz, DMSO-d6):
;
dried over sodium sulfate and the solvent was evaporated 1.86–1.91 (m, 2H, CH2), 2.30 (t, J = 7.5 Hz, 2H, CH2), 2.34 (s,
in vacuo. Purification of the residue by silica gel chromato- 3H, indole ring CH3), 2.62 (t, J = 7.6 Hz, 2H, CH2), 6.03 (s, 1H,
graphy afforded the title compound as a brown solid (0.26 g, ArH), 7.07–7.32 (m, 7H, ArH), 7.70 (s, 1H, ArH), 9.58 (s, 1H,
57%); mp 108 °C; UV (MeOH): λmax 238 nm (ε 36 462 M−1 NH), 10.75 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO-d6): δ
cm−1); IR (neat): νmax 3404, 3223, 3044, 2912, 2846, 2340, 1638, 13.9 (CH3), 27.5 (CH2), 35.2 (CH2), 36.3 (CH2), 99.6 (ArCH),
1539, 1468, 1303, 1226, 1094, 1028, 971, 882, 775, 698 cm−1
;
110.3 (ArCH), 110.5 (ArCH), 114.1 (ArCH), 126.2 (ArCH), 128.8
1H NMR (300 MHz, DMSO-d6): δ 0.86 (t, J = 7.0 Hz, 3H, CH3), (4 × ArCH), 128.9 (ArCH), 128.9 (ArC), 131.6 (ArC), 133.2 (ArC),
1.24–1.29 (m, 16H, CH2), 1.59 (t, J = 7.1 Hz, 2H, CH2), 2.27 (t, 136.5 (ArC), 170.6 (CO); HRMS (+ESI): Found m/z 315.1468
J = 7.2 Hz, 2H, CH2), 2.35 (s, 3H, indole ring CH3), 6.03 (s, 1H, [M + Na]+, C19H20N2ONa requires 315.1469.
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N-(2-Methyl-1H-indol-5-yl)hexanamide (16d). To a solution the residue was dissolved in DMF (7.0 ml). 2-Methyl-1H-indol-
of 2-methyl-1H-indol-5-amine (0.18 g, 1.24 mmol) in dry 5-amine (0.20 g, 1.38 mmol) was added and the mixture was
dichloromethane (15 ml) was added hexanoic acid (0.16 g, heated at 60 °C for 24 h. After cooling to room temperature,
1.36 mmol) and EDC·HCl (0.38 g, 1.98 mmol). The reaction water (20 ml) was added and the mixture was extracted with
mixture was stirred at room temperature for 21 h and then ethyl acetate (30 ml × 3), washed with brine, dried over sodium
further diluted with dichloromethane (20 ml). The mixture sulfate and the solvent was evaporated in vacuo. Purification of
was washed with water (40 ml × 3), saturated potassium bisul- the residue by silica gel chromatography afforded the title
fate solution, sodium bicarbonate solution and then brine, compound an off-white powder (0.09 g, 20%); mp 88 °C; UV
dried over sodium sulfate and the solvent was evaporated (MeOH): λmax 213 nm (ε 33 768 M−1 cm−1); IR (neat): νmax
in vacuo. Purification of the residue by silica gel chromato- 3381, 3276, 2915, 2847, 2341, 2114, 1867, 1719, 1626, 1544,
