Improved Potency of Indole-Based NorA Efflux Pump Inhibitors: From Serendipity toward Rational Design and Development

Article abstract of DOI:10.1021/acs.jmedchem.6b01281

The NorA efflux pump is a potential drug target for reversal of resistance to selected antibacterial agents, and recently we described indole-based inhibitor candidates. Herein we report a second class of inhibitors derived from them but with significant differences in shape and size. In particular, compounds 13 and 14 are very potent inhibitors in that they demonstrated the lowest IC₅₀ values (2 μM) ever observed among all indole-based compounds we have evaluated.

Full text of DOI:10.1021/acs.jmedchem.6b01281

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Brief Article
Improved potency of indole-based NorA efflux pump inhibitors:
from serendipity towards rational design and development.
Federica Buonerba, Susan Lepri, Laura Goracci, Bryan D.
Schindler, Susan M. Seo, Glenn W Kaatz, and Gabriele Cruciani
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01281 • Publication Date (Web): 02 Dec 2016
Downloaded from http://pubs.acs.org on December 3, 2016
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Journal of Medicinal Chemistry
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Improved potency of indole-based NorA efflux pump inhibitors:
from serendipity towards rational design and development.
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Federica Buonerba, Susan Lepri, Laura Goracci, Bryan D. Schindler, Susan M. Seo, Glenn W.
§
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ζ
ζ
‡
‡
¥
Kaatz, * and Gabriele Cruciani. *
ζ
§
§
Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy.
ζ
The John D. Dingell Department of Veterans Affairs Medical Center, Detroit, MI 48201, USA
Department of Internal Medicine, Division of Infectious Diseases, Wayne State University School of Medicine, Detroit, MI
¥
48201, USA
KEYWORDS NorA inhibitors; scaffold hopping approach; SAR study; indole
ABSTRACT: The NorA efflux pump is a potential drug target for reversal of resistance to selected antibacterial agents, and recent-
ly we described indole-based inhibitor candidates. Herein we report a second class of inhibitors derived from them, but with signifi-
cant differences in shape and size. In particular, compounds 13 and 14 were very potent inhibitors in that they demonstrated the
lowest IC50 values (2 ꢀM) ever observed among all indole-based compounds we have evaluated.
al NorA inhibitors belonging to different chemical classes
3
have been identified including natural and synthetic ones. In
INTRODUCTION
The decreasing effectiveness of antibiotics in the treatment
of common infections has quickened in recent years with
worldwide consequences. Of particular concern are globally
disseminated methicillin-resistant Staphylococcus aureus
particular, among heterocyclic compounds, the indole scaffold
is typical of some potent NorA inhibitors such as the alkaloid
9
reserpine, 5-nitro-2-phenyl-1H-indole (INF55) and ana-
10
logues, and aldonitrones.
11
(
MRSA) clones that cause healthcare-associated infections
Starting from these literature data about known indole-based
NorA inhibitors, we have recently identified a novel class of
NorA EPIs with a polysubstituted indole moiety; their activity
was evaluated in terms of EtBr efflux inhibition and intrinsic
such as pneumonia, endocarditis, sepsis and complicated skin
structure infections. A recent report by the US Centers for
Disease Control and Prevention has estimated 80461 severe
1
12
MRSA infections and 11285 deaths from MRSA per year.
Several agents including vancomycin, linezolid, daptomycin,
fluoroquinolones, and tigecycline are recommended to treat
antimicrobial activity determination (MIC). Twenty of them
were highly active, inhibiting EtBr efflux by more than 90%
(obtained results are briefly summarized in Figure S1). During
the development of the our first class of indole-based NorA
2
this Gram-positive bacterium, although it often expresses
concomitant resistance to many of them.
1
2
inhibitors, the reaction of the epoxide intermediate 1 with
28% NH OH aqueous solution, did not give the desired com-
Among factors triggering the resistance response, overex-
pression of efflux pumps is one of the most studied as it leads
to sub lethal drug concentrations at the active site predisposing
4
1
2
pound 2, while the symmetrical compound 3 was obtained
instead (Scheme 1).
3
to the development of high-level target-based resistance.
Therefore, the susceptibility of resistant strains might be re-
stored by co-administration of efflux pump inhibitors (EPIs)
and antibacterials (efflux pump substrates); thus, efflux pumps
are viable antibacterial targets and identification of potent
EPIs is a promising and valid strategy to fight antibiotic re-
a
Scheme 1.
