New ianthelliformisamine derivatives as antibiotic enhancers against resistant gram-negative bacteria

Article abstract of DOI:10.1021/jm500194e

A series consisting of ianthelliformisamimes A, B, and C as well as its synthetic analogues was prepared in high chemical yield, from 27 to 91%, using peptide coupling as the key step, and the compounds were evaluated for their in vitro antibiotic enhancer properties against resistant Gram-negative bacteria and clinical isolates. The mechanism of action of one of these derivatives against Pseudomonas aeruginosa when combined with doxycycline was precisely evaluated utilizing bioluminescence to measure ATP efflux and fluorescence to evaluate membrane depolarization.

Full text of DOI:10.1021/jm500194e

Article
pubs.acs.org/jmc
New Ianthelliformisamine Derivatives as Antibiotic Enhancers
against Resistant Gram-Negative Bacteria
Cyril Pieri,† Diane Borselli,‡ Carole Di Giorgio,§ Michel De Meo,§ Jean-Michel Bolla,‡ Nicolas Vidal,∥
́
Sebastien Combes,† and Jean Michel Brunel*,†
́
†Centre de Recherche en Cancer
́
ologie de Marseille (CRCM), CNRS, UMR7258, Institut Paoli Calmettes, Aix-Marseille Universite,
UM 105, Inserm, U1068, F-13009 Marseille, France
‡Aix-Marseille Universite,
IRBA, TMCD2 UMR-MD1, Faculte
§Aix-Marseille Universite,
CNRS, UMR 7263/IRD 237, 13385 Marseille Cedex 05, France
∥UPCAM iSm2, Case 342, Aix-Marseille Universite,
Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 13, France
́
́
́
́ ́
de Medecine, 13385 Marseille, France
́
́
S
* Supporting Information
ABSTRACT: A series consisting of ianthelliformisamimes A,
B, and C as well as its synthetic analogues was prepared in high
chemical yield, from 27 to 91%, using peptide coupling as the
key step, and the compounds were evaluated for their in vitro
antibiotic enhancer properties against resistant Gram-negative
bacteria and clinical isolates. The mechanism of action of one
of these derivatives against Pseudomonas aeruginosa when
combined with doxycycline was precisely evaluated utilizing
bioluminescence to measure ATP efflux and fluorescence to
evaluate membrane depolarization.
polyamines,10 which can weaken the binding sites of divalent
cations, can lead to the disruption of the outer membrane
organization, increasing its permeability. In this context, the use
of compounds targeting the membranes of Gram-negative
bacteria and enhancing the sensitivity of bacteria to hydro-
phobic antibiotics represents an attractive approach for the
development of antibacterial agents because they are not
expected to promote resistance. The presence of a polyamino
moiety is crucial for achieving high antimicrobial activities.10
We recently reported the synthesis of substituted polyamino
geranic acid molecules containing a terpene core and various
polyamino groups and investigated their use as potent
activators of various common antibiotics against several
Gram-negative bacteria strains that exhibit a MDR pheno-
type.11 Additionally, in 2012, Xu et al. reported the isolation of
ianthelliormisamines A−C, antibacterial bromotyrosine-derived
metabolites from the marine sponge, Suberea ianthelliformis
(Figure 1).12
1. INTRODUCTION
Because of their increased use for the treatment of numerous
bacterial infections during recent decades, antibiotics represent
one of the biggest healthcare blockbusters by decreasing both
morbidity and mortality. Nevertheless, this success has led to
inappropriate prescribing, thus increasing the number of
antibiotic-resistant bacteria. Because of development costs and
low profitability, novel antibiotic compounds are lacking in the
commercial market.1 The emergence of Gram-negative multi-
drug-resistant (MDR) bacteria, such as Pseudomonas aeruginosa
and Klebsiella pneumoniae, has prompted efforts to develop new
classes of antibiotics to overcome this problem. The global
strategy of identifying compounds that can circumvent the
MDR phenotype is very promising because one molecule could
potentially enhance the activity of numerous antibiotics.
Furthermore, the bacterial membrane, which forms an effective
barrier to many types of antibiotics,2 represents an appealing
target because it is highly conserved among most strains of
Gram-negative bacteria. Thus, resistance to membrane-active
antibiotics would require a major change in membrane
structure, which would influence the permeability barrier and
increase susceptibility to hydrophobic antibiotics. Most
responses to Gram-negative bacteria can be attributed to the
major components of the outer membrane, namely, lip-
opolysaccharides (LPS) and their lipid A anchor.3−8
Here, we describe the design, synthesis, and biological
activity of new ianthelliformisamine derivatives as antibiotic
enhancers against resistant Gram-negative bacteria. We also
analyze the original mechanism of action of this class of
derivatives against Gram-negative bacteria.
The permeability barrier of the outer membrane is due to the
cross-bridging between lipid A molecules and calcium or
magnesium divalent cations.3 Thus, cationic peptides9 and
Received: February 6, 2014
Published: May 6, 2014
© 2014 American Chemical Society
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Figure 1. Structure of natural ianthelliformisamine derivatives.
Scheme 1. Synthesis of Ianthelliformisamine A−C and Tokaridine C
involves a Wittig reaction with ethoxymethylidenetriphenyl-
phosphorane (7) as the key step. Subsequent coupling
involving a dicyclohexylcarbodiimide (DCC)/HOBt reagent
afforded ianthelliformisamine A−C (1−3) and tokaridine C (4)
in isolated yields varying from 32 to 80%. Notably, all analytical
data are in accordance with those previously reported, thus
confirming the assigned structures of these isolated marine
metabolites. This chemical approach was successfully utilized
for the preparation of various non-natural ianthelliformisamine
2. RESULTS AND DISCUSSION
Because of the novel chemical structure of ianthelliformis-
amines A−C and tokaridine C, we decided to synthesize these
natural products (Scheme 1).
The common precursor for these compounds is the use of
(E)-3-(3,5-dibromo-4-methoxyphenyl)acrylic acid, which is
easily prepared in a three-step synthesis from 3,5-dibromo-4-
hydroxybenzaldehyde (5) in 84% overall yield; this route
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Table 1. New Ianthelliformisamine Derivatives 11−22
a
Isolated yield.
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derivatives by varying the structure of the polyamines involved
(Table 1).
Adducts 11−22 were obtained in good to excellent yield,
varying from 27 to 85%, depending on the nature of the
polyamine.
preliminary experiment, doxycycline (2 μg/mL, a concentration
corresponding to its pharmacokinetic properties in humans20)
was used to determine the concentrations of our products that
could achieve this effect. At this concentration of doxycycline,
natural derivatives 2−4 at 3.12−12.5 μM are able to restore the
activity of doxycycline against Enterobacter aerogenes EA289, P.
aeruginosa PAO1, and K. pneumoniae KPC2 ST258. Moreover,
non-natural compounds 17, 19, and 21 were highly efficient in
the same range of concentrations (Table 3).
