Guanidine functionalized anthranilamides as effective antibacterials with biofilm disruption activity

Article abstract of DOI:10.1039/c8ob01699b

We describe a library of amphiphilic anthranilamide compounds as antimicrobial peptide (AMP) mimics. These contain a hydrophobic naphthoyl side chain and different hydrophilic cationic groups such as amino, quaternary ammonium and guanidino groups. These are prepared via the ring-opening of different isatoic anhydrides. The antibacterial activity against S. aureus and E. coli of compounds containing guanidino cationic groups was greater than that for amino and quaternary ammonium cationic groups. The fluoro-substituted guanidinium compound 9b showed a minimum inhibitory concentration (MIC) of 2.0 μM against S. aureus, and reduced established biofilms of S. aureus by 92% at 64 μM concentration. The bromo-substituted guanidinium compound 9d exhibited good MIC against S. aureus (3.9 μM) and E. coli (15.6 μM) and disrupted established biofilms of S. aureus by 83% at 62.4 μM concentration. Cytoplasmic membrane permeability studies suggested that depolarization and disruption of the bacterial cell membrane could be a possible mechanism for antibacterial activity and the in vitro toxicity studies against MRC-5 human lung fibroblast cells showed that the potent compounds are non-toxic against mammalian cells.

Full text of DOI:10.1039/c8ob01699b

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DOI: 10.1039/C8OB01699B
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Guanidine Functionalized Anthranilamides as Effective
Antibacterials with Biofilm Disruption Activity
a
b
a
b
a
Rajesh Kuppusamy , Muhammad Yasir , Eugene Yee , Mark Willcox , David StC Black , and Naresh
Received 00th January 20xx,
Accepted 00th January 20xx
a
Kumar *
DOI: 10.1039/x0xx00000x
Abstract
www.rsc.org/
We describe a library of amphiphilic anthranilamide compounds as antimicrobial peptide (AMP)
mimics. These contain a hydrophobic naphthoyl side chain and different hydrophilic cationic
groups such as amino, quaternary ammonium and guanidino groups. These are prepared via the
ring-opening of different isatoic anhydrides. The antibacterial activity against S. aureus and E. coli
of compounds containing guanidino cationic groups was greater than that for amino and
quaternary ammonium cationic groups. The fluoro-substituted guanidinium compound 9b
showed a minimum inhibitory concentration (MIC) of 2.0 µM against S. aureus, and reduced
established biofilms of S. aureus by 92% at 64 µM concentration. The bromo-substituted
guanidinium compound 9d exhibited good MIC against S. aureus (3.9 µM) and E. coli (15.6 µM)
and disrupted established biofilms of S. aureus by 83% at 62.4 µM concentration. Cytoplasmic
membrane permeability studies suggested that depolarization and disruption of the bacterial cell
membrane could be a possible mechanism for antibacterial activity and the in vitro toxicity
studies against MRC-5 human lung fibroblast cells showed that the potent compounds are non-
toxic against mammalian cells.
Introduction
Multidrug-resistant bacteria pose an increasing threat to
1
human health. Resistant bacterial strains can complicate the
Moreover, the existence of bacterial biofilms, which are
clusters of bacterial cells encased in a self-produced matrix,
increases the resistance of microbes to antibiotics and
treatment of urinary tract infections, pneumonia, and
gastroenteritis, and can also contaminate medical devices.2
Bacterial resistance to antibiotics can occur through several
different mechanisms, such as altering the permeability of
cell membranes, altering drug target binding sites, utilization
of enzymes to destroy antibiotics, and use of efflux pumps to
remove antibiotics.3
5
, 6
immune defenses. Biofilm-encased bacteria can be 10 to
1
000 times more resistant to conventional antibiotics than
7
planktonic cells. Therefore, research and development of
new antibacterial drugs must focus not only on planktonic
bacteria but also on potential disruption of the biofilm.
, 4
In recent decades, cationic antimicrobial peptides (AMPs)
have emerged as an alternative approach to small molecule
8
-10
antibiotics to combat bacteria resistance.
AMPs usually
a.
School of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia.
School of Optometry and Vision Science, UNSW Australia, Sydney, NSW 2052,
Australia.
b.
act against microbial membranes via non-receptor
interactions, a principal role in the defence against local and
*
E‒mail: n.kumar@unsw.edu.au* Tel: +61 29385 4698; Fax: +61 29385 6141.
Electronic Supplementary Information (ESI) available:
DOI: 10.1039/x0xx00000x
See
11
systemic
infections.
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Fig. 1. Peptidomimetic compounds in clinical trials.
AMPs usually act against microbial membranes via non- In our previous research, we reported biphenyl backbone
receptor interactions, a principal role in the defence against cationic peptidomimetic derivatives with good antibacterial
1
1
27
local and systemic infections. However, only a few naturally activity against Staphylococcus aureus and Escherichia coli.
occurring AMPs have been used clinically, with the broad- We also demonstrated the importance of having a
1
2
spectrum Pexiganan (MSI-78) being one notable example. tryptophan residue for antibacterial activity, as well as
The use of AMPs as antimicrobial agents is limited by their having simple amine or guanidine groups to mimic lysine and
low proteolytic stability, poor oral bioavailability, and arginine amino acids. Our group has also previously
27
multistep synthesis.
reported glyoxamide derivatives as antibacterial agents and
biofilm disruptors by utilizing the ring-opening of isatins with
amines. However, the ring-opening of isatoic anhydrides to
form carboxamides has not yet been explored as an avenue
to generate peptidomimetics.
Peptidomimetics are small protein-like molecules containing
natural or unnatural amino acids to mimic the properties of
natural peptides. The various types of peptidomimetics
include α-peptides,
peptoids,18 β-turn mimetics,19 and lipopeptides.20 The
peptidomimetics LTX-109 (1) and PMX-30063 (2) (Fig. 1) are
25
13
14-16
17
γ-AA peptides,
β-peptides,
2
1-23
currently undergoing human clinical trials.
Several
research groups have studied small molecular mimics of
2
4
25, 26
AMPs, including arylamide foldamers , glyoxamides
,
2
7
biphenyl backbone mimics , binaphthyl based dicationic
28
29
30, 31
32-38
peptoids , aryl-alkyl-lysines , xanthone
, and others.
AMPs and their synthetic mimics possess an overall
amphiphilic scaffold with both cationic and hydrophobic
groups. The cationic charge enhances the specificity of the
molecules for bacterial cells, due to the fact that the
bacterial membranes contains a greater amount of anionic
Fig. 2. Design of peptidomimetics based on the anthranilic acid scaffold.
39, 40
lipids compared to mammalian membranes.
Meanwhile,
In the present work, we describe the ring-opening of isatoic
anhydrides to generate short cationic peptidomimetics
based on the anthranilic acid (2-aminobenzoic acid) scaffold
the hydrophobicity of the molecules forms pore and it is
important for the peptides partitioning into the lipid bilayer
4
1
to cause bacterial death.
(
Fig. 2).
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Scheme 1 General synthetic scheme to synthesize anthranilic acid-based peptidomimetics. Reaction conditions: a) isatoic anhydrides (1.0
equiv), methyl-L-tryptophanate (1.0 equiv), CH CN, reflux, 16 h; b) 2-naphthoyl chloride (1.0 equiv), Et N (3.0 equiv), CH Cl , rt, 4 h; c) 1 N
3
3
2
2
NaOH(aq) (2.0 equiv), THF, MeOH, rt, overnight; d) N-Boc-1,2-diaminoethane, EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv), DMF, rt,
overnight; e) 1-(2-aminoethyl) 2,3-bis(tert-butoxycarbonyl)guanidine, EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv), DMF, rt, overnight; f)
N,N-dimethylethylenediamine, EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv), DMF, rt, overnight; g) 4 N HCl in dioxane, CH
2 2
Cl , rt, 4 h; h)
TFA, CH Cl , rt, 8 h; 4 N HCl in dioxane, CH Cl , rt, 30 min; i) 4 N HCl in dioxane, CH Cl , rt, 30 min; j) CH I (2.0 equiv), CH CN, rt, overnight.
2
2
2
2
2
2
3
3
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The phenyl ring of the isatoic anhydrides was modified with N,N-dimethylethylenediamine
under
EDCI
coupling
electron-withdrawing halogen substitutions (F, Cl and Br) or conditions to afford the corresponding amides 5a-5e, 6a-6e
an electron-donating OMe group to investigate the effect of and 7a-7e respectively in moderate to good yields (50-61%).
electronic character on biological activity. Four series of Compounds 5a-5e were treated with 4 N HCl in dioxane to
compounds were synthesized containing different cationic yield the primary ammonium hydrochloride salts 8a-8e
groups, namely primary amine (series I), guanidine (series II), (series I) in good yields (75-81%). Treating compounds 6a-6e
tertiary amine (series III) or quaternary ammonium salts with TFA and dichloromethane as solvent followed by 4 N
(series IV), in order to understand the effect of the cationic HCl in dioxane gave the guanidinium hydrochloride 9a-9e
functionality on the antimicrobial activities of these (series II) in moderate yields (45-59%). The compounds 7a-7e
compounds. All of the compounds contained an N-naphthoyl were treated with 4 N HCl in dioxane to give the tertiary
substituent on the anthranilic acid backbone as the ammonium hydrochloride salts 10a-10e (series III) in 90-91%
hydrophobic group. Moreover, due to the previously yield. Finally, treatment of compounds 7a-7e with methyl
reported efficacy of tryptophan-containing peptidomimetics, iodide in acetonitrile yielded the quaternary ammonium
all of the compounds in this study contained a tryptophan iodide salts 11a-11e (series IV) in 90-94% yield.
residue in the amine side chain.
Antibacterial activity
The antibacterial activity of these compounds was measured
against both Gram-positive and Gram-negative bacterial
The antimicrobial activity of peptidomimetics 8a-11e was
examined by determination of their minimal inhibitory
strains. We also investigated the mechanism of action of the
concentration (MIC) following a previously published
active peptidomimetics using a membrane depolarization
42
protocol. The activities were assessed against the Gram-
positive S. aureus [SA38] and the Gram-negative bacterium
and E. coli [K12]. The MIC values of these compounds 8a-11e
are presented in table 1.
assay. Additionally, the ability of the active compounds to
disrupt pre-formed biofilms of S. aureus and E. coli was
evaluated. Finally, mammalian cell cytotoxicity was
investigated for selected potent antimicrobial compounds to
determine compound specificity.
We compared the MIC data of all our compounds with MSI-
7
8 (Pexiganan) which has a 22-amino acid sequence. Based
on the MIC data, a number of structure-activity relationships
SAR) studies could be determined. Most of the compounds
showed good to excellent antibacterial activity against S.
Results and Discussion
(
Synthesis
The isatoic anhydrides 1a-e were ring-opened with methyl-L- aureus, with MIC values ranging between 2.0 and 31.2 µM.
tryptophanate using acetonitrile as solvent to afford the The replacement of the H at the 5-position of isatoic
corresponding substituted methyl (2-aminobenzoyl)-L- anhydride with halogens such as F, Cl, Br or the electron-
tryptophanate 2a-e in 60-75% yields following trituration donating OCH group both resulted in improvement of MIC
3
from acetonitrile and diethyl ether (Scheme 1). Treating 2a- values. Overall, the bromo-guanidinium compound 9d
2
e with 2-naphthoyl chloride and triethylamine as base displayed the best combination of activity against both S.
furnished the N-naphthoyl compounds 3a-3e in good yields aureus (3.9 µM) and E. coli (15.6 µM). Interestingly, the
62-80%). The ester groups of 3a-3e were hydrolyzed using guanidinium compound 9b containing the fluoro group that
(
43
sodium hydroxide and water yielding the key intermediate is widely used in medicinal chemistry also showed excellent
acids 4a-4e in excellent yields (80-89%).
activity against S. aureus (2.0 µM), but was not as effective
against E. coli (125 µM).
The acids 4a-4e were treated with N-Boc-1,2-diaminoethane,
1-(2-aminoethyl)-2,3-bis(tert-butoxycarbonyl) guanidine, or
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Table 1: Antibacterial activity (MIC) of compounds.
3
1.2 µM) against S. aureus and good activity against E. coli
(
15.6–31.2 µM). Compound 8b, substituted with a fluoro
Cpd
X
MIC (µM)
E. coli
125
moiety showed a four-fold increase in MIC (7.8 µM) against
S. aureus compared to unsubstituted derivative 8a.
Compound 8d with least electronegative atom bromo
showed two-fold increase in MIC (7.8 µM) against S. aureus
compared to fluoro substituted compound 8b. Interestingly,
against E. coli the fluoro substituted compound showed
eight-fold increase in MIC (15.6 µM) activity compared to
bromo compound 8d (>125 µM). However, the MIC of chloro
(31.2 µM) and OMe (125 µM) substituted compound against
E. coli suggests that it could be the steric factor which
influences the activity against Gram-negative bacterium.
S. aureus
8
8
8
8
a
b
c
H
F
31.2
7.8
3.9
3.9
15.6
31.2
>125
Cl
Br
d
8
e
a
OMe
H
31.2
15.6
125
9
62.5
9
9
9
9
1
1
1
1
1
1
1
1
1
1
b
F
2.0
125
The guanidinium compounds (9a-9e, series II), which mimic
the arginine residue, showed excellent activity (2.0–15.6 µM)
against S. aureus and good activity (15.6–62.5 µM) against E.
coli. Compound 9b, substituted with a fluoro moiety showed
a two-fold increase in MIC (2.0 µM) against S. aureus
compared to the chloro-substituted compound 9c (3.9 µM)
and the bromo substituted compound 9d (3.9 µM).
Interestingly, against E. coli the trend was reversed with MIC
values of 125 µM for 9b (fluoro), 62.5 µM for 9c (chloro), and
c
Cl
3.9
62.5
15.6
62.5
125
d
Br
3.9
e
OMe
H
7.8
0a
0b
0c
0d
0e
1a
1b
1c
1d
1e
31.2
125
F
>125
>125
125
Cl
15.6
7.8
1
5.6 µM for 9d (bromo). This result suggests that electronic
Br
and/or steric factors may influence the activity of the
halogenated guanidinium compounds against the two
bacterial strains.
OMe
H
62.5
31.2
125
>125
>125
>125
>125
>125
>125
Interestingly, in the case of the tertiary ammonium
compounds (series III) and the quaternary ammonium
compounds (QACs; series IV), S. aureus (7.8–15.6 µM) was
generally more sensitive than E. coli (>125 µM). This suggests
the length of the alkyl chains in the QACs is important for the
F
Cl
15.6
31.2
31.2
3.2-6.45
Br
46, 47
activity against E. coli.
There was no marked difference
OMe
n/a
in the activity between the tertiary and quaternary salts
(10a-10c) and (11a-11c) against S. aureus., Notably, the
bulkier Br substituent (10d) of tertiary ammonium salt
showed four-fold increase in activity (7.8 µM) compared to
quaternary salt (11d).
MSI-78
3.2-6.45
2
7, 44, 45
MIC values for MSI-78 previously reported.
The nature of the cationic moiety greatly influenced the
antimicrobial activity of the compounds. The primary
ammonium compounds (8a-8e, series I), which mimic a
lysine residue, showed good activity ranging from (3.9 to
Overall, peptidomimetics with primary ammonium and
guanidinium cationic groups showed good to excellent
antimicrobial activity against S. aureus irrespective of the
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halogen substituents. This is likely due to the interaction of bacterial cell viability as shown in Fig. 4 (A-C). The cell
View Article Online
DOI: 10.1039/C8OB01699B
primary amines and guanidines to the phosphate groups in viability of compounds 9b and 9d against S. aureus and 9d
the lipids, through a combination of hydrogen bonding and against E. coli generally resembled the results observed in
electrostatic interactions. Against E. coli the activity membrane depolarization studies. The compound 9d at 4x
depended on both the nature of the cationic group and the MIC showed almost 4 log reductions in bacterial numbers
identity of the halogen (F, Cl, Br). The bulky and electron within 5 minutes and this result coincides with the dye
withdrawing Br substitution with a guanidine cationic group release assay as well. Compounds 9b showed < 1 log
had broad-spectrum activity. The tertiary and quaternary reduction in bacterial numbers at 4x MIC suggests the
ammonium salts were not active against E. coli and bacterial killing increases with concentration. Against E. coli,
moderately active against S. aureus. Out of the four series, compound, 9d reduced the bacterial numbers with increase
the guanidinium peptidomimetics (series II) generally in concentration and time. Overall, these results suggest that
showed the highest broad-spectrum activity.
these peptidomimetic compounds could exert their
bactericidal effect via their membrane depolarization
activity.
Cytoplasmic permeability
The mode of action of cationic AMPs and their mimics is
generally believed to occur through electrostatic interactions
between the positively-charged compounds and the
negatively-charged bacterial membrane.48 To test this
hypothesis, the effect of the antibacterial compounds on the
bacterial cytoplasmic membrane was tested by using the
A
S . a u re u s 3 8 / 9 b
1
50
0
.5x M IC
1
2
4
.0x M IC
100
x
x
M IC
M IC
5
0
0
p ositive control
membrane
potential-sensitive
dye
diSC3-5
(3,3'-
0
3
6
9
12
15
dipropylthiadicarbocyanine iodide). The dye readily
partitions into the bacterial cell membrane and aggregates
within the membrane, leading to self-quenching of
fluorescence.49 However, the alteration of membrane
potential due to membrane destabilization or pore formation
by compounds will lead to an increase in fluorescence due to
release of the dye. The guanidine compounds (9b, 9d)
showing excellent MIC values are selected to understand
their mode of action. As shown in Fig. 3A-B, compounds 9b
and 9d (added at 0.5×, 1×, 2× and 4× MIC) caused release of
the dye from S. aureus in a time and concentration-
dependent manner. In particular, compound 9d showed an
increase in fluorescence at 1x MIC levels within 3 min.