graphy afforded the title compound as an off-white solid 1449, 1408, 1306, 1241, 1183, 1044, 874, 782 cm−1 1H NMR
;
(0.16 g, 53%); mp 144 °C; UV(MeOH): λmax 238 nm (ε 48 486 (300 MHz, DMSO-d6): δ 0.85 (t, J = 7.1 Hz, 3H, CH3), 1.21–1.33
M−1 cm−1); IR (neat): νmax 3302, 3079, 2951, 2852, 2340, 2099, (m, 12H, CH2), 1.48 (t, J = 7.1 Hz, 2H, CH2), 2.34 (s, 3H, CH3),
1838, 1627, 1540, 1481, 1217, 957, 868, 773, 678 cm−1 1H 2.56 (t, J = 7.2, 2H, CH2), 3.48 (s, 2H, CH2), 6.05 (s, 1H, ArH),
;
NMR (300 MHz, DMSO-d6): δ 0.88 (t, J = 7.1 Hz, 3H, CH3), 7.04–7.08 (m, 1H, ArH), 7.16 (d, J = 8.5 Hz, 1H, ArH), 7.69 (s,
1.28–1.32 (m, 4H, CH2), 1.57–1.61 (m, 2H, CH2), 2.26 (t, J = 7.2 1H, ArH), 9.81 (s, 1H, NH), 10.80 (s, 1H, indole NH); 13C NMR
Hz, 2H, CH2), 2.34 (s, 3H, indole ring CH3), 6.04 (t, J = 0.93 Hz, (75 MHz, DMSO-d6): δ 13.9 (CH3), 14.4 (CH3), 22.6 (CH2), 23.4
1H, ArH), 7.06–7.16 (m, 2H, ArH), 7.70 (s, 1H, ArH), 9.55 (s, (CH2), 29.0 (CH2), 29.2 (CH2), 29.3 (CH2), 29.4 (CH2), 31.8
1H, NH), 10.75 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO- (CH2), 42.6 (CH2), 52.1 (CH2), 99.7 (ArCH), 110.4 (ArCH), 110.6
d6): δ 13.9 (CH3), 14.4 (CH3), 22.4 (CH2), 25.5 (CH2), 31.5 (CH2), (ArCH), 114.0 (ArCH), 128.9 (ArC), 131.2 (ArC), 133.4 (ArC),
36.8 (CH2), 99.6 (ArCH), 110.3 (ArCH), 110.5 (ArCH), 114.1 136.7 (ArC), 164.7(CO), 205.6 (CO); HRMS (+ESI): Found m/z
(ArCH), 128.9 (ArC), 131.6 (ArC), 133.2 (ArC), 136.5 (ArC), 171 365.2206 [M + Na]+, C21H30N2O2Na requires 365.2200.
(CO); HRMS (+ESI): Found m/z 267.1467 [M
+
Na]+,
N-(2-Methyl-1H-indol-5-yl)-3-oxooctanamide (17b). To
a
C15H20N2ONa requires 267.1468.
solution of hexanoic acid (0.24 g, 2.07 mmol) in dry dichloro-
3-(1H-Indol-3-yl)-N-(2-methyl-1H-indol-5-yl)propanamide (16e). methane (20 ml) was added DCC (0.49 g, 2.35 mmol), DMAP
To solution of 2-methyl-1H-indol-5-amine (0.20 g, (0.27 g, 2.21 mmol) and Meldrum’s acid (0.30 g, 2.07 mmol)
a
1.38 mmol) in dry dichloromethane (15 ml) was added 3-(1H- under a N2 atmosphere. The reaction mixture was stirred at
indol-3-yl)propanoic acid (0.29 g, 1.52 mmol) and EDC·HCl room temperature for 24 h and then filtered through filter
(0.42 g, 2.21 mmol). The reaction mixture was stirred at room paper. The filtrate was evaporated by rotary evaporation and
temperature for 22 h and then further diluted with dichloro- the residue was dissolved in DMF (7 ml). 2-Methyl-1H-indol-5-
