Exploration of the antibiotic potentiating activity of indolglyoxylpolyamines

Article abstract of DOI:10.1016/j.ejmech.2019.111708

A series of substituted di-indolglyoxylamido-spermine analogues were prepared and evaluated for intrinsic antimicrobial properties and the ability to enhance antibiotic action. As a compound class, intrinsic activity was typically observed towards Gram-positive bacteria and the fungus Cryptococcus neoformans, with notable exceptions being the 5-bromo- and 6-chloro-indole analogues which also exhibited modest activity (MIC 34–50 μM) towards the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Several analogues enhanced the activity of doxycycline towards the Gram-negative bacteria Pseudomonas aeruginosa, E. coli, K. pneumoniae and Acinetobacter baumannii. Of particular note was the identification of five antibiotic enhancing analogues (5-Br, 7-F, 5-Me, 7-Me, 7-OMe) which also exhibited low to no cytotoxicity and red blood cell haemolytic properties. The mechanisms of action of the 5-Br and 7-F analogues were attributed to the ability to disrupt the integrity of, and depolarize, bacterial membranes.

Full text of DOI:10.1016/j.ejmech.2019.111708

European Journal of Medicinal Chemistry 183 (2019) 111708
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Exploration of the antibiotic potentiating activity of
indolglyoxylpolyamines
,
Melissa M. Cadelis a, Elliot I.W. Pike a, Weirong Kang b 1, Zimei Wu b,
Marie-Lise Bourguet-Kondracki c, Marine Blanchet d, Nicolas Vidal e, Jean Michel Brunel d,
,
*
Brent R. Copp a
a School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand
b School of Pharmacy, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand
c
ꢀ
ꢀ
Laboratoire Molecules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Museum National d’Histoire Naturelle, 57 Rue Cuvier
(C.P. 54), 75005, Paris, France
d
ꢀ
Aix Marseille Univ, INSERM, SSA, MCT, Faculte de Pharmacie, 27 bd Jean Moulin, 13385, Marseille, France
e
^
YELEN, 10 bd Tempete, 13820, Ensues la Redonne, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2019
Received in revised form
29 August 2019
Accepted 15 September 2019
Available online 17 September 2019
A series of substituted di-indolglyoxylamido-spermine analogues were prepared and evaluated for
intrinsic antimicrobial properties and the ability to enhance antibiotic action. As a compound class,
intrinsic activity was typically observed towards Gram-positive bacteria and the fungus Cryptococcus
neoformans, with notable exceptions being the 5-bromo- and 6-chloro-indole analogues which also
exhibited modest activity (MIC 34e50 mM) towards the Gram-negative bacteria Escherichia coli and
Klebsiella pneumoniae. Several analogues enhanced the activity of doxycycline towards the Gram-
negative bacteria Pseudomonas aeruginosa, E. coli, K. pneumoniae and Acinetobacter baumannii. Of
particular note was the identification of five antibiotic enhancing analogues (5-Br, 7-F, 5-Me, 7-Me, 7-
OMe) which also exhibited low to no cytotoxicity and red blood cell haemolytic properties. The mech-
anisms of action of the 5-Br and 7-F analogues were attributed to the ability to disrupt the integrity of,
and depolarize, bacterial membranes.
Keywords:
Polyamine
Indole
Indolglyoxylamide
Potentiation
Antimicrobial
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
Two such examples include the marine natural product ianthelli-
formisamine C 1 and 2-aminoimidazole analogues such as 2 (Fig. 1)
Given the noted difficulties associated with the discovery and
development of novel antibiotics [1e3], and particularly those with
activity towards Gram-negative bacteria [4,5], an attractive
approach for overcoming antimicrobial resistance is the identifi-
cation of antibiotic adjuvants [6e8]. Such compounds, when co-
dosed with antibiotics, have the potential to restore the action of
an antibiotic towards bacteria that have developed resistance to
the former which acts via bacterial membrane depolarization [11]
while the latter compound class causes potentiation by disruption
of the VraSR two-component system associated with cell wall
biosynthesis [12].
We recently reported polyamine functionalised indolglyox-
ylamide 3 (Fig. 1) as a potentiator of antibiotic activity towards
several examples of clinically important Gram-negative bacteria
including Pseudomonas aeruginosa [13]. That same study provided
insight into some of the structural elements required for potenti-
ation, namely an intact polyamine core of suitable overall length.
The biological activities of 3, which also included intrinsic anti-
bacterial activity towards Gram-positive bacteria and fungi, were
attributed to the molecules ability to disrupt the integrity of, and
depolarize, bacterial membranes. This ability to disrupt membranes
also extended to mammalian cells, with 3 exhibiting cytotoxicity
and also strong red blood cell haemolytic activities. We used 3 as a
that antibiotic. Beyond the classical adjuvant example, the
b-lac-
tamase inhibitor clavulanic acid co-dosed with the -lactam anti-
b
biotic amoxicillin, a growing number of chemical scaffolds have
been identified as being able to enhance antibiotic action [9,10].
* Corresponding author.
E-mail address: b.copp@auckland.ac.nz (B.R. Copp).
Current Address: School of Chinese Medicine, Hong Kong Baptist University,
1
Kowloon, 999077, Hong Kong
https://doi.org/10.1016/j.ejmech.2019.111708
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
Fig. 1. Structures of antibiotic potentiators 1e3.
starting point for a new study, reported herein, that explored the
effect of variation of substitution on the indolglyoxylamide frag-
ment in search for new potentiators that were devoid of cytotoxic/
haemolytic liabilities.
2. Chemistry
A library of analogues of were prepared, exploring the influence
of the 6-bromo substituent present in 3. The method used for the
synthesis of target compounds 4e19, summarised in Scheme 1, was
comprised of a three-step sequence starting with conversion of
commercially available or previously reported substituted indoles
to their corresponding 3-glyoxylchloride analogue. Subsequent
reaction with the Boc-protected PA3-4-3 di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carboxylate) afforded Boc protected in-
termediates that were then directly subjected to Boc group
deprotection (with TFA in CH2Cl2 or MeOH) to give tetramine di-
amides 4e19 as their di-TFA salts. Preparation of the 4-fluoroindole
analogue by this glyoxylchloride method gave the desired product
(20) in very low (<10%) yield e an alternative preparation via re-
action of 2-(4-fluoro-1H-indol-3-yl)-2-oxoacetic acid [14] with
spermine mediated by the coupling agent PyBOP in DMF afforded
20 in a marginally better yield of 14% (Scheme 2). The set of ana-
logues was rounded off with des-bromo 21 and three methoxy
derivatives (22e24) previously reported by us (Fig. 2) [15].
Scheme 1. General synthesis of indolglyoxylamide analogues 4e19. Reagents and
conditions: a) Oxalyl chloride (2 eq.), Et2O, 0 ꢀC, 3 h; b) Di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (0.5 eq.), DIPEA (3 eq.), DMF, r.t., 48 h; c) TFA
(0.2 mL), CH2Cl2/MeOH (2 mL), r.t., 3 h.
3. Results and discussion
Scheme 2. Synthesis of 4-fluoroindolglyoxylamide analogue 20. Reagents and condi-
tions: a) PyBOP (1 eq.), triethylamine (2.5 eq.), DMF, N2, r.t., 48 h.
We first evaluated the intrinsic antimicrobial activity of each
compound against a range of Gram-positive (Staphylococcus aureus
and Staphylococcus intermedius) and Gram-negative (Pseudomonas
aeruginosa, Escherichia coli, Klebsiella pneumoniae and Acinetobacter
baumannii) bacteria and two fungal strains (Candida albicans and
Cryptococcus neoformans) (Table 1). As with the original 6-
bromoindole hit compound 3, a number of the indolglyoxylamide
analogues demonstrated some level of antimicrobial activity
against Gram-positive bacteria and the fungus Cryptococcus neo-
formans. Notably analogues 4 (5-Br) and 10 (6-Cl) were the most
potent, exhibiting broad spectrum antimicrobial activity against
The ability of compounds 3e24 to enhance the antibiotic ac-
tivity of doxycycline against P. aeruginosa ATCC27853 was deter-
mined. These tests used a fixed concentration of doxycycline of
2
m
g/mL (4.5
[40 g/mL (90
pounds were then evaluated at a range of concentrations varying
from 3.125 to 50e100 M, with the upper concentration dependent
m
M), which is twenty-fold lower than the intrinsic MIC
m
mM)] against this organism. Each of the test com-
m
upon the compounds intrinsic MIC towards P. aeruginosa. From all
the compounds examined (Table 2), 5-bromoindole 4 and 7-
fluoroindole 8 analogues were the most effective potentiators
S. aureus (MIC 3.125e6.25
(MIC 25e50 M), K. pneumoniae (MIC 34.4e38.1
C. neoformans (MIC 2.2e4.8
mM), S. intermedius (MIC 3.125 mM), E. coli
(restoring the action of doxycycline at 3.125
by 7-methoxy 24 (3.75 M) with 6-chloroindole 10, 7-
methylcarboxylateindole 17, 5-methylindole 18, and 7-
mM), followed closely
m
mM) and
m
m
M). The results indicated that
C. neoformans was the most susceptible test organism, with ten
analogues exhibiting MIC <10 M, while the compound class was
only poorly active or inactive towards Gram-negative bacteria.
methylindole 19 analogues being equipotent (6.25
original hit compound 3.
mM) to our
m

M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
3
acid against four Gram-negative ESKAPE pathogens P. aeruginosa
ATCC27853, E. coli ATCC25922, K. pneumoniae ATCC13443 and
A. baumannii AYE. In addition to original hit compound 3, both
compounds enhanced the action of doxycycline towards E. coli
and A. baumannii (Table 3). Interestingly the two bromo ana-
logues (6-bromo 3 and 5-bromo 4) enhanced the action of
doxycycline, and to a lesser degree nalidixic acid, towards
K. pneumoniae.
The cytotoxicity of compounds 3e24 were evaluated against rat
skeletal muscle (L6) and human embryonic kidney (HEK-293) cell
lines. Other than the previously reported lead compound 3, only
the 5,6-dibromoindole (5) and 7-chloroindole (11) analogues
exhibited cytotoxicity against L6 cells (IC50 6.03 and 7.47
respectively) with all other compounds being weakly cytotoxic
with IC50 values ranging from 19.8 to >100 M (Table 4). The HEK-
293 cell line testing identified 5-chloroindole 9, 6-
mM,
m
trifluoromethylindole 12 and 7-methylcarboxylateindole 17 as
also exhibiting cytotoxicity. Pronounced haemolytic activity to-
wards rat red blood cells was observed for 6-bromoindole 3 with an
HC50 (concentration at which 50% of red blood cells were lysed) of
Fig. 2. Des-bromo (21) and methoxy analogues 22e24 [15].