Infections caused by MDR Gram-negative bacteria are
increasing worldwide,13,14 and the emergence of bacteria
resistant to all classes of antibiotics (namely, pandrug-resistant
(PDR) bacteria) appears as a frightening end point in the
development of antimicrobial resistance.15,16 Despite the small
number of strains of such bacteria, there is great concern in the
medical community because clinicians have no options for
treating patients with PDR bacterial infections. Recently,
numerous clinical reports confirmed that Gram-negative
bacteria have developed resistance to polymyxins,17,18 which
have become widely used in several countries as the last
therapeutic barrier against PDR Gram-negative bacteria.19
Thus, we investigated the potency of our new class of
derivatives against two Gram-negative bacteria, P. aeruginosa
and K. pneumoniae, that are commonly observed in hospitals.
First, the minimum inhibitory concentrations (MICs) of
molecules 1−4 and 11−22 were evaluated against Gram-
positive and Gram-negative bacterial strains to determine the
concentrations to which each strain could be exposed to
produce direct antibacterial activity.
Table 3. Concentration of Ianthelliformisamine Derivatives
1−4 and 11−22 Necessary To Restore Doxycycline Activity
(2 μg/mL) against EA289, PAO1, and KPC2 ST258 Gram-
Negative Bacterial Strains
concentration of ianthelliformisamine derivative used
(μM)
compound
EA289
PAO1
KPC2 ST258
1
>100
25
12.5
12.5
3.12
3.12
>100
100
>100
3.12
12.5
3.12
50
2
3
12.5
6.25
>100
>100
>100
>100
>100
>100
12.5
>100
12.5
>100
6.25
>100
4
11
12
13
14
15
16
17
18
19
20
21
22
100
>100
>100
25
>100
6.25
>100
>100
3.12
>100
3.12
>100
3.12
>100
As summarized in Table 2, all compounds demonstrated
MICs over 200 μM except for the natural derivative,
>100
6.25
>100
6.25
>100
3.12
>100
Table 2. MIC of Antibiotics and Compounds 1−4 and 11−
22 against Various Bacterial Strains
MIC μM (μg/mL)
compound
EA289
SA DSM 799
PAO1
>200
KPC2 ST258
chloramphenicol
>200
52 (25)
56 (25)
>200
100
ND
ND
cefepime
ND
26 (12.5)
112 (50)
200
ND
doxycycline
<3
ND
We also observed that after the MIC of doxycycline was
decreased by treatment with a compound that later it was also
particularly active with cefepime. Thus, we identified two
groups of compounds, one consisting of derivatives 1, 11−16,
18, 20, and 22, which displayed weak or no activity, and a
second group consisting of compounds 2−4, 17, 19, and 21,
which increased the antibiotic susceptibility effectively against
PAO1. Similar dose-dependent effects were observed for
ianthelliformisamine derivatives 1−4 and 11−22 in combina-
tion with doxycycline against P. aeruginosa PA01 (Figure 2).
1
>200
>200
12.5
200
>200
>200
12.5
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
2
>200
25
3
100
4
>200
>200
>200
>200
>200
>200
>200
>200
>200
200
200
11
12
13
14
15
16
17
18
19
20
21
22
>200
>200
>200
>200
>200
>200
>200
>200
50
>200
100
>200
>200
>200
>200
200
>200
100
>200
>200
>200
>200
50
>200
100
6.25
>200
ianthelliformisamine C (3), which presented MICs varying
from 12.5 to 100 μg/mL depending on the Gram-positive and
Gram-negative bacterial strains.12
As previously described, the development of a chemosensitiz-
ing agent to increase the internal antibiotic concentration in
resistant strains is an attractive approach to circumvent bacterial
resistance. Thus, we envisioned the use of our ianthelliformis-
amine derivatives in combination with classic antibiotics to
bypass the mechanistic and enzymatic barriers that reduce the
intracellular concentrations of active antibacterial drugs. In a
Figure 2. Dose-dependent effect of ianthelliformisamine derivatives
1−4 and 11−22 in combination with doxycycline against P. aeruginosa
PA01.
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Notably, the chloramphenicol activity was improved in the
presence of high concentrations of these derivatives, whereas
the effect on cefepime resistance was stronger at lower
concentrations of these later (Table 4).
the bacteria and (ii) efflux pump inhibition. Recently, Katsu et
al. investigated the structure−activity relationship between the
outer membrane of Gram-negative bacteria and original
polyamines such as naphthylacetylspermine and methoctr-
amine.23 These studies demonstrated that the presence of
lipophilic moieties as well as a number of amino groups on
polyamines was important for permeabilization.23
Thus, the effect of 21 on the intracellular pool of bacterial
ATP was determined by using a bioluminescence method.
Thus, the external concentration of ATP was used as an
indicator of the dose-dependent permeabilizing effect of 21. For
P. aeruginosa, no ATP release was observed (less than 16% of
maximal efflux) using 2 mM compound 21 (Figure 4).
Table 4. Concentration of the Select Ianthelliformisamine
Derivatives Required To Restore Chlorampheninol,
Doxycycline, and Cefepime Activity (2 μg/mL) against the
PAO1 Bacterial Strain and Their Associated Cytotoxicity
P. aeruginosa PAO1
IC50 (μM)
a
a
a
compound
CHL
DOX
12.5
FEP
CHO
fibroblasts
2
200
12.5
100
200
100
100
0.8
1665
663
108
174
573
846
129
237
33
3
3.12
3.12
6.25
6.25
3.12
1.6
4
3.125
6.25
6.25
0.4
17
19
21
24
237
345
a
CHL, choramphenicol; DOX, doxycycline; FEP, cefepime.
Compound 21 was subsequently applied to restore
doxycycline activity against clinical MDR isolates of P.
aeruginosa (Figure 3). Five strains remained insensitive to
Figure 4. Effect of compound 21 on ATP efflux in P. aeruginosa
(PAO1) in the presence or absence of doxycycline.
Furthermore, no ATP efflux was observed using a synergistic
combination of 21 and doxycycline, even at high concen-
trations. Thus, the integrity of the outer membrane is not
affected. Next, nitrocefin, a chromogenic β-lactam that is
efficiently hydrolyzed by periplasmic β-lactamases, was used to
measure the cell outer membrane integrity and to determine if
our compounds possess outer membrane-permeabilizing
activity.24,25 As shown in Figure 5, even at a high concentration
Figure 3. Concentration of ianthelliformisamine derivative 21 used to
restore doxycycline activity (2 μg/mL) against P. aeruginosa clinical
isolates.
compound 21, and all of these strains were resistant to
gentamycin. Some strains of P. aeruginosa exhibit aminoglyco-
side resistance related to a transport defect or membrane
impermeabilization. This mechanism is most probably
chromosomally mediated, and it results in cross-reactivity to
all aminoglycosides, which could explain the ineffectiveness of
compound 21 against these five strains.21,22 All other strains
presented similar antibiograms (data not shown), providing
good to moderate results at a concentration of derivative 21
(12.5−50 μM) necessary to restore doxycycline activity at 2
μg/mL.
Moreover, nondetrimental cytotoxicities against Chinese
hamster ovary cells (CHO) and human fibroblasts, with IC50
values up to 100 μM, were noted for the best derivatives,
suggesting their potential therapeutic use for circumventing
MDR.