methane (20 ml). The mixture was washed with water (40 ml × 3), amine (0.20 g, 1.38 mmol) was added and the mixture was
saturated potassium bisulfate solution, sodium bicarbonate heated at 60 °C for 24 h. After cooling to room temperature,
solution and then brine, dried over sodium sulfate and the water (20 ml) was added and the mixture was extracted with
solvent was evaporated in vacuo. Purification of the residue by ethyl acetate (30 ml × 3), washed with brine, dried over sodium
silica gel chromatography afforded the title compound as light sulfate and the solvent was evaporated in vacuo. Purification of
purple solid (0.24 g, 54%); mp 179 °C; UV (MeOH): λmax the residue by silica gel chromatography afforded the title
226 nm (ε 11 496 M−1 cm−1); IR (neat): νmax 3387, 3183, 3034, compound as an off-white powder (0.11 g, 30%); mp 92–94 °C;
2913, 2343, 2118, 1877, 1635, 1543, 1452, 1337, 1218, 1094, UV (MeOH): λmax 216 nm (ε 50 066 M−1 cm−1); IR (neat): νmax
1007, 879, 799, 740 cm−1; 1H NMR (300 MHz, DMSO-d6): δ 2.34 3380, 3269, 3085, 2919, 2341, 2115, 1906, 1634, 1542, 1442,
(s, 3H, indole ring CH3), 2.63 (t, J = 7.2 Hz, 2H, CH2), 3.02 (t, 1345, 1246, 1182, 1112, 1033, 965, 880, 777 cm−1 1H NMR
;
J = 7.3 Hz, 2H, CH2), 6.04 (s, 1H, ArH), 6.97–7.16 (m, 5H, ArH), (300 MHz, DMSO-d6): δ 1.11–1.14 (m, 3H, CH3), 1.16–1.29 (m,
7.32 (d, J = 7.4 Hz, 1H, ArH), 7.58 (d, J = 7.5 Hz, 1H, ArH), 7.71 4H, CH2), 1.49–1.74 (m, 2H, CH2), 2.36 (s, 3H, CH3), 3.43 (s,
(s, 1H, ArH), 9.62 (s, 1H, NH), 10.76 (s, 1H, indole NH); 13C 2H, CH2), 6.07 (s, 1H, ArH), 7.14–7.20 (m, 2H, ArH), 7.74 (s,
NMR (75 MHz, DMSO-d6): δ 13.9 (CH3), 21.5 (CH2), 37.7 (CH2), 1H, ArH), 9.90 (s, 1H, NH), 10.82 (s, 1H, indole NH); 13C NMR
99.6 (ArCH), 110.3 (ArCH), 110.5 (ArCH), 111.8 (ArC), 114.1 (75 MHz, DMSO-d6): δ 13.9 (2 × CH3), 24.9 (CH2), 25.8 (CH2),
(ArC), 114.4 (ArC), 118.6 (ArC), 118.9 (ArC), 121.4 (ArC), 122.6 33.8 (2 × CH2), 48.0 (CH2), 99.7 (ArCH), 110.2 (ArCH), 110.7
(ArC), 127.6 (ArC), 128.9 (ArC), 131.6 (ArC), 133.2 (ArC), 136.6 (ArCH), 113.9 (ArCH), 128.9 (ArC), 131.3 (ArC), 133.4 (ArC),
(ArC), 136.7 (ArC), 170.7 (CO); HRMS (+ESI): Found m/z 157.1 (CO), 165.5 (CO); HRMS: (+ESI): Found m/z 309.3598
340.1433 [M + Na]+, C20H19N3ONa requires 340.1430.
[M + Na]+, C17H22N2O2Na requires 309.3596.
N-(2-Methyl-1H-indol-5-yl)-3-oxododecanamide (17a). To a
N-(2-Methyl-1H-indol-5-yl)-3-oxo-6-phenylhexanamide (17c).
