N,N′-disubstituted cinnamamide derivatives potentiate ciprofloxacin activity against overexpressing NorA efflux pump Staphylococcus aureus 1199B strains

Article abstract of DOI:10.1016/j.ejmech.2018.03.028

A multi-step procedure has been described which afforded satisfactory yields of N,N′-disubstituted cinnamamides derived from N-Boc-protected amino acids (Boc-Gly, Boc-Val, Boc-Phe). The key step of this synthesis was a regioselective RedAl reduction of an

Full text of DOI:10.1016/j.ejmech.2018.03.028

European Journal of Medicinal Chemistry 150 (2018) 900e907
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
N,N0-disubstituted cinnamamide derivatives potentiate ciprofloxacin
activity against overexpressing NorA efflux pump Staphylococcus
aureus 1199B strains
,
*
b,
Sylvie Radix a , Anne Doleans Jordheim c, Luc Rocheblave a, Serge N'Digo a,
ꢀ
Anne-Laure Prignon a, Carine Commun b, Serge Michalet b,
b
a
a, **
ꢁ
ꢀ
Marie-Genevieve Dijoux-Franca , Angelique Mularoni , Nadia Walchshofer
a
ꢀ
ꢀ
Univ Lyon, Universite Claude Bernard Lyon 1, ISPB-Faculte de Pharmacie, EA 4446, B2MC, F-69373, Lyon Cedex 08, France
Univ Lyon, Universite Claude Bernard Lyon 1, VetAgro Sup, UMR CNRS 5557, Ecologie Microbienne, F-69622, Villeurbanne, France
Laboratoire de Bacteriologie, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon (HCL), F-69, Bron, France
b
c
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
A multi-step procedure has been described which afforded satisfactory yields of N,N0-disubstituted
cinnamamides derived from N-Boc-protected amino acids (Boc-Gly, Boc-Val, Boc-Phe). The key step of
this synthesis was a regioselective RedAl reduction of an amide function in presence of a carbamate
group. Next, these cinnamamides were evaluated in co-admnistration with ciprofloxacin as efflux pump
inhibitors against two S. aureus strains, NorA overexpressing SA1199B and wild type SA1199. In parallel,
their intrinsic toxicity was appreciated on human lung fibroblast MRC5 cells. Therefore, the cinnamamide
combining both carbamate and indol-3-yl groups, was found to be the most active and one of the less
toxic EPI and constituted a promising hit.
Article history:
Received 5 December 2017
Received in revised form
2 March 2018
Accepted 8 March 2018
Available online 10 March 2018
Keywords:
Cinnamamides
Regioselective RedAl-reduction
Staphylococcus aureus
NorA efflux pump inhibitors
Antibiotic resistance
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
makes it difficult to design EPIs as antibiotic adjuvants in resistant
S. aureus infections with a receptor approach. However, various
It is now well recognized that the increasing prevalence of drug-
resistant bacteria represents a major risk for public health [1].
Different factors contribute to this resistance [2]. Among them,
numerous efflux systems, which reduce the effective intracellular
antibiotic concentration, have been described both in Gram-
positive and Gram-negative bacteria. The Staphylococcus aureus
efflux pump NorA actively transports various antibiotics including
fluoroquinolones (i.e ciprofloxacin and norfloxacin) and contributes
to the multidrug resistance (MDR) phenotype in these bacteria [3].
Co-administration of an efflux pump inhibitor (EPI) with antibiotics
is therefore a promising strategy to fight against clinical resistant
strains [4,5].
scaffolds have been described [6], including flavones [7], benzo-
thiazines [8], chalcones [9,10], indoles [11,12], or quinolones [13]
derivatives. Regarding the efflux pump inhibitory activity of chal-
cones and as part of our project to identify EPIs such as mammalian
P-glycoprotein inhibitors [14] or bacterial NorA inhibitors [15], we
decide to explore the potential of N,N0-disubstituted cinnamamides
3a-f and 4a-f. We report here the synthesis of these compounds,
and their evaluation against two S. aureus strains, NorA over-
expressing SA1199B and the wild type SA1199. In parallel, the
in vitro cytotoxicity of these cinnamamides was appreciated on
human lung fibroblast MRC5 cells.
The lack of exhaustive crystallographic data on NorA pump
2. Results and discussion
2.1. Chemistry
* Corresponding author.
** Corresponding author.
E-mail addresses: sylvie.radix@univ-lyon1.fr (S. Radix), nadia.walchshofer@
univ-lyon1.fr (N. Walchshofer).
A straightforward synthetic pathway has been constructed for
the synthesis of N,N0-disubstituted cinnamamides 3a-f and 4a-f
https://doi.org/10.1016/j.ejmech.2018.03.028
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

S. Radix et al. / European Journal of Medicinal Chemistry 150 (2018) 900e907
901
derived from N-Boc-protected amino acids such as glycine (Boc-
Gly), valine (Boc-Val) or phenylalanine (Boc-Phe) (Scheme 1).
Actually, these widely used protected amino acids were inexpen-
sive and readily purchased from commercial sources unlike many
other carbamate-protected amino acids, which should be synthe-
sized beforehand [16a]. The synthesis started with a coupling re-
action between a Boc amino acid and phenethylamine (R1 ¼ Ph) or
tryptamine (R1 ¼ indol-3-yl) under a standard peptide synthesis
procedure
using
2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethylaminium tetrafluoroborate (TBTU) and triethylamine
(TEA) in dimethylformamide (DMF) to obtain amides 1a-f in good
to excellent yields after aqueous workup and without further pu-
rification (except for Boc-Gly derivatives 1a and 1d which were
purified by silica gel flash chromatography).
These amides 1a-f were hence selectively reduced with sodium
bis(2-methoxyethoxy)aluminium hydride (RedAl, 65 wt% in
toluene) in anhydrous tetrahydrofuran (THF) [16b] either under
microwave irradiation (Gly- and Phe-compound series) or at room
temperature (Val-compound series). In all cases, low to average
yields of isolated mono-Boc protected diamines 2a-f were observed
after aqueous workup and column chromatography. Nevertheless,
it appeared that this selective RedAl-reduction method afforded
mono-Boc diamines 2a-f in satisfactory yields while poorer results
were previously observed when these compounds were synthe-
sized by reductive amination [17] or nucleophilic substitution [18]
(data not shown). In the case of Val derivative 1b, it is noteworthy
that increasing reaction temperature and/or reaction time dis-
favored the production of 2b and promoted the formation of
byproducts as hydantoin 5 [16] (66% at 40 ꢀC overnight) or cyclic
urea 6 [19] (17% at 66 ꢀC for 1h30) (Scheme 2).
Scheme 2. RedAl-reduction of amide 1b.
addition of acetyl chloride to methanolic solution in an inert sol-
vent [20]. In these mild conditions, amine hydrochlorides 4a-f were
isolated with satisfactory yields.
Next cinnamamides 3a-f and 4a-f were evaluated in co-
admnistration with ciprofloxacin (CFX) as efflux pump inhibitors
against two S. aureus strains, NorA overexpressing SA1199B and
wild type SA1199. In parallel, their intrinsic toxicity was appraised
on human lung fibroblast MRC5 cells.