From a mechanistic point of view, even if these compounds
exhibit low antibacterial activity, their synergism with different
antibiotics against Gram-negative bacteria remains question-
able. Thus, two main modes of action are envisioned: (i)
permeabilization and/or disruption of the outer membrane of
Figure 5. Effect of polymyxin B and compound 21 (500 μM) on
nitrocefin hydrolysis in the periplasmic space of P. aeruginosa (PA01).
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1
mesh). H and 13C NMR spectra were recorded in MeOD using a
Bruker AC 300 or AC 400 spectrometer (abbreviations: s, singlet; d,
doublet; t, triplet; q, quadruplet; m, multiplet). Tetramethylsilane was
used as the internal standard. All chemical shifts are presented in parts
per million (ppm). Mass spectroscopy analysis was performed using
the Spectropole (Analytical Laboratory) at the University Paul
(500 μM), the efflux pump inhibitor 21 did not increase the
rate of nitrocefin hydrolysis compared to that of the untreated
control. Lower concentrations were also tested under the same
conditions and resulted in a similar response (see the
Supporting Information). In contrast, polymyxin B (PMB)
treatment dramatically increased the rate of nitrocefin
hydrolysis.
Efflux pumps can expel a wide variety of compounds from
bacterial cells, including antibiotics and dyes. In this context,
the transport measurement of a dye, known as a substrate, can
be used to directly monitor the function of efflux pumps.
Treatment with 200 μM 21 resulted in dose-dependent
depolarization of the PAO1 membrane, as indicated by a
rapid and important increase in relative fluorescence units
(ΔRFU values); 60% of the maximal RFU was attained in less
than 3 min (Figure 6), indicating disruption of the proton
́
Cezanne (Marseille). The purity of the compounds was verified by
analytical HPLC (C18 column, eluent CH3CN/water/TFA, 2.3 mL/
min) with a PDA detector from 210 to 310 nm. All compounds
showed a purity greater than 95%, as determined by analytical HPLC-
PDA at 214 and 254 nm.
4.2. Preparation of Compounds 1−4 and 11−22. Synthesis of
3,5-Dibromo-4-methoxybenzaldehyde 6. To a stirred suspension of
3,5-dibromo-4-hydroxybenzaldehyde (5.1 g, 18.2 mmol) and K2CO3
in DMF (15 mL) was added iodomethane (1.4 mL, 22.7 mmol), and
the resulting mixture was stirred at room temperature for 72 h. Then,
the mixture was diluted with EtOAc (200 mL) and washed with water
(2 × 60 mL) and brine (2 × 60 mL). The organic layers were
combined and dried (Na2SO4), and the solvents were removed in
vacuo to afford pure 3,5-dibromo-4-methoxybenzaldehyde (5.1 g,
1
96%) as a pale yellow solid. H NMR (400 MHz, MeOD): δ 9.86 (s,
1H), 8.03 (s, 2H), 3.97 (s, 3H). 13C NMR (75 MHz, MeOD): δ
188.33, 159.08, 34.19, 133.87, 119.26, 60.84.
Synthesis of (E)-Ethyl 3-(3,5-dibromo-4-methoxyphenyl)acrylate.
A solution of 3,5-dibromo-4-methoxybenzaldehyde (900 mg, 3.1
mmol) and ethoxymethylidenetriphenylphosphorane (1.4 g, 4 mmol)
in toluene (20 mL) was heated at 70 °C for 15 h. The mixture was
evaporated to dryness, and the resulting residue was purified by
column chromatography (eluent 100% Et2O) to yield pure (E)-ethyl
3-(3,5-dibromo-4-methoxyphenyl)acrylate (only the (E) isomer was
obtained; 1.01 g, 91%). 1H NMR (400 MHz, MeOD): δ 7.66 (s, 2H),
7.5 (d, J = 15.8 Hz, 1H), 6.36 (d, J = 15.8 Hz, 1H), 4.26 (q, J = 7.3 Hz,
2H), 3.91 (s, 3H), 1.33 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz,
MeOD): δ 166.1, 155.35, 140.82, 133.09, 131.89, 120.10, 118.63,
60.68, 14.21.
Figure 6. Dose-dependent depolarization of the bacterial membrane of
PAO1 in the presence of compound 21.
Synthesis of (E)-3-(3,5-Dibromo-4-methoxyphenyl)acrylic Acid.
To a solution of (E)-ethyl 3-(3,5-dibromo-4-methoxyphenyl)acrylate
(350 mg, 0.97 mmol) in a mixture of THF/H2O (7 mL/2 mL) was
added LiOH·H2O (160 mg, 3.9 mmol), and the solution was stirred at
room temperature for 72 h. The mixture was acidified with
concentrated HCl (approximately 0.3 mL) until pH 1, and the
aqueous layer was extracted with EtOAc (3 × 15 mL). The organic
layers were combined, washed with brine (20 mL), and dried
(MgSO4), and the solvent was removed in vacuo. The pure desired
gradient. Efflux pumps use energy generated by the proton
gradient across the inner membrane for drug extrusion. Taken
together, these data indicate that changes in the transmembrane
electrical potential in P. aeruginosa are not correlated with the
permeabilization of cell membranes by ianthelliformisamines,
suggesting altered proton homeostasis. Finally, ianthelliformis-
amines that disrupt the proton gradient may be considered to
be efflux pump inhibitors.
1
compound was obtained as a white solid (310 mg, 96%). H NMR
(400 MHz, MeOD): δ 7.69 (s, 2H), 7.60 (d, J = 15.8 Hz, 1H), 6.37 (d,
J = 15.8 Hz, 1H), 3.92 (s, 3H).
3. CONCLUSIONS
Synthesis of Ethoxymethylidenetriphenylphosphorane 7. A
solution of triphenylphosphine (4.93 g, 18.8 mmol) and ethyl
chloroacetate (2 mL, 18.7 mmol) in toluene (40 mL) was stirred at
110 °C for 72 h. The precipitate was filtered and washed with ethyl
acetate (2 × 15 mL) to yield 8.8 g of a white solid (pure
An original chemical strategy was developed to produce new
ianthelliformisamine derivatives in moderate to good yield.26
Among the synthesized derivatives, compounds 2−4, 17, 19,
and 21 dramatically affected the antibiotic susceptibility of E.
aerogenes, P. aeruginosa, and K. pneumoniae MDR strains. This
efficiency was correlated with the inhibition of a dye transport,
suggesting that these molecules can act on the activity of drug
transporters. Studies are now under current investigation to
elucidate whether this restoration of antibiotic susceptibility
occurs via direct interaction of the molecule with the efflux
pump or by deenergization of the efflux pump later. Finally,
these derivatives may prove to be useful for studying the
importance of efflux in acquired and natural resistance to
antibiotics in Gram-negative bacteria.
