Spermine Derivatives of Indole-3-carboxylic Acid, Indole-3-acetic Acid and Indole-3-acrylic Acid as Gram-Negative Antibiotic Adjuvants

Article abstract of DOI:10.1002/cmdc.202000359

The discovery of new antibiotic adjuvants is an attractive option for overcoming antimicrobial resistance. We have previously reported the discovery of a bis-6-bromoindolglyoxylamide derivative of spermine as being able to enhance the action of antibiotics against Gram-negative bacteria but suffers from being cytotoxic and red-blood cell haemolytic. A series of analogues was prepared exploring variation of the indolglyoxylamide unit, to include indole-3-acrylic, indole-3-acetic and indole-3-carboxylate units, and evaluated for antibiotic enhancing properties against a range of Gram-negative bacteria, and for intrinsic antimicrobial, cytotoxic and haemolytic properties. Two spermine derivatives, bearing 5-bromo-indole-3-acetic acid (17) and 5-methoxy-indole-3-acrylic acid (14) end groups were found to exhibit good to moderate antibiotic adjuvant activities for doxycycline towards the Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae, but with more modest intrinsic antimicrobial activity and greatly reduced cytotoxic and haemolytic properties. The mechanism of action of the latter derivative identified its ability to disrupt the outer membranes of bacteria and to inhibit the AcrAB-TolC efflux pump directly or by inhibiting the proton gradient.

Full text of DOI:10.1002/cmdc.202000359

Accepted Article
Title: Spermine derivatives of indole-3-carboxylic acid, indole-3-
acetic acid and indole-3-acrylic acid as Gram-negative antibiotic
adjuvants
Authors: Melissa M. Cadelis, Steven A. Li, Marie-Lise Bourguet-
Kondracki, Marine Blanchet, Hana Douafer, Jean Michel
Brunel, and Brent Copp
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000359
Link to VoR: https://doi.org/10.1002/cmdc.202000359
01/2020

10.1002/cmdc.202000359
ChemMedChem
FULL PAPER
Spermine derivatives of indole-3-carboxylic acid, indole-3-acetic
acid and indole-3-acrylic acid as Gram-negative antibiotic
adjuvants
Melissa M. Cadelis,[a] Steven A. Li,[a] Marie-Lise Bourguet-Kondracki,[b] Marine Blanchet,[c] Hana
Douafer,[c] Jean Michel Brunel[c] and Brent R. Copp*[a]
[a]
[b]
[c]
Dr M. M. Cadelis, Mr S. Li, Prof Dr. B. R. Copp
School of Chemical Sciences,
The University of Auckland, Private Bag 92019, Auckland 1142 (New Zealand)
E-mail: b.copp@auckland.ac.nz
Dr M.-L. Bourguet-Kondracki
Laboratoire Molécules de Communication et Adaptation des Micro-organismes,
UMR 7245 CNRS,
Muséum National d’Histoire Naturelle, 57 rue Cuvier (C.P. 54), 75005 Paris (France)
Dr. M. Blanchet, Ms H. Douafer, Dr. J. M. Brunel
Aix-Marseille Université,
INSERM, SSA, MCT, 13385 Marseille (France)
Supporting information for this article is given via a link at the end of the document.
Abstract: The discovery of new antibiotic adjuvants is an attractive
option for overcoming antimicrobial resistance. We have previously
reported the discovery of a bis-6-bromoindolglyoxylamide derivative
of spermine as being able to enhance the action of antibiotics against
Gram-negative bacteria but suffers from being cytotoxic and red-blood
cell haemolytic. A series of analogues was prepared exploring
variation of the indolglyoxylamide unit, to include indole-3-acrylic,
indole-3-acetic and indole-3-carboxylate units, and evaluated for
antibiotic enhancing properties against a range of Gram-negative
bacteria, and for intrinsic antimicrobial, cytotoxic and haemolytic
properties. Two spermine derivatives, bearing 5-bromo-indole-3-
acetic acid (17) and 5-methoxy-indole-3-acrylic acid (14) end groups
were found to exhibit good to moderate antibiotic adjuvant activities
for doxycycline towards the Gram-negative bacteria Pseudomonas
aeruginosa, Escherichia coli and Klebsiella pneumoniae, but with
more modest intrinsic antimicrobial activity and greatly reduced
cytotoxic and haemolytic properties. The mechanism of action of the
latter derivative identified its ability to disrupt the outer membranes of
bacteria and to inhibit the AcrAB-TolC efflux pump directly or by
inhibiting the proton gradient.
through the bacterial membrane. To avoid further undue selective
drug resistance pressure, antibiotic adjuvants should preferably
lack their own antibiotic activity.[12]
Marine natural products represent an excellent resource for the
discovery of new biologically active therapeutic lead-like
compounds.[13] One such scaffold is the indole ring, of which over
1500 examples have been reported from the marine
environment.[14] A diverse array of biological activities have been
observed for these compounds including anti-infective,[15] the
control of depression and anxiety,[16] cytotoxicity and antibacterial
[17]
properties. Our screening for natural products and natural
product-like compounds for the ability to enhance the action of
doxycycline against the Gram negative bacterium Pseudomonas
aeruginosa identified the 6-bromoindoleglyoxylamide-polyamine
1 (Figure 1), a synthetic compound related to a reported marine
natural product,[18] as an antibiotic adjuvant and intrinsic
antimicrobial agent that unfortunately also suffers from being
cytotoxic and haemolytic.[19]
6-Br active but cytotoxic/haemolytic
5-Br active, weak cytotoxic/haemolytic
F/OMe less active, not cytotoxic/haemolytic
Introduction
At least two N's for activity
Activity varies with chain length
With the continual emergence of bacteria that are resistant to
many common antibiotics, there is a well-documented need for
the discovery and development of new antibacterial therapies.[1-3]
With the development of new antibiotics being a difficult and, in
the case of Gram-negative antibacterials, unforthcoming
process,[4-5] there is an increasing effort directed towards the
discovery of adjuvants (enhancers, potentiators) that can restore
the action of antibiotics towards drug-resistant bacteria.[6-11]
Antibiotic adjuvants in current clinical use (e.g. Augmentin,
combination of amoxicillin and clavulanic acid) achieve their
effects by inhibiting the resistance mechanism. An alternative
pathway to enhancing antibiotic action is to increase drug uptake
NH
O
O
H
H
Br
N
N
N
H
N
H
Br
O
O
HN
importance?
Figure 1. Structure of polyamine derivative 1 with summary of SAR for adjuvant
activities.
Further exploration of 1 identified the 5-bromo analogue to be a
more potent enhancer of antibiotic action towards Gram-negative
1
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10.1002/cmdc.202000359
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O
bacteria, and to be less cytotoxic and haemolytic than the original
6-bromoindole hit compound.[20] Other substituted variants,
including 5-/6-fluoro and 5-/6-methoxy analogues exhibited less
effective enhancement of antibiotic action but were notable for the
absence of cytotoxic and haemolytic properties (Figure 1). In an
attempt to ascertain the importance of the glyoxylamide moiety of
1 on biological activity, we have prepared a set of indole-3-acrylic
acid, indole-3-acetic acid and indole-3-carboxylate analogues that
maintain the more biologically active spermine core[19] and that
contain variation at the indole 5- and 6- positions with bromo,
fluoro or methoxy substituents. Herein we report these preliminary
data, exploring the effect of structure on intrinsic antimicrobial
properties, the ability to enhance the action of doxycycline and
other antibiotics against Pseudomonas aeruginosa and other
Gram-negative bacteria and evaluate the ability of one of the
enhancers to influence bacterial membrane permeability and
polarization and to the inhibit AcrAB-TolC efflux pump.
OH
O
R1
R2
R1
R2
(a)
N
H
N
H
2
R1 = R2 = H
R1 = R2 = H
R
R1 = H, R2 = Br
R
3 R1 = Br, R2 = H
R1 = H, R2 = Br
5 R1 = F, R2 = H
R1 = H, R2 = F
R1 = OMe, R2 = H
R1 = H, R2 = OMe
1 = Br, R2 = H
4
1 = F, R2 = H
1 = H, R2 = F
R
6
7
8
R1 = OMe, R2 = H
1 = H, R2 = OMe
R
(b)
H
N
H
N
O
12
2
9
3a
5
R1
2
N
7a
Results and Discussion
R2
H
9
R1 = R2 = H
10 R1 = Br, R2 = H
11
Synthesis of polyamine derivatives 9–20
R
1 = H, R2 = Br
12 R1 = F, R2 = H
Synthesis of the acrylamide series commenced with the
preparation of suitably substituted indole-3-acrylic acids from the
corresponding indole-3-carbaldehydes. Commercially available
indole-3-carbaldehyde and the 5-bromo, 6-bromo, 5-fluoro, 6-
fluoro, 5-methoxy and 6-methoxy substituted analogues were
reacted with malonic acid and piperidine in pyridine at 80 oC for 6
h to afford the respective indole-3-acrylic acids 2–8 after
purification (Scheme 1). Subsequent incubation of indole-3-
acrylic acids with the peptide coupling agent 1,1'-
carbonyldiimidazole (CDI) for 3 h followed by addition of spermine
and reaction for a further 72 h afforded the desired acrylamide
analogues 9–15.
13
14
15
R
R
R
1 = H, R2 = F
1 = OMe, R2 = H
1 = H, R2 = OMe
Scheme 1. Reagents and conditions: (a) Malonic acid (1.5 eq.), piperidine,
pyridine, 80 oC, 6 h, 2 34 %, 3 53 %, 4 54 %, 5 60 %, 6 68 %, 7 48 %, 8 35 %;
(b) spermine (0.5 eq.), CDI (1 eq.), DMF, r.t., 72 h, 9 62 %, 10 65 %, 11 60 %,
12 76 %, 13 54 %, 14 75 %, 15 72 %.