However, 9b did not show much increase in fluorescence at
T im e (m in )
B
S . a u re u s 3 8 / 9 d
1
50
0
1
2
4
.5x M IC
x
x
x
M IC
M IC
M IC
100
5
0
0
p ositive control
0
3
6
9
12
15
T im e (m in )
C
E . c o li K 1 2 / 9 d
5
4
3
2
1
00
00
00
00
00
0
0
.5x M IC
1
x MIC.
1x M IC
2
4
x
x
M IC
M IC
Against E. coli, the active compound 9d showed an increase
in fluorescence intensity even at sub-MIC levels within 3 min
as shown in Fig. 3C. Similarly, 8b and 8c also perturbs the cell
membrane led to similar disruption of diSC3-5 fluorescence
intensity (supplementary material Fig. S1). Taken together,
these results indicated that the isatoic anhydride-derived
p ositive control
0
3
6
9
12
15
T im e (m in )
Fig. 3. S. aureus cytoplasmic membrane disruption promoted by (A) 9b (1.0 µM, 2.0
µM, 4.0 µM, 8.0 µM) and (B) 9d (1.95 µM, 3.9 µM, 7.8 µM, 15.6 µM) at different
cationic
peptidomimetic
compounds
can
readily
permeabilize the bacterial membrane, which may then result concentrations. (C) E. coli cytoplasmic membrane disruption promoted by 9d (7.8 µM,
1
5.6 µM, 31.2 µM, 62.4 µM) at different concentrations. 20% DMSO was used as
in bacterial cell death.
positive control.
To further explore the mechanism responsible for cell killing,
we analyzed the effect of the active peptidomimetics on
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A
S . a u re u s 3 8
/
9 b
B
S . a u re u s 3 8 / 9 d
C
E . c o li K 1 2 / 9 d
7
6
5
4
10
10
10
7
6
5
10
7
6
5
4
1
1
1
1
0
0
0
0
0
1
.5x M IC
M IC
0.5x M IC
1x M IC
2x M IC
4x M IC
B u ffe r
0.5x M IC
1x M IC
2x M IC
4x M IC
B u ffe r
x
1
0
2x M IC
M IC
B u ffe r
4
x
10
1
0
0
5
0
5
0
5
T im e (m in )
T im e (m in )
T im e (m in )
Fig. 4. S. aureus cell viability count in the presence of 9b (A), 9d (B), and E. coli cell viability count in the presence of 9d (C) at same concentrations used for cytoplasmic membrane
disruption.
Anti-biofilm activity
A
B io film d isru p tio n o f 9 b
/
S . a u re u s 3 8
1
00
p ositive control
1
x
The most active antibacterial peptidomimetics were tested
against pre-formed biofilms of both bacterial strains using
the crystal violet staining assay. The ability of compounds 9b
and 9d to disrupt established S. aureus biofilms was
measured at increasing MIC concentrations 1×, 2×, 4×, 8×,
7
5
2
5
0
5
0
2x
4x
8
x
1
6x
32x
l
x
x
x
x
x
x
2
tro
1
2
4
8
6
1
3
n
o
c
16× and 32× (Fig. 5). The most active fluoro substituted
e
iv
it
s
o
p
compound 9b showed 35% disruption at 1x MIC. Whereas 9d
with bromo substituent disrupted 58% of S. aureus biofilms
at 1× MIC.
c o m p o u n d c o n c e n tra tio n s  M
B
B io film d isru p tio n o f 9 d
/
S . a u re u s 3 8
1
00
p ositive control
1
x
7
5
2
5
0
5
0
2x
At the maximum concentration of 32× MIC, the compounds
4
x
9
b and 9d disrupted S. aureus biofilms by 83-93%.
Interestingly, the bromo quaternary ammonium compound
0d showed 72% disruption at 1x MIC concentration despite
8x
1
6x
32x
1
tro l
1 x
x
x
8 x
6 x
2 x
2
4
1
3
n
o
c
e
having moderate MIC values compared to the fluoro
guanidinium compound 9b as shown in supplementary
information Fig. S3.
itiv
s
o
p
c o m p o u n d c o n c e n tra tio n s  M
B io film d isru p tio n o f 9 d
/
E .c o li K 1 2
Compound 9d which showed good antibacterial activity
against E. coli were also examined for their ability to disrupt
established biofilms of E. coli. 9d exhibited low levels of
disruption against the established biofilm of E. coli. The
bromo substituted guanidine compound 9d disrupted 36% of
the established biofilm of E. coli at 1x MIC concentration.
Overall, these compounds are effectively disrupting the
established biofilms of S. aureus.
C
p ositive control
1
00
75
1
x
2x
4
x
5
2
0
5
0
8
x
1
3
6x
2x
l
x
x
x
x
x
x
2
tro
1
2
4
8
1
6
3
n
o
c
e
itiv
s
o
p
c o m p o u n d c o n c e n tra tio n s  M
Fig. 5. Disruption of established biofilm of S. aureus after 24 h with 1× to 32× MIC
concentrations of compounds 9b (A) and 9d (B). Disruption of established biofilm of E.
coli after 24 h with 1× to 32× MIC concentrations of compound 9d (C). The positive
control represents the pre-established biofilms without any compounds. Error bars
indicate the standard error of the mean (SEM) of three independent experiments.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Page 8 of 23
ARTICLE
Journal Name
influenced biological activity. For instance, the bromo-
View Article Online
DOI: 10.1039/C8OB01699B
substituted primary ammonium compound 8d showed a
Cytotoxicity
four-fold increase in antibacterial activity compared to the
corresponding fluoro compound 8b against E. coli, however
interestingly this trend was reversed for the bromo (9d) and
fluoro (9b) guanidinium compounds. The methoxy
substituted compounds are not as effective antibacterials as
the halo derivatives. Cytoplasmic membrane permeability
studies of active and non-toxic compounds 9b and 9d
suggested that depolarization and disruption of the bacterial
cell membrane could be a possible mechanism for
antibacterial activity. In addition, these compounds showed
effective biofilm disruption. The most active compounds 9b
The most active peptidomimetics (8b-8d, 9b-9d, 10d, 11c)
were assessed for mammalian cell cytotoxicity against MRC-5
normal human lung fibroblasts using the Alamar Blue
5
0
(Resazurin) assay. The results showed that the guanidinium
compounds with fluoro (9b) and bromo (9d) substituents
were not cytotoxic at 50 µM. Even at 100 µM, 9b and 9d
showed only 6.5% and 20% decrease in cell viability as shown
in Table 2. These results indicate that compounds 9b and 9d
are essentially non-toxic at the dosages needed to suppress
S. aureus bacterial growth (MICs of 2.0 µM and 3.9 µM
respectively).
(MIC: 2.0 µM) and 9d (MIC: 3.9 µM) showed the highest
biofilm disruption activity of 92% and 83% respectively at
Similarly, the chloro-substituted quaternary ammonium concentrations of 64 µM and 62.4 µM in established S.
compound 11c was not cytotoxic at 50 µM, which again was aureus biofilms. Furthermore, in vitro toxicity assays against
much higher than its MIC of 15.6 µM against S. aureus. All human MRC-5 fibroblast cells demonstrated that the
the primary ammonium compounds (8b-8d) with halogen guanidine containing compounds 9b and 9d were highly
substituents F (7.8 µM), Cl (3.9 µM), Br (3.9 µM) were selective for bacterial cells over mammalian cells. Overall,
cytotoxic at 50 µM concentration even though the MIC this
values are good against S. aureus.
work
suggests
that
anthranilic
acid-based
peptidomimetics bearing primary ammonium or guanidinium
cationic groups could be a new avenue for the development
of membrane-disrupting antibacterial agents with biofilm
disruption activity.
a
Table 2 Percentage cell viability of MRC-5 after 72 h incubation with 100 µM and 50
µM of compounds.
Compounds
MRC-5 cell viability after 72 h
% cell viability)
(
1
00 µM
50 µM
99 ± 3
93 ± 5
97 ± 3
0
Experimental
9
9
b
d
99 ± 7
79 ± 14
0
Chemistry
11c
8
8
b
c
All chemical reagents were purchased from commercial
sources (Combi-Blocks, Chem-Impex and Sigma Aldrich) and
used without further purification. Solvents were commercial
and used as obtained. Reactions were performed using oven-
dried glassware under an atmosphere of nitrogen and in
anhydrous conditions (as required). Room temperature
refers to the ambient temperature. Yields refer to
0
0
8d
a
Data displayed are averages of three independent experiments
with ± values representing standard deviation.
Conclusion
A library of amphiphilic compounds derived from 5- chromatographically and spectroscopically pure compounds
substititued isatoic anhydride was prepared. These unless otherwise stated. Reactions were monitored by thin
peptidomimetic compounds contain
a
hydrophobic layer chromatography (TLC) plates pre-coated with Merck
naphthoyl group and different hydrophilic groups, including silica gel 60 F254. Visualization was accomplished with UV
containing primary ammonium, guanidinium, tertiary light, a ninhydrin staining solution in n-butanol. Flash
ammonium or quaternary ammonium, as a source of cationic chromatography and silica pipette plugs were performed
charge. In the antibacterial assay, the primary ammonium under positive air pressure using Silica Gel 60 of 230–400
compounds (8b-8d) and guanidinium compounds (9b-9d) mesh (40–63 μm) and using Grace Davison LC60A 6-μm for
showed excellent antibacterial activity against S. aureus reverse phase chromatography. Infrared spectra were
compared to the tertiary and quaternary ammonium recorded using a Cary 630 ATR spectrophotometer. High-
counterparts. Additionally, the nature of the substituents resolution mass spectrometry was performed by the
(such as fluoro, chloro, bromo) in the aromatic ring also Bioanalytical Mass Spectrometry facility, UNSW (The
8
| J. Name., 2012, 00, 1-3
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Journal Name
ARTICLE
supplementary HRMS data reports both the measured and J = 2.4 Hz, 1H), 7.10-6.98 (m, 3H), 6.70 (dd, J = 4.0, 7.6 Hz,
View Article Online
DOI: 10.1039/C8OB01699B
calculated mass. The calculated mass is below the found 1H), 6.25 (br s, 2H), 4.66-4.60 (m, 1H), 3.64 (s, 3H) 3.27-3.19
1
3
mass). Proton and Carbon NMR spectra were recorded in the (m, 2H),; C NMR (100 MHz, DMSO-d
solvents specified using a Bruker DPX 300 or a Bruker Avance 136.5, 132.4, 128.9, 127.5, 124.1, 121.4, 118.9, 118.4, 116.8,
00 or 600 MHz spectrometer as designated. Chemical shifts 114.8, 114.1, 111.9, 110.5, 53.9, 52.3, 26.9 ; IR (ATR): νmax
δ) are quoted in parts per million (ppm), to the nearest 0.01 3415, 3327, 3233, 3057, 2937, 2341, 2109, 1739, 1642, 1563,
ppm and internally referenced relative to the solvent nuclei. 1510, 1421, 1234, 1203, 1126, 1017, 946, 891, 826, 745;
6
): δ 173.2, 169.4, 150.2,
4
(
1
+
HNMR spectral data are reported as follows [chemical shift HRMS (ESI): m/z calcd for C19
H
18FN
3
O [M + H] : 356.1405;
3
in ppm; multiplicity in br, broad; s, singlet; d, doublet; t, found: 356.1400.
triplet; q, quartet; quint, quintet; sext, sextet; sept, septet;
Methyl (2-amino-5-chlorobenzoyl)-L-tryptophanate (2c).
The title compound 2c was prepared from compound 1c (2.0
g, 10.15 mmol) and L-tryptophan methyl ester (2.21 g, 10.15
mmol) according to the general procedure A. The product 2c
m, multiplet; or as a combination of these (e.g. dd, dt etc.)];
coupling constant (J) in hertz, integration, proton count and
assignment.
was obtained as a grey solid (2.8 g, 75%); mp 135.6–136.2 °C;
The compound L-tryptophan methyl ester and 1-(2-
1
H NMR (400 MHz, DMSO-d ): δ 10.85 (s, 1H), 8.67 (d, J = 8.0
6
aminoethyl)2,3-Bis(tert-butoxycarbonyl) guanidine was
Hz, 1H), 7.59-7.55 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.20-7.15
2
7
synthesized by using the previously reported procedure .
(m, 2H), 7.06 (t, J = 8.0 Hz, 1H), 6.99 (t, J = 8.0 Hz, 1H), 6.70
(
d, J = 12.0 Hz, 1H), 6.49 (br s, 2H), 4.65-4.59 (m, 1H), 3.64 (s,
General Procedure (A) for the synthesis of compounds 2a-
13
3
1
1
2
1
8
H), 3.21-3.18 (m, 2H) ; C NMR (100 MHz, DMSO-d ): δ
6
2
e from Isatoic anhydrides. The suspension of isatoic
73.0, 168.2, 149.2, 136.5, 132.2, 128.1, 127.5, 124.0, 121.4,
18.9, 118.5, 118.4, 118.0, 114.8, 111.9, 110.5, 54.0, 52.3,
6.9 ; IR (ATR): νmax 3417, 3326, 3259, 2938, 2341, 2097,
890, 1726, 1572, 1508, 1423, 1230, 1174, 1101, 1017, 897,
anhydride (1 mmol) and L-tryptophan methyl ester (1 mmol)
in anhydrous acetonitrile (20 mL) was refluxed under an
argon atmosphere for 16 h. After completion of the reaction
the mixture was concentrated in vacuo to yield the crude
compound, which was subjected to trituration using
acetonitrile and diethyl ether. The solid was filtered out and
dried under vacuo.
22, 742; HRMS (ESI): m/z calcd for C19
H
18ClN
3
O Na [M +
4
+
Na] : 394.0929; found: 394.0929.
Methyl (2-amino-5-bromobenzoyl)-L-tryptophanate (2d).
The title compound 2d was prepared from compound 1d (2.0
g, 8.30 mmol) and L-tryptophan methyl ester (1.81 g, 8.30
mmol) according to the general procedure A. The product 2d
was obtained as a grey solid (2.48 g, 72%); mp 212.5–213.5
°
Methyl (2-aminobenzoyl)-L-tryptophanate (2a). The title
compound 2a was prepared from compound 1a (2.0 g, 12.26
mmol) and L-tryptophan methyl ester (2.67 g, 12.26 mmol)
according to the general procedure A. The product 2a was
1
C; H NMR (400 MHz, DMSO-d
6
): δ 10.86 (s, 1H), 8.68 (d, J =
obtained as a grey solid (2.89 g, 69%); mp 139.0–140.3 °C;
8
8
.0 Hz, 1H), 7.71 (br s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.34 (d, J =
.0 Hz, 1H), 7.27 (dd, J = 1.6, 8.8 Hz, 1H), 7.20 (br s, 1H), 7.07
1
H NMR (400 MHz, DMSO-d
H), 8.46 (d, J = 8.0 Hz, 1H), 7.57-7.50 (m, 2H), 7.34 (d, J = 8.0
Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), ( 7.14 (td, J = 4.0, 10.6 Hz,
6
): 400 MHz, DMSO-d
6
: δ 10.84 (s,
1
(t, J = 8.0 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.66 (d, J = 12.0 Hz,
1
3
1
1
3
1
H), 6.52 (br s, 2H), 4.63 (q, J = 8.0 Hz, 1H), 3.63 (s, 3H), 3.26-
1
H), 7.07 (td, J = 1.2, 10.8 Hz, 1H), 7.00 (td, J = 0.8, 11.0 Hz,
13
.22 (m, 2H) ; C NMR (100 MHz, DMSO-d ): δ 173.0, 168.2,
6
1
H), 6.67 (dd, J = 1.2, 8.2 Hz, 1H), 6.52-6.48 (m, 1H), 6.35 (br
49.5, 136.5, 134.9, 130.9, 127.5, 124.0, 121.4, 118.9, 118.4,
15.4, 111.9, 110.5, 105.2, 54.0, 52.3, 26.9 ; IR (ATR): νmax
471, 3319, 2298, 2112, 1629, 1521, 1362, 1248, 1161, 1097,
1
3
s, 2H), 4.66-4.60 (m, 1H), 3.26-3.23 (m, 2H), 3.63 (s, 3H) ; C
NMR (100 MHz, DMSO-d ): δ173.3, 169.4, 150.2, 136.5,
32.4, 128.9, 127.5, 124.1, 121.5, 118.9, 118.5, 116.8, 114.8,
6
1
1
3
1
C
040, 817, 734; HRMS (ESI): m/z calcd for C19
H
18BrN
3
O [M +
3
14.1, 111.9, 110.5, 53.9, 52.3, 26.9; IR (ATR): νmax 3420,
326, 3239, 2950, 2341, 2114, 1920, 1739, 1640, 1508, 1433,
+
H] : 416.0604; found: 416.0602.
233, 1176, 1133, 1017, 901, 742; HRMS (ESI): m/z calcd for
Methyl (2-amino-5-methoxybenzoyl)-L-tryptophanate (2e).
The title compound 2e was prepared from compound 1e (2.0
g, 10.36 mmol) and L-tryptophan methyl ester (2.26 g, 10.36
mmol) according to the general procedure A. The product 2e
was obtained as a grey solid (2.28 g, 60%); mp 144.0–144.1
°
+
19
H
19
N
3
O
3
[M + H] : 338.1499; found: 338.1494.
Methyl (2-amino-5-fluorobenzoyl)-L-tryptophanate (2b).