1
bromophosphonium salt; 100%). H NMR (400 MHz, MeOD): δ
7.96−7.65 (m, 15H), 5.66 (d, J = 14 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H),
1.04 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, MeOD): δ 164.38,
164.33, 134.86, 133.77, 133.63, 130.07, 129.89, 118.46, 117.28, 62.43,
32.84, 32.10, 20.73, 13.89. 31P NMR: 20.85. To a solution of the
previous bromophosphonium salt (4.2 g, 9.8 mmol) was added 2 M
NaOH (200 mL). The mixture was vigorously shaken several times
and extracted with CH2Cl2 (2 × 100 mL). The organic layers were
combined, washed with water (200 mL) and brine (200 mL), and
dried (MgSO4), and the solvent removed in vacuo. The desired
1
phosphonium ylide was obtained as a white powder (2.8 g, 82%). H
NMR (400 MHz, MeOD): δ 7.69−7.62 (m, 6H), 7.56−7.42 (m, 9H),
3.96 (m, 2H), 2.88 (s, 1H), 1.06 (m, 3H). 13C NMR (75 MHz,
MeOD): δ 132.98, 132.95, 131.83, 128.69, 128.53, 57.76, 30.87, 29.21,
14.75. 31P NMR: 17.59.
Preparation of Ianthelliformisamine A 1 and C 3. To a solution of
(E)-3-(3,5-dibromo-4-methoxyphenyl)acrylic acid (200 mg, 0.6
mmol) in CH2Cl2 (15 mL) were added HOBt (86 mg, 0.66 mmol)
4. EXPERIMENTAL SECTION
4.1. Materials. All solvents were purified according to previously
reported procedures, and the reagents used are commercially available.
Methanol, ethyl acetate, and dichloromethane were purchased from
Fisher Scientific and used without further purification. Column
chromatography was performed using Merck silica gel (70−230
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and DCC (135 mg, 0.65 mmol). After stirring at room temperature for
5 h, the precipitate was filtered and washed with CH2Cl2. Spermine
(135 mg, 0.64 mmol) in CH2Cl2 (2 mL) was added to the filtrate, and
the resulting solution was stirred at room temperature for 15 h.
Evaporation of solvents in vacuo afforded an oil that was purified by
column chromatography (eluent CH2Cl2/MeOH/NH4OH, 7:3:1) to
afford monoalkylated ianthelliformisamine A (1) (100 mg, 30%) and
ianthelliformisamine C (3) as a side product (180 mg, 70%).
Ianthelliformisamine C 3. 1H NMR (400 MHz, MeOD): δ 7.67 (s,
4H), 7.28 (d, J = 15.8 Hz, 2H), 6.46 (d, J = 15.8 Hz, 2H), 3.77 (s, 6H),
3.36−3.18 (m, 4H), 2.65−2.49 (m, 8H), 1.7−1.2 (m, 8H). 13C NMR
(75 MHz, MeOD): δ 168.80, 157.11, 139.01, 136.14, 133.83, 124.79,
120.38, 62.10, 50.54, 48.18, 39.08, 35.60, 26.90. MS (ESI)
C30H38N4O4Br4 m/z 839.0 (100%, (M + H+)), 837.0 (100%, (M −
H−)).
Synthesis of (4-tert-Butoxycarbonylamino-butyl)-(2-cyano-
ethyl)-carbamic Acid tert-Butyl Ester. To a 0 °C solution of [4-(2-
cyano-ethylamino)-butyl]-carbamic acid tert-butyl ester (750 mg, 3.1
mmol) in CH2Cl2 (15 mL) was added a solution of tert-butyl
dicarbonate (0.85 mL, 3.7 mmol) in CH2Cl2 (10 mL) dropwise. After
stirring at room temperature for 15 h, the solvents were removed in
vacuo, and the crude residue was dissolved in CH2Cl2 (50 mL) and
washed with 10% Na2CO3 (2 × 30 mL) and brine (30 mL). The
organic layer was dried (MgSO4), and the solvent was removed to
afford a colorless oil (1 g, 94%). 1H NMR (400 MHz, MeOD): δ 4.64
(s large, 1H), 3.41 (t, J = 6.7 Hz, 2H), 3.23 (t, J = 6.8 Hz, 2H), 3.09−
3.07 (m, 2H), 2.57 (s large, 2H), 1.51−1.39 (m, 22 H). 13C NMR (75
MHz, MeOD): δ 155.91, 146.62, 118.22, 80.37, 78.95, 53.33, 39.88,
28.28, 27.27, 25.78, 25.36.
Synthesis of {4-[3-Amino-propyl)-tert-butoxycarbonyl-amino]-
butyl}-carbamic Acid tert-Butyl Ester. A 0 °C solution of (4-tert-
butoxycarbonylamino-butyl)-(2-cyano-ethyl)-carbamic acid tert-butyl
ester (1 g, 2.9 mmol) in distilled Et2O (20 mL) was carefully added to
a suspension of LiAlH4 (480 mg, 12.6 mmol) in distilled Et2O (20
mL). After stirring at 0 °C for 3 h, 5% NaOH (10 mL) was added
dropwise, and the white precipitate was removed by filtration and
washed with EtOAC (20 mL). The aqueous layer was extracted with
EtOAc (2 × 20 mL), and the organic layers were combined, washed
with brine (2 × 10 mL), and dried (MgSO4); the solvent was removed
in vacuo to afford the desired compound as a colorless oil (760 mg,
75% yield). 1H NMR (400 MHz, MeOD): δ 4.73 (s large, 1H), 3.18−
3.03 (m, 6H), 2.61 (t, J = 6.7 Hz, 2H), 1.44−1.36 (m, 24H). 13C NMR
(75 MHz, MeOD): δ 155.87, 155.55, 79.16, 46.36, 40.02, 28.25, 27.26,
25.51.
Ianthelliformisamine A 1. 1H NMR (400 MHz, MeOD): δ 7.83 (s,
2H), 7.42 (d, J = 15.8 Hz, 1H), 6.61 (d, J = 15.8 Hz, 1H), 3.90 (s, 3H),
2.94−2.78 (m, 12 H), 1.90−1.85 (m, 4H), 1.47−1.70 (m, 4H). 13C
NMR (75 MHz, MeOD): δ 168.96, 157.12, 138.99, 136.23, 133.89,
124.92, 120.37, 62.11, 47.96, 47.55, 40.14, 38.68, 29.21, 28.85, 27.25,
26.87. MS (ESI) C20H32N4O2Br2 m/z 521.1 (100%, (M + H+)), 519.1
(100%, (M − H−)).
Preparation of Ianthelliformisamine B Hydrochloride Salt 2. To a
solution of (E)-3-(3,5-dibromo-4-methoxyphenyl)acrylic acid (150
mg, 0.45 mmol) in CH2Cl2 (10 mL) were added HOBt (65 mg, 0.48
mmol) and DCC (100 mg, 0.47 mmol). After stirring at room
temperature for 1 h, the white precipitate was filtered, washed with
CH2Cl2 (2 mL), and {4-[3-amino-propyl)-tert-butoxycarbonyl-amino]-
butyl}-carbamic acid tert-butyl ester (160 mg, 0.46 mmol) in CH2Cl2
(2 mL) was added to the filtrate. After 3 h, the solution was evaporated
to dryness, and the crude residue (240 mg, 80%) was used in the next
step.