11
HN
N
OH
O
2
H
8
O
3a
5
R1
R2
R1
R2
2
The acetamide analogues 16–19 were synthesized in a similar
manner, by reaction of the appropriate indole-3-acetic acid with
CDI for 3 h followed by reaction with spermine for 72 h (Scheme
2). A single example of an indolcarboxamide analogue (20) was
prepared in a similar manner starting from 5-bromoindole-3-
carboxylic acid (Scheme 3).
N
N
H
H
16
17
18
R1 = R2 = H
R1 = R2 = H
R1 = Br, R2 = H
R1 = H, R2 = F
19 R1 = OMe, R2 = H
R1 = Br, R2 = H
R1 = H, R2 = F
R1 = OMe, R2 = H
Scheme 2. Reagents and conditions: Spermine (0.5 eq.), CDI (1 eq.), DMF, r.t.,
72 h, 16 42 %, 17 38 %, 18 14 %, 19 33 %.
Biological activities
The intrinsic antimicrobial activity of each compound was
evaluated against a panel of Gram-positive (Staphylococcus
aureus and methicillin-resistant S. aureus (MRSA)) and Gram-
negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella
pneumoniae and Acinetobacter baumannii) bacteria and two
fungal strains (Candida albicans and Cryptococcus neoformans)
(Table 1). Several of the analogues exhibited antimicrobial activity
towards Gram-positive bacteria and the fungus Cryptococcus
neoformans, with 5-bromocarboxamide 20 exhibiting strong
activity against S. aureus (MIC 4.8 M), S. aureus MRSA (MIC
12.4 M) and C. neoformans (MIC 12.4 M). Similar selectivity’s
were observed for 10, 11 and to a less active extent 12 and 13.
As noted previously for indole-linked polyamine derivatives, C.
neoformans was particularly susceptible, with notable potency
observed for the 10–13 analogues (MIC 2.9–6.9 M). Little to no
activity was observed towards Gram-negative bacteria.
H
N
H
O
O
N
OH
8
10
3a
2
5
Br
Br
2
N
N
H
20
H
Scheme 3. Reagents and conditions: Spermine (0.5 eq.), CDI (1 eq.), DMF, r.t.,
72 h, 28%.
In particular, it is relevant to note the exceptionally weak activity
(MIC 100 to >200 M) of the series of compounds towards
Pseudomonas aeruginosa ATCC27853.
The set of compounds were next evaluated for the ability to
enhance the antibiotic activity of doxycycline towards P.
2
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aeruginosa ATCC27853 (Table 2). The assay used a fixed
concentration of doxycycline [2 g/mL (4.5 M)] which is
ineffective as the intrinsic MIC of doxycycline towards this
bacterium was twenty-fold higher [MIC 40 g/mL (90 M)]. Two-
than either their methoxyl or non-substituted variants, with
brominated analogues (10, 11, 17, 20, 3.12–6.25 M) being more
effective again than the fluorine-containing analogues (12, 13, 18,
6.25–50 M). The indole-3-carboxamide analogue 20 was
identified as the most effective enhancer of doxycycline activity
towards P. aeruginosa in the compound test set.
fold serial dilutions of the test compounds covering
a
concentration range of 3.125 to 200 M were examined for the
ability to completely inhibit visible growth of the bacterium.
Halogen-substituted compounds were found to be more effective
Table 1. Antimicrobial and antifungal activities (MIC, M) of compounds 1, 9–20.
Cmpd
1
S. aureus[a]
6.25[i]
50
S. aureus MRSA[b]
P. aeruginosa[c]
100[i]
E. coli[d]
100[i]
200
K. pneumoniae[e]
>34[i] [j]
>59[j]
A. baumannii[f]
>34[i] [j]
>59[j]
C. albicans[g]
17.2[i]
>59[j]
45.8
C. neoformans[h]
1.1[i]
2.16
59
9
>200[k]
100
14.8
10
11
12
13
14
15
16
17
18
19
20
12.5
12.5
50
11.4
11.4
27.7
27.7
>53[j]
>53[j]
>62[j]
47.4
>58[j]
>55[j]
12.4
50
>46[j]
>46[j]
5.7
200
50
>46[j]
>46[j]
45.8
2.9
>200[k]
200
200
>55[j]
>55[j]
55.5
6.9
25
200
>55j
>55j
55.5
6.9
100
100
200
25
>200[k]
>200[k]
>200[k]
200
>200[k]
>200[k]
>200[k]
200
>53[j]
>53[j]
>53[j]
53.3
13.3
>53[j]
>53[j]
13.3
>62[j]
>62[j]
>62[j]
>48[j]
>58[j]
>55[j]
49.5
>62[j]
11.9
>48[j]
>48[j]
100
200
4.8
>200[k]
>200[k]
200
>200[k]
>200[k]
200
>58[j]
>58[j]
>58[j]
>55[j]
12.4
>55[j]
>55[j]
>50[j]
>50[j]
[a] Staphylococcus aureus ATCC25923 with streptomycin (MIC 21.5 μM) and chloramphenicol (MIC 1.5–3 μM) used as positive controls and values presented as
the mean (n = 3). [b] Staphylococcus aureus ATCC43300 (MRSA) with vancomycin (MIC 1 μg/mL) used as a positive control and values presented as the mean (n
= 2). [c] Pseudomonas aeruginosa ATCC27853 with streptomycin (MIC 21.5 μM) and colistin (MIC 1 μM) used as positive controls and values presented as the
mean (n = 3). [d] Escherichia coli ATCC25922 with streptomycin (MIC 21.5 μM) and colistin (MIC 2 μM) used as positive controls and values presented as the mean
(n = 3). [e] Klebsiella pneumoniae ATCC700603 with colistin (MIC 0.25 μg/mL) as a positive control and values presented as the mean (n = 2). [f] Acinetobacter
baumannii ATCC19606 with colistin (MIC 0.25 μg/mL) as a positive control and values presented as the mean (n = 2). [g] Candida albicans ATCC90028 with
fluconazole (MIC 0.125 μg/mL) as a positive control and values presented as the mean (n = 2). [h] Cryptococcus neoformans ATCC208821 with fluconazole (MIC
8 μg/mL) as a positive control and values presented as the mean (n = 2). [i] Data taken from Li et. al [19] [j] Not active at a single dose test of 32 µg/mL. [k] Not
active at a single dose test of 200 µM.
Next, four of the more active doxycycline/P. aeruginosa
enhancers, 11, 14, 17 and 20, were evaluated for their abilities to
restore the action of an expanded set of antibiotics (doxycycline,
erythromycin, chloramphenicol and nalidixic acid) against four
Gram-negative ESKAPE[21] pathogens (P. aeruginosa, E. coli, K.
pneumoniae and A. baumannii) (Table 3).
determined against the same four pathogens but this time in the
presence of increasing concentrations (2, 4 or 8 g/mL) of
antibiotics (Table 4). In the presence of doxycycline and nalidixic
acid, 17 demonstrated dose-dependent antibiotic enhancement,
with notable effectiveness for doxycycline against P. aeruginosa,
E. coli and K. pneumoniae and for nalidixic acid against P.
aeruginosa and K. pneumoniae. Even at the highest test dose (8
g/mL) limited or no enhancement was observed in the presence
of erythromycin or chloramphenicol.
In addition to P. aeruginosa, all four analogues were generally
able to strongly enhance the action of doxycycline towards Gram-
negative bacteria (1.56–12.5 M), with less effectiveness
observed for 20 towards K. pneumoniae (100 M), for 14 towards
E. coli (25 M), and for 11, 14 and 17 towards A. baumannii (25–
50 M). Three analogues were good enhancers of nalidixic acid
against P. aeruginosa, and in the cases of 11 and 17 also
exhibiting enhanced activity against K. pneumoniae.
The antibiotic enhancing properties of 17 were further explored in
a
dose-dependent study, where enhancing activity was
3
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Low levels of cytotoxicity, towards the rat skeletal muscle (L6) and
human embryonic kidney (HEK-293) cell lines (IC50 / CC50 values
>46 µM), and weak human red blood cell haemolytic properties
(HC10 >50 µM) were observed for the compound set. When
compared to the cytotoxicity observed for the original 6-
bromoindole-glyoxylamido lead compound (1) in the series, the
results clearly identify the indole-3-acrylic acid, indole-3-acetic
acid and indole-3-carboxylic analogues as being more selective
as antimicrobial agents with little to no effect on mammalian cells
and red blood cell membranes.
Table 2. Doxycycline potentiating activity of compounds 1, 9–20.
Compound
Potentiation[a]
6.25[b]
25
Compound
Potentiation[a]
1[b]
9
15
16
17
18
19
20
50
50
10
11
12
13
14
6.25
6.25
50
6.25
6.25
200
3.125
12.5
12.5
[a] Concentration (µM) required to restore doxycycline activity at 2 µg/mL (4.5
µM) against P. aeruginosa ATCC27853. MIC of doxycycline alone is 40 g/mL
(90 M). Values presented as the mean (n = 3). [b] Data for compound 1 taken
from Li et. al [19].
Table 3. Antibiotic potentiating activity of analogues 11, 14, 17 and 20.