Table 1
Antimicrobial and antifungal activities of analogues 3e24.
20 mM, and for 5,6-dibromoindole 5 (HC50 17 mM), with weaker
activity observed for 5-bromoindole 4, 6-chloroindole 10 and 7-
methylcarboxylateindole 17 (Table 4). Modest to no haemolytic
activities were observed for the other compounds in the library.
Combined analysis of doxycycline potentiating activity with cyto-
toxicity and haemolytic data for the test compounds identified five
analogues of particular interest (i.e. antibiotic potentiating with
limited/no cytotoxicity and haemolytic properties): 5-bromoindole
MIC (
S. aa
mM)
S. ib
P. ac
E. cd
K. pe
A. bf
C. ag
17.2
>34.4
>29.4
>39.6
>39.6
>39.6
38.0
C. nh
i
i
i
i
i
i
i
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
6.25
3.125 i
3.125
n.t. j
50
>200
>200
n.t.
3.125
n.t.
50
100
50
100
200
>200
200
>100
100
100
25
50
200
>200
200
>200
50
200
100
>200
200
200
>200
200
200
>200
100
>100
>200
>100
>200
>34
>34
1.1
3.125
25
34.4
>34.4
>29.4
>39.6
>39.6
>39.6
>38.0
38.1
>38.1
>35.2
>38.9
>38.9
>36.0
>36.0
>36.0
>40.0
>40.0
>55.1
>41.4
>38.4
>38.4
>38.4
2.2
3.65
9.9
19.8
4.9
>38.0
4.8
19.1
4.40
38.9
38.9
>36.0
9.0
>29.4
>39.6
>39.6
>39.6
>38.0
38.1
25
100
25
4 [3.125
modestly haemolytic (34.7% haemolysis at 50
8 (3.125 M, IC50 18.7e38.7 M, 8.4% haemolysis at 50
methylindole 18 (6.25 M, IC50 63.8 M, 0.3% at 50
methylindole 19 (6.25 M, IC50 59.6 M, 0.1% at 50 M) and 7-
methoxyindole analogue 24 [3.75 M, IC50 27e62 M and not
haemolytic (0% at 50 M)].
m
M potentiation, weakly cytotoxic IC50 14.1e19.8
M)], 7-fluoroindole
M), 5-
M), 7-
mM, and
m
>200
6.25
50
m
m
m
m
38.1
>38.1
35.2
m
m
200
200
>38.1
35.2
50
m
m
m
100
100
100
>200
25
>200
25
>200
>200
200
>200
200
100
>200
140
>100
>200
>100
>200
>38.9
>38.9
>36.0
>36.0
>36.0
>40.0
>40.0
>55.1
>41.4
>38.4
>38.4
>38.4
>38.9
>38.9
>36.0
>36.0
>36.0
>40.0
>40.0
>55.1
>41.4
>38.4
>38.4
38.4
m
m
m
6.25
12.5
>200
>200
12.5
35
>100
50
>100
n.t.
Potential mechanisms of antibiotic potentiation include per-
meabilization of the outer membrane or membrane depolariza-
tion [9,10]. Compounds 4 and 8 were selected as the model
compounds for these studies due to their abilities to potently
enhance the action of antibiotics but with only limited cytotoxic/
haemolytic properties. In the first set of studies, a biolumines-
cence method was used to detect extra-cellular concentrations of
ATP that resulted from membrane permeabilization. Brief expo-
sure (1 min) of either compound to bacterial cells induced dose-
dependent increases in extracellular ATP levels in both S. aureus
(Fig. 3 left) and, to a lesser extent, in P. aeruginosa (Fig. 3 right)
suggesting both polyamine derivatives can disrupt the integrity
of bacterial membranes. Both compounds were less effective
than the known membrane disrupting steroidal-polyamine
squalamine [16].
9.0
25
25
10.0
10.0
>55.1
10.3
19.2
19.2
>38.4
>70
>100
100
>100
15
a
Staphylococcus aureus ATCC25923 with streptomycin (MIC 21.5
chloramphenicol (MIC 1.5e3 mM) used as positive controls and values presented as
m
M) and
the mean (n ¼ 3).
b
Staphylococcus intermedius 1051997 with streptomycin (MIC 10.7
m
M) and
chloramphenicol (MIC 3e6
m
M) used as positive controls and values presented as
the mean (n ¼ 3).
c
Pseudomonas aeruginosa ATCC27853 with streptomycin (MIC 21.5
m
M) and
colistin (MIC 1
m
M) used as positive controls and values presented as the mean
(n ¼ 3).
Further evidence for the ability of the test compounds to disrupt
the outer bacterial membrane was determined in the Gram-
negative bacterium Enterobacter aerogenes EA289 using a nitro-
cefin colorimetric assay. The basis of this assay is that membrane
disruption leads to ingress of a chromogenic cephalosporin that
d
Escherichia coli ATCC25922 with streptomycin (MIC 21.5
m
M) and colistin (MIC
2
m
M) used as positive controls and values presented as the mean (n ¼ 3).
e
Klebsiella pneumoniae ATCC700603 with colistin (MIC 0.25
m
g/mL) as a positive
g/mL) as a positive
g/mL) as a positive
g/mL) as a
control and values presented as the mean (n ¼ 2).
f
Acinetobacter baumannii ATCC19606 with colistin (MIC 0.25
m
control and values presented as the mean (n ¼ 2).
acts as a substrate for periplasmic b-lactamase, whereupon b-lac-
g
Candida albicans ATCC90028 with fluconazole (MIC 0.125
m
tam hydrolysis leads to a color change from yellow to red. As pre-
sented in Fig. 4, dose-dependent effect on the rate of nitrocefin
hydrolysis was observed for both 4 and 8, with the former being
more potent.
control and values presented as the mean (n ¼ 2).
h
Cryptococcus neoformans ATCC208821 with fluconazole (MIC 8
positive control and values presented as the mean (n ¼ 2).
m
i
Data taken from Li et al. [13].
j
Not tested.
Many drug efflux pumps function via an energy-dependent
mechanism that makes use of membrane proton gradients [17,18].
Compounds 4 and 8 were investigated for their ability to act as
disruptors of the bacterial transmembrane potential by using the
membrane-potential-sensitive probe DiSC3(5) which concentrates
The spectrum of potentiation of the 5-bromoindole 4 and 7-
methoxyindole 24 analogues was then evaluated for the antibi-
otics doxycycline, erythromycin, chloramphenicol and nalidixic

4
M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
Table 2
Doxycycline potentiation activity of analogues 3e24.
Compound
Conc (
m
M) for potentiationa
Compound
Conc (m
M) for potentiationa
3
4
5
6
7
8
9
10
11
12
13
6.25 b
3.125
25
12.5
25
3.125
14.9
6.25
25
100
50
14
15
16
17
18
19
20
21
22
23
24
50
25
25
6.25
6.25
6.25
35
>50
12.5
50
3.75
a
Concentration (
Data taken from Li et al. [13].
m
M) required to restore doxycycline activity at 2
m
g/mL (4.5
m
M) against P. aeruginosa ATCC27853.
b
Table 3
Antibiotic potentiating activity of analogues 3, 4 and 24.
Antibiotic
Compound
Concentration (
m
M) for potentiationa
P. aeruginosab
E. colic
K. pneumoniaed
A. baumanniie
f
f
Doxycycline
Erythromycin
Chloramphenicol
Nalidixic acid
3
4
24
3
4
24
3
4
24
3
12.5 f
6.25
7.50
200
>200
6.25
6.25 f
13.4
25
1.68
3.75
6.72
15
>200
f
f
f
f
50
50
25
n.t.
g
n.t.
n.t.
100
>200
>200
>200
>200
>200
>200
f
f
f
f
200
26.9
30.0
n.t.
n.t.
n.t.
>200
>200
>200
>200
200
f
f
f
f
50
25
4
>200
100
12.5
100
24
60
>200
>200
>200
a
Concentration (
m
M) of compound required to restore antibiotics activity at 2
m
g/mL concentration of antibiotic.
b
Pseudomonas aeruginosa ATCC27853 against doxycycline (MIC 90
m
M), erythromycin (MIC >200
m
M), chloramphenicol (MIC >200
m
M) and nalidixic acid (MIC >200 mM)
and values presented as the mean (n ¼ 3).
c
Escherichia coli ATCC25922 against doxycycline (MIC 25
m
M) erythromycin (MIC >200
m
M), chloramphenicol (MIC >200
M) erythromycin (MIC >200 M), chloramphenicol (MIC 50
M), chloramphenicol (MIC >200 m
m
M) and nalidixic acid (MIC >200
M) and nalidixic acid (MIC 100
M) and nalidixic acid (MIC >200
mM) and values
presented as the mean (n ¼ 3).
d
Klebsiella pneumoniae ATCC13443 against doxycycline (MIC 25
m
m
m
m
M) and
M) and
values presented as the mean (n ¼ 3).
e
Acinetobacter baumannii AYE against doxycycline (MIC 12.5
values presented as the mean (n ¼ 3).
m
M) erythromycin (MIC 200
m
m
f
Data taken from Li et al. [13].
Not tested.
g
at the inner membrane level and quenches its own fluorescence.
When a compound impairs the membrane potential, the dye is
released leading to an increase in fluorescence. After 15 min incu-
bation, modest membrane depolarization was observed against
on the indolglyoxylamide head-group. Of note was the discovery of
four analogues, bearing 7-methoxy, 7-fluoro, 7-methyl or 5-methyl
indole substitution, that were either equipotent or more potent in
their ability to enhance the action of doxycycline than the original
hit compound. The relative location of these substituents appears
to be a critical factor for potentiating activity. Of these four new
potentiators, the 7-methoxy, 5-methyl and 7-methyl substituted
variants were 3-10-fold less cytotoxic and had little or negligible
haemolytic effect on red blood cells. Cell-based assays identified
the 5-bromo and 7-fluoro analogues as being capable of per-
meabilizing bacterial membranes, likely having a bearing on their
intrinsic antibacterial properties, while both were also capable of
depolarizing membranes, a possible cause of their antibiotic
potentiating activities. Current studies are directed towards the
discovery of more potent antibiotic enhancers and their evaluation
in in vivo models of infection.
Gram-positive S. aureus (Fig.
5
left) and Gram-negative
P. aeruginosa (Fig. 5 right) strains for both 4 and 8.
Collectively the ATP release, nitrocefin hydrolysis and DiSC3(5)
membrane potential assay results suggest that the intrinsic anti-
microbial activities of compounds 4 and 8 could be due to their
abilities to disrupt bacterial membranes while their antibiotic
potentiating activities may stem from their ability to depolarize
membranes leading to efflux pump inhibition.