The synthesis was completed by a coupling reaction between
(E)-cinnamic acid and mono-Boc diamines 2a-f in presence of
benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexa-
fluorophosphate (BOP) and TEA in DMF at room temperature to
afford N-Boc protected cinnamamides 3a-e with good to excellent
yields as pure single (E)-isomers but as a mixture of amide rotamers
cis/trans. Only 3f, derived from Boc-Phe and tryptamine, was ob-
tained with a slightly lower yield than the other examples. Finally,
we decided to improve the hydrosolubility of the N,N0-disubsti-
tuted cinnamamides 3a-f by acidic N-Boc deprotection of the pri-
mary amine with hydrogen chloride generated in situ by the
2.2. Biological activity
Results of potentiation study of cinnamamides 3a-f and 4a-f
with ciprofloxacin (CFX) against two S. aureus strains, SA1199B
(NorA overproducing phenotype) and SA1199 (wild type) are
shown in Table 1. Along with these synthetic molecules, reserpine
Scheme 1. Synthesis of cinnamamides 3a-f and 4a-f. Reagents and conditions: (a) TBTU, TEA, DMF, RT overnight; (b) RedAl (65 wt% toluene), THF, from 0 ꢀC to 70 ꢀC, MW, 10 mn for
2a,2c,2d,2f or from 0 ꢀC to RT, overnight for 2b,2e; (c) BOP, TEA, DMF, RT, overnight; (d) AcCl, MeOH, EtOAc, from 0 ꢀC to RT.

902
S. Radix et al. / European Journal of Medicinal Chemistry 150 (2018) 900e907
Table 1
Effect of compounds 3a-f and 4a-f on MIC reduction of ciprofloxacin in S. aureus (SA1199 and SA1199B strains) and intrinsic toxicities on MRC5 cells.
Compd
R1
R2
Concentration
m
Ma
SA1199 MIC reductionc
SA1199B MIC reductionc
Intrinsic toxicity GI50 mMd
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
Ph
Ph
Ph
indol-3-yl
indol-3-yl
indol-3-yl
Ph
Ph
Ph
indol-3-yl
indol-3-yl
indol-3-yl
e
H
25.3
22.9
20.6
23.0
21.0
19.1
90.9
80.6
37.1b
81.3
73.0
8.5b
32.9
1
1e2
1
2
2
1e2
4
4
4e8
1
2
2
2
54.70
40.70
17.30
35.30
43.70
70.00 10
73.30 10.90
52.40
6.50 0.71
20.70 4.50
25.30 1.53
6.33 0.29
4
4
3
3
7
CH(CH3)2
CH2Ph
H
CH(CH3)2
CH2Ph
H
CH(CH3)2
CH2Ph
H
CH(CH3)2
CH2Ph
e
2
1e2
1e2
1e2
1e2
2e4
2
2e4
2
2
8
4
2
4
reserpine
14.70
5
At least three independent experiments were performed for each measurement.
a
Final concentrations used for compounds 3a-f and 4a, 4b, 4d-e (10
m
g/mL and 30
g/mL respectively). Reserpine was tested at a final concentration of 20
g/mL and 4 g/mL for SA1199 and SA1199B strains respectively) at the given concentration (range of values
m
g/mL respectively) in the MIC reduction evaluation on both strains.
b
c
Compounds 4c and 4f were tested at 1/4 MIC (15.6
m
g/mL and 3.9
m
mg/mL.
Fold reduction of ciprofloxacin (CFX) MIC (CFX MICs are 0.25
m
m
for the three experiments).
d
GI50 (mM) in a human lung fibroblast MRC5 cell line, all values are expressed as the mean SE.
was used as a known EPI for comparative studies [21]. The de-
rivatives 3a-f and 4a-f were safely tested as efflux pump inhibitors
at a concentration devoid of antibacterial activity (data not shown).
Actually, most compounds 3 and 4 did not have bactericidal ac-
tivities at a concentration chosen to avoid precipitation of these
3. Conclusions
We have described herein the synthesis and the biological
evaluation as NorA efflux pump inhibitors of 12 new amino acid
derived cinnamamides. These compounds were prepared accord-
ing to an efficient procedure developed from N-Boc protected
amino acids (Boc-Gly, Boc-Val and Boc-Phe). The key step was a
regioselective hydride reduction of an amide function with RedAl in
presence of a carbamate group.
Direct comparison of the results obtained by in vitro ciproflox-
acin potentiation study with cinnamamides 3a-f and 4a-f sug-
gested the indol-3-yl group afforded the most potent NorA
inhibitors. Phenethylamine-derived compounds (R1 ¼ Ph) only
slightly reduced ciprofloxacin MIC for both SA1199 and SA1199B
strains. Moreover, it appeared that the presence of a N-Boc group
reduced the cytotoxicity of these derivatives. Therefore, cinnama-
mide 3f, combining both carbamate and indol-3-yl groups, was
found to be the most active and one of the less toxic EPI in this work
and constituted a promising hit to further studies.
DMSO soluble compounds in water based culture media (10
or 30 g/mL respectively). However, as cinnamamides 4c and 4f
have showed an intrinsic antibacterial activity, both were tested at
mg/mL
m
one-quarter of their MIC concentration (15.6
respectively) [22].
mg/mL and 3.9 mg/mL
Co-administration of phenethylamine-derived compounds 3a-c
and 4a-c (R1 ¼ Ph) did not potentiate CFX activity against both
strains. On the other hand, tryptamine-derived cinnamamides
(R1 ¼ indol-3-yl) 3d-f and 4e exerted at least fourfold reduction of
CFX MIC against NorA overexpressed strain (SA1199B) but com-
pounds 4d and 4f. It is noteworthy that, unlike amine hydrochlo-
rides 4d-f, the combination of corresponding N-Boc protected
derivatives 3d-f with CFX led to significant differences in fold
reduction between both S. aureus strains. NorA efflux pump
inhibitory activity of these latter cinnamamides 3d-f could explain
these results [23].
Finally, compounds 4c and 4f have showed intrinsic antibacte-
rial activity with a MIC at 62.5 mg/mL and 15.7 mg/mL respectively.
Moreover, the intrinsic toxicity of all synthetic compounds was
evaluated on human lung fibroblast MRC5 cells. As shown in Table 1
and except for compounds 3a/4a and 3b/4b, the presence of a N-
Boc group on the primary amine of the side chain led to increased
These bactericidal effects must be explored to evaluate the mech-
anism of action of both compounds and to identify potential targets
for new bactericidal drugs.
half-maximal inhibitory growth concentration (GI50
compared to the corresponding hydrochloride salt.
) when
4. Experimental section
Compound 3f was found to be the most active EPI in this study
showing a fourfold to eightfold reduction in the MIC of ciproflox-
4.1. Chemistry
acin at 10
approximately twice as potent as reserpine tested at 20
32.9 M). Furthermore, the required concentration of reserpine for
M), whereas
this N-Boc cinnamamide 3f did not show any intrinsic toxicity at
the concentration used in the EPI test.
m
g/mL conc. (i.e. 19.1
m
M) against S. aureus 1199B. 3f was
4.1.1. General
mg/mL (i.e.