The title compound 2b was prepared from compound 1b (2.0
g, 11.04 mmol) and L-tryptophan methyl ester (2.41 g, 11.04
mmol) according to the general procedure A. The product 2b
1
C; H NMR (400 MHz, DMSO-d
6
): δ 10.86 (s, 1H), 8.50 (d, J =
8
.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H),
was obtained as a grey solid (2.43 g, 62%); mp 151.5–152.9 7.23 (d, J = 1.6 Hz, 1H), 7.08-7.06 (m, 2H), 6.98 (t, J = 8.0 Hz,
1
1H), 6.84 (dd, J = 4.0, 8.0 Hz, 1H), 6.63 (d, J = 12.0 Hz, 1H),
°
1
7
C; H NMR (400 MHz, DMSO-d
6
): 400 MHz, DMSO-d : δ
6
5
3
.89 (br s, 2H), 4.65-4.59 (m, 1H), 3.68 (s, 3H), 3.66 (s, 3H),
0.86 (s, 1H), 8.59 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H),
.39 (dd, J = 0.80, 9.2 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.22 (d,
13
.29-3.18 (m, 2H) ; C NMR (100 MHz, DMSO-d ): δ 173.2,
6
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
Journal Name
1
1
2
1
1
69.1, 149.6, 144.4, 136.5, 127.5, 124.1, 121.4, 120.4, 118.9, 128.1, 127.58, 127.5, 124.2, 123.6, 123.3, 122.5, 121.4,
18.4, 118.1, 114.4, 112.6, 111.9, 110.5, 56.0, 54.0, 52.3, 119.8, 118.9, 118.4, 115.6, 115.4, 111.9, 110.1, 54.4, 52.5,
7.0 ; IR (ATR): νmax 3416, 3334, 3227, 2990, 2833, 2631, 26.9, 31.4, 22.5, 14.4 ; IR (ATR): νmax 3308, 3054, 2949, 1904,
639, 1569, 1509, 1741, 1485, 1423, 1343, 1236, 1174, 1146, 1734, 1647, 1605, 1510, 1410,1351, 1301, 1205, 1116, 1009,
View Article Online
DOI: 10.1039/C8OB01699B
043, 1019, 834, 754; HRMS (ESI): m/z calcd for C20
H
21
N
3
O
4
960, 822, 740; HRMS (ESI): m/z calcd for C30
Na] : 532.1643; found: 532.1639.
H
24FN
3
O Na [M +
4
+
+
Na [M + Na] : 390.1424; found: 390.1424.
General Procedure (B) for the synthesis of compounds 3a- Methyl
(2-(2-Naphthamido)-5-chlorobenzoyl)-L-
3e. To a suspension of the anthranilate (1 mmol) in tryptophanate (3c). The title compound 3c was prepared
anhydrous dichloromethane (15 mL) triethylamine (3 mmol) from compound 2c (1.5 g, 4.03 mmol) and 2- naphthoyl
was added at 0 ° C and stirred for 5 min. 2-naphthoyl chloride (0.923 g, 4.84 mmol) according to the general
chloride (1.2 mmol) in dichloromethane (15 mL) was added procedure B. The product 3c was obtained as a grey solid
1
dropwise for 10 min. The reaction mixture was warmed to (1.48 g, 70%); mp 208.6–209.1 °C; H NMR (400 MHz, DMSO-
room temperature and stirred for 6 h. After completion, the
6
d ): δ 12.11 (s, 1H), 10.87 (s, 1H), 9.41 (d, J = 8.0 Hz, 1H), 8.65
reaction mixture was diluted with dichloromethane and (d, J = 12.0 Hz, 1H), 8.46 (s, 1H), 8.10-8.01 (m, 3H), 7.89-7.86
washed with water, saturated sodium bicarbonate and then (m, 2H), 7.70-7.58 (m, 4H), 7.30 (d, J = 8.0 Hz, 1H), 7.25 (d, J =
saturated brine solution. The organic layer was separated, 4.0 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H), 6.97 (t, J = 12.0 Hz, 1H),
1
3
dried over sodium sulfate, filtered and evaporated under 4.80-4.77 (m, 1H), 3.64 (s, 3H), 3.30-3.28 (m, 2H) ; C NMR
reduced pressure. The crude compound was triturated with (100 MHz, DMSO-d ): δ 172.3, 168.0, 165.0, 138.5, 136.5,
diethylether and the solid was filtered and dried under 134.9, 132.69, 132.6, 131.9, 129.5, 129.2, 128.7, 128.6,
6
vacuo.
128.3, 128.2, 127.6, 127.5, 127.2, 124.1, 123.6, 122.7, 122.2,
21.5, 118.9, 118.4, 111.9, 110.1, 54.5, 52.5, 26.9; IR (ATR):
max 3308, 3054, 2947, 2341, 2111, 1917, 1735, 1668, 1628,
1
ν
Methyl (2-(2-Naphthamido)benzoyl)-L-tryptophanate (3a).
The title compound 3a was prepared from compound 2a (1.5
g, 4.44 mmol) and 2- naphthoyl chloride (1.01 g, 5.33 mmol)
according to the general procedure B. The product 3a was
1
7
5
584, 1512, 1436, 1303, 1207, 1096, 1008, 912, 960, 824,
+
35; HRMS (ESI): m/z calcd for C30
H
24ClN
3
O
4
Na [M + Na] :
48.1348; found: 548.1346.
obtained as a off-white solid (1.41 g, 65%); mp 173.9–174.7
1
°
C; H NMR (400 MHz, CDCl
3
): δ 12.22 (s, 1H), 8.87 (d, J = 8.0 Methyl
(2-(2-Naphthamido)-5-bromobenzoyl)-L-
Hz, 1H), 8.58 (s, 1H), 8.20 (br s, 1H), 8.11-8.09 (m, 1H), 8.05- tryptophanate (3d). The title compound 3d was prepared
8
.02 (m, 1H), 7.99-7.97 (m, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.62- from compound 2d (1.5 g, 3.60 mmol) and 2- naphthoyl
7
.52 (m, 4H), 7.37-7.31 (m, 2H), 7.18 (t, J = 8.0 Hz, 1H), 7.10 chloride (0.824 g, 4.32 mmol) according to the general
(
5
t, J = 8.0 Hz, 1H), 7.03-7.01 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H), procedure B. The product 3d was obtained as an off-white
.20-5.18 (m, 1H), 3.77 (s, 3H), 3.51-3.47 (m, 2H) ; C NMR solid (1.27 g, 62%); mp 216.6–217.2 °C; H NMR (400 MHz,
1
3
1
(100 MHz, CDCl
3
): δ 172.0, 168.7, 165.6, 140.1, 136.1, 134.9, DMSO-d ): δ 12.11 (s, 1H), 10.86 (s, 1H), 9.42 (d, J = 8.0 Hz,
6
1
1
1
3
1
33.0, 132.7, 132.0, 129.4, 128.6, 128.4, 127.8, 127.7, 127.5, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.45 (s, 1H), 8.09-7.88 (m, 4H),
26.8, 126.7, 123.7, 122.9, 122.8, 122.4, 121.6, 119.88, 7.88-7.78 (m, 2H), 7.67-7.57 (m, 3H), 7.30-7.23 (m, 2H), 7.05-
19.87, 118.5, 111.4, 109.8, 53.4, 52.6, 27.6 ; IR (ATR): νmax 6.94 (m, 2H), 4.80-4.75 (m, 1H), 3.63 (s, 3H), 3.31-3.30 (m,
13
308, 3050, 2945, 2319, 2112, 1909, 1733, 1661, 1585, 1516, 2H) ; C NMR (100 MHz, DMSO-d ): δ 172.3, 167.9, 165.0,
6
430, 1299, 1218, 1092, 1027, 858, 811, 754, 667; HRMS 138.9, 136.5, 135.6, 134.9, 132.6, 132.0, 131.4, 129.6, 129.2,
+
(ESI): m/z calcd for C30
H
25
N
3
O
4
Na [M + Na] : 514.1737; 128.7, 128.3, 128.2, 127.6, 127.5, 124.2, 123.6, 123.0, 122.4,
121.5, 118.9, 118.5, 115.1, 111.9, 110.2, 54.5, 52.6, 26.9 ; IR
found: 514.1736.
(
ATR): νmax 3448, 3286, 2341, 2105, 1918, 1736, 1649, 1594,
Methyl
(2-(2-Naphthamido)-5-fluorobenzoyl)-L-
1
509, 1430, 1392, 1299, 1098, 972, 912, 823, 739; HRMS
tryptophanate (3b). The title compound 3b was prepared
from compound 2b (1.5 g, 4.22 mmol) and 2- naphthoyl
chloride (0.965 g, 5.06 mmol) according to the general
+
(ESI): m/z calcd for C30
H
24BrN
3
O
4
Na [M + Na] : 592.0842;
found: 592.0842.
procedure B. The product 3b was obtained as an off-white Methyl
solid (1.43 g, 67%); mp 125.1–125.6 °C; H NMR (400 MHz, tryptophanate (3e). The title compound 3e was prepared
(2-(2-Naphthamido)-5-methoxybenzoyl)-L-
1
DMSO-d
6
): δ 11.97 (s, 1H), 10.85 (br s, 1H), 9.30 (d, J = 8.0 Hz, from compound 2e (1.0 g, 4.08 mmol) and 2- naphthoyl
1
H), 8.60 (dd, J = 4.0, 16.0 Hz, 1H), 8.46 (br s, 1H), 8.09-8.00 chloride (0.933 g, 4.89 mmol) according to the general
(
m, 3H), 7.88 (dd, J = 4.0, 8.0 Hz, 1H), 7.66-7.57 (m, 3H), 7.56 procedure B. The product 3e was obtained as a grey solid
1
(d, J = 8.0 Hz, 1H), 7.52-7.47 (m, 1H), 7.30 (d, J = 8.0 Hz, 1H), (1.59 g, 75%); mp 178.9–179.3 °C; H NMR (400 MHz, DMSO-
7
.24 (d, J = 4.0 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H), 6.96 (t, J = 8.0
d
6
): δ 11.77 (s, 1H), 8.74 (d, J = 8.0 Hz, 1H), 8.55 (s, 1H), 8.18
Hz, 1H), 4.77-4.75 (m, 1H), 3.62 (s, 3H), 3.31-3.27 (m, 2H) ; C (br s, 1H), 8.07-7.91 (m, 4H), 7.62-7.57 (m, 3H), 7.30 (t, J =
NMR (100 MHz, DMSO-d ): δ 172.3, 168.0, 164.9, 158.7, 8.0 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.12-7.07 (m, 2H), 7.01 (br
36.5, 136.0, 134.9, 132.6, 132.1, 129.5, 129.1, 128.6, 128.2, s, 1H), 6.82-6.78 (m, 2H), 5.16 (q, J = 4.0 Hz, 1H), 3.77 (s, 3H),
13
6
1
1
0 | J. Name., 2012, 00, 1-3
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Journal Name
ARTICLE
1
3
3
d
1
.68 (s, 3H), 3.55-3.42 (m, 2H) ; C NMR (100 MHz, DMSO- g, 1.90 mmol) according to the general procedure C. The
6
View Article Online
DOI: 10.1039/C8OB01699B
): δ 171.9, 168.4, 165.3, 154.9, 136.1, 134.9, 133.1, 132.8, product 4c was obtained as an off-white solid (0.86 g, 89%);
32.1, 129.3, 128.6, 128.2, 127.76, 127.7, 127.5, 126.6, mp 222.3–222.9 °C; H NMR (400 MHz, DMSO-d
1
6
): δ 12.86
1
1
2
1
23.6, 123.3, 122.8 122.4, 121.5, 119.9, 118.4, 118.1, 112.1, (br s, 1H), 12.23 (s, 1H), 10.85 (br s, 1H), 9.30 (d, J = 8.0 Hz,
11.4, 109.7, 55.6, 53.5, 52.6, 27.5 ; IR (ATR): νmax 3330, 1H), 8.67 (d, J = 8.0 Hz, 1H), 8.45 (br s, 1H), 8.09-8.01 (m, 3H),
990, 2941, 1742, 1662, 1595, 1509, 1438, 1356, 1310, 1208, 7.92-7.86 (m, 2H), 7.69-7.62 (m, 4H), 7.29 (d, J = 8.0 Hz, 1H),
096, 1032, 902, 866, 813, 760, 684; HRMS (ESI): m/z calcd 7.25 (d, J = 4.0 Hz, 1H), 7.06-6.96 (m, 2H), 4.78-4.72 (m, 1H),
+
13
for C31
H
27
N
3
O
5
Na [M + Na] : 544.1843; found: 544.1841.
3.40-3.25 (m, 2H) ; C NMR (100 MHz, DMSO-d
6
): δ 173.3,
1
1
1
2
2
1
C
68.0, 165.0, 138.6, 136.5, 134.9, 132.6, 132.0, 129.6, 129.2,
General Procedure (C) for the hydrolysis of esters 4a-4e. To
the solution of methyl ester (1 mmol) in THF (2.0 mL) and
MeOH (2.0 mL) 1 N NaOH(aq) (2 mmol) was added and stirred
at room temperature for 8 h. After completion, the reaction
mixture was acidified with 1.5 N HCl and then extracted with
ethylacetate. The organic layer was washed with water, and
saturated brine. The organic layer was separated and dried
28.7, 128.6, 128.3, 128.1, 127.5, 127.1, 124.0, 123.6, 122.5,
22.1, 121.4, 118.8, 118.5, 111.9, 110.6, 60.2, 54.4, 49.0,
6.9, 21.5, 21.3 ; IR (ATR): νmax 3392, 3316, 3056, 2930, 2342,
097, 1912, 1725, 1662, 1589, 1521, 1452, 1305, 1396, 1222,
092, 999, 884, 810, 648, 735; HRMS (ESI): m/z calcd for
+
29
H
22ClN
3
O
4
Na [M + Na] : 534.1191; found: 534.1190.
over sodium sulfate, filtered and evaporated under reduced (2-(2-Naphthamido)-5-bromobenzoyl)-L-tryptophan (4d).
pressure to yield the product.
The title compound 4d was prepared from compound 3d (1.0
g, 1.75 mmol) according to the general procedure C. The
(
2-(2-Naphthamido)benzoyl)-L-tryptophan (4a). The title
product 4d was obtained as an off-white solid (0.799 g, 82%);
compound 4a was prepared from compound 3a (1.0 g, 2.03
mmol) according to the general procedure C. The product 4a
was obtained as an off-white solid (0.825 g, 85%); mp 216.0–
1
mp 193.6–194.0 °C; H NMR (400 MHz, DMSO-d
6
): δ 13.0 (br
s, 1H), 12.25 (s, 1H), 10.85 (br s, 1H), 9.30 (d, J = 12.0 Hz, 1H),
8
7
7
4
d
1
1
1
2
1
C
.62 (d, J = 12.0 Hz, 1H), 8.45 (br s, 1H), 8.10-8.01 (m, 4H),
.87 (dd, J = 4.0, 12.0 Hz, 1H), 7.79 (dd, J = 4.0, 12.0 Hz, 1H),
.70-7.62 (m, 3H), 7.30-7.23 (m, 2H), 7.06-6.94 (m, 2H), 4.77-
1
2
1
=
4
4
3
1
1
1
17.4 °C; H NMR (400 MHz, DMSO-d
6
): δ 12.86 ( br s, 1H),
2.32 (s, 1H), 10.82 (s, 1H), 9.11 (d, J = 8.0 Hz, 1H), 8.65 (d, J
8.0 Hz, 1H), 8.46 (br s, 1H), 8.09-8.01 (m, 3H), 7.89 (dd, J = -
.0, -12.0 Hz, 1H), 7.84 (dd, J = 4.0, 8.0 Hz, 1H), 7.67-7.57 (m,
13
.70 (m, 1H), 3.29-3.21 (m, 2H) ; C NMR (100 MHz, DMSO-
6
): δ 173.3, 167.8, 165.0, 139.0, 136.5, 135.5, 134.9, 132.6,
H), 7.30-7.19 (m, 3H), 7.03-6.95 (m, 2H), 4.76-4.72 (m, 1H),
32.0, 131.4, 129.6, 129.2, 128.7, 128.3, 128.1, 127.6, 127.5,
24.0, 123.6, 122.7, 122.4, 121.4, 118.8, 118.6, 115.0, 111.9,
10.7, 54.5, 26.9 ; IR (ATR): νmax 3382, 3313, 3052, 2928,
342, 2110, 1907, 1737, 122, 1580, 1501, 1437, 1388, 1308,
.32-3.27 (m, 2H) ; 1 C NMR (100 MHz, DMSO-d
3
6
): δ 173.5,
69.3, 164.9, 139.7, 136.5, 134.8, 132.9, 132.6, 132.3, 129.6,
29.1, 128.9, 128.6, 128.2, 128.1, 127.5, 124.0, 123.6, 123.3,
21.4, 120.7, 120.5, 118.8, 118.5, 111.9, 110.7, 54.3, 26.8 ; IR
162, 1072, 1092, 912, 818, 736; HRMS (ESI): m/z calcd for
(ATR): νmax 3508, 3071, 2564, 2445, 2188, 2067, 1984, 1728,
+
29
H
22BrN
3
O
4
Na [M + Na ] : 578.0686; found: 578.0686.
1
C
629, 1526, 1434, 1328, 1219, 746; HRMS (ESI): m/z calcd for
+
29
H
23
N
3
O
4
Na [M + Na] : 500.1581; found: 500.1573.
(2-(2-Naphthamido)-5-methoxybenzoyl)-L-tryptophan (4e).
The title compound 4e was prepared from compound 3e (1.0
g, 1.91 mmol) according to the general procedure C. The
(
2-(2-Naphthamido)-5-fluorobenzoyl)-L-tryptophan
(4b).