4. Conclusions
Our
previous
screening
identified
N1,N14-di-(6-
bromoindolglyoxylamido)-spermine to be a potent enhancer of
the action of doxycycline towards P. aeruginosa and that the poly-
amine core of the molecule was essential for activity. A limitation of
the compound however was the observation of cytotoxicity and
haemolytic properties. The present study has expanded this
structure-activity relationship to include variation of substituents
5. Experimental
5.1. Chemistry: general methods
Infrared spectra were recorded on a PerkinElmer spectrometer

M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
5
Table 4
Cytotoxic and haemolytic properties of analogues 3e24.
Compound
Cytotoxicity
Haemolysis
L6 a IC50
(m
M)
HEK-293 b CC50
(
mM)
% haemolysis at 50 mM (HC50 m
M) c
d
e
3
7.7
5.06
91 (20)
4
5
6
7
8
9
19.8
6.03
71.1
69.1
38.7
19.8
33.2
7.47
31.4
37.1
46.9
41.8
63.6
34.4
63.8
59.6
167
14.1
1.62
>39.6
12.5
18.7
4.73
19.1
31.6
3.36
>38.9
>38.9
>36.0
>36.0
3.78
>40.0
>40.0
>55
>41.5
>38.4
>38.4
27.1
34.7
88.7 (17)
0
0.3
8.4
0
11.6
1.1
3.2
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
0
0.6
15.0
0.3
0.1
0.3
0
0
0
0
d
60
56
54
d
d
d
62
a
L6 rat skeletal myoblast cell line with podophyllotoxin as the positive control (IC50 0.018
m
M) and values presented as the mean (n ¼ 2).
Concentration of compound at 50% cytotoxicity on HEK293 human embryonic kidney cells and values presented as the mean (n ¼ 2). Highest dose tested was 32
Percentage haemolysis detected at 540 nm at 50 M concentration of compound with 0.1% Triton X-100 as the positive control (100% hemolysis) with values presented
b
c
m
g/mL.
m
as the mean (n ¼ 3). Where given the value in parenthesis is the concentration (
mM) of compound required to induce 50% haemolysis.
d
Data taken from Wang et al. [15].
e
Data taken from Li et al. [13].
Fig. 3. Dose-dependent ATP release for S. aureus (left) and P. aeruginosa (right) exhibited by 4 and 8 with squalamine as the positive control.
Bruker Avance DRX-400 spectrometer operating at 400 MHz for 1H
nuclei and 100 MHz for 13C nuclei. Proto-deutero solvent signals
were used as internal references (DMSO‑d6: dH 2.50, dC 39.52). For
1H NMR, the data are quoted as position (
d), relative integral,
multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
m ¼ multiplet, br ¼ broad), coupling constant (J, Hz), and assign-
ment to the atom. The 13C NMR data are quoted as position (
d), and
assignment to the atom. Flash column chromatography was carried
out using either Davisil silica gel (40e60 m) or Merck LiChroprep
RP-8 (40e63 m). Thin layer chromatography was conducted on
m
m
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. Di-tert-butyl butane-
1,4-diylbis((3-aminopropyl)carbamate) [19], 5,6-dibromoindole
[20], 2-(4-fluoro-1H-indol-3-yl)-2-oxoacetic acid [14], and 21e24
[15] were prepared according to a literature procedures.
Fig. 4. Dose-dependent effect of 4 and 8 on the rate of nitrocefin hydrolysis in
E. aerogenes EA289 resulting from outer membrane permeabilization.
100 Fourier Transform infrared spectrometer equipped with a
universal ATR accessory. HRMS data were acquired on a Bruker
micrOTOF QII spectrometer. NMR spectra were recorded on a

6
M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
Fig. 5. Dose-dependent depolarization by 4 and 8 of the bacterial membranes of S. aureus (left) and P. aeruginosa (right) with cetyltrimethylammonium bromide (CTAB) (0.01%) as
the positive control.
5.2. Synthesis of compounds
H2-15, H2-150), 1.89e1.81 (4H, m, H2-12, H2-120), 1.67e1.57 (4H, m,
H2-16, H2-160); 13C NMR (DMSO‑d6, 100 MHz)
d
181.7 (C-8, C-80),
5.2.1. General procedure A e synthesis of substituted 3-
indolglyoxylyl chlorides
163.4 (C-9, C-90), 139.5 (C-2, C-20), 135.1 (C-7a, C-7a0), 128.1 (C-3a, C-
3a0), 126.2 (C-6, C-60), 123.4 (C-4, C-40), 115.4 (C-5, C-50), 114.8 (C-7,
C-70), 111.6 (C-3, C-30), 46.1 (C-15, C-150), 44.8 (C-13, C-130), 35.9 (C-
To a solution of indole (1 equiv.) in anhydrous diethyl ether
(12 mL) was added oxalyl chloride (1.15 equiv.) at 0 ꢀC under N2
atmosphere. The reaction mixture was stirred for 3 h before solvent
removal under reduced pressure. The product was used in the next
step without purification.
11, C-110), 25.7 (C-12, C-120), 22.8 (C-16, C-160); (þ)-HRESIMS
79
[MþNa]þ m/z 723.0912 (calcd for C30
H Br2N6O4Na, 723.0900),
34
79
725.0909 (calcd for C
H
Br81BrN6O4Na, 725.0883), 727.0885
30 34
81
(calcd for C
H Br2N6O4Na, 727.0869).
30 34
5.2.3.2. N1,N4 -Bis(3-(2-(5,6-dibromo-1H-indol-3-yl)-2-
oxoacetamido) propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate
(5). Using general procedure A, reaction of 5,6-dibromo-1H-indole
[20] (207 mg, 0.75 mmol) and oxalyl chloride (0.087 mL, 0.90 mmol)
afforded 2-(5,6-dibromo-1H-indol-3-yl)-2-oxoacetyl chloride as a
yellow powder. Using general procedure B, a sub-sample of the
glyoxylyl chloride (105 mg, 0.289 mmol) was reacted with di-tert-
5.2.2. General procedure B e coupling of 3-indolglyoxylyl chlorides
with Boc protected polyamine
To a solution of 3-indolglyoxylyl chloride (2 equiv.) in DMF
(1 mL) was added DIPEA (6 equiv.) and di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (1 equiv.) in DMF (1 mL). Sub-
sequently, the reaction mixture was stirred for 48 h before solvent
removal under reduced pressure. The crude product was purified
using silica gel flash column chromatography (3% MeOH:CH2Cl2).
butyl
butane-1,4-diylbis((3-aminopropyl)carbamate)
(50 mg,
0.124 mmol) and DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to
afford, after chromatography, di-tert-butyl butane-1,4-diylbis((3-(2-
(5,6-dibromo-1H-indol-3-yl)-2-oxoacetamido)propyl)carbamate) as
a yellow oil (39 mg, 41%). Using general procedure C, this material
(39 mg, 0.037 mmol) wasdeprotected to afford thedi-TFA salt of 5 asa
red oil (25 mg, 78%). Rf (MeOH/10% HCl, 7:3) 0.15; IR (ATR) nmax 3350,
5.2.3. General procedure C eBoc deprotection
A solution of tert-butyl-carbamate derivative in CH2Cl2 (2 mL)
and TFA (0.2 mL) was stirred at room temperature under N2 for 2 h
followed by solvent removal under reduced pressure. The crude
product was purified using C8 reversed-phase flash column chro-
matography eluting with 0%e50% MeOH/H2O (0.05% TFA) to afford
the corresponding polyamine as a TFA salt.
3104, 2259, 1672, 1623, 1439, 1199, 1180, 1127 cmꢁ1 1H NMR
;
(DMSO‑d6, 400 MHz)
d
12.57 (2H, d, J ¼ 2.7 Hz, NH-1, NH-10), 8.95 (2H,
t, J ¼ 5.9 Hz, NH-10, NH-100), 8.84 (2H, d, J ¼ 3.1 Hz, H-2, H-20), 8.61
(4H, br s, NH2-14, NH2-140), 8.53 (2H, s, H-4, H-40), 7.99 (2H, s, H-7, H-
70), 3.34e3.26 (4H, m, H2-11, H2-110), 3.00e2.90 (8H, m, H2-13, H2-130,
H2-15, H2-150),1.91e1.81 (4H, m, H2-12, H2-120),1.67e1.60 (4H, m, H2-
5.2.3.1. N1,N4-Bis(3-(2-(5-bromo-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (4). Using gen-
eral procedure A, reaction of 5-bromo-1H-indole (392 mg, 2 mmol)
and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(5-bromo-1H-
indol-3-yl)-2-oxoacetyl chloride as a yellow powder. Using general
procedure B, a sub-sample of the glyoxylyl chloride (71.1 mg,
0.248 mmol) was reacted with di-tert-butyl butane-1,4-diylbis((3-
aminopropyl)carbamate) (50 mg, 0.124 mmol) and DIPEA
(0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chromatog-
raphy, di-tert-butyl butane-1,4-diylbis((3-(2-(5-bromo-1H-indol-3-
yl)-2-oxoacetamido)propyl)carbamate) as a yellow gum (36 mg,
38%). Using general procedure C, a sub-sample of this material
(36 mg, 0.040 mmol) was deprotected to afford the di-TFA salt of 4
as a brown oil (36 mg, quant. yield) which required no further
purification. Rf (MeOH/10% HCl, 7:3) 0.63; IR (ATR) nmax 3152, 2981,
16, H2-160); 13C NMR (DMSO‑d6,100 MHz) 181.6 (C-8, C-80),163.1 (C-
d
9, C-90), 140.4 (C-2, C-20), 136.1 (C-7a, C-7a0), 127.1 (C-3a, C-3a0), 125.2
(C-4, C-40), 117.6 (C-7, C-70, C-5/C-6, C-5'/C-60), 117.3 (C-5/C-6, C-5'/C-
60),111.4 (C-3, C-30), 46.1 (C-15, C-150), 44.7 (C-13, C-130), 35.9 (C-11, C-
110), 25.6 (C-12, C-120), 22.7 (C-16, C-160); (þ)-HRESIMS [MþH]þ m/z
79
856.9308 (calcd for C
H Br4N6O4, 856.9291), 858.9293 (calcd for
30 33
79
79
C
H
Br831BrN6O4, 858.9272), 860.9278(calcdforC30 Br821Br2N6O4,
H
33
30 33
79
860.9254), 862.9261 (calcd for
864.9248 (calcd for C
C H
Br81Br3N6O4, 862.9237),
30 33
81
H Br4N6O4, 864.9226).