4
sistance. The most studied efflux system in S. aureus is NorA,
which is able to extrude multiple structurally dissimilar sub-
strates such as hydrophilic fluoroquinolones (ciprofloxacin,
norfloxacin), biocides (acriflavine, cetrimide, benzalkonium
chloride), and dyes (ethidium bromide [EtBr]), through an an-
a
Reagents and conditions: aq. NH OH 28% 25 eq., MeOH, r.t.
4
Testing 3 as a NorA inhibitor, we found it to be very active
with 78% inhibition of EtBr efflux and an IC50 value of 19.9
µM, comparable with compound 2 (100% EtBr efflux inhibi-
tion; IC50 of 17.1 µM). This finding prompted us to investigate
not only larger compounds characterized by a symmetrical in-
dole scaffold, but also unsymmetrical compounds similar in
size, shape and ability to form chemical interactions with the
5
-7
tiport mechanism driven by the proton-motive force.
To date, no crystal structure of NorA has been determined.
Nevertheless, sequence homology and sharing of several sub-
strates with other MDR pumps have suggested that NorA may
have a large hydrophobic binding site which may explain the
8
broad substrate specificity of MDR pumps. In the past, sever-
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NorA protein, as predicted by in silico scaffold hopping analy- zylamino moiety (as in compound 6a, Table 1) was shown to
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sis. As a result, four out of eleven compounds tested showed
be a valid scaffold to obtain reasonable NorA inhibitors (EtBr
efflux inhibition = 45%); thus compound 12 was synthesized
accordingly (Table 1, Figure S2). Finally, being that the linker
is an important feature for potency, we focused our attention
on the synthesis of novel derivatives of compounds 7 (8-11)
and 12 (13-16) (Table 1).
improved potency (IC50 ≤ 4.9 ꢀM).
RESULTS AND DISCUSSION
Design. On the basis of the unexpected but exciting poten-
cy of compound 3, we were encouraged to further examine
structure-activity relationships. Therefore, we were interested
to observe how the activity may change in case of unsymmet-
rical derivatives of compound 3, where one of the two indoles
was replaced with a molecular fragment similar in shape, size
Figure 1. Structures 2, 4, 5 (A) and superimposition of 4 or
a
5 on 2 in the FLAP ligand based virtual screening (B-I).
0
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and chemical properties.
Thus, a virtual screening campaign on the SPECS database
was performed to search for novel scaffolds using the FLAP
1
3,14
software.
has been reported elsewhere.
common reference framework for comparing molecules, using
An extensive description of the FLAP algorithm
13,15
Briefly, FLAP provides a
1
6
GRID Molecular Interaction Fields (MIFs), and the FLAP
1
7,18
fingerprints can be used for ligand-ligand,
ligand-
and receptor-receptor
In particular, the FLAP ligand-based virtual
screening approach has already proved to be a good strategy
19,20
21-23
pharmacophore,
ligand-receptor,
2
4,25
comparison.
1
8
for the discovery of novel NorA EPIs, and for scaffold-
2
6
hopping applications. Thus, in this study, a ligand-based ap-
proach was applied to identify compounds highly similar to
compound 2 (template), to be used to design and synthesize
unsymmetrical analogues of 3. Details of the virtual screening
procedure are reported in the Supporting Information. Starting
from more than 16500 structures, a final list of the top-ranked
4
4 compounds with high global similarity to the template 2
was generated. Compounds 4 and 5 (Figure 1) were selected
as possible scaffolds to replace the indole moiety, based on the
high FLAP similarity score, the good synthetic accessibility,
and commercial availability. The optimal superimpositions,
according to FLAP, of compounds 4 and 5 on the template 2
are reported in Figure 1 (B and C, respectively). The phe-
nylphenol (4) and the phenyltetrazole (5) pose on the ben-
zylindole ring (Figure 1B and Figure 1C). Concerning the ter-
minal amino group of 2, one of the two piperazine nitrogens of
a
4
perfectly matches it, while for compound 5 the hydroxyl
In FLAP poses (B-I) the template 2 is depicted by yellow sticks,
group instead of the amino group are aligned. However, this
preliminary analysis can be misleading. Indeed, the power of
using a GRID-MIFs-based similarity software is that the con-
cept of similarity is translated from chemical groups to chemi-
cal interactions. Thus, the GRID fields for the aligned struc-
tures are reported in Figure 2D-I. Concerning the hydrophobic
interactions, both compounds 4 and 5 seem to display a re-
duced hydrophobicity with respect to the template 2, but com-
pound 5 shows a more efficient overlap of the hydrophobic
portions (Figure 1D-E). Also for polar interactions (HBA and
HBD, Figures 1F-G and 1H-I, respectively) a higher similarity
was found between 5 and 2. In summary, according to GRID
MIFs analysis, compound 5 was a better candidate than 4 in
mimicking the chemical features of 2. Thus, we decided to de-
sign the new unsymmetrical analogues 7 and 12, where one of
the indole moieties was replaced with 4 (best match of the
amino group) or 5 (best match of GRID-MIFs), respectively
while the candidates 4-5 are shown in green sticks. The simple struc-
ture overlays of candidates 4 and 5 are shown in panels B and C, re-
spectively. A candidate-template comparison of the GRID hydropho-
bic (green), HBA (red) and HBD (blue) fields is reported for 4 and for
5
in panels D-F-H and E-G-I, respectively. MIFs for the template are
reported in mesh style, while candidate MIFs are shown in wireframe
style.