Antibiotic
Compound
Potentiation[a]
E. coli[c]
3.125
25
P. aeruginosa[b]
6.25
12.5
6.25
3.125
100
K. pneumoniae[d]
A. baumannii[e]
11
14
17
20
11
14
17
20
11
14
17
20
11
14
17
20
6.25
6.25
6.25
100
100
>200
100
200
200
>200
50
25
25
Doxycycline
Erythromycin
Chloramphenicol
Nalidixic acid
3.125
1.56
50
50
6.25
100
50
>200
50
100
100
50
100
100
100
100
>200
200
200
100
>200
200
200
200
100
100
200
200
200
12.5
12.5
200
>200
100
200
12.5
>200
12.5
200
100
6.25
12.5
200
200
[a] Concentration (µM) of compound required to restore antibiotics activity at 2 µg/mL concentration of antibiotic. [b] Pseudomonas aeruginosa ATCC27853 against
doxycycline (MIC 50 μM), erythromycin (MIC >200 µM), chloramphenicol (MIC >200 µM) and nalidixic acid (MIC >200 μM) and values presented as the mean (n =
3). [c] Escherichia coli ATCC25922 against doxycycline (MIC 25 μM) erythromycin (MIC >200 µM), chloramphenicol (MIC >200 µM) and nalidixic acid (MIC >200
μM) and values presented as the mean (n = 3). [d] Klebsiella pneumoniae ST258 against doxycycline (MIC 25 μM) erythromycin (MIC >200 µM), chloramphenicol
(MIC 50 µM) and nalidixic acid (MIC 100 μM) and values presented as the mean (n = 3). [e] Acinetobacter baumannii AYE against doxycycline (MIC 12.5 μM)
erythromycin (MIC 200 µM), chloramphenicol (MIC >200 µM) and nalidixic acid (MIC >200 μM) and values presented as the mean (n = 3).
4
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Previous studies have noted the ability of antimicrobial polyamine
analogues to disrupt the outer membrane of bacteria, resulting in
permeabilisation and increased antibiotic influx leading to cell
death.[10][22][23] To examine whether such effects are applicable to
the current set of compounds, we selected 14, being non-
cytotoxic, non-haemolytic, as exhibiting limited intrinsic
antibacterial properties and as being a moderate antibiotic
adjuvant, for further study. A bioluminescence method was used
to determine the effect of 14 on the intracellular pool of bacterial
ATP – the detection of extra-cellular ATP was used as a reporter
reflecting the permeabilising effect of the compound. After a 1 sec
exposure to the test compound, rapid dose-dependent
intracellular ATP leakage from S. aureus was observed over a 5
sec period. The response was lower than that observed for the
positive control, squalamine (Figure 2). Significantly lower ATP
efflux was observed for P. aeruginosa, both in the presence and
absence of doxycycline. This suggests that a disruption of the
membrane barrier occurs in a weak manner but is sufficient to
enhance the entrance of doxycycline into the cytoplasm.
measure of outer membrane permeabilisation. As shown in
Figure 3, 14 demonstrated a dose-dependent effect, identifying it
to be capable of permeabilising the outer membrane of Gram-
negative bacteria at high concentrations.
We next investigated the ability of 14 to disrupt transmembrane
potential, an energy source for drug efflux pumps.[10] Use was
made of the membrane-potential-sensitive probe DiSC3(5), which
when preloaded into bacteria, concentrates in the inner
membrane and self-quenches its fluorescence. If the test
compound effects membrane depolarisation, the dye is released
and fluorescence increases. In a 15 min incubation experiment,
with compound doses ranging from 15.625–125 M, only weak
levels of membrane depolarisation were observed for both of the
test bacteria, S. aureus (Figure 4, upper) and P. aeruginosa
(PAO1) (Figure 4, lower). These results are in stark contrast to
the ability of the original hit compound 1, which we have
previously found to exhibit potent ability to depolarise bacterial
membranes.[19]
Next, 14 was investigated for its ability to alter the outer
membrane integrity of Enterobacter aerogenes (EA289) using a
chromogenic -lactam (nitrocefin) colorimetric assay where the
rate of nitrocefin hydrolysis by periplasmic -lactamases is a
Table 4. Dose-dependent antibiotic potentiating activity of 17.
Antibiotic
Conc. [a]
Potentiation[b]
E. coli[d]
3.125
1.56
0.78
>50
P. aeruginosa[c]
K. pneumoniae[e]
A. baumannii[f]
>50
2
4
8
2
4
8
2
4
8
2
4
8
6.25
3.125
1.56
50
6.25
3.125
0.78
>50
>50
50
Doxycycline
Erythromycin
Chloramphenicol
Nalidixic acid
>50
50
50
50
50
n.t.[g]
n.t. [g]
>50
50
50
>50
50
>50
50
>50
25
>50
50
>50
12.5
12.5
3.125
0.78
>50
6.25
3.125
1.56
>50
>50
>50
n.t. [g]
n.t. [g]
>50
[a] Concentration of antibiotic used, in g/mL. [b] Concentration (µM) of compound required to restore antibiotic activity at varying concentrations of antibiotic. [c]
Pseudomonas aeruginosa ATCC27853 against doxycycline (MIC 50 μM), erythromycin (MIC >200 µM), chloramphenicol (MIC >200 µM) and nalidixic acid (MIC
>200 μM) and values presented as the mean (n = 3). [d]Escherichia coli ATCC25922 against doxycycline (MIC 25 μM) erythromycin (MIC >200 µM), chloramphenicol
(MIC >200 µM) and nalidixic acid (MIC >200 μM) and values presented as the mean (n = 3). [e] Klebsiella pneumoniae ST258 against doxycycline (MIC 25 μM)
erythromycin (MIC >200 µM), chloramphenicol (MIC 50 µM) and nalidixic acid (MIC 100 μM) and values presented as the mean (n = 3). [f] Acinetobacter baumannii
AYE against doxycycline (MIC 12.5 μM) erythromycin (MIC 200 µM), chloramphenicol (MIC >200 µM) and nalidixic acid (MIC >200 μM) and values presented as
the mean (n = 3). [g] Not tested.
5
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Infrared spectra were recorded on a Perkin-Elmer spectrometer 100
Fourier Transform infrared spectrometer equipped with a universal ATR
accessory. Melting points were determined on a Reichert-Hofler block and
are uncorrected. Mass spectra were acquired on a Bruker micrOTOF Q II
spectrometer. 1H and 13C NMR spectra were recorded at 298 K on Bruker
AC300, AVANCE 400 or 500 spectrometer using standard pulse
To specifically address whether derivative 14 could inhibit efflux
pumps, we investigated its ability to alter the transport of the
fluorescent
AcrAB-TolC
efflux
pump
substrate
1,2’-
dinaphthylamine (1,2’-diNA) in EA289 bacteria. This bacterial
species overexpresses the AcrAB-Tolc C pump which uses the
proton gradient across the inner membrane as an energy source.
By loading EA289 bacteria with the 1,2'-diNA dye and de-
sequences.
Proto-deutero solvent signals were used as internal
references (DMSO-d6: δH 2.50, δC 39.52; CDCl3: δH 7.26, δC 77.16). For
1H NMR, the data are quoted as position (δ), relative integral, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad),
coupling constant (J, Hz), and assignment to the atom. The 13C NMR data
are quoted as position (δ), and assignment to the atom. Silica gel column
chromatography was carried out using Davisil silica gel (40–60 μm) or
Merck silica gel (15–40 μm). Column chromatography was also conducted
on Merck Diol bonded silica (40–63 µm) and Luknova amino-bonded silica
(50 µm). Gel filtration flash chromatography was carried out on Sephadex
LH-20 (Pharmacia). Thin layer chromatography was conducted on Merck
DC-plastikfolien Kieselgel 60 F254 plates or on Merck DC Kieselgel 60
RP-18 F254S plates. All solvents used were of analytical grade or better
and/or purified according to standard procedures. Chemical reagents used
were purchased from standard chemical suppliers and used as purchased.
energization
by
adding
carbonyl
cyanide
m-
chlorophenylhydrazone (CCCP), the bacteria are rendered
fluorescent. Bacteria were then incubated with and without 14 at
different concentrations (16–256 µM) before addition of glucose
as an energy source. In the absence of 14, a rapid decrease of
the fluorescence (i.e. active transport of the dye) was observed
(Figure 5, purple line) whereas in the presence of 14, significant
dose-dependent inhibition was observed resulting in retention of
the dye at all test concentrations (Figure 5, blue lines). These
results suggest that this class of compounds may act as an
inhibitor of the AcrAB-TolC efflux pump directly or by inhibiting the
proton gradient.
Indole-3-acrylic acid (2) was prepared according to a literature procedure.
[24]
Taken
together
these
results
identify
the
methoxyindoleacrylamide 14 to be an antibiotic adjuvant, that can
increase the permeability of bacterial outer membranes and
inhibit the AcrAB-TolC efflux pump, but with little to no membrane
depolarisation.
Table 5. Cytotoxicity (IC50, µM) and haemolytic activity (HC10, µM) of analogues
1, 9–20.
Compound
Cytotoxicity
L6[b]
Haemolysis[a]
HC10
HEK-293[c]
5.06 [d]
>59[e]
>46 [e]
>46 [e]
>55 [e]
>55 [e]
>53[e]
>53 [e]
n.t. [f]
Conclusion
1
7.7 [d]
102
>34 [d][e]
>59 [e]
>46 [e]
>46 [e]
>55 [e]
>55 [e]
>53 [e]
>53 [e]
n.t. [f]
In our search for new examples of antibiotic enhancers, we have
9
previously reported that 6-bromoindolglyoxylamide
1 can
enhance the activity of doxycycline towards a panel of Gram-
negative bacteria, including P. aeruginosa, but unfortunately the
compound also exhibits cytotoxicity and is haemolytic. A targeted
series of analogues of the hit, identified that changing substituents
on the indole ring system and varying the length of the polyamine
core could afford antibiotic adjuvants that had reduced levels of
cytotoxicity and haemolytic properties. To explore further variation
on the indole moiety, analogues bearing indole-3-carboxylic acid,
indole-3-acetic acid and indole-3-acrylic acid residues were
synthesised and evaluated for antibiotic enhancing activity.