30 33
5.2.3.3. N1,N4-Bis(3-(2-(5-fluoro-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (6). Using gen-
eral procedure A, reaction of 5-fluoro-1H-indole (270 mg, 2 mmol)
and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(5-fluoro-1H-
indol-3-yl)-2-oxoacetyl chloride as a yellow powder. Using general
procedure B, a sub-sample of the glyoxylyl chloride (52 mg,
0.251 mmol) was reacted with di-tert-butyl butane-1,4-diylbis((3-
aminopropyl)carbamate) (50 mg, 0.124 mmol) and DIPEA
1671, 1623, 1501, 1430, 1199, 1130 cmꢁ1
;
1H NMR (DMSO‑d6,
400 MHz)
d
12.49 (2H, br s, NH-1, NH-10), 8.92 (2H, t, J ¼ 6.1 Hz, NH-
10, NH-100), 8.80 (2H, d, J ¼ 3.3 Hz, H-2, H-20), 8.55 (4H, br s, NH2-
14, NH2-140), 8.35 (2H, d, J ¼ 1.9 Hz, H-4, H-40), 7.53 (2H, d,
J ¼ 8.7 Hz, H-7, H-70), 7.42 (2H, dd, J ¼ 4.3, 1.7 Hz, H-6, H-60),
3.33e3.26 (4H, m, H2-11, H2-110), 3.00e2.88 (8H, m, H2-13, H2-130,

M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
7
(0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after silica gel
(39 mg, 0.050 mmol) was deprotected to afford the di-TFA salt of 8
column chromatography, di-tert-butyl butane-1,4-diylbis((3-(2-(5-
as a brown oil (17 mg, 44%). Rf (MeOH/10% HCl, 7:3) 0.43; IR (ATR)
fluoro-1H-indol-3-yl)-2-oxoacetamido)propyl)carbamate) as
a
nmax 3250, 2840, 1667, 1606, 1438, 1200, 1176, 1126 cmꢁ1 1H NMR
;
yellow oil (72 mg, 74%). Using general procedure C, a sub-sample of
this material (37 mg, 0.047 mmol) was then deprotected to afford
the di-TFA salt of 6 as a yellow oil (28 mg, 76%). Rf (MeOH/10% HCl,
7:3) 0.42; IR (ATR) nmax 3384, 2973, 2109, 1702, 1647, 1368,
(DMSO‑d6, 400 MHz)
d
12.90 (2H, d, J ¼ 7.6 Hz, NH-1, NH-10), 8.94
(2H, t, J ¼ 6.0 Hz, NH-10, NH-100), 8.78 (2H, d, J ¼ 3.1 Hz, H-2, H-20),
8.59 (4H, br s, NH2-14, NH2-140), 8.04 (2H, d, J ¼ 8.1 Hz, H-4, H-40),
7.25 (2H, ddd, J ¼ 7.9, 7.9, 4.8 Hz, H-5, H-50), 7.13 (2H, dd, J ¼ 11.2,
7.9 Hz, H-6, H-60), 3.34e3.27 (4H, m, H2-11, H2-110), 3.00e2.90 (8H,
m, H2-13, H2-130, H2-15, H2-150), 1.91e1.81 (4H, m, H2-12, H2-120),
1.67e1.58 (4H, m, H2-16, H2-160); 13C NMR (DMSO‑d6, 100 MHz)
1231 cmꢁ1 1H NMR (DMSO‑d6, 400 MHz)
; d 12.39 (2H, br s, NH-1,
NH-10), 8.92 (2H, t, J ¼ 6.0 Hz, NH-10, NH-100), 8.81 (2H, d,
J ¼ 3.2 Hz, H-2, H-20), 8.49 (4H, br s, NH2-14, NH2-140), 7.89 (2H, dd,
J ¼ 8.5, 2.6 Hz, H-4, H-40), 7.57 (2H, dd, J ¼ 8.9, 4.3 Hz, H-7, H-70), 7.13
(2H, ddd, J ¼ 9.2, 6.6, 2.7 Hz, H-6, H-60), 3.32e3.27 (4H, m, H2-11,
H2-110), 3.00e2.88 (8H, m, H2-13, H2-130, H2-15, H2-150), 1.89e1.82
(4H, m, H2-12, H2-120), 1.67e1.53 (4H, m, H2-16, H2-160); 13C NMR
d
181.9 (C-8, C-80), 163.4 (C-9, C-90), 158.4 (q, 2JCF ¼ 35.5 Hz, C-18, C-
180), 149.2 (d, 1JCF ¼ 245.7 Hz, C-7, C-70), 138.8 (C-2, C-20), 129.8 (d,
3JCF ¼ 4.3 Hz, C-3a, C-3a0), 124.0 (d, 2JCF ¼ 13.3 Hz, C-7a, C-7a0), 123.4
3
(d, JCF ¼ 5.9 Hz, C-5, C-50), 117.4 (C-4, C-40), 112.8 (C-3, C-30), 108.7
2
(DMSO‑d6, 100 MHz)
d
181.7 (C-8, C-80), 163.6 (C-9, C-90), 159.2 (d,
(d, JCF ¼ 15.5 Hz, C-6, C-60), 46.1 (C-15, C-150), 44.7 (C-13, C-130),
1JCF ¼ 251.2 Hz, C-5, C-50), 158.8 (q, 2JCF ¼ 25.5 Hz, C-18, C-180),139.9
(C-2, C-20), 133.0 (C-7a, C-7a0), 127.0 (C-3a, C-3a0), 114.1 (d,
3JCF ¼ 10.2 Hz, C-7, C-70), 112.3 (C-3, C-30), 111.7 (d, 2JCF ¼ 25.6 Hz, C-
35.9 (C-11, C-110), 25.6 (C-12, C-120), 22.7 (C-16, C-160); (þ)-HRE-
SIMS [MþH]þ m/z 581.2679 (calcd for C30H35F2N6O4, 581.2682).
2
6, C-60), 106.3 (d, JCF ¼ 25.5 Hz, C-4, C-40), 46.2 (C-15, C-150), 44.9
5.2.3.6. N1,N4-Bis(3-(2-(5-chloro-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (9). Using gen-
eral procedure A, reaction of 5-chloro-1H-indole (303 mg, 2 mmol)
and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(5-chloro-1H-
indol-3-yl)-2-oxoacetyl chloride as a yellow powder. Using general
procedure B, a sub-sample of the glyoxylyl chloride (60 mg,
0.248 mmol) was reacted with di-tert-butyl butane-1,4-diylbis((3-
aminopropyl)carbamate) (50 mg, 0.124 mmol) and DIPEA
(0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chromatog-
raphy, di-tert-butyl butane-1,4-diylbis((3-(2-(5-chloro-1H-indol-3-
yl)-2-oxoacetamido)propyl)carbamate) as a yellow gum (10 mg,
10%). Using general procedure C, this material (10 mg, 0.012 mmol)
was deprotected to afford the di-TFA salt of 9 as a brown oil (5.0 mg,
67%). Rf (MeOH/10% HCl, 7:3) 0.43; IR (ATR) nmax 3374, 2977, 1676,
1426, 1132, 1034, 953, 817, 723 cmꢁ1; 1H NMR (400 MHz, DMSO‑d6)
(C-13, C-130), 35.9 (C-11, C-110), 25.8 (C-12, C-120), 22.8 (C-16, C-160);
(þ)-HRESIMS [MþH]þ m/z 581.2682 (calcd for C30H35F2N6O4,
581.2682).
5.2.3.4. N1,N4-Bis(3-(2-(6-fluoro-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (7). Using gen-
eral procedure A, reaction of 6-fluoro-1H-indole (270 mg, 2 mmol)
and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(6-fluoro-1H-
indol-3-yl)-2-oxoacetyl chloride as a yellow powder. Using general
procedure B, a sub-sample of the glyoxylyl chloride (52 mg,
0.251 mmol) was reacted with di-tert-butyl butane-1,4-diylbis((3-
aminopropyl)carbamate) (50 mg, 0.124 mmol) and DIPEA
(0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chromatog-
raphy, di-tert-butyl butane-1,4-diylbis((3-(2-(6-fluoro-1H-indol-3-
yl)-2-oxoacetamido)propyl)carbamate) as a yellow gum (62 mg,
64%). Using general procedure C, a sub-sample of this material
(26 mg, 0.033 mmol) was deprotected to afford the di-TFA salt of 7
as a yellow oil (16 mg, 65%). Rf (MeOH/10% HCl, 7:3) 0.41; IR (ATR)
d
12.52 (2H, d, J ¼ 3.3 Hz, NH-1, NH-10), 8.92 (2H, t, J ¼ 5.5 Hz, NH-
10, NH-100), 8.81 (2H, d, J ¼ 3.3 Hz, H-2, H-20), 8.66e8.58 (4H, m,
NH2-14, NH2-140), 8.20 (2H, d, J ¼ 2.0 Hz, H-4, H-40), 7.58 (2H, d,
J ¼ 8.7 Hz, H-7, H-70), 7.30 (2H, dd, J ¼ 8.7, 2.0 Hz, H-6, H-60),
3.33e3.28 (4H, m, H2-11, H2-110), 2.98e2.90 (8H, m, H2-13, H2-130,
H2-15, H2-150), 1.90e1.82 (4H, m, H2-12, H2-120), 1.66e1.60 (4H, m,
nmax 2963, 2850, 1672, 1624, 1513, 1496, 1447, 1200, 1131 cmꢁ1
;
1H
NMR (DMSO‑d6, 400 MHz)
d
12.35 (2H, br s, NH-1, NH-10), 8.91 (2H,
t, J ¼ 6.1 Hz, NH-10, NH-100), 8.77 (2H, d, J ¼ 3.0 Hz, H-2, H-20), 8.57
(4H, br s, NH2-14, NH2-140), 8.20 (2H, dd, J ¼ 8.7, 5.5 Hz, H-4, H-40),
7.35 (2H, dd, J ¼ 9.6, 2.4 Hz, H-7, H-70), 7.12 (2H, td, J ¼ 9.1, 2.3 Hz, H-
5, H-50), 3.34e3.27 (4H, m, H2-11, H2-110), 3.00e2.90 (8H, m, H2-13,
H2-130, H2-15, H2-150), 1.90e1.80 (4H, m, H2-12, H2-120), 1.66e1.57
H2-16, H2-160); 13C NMR (100 MHz, DMSO‑d6) 181.7 (C-8, C-80),
d
163.4 (C-9, C-90), 139.7 (C-2, C-20), 134.8 (C-7a, C-7a0), 127.5 (C-3a, C-
3a'/C-5, C-50), 127.3 (C-3a, C-3a'/C-5, C-50), 123.5 (C-6, C-60), 120.3
(C-4, C-40), 114.3 (C-7, C-70), 111.7 (C-3, C-30), 46.1 (C-15, C-150), 44.7
(C-13, C-130), 35.9 (C-11, C-110), 25.7 (C-12, C-120), 22.7 (C-16, C-160);
35
(4H, m, H2-16, H2-160); 13C NMR (DMSO‑d6, 100 MHz)
d
181.8 (C-8,
(þ)-HRESIMS [MþNa]þ m/z 635.1902 (calcd for C30
H Cl2N6O4Na,
34
35
C-80), 163.5 (C-9, C-90), 160.0 (d, 1JCF ¼ 237.1 Hz, C-6, C-60), 158.5 (q,
635.1911), 637.1887 (calcd for C H
Cl37ClN6O4Na, 637.1890),
30 34
2JCF ¼ 34.5 Hz, C-18, C-180),139.3 (C-2, C-20), 136.5 (d, 3JCF ¼ 12.0 Hz,
639.1852 (calcd for C
H Cl2N6O4Na, 639.1877).