Preparation of Tokaridine C Hydrochloride Salt 4. To a solution
of (E)-3-(3,5-dibromo-4-methoxyphenyl)acrylic acid (152 mg, 0.45
mmol) in CH2Cl2 (10 mL) were added HOBt (67 mg, 0.48 mmol)
and DCC (102 mg, 0.47 mmol). After stirring at room temperature for
1 h, the white precipitate was removed by filtration and washed with
CH2Cl2 (2 mL). A solution of (4-amino-butyl)-(3-tert-butoxycarbo-
nylamino-propyl)-carbamic acid tert-butyl ester (158 mg, 0.45 mmol)
in CH2Cl2 (2 mL) was added to the filtrate. After 3 h, the solution was
evaporated to dryness, and the crude residue (275 mg, 91%) was used
in the next step.
BOC-protected ianthelliformisamine B (50 mg, 0.15 mmol) was
dissolved in MeOH (1 mL), and 1 M HCl in Et2O (1 mL) was added.
After stirring at room temperature for 15 h, a white precipitate
appeared, and Et2O (10 mL) was added to complete the precipitation
of ianthelliformisamine B hydrochloride salt 3. This compound was
then filtered to yield the desired compound as a white solid (40 mg,
1
100%). H NMR (400 MHz, D2O): δ 7.44 (s, 2H), 7.01 (d, J = 15.8
BOC-protected tokaridine C (100 mg, 0.07 mmol) dissolved in
MeOH (1 mL) and 1 M HCl in Et2O (1 mL) were added. After
stirring at room temperature for 15 h, a white precipitate appeared,
and Et2O (10 mL) was added to completely precipitate the tokaridine
C hydrochloride salt. This suspension was then filtered to yield the
Hz, 1H), 6.25 (d, J = 15.8 Hz, 1H), 3.73 (s, 3H), 3.29 (m, 2H), 3.01−
2.97 (m, 6H), 1.71−1.63 (m, 6H). 13C NMR (75 MHz, D2O): δ
168.10, 153.88, 137.98, 133.24, 131.83, 121.44, 117.74, 60.88, 46.93,
45.16, 38.76, 36.35, 25.64, 23.88, 22.79. MS (ESI) C17H27N3O2Cl2Br2
m/z 464.1 (100%, (M + H+)), 462.0 (100%, (M − H−)).
1
desired compound as a white solid (120 mg, 100%). H NMR (400
Preparation of {4-[3-Amino-propyl)-tert-butoxycarbonyl-amino]-
butyl}-carbamic acid tert-Butyl Ester 9. Synthesis of tert-Butyl-4-
aminobutylcarbamate. To a 0 °C solution of 1,4-diaminobutane (12
mL, 119.4 mmol) in chloroform (100 mL) was added tert-butyl
dicarbonate (5 mL, 21.7 mmol) dropwise over 1.5 h. After stirring at
room temperature for 15 h, the solvents were removed in vacuo, water
was added (150 mL), and the insoluble bis-substituted product was
removed by filtration. The filtrate was extracted with CH2Cl2 (3 × 100
mL), and the organic layers were combined, washed with brine (100
mL), and dried over MgSO4; the solvents were removed in vacuo to
afford a pale yellow oil (4.2 g, 100%). 1H NMR (400 MHz, MeOD): δ
4.72 (s large, 1H), 3.09 (t, J = 6.2 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H),
1.52−1.4 (m, 13H). 13C NMR (75 MHz, MeOD): δ 155.81, 41.53,
30.64, 28.15.
MHz, D2O): δ 7.56 (s, 2H), 7.12 (d, J = 15.8 Hz, 1H), 6.35 (d, J =
15.8 Hz, 1H), 3.84 (s, 3H), 3.30 (t, J = 6.8 Hz, 2H), 3.13−3.06 (m,
6H), 2.10−2.04 (m, 2H), 1.72−1.62 (m, 4H). 13C NMR (75 MHz,
D2O): δ 167.81, 153.88, 137.46, 133.40, 131.84, 121.82, 117.78, 60.94,
47.38, 44.46, 38.83, 36.57, 25.63, 23.73, 23.08. MS (ESI)
C17H25N3O2Br2 m/z 464.0 (100%, (M + H+)), 462.3 (100%, (M −
H−)).
Preparation of (4-Amino-butyl)-(3-tert-butoxycarbonylamino-
propyl)-carbamic Acid tert-Butyl Ester 10. Synthesis of tert-Butyl-
3-aminopropylcarbamate. To a 0 °C solution of 1,4-diaminopropane
(10 mL, 120 mmol) in chloroform (100 mL) was added tert-butyl
dicarbonate (5 mL, 21.7 mmol) in chloroform (100 mL) dropwise
over 1.5 h. After stirring at room temperature for 15 h, the solvents
were removed in vacuo, water was added (150 mL), and the insoluble
bis-substituted product was removed by filtration. The filtrate was
extracted with Et2O (3 × 100 mL), the organic layers were combined,
washed with brine (100 mL), and dried (MgSO4), and the solvents
were removed in vacuo to afford the desired product as a pale yellow
oil (90% yield (3.4 g)). 1H NMR (400 MHz, MeOD): δ 4.90 (s large,
1H), 3.20 (q, J = 6.3 Hz, 2H), 2.74 (t, J = 6.5 Hz, 2H), 1.59 (m, 2H),
1.43 (s, 9H).
Synthesis of [4-(2-Cyano-ethylamino)-butyl]-carbamic Acid tert-
Butyl Ester. To a stirred suspension of tert-butyl-4-aminobutylcarba-
mate (2 g, 10.6 mmol) and K2CO3 (5 g, 36.1 mmol) in acetonitrile (50
mL) was added 3-bromopropionitrile (0.9 mL, 10.8 mmol), and the
mixture was stirred at 50 °C for 15 h. The solvents were removed in
vacuo, and the crude oil was purified by column chromatography (40 g
SiO2, eluent MeOH/CH2Cl2, 9:1) to yield the desired compound as a
1
colorless oil (820 mg, 32%). H NMR (400 MHz, MeOD): δ 4.81 (s
large, 1H), 3.06 (m, 2H), 2.86 (t, J = 6.7 Hz, 2H), 2.60 (t, J = 6.4 Hz,
2H), 2.46 (t, J = 6.5 Hz, 2H), 1.48−1.38 (m, 13H). 13C NMR (75
MHz, MeOD): δ 155.88, 118.58, 78.84, 48.55, 44.86, 40.19, 28.27,
27.08, 18.53.
Synthesis of [3-(3-Cyano-propylamino)-propyl]-carbamic Acid
tert-Butyl Ester. To a stirred suspension of tert-butyl-3-amino-
propylcarbamate (1.5 g, 8.6 mmol) and K2CO3 (4 g, 28.9 mmol) in
acetonitrile (50 mL) was added 3-bromopropionitrile (0.9 mL, 9
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Journal of Medicinal Chemistry
Article
mmol), and the mixture was stirred at 50 °C for 15 h. The solvents
were removed in vacuo, and the crude oil (100%) was used without
further purification for the next step. 1H NMR (400 MHz, MeOD): δ
5.10 (s large, 1H), 3.20 (m, 2H), 2.72 (t, J = 6.5 Hz, 2H), 2.65 (t, J =
6.5 Hz, 2H), 2.44 (t, J = 7.3 Hz, 2H), 1.80 (m, 2H), 1.63 (t, J = 6.5 Hz,
2H), 1.42 (s, 9H). 13C NMR (75 MHz, MeOD): δ 155.97, 119.61,
47.73, 47.13, 38.76, 29.66, 28.22, 25.54, 14.70.