solution of decanoic acid (0.33 g, 1.93 mmol) in dry dichloro- To a solution of 4-phenyl butyric acid (0.34 g, 2.07 mmol) in
methane (20 ml) was added DCC (0.46 g, 2.21 mmol), DMAP dry dichloromethane (20 ml) was added DCC (0.49 g,
(0.25 g, 2.07 mmol) and Meldrum’s acid (0.28 g, 1.93 mmol) 2.35 mmol), DMAP (0.27 g, 2.21 mmol) and Meldrum’s acid
under a N2 atmosphere. The reaction mixture was stirred at (0.30 g, 2.07 mmol) under a N2 atmosphere. The reaction
room temperature for 24 h and then filtered through filter mixture was stirred at room temperature for 24 h and then fil-
paper. The filtrate was evaporated by rotary evaporation and tered through filter paper. The filtrate was evaporated by rotary
934 | Org. Biomol. Chem., 2015, 13, 925–937
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evaporation and the residue was dissolved in DMF (7 ml). J = 7.8 Hz, 1H, ArH), 7.29–7.35 (m, 2H, ArH), 7.41 (d, J =
2-Methyl-1H-indol-5-amine (0.20 g, 1.38 mmol) was added and 7.6 Hz, 1H, ArH), 9.66 (s, 1H, NH), 10.73 (s, 1H, indole NH);
the mixture was heated at 60 °C for 24 h. After cooling to room 13C NMR (75 MHz, DMSO-d6): δ 14.4 (CH3), 22.6 (CH2), 25.7
temperature, water (20 ml) was added and the mixture was (CH2), 29.2 (2 × CH2), 29.4 (2 × CH2), 31.8 (CH2), 36.7 (CH2),
extracted with ethyl acetate (30 ml × 3), washed with brine, 102.0 (ArCH), 113.7 (ArCH), 116.7 (ArCH), 119.3 (ArCH), 124.1
dried over sodium sulfate and the solvent was evaporated (ArC), 125.4 (ArCH), 128.5 (ArC), 129.5 (ArC), 171.8 (CO);
in vacuo. Purification of the residue by silica gel chromato- HRMS (+ESI): Found m/z 309.1941 [M + Na]+, C18H26N2ONa
graphy afforded the title compound as a brown oil (0.08 g, requires 309.1939.
20%); UV (MeOH): λmax 262 nm (ε 35 299 M−1 cm−1); IR (neat):
νmax 3247, 2923, 2848, 2328, 2116, 1644, 1569, 1425, 1363, 1H-indol-7-amine (0.16 g, 1.21 mmol) in dry dichloromethane
1272, 1200, 1084, 1018, 928, 826, 777, 699 cm−1 1H NMR (15 ml) was added hexanoic acid (0.16 g, 1.33 mmol) and
N-(1H-Indol-7-yl)hexanamide (20b). To
a
solution of
;
(300 MHz, DMSO-d6): δ 1.74–1.84 (m, 2H, CH2), 2.35 (s, 3H, EDC·HCl (0.37 g, 1.94 mmol). The reaction mixture was stirred
CH3) 2.54–2.61 (m, 4H, CH2), 3.49 (s, 2H, CH2), 6.06 (s, 1H, at room temperature for 18 h and then further diluted with
ArCH), 7.04–7.29 (m, 9H, ArH), 9.81 (s, 1H, NH), 10.80 (s, 1H, dichloromethane (20 ml). The mixture was washed with water
indole NH); 13C NMR (75 MHz, DMSO-d6): δ 13.9 (CH3), 25.3 (40 ml × 3), saturated potassium bisulfate solution, sodium
(CH2), 34.8 (CH2), 42.1 (CH2), 52.1 (CH2), 99.7 (ArCH), 110.4 bicarbonate solution and then brine, dried over sodium