30 34
37
3
C-7a, C-7a0), 122.9 (C-3a, C-3a0), 122.4 (d, JCF ¼ 9.9 Hz, C-4, C-40),
2
112.1 (C-3, C-30), 110.8 (d, JCF ¼ 24.5 Hz, C-5, C-50), 99.1 (d,
5.2.3.7. N1,N4-Bis(3-(2-(6-chloro-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (10). Using
general procedure A, reaction of 6-chloro-1H-indole (303 mg,
2 mmol) and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(6-
chloro-1H-indol-3-yl)-2-oxoacetyl chloride as a yellow powder.
Using general procedure B, a sub-sample of the glyoxylyl chloride
(60 mg, 0.248 mmol) was reacted with di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (50 mg, 0.124 mmol) and
DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chro-
matography, di-tert-butyl butane-1,4-diylbis((3-(2-(6-chloro-1H-
2JCF ¼ 26.0 Hz, C-7, C-70), 46.1 (C-15, C-150), 44.7 (C-13, C-130), 35.8
(C-11, C-110), 25.7 (C-12, C-120), 22.7 (C-16, C-160); (þ)-HRESIMS
[MþH]þ m/z 581.2690 (calcd for C30H35F2N6O4, 581.2682).
5.2.3.5. N1,N4-Bis(3-(2-(7-fluoro-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (8). Using gen-
eral procedure A, reaction of 7-fluoro-1H-indole (270 mg, 2 mmol)
and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(7-fluoro-1H-
indol-3-yl)-2-oxoacetyl chloride as a yellow powder. Using general
procedure B, a sub-sample of the glyoxylyl chloride (62 mg,
0.300 mmol) was reacted with di-tert-butyl butane-1,4-diylbis((3-
aminopropyl)carbamate) (50 mg, 0.124 mmol) and DIPEA
(0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chromatog-
raphy, di-tert-butyl butane-1,4-diylbis((3-(2-(7-fluoro-1H-indol-3-
yl)-2-oxoacetamido)propyl)carbamate) as a yellow gum (62 mg,
64%). Using general procedure C, a sub-sample of this material
indol-3-yl)-2-oxoacetamido)propyl)carbamate) as
a yellow oil
(53 mg, 55%). Using general procedure C, a sub-sample of this
material (20 mg, 0.025 mmol) was deprotected to afford the di-TFA
salt of 10 as a brown oil (15 mg, 75%). Rf (MeOH/10% HCl, 7:3) 0.67;
IR (ATR) nmax 3420, 2254, 1672, 1632, 1444, 1179, 1023, 1002 cmꢁ1
;
1H NMR (DMSO‑d6, 400 MHz)
d
12.43 (2H, br s, NH-1, NH-10), 8.94
(2H, t, J ¼ 6.1 Hz, NH-10, NH-100), 8.80 (2H, d, J ¼ 3.3 Hz, H-2, H-20),

8
M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
8.59 (4H, m, NH2-14, NH2-140), 8.20 (2H, d, J ¼ 8.5 Hz, H-4, H-40),
7.61 (2H, d, J ¼ 1.8 Hz, H-7, H-70), 7.29 (2H, dd, J ¼ 8.5, 6.6 Hz, H-5, H-
50), 3.34e3.25 (4H, m, H2-11, H2-110), 3.00e2.90 (8H, m, H2-13, H2-
130, H2-15, H2-150), 1.90e1.80 (4H, m, H2-12, H2-120), 1.66e1.57 (4H,
158.3 (q, 2JCF ¼ 35.0 Hz, C-19, C-19’), 140.9 (C-2, C-20), 135.4 (C-7a, C-
7a0), 129.0 (C-3a, C-3a0), 124.1 (q, 1JCF ¼ 257.3 Hz, C-17, C-170), 123.6
(q, 2JCF ¼ 31.7 Hz, C-6, C-60), 122.0 (C-4, C-40), 118.9 (C-5, C-50), 112.1
(C-3, C-30), 110.1 (C-7, C-70), 46.1 (C-15, C-150), 44.7 (C-13, C-130),
35.9 (C-11, C-110), 25.7 (C-12, C-120), 22.7 (C-16, C-160); (þ)-HRE-
SIMS [MþH]þ m/z 681.2625 (calcd for C32H35F6N6O4, 681.2618).
m, H2-16, H2-160); 13C NMR (DMSO‑d6, 100 MHz) 181.8 (C-8, C-80),
d
163.4 (C-9, C-90), 158.4 (q, JCF ¼ 36.1 Hz, C-17, C-170), 139.4 (C-2, C-
20), 136.8 (C-7a, C-7a0), 128.0 (C-6, C-60), 125.0 (C-3a, C-3a0), 122.9
(C-4, C-40), 122.5 (C-5, C-50), 112.5 (C-7, C-70), 112.1 (C-3, C-30), 46.1
(C-15, C-150), 44.7 (C-13, C-130), 35.8 (C-11, C-110), 25.7 (C-12, C-120),
22.7 (C-16, C-160); (þ)-HRESIMS [MþH]þ m/z 613.2070 (calcd for
5.2.3.10. N1,N4-Bis(3-(2-(5-cyano-1H-indol-3-yl)-2-oxoacetamido)
propyl) butane-1,4-diaminium 2,2,2-trifluoroacetate (13). Using
general procedure A, reaction of 5-cyano-1H-indole (142 mg,
1 mmol) and oxalyl chloride (0.097 mL, 1.15 mmol) afforded 2-(5-
cyano-1H-indol-3-yl)-2-oxoacetyl chloride as a yellow powder.
Using general procedure B, a sub-sample of the glyoxylyl chloride
(58 mg, 0.248 mmol) was reacted with di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (50 mg, 0.124 mmol) and
DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chro-
matography, di-tert-butyl butane-1,4-diylbis((3-(2-(5-cyano-1H-
indol-3-yl)-2-oxoacetamido)propyl)carbamate) as a white gum
(24 mg, 26%). Using general procedure C, a sub-sample of this
material (16 mg, 0.020 mmol) was deprotected to afford the di-TFA
salt of 13 as a brown oil (9.0 mg, 56%). Rf (MeOH/10% HCl, 7:3) 0.63;
IR (ATR) nmax 3312, 2964, 2225, 1732 1668, 1448, 1209, 1126,
35
35
C
H Cl2N6O4, 613.2091), 615.2044 (calcd for C30H
Cl37ClN6O4,
30 35 35
37
615.2070), 617.2023 (calcd for C
H Cl2N6O4, 617.2058).
30 35
5.2.3.8. N1,N4-Bis(3-(2-(7-chloro-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (11). Using
general procedure A, reaction of 7-chloro-1H-indole (303 mg,
2 mmol) and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(7-
chloro-1H-indol-3-yl)-2-oxoacetyl chloride as a yellow powder.
Using general procedure B, a sub-sample of the glyoxylyl chloride
(52 mg, 0.248 mmol) was reacted with di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (50 mg, 0.124 mmol) and
DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chro-
matography, di-tert-butyl butane-1,4-diylbis((3-(2-(7-chloro-1H-
1037 cmꢁ1; 1H NMR (DMSO‑d6, 400 MHz)
d
12.79 (2H, d, J ¼ 2.5 Hz,
indol-3-yl)-2-oxoacetamido)propyl)carbamate) as
a yellow oil
NH-1, NH-10), 8.98 (2H, t, J ¼ 5.9 Hz, NH-10, NH-100), 8.94 (2H, d,
J ¼ 2.5 Hz, H-2, H-20), 8.59e8.54 (6H, m, NH2-14, NH2-140, H-4, H-
40), 7.75 (2H, d, J ¼ 8.5 Hz, H-7, H-70), 7.67 (2H, dd, J ¼ 8.5, 1.5 Hz, H-
6, H-60), 3.34e3.25 (4H, m, H2-11, H2-110), 3.03e2.87 (8H, m, H2-13,
H2-130, H2-15, H2-150), 1.92e1.80 (4H, m, H2-12, H2-120), 1.68e1.58
(53 mg, 55%). Using general procedure C, a sub-sample of this ma-
terial (30 mg, 0.037 mmol) was deprotected to afford the di-TFA salt
of 11 as a brown oil (20 mg, 65%). Rf (MeOH/10% HCl, 7:3) 0.48; IR
(ATR) nmax 2927, 1674, 1638, 1505, 1430, 1203, 1135, 1026, 835, 799,
722 cmꢁ1
;
1H NMR (400 MHz, DMSO‑d6)
d
12.71 (2H, d, J ¼ 3.3 Hz,
(4H, m, H2-16, H2-160); 13C NMR (DMSO‑d6, 100 MHz)
d 181.9 (C-8,
NH-1, NH-10), 8.95 (2H, t, J ¼ 5.9 Hz, NH-10, NH-100), 8.77 (2H, d,
J ¼ 3.3 Hz, H-2, H-20), 8.55e8.50 (4H, m, NH2-14, NH2-140), 8.19 (2H,
d, J ¼ 7.9 Hz, H-4, H-40), 7.38 (2H, d, J ¼ 7.9 Hz, H-6, H-60), 7.28 (2H, t,
J ¼ 7.9 Hz, H-5, H-50), 3.33e3.28 (4H, m, H2-11, H2-110), 2.98e2.92
(8H, m, H2-13, H2-130, H2-15, H2-150), 1.90e1.83 (4H, m, H2-12, H2-
120),1.65e1.57 (4H, m, H2-16, H2-160); 13C NMR (100 MHz, DMSO‑d6)
C-80), 163.0 (C-9, C-90), 158.5 (q, JCF ¼ 34.0 Hz, C-19, C-19’), 140.7 (C-
2, C-20), 138.2 (C-7a, C-7a0), 126.4 (C-6, C-60), 126.05 (C-3a, C-3a'/C-
4, C-40), 125.99 (C-3a, C-3a'/C-4, C-40), 120.0 (C-17, C-170), 114.2 (C-7,
C-70), 112.2 (C-3, C-30), 104.8 (C-5, C-50), 46.0 (C-15, C-150), 44.7 (C-
13, C-130), 35.9 (C-11, C-110), 25.6 (C-12, C-120), 22.7 (C-16, C-160);
(þ)-HRESIMS [MþH]þ m/z 595.2766 (calcd for C32H35N8O4,
595.2776).
d
181.8 (C-8, C-80), 163.3 (C-9, C-90), 139.0 (C-2, C-20), 133.2 (C-7a, C-
7a0),128.1 (C-3a, C-3a0),123.8 (C-5, C-50),123.2 (C-6, C-60),120.2 (C-4,
C-40),116.9 (C-7, C-70),112.9 (C-3, C-30), 46.1 (C-15, C-150), 44.7 (C-13,
C-130), 35.9 (C-11, C-110), 25.6 (C-12, C-120), 22.7 (C-16, C-160);
5.2.3.11. N1,N4-Bis(3-(2-(6-cyano-1H-indol-3-yl)-2-oxoacetamido)
propyl) butane-1,4-diaminium 2,2,2-trifluoroacetate (14). Using
general procedure A, reaction of 6-cyano-1H-indole (142 mg,
1 mmol) and oxalyl chloride (0.097 mL, 1.15 mmol) afforded 2-(6-
cyano-1H-indol-3-yl)-2-oxoacetyl chloride as a yellow powder.