Chemistry. The compounds reported herein were obtained
through different chemical synthetic approaches (Schemes 1-
4
bromo-indoles 20a-b have been described previously.
). The synthesis of epoxide 1, amino derivatives 6a-e, and
1
2
As shown in Scheme 2, refluxing 2-phenylphenol with an
excess of 1,2-dibromoethane or 1,3-dibromopropane in the
presence of potassium carbonate in absolute ethanol gave ha-
lo-derivatives 18a-b, that were used to give piperazine ethers
1
9a-b. Final compounds 7-10 were prepared analogously by
combining halo-ethers 20a-b with piperazine 19a-b (Scheme
). Similarly, compound 11 was obtained from 4-fluorophenol
and 1-bromo-3-chloropropane to prepare 1-(3-chloropropoxy)-
(
preserved (with respect to 3) with the piperazine being the new
Table 1). In particular, in compound 7 both the spacers were
2
1
asymmetric center. From our previous paper, a terminal ben-
2
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Table 1. Biological activity of compound 3 analogues.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
a
% EtBr efflux
inhibition @50 µM
MIC
(µg/mL)
d
Comp.
X
LogP
pK
a
b
c
IC50 (µM)
FIC index
6
a
c
6.0
4.0
6.0
8.81 (N)
10.1 (N)
9.6 (N)
45.5
100.0
98.5
>100
>100
>100
_
_
_
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
12.5
6.0
6f
0.28
3.82 (N)
8
7
8.6
7.6
8.1
12.1 ± 2.1
24.3 ± 6.5
27.1 ± 7.9
_
_
_
_
_
_
_
_
_
.39 (N)
7
3
.62 (N)
.05 (N)
8
9
3.82 (N)
8.39 (N)
8
3
.39 (N)
.82 (N)
1
0
1
8.1
7.1
23.9 ± 5.5
53.7 ± 1.6
_
_
_
_
_
_
F
3.82 (N)
8.39 (N)
1
N
O
O
N
O
OCH
O
3
N
H
12
5.8
6.3
6.8
7.00
7.21
8.20
50.3 ± 2.0
96.5 ± 0.3
100.8 ± 2.1
_
_
_
N
N
N
N
OCH
3
O
N
>100
>100
1.82 ± 0.21
2.02 ± 0.27
0.27
0.25
1
1
3
4
H
N
N
O
N
N
N
N
H
N
N
O
N
15
4.6
6.0
5.2
6.93
8.54
22.8 ± 3.4
67.0 ± 1.4
96.9 ± 2.5
_
_
_
_
F
H
N
1
1
6
7
O
6.3
3.83 ± 0.45
4.93 ± 0.82
OCH
3
OCH
3
6.73 (N)
14.83 (OH)
O
N
>100
0.13
OH
H
N
N
O
N
N
2
7,28
a) predicted by MoKa;
Biological data were generated employing SA-1199B, which overexpresses norA; b) a compound was considered inhib-
itor if showing an EtBr efflux inhibition > 60%; c) inhibitors with a MIC >100 do not have intrinsic antimicrobial activity; d) FIC indices were for
the combination of ciprofloxacin (CPX) with NorA inhibitors by using strain SA1199B. A FIC index of 0.5 was considered to be an indicator of
synergistic activity; more details are given in Supplementary Information.