Examples of each compound class were found to be as potent, or
more so, as enhancers of doxycycline towards a range of Gram-
negative bacteria compared to 1. Gratifyingly, the new adjuvants
were found to be non-cytotoxic towards two mammalian cell lines
and non-haemolytic towards human red blood cells, highlighting
their selectivity and identifying them as leads for further
exploration. Mechanistically, 14 targets bacterial membrane
integrity and also inhibits drug efflux, both effects presumably
leading to heightened antibiotic ingress and retention, and
subsequent cell death. The finding that several of the analogues,
including 14, can act as an antibiotic enhancer but with attenuated
intrinsic antimicrobial activities, suggests the indole-3-acrylamido-
10
11
12
13
14
15
16
17
18
19
20
67.4
78.9
70.2
56.4
93.2
100
95.9
59.8
79.3
78.9
58.9
>48 [e]
n.t. [f]
>48 [e]
n.t. [f]
n.t. [f]
n.t. [f]
>50 [e]
>50 [e]
[a]Concentration (HC10, M) of compound at 10% haemolytic activity on human
red blood cells and values presented as the mean (n=2). Melittin was the
positive control (HC10 2.7 μg/mL). [b] L6 rat skeletal myoblast cell line with
podophyllotoxin as the positive control (IC50 0.018 µM) and values presented as
the mean (n = 2). [c] Concentration of compound at 50% cytotoxicity on HEK-
293 human embryonic kidney cells and values presented as the mean (n=2).
Tamoxifen was the positive control (IC50 9 μg/mL, 24μM). [d] Data taken from Li
et. al [19] [e] Not active at a single dose test of 32 µg/mL. [f] Not tested.
polyamine scaffold could be approaching that of
adjuvant.[12]
a true
Experimental Section
General methods
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000359
ChemMedChem
FULL PAPER
Figure 5. Dose-dependent inhibition by 14 of glucose-triggered 1,2’-diNA
release via efflux pumps in E. aerogenes EA289.
General procedure A – Acrylic acid formation
Figure 2. Dose-dependent ATP release for S. aureus (upper) and P. aeruginosa
(lower) exhibited by 14 alone or in combination with doxycycline (50 μg/mL) with
squalamine as the positive control.
To a stirred solution of indole-3-carbaldehyde (1 eq.) and malonic acid (1.5
eq.) in pyridine was added piperidine. The reaction mixture was allowed to
stir at 80 oC for 6 h under N2 atmosphere. Upon cooling, the crude reaction
product was concentrated under reduced pressure and subjected to
purification by silica gel column chromatography (EtOAc/n-hexane, 1:1) to
afford the corresponding indole acrylic acid.
General procedure B – Coupling with spermine
To a stirred solution of indole acid (2 eq.) in DMF (1 mL) was added CDI
(2 eq.) and stirred at room temperature for 3 h under N2 atmosphere.
Spermine (1 eq.) in DMF (1 mL) was then added and the reaction mixture
was stirred for a further 72 h. Solvent was removed under reduced
pressure and the crude product subjected to purification by amino-bonded
silica gel (CH2Cl2/MeOH, 9:1) and diol-bonded silica gel (CH2Cl2/MeOH,
9:1) column chromatography to afford indole acrylamide, acetamide and
carboxamide analogues.
Figure 3. Dose-dependent effect of 14 on the rate of nitrocefin hydrolysis in E.
aerogenes EA289.
(E)-3-(5-Bromo-1H-indol-3-yl)acrylic acid (3): Following general
procedure A, 5-bromoindole-3-carbaldehyde (0.783 g, 3.2 mmol) was
reacted with malonic acid (0.502 g, 4.8 mmol) and piperidine (100 µL) in
pyridine (20 mL) to afford the title compound as a white solid (0.450 g,
53%). Rf (EtOAc) 0.57; m.p 227.4–228.2 °C; IR (ATR) vmax 3222, 2901,
2532, 1640, 1611, 1421, 1231, 791, 701 cm-1; 1H NMR (DMSO-d6, 400
MHz) δ 11.92 (1H, s, OH), 11.90 (1H, br s, NH-1), 7.99 (1H, d, J = 1.5 Hz,
H-4), 7.97 (1H, d, J = 2.2 Hz, H-2), 7.79 (1H, d, J = 16.1 Hz, H-8), 7.43 (1H,
d, J = 8.5 Hz, H-7), 7.32 (1H, dd, J = 8.5, 1.5 Hz, H-6), 6.31 (1H, d, J =
16.1 Hz, H-9); 13C NMR (DMSO-d6, 100 MHz) δ 168.4 (C-10), 137.6 (C-8),
136.0 (C-7a), 132.1 (C-2), 126.8 (C-3a), 124.9 (C-6), 121.9 (C-4), 114.3
(C-7), 113.4 (C-5), 113.0 (C-9), 111.3 (C-3); (–)-HRESIMS [M–H]– m/z
263.9668 (calcd for
11H781BrNO2, 265.9646).
C
11H779BrNO2, 263.9666), 265.9649 (calcd for
C
(E)-3-(6-Bromo-1H-indol-3-yl)acrylic acid (4): Following general
procedure A, 6-bromoindole-3-carbaldehyde (0.200 g, 0.893 mmol) was
reacted with malonic acid (0.139 g, 1.34 mmol) and piperidine (30 µL) in
pyridine (6 mL) to afford the title compound as a pale yellow solid (0.130
g, 54%). Rf (EtOAc) 0.57; m.p 233.3–234.3 °C; IR (ATR) vmax 3253, 2971,
2528, 1637, 1609, 1568, 955 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ 11.82
(1H, br s, NH-1), 7.93 (1H, s, H-2), 7.81 (1H, d, J = 8.5 Hz, H-4), 7.77 (1H,
d, J = 16.1 Hz, H-8), 7.64 (1H, d, J = 1.8 Hz, H-7), 7.27 (1H, dd, J = 8.5,
1.8 Hz, H-5), 6.31 (1H, d, J = 16.1 Hz, H-9), not observed (OH); 13C NMR
(DMSO-d6, 100 MHz) δ 168.4 (C-10), 138.2 (C-7a), 137.7 (C-8), 131.8 (C-
2), 124.0 (C-3a), 123.5 (C-5), 121.5 (C-4), 115.0 (C-6), 114.9 (C-7), 113.3
(C-9), 111.8 (C-3); (+)-HRESIMS [M+Na]+ m/z 287.9626 (calcd for
Figure 4. Depolarisation of the bacterial membrane of S. aureus (upper) and P.
aeruginosa (lower) exhibited by 14 with cetyltrimethylammonium bromide
(CTAB) (0.01%) as the positive control.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000359
ChemMedChem
FULL PAPER
C11H879BrNNaO2, 287.9631), 289.9614 (calcd for C11H881BrNNaO2,
289.9611).
1605, 1438, 1023, 747 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ 11.59 (2H,
br s, NH-1), 7.99 (2H, t, J = 5.5 Hz, NH-11), 7.89 (2H, d, J = 7.3 Hz, H-4),
7.73 (2H, s, H-2), 7.61 (2H, d, J = 15.9 Hz, H-8), 7.45 (2H, d, J = 7.3 Hz,
H-7), 7.17 (2H, ddd, J = 18.5, 7.3, 1.0 Hz, H-6), 7.14 (2H, ddd, J = 18.5,
7.3, 1.0 Hz, H-5), 6.62 (2H, d, J = 15.9 Hz, H-9), 3.28–3.21 (4H, m, H2-12),
2.65–2.51 (8H, m, H2-14, H2-16), 1.69–1.58 (4H, m, H2-13) 1.53–1.45 (4H,
m, H2-17), not observed (NH-15); 13C NMR (DMSO-d6, 100 MHz) δ 166.4
(C-10), 137.4 (C-7a), 132.8 (C-8), 130.1 (C-2), 124.9 (C-3a), 122.1 (C-6),
120.2 (C-5), 119.9 (C-4), 116.3 (C-9), 112.2 (C-3 or C-7), 112.1 (C-7 or C-
3), 48.6 (C-16), 46.3 (C-14), 36.6 (C-12), 29.0 (C-13), 26.7 (C-17); (+)-
HRESIMS [M+Na]+ m/z 563.3099 (calcd for C32H40N6NaO2, 563.3105).