Using general procedure B, a sub-sample of the glyoxyl chloride
(58 mg, 0.248 mmol) was reacted with di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (50 mg, 0.124 mmol) and
DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after chro-
matography, di-tert-butyl butane-1,4-diylbis((3-(2-(6-cyano-1H-
indol-3-yl)-2-oxoacetamido)propyl)carbamate) as a white gum
(43 mg, 45%). Using general procedure C, a sub-sample of this
material (15 mg, 0.019 mmol) was deprotected to afford the di-TFA
salt of 14 as a brown oil (10 mg, 66%). Rf (MeOH/10% HCl, 7:3) 0.57;
IR (ATR) nmax 3040, 2852, 2223, 1673, 1635, 1490, 1200, 1128,
35
(þ)-HRESIMS [MþNa]þ m/z 635.1898 (calcd for C30
H Cl2N6O4Na,
34
35
635.1911), 637.1888 (calcd for
C H
Cl37ClN6O4Na, 637.1890),
30 34
37
639.1869 (calcd for C
H Cl2N6O4Na, 639.1877).
30 34
5.2.3.9. N1,N4-Bis(3-(2-(6-trifluoromethyl-1H-indol-3-yl)-2-oxoacet
amido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (12).
Using general procedure A, reaction of 6-trifluoromethyl-1H-indole
(278 mg, 1.5 mmol) and oxalyl chloride (0.146 mL, 1.7 mmol) affor-
ded 2-(6-trifluoromethyl-1H-indol-3-yl)-2-oxoacetyl chloride as a
yellow powder. Using general procedure B, a sub-sample of the
glyoxylyl chloride (68 mg, 0.248 mmol) was reacted with di-tert-
butyl
butane-1,4-diylbis((3-aminopropyl)carbamate)
(50 mg,
0.124 mmol) and DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to
afford, after chromatography, di-tert-butyl butane-1,4-diylbis((3-
(2-oxo-2-(6-(trifluoromethyl)-1H-indol-3-yl)acetamido)propyl)
carbamate) as a yellow oil (34 mg, 36%). Using general procedure C,
a sub-sample of this material (24 mg, 0.027 mmol) was deprotected
to afford the di-TFA salt of 12 as a brown oil (14 mg, 58%). Rf (MeOH/
10% HCl, 7:3) 0.48; IR (ATR) nmax 3017, 2957, 1671, 1628, 1494, 1329,
1024 cmꢁ1 1H NMR (DMSO‑d6, 400 MHz)
; d 12.81 (2H, br s, NH-1,
NH-10), 9.00e8.93 (4H, m, H-2, H-20, NH-10, NH-100), 8.64 (4H, br
s, NH2-14, NH2-140), 8.36 (2H, d, J ¼ 8.3 Hz, H-4, H-40), 8.07 (2H, br s,
H-7, H-70), 7.63 (2H, dd, J ¼ 8.4, 1.3 Hz, H-5, H-50), 3.34e3.27 (4H, m,
H2-11, H2-110), 3.00e2.90 (8H, m, H2-13, H2-130, H2-15, H2-150),
1.90e1.82 (4H, m, H2-12, H2-120), 1.65e1.56 (4H, m, H2-16, H2-160);
1199, 1109 cmꢁ1 1H NMR (DMSO‑d6, 400 MHz)
; d 12.67 (2H, br s,
NH-1, NH-10), 8.98e8.93 (4H, m, H-2, H-20, NH-10, NH-100), 8.56
(4H, br s, NH2-14, NH2-140), 8.41 (2H, d, J ¼ 8.4 Hz, H-4, H-40), 7.83
(2H, s, H-7, H-70), 7.59 (2H, d, J ¼ 8.4 Hz, H-5, H-50), 3.34e3.27 (4H,
m, H2-11, H2-110), 3.00e2.90 (8H, m, H2-13, H2-130, H2-15, H2-150),
1.90e1.80 (4H, m, H2-12, H2-120), 1.67e1.57 (4H, m, H2-16, H2-160);
13C NMR (DMSO‑d6, 100 MHz) 181.9 (C-8, C-80), 163.2 (C-9, C-90),
d
141.5 (C-2, C-20), 135.3 (C-7a, C-7a0), 129.6 (C-3a, C-3a0), 125.5 (C-5,
C-50), 122.1 (C-4, C-40), 119.7 (C-17, C-170), 117.5 (C-7, C-70), 112.2 (C-
3, C-30), 105.1 (C-6, C-60), 46.1 (C-15, C-150), 44.7 (C-13, C-130), 35.9
(C-11, C-110), 25.6 (C-12, C-120), 22.7 (C-16, C-160); (þ)-HRESIMS
[MþH]þ m/z 595.2772 (calcd for C32H35N8O4, 595.2776).
13C NMR (DMSO‑d6, 100 MHz)
d
182.0 (C-8, C-80), 163.3 (C-9, C-90),

M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
9
5.2.3.12. N1,N4-Bis(3-(2-(5-(methoxycarbonyl)-1H-indol-3-yl)-2-
oxoacet amido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate
(15). Using general procedure A, reaction of 5-methyl-1H-indole
carboxylate (176 mg, 1 mmol) and oxalyl chloride (0.097 mL,
1.15 mmol) afforded methyl 3-(2-chloro-2-oxoacetyl)-1H-indole-5-
carboxylate as a yellow powder. Using general procedure B, a sub-
sample of the glyoxylyl chloride (77 mg, 0.289 mmol) was reacted
with di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate)
(50 mg, 0.124 mmol) and DIPEA (0.130 mL, 0.744 mmol) in DMF
(2 mL) to afford, after chromatography, dimethyl 3,3'-(7,12-bis(tert-
butoxycarbonyl)-2,17-dioxo-3,7,12,16-tetraazaoctadecanedioyl)
bis(1H-indole-5-carboxylate) as a yellow oil (36 mg, 38%). Using
general procedure C, this material (36 mg, 0.042 mmol) was
deprotected to afford the di-TFA salt of 15 as a brown oil (9.2 mg,
25%). Rf (MeOH/10% HCl, 7:3) 0.44; IR (ATR) nmax 3345, 3040, 1677,
carboxylate as a yellow powder. Using general procedure B, a sub-
sample of the glyoxylyl chloride (77 mg, 0.289 mmol) was reacted
with di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate)
(50 mg, 0.124 mmol) and DIPEA (0.130 mL, 0.744 mmol) in DMF
(2 mL) to afford, after chromatography, dimethyl 3,3'-(7,12-bis(tert-
butoxycarbonyl)-2,17-dioxo-3,7,12,16-tetraazaoctadecanedioyl)
bis(1H-indole-7-carboxylate) as a yellow oil (65 mg, 67%). Using
general procedure C,
a sub-sample of this material (60 mg,
0.070 mmol) was deprotected to afford the di-TFA salt of 17 as a
brown oil (26 mg, 43%). Rf (MeOH/10% HCl, 7:3) 0.32; IR (ATR) nmax
3321, 2958, 1670, 1625, 1505, 1280, 1202, 1130 cmꢁ1 1H NMR
;
(DMSO‑d6, 400 MHz)
d
12.17 (2H, s, NH-1, NH-10), 8.96 (2H, t,
J ¼ 6.0 Hz, NH-10, NH-100), 8.81 (2H, d, J ¼ 3.2 Hz, H-2, H-20), 8.58
(4H, br s, NH2-14, NH2-140), 8.54 (2H, dd, J ¼ 8.0, 0.7 Hz, H-4, H-40),
7.92 (2H, dd, J ¼ 7.4, 1.1 Hz, H-6, H-60), 7.44e7.39 (2H, m, H-5, H-50),
3.97 (6H, s, H3-18, H3-180), 3.34e3.27 (4H, m, H2-11, H2-110),
3.00e2.90 (8H, m, H2-13, H2-130, H2-15, H2-150), 1.91e1.82 (4H, m,
H2-12, H2-120), 1.66e1.58 (4H, m, H2-16, H2-160); 13C NMR
1619, 1497, 1433, 1200, 1111 cmꢁ1
;
1H NMR (DMSO‑d6, 400 MHz)
d
12.61 (2H, s, NH-1, NH-10), 8.95e8.89 (4H, m, H-4, H-40, NH-10,
NH-100), 8.87 (2H, d, J ¼ 2.8 Hz, H-2, H-20), 8.56 (4H, br s, NH2-14,
NH2-140), 7.89 (2H, dd, J ¼ 8.5, 7.0 Hz, H-6, H-60), 7.65 (2H, d,
J ¼ 8.5 Hz, H-7, H-70), 3.88 (6H, s, H3-18, H3-180), 3.36e3.28 (4H, m,
H2-11, H2-110), 3.00e2.90 (8H, m, H2-13, H2-130, H2-15, H2-150),
1.91e1.82 (4H, m, H2-12, H2-120), 1.67e1.58 (4H, m, H2-16, H2-160);
(DMSO‑d6, 100 MHz) d
181.9 (C-8, C-80), 165.9 (C-17, C-170), 163.2 (C-
9, C-90), 139.7 (C-2, C-20), 134.4 (C-7a, C-7a0), 127.7 (C-3a, C-3a0),
126.9 (C-4, C-40), 125.6 (C-6, C-60), 122.5 (C-5, C-50), 114.0 (C-7, C-70),
112.0 (C-3, C-30), 52.2 (C-18, C-180), 46.1 (C-15, C-150), 44.7 (C-13, C-
130), 35.9 (C-11, C-110), 25.6 (C-12, C-120), 22.7 (C-16, C-160);
(þ)-HRESIMS [MþH]þ m/z 661.2961 (calcd for C34H41N6O8,
661.2980).