All air- and moisture-sensitive reactions were carried out under
argon atmosphere. All commercially available chemicals were used
as received. Anhydrous tetrahydrofurane was purchased from
ACROS and used as received. Microwave irradiation-assisted re-
actions were performed in a monomode Biotage® Initiator micro-
wave synthesizer. Melting points were determined with an
m
the observed EPI effect was cytotoxic (GI50 ¼ 14.7
m

S. Radix et al. / European Journal of Medicinal Chemistry 150 (2018) 900e907
903
Electrothermal 9300 capillary apparatus. 1H spectra were recorded
with Bruker DRX300 or DRX400 Fourier transform spectrometers,
2.62e2.50 (m, 2H, CH2), 1.31 (s, 9H, (CH3)3C); 13C NMR (CDCl3,
100 MHz): 171.54 (amide CO), 155.79 (Boc CO), 139.02 (Cquat
arom), 137.26 (Cquat arom), 129.78 (2*CHarom), 129.08
(2*CHarom), 129.02 (4*CHarom), 127.32 (CHarom), 126.92
(CHarom), 80.44 ((CH3)3C), 56.43 (NHCH), 41.02 (NHCH2), 39.31
(CH2), 35.97 (CH2), 28.70 ((CH3)3C); HRMS (ESI) m/z calcd for
d
using an internal deuterium lock, operating at 300 or 400 MHz. 13
C
NMR spectra were recorded with Bruker DRX400 Fourier transform
spectrometer, using an internal deuterium lock, operating at
100 MHz. Chemical shifts are reported in part per million (ppm)
relative to residual protons of CDCl3 or DMSO-d6 (CDCl3,
C22H28N2O3þH, 369.2173; found: 369.2164.
d
¼ 7.26 ppm for 1H NMR and
d
¼ 77.16 ppm for 13C NMR; DMSO-d6,
d
¼ 2.54 ppm for 1H NMR and
d
¼ 39.52 ppm for 13C NMR) and
4.1.2.4. Tert-butyl (2-((2-(1H-indol-3-yl)ethyl)amino)-2-oxoethyl)
coupling constants J are reported in hertz (Hz). Carbon multiplicity
was determined by DEPT 135ꢀ experiments. The following abbre-
viations are used: s: singlet; br s: broad signal; d: doublet; t: triplet;
m: multiplet. Chromatographic separations were performed on
silica gel columns (Kieselgel 300e400 mesh). All reactions were
monitored by TLC on GF254 plates and spots were detected under a
UV lamp (254 nm) or by spraying plates with 10% v/v aqueous
H2SO4 or with cerium-ammonium-molybdate (CAM) in diluted
sulfuric acid solution, followed by drying with heat gun. High-
resolution mass spectra were performed on a ThermoFinnigan
MAT 95 XL apparatus operating at 70eV.
carbamate 1d. Silica gel flash chromatography (cyclohexane/EtOAc,
1:3, Rf 0.28). Pale yellow oil (1.25 g, 69%). 1H NMR (CDCl3,
300 MHz):
d
8.74 (bs,1H, NH indole), 7.56 (d,1H, J ¼ 7.6 Hz, CH), 7.34
(d, 1H, J ¼ 7.6 Hz, CH), 7.20 (t, 1H, J ¼ 7.6 Hz, CH), 7.10 (t, 1H,
J ¼ 7.6 Hz, CH), 6.95 (s, 1H, CH), 6.44 (m, 1H, NH), 5.40 (m, 1H, NH),
3.74e3.69 (m, 2H, CH2), 3.55e3.51 (m, 2H, CH2), 2.93 (t, 2H,
J ¼ 6.6 Hz, CH2), 1.43 (s, 9H, (CH3)3C); 13C NMR (CDCl3, 100 MHz):
d
169.66 (amide CO), 156.22 (Boc CO), 136.42 (Cquat arom), 127.25
(Cquat arom), 122.40 (CHarom), 121.98 (CHarom), 119.26 (CHarom),
118.51 (CHarom), 112.34 (Cquat arom), 111.45 (CHarom), 80.17
((CH3)3C), 44.19 (CH2), 39.77 (CH2), 28.31 ((CH3)3C), 25.14 (CH2).
4.1.2. General procedure for syntheses of amides (1)
4.1.2.5. Tert-butyl (S)-(1-((2-(1H-indol-3-yl)ethyl)amino)-3-methyl-
1-oxobutan-2-yl)carbamate 1e [27]. White solid (3.09 g, 86%). Rf
0.49 (cyclohexane/EtOAc, 1:1); Mp 130.8 ꢀC; 1H NMR (CDCl3,
TBTU (1.1 eq.) and triethylamine (1.5 eq.) were successively
added to a solution of N-Boc amino acid (1 eq.) in DMF (30 mL,
0.2 Me0.3 M of amino acid). The reaction mixture was stirred for
30 min at room temperature. Phenethylamine or tryptamine (1.1
eq.) was then added and stirring was pursued overnight at room
temperature. After dilution with EtOAc (60e100 mL), the organic
phase was successively washed with a saturated aqueous NaHCO3,
a 10% aqueous citric acid solution and finally brine. Organic extracts
were dried over Na2SO4, filtered and concentrated under vacuum.
The title compound 1a-f was obtained as a white solid pure enough
to be used without further purification. For 1a and 1d derivatives,
the residue was purified by silica gel flash chromatography.
400 MHz):
d
8.47 (bs, 1H, NH indole), 7.50 (d, 1H, J ¼ 7.55 Hz, CH),
7.27 (d, 1H, J ¼ 7.55 Hz, CH), 7.11 (t, 1H, J ¼ 7.55 Hz, CH), 7.03 (t, 1H,
J ¼ 7.55 Hz, CH), 6.90 (d, 1H, J ¼ 2 Hz, CH), 6.10 (m, 1H, NH), 5.04 (d,
1H, J ¼ 8.6 Hz, BocNH), 3.79 (dd, 1H, J ¼ 6.6, 8.6 Hz, BocNHCH),
3.52e3.47 (m, 2H, NHCH2), 2.86 (t, 2H, J ¼ 6.8 Hz, CH2Ind), 1.98 (m,
1H, (CH3)2)CH), 1.33 (s, 9H, (CH3)3C), 0.82 (d, 3H, J ¼ 6.7 Hz, CH3),
0.77 (d, 3H, J ¼ 6.8 Hz, CH3); 13C NMR (CDCl3, 100 MHz):
d 171.62
(amide CO), 155.95 (Boc CO), 136.43 (Cquat arom), 127.23 (Cquat
arom), 122.17 (CHarom), 122.13 (CHarom), 119.40 (CHarom), 118.62
(CHarom), 112.62 (Cquat arom), 111.33 (CHarom), 79.82 ((CH3)3C),
60.04 (CH), 39.66 (CH2), 31.00 (CH), 28.31 ((CH3)3C), 25.37 (CH2),
19.29 (CH3), 17.73 (CH3); HRMS (ESI) m/z calcd for C20H29N3O3þH,
360.2282; found: 360.2272.
4.1.2.1. Tert-butyl (2-oxo-2-(phenethylamino)ethyl)carbamate 1a
[24]. Silica gel flash chromatography (cyclohexane/EtOAc, 1:3, Rf
0.38). Pale yellow oil (1.15 g, 72%). 1H NMR (CDCl3, 300 MHz):
d
7.49e7.00 (m, 5H, Ph), 6.36 (m, 1H, NH), 5.29 (m, 1H, NHBoc), 3.74
4.1.2.6. Tert-butyl (S)-(1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)carbamate 1f. White solid (3.05 g, 75%). Rf 0.65
(cyclohexane/EtOAc, 6:4); Mp 137.5 ꢀC; 1H NMR (CDCl3, 300 MHz):
(d, 2H, J ¼ 6 Hz, BocNHCH2), 3.52 (q, 2H, J ¼ 7 Hz, NHCH2), 2.80 (t,
2H, J ¼ 7 Hz, CH2Ph), 1.43 (s, 9H, (CH3)3C); 13C NMR (CDCl3,
100 MHz):
d 169.45 (amide CO), 156.03 (Boc CO), 138.73 (Cquat
d
8.17 (bs, 1H, NH indole), 7.40 (d, 1H, J ¼ 7.6 Hz, CH), 7.27 (d, 1H,
arom), 128.74 (2*CHarom), 128.64 (2*CHarom), 126.53 (CHarom),
80.14 ((CH3)3C), 44.33 (CH2), 40.56 (CH2), 35.63 (CH2), 28.31
((CH3)3C).