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4
-fluorobenzene, further condensed with the piperazine de- having 4-fluorophenoxy group instead of a 2-phenylphenoxy
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rivative 6b (Scheme 3). According to Scheme 3, tetrazole de-
rivatives 12-14 were prepared from the suitable amino inter-
mediate 6c-e and 3-methoxy-4-((1-phenyl-1H-tetrazol-5-
yl)oxy)benzaldehyde (21), in turn prepared by O-alkylation of
vanillin with 1-phenyl-5-chloro-1H-tetrazole in the presence
of potassium tert-butoxyde. The amine 22 was obtained by
conversion of the aldehyde 21 into the corresponding oxime,
followed by reduction with zinc and hydrochloric acid. Amine
group, as for compounds 7-10, showed an improved inhibitory
effect with the percentage of efflux inhibition between 10 and
30%. This finding might suggest that the hydrophobicity and
the rigidity of the 2-phenylphenoxy group were detrimental for
the activity and a more polar moiety was preferable. Overall
according to the data reported in Table 1, the NorA binding
site seems to be sensitive to how hydrophobic the inhibitor is:
compounds with a LogP value > 8 were not active (e.g. 7, 9-
10). On the contrary, the lack of inhibition of 5 is probably
related to its very high polarity (LogP = 0.9). Inhibitor 12 con-
taining fragment 5 inhibited EtBr efflux by 50%. Between the
two derivatives with three (13) and four (14) carbon atoms
chain, the first was a very strong inhibitor (% inhibition =
2
2 was converted into final compound 17 by reaction with the
epoxide 1 (Scheme 4). Amine 6c was used as starting material
to give compounds 15-16 upon reaction with 5-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
chloromethyl)-1-phenyl-1H-tetrazole
and
methoxybenzaldehyde, respectively (Scheme 3).
4-fluoro-3-
9
6%), having the best IC50 value ever observed (1.8 ꢀM1)2
a
Scheme 2.
among all indole-based compounds that we have reported.
Indeed, comparing the biological profile of 13 with those pre-
viously published, it exhibited a 2-fold lower IC50 compared to
the best inhibitor found in that series (compound 6f) and a 6-
fold lower IC50 compared to 6c, that has the same alkyl
a
12
Reagents and conditions: (i) 1,3-dibromopropane or 1,2-
CO , ethanol reflux; (ii) 1. NaI, acetonitrile reflux;
chain. Moreover 13 had a better activity than that of the
symmetrical compound 3, being 10-fold more active at the 50
dibromoethane, K
. K
2
3
2
9
2
2
CO
3
2 3
, piperazine; (iii) 1. NaI, acetonitrile reflux; 2. K CO .
ꢀM screening concentration.
Concerning the length of the alkyl spacer, the analogue 14,
a
Scheme 3.
having a four carbon atoms chain, exhibited the same % inhi-
bition and IC50 value of 13, 100% and 2 ꢀM respectively; the
shorter chain (2 carbon atoms) of 12 produced a 2-fold activity
decrease (50% efflux inhibition). Thus, three or four carbon
atoms seem to confer the right distance between the indole
scaffold and the basic center to obtain the best inhibitory ef-
fect. The insertion of the hydroxyl groups in 13 to give 17 was
detrimental producing a 2-fold activity decrease with respect
to the IC .
50
Compounds 15 and 16 have been synthetized as analogues
of 13 to study the importance of the 3-methoxybenzyl moiety
bound to the amino group: compound 15 that lacks this group
was inactive, while compound 16 that preserves the 3-
methoxybenzyl but lacks the tetrazole moiety had a lower in-
hibitory activity (67% efflux inhibition) and an intrinsic anti-
microbial activity (MIC = 6.3 ꢀg/mL).
Although the promiscuous activity of 16 as NorA inhibitor
and antibacterial compound might be interesting, to avoid mis-
leading results we preferred to further investigate only com-
pounds 13 and 14, whose activity was exclusively due to
NorA inhibition contribution. Indeed, synergistic studies were
performed to evaluate their ability to restore antibiotic activity
versus SA-1199B, a strain that overexpresses norA. As a re-
sult, compounds 13 and 14 were also able to restore ciproflox-
acin (a commonly used antibiotic) activity according to the
FIC index values observed (Table 1).
a
Reagents and conditions: i) 1. 1-(3-chloropropoxy)-4-fluorobenzene,
29
NaI, acetonitrile reflux; 2. K
5
K
2
4
CO3; ii) 21, NaBH , MeOH, r.t.; iii) 1.