The title compound 4b was prepared from compound 3b (1.0
g, 1.96 mmol) according to the general procedure C. The
product 4e was obtained as an off-white solid (0.78 g, 81%);
1
mp 150.8–151.0 °C; H NMR (400 MHz, DMSO-d
6
): δ 11.96 (s,
product 4b was obtained as an off-white solid (0.77 g, 80%);
1
1
7
6
3
1
1
1
5
2
1
H), 10.86 (s, 1H), 9.10 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 12.0 Hz,
H), 8.45 (br s, 1H), 8.11-7.97 (m, 3H), 7.88 (d, J = 8.0 Hz, 1H),
.68-7.62 (m, 3H), 7.31-7.18 (m, 4H), 7.04 (t, J = 8.0 Hz, 1H),
1
mp 171.9–172.9 °C; H NMR (400 MHz, DMSO-d
6
): δ 12.80
(
br s, 1H), 12.11 (s, 1H), 10.84 (d, J = 4.0 Hz, 1H), 9.19 (d, J =
8
1
8
.0 Hz, 1H), 8.64 (dd, J = 8.0, 10.0 Hz, 1H), 8.46 (d, J = 4.0 Hz,
H), 8.08-8.06 (m, 2H), 8.02-8.00 (m, 1H), 7.88 (dd, J = 4.0,
.0 Hz, 1H), 7.69-7.60 (m, 4H), 7.51-7.47 (m, 1H), 7.30-7.24
.96 (t, J = 8.0 Hz, 1H), 4.75-4.74 (m, 1H), 3.83 (s, 3H), 3.28-
13
.22 (m, 2H) ; C NMR (100 MHz, DMSO-d
6
): δ 173.4, 168.9,
64.5, 154.9, 136.5, 134.8, 132.8, 132.7, 132.4, 129.5, 129.1,
28.5, 128.1, 128.0, 127.6, 127.4, 125.6, 124.1, 123.6, 122.6,
22.3, 121.4, 118.8, 118.5, 118.4, 113.8, 111.9, 110.7, 56.0,
4.3, 26.9 ; IR (ATR): νmax 3491, 3430, 3254, 2917, 2734,
540, 2082, 1919, 1707, 1647, 1600, 1520, 1451, 1432, 1345,
(
m, 2H), 7.05-6.94 (m, 2H), 4.76-4.70 (m, 1H), 3.37-3.27 (m,
2
1
1
1
H) ; 13C NMR (100 MHz, DMSO-d
): δ 173.3, 168.0, 164.9,
6
56.3, 136.5, 136.2, 134.9, 132.6, 132.1, 129.6, 129.1, 128.6,
28.2, 128.1, 127.5, 124.1, 123.6, 123.0, 122.4, 122.3, 121.4,
19.7, 119.5, 118.5, 115.3, 111.9, 110.6, 79.6, 54.4, 26.9 ; IR
097, 1044, 926, 864, 823, 737, 682; HRMS (ESI): m/z calcd
(ATR): νmax 3296, 3053, 2921, 2341, 2106, 1918, 1718, 1640,
+
for C30
H
25
N
3
O
5
Na [M + Na] : 530.1686; found: 530.1686.
1
7
5
604, 1509, 1409, 1305, 1203, 1094, 1010, 948, 864, 821,
39; HRMS (ESI): m/z calcd for C29
18.1487; found: 518.1479.
+
H
22FN
3
O
4
Na [M + Na] : General Procedure (D) for the synthesis of amides 5a-5e,
6a-6e, 7a-7e. To the stirred solution of an acid (1.0 mmol),
amine (1.0 mmol), HOBt (1.0 mmol), DIEA (2.5 mmol) in DMF
(
2-(2-Naphthamido)-5-chlorobenzoyl)-L-tryptophan
(4c).
(5 – 10 mL) EDCI (1.2 mmol) was added portion-wise. The
The title compound 4c was prepared from compound 3c (1.0
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Page 12 of 23
ARTICLE
Journal Name
reaction was stirred for 16 h before the solvent was removed 1H), 8.25 (t, J = 8.0 Hz, 1H), 8.07-8.02 (m, 3H), 7.90-7.71 (m,
View Article Online
DOI: 10.1039/C8OB01699B
under reduced pressure and the resultant residue subjected 2H), 7.69-7.61 (m, 4H), 7.26-7.21 (m, 2H), 7.03-6.94 (m, 2H),
to flash chromatography (2-5% MeOH/CH
to afford the desired compounds.
2
Cl as the eluent) 6.78 (t, J = 4.0 Hz, 1H), 4.76-4.71 (m, 1H), 3.29-3.01 (m, 6H),
2
1
3
1.33 (s, 9H), ; C NMR (100 MHz, DMSO-d
6
): δ 171.5, 167.8,
1
1
1
4
1
9
65.0, 156.1, 138.5, 136.5, 134.9, 132.6, 132.3, 132.0, 129.5,
tert-Butyl (S)-(2-(2-(2-(2-naphthamido)benzamido)-3-(1H-
indol-3-yl)propanamido)ethyl)carbamate (5a). The title
compound 5a was prepared from compound 4a (0.1 g,
29.1, 128.8, 128.6, 128.4, 128.1, 127.6, 127.5, 127.1, 124.0,
23.6, 122.7, 122.5, 121.3, 118.9, 118.6, 111.8, 110.9, 78.1,
0.0, 39.4, 28.6, 27.8 ; IR (ATR): νmax 3298, 3054, 2342, 2112,
910, 1655, 1584, 1506, 1451, 1392, 1301, 1230, 1166, 1096,
0.2094 mmol) and N-Boc-1,2-diaminoethane (33 mg, 0.2094
mmol) according to the general procedure D. The product 5a
was obtained as an off-white solid (91 mg, 70%); mp 223.7–
70, 913, 826, 734; HRMS (ESI): m/z calcd for C36
H
36ClN
5
5
O Na
+
[M + Na] : 676.2297; found: 676.2295.
2
8
8
1
3
1
25.1 °C; 400 MHz, DMSO-d : δ 12.30 (s, 1H), 10.78 (s, 1H),
6
tert-Butyl
bromobenzamido)-3-(1H-indol-3-
yl)propanamido)ethyl)carbamate (5d). The title compound
(S)-(2-(2-(2-(2-naphthamido)-5-
.92 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 8.0 Hz, 1H), 8.46 (s, 1H),
.26 (t, J = 4.0 Hz, 1H), 8.07-8.00 (m, 3H), 7.88 (d, J = 8.0 Hz,
H), 7.82 (d, J = 8.0 Hz, 1H), 7.71-7.56 (m, 4H), 7.28-7.20 (m,
H), 7.04-6.95 (m, 2H), 6.77 (t, J = 4.0 Hz, 1H), 4.76-4.73 (m,
5d was prepared from compound 4d (0.1 g, 0.1797 mmol)
and a N-Boc-1,2-diaminoethane (28 mg, 0.1797 mmol)
according to the general procedure D. The product 5d was
obtained as an off-white solid (81 mg, 65%); mp 189.5–190.9
13
H), 3.29-3.01 (m, 6H), 1.33 (s, 9H) ; C NMR (100 MHz,
): δ 171.7, 169.1, 165.0, 156.1, 139.6, 136.5, 134.8,
32.7, 132.6, 132.3, 129.5, 129.0, 128.5, 128.3, 128.1, 127.6,
DMSO-d
6
1
1
1
3
9
6
1
°
C; H NMR 400 MHz, DMSO-d
6
: δ 12.22 (s, 1H), 10.80 (s, 1H),
27.5, 124.1, 123.7, 123.2, 121.3, 121.2, 120.8, 118.9, 118.6,
11.8, 110.9, 78.1, 54.9, 40.0, 39.4, 28.6, 27.8 ; IR (ATR): νmax
9
8
7
1
.16 (d, J = 8.0 Hz, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.43 (s, 1H),
.27 (br s, 1H), 8.06-8.00 (m, 4H), 7.86-7.61 (m, 5H), 7.26-
.21 (m, 2H), 7.03-6.95 (m, 2H), 6.78 (br s, 1H), 4.75-4.71 (m,
300, 3054, 2112, 1656, 1568, 1516, 1447, 1232, 1165, 971,
+
11, 862; HRMS (ESI): m/z calcd for C36
H
37
N
5
O
5
Na [M + Na] :
13
H), 3.29-3.01 (m, 6H), 1.33 (s, 9H), ; C NMR (100 MHz,
42.2687; found: 642.2678.
DMSO-d
34.9, 132.6, 132.0, 131.6, 129.5, 129.1, 128.6, 128.4, 128.1,
-(1H-indol-3-yl)propanamido)ethyl)carbamate (5b). The 127.6, 127.5, 124.0, 123.6, 122.9, 122.7, 121.3, 118.9, 118.6,
title compound 5b was prepared from compound 4b (0.1 g, 115.0, 111.8, 110.9, 78.1, 55.1, 40.0, 39.4, 28.6, 27.8 ; IR
ATR): νmax 3390, 1740, 1636, 1578, 1498, 1437, 1388, 1299,
6
): δ 171.5, 167.7, 165.0, 156.1, 138.9, 136.5, 135.2,
tert-Butyl (S)-(2-(2-(2-(2-naphthamido)-5-fluorobenzamido)-
3
1
(
1
C
0.2018 mmol) and N-Boc-1,2-diaminoethane (32 mg, 0.2018
225, 1164, 1093, 1010, 912, 819; HRMS (ESI): m/z calcd for
mmol) according to the general procedure D. The product 5b
+
36
H
36BrN
5
O
5
Na [M + Na] : 720.1792; found: 720.1792.
was obtained as an off-white solid (91 mg, 71%); mp 241.4–
tert-Butyl
methoxybenzamido)-3-(1H-indol-3-
yl)propanamido)ethyl)carbamate (5e). The title compound
(S)-(2-(2-(2-(2-naphthamido)-5-
1
2
42.4 °C; H NMR (400 MHz, DMSO-d
6
): δ 12.05 (s, 1H), 10.79
(s, 1H), 9.03 (d, J = 4.0 Hz, 1H), 8.58-8.55 (m, 1H), 8.45 (s,
1
7
7
3
1
1
1
1
H), 8.25 (s, 1H), 8.06-8.00 (m, 3H), 7.86 (d, J = 8.0 Hz, 1H), 5e was prepared from compound 4e (0.1 g, 0.197 mmol) and
N-Boc-1,2-diaminoethane (31 mg, 0.197 mmol) according to
the general procedure D. The product 5e was obtained as an
off-white solid (77 mg, 60%); mp 244.1–244.6 °C; H NMR
.69-7.62 (m, 4H), 7.47 (t, J = 4.0 Hz, 1H), 7.27-7.22 (m, 2H),
.03-6.94 (m, 2H), 6.78 (br s, 1H), 4.73-4.69 (m, 1H), 3.17-
1
1
3
.00 (m, 6H), 1.33 (s, 9H) ; C NMR (100 MHz, DMSO-d ): δ
6
(
=
3
400 MHz, DMSO-d
6
): δ 11.87 (s, 1H), 10.83 (s, 1H), 8.95 (d, J
8.0 Hz, 1H), 8.44-8.40 (m, 2H), 8.28 (br s, 1H), 8.06-7.99 (m,
H), 7.88-7.86 (m, 1H), 7.71-7.60 (m, 3H), 7.29-7.24 (m, 3H),
71.5, 167.8, 165.0, 158.7, 156.3, 156.1, 136.5, 136.0, 134.8,
32.6, 132.1, 129.5, 129.0, 128.5, 128.3, 128.1, 127.6, 127.5,
24.1, 123.7, 123.2, 121.3, 119.4, 119.2, 118.9, 118.6, 115.8, 7.17 (dd, J = 4.0, 8.0 Hz, 1H), 7.04-6.44 (m, 2H), 6.78 (t, J =
15.5, 111.8, 110.9, 78.1, 55.0, 40.0, 39.5, 28.6, 27.8 ; IR 8.0 Hz, 1H), 4.74-4.68 (m, 1H), 3.82 (s, 3H), 3.34-3.01 (m, 6H),
1
3
1
1
1
1
5
1
8
6
.33 (s, 9H) ; C NMR (100 MHz, DMSO-d ): δ 171.7, 168.7,
6
(
1
ATR): νmax 3304, 3057, 2928, 1657, 1596, 1520, 1285, 1232,
64.7, 156.0, 155.0, 136.5, 134.7, 132.6, 132.5, 132.4, 129.5,
28.9, 128.4, 128.1, 127.6, 127.4, 124.2, 123.7, 123.3, 122.8,
21.3, 118.9, 118.6, 118.1, 113.9, 111.8, 110.9, 78.1, 55.9,
4.8, 40.0, 39.4, 28.6, 27.8 ; IR (ATR): νmax 3302, 2288, 1655,
592, 1519, 1455, 1271, 1225, 1166, 1095, 1042, 991, 859,
19, 735; HRMS (ESI): m/z calcd for C37
168, 1094, 946, 866, 757, 724, 660; HRMS (ESI): m/z calcd
+
for C36
H
36FN
5
O
5
Na [M + Na] : 660.2593; found: 660.2590.
tert-Butyl
(S)-(2-(2-(2-(2-naphthamido)-5-
chlorobenzamido)-3-(1H-indol-3-
yl)propanamido)ethyl)carbamate (5c). The title compound
c was prepared from compound 4c (0.1 g, 0.1953 mmol)
+
H
39
N
5
O
6
Na [M + Na] :
72.2793; found: 672.2791.
5
and N-Boc-1,2-diaminoethane (31 mg, 0.1953 mmol)
according to the general procedure D. The product 5c was
(
S)-N-(2-((1-((2-diBocguanidinoethyl)amino)-3-(1H-indol-3-
yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-naphthamide
6a). The title compound 6a was prepared from compound
a (0.2 g, 0.4188 mmol) and amine (126 mg, 0.4188 mmol)
obtained as an off-white solid (89 mg, 69%); mp 189.6–191.2
(
4
1
°
C; H NMR (400 MHz, DMSO-d
6
): δ 12.19 (s, 1H), 10.80 (s,
1
H), 9.13 (d, J = 8.0 Hz, 1H), 8.61 (d, J = 8.0 Hz, 1H), 8.44 (s,
according to the general procedure D. The product 6a was
1
2 | J. Name., 2012, 00, 1-3
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Page 13 of 23
O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Journal Name
ARTICLE
+
obtained as an off-white solid (0.172 mg, 54%); mp 243.8– HRMS (ESI): m/z calcd for C42
H
46ClN
7
O
7
Na [M + Na] :
View Article Online
1
DOI: 10.1039/C8OB01699B
2
45.1 °C; H NMR (400 MHz, DMSO-d
6
): δ 12.56 (s, 1H), 11.35 818.3039; found: 818.3042.
(
(
(
br s, 1H), 8.91 (dd, J = 4.0, 8.0 Hz, 1H), 8.63 (br s, 1H), 8.44
br s, 1H), 8.22 (br s, 1H), 8.14 (dd, J = 4.0, 8.0 Hz, 1H), 8.05
d, J = 8.0 Hz, 1H), 7.99-7.92 (m, 2H), 7.70-7.55 (m, 6H), 7.36-
(
S)-N-(4-bromo-2-((1-((2-diBocguanidinoethyl)amino)-3-
(
1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
naphthamide (6d). The title compound 6d was prepared
7
.31 (m, 2H), 7.19-7.09 (m, 4H), 4.92-4.91 (m, 1H), 3.50-3.21
13
from compound 4d (0.2 g, 0.3594 mmol) and amine (108 mg,
(m, 6H), 1.57 (s, 9H), 1.52 (s, 9H) ; C NMR (100 MHz, DMSO-
0
.3594 mmol) according to the general procedure D. The
d
1
1
1
2
1
6
): δ 170.8, 168.5, 165.6, 157.0, 152.8, 140.3, 136.0, 134.9,
product 6d was obtained as an off-white solid (150 mg, 50%);
32.9, 132.8, 132.3, 129.4, 128.4, 128.3, 127.7, 127.6, 127.1,
26.5, 123.8, 123.1, 122.8, 122.2, 121.4, 119.7, 119.6, 118.8,
11.1, 110.4, 84.0, 80.5, 60.4, 54.5, 41.1, 40.2, 29.08, 28.2,
8.0, 21.0, 14.2 ; IR (ATR): νmax 3280, 2972, 2330, 2113, 1719,
1
mp 259.5–260.8 °C; H NMR (400 MHz, CDCl
3
): δ 12.37 (s,
1
1
8
H), 11.33 (s, 1H), 8.80 (dd, J = 4.0, 1.8 Hz, 1H), 8.57 (br s,
H), 8.43 (t, J = 4.0 Hz, 1H), 8.23 (br s, 1H), 8.09-8.06 (m, 1H),
.01 (d, J = 8.0 Hz, 1H), 7.95-7.89 (m, 2H), 7.73 (t, J = 4.0 Hz,
615, 1507, 1430, 1304, 1925, 1128, 1047, 880, 739; HRMS
+
1H), 7.58-7.54 (m, 5H), 7.33-7.28 (m, 2H), 7.16-7.07 (m, 3H),
(ESI): m/z calcd for C42
H
47
N
7
O
7
[M +H] : 762.3610; found:
4
9
.90-4.85 (m, 1H), 3.45-3.25 (m, 6H), 1.54 (s, 9H), 1.50 (s,
H); C NMR (100 MHz, CDCl ): δ 170.4, 167.2, 165.5, 162.7,
7
62.3605.
13
3
(
S)-N-(4-fluoro-2-((1-((2-diBocguanidinoethyl)amino)-3-(1H- 157.3, 152.9, 139.3, 136.0, 135.6, 135.0, 132.7, 131.9, 129.8,
indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2- 129.3, 128.5, 128.4, 127.9, 127.7, 127.5, 126.6, 123.7, 123.1,
naphthamide (6b). The title compound 6b was prepared 123.0, 122.3, 121.5, 119.8, 118.7, 115.3, 111.2, 110.3, 83.8,
from compound 4b (0.2 g, 0.4039 mmol) and amine (122 mg, 80.0, 54.6, 41.5, 40.0, 29.0, 28.3, 28.0 ; IR (ATR): νmax 3413,
0
.4039 mmol) according to the general procedure D. The 3353, 3276, 2984, 2763, 1769, 1679, 1646, 1551, 1459, 1405
product 6b was obtained as an off-white solid (160 mg, 52%); 1328, 1256, 1128, 1019, 831, 801; HRMS (ESI): m/z calcd for
1
+
mp 208.4–209.9 °C; H NMR (400 MHz, CDCl
3
): δ 12.30 (s,
42
C H
46BrN
7
O
7
[M + H] : 840.2715; found: 840.2712.