J ¼ 8.1 Hz, CH), 7.19e6.99 (m, 7H), 6.73 (s, 1H, CH), 5.76 (m, 1H, NH),
5.01 (m, 1H, BocNH), 4.17 (m, 1H, BocNHCH), 3.48e3.39 (m, 2H,
CH2), 2.95e2.85 (m, 2H, CH2), 2.81e2.65 (m, 2H, CH2), 1.29 (s, 9H,
(CH3)3C); 13C NMR (CDCl3, 100 MHz):
d 171.00 (amide CO), 155.33
4.1.2.2. Tert-butyl (S)-(3-methyl-1-oxo-1-(phenethylamino)butan-2-
yl)carbamate 1b [25]. White solid (2.88 g, 98%). Rf 0.80 (cyclo-
hexane/EtOAc/TEA, 1:1:10%); Mp 115.3 ꢀC; 1H NMR (CDCl3,
(Boc CO), 136.83 (Cquat arom), 136.39 (Cquat arom), 129.37
(2*CHarom), 128.63 (2*CHarom), 127.18 (Cquat arom), 126.91
(CHarom), 122.18 (CHarom), 122.02 (CHarom), 119.45 (CHarom),
118.59 (CHarom), 112.58 (Cquat arom), 111.29 (CHarom), 80.07
((CH3)3C), 56.09 (CH), 39.62 (CH2), 38.95 (CH2), 28.36 ((CH3)3C),
25.10 (CH2); HRMS (ESI) m/z calcd for C24H29N3O3þH, 408.2282;
found: 408.2271.
400 MHz):
d 7.24e7.10 (m, 5H, Ph), 6.21 (m, 1H, NH), 5.08 (d, 1H,
J ¼ 9.0 Hz, NHBoc), 3.78 (dd, 1H, J ¼ 6.4, 9.0 Hz, NHCH), 3.54e3.33
(m, 2H, NHCH2), 2.73 (t, 2H, J ¼ 7.3 Hz, CH2Ph), 2.00 (m, 1H,
(CH3)2CH), 1.35 (s, 9H, (CH3)3C), 0.83 (d, 3H, J ¼ 6.8 Hz, CH3), 0.80 (d,
3H, J ¼ 6.8 Hz, CH3); 13C NMR (CDCl3, 100 MHz):
d 172.07 (amide
CO), 156.32 (Boc CO), 139.09 (Cquat arom), 129.12 (2*CHarom),
129.03 (2*CHarom), 126.93 (CHarom), 80.19 ((CH3)3C), 60.48
(NHCH), 41.02 (NHCH2), 36.16 (CH2), 31.27 ((CH3)2CH), 28.73
((CH3)3C), 19.67 (CH3), 18.26 (CH3); HRMS (ESI) m/z calcd for
4.1.3. General procedure for the syntheses of diamines (2)
4.1.3.1. Microwave-assisted syntheses of diamines derived from Gly
and Phe (2a,2c,2d,2f). RedAl (65 wt% in toluene, 1.6 mL, 5.1 mmol)
was added dropwise to a solution of amide 1a,c,d,f (1 mmol) in
anhydrous THF (4 mL) at 0 ꢀC. The reaction mixture was heated
under microwave irradiation at 70 ꢀC for 10 min. A saturated solu-
tion of Rochelle's salt (4 mL) was added and the aqueous phase was
extracted with EtOAc. The combined organic phases were dried
over Na2SO4, filtered and concentrated under vacuum. The residue
was purified by silica gel flash chromatography.
C
18H28N2O3þNa, 343.1992; found: 343.1982.
4.1.2.3. Tert-butyl (S)-(1-oxo-1-(phenethylamino)-3-phenylpropan-
2-yl)carbamate 1c [26]. White solid (2.98 g, 81%). Rf 0.28 (cyclo-
hexane/EtOAc, 8:2); Mp 130 ꢀC; 1H NMR (CDCl3, 400 MHz):
d
7.22e6.95 (m, 10H, 2*Ph), 6.96 (m, 1H, NH), 5.09 (m, 1H, NHBoc),
4.21 (m, 1H, NHCH), 3.43e3.25 (m, 2H, CH2), 2.93 (m, 2H, CH2),

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S. Radix et al. / European Journal of Medicinal Chemistry 150 (2018) 900e907
4.1.3.2. Room-temperature syntheses of diamines derived from Val
(2b,2e). RedAl (65 wt% in toluene, 1.6 mL, 5.1 mmol) was added
dropwise to a solution of amide 1b,e (1 mmol) in anhydrous THF
(4 mL) at 0 ꢀC. The reaction mixture was stirred at room tempera-
ture overnight. A saturated solution of Rochelle's salt (4 mL) was
added and the aqueous phase was extracted with EtOAc. The
combined organic phases were dried over Na2SO4, filtered and
concentrated under vacuum. The residue was purified by silica gel
flash chromatography.
(d, 1H, J ¼ 7.9 Hz, CH), 7.10 (s, 1H, CH), 7.04 (t, 1H, J ¼ 7.9 Hz, CH),
6.94 (t, 1H, J ¼ 7.9 Hz, CH), 6.49 (d, 1H, J ¼ 9.2 Hz, NHBoc), 3.30 (m,
1H, BocNHCH), 2.83 (m, 4H, CH2NHCH2), 2.50 (m, 2H, CH2Ind), 1.65
(m, 1H, (CH3)2CH), 1.50 (bs, 1H, NH), 1.37 (s, 9H, (CH3)3C), 0.82e0.77
(m, 6H, (CH3)2CH); 13C NMR (CDCl3, 100 MHz):
d 156.29 (Boc CO),
136.34 (Cquat arom), 127.38 (Cquat arom), 121.97 (2*CHarom),
119.24 (CHarom), 118.80 (CHarom), 113.65 (Cquat arom), 111.13
(CHarom), 79.11 ((CH3)3C), 55.21 (CH), 51.17 (CH2), 49.77 (CH2),
45.87 (CH2), 30.53 ((CH3)2CH), 28.36 ((CH3)3C), 25.52 (CH2), 19.24
(CH3), 18.06 (CH3); HRMS (ESI) m/z calcd for C20H31N3O2þH,
346.2489; found: 346.2477.
4.1.3.3. Tert-butyl (2-(phenethylamino)ethyl)carbamate 2a [17,18].
Cyclohexane/EtOAc/TEA, 1:1:10%, Rf 0.56. Colorless oil (124 mg,
47%). 1H NMR (DMSO-d6, 400 MHz):
d
7.30e7.15 (m, 5H, Ph), 6.70
4.1.3.8. Tert-butyl (S)-(1-((2-(1H-indol-3-yl)ethyl)amino)-3-
phenylpropan-2-yl)carbamate 2f. Cyclohexane/EtOAc/TEA, 1:1:10%,
Rf 0.17. Yellow sticky oil (153 mg, 39%). 1H NMR (DMSO-d6,
(m, 1H, NHBoc), 2.98 (q, 2H, J ¼ 6.2 Hz, BocNHCH2), 2.74e2.63 (m,
4H, CH2NHCH2), 2.55 (t, 2H, J ¼ 6.4 Hz, CH2Ph), 1.69 (bs, 1H, NH),
1.37 (s, 9H, (CH3)3C); 13C NMR (CDCl3, 100 MHz):
d
156.09 (Boc CO),
300 MHz):
d
10.81 (s, 1H, NH indole), 7.51 (d, 1H, J ¼ 7.6 Hz, CH),
139.70 (Cquat arom), 128.67 (2*CHarom), 128.48 (2*CHarom),
126.20 (CHarom), 79.16 ((CH3)3C), 50.57 (CH2), 48.85 (CH2), 40.08
(CH2), 36.13 (CH2), 28.37 ((CH3)3C); HRMS (ESI) m/z calcd for
7.35e6.94 (m, 9H), 6.67 (d, 1H, J ¼ 8.2 Hz, NHBoc), 3.69 (m, 1H,
BocNHCH), 2.80e2.55 (m, 8H), 1.33 (s, 9H, (CH3)3C); 13C NMR
(CDCl3, 100 MHz):
d 156.62 (Boc CO), 138.00 (Cquat arom), 136.35
C
15H24N2O2þH, 265.1911; found: 265.1907.