(E)-3-(5-Fluoro-1H-indol-3-yl)acrylic acid (5): Following general
procedure A, 5-fluoroindole-3-carbaldehyde (0.200 g, 1.23 mmol) was
reacted with malonic acid (0.193 g, 1.85 mmol) and piperidine (40 µL) in
pyridine (8 mL) to afford the title compound as a yellow solid (0.152 g,
60%). Rf (EtOAc) 0.37; m.p 208.5–209.7 °C; IR (ATR) vmax 3257, 2964,
2843, 1634, 1611, 1424, 1243, 670 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ
11.88 (1H, s, OH), 11.81 (1H, br s, NH-1), 7.98 (1H, d, J = 2.9 Hz, H-2),
7.78 (1H, d, J = 16.0 Hz, H-8), 7.60 (1H, dd, J = 10.2, 2.5 Hz, H-4), 7.46
(1H, dd, J = 8.8, 4.7 Hz, H-7), 7.05 (1H, ddd, J = 9.1, 9.1, 2.5 Hz, H-6),
6.28 (1H, d, J = 16.0 Hz, H-9); 13C NMR (DMSO-d6, 100 MHz) δ 168.5 (C-
10), 157.9 (d, 1JFC = 233.5 Hz, C-5), 137.9 (C-8), 133.9 (C-7a), 132.6 (C-
2), 125.3 (d, 3JFC = 9.8 Hz, C-3a), 113.4 (d, 3JFC = 8.5 Hz, C-7), 112.5 (C-
9), 111.8 (d, 4JFC = 5.1 Hz, C-3), 110.4 (d, 2JFC = 25.8, C-6), 104.8 (d, 2JFC
(2E,2'E)-N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-
diyl))bis(3-(5-bromo-1H-indol-3-yl)acrylamide) (10): Following general
procedure B, reaction of (E)-3-(5-bromo-1H-indol-3-yl)acrylic acid (3)
(0.076 g, 0.286 mmol) was reacted with CDI (0.046 g, 0.286 mmol) and
spermine (0.029 g, 0.143 mmol) afforded the title compound as a yellow
gum (0.065 g, 65%). Rf (MeOH/10% HCl, 7:3) 0.14; IR (ATR) vmax 3388,
3261, 2932, 2857, 1650, 1600, 1455, 1276, 1023, 760 cm-1;1H NMR
(DMSO-d6, 400 MHz) δ 8.03 (2H, d, J = 1.7 Hz, H-4), 7.94 (2H, t, J = 5.0
Hz, NH-11), 7.80 (2H, s, H-2), 7.55 (2H, d, J = 16.0 Hz, H-8), 7.42 (2H, d,
J = 8.7 Hz, H-7), 7.31 (2H, dd, J = 8.7, 1.7 Hz, H-6), 6.57 (2H, d, J = 16.0
Hz, H-9), 3.25–3.18 (4H, m, H2-12), 2.55–2.45 (8H, m, H2-14, H2-16),
1.63–1.54 (4H, m, H2-13), 1.45–1.37 (4H, m, H2-17), not observed (NH-1,
NH-15); 13C NMR (DMSO-d6, 100 MHz) δ 166.1 (C-10), 136.1 (C-7a),
132.1 (C-8), 131.6 (C-2), 126.5 (C-3a), 124.7 (C-6), 122.1 (C-4), 117.0 (C-
9), 114.3 (C-7), 113.1 (C-5), 111.8 (C-3), 49.5 (C-16), 47.1 (C-14), 36.9
(C-12), 29.8 (C-13), 27.6 (C-17); (–)-HRESIMS [M–H]– m/z 695.1377
=
24.2 Hz, C-4); (+)-HRESIMS [M+Na]+ m/z 228.0435 (calcd for
C11H8FNNaO2, 228.0431).
(E)-3-(6-Fluoro-1H-indol-3-yl)acrylic acid (6): Following general
procedure A, 6-fluoroindole-3-carbaldehyde (0.200 g, 1.23 mmol) was
reacted with malonic acid (0.193 g, 1.85 mmol) and piperidine (40 µL) in
pyridine (8 mL) to afford the title compound as a yellow solid (0.172 g,
68%). Rf (EtOAc) 0.69; m.p 198.5–199.7 °C; IR (ATR) vmax 3279, 2934,
2867, 1637, 1611, 1420, 1231, 669 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ
11.90 (1H, s, OH), 11.75 (1H, br s, NH-1), 7.90 (1H, d, J = 2.7 Hz, H-2),
7.85 (1H, dd, J = 8.8, 5.2 Hz, H-4), 7.78 (1H, d, J = 16.0 Hz, H-8), 7.25 (1H,
dd, J = 9.8, 2.4 Hz, H-7), 7.00 (1H, ddd, J = 9.6, 9.6, 2.4 Hz, H-5), 6.31
(1H, d, J = 16.0 Hz, H-9); 13C NMR (DMSO-d6, 100 MHz) δ 168.5 (C-10),
159.1 (d, 1JFC = 236.0 Hz, C-6), 138.0 (C-8), 137.4 (d, 3JFC = 12.5 Hz, C-
7a), 131.8 (C-2), 121.8 (C-3a), 120.9 (d, 3JFC = 10.1 Hz, C-4), 112.7 (C-9),
111.8 (C-3), 109.0 (d, 2JFC = 24.1 Hz, C-5), 98.4 (d, 2JFC = 25.3, C-7); (+)-
HRESIMS [M+Na]+ m/z 228.0425 (calcd for C11H8FNNaO2, 228.0431).
(calcd for
32H3779Br81BrN6O2, 697.1332), 699.1352 (calcd for C32H3781Br2N6O2,
699.1320).
C
32H3779Br2N6O2, 695.1350), 697.1377 (calcd for
C
(2E,2'E)-N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-
diyl))bis(3-(6-bromo-1H-indol-3-yl)acrylamide) (11): Following general
procedure B, reaction of (E)-3-(6-bromo-1H-indol-3-yl)acrylic acid (4)
(0.076 g, 0.286 mmol) was reacted with CDI (0.046 g, 0.286 mmol) and
spermine (0.029 g, 0.143 mmol) afforded the title compound as a yellow
gum (0.060 g, 60%). Rf (MeOH/10% HCl, 7:3) 0.26; IR (ATR) vmax 3391,
3265, 2933, 2863, 1652, 1605, 1450, 1254, 1001, 759 cm-1; 1H NMR
(DMSO-d6, 400 MHz) δ 11.69 (NH-1), 8.00 (2H, t, J = 5.5 Hz, NH-11), 7.82
(2H, d, J = 8.6 Hz, H-4), 7.79 (2H, s, H-2), 7.65 (2H, d, J = 1.7 Hz, H-7),
7.58 (2H, d, J = 15.9 Hz, H-8), 7.29 (2H, dd, J = 8.6, 1.7 Hz, H-5), 6.61 (2H,
d, J = 15.9 Hz, H-9), 3.28–3.19 (4H, m, H2-12), 2.67–2.54 (8H, m, H2-14,
H2-16), 1.70–1.60 (4H, m, H2-13), 1.54–1.46 (4H, m, H2-17); 13C NMR
(DMSO-d6, 100 MHz) δ 166.2 (C-10), 138.2 (C-7a), 132.2 (C-8), 130.8 (C-
2), 123.9 (C-3a), 123.0 (C-5), 121.5 (C-4), 117.1 (C-9), 114.9 (C-6 or C-7),
114.8 (C-7 or C-6), 112.2 (C-3), 48.4 (C-16), 46.1 (C-14), 36.5 (C-12), 28.8
(C-13), 26.5 (C-17); (+)-HRESIMS [M+Na]+ m/z 719.1339 (calcd for
(E)-3-(5-Methoxy-1H-indol-3-yl)acrylic acid (7): Following general
procedure A, 5-methoxyindole-3-carbaldehyde (0.200 g, 1.14 mmol) was
reacted with malonic acid (0.178 g, 1.71 mmol) and piperidine (40 µL) in
pyridine (7 mL) to afford the title compound as a white solid (0.118 g, 48%).
Rf (EtOAc) 0.86; m.p 208.7–210.1 °C; IR (ATR) vmax 3427, 3123, 2953,
1652, 1596, 1407, 1216, 670 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ 11.81
(1H, s, OH), 11.59 (1H, br s, NH-1), 7.85 (1H, d, J = 2.8 Hz, H-2), 7.80 (1H,
d, J = 15.9 Hz, H-8), 7.35 (1H, d, J = 8.8 Hz, H-7), 7.26 (1H, d, J = 2.4 Hz,
H-4), 6.84 (1H, dd, J = 8.8, 2.4 Hz, H-6), 6.24 (1H, d, J = 15.9 Hz, H-9),
3.82 (3H, s, 5-OMe); 13C NMR (DMSO-d6, 100 MHz) δ 168.6 (C-10), 154.7
(C-5), 138.5 (C-8), 132.1 (C-7a), 131.3 (C-2), 125.6 (C-3a), 113.0 (C-7),
112.52 (C-6), 112.48 (C-3), 111.5 (C-9), 101.6 (C-4), 55.4 (OMe); (+)-
HRESIMS [M+Na]+ m/z 240.0634 (calcd for C12H11NNaO3, 240.0631).
C
C
C
32H3879Br2N6NaO2,
719.1315),
721.1297),
721.1334
723.1316
(calcd
(calcd
for
for
(E)-3-(6-Methoxy-1H-indol-3-yl)acrylic acid (8): Following general
procedure A, 6-methoxyindole-3-carbaldehyde (0.200 g, 1.14 mmol) was
reacted with malonic acid (0.178 g, 1.71 mmol) and piperidine (40 µL) in
pyridine (7 mL) to afford the title compound as a pale yellow solid (0.086
g, 35%). Rf (EtOAc) 0.60; m.p 186.6–187.5 °C; IR (ATR) vmax 3427, 3383,
2944, 2836, 1655, 1599, 1403, 1228, 664 cm-1; 1H NMR (DMSO-d6, 400
MHz) δ 11.81 (1H, s, OH), 11.50 (1H, br s, NH-1), 7.75 (1H, d, J = 2.7 Hz,
H-2), 7.74 (1H, d, J = 15.9 Hz, H-8), 7.71 (1H, d, J = 8.7 Hz, H-4), 6.95 (1H,
d, J = 2.3 Hz, H-7), 6.80 (1H, dd, J = 8.7, 2.3 Hz, H-5), 6.24 (1H, d, J =
15.9 Hz, H-9), 3.79 (3H, s, 5-OMe); 13C NMR (DMSO-d6, 100 MHz) δ 168.6
(C-10), 156.1 (C-6), 138.6 (C-8), 138.3 (C-7a), 130.5 (C-2), 120.4 (C-4),
119.0 (C-3a), 111.8 (C-3, C-9), 110.5 (C-5), 95.4 (C-7), 55.2 (OMe); (+)-
HRESIMS [M+Na]+ m/z 240.0637 (calcd for C12H11NNaO3, 240.0631).