13C NMR (DMSO‑d6, 100 MHz) 182.0 (C-8, C-80), 166.8 (C-17, C-170),
d
163.4 (C-9, C-90),140.2 (C-2, C-20),138.9 (C-7a, C-7a0),125.8 (C-3a, C-
3a0), 124.4 (C-6, C-60), 123.9 (C-5, C-50), 123.4 (C-4, C-40), 112.8 (C-3,
C-30, C-7, C-70), 52.0 (C-18, C-180), 46.1 (C-15, C-150), 44.7 (C-13, C-
130), 35.9 (C-11, C-110), 25.7 (C-12, C-120), 22.7 (C-16, C-160);
(þ)-HRESIMS [MþH]þ m/z 661.2963 (calcd for C34H41N6O8,
661.2980).
5.2.3.15. N1,N4-Bis(3-(2-(5-methyl-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (18). Using
general procedure A, reaction of 5-methyl-1H-indole (262 mg,
2 mmol) and oxalyl chloride (0.195 mL, 2.3 mmol) afforded 2-(5-
methyl-1H-indol-3-yl)-2-oxoacetyl chloride as a yellow powder.
Using general procedure B, a sub-sample of the glyoxylyl chloride
(55.2 mg, 0.248 mmol) was reacted with di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (50 mg, 0.124 mmol) and
DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to afford di-tert-butyl
butane-1,4-diylbis((3-(2-(5-methyl-1H-indol-3-yl)-2-
5.2.3.13. N1,N4-Bis(3-(2-(6-(methoxycarbonyl)-1H-indol-3-yl)-2-
oxoacet amido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate
(16). Using general procedure A, reaction of 6-methyl-1H-indole
carboxylate (176 mg, 1 mmol) and oxalyl chloride (0.097 mL,
1.15 mmol) afforded methyl 3-(2-chloro-2-oxoacetyl)-1H-indole-6-
carboxylate as a yellow powder. Using general procedure B, a sub-
sample of the glyoxylyl chloride (77 mg, 0.289 mmol) was reacted
with di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate)
(50 mg, 0.124 mmol) and DIPEA (0.130 mL, 0.744 mmol) in DMF
(2 mL) to afford, after chromatography, dimethyl 3,3'-(7,12-bis(tert-
butoxycarbonyl)-2,17-dioxo-3,7,12,16-tetraazaoctadecanedioyl)
bis(1H-indole-6-carboxylate) as a yellow oil (43 mg, 45%). After a
repeated synthesis, this material (61 mg, 0.071 mmol) was depro-
tected to afford the di-TFA salt of 16 as a brown oil (59 mg, 97%). Rf
(MeOH/10% HCl, 9:1) 0.87; IR (ATR) nmax 3314, 2944, 2261, 1671,
oxoacetamido)propyl)carbamate) as a white gum (40 mg, 42%).
Using general procedure C, a sub-sample of this material (34 mg,
0.043 mmol) was deprotected to afford the di-TFA salt of 18 as a
brown oil (34 mg, quant. yield). Rf (MeOH/10% HCl, 7:3) 0.65; IR
(ATR) nmax 3029, 1672, 1621, 1428, 1199, 1129, 1024, 1004 cmꢁ1
;
1H
NMR (DMSO‑d6, 400 MHz)
d
12.28 (2H, br s, NH-1, NH-10), 8.87 (2H,
t, J ¼ 6.1 Hz, NH-10, NH-100), 8.70 (2H, d, J ¼ 3.3 Hz, H-2, H-20), 8.67
(4H, m, NH2-14, NH2-140), 8.04 (2H, s, H-4, H-40), 7.42 (2H, d,
J ¼ 8.4 Hz, H-7, H-70), 7.09 (2H, dd, J ¼ 8.3, 7.2 Hz, H-6, H-60),
3.32e3.27 (4H, m, H2-11, H2-110), 2.98e2.91 (8H, m, H2-13, H2-130,
H2-15, H2-150), 2.42 (6H, s, H3-17, H3-170), 1.90e1.83 (4H, m, H2-12,
H2-120), 1.65e1.61 (4H, m, H2-16, H2-160); 13C NMR (DMSO‑d6,
1621, 1490, 1284, 1200, 1133 cmꢁ1 1H NMR (DMSO‑d6, 400 MHz)
;
d
12.65 (2H, d, J ¼ 2.7 Hz, NH-1, NH-10), 8.98e8.90 (4H, m, H-2, H-20,
NH-10, NH-100), 8.62 (4H, br s, NH2-14, NH2-140), 8.31 (2H, d,
J ¼ 8.3 Hz, H-4, H-40), 8.18 (2H, d, J ¼ 1.1 Hz, H-7, H-70), 7.87 (2H, dd,
J ¼ 8.4, 7.0 Hz, H-5, H-50), 3.88 (6H, s, H3-18, H3-180), 3.34e3.27 (4H,
m, H2-11, H2-110), 3.00e2.90 (8H, m, H2-13, H2-130, H2-15, H2-150),
1.92e1.82 (4H, m, H2-12, H2-120), 1.67e1.58 (4H, m, H2-16, H2-160);
100 MHz)
d
181.6 (C-8, C-80),163.9 (C-9, C-90),158.6 (q, JCF ¼ 35.7 Hz,
C-19, C-190), 138.4 (C-2, C-20), 134.6 (C-7a, C-7a0), 131.5 (C-5, C-50),
126.5 (C-3a, C-3a0), 124.9 (C-6, C-60), 121.0 (C-4, C-40), 112.2 (C-7, C-
70), 111.8 (C-3, C-30), 46.0 (C-15, C-150), 44.7 (C-13, C-130), 35.8 (C-11,
C-110), 25.7 (C-12, C-120), 22.7 (C-16, C-160), 21.3 (C-17, C-170);
(þ)-HRESIMS [MþH]þ m/z 573.3174 (calcd for C32H41N6O4,
573.3184).
13C NMR (DMSO‑d6, 100 MHz) 181.9 (C-8, C-80), 166.6 (C-17, C-170),
d
163.4 (C-9, C-90), 141.1 (C-2, C-20), 135.7 (C-7a, C-7a0), 129.9 (C-3a, C-
3a0), 124.5 (C-6, C-60), 123.2 (C-5, C-50), 121.0 (C-4, C-40), 114.3 (C-7,
C-70), 112.2 (C-3, C-30), 52.0 (C-18, C-180), 46.0 (C-15, C-150), 44.7 (C-
13, C-130), 35.8 (C-11, C-110), 25.6 (C-12, C-120), 22.7 (C-16, C-160);
(þ)-HRESIMS [MþH]þ m/z 661.2959 (calcd for C34H41N6O8,
661.2980).
5.2.3.16. N1,N4-Bis(3-(2-(7-methyl-1H-indol-3-yl)-2-oxoacetamido)
propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (19). Using
general procedure A, reaction of 7-methyl-1H-indole (131 mg,
1 mmol) and oxalyl chloride (0.097 mL, 1.15 mmol) afforded 2-(7-
methyl-1H-indol-3-yl)-2-oxoacetyl chloride as a yellow powder.
Using general procedure B, a sub-sample of the glyoxylyl chloride
(55.2 mg, 0.248 mmol) was reacted with di-tert-butyl butane-1,4-
diylbis((3-aminopropyl)carbamate) (50 mg, 0.124 mmol) and
DIPEA (0.130 mL, 0.744 mmol) in DMF (2 mL) to afford, after
5.2.3.14. N1,N4-Bis(3-(2-(7-(methoxycarbonyl)-1H-indol-3-yl)-2-
oxoacet amido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate
(17). Using general procedure A, reaction of 7-methyl-1H-indole
carboxylate (176 mg, 1 mmol) and oxalyl chloride (0.097 mL,
1.15 mmol) afforded methyl 3-(2-chloro-2-oxoacetyl)-1H-indole-7-

10
M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
chromatography, di-tert-butyl butane-1,4-diylbis((3-(2-(7-methyl-
1H-indol-3-yl)-2-oxoacetamido)propyl)carbamate) as a yellow oil
(30 mg, 32%). Using general procedure C, a sub-sample of this
material (18 mg, 0.023 mmol) was deprotected to afford the di-TFA
salt of 19 as a brown oil (12 mg, 65%). Rf (MeOH/10% HCl, 7:3) 0.40;
IR (ATR) nmax 3052, 1671, 1619, 1501, 1445, 1199, 1126, 1024,
their standard protocols [21]. For antimicrobial assays, the tested
strains were cultured in either Luria broth (LB) (In Vitro Technol-
ogies, USB75852), nutrient broth (NB) (Becton Dickson, 234000), or
MHB at 37 ꢀC overnight. A sample of culture was then diluted 40-
fold in fresh MHB and incubated at 37 ꢀC for 1.5e2 h. The com-
pounds were serially diluted 2-fold across the wells of 96-well
plates (Corning 3641, nonbinding surface), with compound con-
1002 cmꢁ1 1H NMR (DMSO‑d6, 400 MHz)
; d 12.33 (2H, br s, NH-1,
NH-10), 8.89 (2H, t, J ¼ 5.7 Hz, NH-10, NH-100), 8.74 (2H, d,
J ¼ 3.0 Hz, H-2, H-20), 8.58e8.46 (4H, m, NH2-14, NH2-140), 8.06 (2H,
d, J ¼ 7.8 Hz, H-4, H-40), 7.16 (2H, t, J ¼ 7.5 Hz, H-5, H-50), 7.07 (2H, d,
J ¼ 7.2 Hz, H-6, H-60), 3.34e3.26 (4H, m, H2-11, H2-110), 3.00e2.88
(8H, m, H2-13, H2-130, H2-15, H2-150), 2.51 (6H, s, H3-17, H3-170),
1.90e1.81 (4H, m, H2-12, H2-120), 1.66e1.57 (4H, m, H2-16, H2-160);
centrations ranging from 0.015 to 64 mg/mL, plated in duplicate. The
resultant mid log phase cultures were diluted to the final concen-
tration of 1 ꢂ 106 CFU/mL; then, 50
mL was added to each well of the
compound containing plates giving a final compound concentra-
tion range of 0.008e32
m
g/mL and a cell density of 5 ꢂ 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.