(ArCH), 110.7 (ArCH), 114.0 (ArCH), 126.3 (ArCH), 128.8 (4 × sulfate and the solvent was evaporated in vacuo. Purification of
ArCH), 128.9 (ArC), 131.2 (ArC), 133.4 (ArC), 136.8 (ArC), 142.2 the residue by silica gel chromatography afforded the title
(ArC) 164.7 (CO), 205.4 (CO); HRMS (+ESI): Found m/z compound as
a light brown solid (0.11 g, 61%); mp
357.1569 [M + Na]+, C21H22N2O2Na requires 357.1573. 108–110 °C; UV (MeOH): λmax 256 nm (ε 11 791 M−1 cm−1); IR
1H-Indol-7-amine (19). To a boiling solution of 7-nitroin- (neat): νmax 3363, 3275, 3068, 2921, 2856, 2339, 2108, 1891,
dole (1.00 g, 6.17 mmol) in ethanol (40 ml) was added Pd/C 1647, 1541, 1432, 1341, 1256, 1198, 1052, 949, 844, 769,
1
(10%) (0.18 g) and hydrazine hydrate (3.09 g, 61.73 mmol) 723 cm−1; H NMR (300 MHz, DMSO-d6): δ 0.89 (t, J = 7.3 Hz,
under inert atmosphere. The reaction mixture was heated 3H, CH3), 1.32–1.35 (m, 4H, CH2), 1.64–1.68 (m, 2H, CH2),
under reflux for 2 h, cooled, and filtered through a small 2.40 (t, J = 7.5 Hz, 2H, CH2), 6.43 (dd, J = 3.1, 2.1 Hz, 1H, ArH),
amount of silica in a filter column. The ethanol was evapor- 6.93 (t, J = 7.6 Hz, 1H, ArH), 7.30–7.40 (m, 3H, ArH), 9.62 (s,
ated, dichloromethane (20 ml) was added and the solution 1H, NH), 10.66 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO-
was washed with water (40 ml × 3). The organic layer was then d6): δ 14.4 (CH3), 22.4 (CH2), 25.4 (CH2), 31.4 (CH2), 36.6 (CH2),
washed with brine and dried over sodium sulfate. Evaporation 102.0 (ArCH), 113.8 (ArCH), 116.7 (ArCH), 119.3 (ArCH), 124.1
of the solvent afforded the title compound as a red-brown (ArC), 125.4 (ArCH), 128.6 (ArC), 129.6 (ArC), 171.7 (CO);
solid (0.57 g, 70%); mp 95–96 °C; UV (MeOH): λmax 278 nm HRMS (+ESI): Found m/z 253.1315 [M + Na]+, C18H26N2ONa
(ε 24 231 M−1 cm−1), 286 (30 120); IR(neat): νmax 3300, 3000, requires 253.1311.
1
1850, 1610 cm−1; H NMR (300 MHz, DMSO-d6): δ 4.99 (s, 2H,
N-(1H-Indol-7-yl)-4-phenylbutanamide (20c). To a solution
NH2), 6.29–6.33 (m, 2H, ArH), 6.73 (t, J = 7.6 Hz, 1H, ArH), of 1H-indol-7-amine (0.16 g, 1.21 mmol) in dry dichloro-
6.82–6.99 (m, 1H, ArH), 7.23 (t, J = 2.8 Hz, 1H, ArH), 10.61 (s, methane (15 ml) was added 4-phenyl butyric acid (0.22 g,
1H, indole NH); 13C NMR (75 MHz, DMSO-d6): δ 101.9 (ArCH), 1.33 mmol) and EDC·HCl (0.37 g, 1.94 mmol). The reaction
105.1 (ArCH), 109.1 (ArCH), 120.4 (ArCH), 124.2 (ArCH), 126.1 mixture was stirred at room temperature for 18 h and then
(ArC), 128.7 (ArC), 134.2 (ArC); HRMS (+ESI): Found m/z further diluted with dichloromethane (20 ml). The mixture
165.1419[M + Na]+, C8H8N2Na requires 165.1418.