1
2
1
7
4
H), 11.35 (s, 1H), 8.88 (dd, J = 4.0, 8.0 Hz, 1H), 8.60 (br s,
(
S)-N-(4-methoxy-2-((1-((2-diBocguanidinoethyl)amino)-3-
H), 8.46 (br s, 1H), 8.21 (br s, 1H), 8.11 (dd, J = 4.0, 8.0 Hz,
H), 8.05-8.03 (m, 1H), 7.98-7.92 (m, 2H), 7.76 (br s, 1H),
.68-7.56 (m, 3H), 7.31-7.28 (m, 2H), 7.25-7.09 (m, 5H), 4.90-
(
1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
naphthamide (6e). The title compound 6e was prepared
from compound 4e (0.2 g, 0.394 mmol) and amine (119 mg,
13
.89 (m, 1H), 3.49-3.21 (m, 6H), 1.57 (s, 9H), 1.53 (s, 9H) ; C
): δ 170.6, 167.3, 165.4, 156.5, 152.8,
36.4, 136.0, 134.9, 132.7, 132.0, 129.3, 128.5, 128.3, 127.8,
0
.394 mmol) according to the general procedure D. The
NMR (100 MHz, CDCl
3
product 6e was obtained as an off-white solid (159 mg, 51%);
1
1
1
4
1
8
7
1
mp 249.1–249.8 °C; H NMR (400 MHz, DMSO-d
6
): δ 11.90 (s,
27.7, 127.5, 126.6, 123.7, 123.4, 123.3, 123.1, 122.2, 119.7,
19.5, 118.7, 113.8, 113.6, 111.2, 110.3, 84.1, 54.6, 41.3,
0.3, 29.0, 28.2, 28.0 ; IR (ATR): νmax 3278, 2974, 2327, 2119,
1
8
H), 11.48 (s, 1H), 10.80 (s, 1H), 8.95 (d, J = 8.0 Hz, 1H), 8.44-
.38 (m, 4H), 8.04-8.02 (m, 3H), 7.87-7.84 (m, 1H), 7.72-7.61
(m, 3H), 7.29-7.25 (m, 3H), 7.16 (dd, J = 4.0, 12.0 Hz, 1H),
908, 1719, 1607, 1509, 1409, 1322, 1226, 1128, 1047, 958,
+
7.02-6.96 (m, 2H), 4.74-4.68 (m, 1H), 3.83 (s, 3H), 3.40-3.27
09, 738; HRMS (ESI): m/z calcd for C42
H
46FN
7
O
7
[M + H] :
1
3
(
(
m, 2H), 3.26-3.17 (m, 4H), 1.41 (s, 9H), 1.35 (s, 9H) ; C NMR
100 MHz, DMSO-d ): δ 172.0, 168.7, 164.6, 163.5, 156.0,
80.3516; found: 780.3498.
6
(
S)-N-(4-chloro-2-((1-((2-diBocguanidinoethyl)amino)-3-
154.9, 152.3, 136.5, 134.7, 132.68, 132.6, 132.4, 129.4,
128.9, 128.4, 128.1, 127.6, 127.4, 124.1, 123.7, 123.1, 122.7,
(
1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
naphthamide (6c). The title compound 6c was prepared from 121.3, 118.9, 118.6, 118.1, 113.9, 111.7, 111.0, 83.2, 78.6,
compound 4c (0.2 g, 0.3906 mmol) and amine (118 mg, 55.9, 54.9, 40.5, 38.4 28.4, 28.0, 27.8; IR (ATR): νmax 3292,
0
.3906 mmol) according to the general procedure D. The 2976, 2088, 1720, 1612, 1517, 1412, 1326, 1227, 1132, 1047,
+
product 6c was obtained as an off-white solid (155 mg, 54%); 740; HRMS (ESI): m/z calcd for C43
mp 235.1–236.0 °C; H NMR (300 MHz, CDCl
H
49N7O
8
Na [M + Na] :
1
3
): δ 12.39 (s, 814.3535; found: 814.3533.
1
(
(
H), 11.35 (s, 1H), 8.88 (d, J = 9.0 Hz, 1H), 8.59 (br s, 1H), 8.46
t, J = 3.0 Hz, 1H), 8.28 (br s, 1H), 8.04-8.01 (m, 1H), 7.98-7.91
m, 2H), 7.76-7.69 (m, 1H), 7.66-7.55 (m, 3H), 7.51-7.48 (m, 3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-naphthamide
(
S)-N-(2-((1-((2-(dimethylamino)ethyl)amino)-3-(1H-indol-
2
3
H), 7.35-7.28 (m, 2H), 7.18-7.09 (m, 3H), 4.94-4.87 (m, 1H),
(
4
(
7a). The title compound 7a was prepared from compound
a (0.2 g, 0.4188 mmol) and N,N-dimethylethylenediamine
37 mg, 0.4188 mmol) according to the general procedure D.
13
.45-3.37 (m, 6H), 1.56 (s, 9H), 1.52 (s, 9H) ; C NMR (75
): δ 170.4, 167.3, 165.5, 162.7, 157.3, 152.9,
38.8, 136.0, 135.0, 132.76, 132.7, 131.9, 129.3, 128.5,
MHz, CDCl
3
1
1
1
4
1
The product 7a was obtained as an off-white solid (158 mg,
6
1
28.4, 127.9, 127.7, 127.5, 126.9, 126.6, 123.7, 123.1, 122.7,
22.3, 121.1, 119.7, 118.7, 111.2, 110.3, 83.8, 80.0, 54.6,
1.5, 40.06, 29.0, 28.3, 28.0 IR (ATR): νmax 3277, 2977, 1728,
615, 1502, 1397, 1305, 1226, 1128, 1048, 912, 807, 739; = 6.0 Hz, 1H), 8.47 (s, 1H), 8.09-8.01 (m, 4H), 7.89 (d, J = 12.0
Hz, 1H), 7.80 (d, J = 12.0 Hz, 1H), 7.71-7.56 (m, 4H), 7.29-7.25
1
9%); mp 87.0–88.2 °C; H NMR (400 MHz, DMSO-d
6
): δ
2.28 (s, 1H), 10.80 (s, 1H), 8.94 (d, J = 12.0 Hz, 1H), 8.57 (d, J
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Page 14 of 23
ARTICLE
Journal Name
(
4
m, 2H), 7.05-6.96 (m, 2H), 4.74-4.71 (m, 1H), 3.20-3.16 (m, naphthamide (7d). The title compound 7d was prepared
View Article Online
DOI: 10.1039/C8OB01699B
13
from compound 4d (0.2 g, 0.3594 mmol) and N,N-
dimethylethylenediamine (30 mg, 0.3594 mmol) according to
H), 2.23-2.18 (m, 2H), 2.02 (s, 6H); C NMR (150 MHz,
): δ 171.4, 169.1, 165.0, 139.5, 136.5, 134.8, 132.6,
32.3, 129.5, 129.0, 128.5, 128.2, 128.1, 127.6, 127.5, 124.2,
23.7, 123.3, 121.4, 121.3, 120.9, 118.9, 118.6, 111.7, 110.9,
8.4, 54.9, 45.4, 37.4, 27.7 ; IR (ATR): νmax 3282, 3053, 2942,
DMSO-d
6
the general procedure D. The product 7d was obtained as an
1
1
5
2
1
1
off-white solid (146 mg, 65%); mp 114.9–116.3 °C; H NMR
(
8
400 MHz, CDCl
.57 (br s, 1H), 8.17 (br s, 1H), 8.09-8.03 (m, 2H), 7.97 (d, J =
3
) : δ 12.26 (s, 1H), 8.80 (d, J = 8.0 Hz, 1H),
341, 2110, 1921, 1638, 1594, 1507, 1441, 1304, 1229, 1041, 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H),
096, 862, 911, 740; HRMS (ESI): m/z calcd for C33
H
33
N
5
O
3
7.67-7.56 (m, 4H), 7.44 (d, J = 8.0 Hz, 1H), 7.35-7.33 (m, 1H),
7.23-7.15 (m, 3H), 6.25 (br s, 1H), 4.97-4.91 (m, 1H), 3.51-
+
[M + H] : 548.2656; found: 548.2647.
3
.10 (m, 1H), 3.31-3.24 (m, 2H), 3.19-3.12 (m, 1H), 2.22-2.19
1
3
(m, 1H), 2.08-2.06 (m, 1H), 2.00 (s, 6H) ; C NMR (100 MHz,
(
3
S)-N-(2-((1-((2-(dimethylamino)ethyl)amino)-3-(1H-indol-
-yl)-1-oxopropan-2-yl)carbamoyl)-4-fluorophenyl)-2-
CDCl ): δ 170.4, 167.4, 165.5, 139.2, 136.2, 135.7, 135.0,
3
1
1
1
2
32.7, 131.8, 129.7, 129.4, 128.6, 128.4, 127.9, 127.7, 127.4,
26.7, 123.6, 123.1, 122.5, 121.4, 120.0, 118.8, 115.3, 111.3,
10.7, 57.1, 54.6, 44.6, 36.7, 29.0 ; IR (ATR): νmax 3055, 2934,
naphthamide (7b). The title compound 7b was prepared
from compound 4b (0.2 g, 0.4036 mmol) and N,N-
dimethylethylenediamine (36 mg, 0.4036 mmol) according to
the general procedure D. The product 7b was obtained as an
319, 1736, 1637, 1580, 1431, 1389, 1303, 1223, 1177, 912,
+
1
819; HRMS (ESI): m/z calcd for C H BrN O [M + H] :
33
32
5
3
off-white solid (161 mg, 71%); mp 148.4–149.9 °C; H NMR
6
25.1761; found: 626.1759.
(400 MHz, DMSO-d
6
): δ 12.03 (s, 1H), 10.81 (s, 1H), 9.03 (d, J
=
8
7
8.0 Hz, 1H), 8.53 (q, J = 8.0 Hz, 1H), 8.45 (br s, 1H), 8.09-
.01 (m, 4H), 7.87 (d, J = 8.0 Hz, 1H), 7.70-7.61 (m, 4H), 7.49-
.44 (m, 1H), 7.28-7.24 (m, 2H), 7.04-6.94 (m, 2H), 4.75-4.69
(
S)-N-(2-((1-((2-(dimethylamino)ethyl)amino)-3-(1H-indol-
3-yl)-1-oxopropan-2-yl)carbamoyl)-4-methoxyphenyl)-2-
naphthamide (7e). The title compound 7e was prepared
from compound 4e (0.2 g, 0.394 mmol) and N,N-
dimethylethylenediamine (32 mg, 0.394 mmol) according to
the general procedure D. The product 7e was obtained as an
off-white solid (154 mg, 68%); mp 98.8–99.6 °C; H NMR (400
MHz, DMSO-d ): δ 11.84 (s, 1H), 10.84 (s, 1H), 8.95 (d, J = 8.0
(m, 1H), 3.29-3.26 (m, 1H), 3.18-3.10 (m, 3H), 2.22-2.10 (m,
H), 2.03 (s, 6H) ; 1 C NMR (100 MHz, DMSO-d
3
2
1
1
1
6
): δ 171.2,
67.8, 165.0, 156.3, 136.5, 135.8, 134.8, 132.6, 132.1, 129.5,
29.0, 128.6, 128.3, 128.1, 127.6, 127.5, 124.2, 123.7, 121.3,
18.9, 118.6, 111.8, 110.8, 58.4, 55.0, 45.5, 37.4, 27.7 ; IR
1
6
(ATR): νmax 3390, 3318, 3050, 2918, 2599, 2119, 1724, 1639,
Hz, 1H), 8.45 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.10-8.00 (m,
4H), 7.89-7.86 (m, 1H), 7.71-7.61 (m, 3H), 7.30-7.24 (m, 3H),
1
589, 1520, 1447, 1326, 1213, 1154, 1094, 897, 759; HRMS
+
(ESI): m/z calcd for C33
H
32FN
5
O
3
[M + H] : 566.2562; found:
7
.16 (dd, J = 4.0, 12.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.96 (t,
5
66.2559.
J = 12.0 Hz, 1H), 4.74-4.68 (m, 1H), 3.82 (s, 3H), 3.27-3.21 (m,
1
3
2
H), 3.17-3.10 (m, 3H), 2.26-2.23 (m, 2H), 2.00 (s, 6H);
C
(
S)-N-(4-chloro-2-((1-((2-(dimethylamino)ethyl)amino)-3-
NMR (10 MHz, DMSO-d ): δ 171.4, 168.7, 164.7, 155.0,
6
(
1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
1
1
1
5
36.5, 134.8, 132.6, 132.4, 132.3, 129.5, 128.9, 128.4,
naphthamide (7c). The title compound 7c was prepared from
compound 4c (0.2 g, 0.3906 mmol) and N,N-
dimethylethylenediamine (35 mg, 0.3906 mmol) according to
the general procedure D. The product 7c was obtained as an
28.16, 128.1, 127.6, 127.4, 124.2, 123.7, 123.6, 122.9,
21.3, 118.9, 118.7, 118.1, 113.8, 111.8, 110.9, 58.4, 55.9,
4.9, 45.5, 37.4, 27.7 ; IR (ATR): νmax 3281, 3056, 2937, 2317,
1
2098, 1908, 1649, 1595, 1516, 145, 1224, 1097, 1036, 910,
off-white solid (158 mg, 70%); mp 131.2–132.6 °C; H NMR
+
8
5
20, 741; HRMS (ESI): m/z calcd for C34
78.2762; found: 578.2759.
H
35
N
5
O
4
[M + H] :
(
8
(
400 MHz, CDCl
.55 (br s, 1H), 8.22 (br s, 1H), 8.07-8.01 (m, 2H), 7.97-7.90
m, 2H), 7.79 (d, J = 8.0 Hz, 1H), 7.62-7.48 (m, 5H), 7.31 (d, J =
3
): δ 12.25 (s, 1H), 8.83 (d, J = 8.0 Hz, 1H),
General Procedure (E) for the N-Boc deprotection of
compounds 8a-8e. To a solution of 5a-5e (0.1 mmol) in
dichloromethane (4.0 mL) was added HCl in dioxane (4 N
solution) (1.0 mL) at 0 °C. The reaction mixture was warmed
to room temperature and stirred for 6 h. After completion of
the reaction, solvent was removed under reduced pressure
and treated with diethylether and the solid filtered out and
dried under high vacuum to yield the product.
8
4
3
.0 Hz, 1H), 7.28 (s, 1H),7.21-7.15 (m, 3H), 6.59 (br s, 1H),
.98-4.96 (m, 1H),3.52-3.47 (m, 1H), 3.36-3.27 (m, 2H), 3.19-
13
.16 (m, 2H), 2.35-2.16 (m, 6H), 2.08 (s, 6H) ; C NMR (100
): δ 170.6, 167.6, 165.5, 138.7, 136.2, 135.0,
32.79, 132.7, 131.8, 129.4, 128.6, 128.3, 127.96, 127.9,
MHz, CDCl
3
1
1
1
27.7, 127.4, 127.0, 126.7, 123.6, 123.2, 122.8, 122.4, 121.1,
19.9, 118.9, 111.3, 110.7, 57.1, 54.6, 44.4, 36.3, 28.9; IR
(ATR): νmax 3275, 3053, 2933, 2320, 2105, 2105, 1908, 1651,
1
7
5
581, 1500, 1430, 1396, 1297, 1227, 104, 1038, 912, 823,
(
S)-N-(2-((1-((2-aminoethyl)amino)-3-(1H-indol-3-yl)-1-
oxopropan-2-yl)carbamoyl)phenyl)-2-naphthamide
The title compound 8a was prepared from compound 5a
0.05 g, 0.08 mmol) according to the general procedure E.
+
36; HRMS (ESI): m/z calcd for C33
H
32ClN
5
O
3
[M + H] :
(8a).
82.2266; found: 582.2264.
(
(
S)-N-(4-bromo-2-((1-((2-(dimethylamino)ethyl)amino)-3-
The product 8a was obtained as a grey solid (33 mg, 79%);
(
1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
1
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 15 of 23
O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Journal Name
mp 249.4–250.0 °C; H NMR (400 MHz, DMSO-d
ARTICLE
): δ 12.28 (s, naphthamide (8d). The title compound 8d was prepared
1
6
View Article Online
1
1
2
4
2
1
1
1
5
1
8
5
H), 10.82 (s, 1H), 9.05 (d, J = 8.0 Hz, 1H), 8.59 (d, J = 8.0 Hz, from compound 5d (0.075 g, 0.107 mmDOoIl:)10a.c10co39r/dCi8nOgBt0o16t9h9Be
H), 8.49-8.46 (m, 2H), 8.09-8.02 (m, 6H), 7.87 (d, J = 8.0 Hz, general procedure E. The product 8d was obtained as an off-
H), 7.72-7.56 (m, 4H), 7.27-7.18 (m, 3H), 7.03-6.94 (m, 2H), white solid (45 mg, 70%); mp 131.9–132.7 °C; H NMR (400
1
.82-4.77 (m, 1H), 3.42-3.38 (m, 1H), 3.25-3.19 (m, 3H), 2.88- MHz, DMSO-d
6
): δ 12.17 (s, 1H), 10.83 (s, 1H), 9.25 (d, J = 8.0
.83 (m, 2H); 13C NMR (100 MHz, DMSO-d
6
): δ 172.2, 169.0, Hz, 1H), 8.55-8.44 (m, 3H), 8.08-8.02 (m, 7H), 7.84 (d, J = 8.0
65.0, 139.6, 136.5, 134.8, 132.7, 132.6, 132.3, 129.5, Hz, 1H), 7.78-7.62 (m, 4H), 7.25-7.21 (m, 2H), 7.02-6.94 (m,
29.18, 129.1, 128.5, 128.3, 128.1, 127.7, 127.5, 124.1, 2H), 4.79-4.77 (m, 1H), 3.40-3.34 (m, 3H), 3.22-3.16 (m, 1H),
23.6, 123.3, 121.3, 121.2, 120.9, 118.9, 118.6, 111.8, 110.9, 2.87-2.83 (m, 2H) ; C NMR (100 MHz, DMSO-d ): δ 172.0,
4.8, 38.8, 37.0, 27.7 ; IR (ATR): νmax 3236, 3048, 2341, 2109, 167.7, 165.0, 138.8, 136.5, 135.3, 134.9, 132.6, 132.0, 131.6,
919, 1638, 1597, 1505, 1403, 1396, 1227, 1095, 911, 863, 129.6, 129.1, 128.6, 128.4, 128.1, 127.7, 127.5, 124.0, 123.6,
1
3
6
+
21, 741; HRMS (ESI): m/z calcd for C31
H
29
N
5
O
3
[M + H] : 123.0, 122.8, 121.3, 118.9, 118.7, 115.1, 111.8, 110.9, 66.8,
20.2343; found: 520.2335.