(Cquat arom), 129.28 (2*CHarom), 128.33 (2*CHarom), 127.40
(Cquat arom), 126.26 (CHarom), 122.02 (CHarom), 121.89 (CHarom),
119.27 (CHarom), 118.85 (CHarom), 113.85 (Cquat arom), 111.13
(CHarom), 79.10 ((CH3)3C), 51.59 (CH2), 49.83 (CH2), 45.94 (CH2),
38.99 (CH2), 28.35 (3*CH3), 28.22 (CH), 25.66 (CH2); HRMS (ESI) m/z
calcd for C24H31N3O2þH, 394.2489; found: 394.2481.
4.1.3.4. Tert-butyl (S)-(3-methyl-1-(phenethylamino)butan-2-yl)
carbamate 2b. Cyclohexane/EtOAc/TEA, 1:1:10%, Rf 0.62. Colorless
oil (133 mg, 43%). 1H NMR (DMSO-d6, 300 MHz):
7.31e7.14 (m,
d
5H, Ph), 6.48 (d, 1H, J ¼ 9.3 Hz, NHBoc), 3.33 (m, 1H, BocNHCH),
2.77e2.63 (m, 4H, CH2NHCH2), 2.53 (m, 2H, CH2Ph), 1.66 (m, 1H,
(CH3)2CH), 1.48 (bs, 1H, NH), 1.37 (s, 9H, (CH3)3C), 0.80 (d, 3H,
J ¼ 7.0 Hz, CH3), 0.78 (d, 3H, J ¼ 7.0 Hz, CH3); 13C NMR (DMSO-d6,
4.1.4. General procedure for the syntheses of N-Boc-protected
cinnamamides (3)
100 MHz):
d
156.29 (Boc CO), 140.95 (Cquat arom), 129.02
Cinnamic acid (1 eq.), BOP (1.1 eq.) and triethylamine (3 eq.)
were successively added to a solution of diamine 2 (1 eq.) in DMF
(0.2 M of 2). The reaction mixture was stirred at room temperature
overnight. After dilution with EtOAc, the organic phase was suc-
cessively washed with a saturated aqueous NaHCO3 and brine.
Organic extracts were dried over Na2SO4, filtered and concentrated
under vacuum. The residue was purified by silica gel flash chro-
matography. The title compound was obtained as a rotamer
mixture.
(2*CHarom), 128.67 (2*CHarom), 126.24 (CHarom), 77.65 ((CH3)3C),
57.64 (NHCH), 51.24 (CH2), 50.89 (CH2), 36.51 (CH2), 30.63
((CH3)2CH), 28.75 ((CH3)3C), 19.85 (CH3), 18.65 (CH3); HRMS (ESI)
m/z calcd for C18H30N2O2þH, 307.2380; found: 307.2371.
4.1.3.5. Tert-butyl
carbamate 2c. Cyclohexane/EtOAc/TEA, 1:1:10%, Rf 0.70. Colorless
oil (159 mg, 45%). 1H NMR (CDCl3, 300 MHz):
7.26e7.05 (m, 10H,
(S)-(1-(phenethylamino)-3-phenylpropan-2-yl)
d
2*Ph), 4.76 (m, 1H, NHBoc), 3.81 (m, 1H, BocNHCH), 2.86e2.47 (m,
8H), 1.36 (s, 9H, (CH3)3C), 1.30 (bs, 1H, NH); 13C NMR (CDCl3,
4.1.4.1. Tert-butyl (2-(N-phenethylcinnamamido)ethyl)carbamate 3a.
Cyclohexane/EtOAc, 3:1 then pure EtOAc, Rf 0.18 (cyclohexane/
EtOAc, 3:1). Yellow oil (327 mg, 83%). 1H NMR (CDCl3, 400 MHz):
100 MHz):
d 156.06 (Boc CO), 140.30 (Cquat arom), 138.47 (Cquat
arom), 129.74 (2*CHarom), 129.14 (2*CHarom), 128.90 (2*CHarom),
128.81 (2*CHarom), 126.75 (CHarom), 126.60 (CHarom), 79.60
((CH3)3C), 51.96 (CH2), 57.74 (NHCH), 51.39 (CH2), 39.49 (CH2),
36.69 (CH2), 28.81 (3*CH3); HRMS (ESI) m/z calcd for
C22H30N2O2þH, 355.2380; found: 355.2372.
d
7.78e7.19 (m, 11H), 7.09 and 6.61 (d, 1H, J ¼ 15.4 Hz, Ph-CH¼CH-
CO), 5.19 and 4.69 (m,1H, NH), 3.73e3.18 (m, 6H), 2.95 (q, J ¼ 7.3 Hz,
2H), 1.44 and 1.40 (s, 9H, (CH3)3C); 13C NMR (CDCl3, 100 MHz):
d
167.46 and 166.95 (CO), 156.28 and 155.99 (Boc CO), 143.05 and
142.76 (PhCH ¼ CH-CO), 139.29 and 138.01 (Cquat arom), 135.36
and 135.19 (Cquat arom), 129.67, 129.59, 128.95, 128.86, 128.79,
128.71, 128.58, 128.12, 127.82, 126.91 and 126.43 (10*CHarom),
117.48 and 117.16 (PhCH ¼ CH-CO), 79.78 and 79.29 ((CH3)3C), 50.34
and 49.46 (CH2), 48.05 and 46.30 (CH2), 39.74 and 39.41 (CH2),
36.00 and 34.16 (CH2), 28.43 and 28.33 ((CH3)3C); HRMS (ESI) m/z
calcd for C24H30N2O3þH, 395.2329; found: 395.2314.
4.1.3.6. Tert-butyl
(2-((2-(1H-indol-3-yl)ethyl)amino)ethyl)carba-
mate 2d. Cyclohexane/EtOAc, 1:3 then cyclohexane/EtOAc/TEA,
1:3:10%, Rf 0.11. Yellow sticky oil (182 mg, 60%). 1H NMR (DMSO-d6,
300 MHz):
d
10.77 (s, 1H, NH indole), 7.50 (d, 1H, J ¼ 8.0 Hz, CH),
7.32 (d, 1H, J ¼ 8.0 Hz, CH), 7.11 (s, 1H, CH), 7.05 (t, 1H, J ¼ 8.0 Hz,
CH), 6.95 (t, 1H, J ¼ 8.0 Hz, CH), 6.71 (m, 1H, NHBoc), 3.02 (q, 2H,
J ¼ 6.0 Hz, BocNHCH2), 2.78 (m, 4H, CH2NHCH2), 2.57 (t, 2H,
J ¼ 6.6 Hz, CH2Ind), 1.70 (bs, 1H, NH), 1.37 (s, 9H, (CH3)3C); 13C NMR
4.1.4.2. Tert-butyl
(S)-(3-methyl-1-(N-phenethylcinnamamido)
(CDCl3, 100 MHz):
d
156.15 (Boc CO), 136.37 (Cquat arom), 127.29
butan-2-yl)carbamate 3b. Cyclohexane/EtOAc, 5:1 then pure EtOAc,
(Cquat arom), 122.07 (CHarom), 122.01 (CHarom), 119.27 (CHarom),
118.75 (CHarom), 113.40 (Cquat arom), 111.19 (CHarom), 79.21
((CH3)3C), 49.33 (CH2), 48.86 (CH2), 40.00 (CH2), 28.37 (3*CH3),
25.42 (CH2); HRMS (ESI) m/z calcd for C17H25N3O2þH, 304.2020;
found: 304.2014.