3.35−3.30 (m, 2H), 2.76−2.72 (t, J = 7.3 Hz, 2H), 1.63−1.56 (m,
4H), 1.46−1.41 (m, 2H). 13C NMR (MeOD, 75 MHz): δ 168.61,
156.99, 138.82, 163.17, 133.78, 124.89, 120.32, 62.11, 42.76, 41.34,
32.84, 30.93, 25.99. MS (ESI) C15H20N2O2Br2 m/z 421.0 (100%, (M
+ H+)), 419.0 (100%, (M − H−−)).
(E)-3-(3,5-Dibromo-4-methoxy-phenyl)-N-(3-pyrrolidin-1-yl-prop-
1
yl)-acrylamide 15. H NMR (MeOD, 400 MHz): δ 7.81 (s, 2H),
7.42−7.38 (d, J = 15.8 Hz, 1H), 6.59−6.55 (d, J = 15.8 Hz, 1H), 3.90
(s, 3H), 3.38−3.34 (t, J = 7.0 Hz, 2H), 2.61−2.55 (m, 6H), 1.86−1.82
(m, 6H). 13C NMR (MeOD, 75 MHz): δ 168.55, 157.10, 138.89,
136.22, 133.81, 124.86, 120.37, 62.08, 55.85, 39.82, 30.41, 25.03. MS
(ESI) C17H22N2O2Br2 m/z 446.9 (100%, (M + H+)), 445.2 (100%, (M
− H−−)).
(E)-3-(3,5-Dibromo-4-methoxy-phenyl)-N-(3-morpholin-4-yl-
propyl)-acrylamide 16. 1H NMR (MeOD, 400 MHz): δ 7.69 (s, 2H),
7.30−7.26 (d, J = 15.6 Hz,1), 6.47−6.43 (d, J = 15.6 Hz, 1H), 3.78 (s,
3H), 3.62−3.60 (t, J = 4.5 Hz, 4H), 3.27−3.24 (t, J = 6.8 Hz, 2H), 2.42
(m, 4H), 2.38−2.34 (t, J = 7.3 Hz, 2H), 1.70−1.66 (t, J = 7.0 Hz, 2H).
13C NMR (MeOD, 75 MHz): δ 168.55, 157.02, 138.87, 136.14,
Synthesis of (3-[tert-Butoxycarbonyl-(3-cyano-propyl)-amino]-
propyl)-carbamic Acid tert-Butyl Ester. To a 0 °C solution of [3-
(3-cyano-propylamino)-propyl]-carbamic acid tert-butyl ester (2.05 g,
8.5 mmol) in CH2Cl2 (40 mL) was added tert-butyl dicarbonate (1.9
mL, 8.2 mmol) in CH2Cl2 (20 mL) dropwise. After stirring at room
temperature for 15 h, the solvents were removed in vacuo, and the
crude residue was dissolved in CH2Cl2 (100 mL) and washed with
10% Na2CO3 (2 × 50 mL) and brine (50 mL). The organic layer was
dried over MgSO4, and the solvent was removed to afford a colorless
1
oil (2.8 g, 96%). H NMR (400 MHz, MeOD): δ 3.28 (t, J = 7 Hz,
4H), 3.10 (m, 2H), 2.34 (t, J = 7.3 Hz, 2H), 1.88 (t, J = 6.8 Hz, 2H),
1.69−1.61 (m, 2H), 1.51−1.43 (m, 18H). 13C NMR (75 MHz,
MeOD): δ 161.15, 155.88, 105.85, 85.06, 80.18, 79.01, 53.34, 52.02,
45.47, 37.41, 30.61, 28.27, 27.29, 22.99, 15.87, 14.74, 14.59.
133.80, 124.85, 120.33, 68.15, 62.09, 58.11, 55.32, 39.57, 27.11. MS
(ESI) C17H22N2O3Br2 m/z 463.1 (100%, (M + H+)), 461.0 (100%, (M
− H−−)).
Synthesis of (4-Amino-butyl)-(3-tert-butoxycarbonylamino-prop-
yl)-carbamic Acid tert-Butyl Ester. A 0 °C solution of (4-tert-
butoxycarbonylamino-butyl)-(2-cyano-ethyl)-carbamic acid tert-butyl
ester (2.8 g, 8.2 mmol) in distilled Et2O (50 mL) was carefully added
to a suspension of LiAlH4 (1.3 g, 34.2 mmol) in distilled Et2O (50
mL). After stirring at 0 °C for 3 h, 5% NaOH (30 mL) was added
dropwise, and the white precipitate was isolated by filtration and
washed with EtOAC (20 mL). The aqueous layer was extracted with
EtOAc (2 × 50 mL), and the organic layers were combined, washed
with brine (2 × 30 mL), and dried over MgSO4; the solvent was
removed in vacuo to afford the desired compound as a colorless oil
(E)-N-{3-[(3-Amino-propyl)-methylamino]-propyl}-3-(3,5-dibro-
1
mo-4-methoxy-phenyl)-phenyl)-acrylamide 17. H NMR (MeOD,
400 MHz): δ 7.80 (s, 2H), 7.42−7.37 (d, J = 15.7 Hz, 1H), 6.59−6.54
(d, J = 15.7 Hz, 1H), 3.89 (s, 3H), 2.73−2.68 (t, J = 6.8 Hz, 2H),
2.48−2.44 (m, 4H), 2.27 (s, 3H), 1.80−1.66 (m, 4H). 13C NMR
(MeOD, 75 MHz): δ 168.56, 157.05, 138.81, 136,19, 133.75, 124.87,
120.32, 62.06, 57.23, 43.08, 41.70, 39.74, 30.99, 28.57. MS (ESI)
C17H25N3O2Br2 m/z 464.1 (100%, (M + H+)), 462.0 (100%, (M −
H
−−)).
(E)-N-{2-[2-(2-Amino-ethylamino)-ethylamino]-ethyl}-3-(3,5-di-
1
bromo-4-methoxy-phenyl)-acrylamide 18. H NMR (MeOD, 400
MHz): δ 7.82 (s, 2H), 7.43−7.39 (d, J = 15.8 Hz, 1H), 6.62−6.58 (d, J
= 15.8 Hz, 1H), 3.90 (s, 3H), 3.47 (t, J = 7.3 Hz, 2H), 2.85−2.74 (m,
10H). 13C NMR (MeOD, 75 MHz): δ 168.99, 157.13, 139.07, 136.21,
133.87, 124.84, 120.37, 62.12, 51.97, 42.12, 41.2, 34.86, 27.12, 26.84.
MS (ESI) C16H24N4O2Br2 m/z 465.1 (100%, (M + H+)), 463.0
(100%, (M − H−−)).