55.3, 55.0, 38.7, 37.0, 27.6, 15.6 ; IR (ATR): νmax 3639, 3359,
2
1
C
996, 2619, 1795, 1691, 1608, 1552; 1462, 1411, 1347, 1257,
(
S)-N-(2-((1-((2-aminoethyl)amino)-3-(1H-indol-3-yl)-1-
208, 1106, 1061, 931, 838, 805; HRMS (ESI): m/z calcd for
oxopropan-2-yl)carbamoyl)-4-fluorophenyl)-2-naphthamide
+
31
H
28BrN
5
O
3
[M + H] : 598.1448; found: 598.1444.
(8b). The title compound 8b was prepared from compound
5
b (0.075 g, 0.117 mmol) according to the general procedure (S)-N-(2-((1-((2-aminoethyl)amino)-3-(1H-indol-3-yl)-1-
E. The product 8b was obtained as an off-white solid (51 mg, oxopropan-2-yl)carbamoyl)-4-methoxyphenyl)-2-
1
8
0%); mp 229.7–230.8 °C; H NMR (400 MHz, DMSO-d
6
): δ naphthamide (8e). The title compound 8e was prepared
1
2.04 (s, 1H), 10.83 (s, 1H), 9.18 (d, J = 8.0 Hz, 1H), 8.56-8.53 from compound 5e (0.075 g, 0.115 mmol) according to the
(
m, 1H), 8.45 (s, 1H), 8.49-8.45 (m, 2H), 8.08-8.01 (m, 6H), general procedure E. The product 8e was obtained as an off-
.85 (d, J = 8.0 Hz, 1H), 7.75-7.62 (m, 4H), 7.47-7.45 (m, 1H), white solid (44 mg, 69%); mp 129.7–130.3 °C; H NMR (400
1
7
7
1
.26-7.23 (m, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, MHz, DMSO-d
6
): δ 11.88 (s, 1H), 10.85 (s, 1H), 9.09 (d, J = 8.0
H), 4.80-4.75 (m, 1H), 3.41-3.17 (m, 4H), 2.88-2.84 (m, 2H); Hz, 1H), 8.45-8.39 (m, 3H), 8.07-7.61 (m, 9H), 7.34-7.15 (m,
1
3
C NMR (100 MHz, DMSO-d
56.3, 136.5, 135.9, 134.8, 132.6, 132.1, 129.5, 129.1, 128.6, (m, 4H), 2.85 (t, J = 12.0 Hz, 2H) ; C NMR (100 MHz, DMSO-
28.3, 128.1, 127.7, 127.5, 124.1, 123.6, 123.29, 123.2, ): δ 172.1, 168.6, 164.7, 155.0, 136.5, 134.7, 132.6, 132.5,
6
): δ 172.0, 167.8, 165.0, 158.7, 4H), 7.03-6.94 (m, 2H), 4.77 (br s, 1H), 3.84 (s, 3H), 3.22-3.17
13
1
1
1
1
2
d
6
23.1, 121.3, 119.4, 119.2, 118.9, 118.6, 115.9, 115.7, 111.8, 132.4, 129.5, 129.0, 128.4, 128.1, 127.7, 127.5, 124.2, 123.7,
10.8, 66.8, 54.8, 38.8, 37.0, 27.6 ; IR (ATR): νmax 3054, 2610, 123.2, 122.8, 121.3, 118.9, 118.7, 118.3, 113.8, 111.8, 110.9,
113, 1910, 1705, 1658, 1596, 1539, 1385, 1308, 1203, 811; 56.0, 54.8, 38.9, 37.3, 27.7 ; IR (ATR): νmax 3262, 3055, 2952,
+
HRMS (ESI): m/z calcd for C31
found: 538.2242.
H
28FN
5
O
3
[M + H] : 538.2249; 2341, 1645, 1597, 1506, 1430, 1364, 1234, 1201, 132, 1036,
+
913, 823; HRMS (ESI): m/z calcd for C32
H
31
N
5
O
4
[M + H] :
5
50.2449; found: 550.2443.
(
S)-N-(2-((1-((2-aminoethyl)amino)-3-(1H-indol-3-yl)-1-
oxopropan-2-yl)carbamoyl)-4-chlorophenyl)-2-
General Procedure (F) for the N-Boc deprotection of
naphthamide (8c). The title compound 8c was prepared from compounds 9a-9e. To a solution of 6a-6e (0.1 mmol) in
compound 5c (0.075 g, 0.116 mmol) according to the general dichloromethane (1 mL) was added TFA (1 mL). The reaction
procedure E. The product 8c was obtained as an off-white mixture was warmed to room temperature and stirred for 8
1
solid (54 mg, 75%); mp 119.3–120.4 °C; H NMR (400 MHz, h. After completion of the reaction, excess solvent was
DMSO-d
6
): δ 12.16 (s, 1H), 10.84 (s, 1H), 9.25 (d, J = 8.0 Hz, removed under reduced pressure and treated with excess
1
8
7
H), 8.59 (d, J = 8.0 Hz, 1H), 8.50 (t, 1H), 8.44 (s, 1H), 8.09- HCl in dioxane (4 N solution) to exchange the TFA anion with
.02 (m, 6H), 7.96-7.95 (m, 1H), 7.84 (d, J = 4.0 Hz, 1H), 7.73- HCl. The gummy liquid was concentrated under reduced
.62 (m, 4H), 7.26-7.22 (m, 2H), 7.00 (t, J = 8.0 Hz, 1H), 6.95 pressure to yield the gummy solid. The gummy solid was
(
t, J = 8.0 Hz, 1H), 4.82-4.76 (m, 1H), 3.23-3.17 (m, 4H), 2.88- dissolved in minimum amount of MeOH (≤10 drops) and
2
1
1
1
5
1
7
5
.84 (m, 2H); 13C NMR (100 MHz, DMSO-d
): δ 171.9, 167.8, diethylether (5-10 mL) was added to get precipitation of the
6
65.0, 138.4, 136.5, 134.9, 132.6, 132.3, 132.0, 129.6, 129.1, product.
28.9, 128.6, 128.4, 128.1, 127.7, 127.5, 127.5, 127.1, 124.0,
(
S)-N-(2-((1-((2-guanidinoethyl)amino)-3-(1H-indol-3-yl)-1-
23.6, 122.8, 122.6, 121.3, 118.9, 118.6, 111.8, 110.9, 66.8,
5.0, 38.7, 37.0, 27.6 ; IR (ATR): νmax 3278, 3052, 2938, 2111,
oxopropan-2-yl)carbamoyl)phenyl)-2-naphthamide (9a).
The title compound 9a was prepared from compound 6a (0.1
g, 0.131 mmol) according to the general procedure F. The
product 9a was obtained as an off-white solid (44 mg, 59%);
mp 180.8–181.2 °C; H NMR (400 MHz, DMSO-d
581, 1500, 1429, 1395, 1297, 1157, 1222, 1010, 912, 923,
+
33; HRMS (ESI): m/z calcd for C31
H
28ClN
5
O
3
[M + H] :
54.1953; found: 554.1951.
1
6
): δ 12.25 (s,
(
S)-N-(2-((1-((2-aminoethyl)amino)-3-(1H-indol-3-yl)-1-
1H), 10.80 (s, 1H), 8.97 (d, J = 8.0 Hz, 1H), 8.59 (d, J = 4.0 Hz,
1H), 8.45 (br s, 1H), 8.39 (t, J = 4.0 Hz, 1H), 8.06-8.02 (m, 3H),
oxopropan-2-yl)carbamoyl)-4-bromophenyl)-2-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 15
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Page 16 of 23
ARTICLE
Journal Name
1
7
6
.88-7.82 (m, 2H), 7.70-7.63 (m, 3H), 7.59-7.55 (m, 2H), 7.28- solid (34 mg, 45%); H NMR (400 MHz, DMSO-d ): δ 12.14 (s,
6
View Article Online
DOI: 10.1039/C8OB01699B
13
.98 (m, 8H), 4.80-4.76 (m, 1H), 3.29-3.18 (m, 6H) ; C NMR 1H), 10.81 (br s, 1H), 9.17 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 8.0
): δ 172.4, 169.1, 165.0, 158.8, 157.4, Hz, 1H), 8.42-8.38 (m, 2H), 8.05-8.02 (m, 3H), 7.83-7.76 (m,
(100 MHz, DMSO-d
6
1
1
1
5
1
39.6, 136.5, 134.8, 132.8, 132.6, 132.3, 129.5, 129.09, 2H), 7.70-7.62 (m, 4H), 7.47 (t, J = 8.0 Hz, 1H), 7.26-7.20 (m,
29.0, 128.07, 128.6, 128.3, 128.1, 127.6, 127.5, 124.1, 5H), 7.02-6.96 (m, 3H), 4.78-4.73 (m, 1H), 3.28-3.25 (m, 2H),
1
3
23.6, 123.3, 121.4, 121.2, 120.9, 118.8, 118.6, 111.8, 110.8, 3.21-3.13 (m, 4H) ; C NMR (100 MHz, DMSO-d ): δ 172.2,
6
4.8, 40.8, 38.5, 27.8 ; IR (ATR): νmax 3293, 2340, 2113, 1903, 167.8, 165.1, 157.3, 138.8, 136.5, 135.3, 134.9, 132.6, 132.0,
654, 1518, 1431, 1309, 1180, 1127, 912, 837, 742; HRMS 131.6, 129.5, 129.1, 128.7, 128.4, 128.1, 127.6, 127.5, 124.0,
+
(ESI): m/z calcd for C32
H
31
N
7
O
3
[M + H] : 562.2561; found: 123.6, 123.5, 123.0, 122.9, 121.4, 118.7, 115.1, 111.8, 110.8,
5
62.2554.
55.0, 40.8, 38.5, 27.7 ; IR (ATR): νmax 3889, 3761, 3824, 3390,
2
2
7
6
940, 2825, 2437, 2257, 2180, 2092, 2092, 2139, 2127, 2040,
(
S)-N-(4-fluoro-2-((1-((2-guanidinoethyl)amino)-3-(1H-
003, 1966, 1942, 1860, 1655, 1447, 1406, 1113, 995, 824,
indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
naphthamide (9b). The title compound 9b was prepared
from compound 6b (0.1 g, 0.128 mmol) according to the
+
61; HRMS (ESI): m/z calcd for C32
H
30BrN
7
O
3
[M + H] :
40.1666; found: 640.1662.
general procedure F. The product 9b was obtained as gummy (S)-N-(2-((1-((2-guanidinoethyl)amino)-3-(1H-indol-3-yl)-1-
1
solid (40 mg, 54%); H NMR (600 MHz, DMSO-d
6
): δ 12.05 (s, oxopropan-2-yl)carbamoyl)-4-methoxyphenyl)-2-
1
8
H), 10.80 (s, 1H), 9.25 (d, J = 6.0 Hz, 1H), 8.70 (br s, 1H), naphthamide (9e). The title compound 9e was prepared
.58-8.56 (m, 2H), 8.47-8.43 (m, 2H), 8.35 (s, 1H), 8.06-8.01 from compound 6e (0.1 g, 0.126 mmol) according to the
(
m, 3H), 7.85 (d, J = 6.0 Hz, 1H), 7.69-7.62 (m, 7H), 7.48-7.44 general procedure F. The product 9e was obtained as gummy
1
(m, 1H), 7.26-7.22 (m, 2H), 7.00 (t, J = 12.0 Hz, 1H), 6.95 (t, J solid (36 mg, 49%); H NMR (600 MHz, DMSO-d
6
): δ 11.87 (s,
=
3
1
1
1
4
2
1
12.0 Hz, 1H), 4.78-4.75 (m, 1H), 3.27-3.23 (m, 2H), 3.17- 1H), 10.82 (s, 1H), 9.13 (d, J = 12.0 Hz, 1H), 8.62 (br s, 1H),
.13 (m, 4H) ; 13C NMR (150 MHz, DMSO-d
6
): δ 171.9, 167.9, 8.48-8.41 (m, 4H), 8.05-8.00 (m, 3H), 7.86 (dd, J = 1.2, 8.8 Hz,
66.5, 165.0, 157.8, 156.7, 136.5, 136.0, 134.8, 132.6, 132.1, 1H), 7.70-7.61 (m, 6H), 7.30-7.22 (m, 3H), 7.16 (dd, J = 6.0,
29.5, 129.0, 128.6, 128.3, 128.1, 127.6, 127.5, 124.1, 123.6, 12.0 Hz, 1H), 7.03-6.94 (m, 2H), 4.77-4.74 (m, 1H), 3.82 (s,
13
23.19, 123.1, 121.3, 118.8, 118.6, 115.6, 111.8, 110.9, 54.9, 3H), 3.26-3.14 (m, 6H) ; C NMR (150 MHz, DMSO-d ): δ
6
0.5, 38.5, 27.7; IR (ATR): νmax 3750, 3716, 3320, 2947, 2836, 172.1, 168.7, 167.6, 164.7, 157.8, 155.0, 136.5, 134.7, 132.6,
462, 2139, 2048, 1976, 1919, 1890, 1786, 1669, 1519, 1449, 132.5, 129.5, 128.9, 128.4, 128.16, 128.1, 127.6, 127.4,
400, 1312, 1268, 1204, 1142, 1022, 799, 738; HRMS (ESI): 124.1, 123.7, 123.2, 122.8, 121.3, 118.7, 118.2, 113.9, 111.8,
+
m/z calcd for C32
H
30FN
7
O
3
[M + H] : 580.2467; found: 110.9, 55.9, 54.8, 40.7, 38.5, 27.7 ; IR (ATR): νmax 3732, 3350,
5
80.2461.
3150, 2955, 2526, 2313, 1680, 1537, 1355, 1262, 1201, 1135,
+
9
5
84, 826, 730; HRMS (ESI): m/z calcd for C33
92.2667; found: 592.2662.
H
33
N
7
O
4
[M + H] :
(
S)-N-(4-chloro-2-((1-((2-guanidinoethyl)amino)-3-(1H-
indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
naphthamide (9c). The title compound 9c was prepared from General Procedure (G) for the synthesis of compounds 10a-
compound 6c (0.1 g, 0.125 mmol) according to the general 10e.To a solution of 7a-7e (0.1 mmol) in DCM was added 4 N
procedure F. The product 9c was obtained as an off-white HCl/dioxane (1.0 mL) The reaction mixture was stirred at
1
solid (34 mg, 46%); mp 117.8–118.8 °C; H NMR (400 MHz, room temperature for 15 min. After completion of the
DMSO-d
6
): δ 12.14 (s, 1H), 10.81 (s, 1H), 9.15 (d, J = 8.0 Hz, reaction, solvent was removed under reduced pressure and
1
8
7
4
H), 8.59 (d, J = 6.0 Hz, 1H), 8.43 (s, 1H), 8.38-8.37 (m, 2H), treated with diethylether and compound was dried under
.06-8.02 (m, 3H), 7.91-7.90 (m, 1H), 7.84-7.83 (m, 1H), high vacuum to yield the product.
.70-7.64 (m, 4H), 7.46 (t, J = 6.0 Hz, 1H), 7.26-6.96 (m, 8H),
1
3
(S)-2-(2-(2-(2-naphthamido)benzamido)-3-(1H-indol-3-
yl)propanamido)-N,N-dimethylethan-1-aminium chloride
.79-4.75 (m, 1H), 3.29-3.17 (m, 6H) ; C NMR (150 MHz,
): δ 172.4, 169.1, 165.0, 158.8, 157.4, 139.6, 136.5,
34.8, 132.8, 132.6, 132.3, 129.5, 129.09, 129.0, 128.6,
DMSO-d
6
(
7
10a). The title compound 10a was prepared from compound
a (0.05 g, 0.094 mmol) according to the general procedure
1
1
1
28.3, 128.1, 127.6, 127.5, 124.1, 123.6, 123.3, 121.4, 121.2,
G. The product 10a was obtained as an white solid (62 mg,
20.9, 118.8, 118.6, 111.8, 110.8, 54.8, 40.8, 38.5, 27.8; IR
1
9
1
0%); mp 234.4–234.8 °C; H NMR (400 MHz, DMSO-d
2.3 (s, 1H), 10.83 (s, 1H), 10.33 (br s, 1H), 9.11 (d, J = 8.0 Hz,
6
): δ
(ATR): νmax 3310, 3067, 2905, 2664, 2352, 1656, 1507, 1431,
1
C
306, 1198, 1132, 915, 744; HRMS (ESI): m/z calcd for
+
1H), 8.59 (d, J = 8.0 Hz, 1H), 8.51-8.46 (m, 2H), 8.09-8.02 (m,
H), 7.89-7.89 (t, J = 8.0 Hz, 2H), 7.71-7.56 (m, 4H), 7.28-7.18
(m, 3H), 7.04-6.95 (m, 2H), 4.79-4.75 (m, 1H), 3.49-3.36 (m,
32
H
30ClN
7
O
3
[M + H] : 596.2171; found: 596.2167.