32H3879Br81BrN6NaO2,
32H3881Br2N6NaO2, 723.1284).
(2E,2'E)-N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-
diyl))bis(3-(5-fluoro-1H-indol-3-yl)acrylamide) (12): Following general
procedure B, reaction of (E)-3-(5-fluoro-1H-indol-3-yl)acrylic acid (5)
(0.071 g, 0.347 mmol) was reacted with CDI (0.056 g, 0.347 mmol) and
spermine (0.035 g, 0.173 mmol) afforded the title compound as a yellow
gum (0.076 g, 76%). Rf (MeOH/10% HCl, 7:3) 0.56; IR (ATR) vmax 3240,
3167, 3052, 2930, 1654, 1607, 1471, 1275, 1005, 757 cm-1; 1H NMR
(DMSO-d6, 400 MHz) δ 11.65 (2H, br s, NH-1), 7.92 (2H, t, J = 5.6 Hz, NH-
11), 7.82 (2H, s, H-2), 7.63 (2H, dd, J = 10.3, 2.4 Hz, H-4), 7.58 (2H, d, J
= 15.9 Hz, H-8), 7.46 (2H, dd, J = 9.1, 4.4 Hz, H-7), 7.06 (2H, ddd, J = 9.1,
9.1, 2.4 Hz, H-6), 6.58 (2H, d, J = 15.9 Hz, H-9), 3.27–3.19 (4H, m, H2-12),
2.64–2.51 (8H, m, H2-14, H2-16), 1.67–1.59 (4H, m, H2-13), 1.52–1.43 (4H,
(2E,2'E)-N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-
diyl))bis(3-(1H-indol-3-yl)acrylamide) (9): Following general procedure
B, reaction of (E)-3-(1H-indol-3-yl)acrylic acid (2) (0.069 g, 0.367 mmol)
was reacted with CDI (0.060 g, 0.367 mmol) and spermine (0.038 g, 0.185
mmol) afforded the title compound as a yellow gum (0.062 g, 62%). Rf
(MeOH/10% HCl, 7:3) 0.59; IR (ATR) vmax 3412, 3254, 2928, 2863, 1654,
m, H2-17); 13C NMR (DMSO-d6, 100 MHz) δ 166.3 (C-10), 157.7 (d, 1JFC
233.3 Hz, C-5), 134.0 (C-7a), 132.3 (C-8), 131.9 (C-2), 125.0 (d, JFC
=
=
3
10.0 Hz, C-3a), 116.5 (C-9), 113.3 (d, 3JFC = 10.3 Hz, C-7), 112.2 (d, 4JFC
= 4.1 Hz, C-3), 110.2 (d, 2JFC = 26.0 Hz, C-6), 104.9 (d, 2JFC = 24.2 Hz, C-
8
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10.1002/cmdc.202000359
ChemMedChem
FULL PAPER
4), 48.7 (C-16), 46.9 (C-14), 36.6 (C-12), 29.0 (C-13), 26.7 (C-17); (–)-
6.3, 6.0 Hz, H2-11), 2.52–2.48 (4H, m, H2-13), 2.46–2.43 (4H, m, H2-15),
1.58–1.51 (4H, m, H2-12), 1.40–1.37 (4H, m, H2-16); 13C NMR (DMSO-d6,
100 MHz) δ 170.7 (C-9), 136.1 (C-7a), 127.2 (C-3a), 123.8 (C-2), 120.9
(C-6), 118.6 (C-4), 118.2 (C-5), 111.3 (C-7), 108.9 (C-3), 48.4 (C-15), 46.1
(C-13), 36.7 (C-11), 32.8 (C-8), 28.5 (C-12), 26.4 (C-16); (+)-HRESIMS
[M+H]+ m/z 517.3283 (calcd for C30H41N6O2, 517.3286).
HRESIMS [M–H]– m/z 575.2930 (calcd for C32H37F2N6O2, 575.2952).
(2E,2'E)-N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-
diyl))bis(3-(6-fluoro-1H-indol-3-yl)acrylamide) (13): Following general
procedure B, reaction of (E)-3-(6-fluoro-1H-indol-3-yl)acrylic acid (6)
(0.071 g, 0.347 mmol) was reacted with CDI (0.056 g, 0.347 mmol) and
spermine (0.035 g, 0.173 mmol) afforded the title compound as a yellow
gum (0.054 g, 54%). Rf (MeOH/10% HCl, 7:3) 0.54; IR (ATR) vmax 3245,
2926, 2855, 1653, 1607, 1453, 1235, 1005, 758 cm-1; 1H NMR (DMSO-d6,
400 MHz) δ 11.61 (2H, br s, NH-1), 7.95 (2H, t, J = 5.5 Hz, NH-11), 7.87
(2H, dd, J = 8.9, 5.4 Hz, H-4), 7.75 (2H, s, H-2), 7.56 (2H, d, J = 15.9 Hz,
H-8), 7.25 (2H, dd, J = 9.8, 2.4 Hz, H-7), 7.03 (2H, ddd, J = 9.8, 8.9, 2.4
Hz, H-5), 6.62 (2H, d, J = 15.9 Hz, H-9), 3.26–3.18 (4H, m, H2-12), 2.60–
N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-diyl))bis(2-(5-
bromo-1H-indol-3-yl)acetamide) (17): Following general procedure B,
reaction of 5-bromoindole-3-acetic acid (0.050 g, 0.20 mmol) was reacted
with CDI (0.032 g, 0.20 mmol) and spermine (0.020 g, 0.10 mmol) afforded
the title compound as a pale yellow gum (0.025 g, 38%). Rf (CH2Cl2/MeOH,
9:1) 0.44; IR (ATR) vmax 3271, 2932, 1647, 1554, 1453, 1228, 1036, 953,
883, 796 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ 11.12 (2H, br s, NH-1),
8.01 (2H, t, J = 5.2 Hz, NH-10), 7.74 (2H, d, J = 1.9 Hz, H-4), 7.31 (2H, d,
J = 8.6 Hz, H-7), 7.23 (2H, s, H-2), 7.16 (2H, dd, J = 8.6, 1.9 Hz, H-6), 3.46
(4H, s, H2-8), 3.08 (4H, dt, J = 6.2, 5.9 Hz, H2-11), 2.51–2.47 (4H, m, H2-
13), 2.47–2.44 (4H, m, H2-15), 1.58–1.51 (4H, m, H2-12), 1.40–1.37 (4H,
m, H2-16); 13C NMR (DMSO-d6, 100 MHz) δ 170.5 (C-9), 134.8 (C-7a),
129.1 (C-3a), 125.5 (C-2), 123.3 (C-6), 121.1 (C-4), 113.3 (C-7), 111.0 (C-
5), 108.9 (C-3), 48.5 (C-15), 46.1 (C-13), 36.7 (C-11), 32.6 (C-8), 28.6 (C-
12), 26.5 (C-16); (+)-HRESIMS [M+H]+ m/z 673.1497 (calcd for
2.51 (8H, m, H2-14, H2-16), 1.65–1.54 (4H, m, H2-13), 1.49–1.42 (4H, m,
1
H2-17); 13C NMR (DMSO-d6, 100 MHz) δ 166.2 (C-10), 159.0 (d, JFC
=
236.8 Hz, C-6), 137.4 (d, 3JFC = 12.0 Hz, C-7a), 132.3 (C-8), 130.6 (C-2),
121.7 (C-3a), 120.8 (d, 3JFC = 9.3 Hz, C-4), 116.8 (C-9), 112.2 (C-3), 108.5
2
2
(d, JFC = 24.5 Hz, C-5), 98.3 (d, JFC = 25.2 Hz, C-7), 48.9 (C-16), 46.5
(C-14), 36.7 (C-12), 29.2 (C-13), 27.0 (C-17); (–)-HRESIMS [M–H]– m/z
575.2943 (calcd for C32H37F2N6O2, 575.2952).
C
30H3979Br2N6O2, 673.1496), 675.1478 (calcd for C30H3979Br81BrN6O2,
(2E,2'E)-N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-
675.1578), 677.1460 (calcd for C30H3981Br2N6O2, 677.1464).
diyl))bis(3-(5-methoxy-1H-indol-3-yl)acrylamide)
(14):
Following
general procedure B, reaction of (E)-3-(5-methoxy-1H-indol-3-yl)acrylic
acid (7) (0.072 g, 0.330 mmol) was reacted with CDI (0.054 g, 0.330 mmol)
and spermine (0.033 g, 0.165 mmol) afforded the title compound as a
yellow gum (0.075 g, 75%). Rf (MeOH/10% HCl, 7:3) 0.60; IR (ATR) vmax
3244, 3043, 2935, 1651, 1603, 1481, 1218, 1023, 758 cm-1; 1H NMR
(DMSO-d6, 400 MHz) δ 11.43 (2H, br s, NH-1), 7.95 (2H, t, J = 5.6 Hz, NH-
11), 7.70 (2H, s, H-2), 7.58 (2H, d, J = 15.8 Hz, H-8), 7.36 (2H, d, J = 8.7
Hz, H-7), 7.35 (2H, d, J = 2.2 Hz, H-4), 6.86 (2H, dd, J = 8.7, 2.2 Hz, H-6),
6.53 (2H, d, J = 15.8 Hz, H-9), 3.84 (6H, s, 5-OMe), 3.27–3.19 (4H, m, H2-
12), 2.58–2.49 (8H, m, H2-14, H2-16), 1.65–1.55 (4H, m, H2-13), 1.48–1.40
(4H, m, H2-17); 13C NMR (DMSO-d6, 100 MHz) δ 166.4 (C-10), 154.4 (C-
5), 132.9 (C-8), 132.4 (C-7a), 130.5 (C-2), 125.4 (C-3a), 115.7 (C-9), 112.8
(C-7), 111.8 (C-3 or C-6), 111.6 (C-6 or C-3), 102.6 (C-4), 55.6 (OMe),
49.0 (C-16), 46.7 (C-14), 36.7 (C-12), 29.4 (C-13), 27.1 (C-17); (–)-
HRESIMS [M–H]– m/z 599.3361 (calcd for C34H43N6O4, 599.3351).