N-(1H-Indol-7-yl)decanamide (20a). To
was extracted with water (40 ml × 3) and washed with saturated
solution of potassium bisulfate solution, sodium bicarbonate solution
a
1H-indol-7-amine (0.18 g, 1.36 mmol) in dry dichloromethane and then brine, dried over sodium sulfate and the solvent was
(15 ml) was added decanoic acid (0.26 g, 1.50 mmol) and evaporated in vacuo. Purification of the residue by silica gel
EDC·HCl (0.42 g, 2.18 mmol). The reaction mixture was stirred chromatography afforded the title compound as a light brown
at room temperature for 18 h and then further diluted with solid (0.21 g, 39%); mp 130–132 °C; UV (MeOH): λmax 262 nm
dichloromethane (20 ml). The mixture was washed with water (ε 15 030 M−1 cm−1); IR (neat): νmax 3291, 3022, 2918, 2685,
(40 ml × 3), saturated potassium bisulfate solution, sodium 2339, 2112, 1874, 1685, 1623, 1540, 1431, 1339, 1252, 1213,
bicarbonate solution and then brine, dried over sodium 1116, 1022, 947, 841, 770 cm−1; 1H NMR (300 MHz, DMSO-d6):
sulfate and the solvent was evaporated in vacuo. Purification of δ 1.92–2.02 (m, 2H, CH2), 2.43 (t, J = 7.6 Hz, 2H, CH2), 2.68 (t,
the residue by silica gel chromatography afforded the title J = 7.7 Hz, 2H, CH2), 6.43 (dd, J = 3.1, 2.1 Hz, 1H, ArH), 6.94 (t,
compound as a light pink solid (0.36 g, 92%); mp 80 °C; UV J = 7.8 Hz, 1H, ArH), 7.17–7.40 (m, 8H, ArH), 9.66 (s, 1H, NH),
(MeOH): λmax 262 nm (ε 13 174 M−1 cm−1); IR (neat): νmax 10.67 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO-d6): δ 27.4
3389, 3251, 3025, 2916, 2847, 2723, 2340, 2111, 1895, 1655, (CH2), 35.2 (CH2), 36.0 (CH2), 102.0 (ArCH), 114.0 (ArCH),
1581, 1533, 1488, 1430, 1337, 1184, 1105, 960, 887, 795 cm−1
;
116.8 (ArCH), 119.4 (ArCH), 124.0 (ArC), 125.5 (ArCH), 126.3
1H NMR (300 MHz, DMSO-d6): δ 0.86 (t, J = 6.9 Hz, 3H, CH3), (ArCH), 128.7 (ArCH), 128.8 (2 × ArCH), 128.9 (ArCH), 129.6
1.21–1.31 (m, 12H, CH2), 1.65 (t, J = 7.1 Hz, 2H, CH2), 2.41 (t, (ArC), 142 (ArC), 142.2 (ArC), 171.4 (CO); HRMS (+ESI): Found
J = 7.2 Hz, 2H, CH2), 6.42 (dd, J = 3.0, 2.1, 1H, ArH), 6.92 (t, m/z 301.1312 [M + Na]+, C18H18N2ONa requires 301.1311.
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N-(1H-Indol-7-yl)-3-oxododecanamide (21a). To a solution of
N-(1H-Indol-7-yl)-3-oxo-6-phenylhexanamide (21c). To
a
decanoic acid (0.35 g, 2.04 mmol) in dry dichloromethane solution of 4-phenyl butyric acid (0.34 g, 2.04 mmol) in dry
(20 ml) was added DCC (0.48 g, 2.31 mmol), DMAP (0.27 g, dichloromethane (20 ml) was added DCC (0.48 g, 2.31 mmol),
2.18 mmol) and Meldrum’s acid (0.29 g, 2.04 mmol) under a DMAP (0.27 g, 2.18 mmol) and Meldrum’s acid (0.29 g,
N2 atmosphere. The reaction mixture was stirred at room temp- 2.04 mmol) under a N2 atmosphere. The reaction mixture was