1
(2.2 g, 78%). H NMR (400 MHz, MeOD): δ 3.25−3.09 (m, 6H),
2.72−2.69 (m, 2H), 2.03 (br s, 2H), 1.68−1.44 (m, 18H). 13C NMR
(75 MHz, MeOD): δ 155.97, 79.39, 46.75, 43.72, 37.52, 30.86, 25.90,
28.35.
General Procedure for the Synthesis of Compounds 11−22. To a
solution of 8 (100 mg, 0.3 mmol) in CH2Cl2 (5 mL) were added
HOBt (45 mg, 1.1 equiv) and DCC (70 mg, 1.1 equiv). After stirring
at room temperature for 1 h, the white precipitate was removed by
filtration, and a solution of amine (1.1 equiv) in dichloromethane (2
mL) was added to the filtrate. The reaction was continued for 5−12 h
at room temperature, and the solvents were removed in vacuo to yield
a crude residue that was purified by column chromatography.
(E)-N-(2-Amino-ethyl)-3-(3,5-dibromo-4-methoxy-phenyl)-acryla-
mide 11. 1H NMR (MeOD, 300 MHz): δ 7.82 (s, 2H), 7.43−7.39 (d,
J = 15.6 Hz, 1H), 6.61−6.57 (d, J = 15.6 Hz, 1H), 3.90 (s, 3H), 3.41−
3.37 (t, J = 6.3 Hz, 2H), 2.83−2.80 (t, J = 7.3 Hz, 2H). 13C NMR
(MeOD, 75 MHz): δ 169.03, 157.16, 139.08, 136.23, 133.87, 124.81,
120.39, 62.09, 43.87, 42.79. MS (ESI) C12H14N2O2Br2 m/z 379.0
(100%, (M + H+)), 376.9 (100%, (M − H−)).
(E)-N-(2-{2-[2-(2-Amino-ethylamino)-ethylamino]-ethylamino}-
1
ethyl)-3-(3,5-dibromo-4-methoxy-phenyl)-acrylamide 19. H NMR
(MeOD, 400 MHz): δ 7.83 (s, 2H), 7.43−7.39 (d, J = 15.6 Hz, 1H),
6.61−6.58 (d, J = 15.6 Hz, 1H), 3.90 (s, 3H), 3.48−3.44 (t, J = 6.3 Hz,
2H), 2.82−2.61 (m, 14H). 13C NMR (MeOD, 75 MHz): δ 168.82,
157.09, 138.94, 136.23, 133.85, 124.94, 120.37, 62.12, 53.27, 42.53,
35.58, 34.87, 26.89. MS (ESI) C18H29N5O2Br2 m/z 508.1(100%, (M +
H+)), 506.2 (100%, (M − H−−)).
(E)-N-{3-[4-(3-Amino-propoxy)-butoxy]-propyl}-3-(3,5-dibromo-
1
4-methoxy-phenyl)-acrylamide 20. H NMR (MeOD, 400 MHz): δ
7.68 (s, 2H), 7.29−7.25 (d, J = 15.8 Hz, 1H), 6.47−6.43 (d, J = 15.8
Hz, 1H), 3.77 (s, 3H), 3.54−3.46 (m, 8H), 3.38 (t, J = 7.0 Hz, 2H),
2.64−2.60 (t, J = 7.0 Hz, 2H), 1.73−1.70 (m, 2H), 163−1.59 (m, 2H),
1.53−1.51 (m, 4H). 13C NMR (MeOD, 75 MHz): δ 168.59, 157.12,
138.84, 136.29, 133.83, 124.97, 120.39, 72.66, 70.89, 70.28, 62.09,
40.99, 38.99, 34.38, 31.41, 28.43. MS (ESI) C20H30N2O4Br2 m/z 523.0
(100%, (M + H+)), 521.2 (100%, (M − H−−)).
(E)-N-(3-Amino-propyl)-3-(3,5-dibromo-4-methoxy-phenyl)-
1
acrylamide 12. H NMR (MeOD, 300 MHz): δ 7.81 (s, 2H), 7.42−
7.38 (d, J = 15.8 Hz, 1H), 6.59−6.55 (d, J = 15.8 Hz, 1H), 3.90 (s,
3H), 3.40−3.37 (t, J = 7.0 Hz, 2H), 2.73−2.70 (t, J = 7.0 Hz, 2H),
1.75−1.72 (M, J = 7.0 Hz, 2H). 13C NMR (MeOD, 75 MHz): δ
168.77, 157.1,1, 138.96, 136.20, 133.82, 124.77, 120.38, 62.09, 40.54,
38.76, 33.99. MS (ESI) C13H16N2O2Br2 m/z 393.0 (100%, (M + H+)),
391.0 (100%, (M − H−−)).
(E)-N-{3-[Bis(3-amino-propyl)-amino]-propyl}-3-(3,5-dibromo-4-
1
methoxy-phenyl)-acrylamide 21. H NMR (MeOD, 400 MHz): δ
7.82 (s, 2H), 7.42−7.38 (d, J = 15.8 Hz, 1H), 6.60−6.56 (d, J = 15.8
Hz, 1H), 3.90 (s, 3H), 2.73−2.69 (m, 4H), 2.56−2.51 (m, 8H), 1.70−
1.63 (m, 6H). 13C NMR (MeOD, 75 MHz): δ 168.56, 157.11, 138.86,
136.26, 133.82, 124.94, 120.39, 62.10, 53.71, 53.38, 41.84, 39.86,
31.22, 28.49. MS (ESI) C19H30N4O2Br2 m/z 507.1 (100%, (M + H+)),
505.1 (100%, (M − H−−)).
(E)-N-{3-[4-(3-Amino-propyl)-piperazin-1-yl]-propyl}-3-(3,5-dibro-
mo-4-methoxy-phenyl)-acrylamide 22. 1H NMR (MeOD, 400
MHz): δ 7.80 (s, 2H), 7.40−7.36 (d, J = 15.6 Hz, 1H), 6.59−6.55
(d, J = 15.6 Hz, 1H), 3.89 (s, 3H), 2.89−2.87 (m, 2H), 2.55−2.48 (m,
14H), 1.78 (m, 4H). 13C NMR (MeOD, 75 MHz): δ 168.62, 157.14,
138.89, 136.25, 133.83, 124.93, 120.39, 62.10, 57.93, 54.76, 54.68,
41.41, 39.84, 28.24, 28.08.
(E)-N-(4-Amino-butyl)-3-(3,5-dibromo-4-methoxy-phenyl)-acryl-
1
amide 13. H NMR (MeOD, 400 MHz): δ 7.80 (s, 2H), 7.41−7.37
(d, J = 15.6 Hz, 1H), 6.59−6.55 (d, J = 15.6 Hz, 1H), 3.89 (s, 3H),
3.36 (m, 2H), 2.71−2.67 (t, J = 7.0 Hz, 2H), 1.62−1.55 (m, 4H). 13C
NMR (MeOD, 75 MHz): δ 168.51, 156.99, 138.77, 136.17, 133.76,
124.92, 120.33, 62.09, 42.95, 41.27, 31.77, 28.63. MS (ESI)
C14H18N2O2Br2 m/z 407.0 (100%, (M + H+)), 405.0 (100%, (M −
H
−−)).