N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-diyl))bis(2-(6-
fluoro-1H-indol-3-yl)acetamide) (18): Following general procedure B,
reaction of 6-fluoroindole-3-acetic acid (0.050 g, 0.26 mmol) was reacted
with CDI (0.042 g, 0.26 mmol) and spermine (0.026 g, 0.13 mmol) afforded
the title compound as a pale yellow gum (0.010 g, 14%). Rf (CH2Cl2/MeOH,
9:1) 0.42; IR (ATR) vmax 3430, 2975, 1679, 1420, 1268, 1130, 952 cm-1; 1H
NMR (DMSO-d6, 400 MHz) δ 10.94 (2H, br s, NH-1), 7.92 (2H, br s, NH-
10), 7.51 (2H, dd, J = 8.4, 5.4 H, H-4), 7.16 (2H, s, H-2), 7.10 (2H, dd, J =
10.5, 2.5 Hz, H-7), 6.82 (2H, ddd, J = 10.5, 8.4, 2.5 Hz, H-5), 3.46 (4H, s,
H2-8), 3.07 (4H, dt, J = 6.4, 5.9 Hz, H2-11), 2.52–2.48 (4H, m, H2-13), 2.48–
2.42 (4H, m, H2-15), 1.54–1.49 (4H, m, H2-12), 1.39–1.36 (4H, m, H2-16);
13C NMR (DMSO-d6, 100 MHz) δ 170.5 (C-9), 158.8 (d, 1JFC = 236.9 Hz,
C-6), 135.9 (d, 3JFC = 12.7 Hz, C-7a), 124.3 (d, 4JFC = 3 Hz, C-3a), 124.1
(C-2), 119.6 (d, 3JFC = 10.5 Hz, C-4), 109.2 (C-3), 106.7 (d, 2JFC = 24.2 Hz,
C-5), 97.2 (d, 2JFC = 25.5 Hz, C-7), 48.6 (C-15), 46.3 (C-13), 36.7 (C-11),
32.7 (C-8), 28.8 (C-12), 26.7 (C-16); (+)-HRESIMS [M+H]+ m/z 553.3090
(calcd for C30H39F2N6O2, 553.3097).
(2E,2'E)-N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-
diyl))bis(3-(6-methoxy-1H-indol-3-yl)acrylamide)
(15):
Following
general procedure B, reaction of (E)-3-(6-methoxy-1H-indol-3-yl)acrylic
acid (8) (0.072 g, 0.330 mmol) was reacted with CDI (0.054 g, 0.330 mmol)
and spermine (0.033 g, 0.165 mmol) afforded the title compound as a
yellow gum (0.072 g, 72%). Rf (MeOH/10% HCl, 7:3) 0.66; IR (ATR) vmax
3202, 2929, 2836, 1651, 1604, 1524, 1162, 1023, 757 cm-1; 1H NMR
(DMSO-d6, 400 MHz) δ 11.36 (2H, br s, NH-1), 7.91 (2H, t, J = 5.6 Hz, NH-
11), 7.76 (2H, d, J = 8.8 Hz, H-4), 7.60 (2H, s, H-2), 7.55 (2H, d, J = 15.7
Hz, H-8), 6.95 (2H, d, J = 2.2 Hz, H-7), 6.80 (2H, dd, J = 8.8, 2.2 Hz, H-5),
6.56 (2H, d, J = 15.7 Hz, H-9), 3.80 (6H, s, 5-OMe), 3.25–3.18 (4H, m, H2-
12), 2.58–2.49 (8H, m, H2-14, H2-16), 1.65–1.54 (4H, m, H2-13), 1.47–1.39
(4H, m, H2-17); 13C NMR (DMSO-d6, 100 MHz) δ 166.4 (C-10), 156.0 (C-
6), 138.3 (C-7a), 132.8 (C-8), 129.1 (C-2), 120.5 (C-4), 119.0 (C-3a), 116.1
(C-9), 112.2 (C-3), 110.1 (C-5), 95.3 (C-7), 55.2 (OMe), 49.1 (C-16), 46.7
(C-14), 36.7 (C-12), 29.4 (C-13), 27.2 (C-17); (–)-HRESIMS [M–H]– m/z
599.3352 (calcd for C34H43N6O4, 599.3351).
N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-diyl))bis(2-(5-
methoxy-1H-indol-3-yl)acetamide) (19): Following general procedure B,
reaction of 5-methoxyindole-3-acetic acid (0.050 g, 0.24 mmol) was
reacted with CDI (0.040 g, 0.24 mmol) and spermine (0.025 g, 0.12 mmol)
afforded the title compound as a pale yellow gum (0.023 g, 33%). Rf
(CH2Cl2/MeOH, 9:1) 0.14; IR (ATR) vmax 3392, 3272, 2972, 2940, 1645,
1560, 1487, 1439, 1406, 1216, 1165, 953 cm-1; 1H NMR (DMSO-d6, 400
MHz) δ 10.73 (2H, br s, NH-1), 7.93 (2H t, J = 5.7 Hz, NH-10), 7.22 (2H, d,
J = 8.8 Hz, H-7), 7.13 (2H, d, J = 1.8 Hz, H-2), 7.04 (2H, d, J = 2.5 Hz, H-
4), 6.70 (2H, dd, J = 8.8, 2.5 Hz, H-6), 3.74 (6H, s, 5-OMe), 3.44 (4H, s,
H2-8), 3.08 (4H, dt, J = 6.1, 6.1 Hz, H2-11), 2.51–2.47 (4H, m, H2-13), 2.44–
2.41 (4H, m, H2-15), 1.57–1.51 (4H, m, H2-12), 1.38–1.35 (4H, m, H2-16);
13C NMR (DMSO-d6, 100 MHz) δ 170.7 (C-9), 153.0 (C-5), 131.3 (C-7a),
127.5 (C-3a), 124.4 (C-2), 111.9 (C-7), 111.0 (C-6), 108.7 (C-3), 100.5 (C-
4), 55.3 (OMe), 48.5 (C-15), 46.2 (C-13), 36.7 (C-11), 32.9 (C-8), 28.7 (C-
12), 26.5 (C-16); (+)-HRESIMS [M+H]+ m/z 577.3501 (calcd for
N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-diyl))bis(2-(1H-
indol-3-yl)acetamide) (16): Following general procedure B, reaction of
indole-3-acetic acid (0.050 g, 0.29 mmol) was reacted with CDI (0.046 g,
0.29 mmol) and spermine (0.029 g, 0.14 mmol) afforded the title compound
as a pale yellow oil (0.030 g, 42%). Rf (CH2Cl2/MeOH, 9:1) 0.28; IR (ATR)
C
32H45N6O4, 577.3497).
N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-diyl))bis(5-
bromo-1H-indole-3-carboxamide) (20): Following general procedure B,
5-bromoindole-3-carboxylic acid (0.050 g, 0.24 mmol) was reacted with
CDI (0.040 g, 0.24 mmol) and spermine (0.025 g, 0.12 mmol) in DMF for
72 hours. Purification by column chromatography afforded the title
compound as a pale brown oil (0.019 g, 28 %). Rf (CH2Cl2/MeOH, 9:1)
0.22; IR (ATR) vmax 3263, 2927, 1627, 1553, 1457, 1379, 1225, 1142, 952,
v
max 3250, 3059, 2931, 1643, 1553, 1401, 1341, 1229, 1049, 1024, 1008,
745 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ 10.89 (2H, br s, NH-1), 7.93
(2H, t, J = 5.5 Hz, NH-10), 7.53 (2H, d, J = 8.0 Hz, H-4), 7.33 (2H, d, J =
8.0 Hz, H-7), 7.17 (2H, d, J = 2.0 Hz, H-2), 7.05 (2H, td, J = 6.9, 0.9 Hz, H-
6), 6.96 (2H, td, J = 6.9, 0.9 Hz, H-5), 3.48 (4H, s, H2-8), 3.08 (4H, dt, J =
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000359
ChemMedChem
FULL PAPER
803, 713 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ 11.96 (2H, br s, NH-1),
8.31 (2H, br s, H-4), 8.18–8.14 (2H, m, NH-9), 8.09 (2H, br s, H-2), 7.42
(2H, d, J = 8.6 Hz, H-7), 7.26 (2H, d, J = 8.6 Hz, H-6), 3.33–3.29 (4H, m,
H2-10), 2.67–2.62 (4H, m, H2-12), 2.61–2.57 (4H, m, H2-14), 1.74–1.69
(4H, m, H2–11), 1.53–1.49 (4H, m, H2-15); 13C NMR (DMSO-d6, 100 MHz)
δ 164.2 (C-8), 134.8 (C-7a), 129.0 (C-2), 127.9 (C-3a), 124.2 (C-6), 123.1
(C-4), 113.9 (C-7), 113.0 (C-5), 110.2 (C-2), 48.3 (C-14), 46.1 (C-12), 36.4
(C-10), 28.6 (C-11), 26.2 (C-15); (+)-HRESIMS [M+H]+ m/z 645.1158
Fluconazole was used as a positive fungal inhibitor standard for C.
albicans and C. neoformans. The antibiotics were provided in
4
concentrations, with 2 above and 2 below its MIC value, and plated into
the first 8 wells of column 23 of the 384-well NBS plates. The quality control
(QC) of the assays was determined by the antimicrobial controls and the
Z'-factor (using positive and negative controls). Each plate was deemed to
fulfil the quality criteria (pass QC), if the Z'-factor was above 0.4, and the
antimicrobial standards showed full range of activity, with full growth
inhibition at their highest concentration, and no growth inhibition at their
lowest concentration.