Rf 0.30 (cyclohexane/EtOAc, 3:1). White solid (180 mg, 61%). Mp
115.5e116.6 ꢀC; 1H NMR (CDCl3, 400 MHz):
d 7.65 and 7.53 (d, 1H,
J ¼ 15.4 Hz, PhCH ¼ CH-CO), 7.36e7.11 (m, 10H), 7.06 and 6.56 (d,
1H, J ¼ 15.4 Hz, Ph-CH¼CH-CO), 4.80 and 4.31 (d, 1H, J ¼ 9.2 Hz,
NH), 3.96 (m, 1H, NHCH), 3.74e2.78 (m, 6H), 1.85 and 1.70 (m, 1H,
(CH3)2CH), 1.33 and 1.26 (s, 9H, (CH3)3C), 0.88e0.81 (m, 6H,
4.1.3.7. Tert-butyl (S)-(1-((2-(1H-indol-3-yl)ethyl)amino)-3-
methylbutan-2-yl)carbamate 2e. Cyclohexane/EtOAc/TEA, 1:1:10%,
Rf 0.18. Yellow sticky oil (128 mg, 37%). 1H NMR (DMSO-d6,
(CH3)2CH); 13C NMR (CDCl3, 100 MHz):
d 167.74 and 166.81 (CO),
156.54 and 155.70 (Boc CO), 142.70 and 142.61 (PhCH ¼ CH-CO),
139.50 and 138.25 (Cquat arom), 135.53 and 135.28 (Cquat arom),
129.60, 129.51, 129.19, 128.93, 128.83, 128.79, 128.54, 128.09, 127.77,
300 MHz):
d
10.76 (s, 1H, NH indole), 7.50 (d,1H, J ¼ 7.9 Hz, CH), 7.31

S. Radix et al. / European Journal of Medicinal Chemistry 150 (2018) 900e907
905
126.84 and 126.31 (10*CHarom), 117.88 and 117.43 (PhCH ¼ CH-CO),
79.70 and 79.06 ((CH3)3C), 55.56 and 54.39 (CH), 50.13 and 49.89
(CH2), 49.26 and 46.86 (CH2), 35.97 and 34.04 (CH2), 30.80
((CH3)2CH), 28.48 and 28.36 ((CH3)3C), 20.04 and 19.21 (CH3), 17.97
and 16.83 (CH3); HRMS (ESI) m/z calcd for C27H36N2O3þH,
437.2799; found: 437.2778.
pure EtOAc, Rf 0.37 (cyclohexane/EtOAc, 3:2). White solid (235 mg,
45%). Mp 112.9e114.1 ꢀC; 1H NMR (CDCl3, 400 MHz):
8.11 and 8.09
d
(bs, 1H, NH indole), 7.69e6.74 (m, 16H), 6.18 (d, 1H, J ¼ 15.4 Hz, Ph-
CH¼CH-CO), 5.26 (d, 1H, J ¼ 7.2 Hz, NH), 4.05e3.78 (m, 2H),
3.65e3.39 (m, 2H), 3.20e2.80 (m, 4H), 2.65 (m,1H), 1.29 and 1.27 (s,
9H, (CH3)3C); 13C NMR (CDCl3, 100 MHz):
d 168.35 (CO), 156.03 (Boc
CO), 142.93 and 142.21 (PhCH ¼ CH-CO), 137.97 (Cquat arom),
136.43 (Cquat arom), 135.05 (Cquat arom), 129.32, 129.29, 128.63,
128.55, 127.69 (9*CHarom), 127.02 (Cquat arom), 126.49, 122.88,
122.32, 119.78, 118.09 and 117.28 (6*CHarom), 111.71 (Cquat arom),
111.64 (PhCH ¼ CH-CO), 79.14 ((CH3)3C), 51.71 (NHCH), 48.32, 48.18
and 39.68 (3*CH2), 28.42 ((CH3)3C), 25.00 (CH2); HRMS (ESI) m/z
calcd for C33H37N3O3þH, 524.2908; found: 524.2883.
4.1.4.3. Tert-butyl (S)-(1-(N-phenethylcinnamamido)-3-
phenylpropan-2-yl)carbamate 3c. Petroleum ether/EtOAc, 3:1, Rf
0.76 (petroleum ether/EtOAc, 1:1). White solid (465 mg, 96%). Mp
135.5e136.7 ꢀC; 1H NMR (CDCl3, 400 MHz):
d 7.73 and 7.63 (d, 1H,
J ¼ 15.3 Hz, PhCH ¼ CH-CO), 7.53e7.18 (m, 13H), 7.09 (d, 2H,
J ¼ 7.0 Hz), 6.92 and 6.65 (d, 1H, J ¼ 15.3 Hz, Ph-CH¼CH-CO), 5.25
and 4.50 (d, 1H, J ¼ 7.4 Hz, NH), 4.07 and 3.96 (m, 1H, NHCH), 3.73
and 3.50 (m, 2H), 3.30e2.69 (m, 6H), 1.39 and 1.35 (s, 9H, (CH3)3C);
4.1.5. General procedure for the syntheses of cinnamamide
hydrochloride salts (4)
13C NMR (CDCl3, 100 MHz):
d 167.84 (CO), 155.97 and 155.29 (Boc
CO), 142.97 and 142.81 (PhCH ¼ CH-CO), 139.32 and 137.97 (Cquat
arom), 137.92 and 137.30 (Cquat arom), 135.52 and 135.20 (Cquat
arom), 129.68, 129.58, 129.35, 129.25, 129.09, 128.93, 128.81, 128.76,
128.69, 128.63, 128.54, 128.10, 127.82, 126.81, 126.49 and 126.32
(15*CHarom), 117.45 and 117.18 (PhCH ¼ CH-CO), 79.85 and 79.14
((CH3)3C), 52.54 and 51.53 (CH), 49.66 and 48.55 (CH2), 39.53 and
38.67 (CH2), 35.84 and 33.98 (CH2), 28.40 and 28.30 ((CH3)3C),
26.93 (CH2); HRMS (ESI) m/z calcd for C31H36N2O3þH, 485.2799;
found: 485.2777.
Acetyl chloride (3eq.) was added dropwise to an ice cold solu-
tion of N-Boc-protected cinnamamide 3 (1eq.) in an excess amount
of CH3OH (0.2 M of 3) and EtOAc (0.4 M of 3). The mixture was
warmed up to room temperature and stirred until completion of
the reaction (1 h-2h, monitored by TLC). After concentration under
vacuum, the residue was crystallized from tert-butyl methyl ether
to obtain the title compound as a rotamer mixture.
4.1.5.1. 2-(N-phenethylcinnamamido)ethan-1-aminium chloride 4a.