(E)-N-(5-Amino-pentyl)-3-(3,5-dibromo-4-methoxy-phenyl)-acryl-
1
amide 14. H NMR (MeOD, 400 MHz): δ 7.81 (s, 2H), 7.41−7.37
(d, J = 15.6 Hz, 1H), 6.60−6.56 (d, J = 15.6 Hz, 1H), 3.90 (s, 3H),
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4.3. Bacterial Strains. Four bacterial strains were used in this
study: S. aureus DSM799, P. aeruginosa PAO1 (ATCC 15692), E.
aerogenes EA289, a Kan derivative of the MDR clinical isolate EA27,27
and K. pneumoniae KPC2 ST258 (Poland). P. aeruginosa clinical
isolates (PA-LAV) were obtained from E. Garnotel (Laveran
Hospital). Strains were maintained at −80 °C in 15% (v/v) glycerol
for cryoprotection. Bacteria were routinely grown in Mueller−Hinton
(MH) broth at 37 °C.
4.4. Antibiotics. Chloramphenicol and cefepime were purchased
from Sigma (St. Quentin Fallavier, France). Doxycycline was
purchased from TCI Europe. All antibiotics were dissolved in dimethyl
sulfoxide (DMSO), and DMSO concentrations up to 5% v/v did not
detrimentally affect bacterial growth.
to 100 μL of the cell suspension to obtain a final concentration varying
from 0.98 to 500 μM. Fifty microliters of nitrocefin was then added to
obtain a final concentration of 50 μg/mL. Nitrocefin hydrolysis was
monitored spectrophotometrically by measuring the increase in
absorbance at 490 nm. Assays were performed in 96-well plates
using a M200 Pro Tecan spectrophotometer.
4.10. Cytotoxicity toward Chinese Hamster Ovary Cells and
Normal Human Fibroblasts. CHO-K1 cells (ATCC-LGC Stand-
ards Sarl, Molsheim, France) and human fibroblasts (Clinisciences,
Paris, France) were maintained in McCoy’s 5A and DMEM media,
respectively, supplemented with 10% bovine calf serum, 2 mM
glutamine, and 100 (U/mL)/10 μg/mL penicillin/streptomycin. They
were incubated at 37 °C in a humidified atmosphere containing 5%
CO2. The cell lines were seeded in 96-well plates and incubated
overnight. Various concentrations of compounds were incorporated in
triplicate cultures, and the cells were incubated at 37 °C for 24 h. At
the end of the incubation period, cells were submitted to three
successive washes in phosphate buffer saline (PBS) and incubated in
PBS containing 10% WST-1 for an additional 30 min. Cell viability
was evaluated by measuring WST-1 absorbance at 450 nm in a
microplate spectrophotometer. The results are expressed as the
percentage of cell viability compared to that of the control (culture
medium only), which corresponded to 100% cell viability. Dose−
response curves were calculated by nonlinear regression analysis using
TableCurve V2 software. The inhibitory concentration 50% (IC50) was
defined as the concentration of saponin that induced a 50% decrease in
cell viability.
4.5. Antibiotic Susceptibility Testing. The susceptibility of
bacterial strains to antibiotics and compounds was determined in
microplates using the standard broth dilution method in accordance
with the recommendations of the Commite
́
de l′AntibioGramme de la
Societ Franca
́
e
́
̧
ise de Microbiologie (CA-SFM).28 Briefly, the MICs
were determined with an inoculum of 105 CFU in 200 μL of MH
broth containing 2-fold serial dilutions of each drug. The MIC was
defined as the lowest concentration of drug that completely inhibited
visible growth after incubation for 18 h at 37 °C. To determine all
MICs, the measurements were independently repeated at least three
times.
4.6. Determination of the MICs of Antibiotics in the
Presence of Synergizing Compounds. Briefly, restoring enhancer
concentrations was determined with an inoculum of 105 CFU in 200
μL of MH broth containing 2-fold serial dilutions of each derivative in
the presence of the desired antibiotic (chloramphenicol, doxycycline,
or cefepime; 2 μg/mL). The lowest concentration of ianthelliformis-
amine derivative that completely inhibited visible growth after
incubation for 18 h at 37 °C was determined. These measurements
were independently repeated at least three times.
4.7. Measurement of ATP Release. Solutions of compound 21
in doubly distilled water were prepared at different concentrations. A
suspension of growing bacteria in MH broth was prepared and
incubated at 37 °C. Nine hundred microliters of this suspension was
added to 100 μL of compound 21 to obtain the indicated final
concentration of 21 in the presence or absence of doxycycline. After 5
min, an aliquot of 100 μL of this mixture was collected and vortexed
for 1 s. Fifty microliters of luciferin−luciferase reagent (Yelen, France)
was immediately added, and the luminescent signal was quantified
using a Lucy luminometer (Yelen, France) for 5 s. The ATP
concentration was quantified by the addition of an internal standard.
Maximum ATP release was obtained with a 0.02% solution of
tetramethylammonium bromide (TMAB).
4.8. Membrane Depolarization Assays. Bacteria were grown in
MH broth for 24 h at 37 °C and centrifuged at 10 000 rpm at 20 °C.
The supernatant was discarded, and the bacteria were washed twice
with buffered sucrose solution (250 mM) and magnesium sulfate
solution (5 mM). The fluorescent dye, 3,3-diethylthiodicarbodyanine
iodide, was added to a final concentration of 3 μM, and it was allowed
to penetrate into bacterial membranes during a 1 h incubation at 37
°C. Compound 21 was then added at different concentrations.
Fluorescence measurements were performed using a Jobin Yvon
Fluoromax 3 spectrofluorometer with slit widths of 5/5 nm. The
relative corrected fluorescence (RCF) was recorded at time intervals of
0, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 min. The maximum RCF was
considered to be that recorded with a pure solution of the fluorescent
dye in buffer (3 μM).
4.9. Nitrocefin Hydrolysis Assay. Outer membrane permeabili-
zation was measured using nitrocefin as a chromogenic substrate of
periplasmic β-lactamase. Ten milliliters of MH broth was inoculated
with 0.1 mL of an overnight culture of PA01 and grown at 37 °C until
the OD600 reached 0.5. The remaining steps were performed at room
temperature. Cells were recovered by centrifugation (4000 rpm for 20
min) and washed once in 20 mM potassium phosphate buffer (pH
7.2) containing MgCl2 (1 mM). After a second centrifugation, the
pellet was resuspended and adjusted to a OD600 of 0.5. Then, 50 μL
of either polymyxin B (positive control) or compound 21 was added
ASSOCIATED CONTENT
* Supporting Information
■
S
Analytical data of all compounds and biological test systems.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: (+33) 491835551. E-mail: bruneljm@yahoo.fr.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the YELEN Company (Marseille) for
assisting with the measurements of ATP release and membrane
depolarization.
ABBREVIATIONS USED
PDR, pandrug resistance; TMAB, tetramethylammonium
bromide
■
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