3
(
S)-N-(4-bromo-2-((1-((2-guanidinoethyl)amino)-3-(1H-
1
3
indol-3-yl)-1-oxopropan-2-yl)carbamoyl)phenyl)-2-
3H), 3.26-3.20 (m, 1H), 3.07-3.06 (m, 2H), 2.68 (s, 6H);
C
naphthamide (9d). The title compound 9d was prepared NMR (150 MHz, DMSO-d ): δ 172.1, 169.1, 165.0, 139.7,
6
from compound 6d (0.1 g, 0.118 mmol) according to the 136.5, 134.8, 132.7, 132.6, 132.4, 129.6, 129.2, 129.1, 128.6,
general procedure F. The product 9d was obtained as gummy 128.3, 128.1, 127.7, 127.5, 124.1, 123.7, 123.3, 121.3, 121.1,
1
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 17 of 23
O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Journal Name
ARTICLE
1
;
1
20.9, 118.9, 118.6, 111.8, 110.9, 55.8, 54.9, 42.6, 34.6, 27.6 Hz, 1H), 7.78 (dd, J = 2.4, 9.0 Hz, 1H), 7.71-7.63 (m, 3H), 7.26-
View Article Online
DOI: 10.1039/C8OB01699B
IR (ATR): νmax 3231, 3049, 2961, 2650, 2455, 2342, 2109, 7.22 (m, 2H), 7.02-6.95 (m, 2H), 4.78-4.74 (m, 1H), 3.47-3.36
1
3
922, 1637, 1595, 1508, 1428, 1302, 910, 863, 822, 743; (m, 3H), 3.21-3.01 (m, 3H), 2.67 (s, 6H); C NMR (150 MHz,
+
HRMS (ESI): m/z calcd for C33
H
33
N
5
O
3
[M + H] : 548.2656; DMSO-d
6
): δ 171.9, 167.8, 165.1, 138.8, 136.5, 135.3, 134.9,
found: 548.2647.
132.6, 132.0, 131.6, 129.6, 129.1, 128.7, 128.4, 128.1,
1
1
27.69, 127.6, 124.1, 123.6, 123.0, 122.9, 121.3, 118.9,
18.7, 115.1, 111.8, 110.8, 55.9, 55.1, 42.8, 34.7, 27.5; IR
(
S)-2-(2-(2-(2-naphthamido)-5-fluorobenzamido)-3-(1H-
indol-3-yl)propanamido)-N,N-dimethylethan-1-aminium
chloride (10b). The title compound 10b was prepared from
compound 7b (0.05 g, 0.08 mmol) according to the general
procedure G. The product 10b was obtained as an white
solid (47 mg, 89%); H NMR (600 MHz, DMSO-d
(ATR): νmax 3230, 3051, 2646, 2320, 2106, 1906, 1638; 1578,
1
499, 1428, 1391, 1297, 1224, 1094, 1010, 912, 820, 738;
+
HRMS (ESI): m/z calcd for C33
found: 626.1758.
H
32BrN
5
O
3
[M + H] : 626.1761;
1
6
): δ 12.06 (s,
1
8
8
8
7
6
3
H), 10.83 (s, 1H), 10.37 (br s, 1H), 9.22 (d, J = 6.0 Hz, 1H), (S)-2-(2-(2-(2-naphthamido)-5-methoxybenzamido)-3-(1H-
.55 (q, J = 6.0 Hz, 1H), 8.50 (t, J = 6.0 Hz, 1H), 8.45 (s, 1H), indol-3-yl)propanamido)-N,N-dimethylethan-1-aminium
.08-8.05 (m, 2H), 8.03 (d, J = 6.0 Hz, 1H), 7.86 (dd, J = 1.8, (10e). The title compound 10e was prepared from compound
.4 Hz, 1H), 7.74 (dd, J = 6.0, 12.0 Hz, 1H), 7.70-7.62 (m, 3H), 7e (0.05 g, 0.09 mmol) according to the general procedure G.
.47 (td, J = 6.0, 12.0 Hz, 1H), 7.27-7.24 (m, 2H), 7.01 (t, J = The product 10e was obtained as an white solid (47 mg,
1
.0 Hz, 1H), 6.96 (t, J = 6.0 Hz, 1H), 4.77-4.74 (m, 1H), 3.50- 90%); mp 201.6–201.9 °C; H NMR (600 MHz, DMSO-d
6
): δ
13
.37 (m, 3H), 3.22-3.18 (m, 3H), 2.68 (s, 6H) ; C NMR (150 11.90 (s, 1H), 10.86 (s, 1H), 10.33 (br s, 1H), 9.16 (d, J = 6.0
): δ 171.9, 167.8, 165.0, 136.5, 136.0, 134.8, Hz, 1H), 8.49 (t, J = 6.0 Hz, 1H), 8.44 (s, 1H), 8.41 (d, J = 6.0
32.6, 132.1, 129.5, 129.1, 128.6, 128.3, 128.1, 127.6, 127.5, Hz, 1H), 8.10-8.04 (m, 1H), 8.01 (d, J = 12.0 Hz, 1H), 7.87 (d, J
MHz, DMSO-d
1
6
1
1
;
1
24.1, 123.7, 123.18, 123.14, 121.3, 119.4, 119.3, 118.8, = 3.6 Hz, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.67-7.62 (m, 2H), 7.37
18.7, 115.9, 115.7, 111.8, 110.8, 55.8, 55.0, 42.7, 34.6, 27.5 (d, J = 6.0 Hz, 1H), 7.29-7.25 (m, 2H), 7.17 (dd, J = 3.0, 9.0 Hz,
IR (ATR): νmax 3217, 3053, 2962, 2593, 2464, 2111, 1902, 1H), 7.04-7.00 (m, 1H), 6.97-6.94 (m, 1H), 4.78-4.74 (m, 1H),
654, 1599, 1518, 1409, 1290, 1246, 940, 869, 824, 752; 3.84 (s, 3H), 3.52-3.49 (m, 1H), 3.40-3.37 (m, 2H), 3.25-3.20
+
13
HRMS (ESI): m/z calcd for C33
found: 566.2555.
H
32FN
5
O
3
[M + H] : 566.2562; (m, 1H), 3.11-3.05 (m, 2H), 2.68 (br s, 6H) ; C NMR (150
MHz, DMSO-d ): δ 172.1, 168.7, 164.7, 155.0, 136.5, 134.7,
32.68, 132.6, 132.5, 129.5, 129.0, 128.4, 128.16, 128.1,
6
1
1
1
(
S)-2-(2-(2-(2-naphthamido)-5-chlorobenzamido)-3-(1H-
27.7, 127.5, 124.2, 123.7, 123.0, 122.8, 121.3, 118.9, 118.7,
18.4, 113.8, 111.8, 110.8, 56.0, 55.8, 54.9, 42.6, 34.6, 27.5;
indol-3-yl)propanamido)-N,N-dimethylethan-1-aminium
chloride (10c). The title compound 10c was prepared from
compound 7c (0.05 g, 0.085 mmol) according to the general
IR (ATR): νmax 3242, 3050, 2958, 2674, 2467, 2342, 2113,
1
9
907, 1639, 1595, 1594, 1509, 1454, 1434, 1222, 109, 1032,
procedure G. The product 10c was obtained as an white solid
10, 819, 742; HRMS (ESI): m/z calcd for C34
H
35
N
5
O
4
[M +
1
(48 mg, 91%); mp 200.3–202.5 °C; H NMR (300 MHz, DMSO-
+
H] : 578.2762; found: 578.2760.
d
6
): δ 12.27 (s, 1H), 8.86 (d, J = 9.0 Hz, 1H), 8.50 (s, 1H), 8.21
(
br s, 1H), 8.10-7.90 (m, 4H), 7.80 (d, J = 6.0 Hz, 1H), 7.64- General Procedure (H) for the synthesis of compounds 11a-
7
1
3
.44 (m, 5H), 7.35-7.30 (m, 1H), 7.24-7.13 (m, 3H), 6.23 (br s, 11e. To a solution of 7a-7e (0.1 mmol) in CH
H), 4.93-4.92(m, 1H), 3.49 (dd, J = 6.0, 15.0 Hz, 1H), 3.32- added CH I (0.1 mmol) The reaction mixture was stirred at
.10 (m, 3H), 2.25-2.20 (m, 3H), 1.99 (s, 6H) ; C NMR (75 room temperature for 8 h. After completion of the reaction,
): δ 170.5, 167.5, 165.5, 138.8, 136.2, 135.0, the solvent was removed under reduced pressure and
32.7, 131.8, 129.4, 128.6, 128.3, 127.9, 127.7, 127.4, 126.9, treated with diethylether and compound was dried under
3
CN (1.0 mL) was
3
13
MHz, DMSO-d
6
1
1
1
ν
1
26.7, 123.6, 123.1, 122.8, 122.5, 121.13, 121.1, 120.0, high vacuum to yield the product.
18.8, 111.3, 110.7, 57.0, 54.6, 44.6, 36.7, 29.0 ; IR (ATR):
(
S)-2-(2-(2-(2-naphthamido)benzamido)-3-(1H-indol-3-
max 3272, 3052, 2938, 2320, 2110, 1907, 1639, 1580, 1498,
yl)propanamido)-N,N,N-trimethylethan-1-aminium iodide
(11a). The title compound 11a was prepared from compound
428, 1296, 1224, 1095, 912, 822, 738; HRMS (ESI): m/z calcd
+
for C33
H
32ClN
5
O
3
[M + H] : 582.2266; found: 582.2262.
7a (0.05 g, 0.094 mmol) according to the general procedure
(
S)-2-(2-(2-(2-naphthamido)-5-bromobenzamido)-3-(1H-
H. The product 11a was obtained as an white solid (58 mg,
1
indol-3-yl)propanamido)-N,N-dimethylethan-1-aminium
90%); mp 240.3–240.6 °C; H NMR (600 MHz, DMSO-d
6
): δ
(10d). The title compound 10d was prepared from 12.28 (s, 1H), 10.83 (s, 1H), 9.05 (d, J = 6.0 Hz, 1H), 8.59 (d, J
compound 7d (0.05 g, 0.08 mmol) according to the general = 6.0 Hz, 1H), 8.48-8.45 (m, 2H), 8.09-8.03 (m, 3H), 7.90-7.86
procedure G. The product 10d was obtained as an white (m, 2H), 7.70-7.59 (m, 4H), 7.31-7.22 (m, 3H), 7.06-6.98 (m,
1
solid (47 mg, 90%); mp 156.8–157.5 °C; H NMR (600 MHz, 2H), 4.74-4.70 (m, 1H), 3.50-3.48 (m, 3H), 3.30-3.17 (m, 3H),
1
3
DMSO-d
6
): δ 12.19 (s, 1H), 10.84 (s, 1H), 10.22 (br s, 1H), 9.28 2.97 (s, 9H) ; C NMR (150 MHz, DMSO-d
6
): δ 172.3, 169.2,
(d, J = 12.0 Hz, 1H), 8.54 (d, J = 6.0 Hz, 1H), 8.48 (t, J = 6.0 Hz, 165.1, 139.6, 136.5, 134.8, 132.9, 132.6, 132.4, 129.5,
1
H), 8.44 (br s, 1H), 8.08-8.02 (m, 4H), 7.85 (dd, J = 1.8, 9.0 129.17, 129.1, 128.6, 128.3, 128.1, 127.6, 127.5, 124.2,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 17
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Page 18 of 23
ARTICLE
Journal Name
1
5
1
8
5
23.6, 123.3, 121.4, 121.08, 121.0, 118.7, 111.9, 110.6, 64.0, 167.9, 165.1, 138.9, 136.5, 135.4, 134.9, 132.6, 132.1, 131.6,
5.0, 52.9, 33.8, 27.4; IR (ATR): νmax 3244, 3050, 2341, 2109, 129.6, 129.1, 128.7, 128.4, 128.2, 127.6, 127.5, 124.1, 123.6,
View Article Online
DOI: 10.1039/C8OB01699B
650, 1596, 1506, 149, 1301, 1228, 1094, 1009, 954, 912, 123.0, 122.8, 121.4, 118.8, 115.1, 111.9, 110.6, 64.0, 55.2,
+
65, 823, 746; HRMS (ESI): m/z calcd for C34
H
36
N
5
O
3
[M] : 52.9, 33.8, 27.4 ; IR (ATR): νmax 3622, 3508, 3427, 3238, 3039,
62.2813; found: 562.2816.
2913, 2763, 1783, 1603, 1679, 1543, 1427, 1359, 1278, 1233,
1
C
103, 1058, 963, 827, 762; HRMS (ESI): m/z calcd for
(
S)-2-(2-(2-(2-naphthamido)-5-fluorobenzamido)-3-(1H-
+
34
H
35BrN
5
O
3
[M] : 640.1918; found: 640.1912.
indol-3-yl)propanamido)-N,N,N-trimethylethan-1-aminium
iodide (11b). The title compound 11b was prepared from (S)-2-(2-(2-(2-naphthamido)-5-methoxybenzamido)-3-(1H-
compound 7b (0.05 g, 0.08 mmol) according to the general indol-3-yl)propanamido)-N,N,N-trimethylethan-1-aminium
procedure H. The product 11b was obtained as an white solid iodide (11e). The title compound 11e was prepared from
1
(
52 mg, 92%); mp 182.1–182.0 °C; H NMR (600 MHz, DMSO- compound 7e (0.05 g, 0.09 mmol) according to the general
d
8
7
7
3
1
1
1
1
3
1
C
6
): 12.04 (s, 1H), 10.84 (s, 1H), 9.12 (d, J = 12.0 Hz, 1H), 8.54- procedure H. The product 11e was obtained as an white solid
.47 (m, 3H), 8.07-8.02 (m, 3H), 7.87 (d, J = 12.0 Hz, 1H), (62 mg, 90%); mp 168.9–171.1 °C; H NMR (400 MHz, DMSO-
1
.68-7.64 (m, 4H), 7.48 (t, J = 12.0 Hz, 1H), 7.30-7.25 (m, 2H),
6
d ): δ 11.85 (s, 1H), 10.86 (s, 1H), 9.04 (d, J = 8.0 Hz, 1H),
.04-6.98 (m, 2H), 4.71 (br m, 1H), 3.48-3.31 (m, 2H), 3.20- 8.48-8.45 (m, 2H), 8.39 (d, J = 8.0 Hz, 1H), 8.09-8.02 (m, 3H),
13
.14 (m, 4H), 2.98 (s, 9H); C NMR (150 MHz, DMSO-d ): δ 7.89-7.86 (m, 1H), 7.69-7.62 (m, 3H), 7.32-7.27 (m, 3H), 7.20
6
72.1, 167.9, 165.1, 158.8, 156.4, 136.5, 135.9, 134.8, 132.6, (dd, J = 4.0, 8.0 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 6.99 (t, J = 4.0
32.2, 129.5, 129.1, 128.6, 128.3, 128.2, 127.6, 127.5, 124.2, Hz, 1H), 4.72-4.67 (m, 1H), 3.84 (s, 3H), 3.49-3.47 (m, 2H),
13
23.7, 123.4, 123.3, 123.0, 121.4, 119.6, 119.4, 118.7, 115.8, 3.26-3.15 (m, 4H), 2.97 (s, 9H); C NMR (100 MHz, DMSO-
15.6, 111.9, 110.5, 64.0, 55.1, 53.0, 33.8, 27.4; IR (ATR): νmax ): δ 172.2, 168.8, 164.8, 155.0, 136.5, 134.7, 132.6, 132.55,
d
6
345, 3244, 2112, 1904, 1605, 1665, 1520, 1434, 1307, 1233, 132.5, 129.5, 129.0, 128.5, 128.1, 127.5, 124.3, 123.7, 123.2,
019, 1092, 956, 870, 826, 754; HRMS (ESI): m/z calcd for 123.0, 121.4, 118.8, 118.2, 114.1, 111.9, 110.6, 65.3, 64.0,
+
34
H
35FN
5
O
3
[M] : 580.2718; found: 580.2712.
56.0, 55.0, 52.9, 33.7, 27.4, 15.6; IR (ATR): νmax 3310, 2341,
1
599, 1654, 1519, 1430, 1235, 1095, 1038, 956, 818, 742;
(
S)-2-(2-(2-(2-naphthamido)-5-chlorobenzamido)-3-(1H-
+
HRMS (ESI): m/z calcd for C35
92.2912.
H
38
N
5
O
4
[M] : 592.2918; found:
indol-3-yl)propanamido)-N,N,N-trimethylethan-1-aminium
iodide (11c). The title compound 11c was prepared from
compound 7c (0.05 g, 0.085 mmol) according to the general Minimum inhibitory concentration (MIC)
5
procedure H. The product 11c was obtained as an white solid
1
The antimicrobial activity of the compounds was evaluated
(56 mg, 91%); mp 194.1–194.2 °C; H NMR (400 MHz, DMSO-
d
6
): δ 12.17 (s, 1H), 10.84 (s, 1H), 9.22 (d, J = 8.0 Hz, 1H), 8.59 by a broth microdilution assay using the procedure described
51
(d, J = 8.0 Hz, 1H), 8.48-8.45 (m, 2H), 8.09-8.02 (m, 3H), 7.94
by Clinical and Laboratory Standards Institute (CLSI).