13C NMR (DMSO‑d6, 100 MHz)
d
181.7 (C-8, C-80), 122.8 (C-5, C-50),
121.9 (C-7, C-70),118.8 (C-4, C-40),112.5 (C-3, C-30), 46.1 (C-15, C-150),
44.7 (C-13, C-130), 35.8 (C-11, C-110), 25.7 (C-12, C-120), 22.7 (C-16,
C-160), 16.6 (C-17, C-170); (þ)-HRESIMS [MþH]þ m/z 573.3170 (calcd
for C32H41N6O4, 573.3184).
For the antifungal assay, fungi strains were cultured for 3 days
on YPD agar at 30 ꢀC. A yeast suspension of 1 ꢂ 106 to 5 ꢂ 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 ꢂ 103 CFU/mL. The compounds
were serially diluted 2-fold across the wells of 96-well plates
(Corning 3641, nonbinding surface), with compound concentra-
5.2.3.17. N,N'-((Butane-1,4-diylbis(azanediyl))bis(propane-3,1-diyl))
bis (2-(4-fluoro-1H-indol-3-yl)-2-oxoacetamide) (20). To a stirred
solution of 2-(4-fluoro-1H-indol-3-yl)-2-oxoacetic acid [14]
(38 mg, 0.18 mmol) and spermine (17 mg, 0.083 mmol) in DMF
(1 mL) was added PyBOP (91 mg, 0.18 mmol) under N2 atmosphere.
Triethylamine (0.069 mL, 0.50 mmol) was then added and the re-
action mixture was stirred for 48 h. Solvent was removed under
reduced pressure to afford a crude product that was triturated with
MeOH:H2O (1 mL, 1:1) and the resulting solid was removed by
filtration, washed and dried to afford 20 as the free base as a white
powder (7.0 mg, 14%). Rf (MeOH/10% HCl, 7:3) 0.57; m.p > 250 ꢀC; IR
tions ranging from 0.015 to 64
mg/mL and final volumes of 50 mL,
plated in duplicate. Then, 50 L of the fungi suspension that was
m
previously prepared in YNB broth to the final concentration of
2.5 ꢂ 103 CFU/mL was added to each well of the compound-
containing plates, giving a final compound concentration range of
0.008e32 m
g/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
(ATR) nmax 3274, 2951, 2742, 1614, 1501, 1427, 1312, 1258 cmꢁ1
;
1H
NMR (DMSO‑d6, 400 MHz)
d
12.54 (2H, br s, NH-1, NH-10), 8.91 (2H,
t, J ¼ 5.9 Hz, NH-10, NH-100), 8.68 (2H, s, H-2, H-20), 7.37 (2H, d,
J ¼ 8.0 Hz, H-7, H-70), 7.26 (2H, td, J ¼ 8.0, 4.7 Hz, H-6, H-60), 6.97
(2H, dd, J ¼ 11.0, 7.9 Hz, H-5, H-50), 3.33e3.26 (4H, m, H2-11, H2-110),
2.94e2.87 (8H, m, H2-13, H2-130, H2-15, H2-150), 1.93e1.84 (4H, m,
H2-12, H2-120), 1.72e1.66 (4H, m, H2-16, H2-160); 13C NMR
absorbance at OD570ꢁ600.
Colistin and vancomycin were used as positive bacterial inhib-
itor standards for Gram-negative and Gram-positive bacteria,
respectively. 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 Z0-factor (using positive and
negative controls). Each plate was deemed to fulfil the quality
criteria (pass QC), if the Z0-factor was above 0.4, and the antimi-
crobial standards showed full range of activity, with full growth
inhibition at their highest concentration, and no growth inhibition
at their lowest concentration.
(DMSO‑d6, 100 MHz)
d
181.0 (C-8, C-80), 164.4 (C-9, C-90), 155.8 (d,
1JCF ¼ 251.2 Hz, C-4, C-40), 139.5 (d, 3JCF ¼ 11.3 Hz, C-7a, C-7a0), 138.9
(C-2, C-20), 124.5 (d, 3JCF ¼ 7.0 Hz, C-6, C-60), 113.4 (d, 2JCF ¼ 21.3 Hz,
C-3a, C-3a0), 111.8 (d, 3JCF ¼ 6.1 Hz, C-3, C-30), 109.0 (C-7, C-70), 108.0
2
(d, JCF ¼ 21.7 Hz, C-5, C-50), 46.0 (C-15, C-150), 44.7 (C-13, C-130),
35.9 (C-11, C-110), 25.7 (C-12, C-120), 22.8 (C-16, C-160); (þ)-HRE-
SIMS [MþH]þ m/z 581.2667 (calcd for C30H35F2N6O4, 581.2682).
5.3. Antimicrobial assays11,21
The susceptibility of bacterial strains S. aureus (ATCC25923),
S. intermedius (1051997), E. coli (ATCC25922) and P. aeruginosa
(ATCC27853) to antibiotics and compounds was determined in
microplates using the standard broth dilution method in accor-
5.4. Determination of the MICs of antibiotics in the presence of
synergizing compounds11
Briefly, restoring enhancer concentrations were determined
dance with the recommendations of the Comite de l’AntibioG-
with an inoculum of 5 ꢂ 105 CFU in 200
mL of MHB containing two-
fold serial dilutions of each derivative in the presence of doxycy-
ꢀ
ꢀ ꢀ
ramme de la Societe Française de Microbiologie (CA-SFM). Briefly,
the minimal inhibitory concentrations (MICs) were determined
cline at 2 mg/mL. The lowest concentration of the polyamine adju-
with an inoculum of 105 CFU in 200
mL of Mueller-Hinton broth
vant that completely inhibited visible growth after incubation for
18 h at 37 ꢀC was determined. These measurements were inde-
pendently repeated in triplicate.
(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.
5.5. Cytotoxicity assays21,22
Additional antimicrobial evaluation against Klebsiella pneumo-
niae (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
The L6 cell line cytotoxicity assays were performed in 96-well
microtiter plates, each well containing 100 mL of RPMI 1640 me-
dium supplemented with 1% L-glutamine (200 mM) and 10% fetal
bovine serum, and 4 ꢂ 104 L6 cells (a primary cell line derived from
rat skeletal myoblasts). Serial drug dilutions of seven 3-fold dilution

M.M. Cadelis et al. / European Journal of Medicinal Chemistry 183 (2019) 111708
11
steps covering a range from 90 to 0.123
m
g/mL were prepared. After
5.8. Membrane depolarization assays11
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 mL) 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 [22].
HEK293 cells were counted manually in a Neubauer haemocy-
tometer and plated at a density of 5,000 cells/well into each well of
the 384-well plates containing the 25x (2 mL) concentrated com-
pounds. 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 ꢀC, 5%
Bacteria were grown in MHB for 24 h at 37 ꢀC and centrifuged at
10,000 rpm at 20 ꢀC. The supernatant was discarded, and the bac-
teria were washed twice with buffered sucrose solution (250 mM)
and magnesium sulfate solution (5 mM). The fluorescent dye 3,30-
diethylthiacarbocyanine iodide was added to a final concentration
of 3 mM, and it was allowed to penetrate into bacterial membranes
during 1 h of incubation at 37 ꢀC. Bacteria were then washed to
remove the unbound dye before adding compound 4 or 8 at
different concentrations. Fluorescence measurements were per-
formed using a Jobin Yvon Fluoromax 3 spectrofluorometer with
slit widths of 5/5 nm. The maximum fluorescence was recorded
with a pure solution of the fluorescent dye in buffer (3
mM).
5.9. Nitrocefin hydrolysis assay11
CO2. To measure cytotoxicity, 5 mL (equals 100 mM final) of resazurin
was added to each well after incubation, and incubated for further
3 h at 37 ꢀC with 5% CO2. After final incubation fluorescence in-
Outer membrane permeabilization was measured using nitro-
cefin as a chromogenic substrate of periplasmic -lactamase. Ten
tensity was measured as Fex 560/10 nm, em 590/10 nm (F560/590
)
b
using a Tecan M1000 Pro monochromator plate reader. CC50 values
(concentration at 50% cytotoxicity) were calculated by normalizing
the fluorescence readout, with 74 mg/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.
milliliters of MHB were inoculated with 0.1 mL of an overnight
culture of EA289 bacteria and grown at 37 ꢀC until the OD600
reached 0.5. The remaining steps were performed at room tem-
perature. Cells were recovered by centrifugation (4,000 rpm for
20 min) and washed once in 20 mM potassium phosphate buffer
(pH 7.2) containing MgCl2 (1 mM). After a second centrifugation,
the pellet was re-suspended and adjusted to OD600 of 0.5. Then,
50
suspension to obtain a final concentration varying from 3.9
250 M. Fifty microliters of nitrocefin were then added to obtain a
final concentration of 50 g/mL. Nitrocefin hydrolysis was moni-
m
L of the desired compound were added to 100
m
L of the cell
mM to
m
5.6. Haemolytic assay
m
tored spectrophotometrically by measuring the increase in absor-
bance at 490 nm. Assays were performed in 96-well plates using a
M200 Pro Tecan spectrophotometer.
Haemolytic activities of the compounds were investigated by
determining the hemoglobin release from erythrocyte suspensions
of fresh Sprague Dawley (SD) rats blood. Fresh blood samples were
obtained from the Vernon Jansen Unit (The University of Auckland)
and stored in heparin-coated blood collection tubes. The blood cells
were centrifuged at low speed for 5 min and the plasma removed.
The blood cells were then washed with saline (10 mL of saline for
1 mL of blood) and re-suspended in saline 2% v/v. Re-suspended
Acknowledgements
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
data. Some of the antimicrobial screening was performed by CO-
ADD (The Community for Antimicrobial Drug Discovery), funded
by the Wellcome Trust (UK) and The University of Queensland
(Australia). We thank Marcel Kaiser (Swiss Tropical Public Health
Institute) for the L6 cytotoxicity data.
blood cells (100
DMSO (20 L) and PBS buffer (80
bated for 1 h at 37 ꢀC. After incubation the plate was centrifuged for
5 min at 200 g, the supernatant (100 L) was transferred into
mL) were added to the compounds, dissolved in
m
mL), in a 96-well plate and incu-
m
another plate and the haemolysis was measured with a microplate
reader at absorbance 540 nm (A). DMSO in PBS buffer was used as
the negative control (0% haemolysis) while 0.1% Triton X-100 was
used as the positive control (100% haemolysis). The percentage
haemolysis was calculated as (AcompoundeAnegative control)/(Apositive
controleAnegative control)x100. All data corresponds to an average of
three independent experiments. HC50 was determined from dose-
response curves generated from 10 points in the 96-well plates.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at https://doi.org/10.1016/j.ejmech.2019.111708.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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