CHCl3/CH3OH, 10:1, Rf 0.13. White solid (152 mg, 73%). Mp 154.0 ꢀC;
4.1.4.4. Tert-butyl
ethyl)carbamate 3d. CH2Cl2/MeOH, 99:1, Rf 0.26 (CH2Cl2/MeOH,
95:5). Yellow oil (333 mg, 77%). 1H NMR (CDCl3, 400 MHz):
8.28
(2-(N-(2-(1H-indol-3-yl)ethyl)cinnamamido)
1H NMR (DMSO-d6, 400 MHz): 8.35 and 8.11 (bs, 3H, þNH3),
d
7.85e7.17 (m, 11H), 6.91 (d, 1H, J ¼ 15.3 Hz, Ph-CH¼CH-CO), 3.77 (m,
d
2H), 3.58 (m, 2H), 3.10e2.75 (m, 4H); 13C NMR (DMSO-d6,
(m, 1H, NH indole), 7.81e6.85 (m, 11H), 6.19 (d, 1H, J ¼ 15.3 Hz, Ph-
100 MHz):
d
166.39 and 165.38 (CO),142.36 and 141.15 (PhCH ¼ CH-
CH¼CH-CO), 5.50e4.50 (bs, 1H, NH), 3.76e3.06 (m, 8H), 1.44 and
CO), 139.07 and 138.38 (Cquat arom), 134.91 (Cquat arom), 129.64,
129.54, 129.05, 128.74, 128.68, 128.64, 128.45, 128.36, 128.30,
127.95, 126.33 and 126.17 (10*CHarom), 118.14 and 117.93
(PhCH ¼ CH-CO), 49.24 and 47.78 (CH2), 44.23 and 43.78 (CH2),
37.67 and 37.01 (CH2), 34.97 and 33.20 (CH2); HRMS (ESI) m/z calcd
for C19H23N2O, 295.1805; found: 295.1807.
1.38 (s, 9H, (CH3)3C); 13C NMR (CDCl3, 100 MHz):
d 167.90 (CO),
156.22 and 155.96 (Boc CO), 142.99 and 142.18 (PhCH ¼ CH-CO),
136.43 and 136.23 (Cquat arom), 135.34 and 134.93 (Cquat arom),
129.53, 129.25, 128.67, 128.56, 128.08 and 127.64 (5*CHarom),
127.38 and 127.00 (Cquat arom), 122.99, 122.29, 122.10, 122.02,
119.77, 119.42, 118.84, 118.03, 117.52 and 117.15 (5*CHarom), 113.18
and 111.55 (Cquat arom), 111.68 and 111.19 (PhCH ¼ CH-CO), 79.74
and 79.32 ((CH3)3C), 48.69 and 48.35 (CH2), 46.09 (CH2), 39.56
(CH2), 28.40 and 28.28 ((CH3)3C), 25.04 and 23.69 (CH2); HRMS
(ESI) m/z calcd for C26H31N3O3þH, 434.2438; found: 434.2422.
4.1.5.2. (S)-3-Methyl-1-(N-phenethylcinnamamido)butan-2-
aminium chloride 4b. CHCl3/CH3OH, 10:1, Rf 0.27. White solid
(197 mg, 76%). Mp 172.0ꢀC-173.0 ꢀC; 1H NMR (DMSO-d6, 400 MHz):
d
8.39 and 8.11 (bs, 3H, þNH3), 7.85e7.14 (m, 11H), 6.94 (d, 1H,
J ¼ 15.3 Hz, Ph-CH¼CH-CO), 3.88e3.39 (m, 4H), 3.31e3.16 (m, 1H,
CHNHþ3 ), 2.95e2.75 (m, 2H), 1.98 (m, 1H, (CH3)2CH), 0.985 (d, 3H,
J ¼ 6.9 Hz, CH3), 0.98 (d, 3H, J ¼ 6.6 Hz, CH3); 13C NMR (DMSO-d6,
4.1.4.5. Tert-butyl (S)-(1-(N-(2-(1H-indol-3-yl)ethyl)cinnamamido)-
3-methylbutan-2-yl)carbamate 3e. Cyclohexane/EtOAc, 5:1 then
cyclohexane/EtOAc, 1:1, Rf 0.48 (cyclohexane/EtOAc, 1:1). White
100 MHz):
d 166.92 and 165.85 (CO), 142.00 and 141.05
solid (304 mg, 64%). Mp 234.7 ꢀC; 1H NMR (CDCl3, 400 MHz):
d
8.42
(PhCH ¼ CH-CO), 139.20 and 138.45 (Cquat arom), 134.97 (Cquat
arom), 129.67, 129.59, 129.12, 128.81, 128.41, 128.33, 127.97, 126.36
and 126.21 (10*CHarom), 118.50 and 118.31 (PhCH ¼ CH-CO), 55.33
and 54.51 (CHNHþ3 ), 49.46 and 48.07 (CH2), 46.51 and 45.70 (CH2),
34.94 and 32.93 (CH2), 28.70 and 28.16 ((CH3)2CH), 18.03 (CH3),
17.59 (CH3); HRMS (ESI) m/z calcd for C22H29N2O, 337.2274; found:
337.2272.
(bs, 1H, NH indole), 7.70e6.74 (m, 11H), 6.21 and 6.09 (d, 1H,
J ¼ 15.4 Hz, Ph-CH¼CH-CO), 5.00 and 4.91 (d, 1H, J ¼ 9.3 Hz, NH),
4.02 (m, 1H, NHCH), 3.78e3.30 (m, 3H), 3.04e2.85 (m, 3H), 1.70 (m,
1H, (CH3)2CH), 1.31 and 1.25 (s, 9H, (CH3)3C), 0.91e0.25 (m, 6H,
(CH3)2)CH); 13C NMR (CDCl3, 100 MHz):
d 171.55 and 168.26 (CO),
156.64 and 155.91 (Boc CO), 142.38 and 142.08 (PhCH ¼ CH-CO),
136.51 and 136.46 (Cquat arom), 135.47 and 135.11 (Cquat arom),
129.50, 129.27, 128.80, 128.64, 127.79 and 127.66 (5*CHarom),
127.25 and 127.06 (Cquat arom), 122.99, 122.23, 122.08, 121.89,
119.69, 119.36, 118.61, 118.12, 117.78 and 117.56 (5*CHarom), 112.56
and 111.68 (Cquat arom), 111.72 and 111.37 (PhCH ¼ CH-CO), 79.80
and 78.93 ((CH3)3C), 54.55 and 49.47 (NHCH), 48.75 and 47.59
(CH2), 46.47 and 39.71 (CH2), 28.39 and 28.33 ((CH3)3C), 27.01 and
26.93 ((CH3)2CH), 25.38 and 25.07 (CH2), 19.18 and 18.00
((CH3)2CH); HRMS (ESI) m/z calcd for C29H37N3O3þH, 476.2908;
found: 476.2892.
4.1.5.3. (S)-1-(N-phenethylcinnamamido)-3-phenylpropan-2-
aminium chloride 4c. Petroleum ether/EtOAc, 1:1, Rf 0.34. White
solid (81 mg, 46%). Mp 198.5ꢀC-200.0 ꢀC; 1H NMR (DMSO-d6,
400 MHz):
d
8.25 (bs, 3H, þNH3), 7.63e7.11 (m, 16H), 6.93 and 6.91
(d,1H, J ¼ 15.3 Hz, Ph-CH¼CH-CO), 3.84e3.35 (m, 5H), 3.17 and 3.07
(m, 1H, CH), 2.86e2.62 (m, 3H); 13C NMR (DMSO-d6, 100 MHz):
d
167.37 and 165.93 (CO),142.65 and 141.65 (PhCH ¼ CH-CO),139.37
and 138.72 (Cquat arom), 136.92 and 136.74 (Cquat arom), 135.42
and 135.24 (Cquat arom), 130.22, 130.07, 129.70, 129.63, 129.45,
129.28, 129.20, 129.11, 128.85, 128.75, 128.72, 128.45, 127.45, 127.33,
126.80 and 126.63 (15*CHarom), 118.67 and 118.30 (PhCH ¼ CH-
CO), 52.51 and 51.10 (CHNH3þ), 50.08 (CH2), 48.44 and 48.28 (CH2),
4.1.4.6. Tert-butyl (S)-(1-(N-(2-(1H-indol-3-yl)ethyl)cinnamamido)-
3-phenylpropan-2-yl)carbamate 3f. Cyclohexane/EtOAc, 5:1 then

906
S. Radix et al. / European Journal of Medicinal Chemistry 150 (2018) 900e907
37.17 and 36.90 (CH2), 35.33 and 33.20 (CH2); HRMS (ESI) m/z calcd
performed in triplicate.
for C26H29N2O, 385.2274; found: 385.2277.