erature for 24 h and then filtered through filter paper. The fil- stirred at room temperature for 24 h and then filtered through
trate was evaporated by rotary evaporation and the residue was filter paper. The filtrate was evaporated by rotary evaporation
dissolved in DMF (7.0 ml). 1H-Indol-7-amine (0.18 g, and the residue was dissolved in DMF (7 ml). 1H-Indol-7-
1.36 mmol) was added and the mixture was heated at 70 °C for amine (0.18 g, 1.36 mmol) was added and the mixture was
48 h. After cooling to room temperature, water (20 ml) was heated at 70 °C for 48 h. After cooling to room temperature,
added and the mixture was extracted with ethyl acetate (30 ml water (20 ml) was added and mixture was extracted with ethyl
× 3), washed with brine, dried over sodium sulfate and the acetate (30 ml × 3), washed with brine, dried over sodium
solvent was evaporated in vacuo. Purification of the residue by sulfate and the solvent was evaporated in vacuo. Purification of
silica gel chromatography afforded the title compound as an the residue by silica gel chromatography afforded the title
off-white powder (0.10 g, 22%); mp 108–110 °C; UV (MeOH): compound as an off-white solid (0.08 g, 19%); mp 130–132 °C;
λmax 263 nm (ε 10 805 M−1 cm−1); IR (neat): νmax 3330, 3082, UV (MeOH): λmax 262 nm (ε 12 601 M−1 cm−1); IR (neat): νmax
2915, 2848, 2338, 2106, 1713, 1646, 1543, 1434, 1329, 1259, 3325, 3025, 2923, 2847, 2329, 2112, 1716, 1627, 1543, 1434,
1116, 1044, 941, 890, 782, 721 cm−1 1H NMR (300 MHz, 1327, 1245, 1117, 953, 889, 781, 723, 676 cm−1 1H NMR
; ;
DMSO-d6): δ 0.86 (t, J = 6.8 Hz, 3H, CH3), 1.21–1.31 (m, 12H, (300 MHz, DMSO-d6): δ 1.77–1.87 (m, 2H, CH2), 2.55–2.67 (m,
CH2), 1.51 (t, J = 6.9 Hz, 2H, CH2), 2.63 (t, J = 7.0 Hz, 2H, CH2), 4H, CH2), 3.62 (s, 2H, CH2), 6.45 (dd, J = 3.0, 2.1 Hz, 1H, ArH),
3.61 (s, 2H, CH2), 6.45 (dd, J = 3.0, 2.1 Hz, 1H, ArH), 6.95 (t, J = 6.95 (t, J = 7.7 Hz, 1H, ArH), 7.10–7.37 (m, 8H, ArH), 9.87 (s,
7.8 Hz, 1H, ArH), 7.34–7.38 (m, 3H, ArH), 9.87 (s, 1H, NH), 1H, NH), 10.59 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO-
10.59 (s, 1H, indole NH); 13C NMR (75 MHz, DMSO-d6): δ 14.4 d6): δ 33.8 (CH2), 34.8 (CH2), 42.2 (CH2), 51.7 (CH2), 102.1
(CH3), 22.6 (CH2), 23.4 (CH2), 26.6 (CH2), 29.0 (CH2), 29.2 (ArCH), 114.3 (ArCH), 117.3 (ArCH), 119.4 (ArCH), 123.5 (ArC),
(CH2), 29.4 (CH2), 31.8 (CH2), 42.7 (CH2), 51.7 (CH2), 102.1 125.7 (ArCH), 126.3 (ArC), 128.7 (2 × ArCH), 128.8 (3 × ArCH),
(ArCH), 114.2 (ArCH), 117.3 (ArCH), 119.4 (ArCH), 123.5 (ArC), 129.6 (ArC), 142.2 (ArC), 165.5 (CO), 205.2 (CO); HRMS (+ESI):
125.6 (ArCH), 128.8 (ArC), 129.6 (ArC), 165.6 (CO), 205.3 (CO); Found m/z 343.1425 [M
HRMS (+ESI): Found m/z 351.2051 [M + Na]+, C20H28N2O2Na 343.1421.
requires 351.2049.