(d, J = 4.0 Hz, 1H), 7.87-7.84 (m, 1H), 7.70-7.62 (m, 4H), 7.29-
Briefly, bacteria were grown to mid-log phase in Muller
Hinton broth (MHB) with shaking at 120 rpm and incubated
at 37°C for 12-16 h. Following incubation, bacteria were
washed three times in PBS pH 7.4 at 3500 g for 10 min. After
7
.23 (m, 2H), 7.05-6.96 (m, 2H), 4.74-4.68 (m, 1H), 3.49-3.46
1
3
(
m, 2H), 3.30-3.14 (m, 4H), 2.97 (s, 9H); C NMR (150 MHz,
DMSO-d ): δ 172.0, 168.0, 165.1, 138.5, 136.5, 134.9, 132.6,
32.5, 132.1, 129.6, 129.1, 128.8, 128.7, 128.4, 128.2, 127.6,
6
1
1
1
3
1
27.5, 127.2, 124.2, 123.6, 122.8, 122.6, 121.4, 118.7, 111.9, washing, bacteria were diluted with fresh MHB. The turbidity
10.6, 64.0, 55.2, 52.9, 33.8, 27.4; IR (ATR): νmax 3626, 3429, of the bacterial suspensions were adjusted so that OD660nm
242, 3055, 2778, 2320, 1649, 1598, 1429, 1504, 1292, 1103,
007, 957, 825, 747; HRMS (ESI): m/z calcd for C34
8
was 0.1, which gave 1×10 CFU/ml, and then further diluted
to achieve 5×10 CFU/ml as a final bacterial concentration.
H
35ClN
5
O
3
5
+
[M] : 596.2423; found: 596.2418.
Each compound was diluted (250-3.9 µM) through two-fold
dilution. Wells in microtiter plates were loaded with 100 µl of
inoculum containing 5×10 CFU/ml bacteria. Wells without
(
S)-2-(2-(2-(2-naphthamido)-5-bromobenzamido)-3-(1H-
5
indol-3-yl)propanamido)-N,N,N-trimethylethan-1-aminium
iodide (11d) The title compound 11d was prepared from
compound 7d (0.05 g, 0.08 mmol) according to the general
any compound and containing only bacteria were used as
negative controls (i.e. no inhibition of growth). Wells with
media only was set as blank. The microtiter plate was
wrapped with paraffin to prevent evaporation and incubated
with shaking at 120 rpm at 37°C for 18-24 h. After
procedure H. The product 11d was obtained as an white solid
1
(
55 mg, 90%); mp 231.6–231.8 °C; H NMR (400 MHz, DMSO-
d
6
): δ 12.20 (s, 1H), 10.85 (s, 1H), 9.24 (d, J = 8.0 Hz, 1H), 8.54
(
=
3
d, J = 8.0 Hz, 1H), 8.48 (s, 2H), 8.09-8.02 (m, 4H), 7.86 (dd, J
2.0, 8.6 Hz, 1H), 7.80 (dd, J = 2.0, 9.0 Hz, 1H), 7.70-7.62 (m, incubation, spectrophotometric reading was taken. The well
H), 7.29 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 4.0 Hz, 1H), 7.06-6.97 at the lowest concentration without any bacterial growth
(
4
m, 2H), 4.74-4.69 (m, 1H), 3.50-3.58 (m, 2H), 3.30-3.27 (m,
and showing zero spectrophotometric reading was regarded
as the MIC of the compounds. We compared the MIC data of
H), 2.98 (s, 9H); 13C NMR (100 MHz, DMSO-d
6
): δ 172.1,
1
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 19 of 23
O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Journal Name
ARTICLE
all our compounds with MSI-78 (also known as pexiganan), amino acids (NEAA) and 1% sodium pyruvate. The cell line
View Article Online
DOI: 10.1039/C8OB01699B
which is an antibiotic currently in Phase-III clinical trials and was maintained at 37 °C in 5% CO as an adherent monolayer
2
has the amino acid sequence of Gly-Ile-Gly-Lys-Phe-Leu-Lys- and was passaged upon reaching confluence by standard cell
4
Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys-
NH
culture techniques. MRC-5 cells were seeded at 2 × 10 cells
2
. Each experiment was performed in triplicate and was per well in 96-well plates to ensure full confluence
repeated in three independent experiments.
(quiescence). Cells were treated 24 h after seeding with 0.1
to 1000 μM of compounds. After 72 h drug incubation, the
treated media was replaced with fresh media containing 10%
Alamar Blue and the cells were incubated for another 6 h.
The metabolic activity was detected by spectrophotometric
analysis by assessing the absorbance of Alamar blue as
Cytoplasmic membrane permeability assay
The method was adopted from Wu et al.52 with slight
modification. Bacterial cytoplasmic membrane permeability
was determined using membrane potential sensitive dye
diSC3-5 (3,3'-dipropylthiadicarbocyanine iodide) which
53
previously described by Pasquier et al. Cell proliferation
penetrates inside bacterial cells depending on the membrane was determined and expressed as a percentage of untreated
potential gradient of the cytoplasmic membrane. Bacteria control cells. The determination of IC50 values was performed
were grown in MHB to mid-log phase by incubating with
shaking at 37 °C for 18-24 h. Following incubation bacteria
were washed with 5 mM HEPES containing 20 mM glucose
using GraphPad Prism 6 (San Diego, CA, USA). Each
experiment was performed in triplicate and was repeated in
three independent experiments.
pH 7.2 and resuspended in the same buffer to an OD600 0.5-
7
0
4
.6 which gave 1× 10 CFU/ml. The dye diSC3-5 was added at Biofilm inhibition assay
µM to the bacterial suspension. The suspensions were
Bacterial cultures (S. aureus and E. coli) were grown in MHB
incubated at room temperature for 1 h in the dark for
maximum dye take-up by the bacterial cells. Then 100 mM media overnight at 37 °C with shaking at 120 rpm. Cultures
KCl was added to balance the K+ outside and inside the were diluted (1:20) in MHB medium and 200 μl aliquots were
bacterial cell to prevent further uptake or outflow of the dye. dispensed to flat bottom 96-well plate wells (Sarstedt
For Gram-negative bacteria, 0.5 mM EDTA was used to
destabilize the lipopolysaccharides-Mg -Ca complex to
help in dye penetration without affecting bacterial growth.
Australia). Cultures were supplemented with varying
concentrations of synthetic compounds dissolved in DMSO.
Biofilm was grown in 96-well plate for 24 h followed by
addition of synthetic compounds and incubated further for
2+
2+
1
00 µl of bacterial suspension was added in 96-well
microtiter plate and with equal volume of antimicrobial
compounds. DMSO (20%) was set as a positive control while
dye and only bacterial cells were set as negative control.
2
4 h. Plates were sealed with self-adhesive microplate
sealers (TopSeal-A, PerkinElmer) to allow air diffusion and to
prevent condensation. Biofilms adhered on polystyrene
substratum were quantified by crystal violet staining as
Fluorescence was measured with
a
luminescence
spectrophotometer at 3 min intervals at an excitation
wavelength of 621m and an emission wavelength of 670 nm.
5
4
described previously. The experiment was performed in
triplicate.
Viable cell count assay
Acknowledgments
The number of viable cells was confirmed by serially diluting
aliquots of bacteria in D/E neutralizing broth (Remel, Lenexa,
KS, USA) and plating these onto Tryptic Soy Agar (Oxoid,
Basingstoke, UK) containing phosphatidylcholine (0.7 g /L)
and Tween 80 (5 ml/L). The plates were incubated at 37 °C
overnight and numbers of live bacteria were enumerated
and expressed as CFU/ml. The experiment was performed in
triplicate.
We thank the NMR and BMSF facilities at UNSW Australia for
supporting the characterization of the synthesized
compounds. This work was supported by a Discovery Project
from Australian Research Council grant (DP 180100845).
Rajesh Kuppusamy is thankful to the University of New South
Wales for a Tuition Fee Scholarship (TFS) and to Naresh
Kumar for a Living Allowance Scholarship.
Toxicity assay
References
Normal human lung fibroblasts MRC-5 were cultured in
minimal essential medium (MEM, Invitrogen) supplemented 1.
E. Tacconelli, E. Carrara, A. Savoldi, S. Harbarth, M.
Mendelson, D. L. Monnet, C. Pulcini, G. Kahlmeter, J.
Kluytmans and Y. Carmeli, The Lancet Infectious
Diseases, 2017, 18, 318-27.
with 10% foetal calf serum (FCS), 1% L-glutamine–penicillin–
streptomycin, 2% sodium bicarbonate, 1% non-essential
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 19
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O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Page 20 of 23
ARTICLE
Journal Name
2
.
N. Palma, M. J. Pons, C. Gomes, J. Mateu, M. 23.
Riveros, W. García, J. Jacobs, C. García, T. J. Ochoa
and J. Ruiz, Journal of global antimicrobial 24.
resistance, 2017, 11, 28-33.
N. Molchanova, P. R. Hansen and H. Franzyk,
Molecules, 2017, 22, 1430.
View Article Online
DOI: 10.1039/C8OB01699B
S. Choi, A. Isaacs, D. Clements, D. Liu, H. Kim, R. W.
Scott, J. D. Winkler and W. F. DeGrado, Proceedings
of the National Academy of Sciences, 2009, 106,
6968-6973.
S. Nizalapur, O. Kimyon, E. Yee, K. Ho, T. Berry, M.
Manefield, C. G. Cranfield, M. Willcox, D. S. Black
and N. Kumar, Organic & biomolecular chemistry,
2017, 15, 2033-2051.
3.
4.
5.
J. F. Fisher and S. Mobashery, Medchemcomm,
2
016, 7, 37-49.
E. D. Brown and G. D. Wright, Nature, 2016, 529, 25.
36-343.
3
H. C. Flemming and J. Wingender, Nat Rev
Microbiol, 2010, 8, 623-633.
6
7
.
.
J. G. Leid, Microbe, 2009, 4, 66-70.
I. Olsen, European Journal of Clinical Microbiology &
Infectious Diseases, 2015, 34, 877-886.
K. L. Brown and R. E. Hancock, Current opinion in
immunology, 2006, 18, 24-30.
26.
27.
28.
S. Nizalapur, K. K. Ho, Ö. Kimyon, E. Yee, T. Berry, M.
Manefield, C. G. Cranfield, M. Willcox, D. S. Black
and N. Kumar, Organic & biomolecular chemistry,
2016, 14, 3623-3637.
R. Kuppusamy, M. Yasir, T. Berry, C. G. Cranfield, S.
Nizalapur, E. Yee, O. Kimyon, A. Taunk, K. K. Ho and
B. Cornell, European journal of medicinal chemistry,
2018, 143, 1702-1722.
J. B. Bremner, P. A. Keller, S. G. Pyne, T. P. Boyle, Z.
Brkic, D. M. David, A. Garas, J. Morgan, M.
Robertson and K. Somphol, Angewandte Chemie
International Edition, 2010, 49, 537-540.
8
9
1
1
.
.
R. E. Hancock and H.-G. Sahl, Nature biotechnology,
2
006, 24, 1551.
0.
1.
D. K. Mercer and D. A. O’Neil, Future medicinal
chemistry, 2013, 5, 315-337.
M. G. Scott, D. J. Davidson, M. R. Gold, D. Bowdish
and R. E. Hancock, The Journal of Immunology,
2
002, 169, 3883-3891.
12.
13.
14.
L. M. Gottler and A. Ramamoorthy, Biochimica et 29.
Biophysica Acta (BBA)-Biomembranes, 2009, 1788,
C. Ghosh, G. B. Manjunath, M. M. Konai, D. S. Uppu,
K. Paramanandham, B. R. Shome, R. Ravikumar and
J. Haldar, ACS infectious diseases, 2015, 2, 111-122.
S. Lin, J.-J. Koh, T. T. Aung, F. Lim, J. Li, H. Zou, L.
Wang, R. Lakshminarayanan, C. Verma and Y. Wang,
Journal of medicinal chemistry, 2017, 60, 1362-
1378.
J.-J. Koh, S. Lin, T. T. Aung, F. Lim, H. Zou, Y. Bai, J. Li,
H. Lin, L. M. Pang and W. L. Koh, Journal of
medicinal chemistry, 2014, 58, 739-752.
1680-1686.
Y. Chen, C. T. Mant, S. W. Farmer, R. E. Hancock, M. 30.
L. Vasil and R. S. Hodges, Journal of Biological
Chemistry, 2005, 280, 12316-12329.
Y. Niu, S. Padhee, H. Wu, G. Bai, Q. Qiao, Y. Hu, L.
Harrington, W. N. Burda, L. N. Shaw and C. Cao, 31.
Journal of medicinal chemistry, 2012, 55, 4003-
4009.
15.
Y. Niu, H. Wu, Y. Li, Y. Hu, S. Padhee, Q. Li, C. Cao 32.
L. Arnt and G. N. Tew, Journal of the American
and J. Cai, Organic & biomolecular chemistry, 2013,
Chemical Society, 2002, 124, 7664-7665.
1
1, 4283-4290.
33.
Y. Ishitsuka, L. Arnt, J. Majewski, S. Frey, M.
Ratajczek, K. Kjaer, G. N. Tew and K. Y. C. Lee,
Journal of the American Chemical Society, 2006,
128, 13123-13129.
1
1
6.
7.
J. Cai, Y. Niu, H. Wu and S. Padhee, Journal, 2016.
A. J. Karlsson, R. M. Flessner, S. H. Gellman, D. M.
Lynn and S. P. Palecek, Biomacromolecules, 2010,
1
1, 2321-2328.
34.
H. D. Thaker, F. Sgolastra, D. Clements, R. W. Scott
and G. N. Tew, Journal of medicinal chemistry, 2011,
54, 2241-2254.
18.
N. P. Chongsiriwatana, J. A. Patch, A. M. Czyzewski,
M. T. Dohm, A. Ivankin, D. Gidalevitz, R. N.
Zuckermann and A. E. Barron, Proceedings of the 35.
National Academy of Sciences, 2008, 105, 2794-
H. D. Thaker, A. Cankaya, R. W. Scott and G. N. Tew,
ACS medicinal chemistry letters, 2013, 4, 481-485.
P. Teng, D. Huo, A. Nimmagadda, J. Wu, F. She, M.
Su, X. Lin, J. Yan, A. Cao and C. Xi, Journal of
medicinal chemistry, 2016, 59, 7877-7887.
J. E. Wynn, W. Zhang, J. O. Falkinham III and W. L.
Santos, ACS medicinal chemistry letters, 2017, 8,
820-823.
2799.
36.
1
9.
0.
N. Srinivas, P. Jetter, B. J. Ueberbacher, M.
Werneburg, K. Zerbe, J. Steinmann, B. Van der
Meijden, F. Bernardini, A. Lederer and R. L. Dias, 37.
Science, 2010, 327, 1010-1013.
A. Makovitzki, D. Avrahami and Y. Shai, Proceedings
2
of the National Academy of Sciences, 2006, 103, 38.
M. Su, D. Xia, P. Teng, A. Nimmagadda, C. Zhang, T.
Odom, A. Cao, Y. Hu and J. Cai, Journal of medicinal
chemistry, 2017, 60, 8456-8465.
15997-16002.
2
1.
2.
R. W. Scott, W. F. DeGrado and G. N. Tew, Current
opinion in biotechnology, 2008, 19, 620-627.
39.
A. Verkleij, R. Zwaal, B. Roelofsen, P. Comfurius, D.
Kastelijn and L. Van Deenen, Biochimica et
Biophysica Acta (BBA)-Biomembranes, 1973, 323,
178-193.
2
X.-Z. Lai, Y. Feng, J. Pollard, J. N. Chin, M. J. Rybak, R.
Bucki, R. F. Epand, R. M. Epand and P. B. Savage,
Accounts of chemical research, 2008, 41, 1233-1240.
2
0 | J. Name., 2012, 00, 1-3
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Please do not adjust margins

Page 21 of 23
O rP gl ea na is ce &d Bo i on mo to al e dc juu l sa tr mC haer mg i ins ts ry
Journal Name
ARTICLE
4
0.
1.
S. Wilkinson and C. Ratledge, Academic Press: San
Diego, 1988, 299.
K. Matsuzaki, O. Murase, N. Fujii and H. Miyajima,
View Article Online
DOI: 10.1039/C8OB01699B
4
Biophys. J, 1995, 68, A126.
4
4
2.
3.
M. A. Wikler, Approved Standard M7-A8, 2009.
H. J. Böhm, D. Banner, S. Bendels, M. Kansy, B.
Kuhn, K. Müller, U. Obst‐Sander and M. Stahl,
ChemBioChem, 2004, 5, 637-643.
4
4.
5.
Y. Ge, D. L. MacDonald, K. J. Holroyd, C.
Thornsberry, H. Wexler and M. Zasloff,
Antimicrobial agents and chemotherapy, 1999, 43,
782-788.
4
A. K. Jepson, J. Schwarz-Linek, L. Ryan, M. G.
Ryadnov and W. C. Poon, in Biophysics of Infection,
Springer, 2016, pp. 33-48.
4
6.
7.
M. Tischer, G. Pradel, K. Ohlsen and U. Holzgrabe,
ChemMedChem, 2012, 7, 22-31.
E. Zhang, P.-Y. Bai, D.-Y. Cui, W.-C. Chu, Y.-G. Hua, Q.
Liu, H.-Y. Yin, Y.-J. Zhang, S. Qin and H.-M. Liu,
European journal of medicinal chemistry, 2018, 143,
4
1
489-1509.
4
4
8.
9.
M. Zasloff, nature, 2002, 415, 389.
P. J. Sims, A. S. Waggoner, C.-H. Wang and J. F.
Hoffman, Biochemistry, 1974, 13, 3315-3330.
J. O'brien, I. Wilson, T. Orton and F. Pognan, The
FEBS Journal, 2000, 267, 5421-5426.
M. A. Wikler, Methods for dilution antimicrobial
susceptibility tests for bacteria that grow
aerobically: approved standard, Clinical and
Laboratory Standards Institute, 2006.
5
0.
1.
5
5
2.
3.
M. Wu, E. Maier, R. Benz and R. E. Hancock,
Biochemistry, 1999, 38, 7235-7242.
E. Pasquier, J. Ciccolini, M. Carre, S. Giacometti, R.
Fanciullino, C. Pouchy, M.-P. Montero, C. Serdjebi,
M. Kavallaris and N. André, Oncotarget, 2011, 2,
5
797.
54.
G. A. O'Toole, Journal of visualized experiments:
JoVE, 2011, 47, 2437.
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Guanidine Functionalized Anthranilamides as Effective Antibacterials with Biofilm Disruption Activity