4.2.2. Evaluation of the efflux-pump inhibition (EPI) activity
This activity was determined by evaluating ciprofloxacin MIC
when this antibiotic was combined with chemical compound used
at a concentration chosen to avoid precipitation of these DMSO
4.1.5.4. 2-(N-(2-(1H-indol-3-yl)ethyl)cinnamamido)ethan-1-
aminium chloride 4d. White solid (109 mg, 92%). Mp 209.0 ꢀC; 1H
NMR (DMSO-d6, 400 MHz): d 10.93 and 10.91 (NH indole), 8.18 (bs,
3H, þNH3), 7.87e6.97 (m, 11H), 6.53 (d, 1H, J ¼ 15.4 Hz, Ph-CH¼CH-
soluble compounds in water based culture media (10
compounds 3a-f and 30 g/mL for compounds 4a, 4b, 4d and 4e).
Compounds 4c and 4f were respectively used at 15.6 g/mL and
3.9 g/mL to avoid an intrinsic antibacterial activity previously
mg/mL for
CO), 3.83e3.63 (m, 4H), 3.03e2.92 (m, 4H); 13C NMR (DMSO-d6,
m
100 MHz):
d 166.58 and 165.47 (CO), 142.40 and 140.88
m
(PhCH ¼ CH-CO), 136.32 and 136.24 (Cquat arom), 135.05 and
134.81 (Cquat arom), 129.68, 129.40, 128.70, 128.49 and 127.69
(5*CHarom), 127.25 and 127.05 (Cquat arom), 123.77, 122.74, 121.09,
120.98, 118.53, 118.28 and 118.06 (5*CH arom indole), 111.63 and
111.40 (nPhCH ¼ CH-CO), 110.42 (Cquat arom), 48.43 and 47.21
(CH2), 44.41 and 43.71 (CH2), 37.81 and 37.13 (CH2), 24.58 and 23.27
(CH2); HRMS (ESI) m/z calcd for C21H24N3O, 334.1914; found:
334.1909.
m
detected. Reserpine was tested as a positive control at 20
Experiments were performed in triplicate.
mg/mL.
4.3. Intrinsic toxicity
Cell viability was evaluated through the MTT colorimetric assay.
The MTT assay was based on the protocol described by Mossmann
[29]. The assay was optimized for the cell lines used in the exper-
iment. MRC5 cells (Lung Fibroblast Human CCL-171 ATCC) were
plated in triplicated at a density of 6000 cells/well into 96 well
culture plates. Cells were incubated overnight at 37 ꢀC in 5% CO2 in
MEM (modified Eagle's medium) medium supplemented with 10%
FBS (fetal bovine serum). The following day, cells were treated with
4.1.5.5. (S)-1-(N-(2-(1H-indol-3-yl)ethyl)cinnamamido)-3-
methylbutan-2-aminium chloride 4e. CHCl3/CH3OH, 10:1, Rf 0.14.
White solid (179 mg, 95%). Mp 176.0 ꢀC; 1H NMR (DMSO-d6,
400 MHz): d
10.92 (bs, 1H, NH indole), 8.37 and 8.09 (bs, 3H, þNH3),
7.84e7.01 (m, 11H), 6.59 (d, 1H, J ¼ 15.4 Hz, Ph-CH¼CH-CO), 4.01
(m,1H), 3.75 (m,1H), 3.65e3.39 (m, 2H), 3.35e3.17 (m,1H, CHNHþ3 ),
3.05e2.82 (m, 2H), 1.96 (m, 1H, (CH3)2CH), 1.05e0.85 (m, 6H,
the compounds (10e100
thylsulfoxide, DMSO). After 72 h, the cells were incubated with
1 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Sigma Aldrich) for 3 h at 37 ꢀC. The
medium was removed and 100 L of 0.01 M HCl in isopropanol was
mM) or with vehicle control (dime-
(CH3)2CH); 13C NMR (DMSO-d6, 100 MHz):
d 167.02 and 165.85
(CO), 141.96 and 140.71 (PhCH ¼ CH-CO), 136.23 (Cquat arom),
134.78 (Cquat arom), 129.61, 129.35, 128.67, 128.31 and 127.63
(5*CHarom), 126.97 (Cquat arom), 123.75, 121.02, 120.92, 118.43,
118.31 and 117.96 (5*CH arom indole), 111.53 and 111.37
(PhCH ¼ CH-CO), 110.34 (Cquat arom), 55.25 and 54.63 (CHNHþ3 ),
48.48, 46.57 and 45.54 (2*CH2), 28.66 and 28.00 ((CH3)2CH), 24.39
and 22.81 (CH2), 17.99 (CH3), 17.56 (CH3); HRMS (ESI) m/z calcd for
m
added in each well for 15 min. Absorbance was measured by a plate
reader at 570 nm and the value measured at 690 nm was sub-
tracted. Data are the mean SD of at least three independent
experiments.
C
24H30N3O, 376.2383; found: 376.2381.
Acknowledgement
4.1.5.6. (S)-1-(N-(2-1H-indol-3-yl)ethyl)cinnamamido)-3-
phenylpropan-2-aminium chloride 4f. CHCl3/CH3OH, 10:1, Rf 0.15.
White solid (137 mg, 57%). Mp 215.0ꢀC-217.0 ꢀC; 1H NMR (DMSO-
ꢀ
^
ꢁ
We thank the Region Rhone-Alpes and the Ministere de
ꢀ
l'Enseignement Superieur et de la Recherche for financial support.
d6, 400 MHz):
d
10.89 (bs, 1H, NH indole), 8.29 (bs, 3H, þNH3),
Appendix A. Supplementary data
7.67e6.94 (m, 16H), 6.56 (d, 1H, J ¼ 15.3 Hz, Ph-CH¼CH-CO),
3.93e3.48 (m, 5H), 3.23e3.08 (m, 1H, CH), 2.81e2.71 (m, 3H); 13C
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.03.028.
NMR (DMSO-d6, 100 MHz):
d 167.06 and 165.58 (CO), 142.24 and
140.84 (PhCH ¼ CH-CO), 136.43 and 136.33 (Cquat arom), 136.27
and 136.19 (Cquat arom), 134.84 (Cquat arom), 129.74, 129.43,
129.24, 129.19, 128.84, 128.74, 128.67, 128.32, 127.69, 127.14 and
126.98 (10*CHarom), 126.89 (Cquat arom), 123.71, 122.76, 121.07,
120.95 118.51, 118,29, 117.96, 111.59 and 111.39 (5*CH arom,
PhCH ¼ CH-CO), 110.25 (Cquat arom), 52.07 and 50.79 (CHNHþ3 ),
48.84 and 47.92 (CH2), 47.70 and 47.37 (CH2), 36.73 and 36.29 (CH2),
24.34 and 22.62 (CH2); HRMS (ESI) m/z calcd for C28H30N3O,
424.2383; found: 424.2370.
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