+
Na]+, C20H20N2O2Na requires
Quorum sensing inhibition assay
N-(1H-Indol-7-yl)-3-oxooctanamide (21b). To a solution of
hexanoic acid (0.24 g, 2.04 mmol) in dry dichloromethane To evaluate the effect of the synthesized compounds on QS sig-
(20 ml) was added DCC (0.48 g, 2.31 mmol), DMAP (0.27 g, naling, the P. aeruginosa MH602 PlasB::gfp(ASV) reporter strain,
2.18 mmol) and Meldrum’s acid (0.29 g, 2.04 mmol) under a which harbors a chromosomal fusion of the lasB promoter to
N2 atmosphere. The reaction mixture was stirred at room temp- an unstable gfp gene and responds to the AHL 3-oxo-dodeca-
erature for 24 h and then filtered through filter paper. The fil- noyl homoserine lactone (3oxo-C12-HSL), was used.35 To each
trate was evaporated by rotary evaporation and the residue was well of the top row in a 96-well plate, 160 μL of Luria-Bertani
dissolved in DMF (7 ml). 1H-Indol-7-amine (0.18 g, (LB10) broth media and 40 μL of 5 mM test compound’s solu-
1.36 mmol) was added and the mixture was heated at 70 °C for tion in DMSO were added. This was followed by 2 times
48 h. After cooling to room temperature, water (20 ml) was dilution each time in LB10 broth media in all subsequent
added and the mixture was extracted with ethyl acetate (30 ml wells. Then 100 μL of a diluted (100 times diluted in LB10
× 3), washed with brine, dried over sodium sulfate and the broth) overnight culture of P. aeruginosa MH602 were added to
solvent was evaporated in vacuo. Purification of the residue by all wells and the final volume in each well was 200 μL. The
silica gel chromatography afforded the title compound as a plates were incubated for 15 h in a microplate reader (Wallac
brown powder (0.08 g, 20%); mp 38–40 °C; UV (MeOH): λmax Victor, Perkin-Elmer) heated at 37 °C and every 30 min were
262 nm (ε 12 028 M−1 cm−1); IR (neat): νmax 3321, 2923, 2254, briefly shaken and measured for GFP expression (fluorescence:
2126, 1649, 1554, 1430, 1341, 1293, 994, 822, 760, 723, excitation 485 nm, emission 535 nm) and cell growth (OD
1
648 cm−1; H NMR (300 MHz, DMSO-d6): δ 0.86 (t, J = 7.0 Hz, 600). The inhibitory effect of a DMSO control (1% of total
3H, CH3), 1.24–1.28 (m, 4H, CH2), 1.47–1.57 (m, 2H, CH2), volume) was examined in similar fashion but no inhibitory
2.62 (t, J = 7.1 Hz, 2H, CH2), 3.62 (s, 2H, CH2), 6.45 (dd, J = 3.0, effect either for GFP or OD was observed.
2.1 Hz, 1H, ArH), 6.95 (t, J = 7.7 Hz, 1H, ArH), 7.31–7.38 (m,
Docking
3H, ArH), 9.86 (s, 1H, NH), 10.59 (s, 1H, indole NH); 13C NMR
(75 MHz, DMSO-d6): δ 14.3 (CH3), 22.4 (CH2), 23.1 (CH2), 31.2 Possible binding sites and poses of the compounds within the
(CH2), 42.7 (CH2), 51.7 (CH2), 102.1 (ArCH), 114.2 (ArCH), quorum sensing receptor LasR were predicted by docking
117.3 (ArCH), 119.4 (ArCH), 123.5 (ArC), 125.6 (ArCH), 128.8 these compounds into the LasR receptor (PDB code, 2UV0,
(ArC), 129.6 (ArC), 165.6 (CO), 205.3 (CO); HRMS (+ESI): Found resolution 1.8 Å) using GOLD (Cambridge Crystallography
m/z 295.1422 [M + Na]+, C16H20N2O2Na requires 295.1420.
Data Centre, UK) in its implementation through the Discovery
936 | Org. Biomol. Chem., 2015, 13, 925–937
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Paper
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Acknowledgements
We thank Marcin Mielczarek for helping with HRMS analysis. 21 N. Amara, R. Mashiach, D. Amar, P. Krief, S. P. A.
We thank the NMR and BMSF facilities at UNSW Australia for
supporting the characterization of the synthesized compounds.
H. Spieser, M. J. Bottomley;, A. Aharoni and M. M. Meijler,
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This work was supported by a Discovery Project from Australian 22 E. A. Yates, B. Philipp, C. Buckley, S. Atkinson, S. R. Chhabra,
Research Council grant (DP 140102195). N.N.B is thankful to
the University of New South Wales for a Tuition Fee Scholarship
(TFS) and to N.K. for a Living Allowance Scholarship.
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