(calcd for
28H3579Br81BrN6O2, 647.1164), 649.1130 (calcd for C28H3581Br2N6O2,
649.1150).
C
28H3579Br2N6O2, 645.1183), 647.1139 (calcd for
C
Determination of the MICs of antibiotics in the presence of
synergising compounds: Briefly, restoring enhancer concentrations
were determined with an inoculum of 5x105 CFU in 200 µL of Mueller-
Hinton Broth (MHB) containing two-fold serial dilutions of each derivative
in the presence of doxycycline at 2 µg/mL.[23] The lowest concentration of
the polyamine adjuvant that completely inhibited visible growth after
incubation for 18 h at 37 °C was determined. These measurements were
independently repeated in triplicate.
Biological evaluation
Antimicrobial assays: The susceptibility of bacterial strains S. aureus
(ATCC25923), E. coli (ATCC25922) and P. aeruginosa (ATCC27853) to
antibiotics and compounds was determined in microplates using the
standard broth dilution method in accordance with the recommendations
of the Comité de l’AntibioGramme de la Société Française de
Microbiologie (CA-SFM) as previously described.[23] Briefly, the minimal
inhibitory concentrations (MICs) were determined with an inoculum of 105
CFU in 200 µL of Mueller-Hinton broth (MHB) containing two-fold serial
dilutions of each drug. The MIC was defined as the lowest concentration
of drug that completely inhibited visible growth after incubation for 18 h at
37 °C. To determine all MICs, the measurements were independently
repeated in triplicate.
Cytotoxicity assays: The L6 cell line cytotoxicity assays were carried out
exactly as previously described.[26] Briefly, each well of
a 96-well
microplate contained 100 µL of RPMI 1640 medium supplemented with
1% L-glutamine (200 mM) and 10% fetal bovine serum, and 4 x 104 L6
cells (a primary cell line derived from rat skeletal myoblasts).Serial drug
dilutions of seven 3-fold dilution steps covering a range from 90 to 0.123
µg/mL were prepared. After 72 h of incubation, the plates were inspected
under an inverted microscope to assure growth of the controls and sterile
conditions. Alamar Blue solution (10 µL) was then added to each well and
the plates incubated for another 2 h. Then the plates were read with a
Spectramax Gemini XS microplate fluorometer using an excitation
wavelength of 536 nm and an emission wavelength of 588 nm. Data were
analysed using the microplate reader software Softmax Pro.
Podophyllotoxin was the reference drug used.
Additional antimicrobial evaluation against Staphylococcus aureus MRSA
(ATCC43300), Klebsiella pneumoniae (ATCC700603), Acinetobacter
baumannii (ATCC19606), Candida albicans (ATCC90028), and
Cryptococcus neoformans (ATCC208821) was undertaken at the
Community for Open Antimicrobial Drug Discovery at The University of
Queensland (Australia) according to their standard protocols.[25] For
antimicrobial assays, the tested strains were cultured in either Luria broth
(LB) (In Vitro Technologies, USB75852), nutrient broth (NB) (Becton
Dickson, 234000), or cation-adjusted MHB at 37 °C overnight. A sample
of culture was then diluted 40-fold in fresh MHB and incubated at 37 °C for
1.5−2 h. The compounds were serially diluted 2-fold across the wells of
96-well plates (Corning 3641, nonbinding surface), with compound
concentrations ranging from 0.015 to 64 μg/mL, plated in duplicate. The
resultant mid log phase cultures were diluted to the final concentration of
1 × 106 CFU/mL; then, 50 μL was added to each well of the compound
containing plates giving a final compound concentration range of 0.008 to
32 μg/mL and a cell density of 5 × 105 CFU/mL. All plates were then
covered and incubated at 37 °C for 18 h. Resazurin was added at 0.001%
final concentration to each well and incubated for 2 h before MICs were
read by eye.
In the case of the HEK-293 cytotoxicity assay, cells were counted manually
in a Neubauer haemocytometer and plated at a density of 5,000 cells/well
into each well of the 384-well plates containing the 25x (2 μL) concentrated
compounds.[25] The medium used was Dulbecco's modified eagle medium
(DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were
incubated together with the compounds for 20 h at 37 oC, 5% CO2. To
measure cytotoxicity, 5 μL (equals 100 μM final) of resazurin was added
to each well after incubation, and incubated for further 3 h at 37 oC with
5% CO2. After final incubation fluorescence intensity was measured as Fex
560/10 nm, em 590/10 nm (F560/590
) using a Tecan M1000 Pro
monochromator plate reader. CC50 values (concentration at 50%
cytotoxicity) were calculated by normalizing the fluorescence readout, with
74 μg/mL tamoxifen as negative control (0%) and normal cell growth as
positive control (100%). The concentration-dependent percentage
cytotoxicity was fitted to a dose response function (using Pipeline Pilot)
and CC50 values determined.
For the antifungal assay, fungi strains were cultured for 3 days on yeast
extract-peptone dextrose (YPD) agar at 30 °C. A yeast suspension of 1 ×
106 to 5 × 106 CFU/mL was prepared from five colonies. These stock
suspensions were diluted with yeast nitrogen base (YNB) (Becton
Dickinson, 233520) broth to a final concentration of 2.5 × 103 CFU/mL. The
compounds were serially diluted 2-fold across the wells of 96-well plates
(Corning 3641, nonbinding surface), with compound concentrations
ranging from 0.015 to 64 μg/mL and final volumes of 50 μL, plated in
duplicate. Then, 50 μL of the fungi suspension that was previously
prepared in YNB broth to the final concentration of 2.5 × 103 CFU/mL was
added to each well of the compound-containing plates, giving a final
compound concentration range of 0.008 to 32 μg/mL. Plates were covered
and incubated at 35 °C for 36 h without shaking. C. albicans MICs were
determined by measuring the absorbance at OD530. For C. neoformans,
resazurin was added at 0.006% final concentration to each well and
incubated for a further 3 h before MICs were determined by measuring the
Haemolytic assay: [25] Human whole blood was washed three times with
3 volumes of 0.9% NaCl and then resuspended in same to a concentration
of 0.5 × 108 cells/mL, as determined by manual cell count in a Neubauer
haemocytometer. The washed cells were then added to the 384-well
compound-containing plates for a final volume of 50 μL. After a 10 min
shake on a plate shaker the plates were then incubated for 1 h at 37 °C.
After incubation, the plates were centrifuged at 1,000 g for 10 min to pellet
cells and debris, 25 μL of the supernatant was then transferred to a
polystyrene 384-well assay plate. Haemolysis was determined by
measuring the supernatant absorbance at 405 mm (OD405). The
absorbance was measured using a Tecan M1000 Pro monochromator
plate reader. HC10 and HC50 (concentration at 10% and 50% haemolysis,
respectively) were calculated by curve fitting the inhibition values vs.
log(concentration) using a sigmoidal dose-response function with variable
fitting values for top, bottom and slope.
absorbance at OD570−600
.
Colistin and vancomycin were used as positive bacterial inhibitor
standards for Gram-negative and Gram-positive bacteria, respectively.
10
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10.1002/cmdc.202000359
ChemMedChem
FULL PAPER
Measurement of ATP efflux: [23] Squalamine solutions were prepared in
doubly distilled water at different concentrations. A suspension of growing
S. aureus or P. aeruginosa PAO1 to be studied in MHB was prepared and
incubated at 37°C. An aliquot of 90 µL of this suspension was added to 10
µL of squalamine solution and vortexed for 1 sec. Luciferin-luciferase
reagent (Yelen, France; 50 µL) was immediately added to the precedent
mix and luminescent signal quantified with an Infinite M200 microplate
reader (Tecan) for five sec. ATP concentration was quantified by internal
sample addition. A similar procedure was used for 14 (4 times the MIC).
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Acknowledgements
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We acknowledge funding from the Auckland Medical Research
Foundation (1116001). We thank Dr. Michael Schmitz and Tony
Chen for their assistance with the NMR and mass spectrometric
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by the Wellcome Trust (UK) and The University of Queensland
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Keywords: Indole • polyamine • adjuvant • enhancer • antibiotic
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Antibiotic adjuvant: Indole-3-acrylamido-spermine conjugates were identified as being able to enhance the action of doxycycline
towards Gram-negative bacteria, including Pseudomonas aeruginosa and Escherichia coli and with attenuated cytotoxic and
haemolytic properties. Mechanism of action experiments suggest enhancement derives from the disruption of bacterial membrane
integrity and inhibition of efflux pumps.
12
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