-(chloromethyl)-1-phenyl-1H-tetrazole, NaI, acetonitrile reflux; 2.
;
29
2
CO
3
4
iv) 1. 4-fluoro-3-methoxybenzaldehyde, Ti(Oi-Pr) , EtOH
30
4
r.t.; 2. NaBH , r.t., 5 h.
a
Scheme 4.
ADME studies. Metabolic stability (in HLM), water solubili-
33
a
ty (by NMR) and permeability in CaCo2 cells (predicted in
silico by VolSurf+ and reported as value of CACO2 de-
Reagents and conditions: (i) 1) Na
31
O, r.t., 24 h; 2) Zn, H
2
CO
3 2
, NH OH ⋅ HCl, EtOH,
31
H
2
2
O, 10% HCl, EtOH reflux, 12 h; (ii) 1,
34
32
scriptor), have been evaluated for the two best compounds 13
and 14 (Table 2). They are metabolically stable, but character-
ized by a quite low solubility and permeability.
4 3
LiClO , CH CN, r.t., 12 h.
Biological evaluation. The biological activity of com-
pounds was evaluated by use of EtBr efflux inhibition assays
and MIC determinations, as described in Supporting Infor-
mation.
Compounds 4 and 5 were inactive as EPIs (EtBr efflux In-
hibition < 10%), suggesting the fundamental role of the indole
scaffold to achieve satisfactory activity. Like fragment 4, also
compounds 7-10 were inactive. Interestingly, compound 11
Table 2. ADME study of the best compounds.
Solub.
a
(mM)
Met
analysis
CACO2
Permeability
Comp.
pK
a
b
c
0
.73
1
3
7.21
< 0.002
stable
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Journal of Medicinal Chemistry
sure. The crude was purified by column chromatography on silica gel
/MeOH/Et
8.20
< 0.002
stable
0
.70
1
4
1
(eluent, CH
Diethyl
2
Cl
2
3
N 95:4:1).
1
a) evaluated by H-NMR in D
2
O solution (containing 0.4% of
1
5,5'-((azanediylbis(2-hydroxypropane-3,1-
diyl))bis(oxy))bis(1-benzyl-2-methyl-1H-indole-3-carboxylate) (3).
28% aquous NH OH (4 mL) was added to a stirred solution of
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
DMSO-d
c) predicted by VolSurf+ (the value +1 is assigned to permeable com-
6
) at 25 °C; b) see experimental section for further details;
4
−6
−1 34
pounds, with Papp. ≥ 8 x 10 cm s ).
epoxyde 1 (515 mg, 1.41 mmol) in methanol (20 mL) and the
mixture was reacted at room temperature overnight. All the volatile
materials were evaporated under reduced pressure and the residue
was taken up with chloroform, washed with water and brine,
dryed under sodium sulfate anydrous and concentrated under
vacuum. The residue was purified by column cromatography on
CONCLUSIONS
In this study, we conducted an investigation on the anti-
efflux pump properties of a series of indole-based derivatives
against a NorA-overexpressing S. aureus strain. During the
development of our first class of NorA inhibitors, we identi-
fied a symmetrical compound that even though being a very
large molecule was able to inhibit EtBr efflux by more than
80%. This unexpected discovery suggested that the NorA ef-
flux pump can bind molecules bigger than the ones we had
previously developed. Therefore, on the basis of a virtual
screening with FLAP, we selected two molecular moieties 4
and 5 to bind to the indole scaffold and to obtain asymmetrical
derivatives similar in size, shape and chemical properties to
the symmetrical compound 3. The combination of 5, disclos-
ing the best GRID-MIFs, with the indole moiety, allowed to
identified compounds 13 and 14 as very strong NorA efflux
pump inhibitors, both with IC50 values of ≤ 2 ꢀM. To con-
clude, within this study we confirmed that the indole is a
promising scaffold for the development of new EPIs and we
have identified two very potent NorA multidrug EPIs with un-
expected features in shape and size.
silica gel eluting with AcOEt/MeOH/Et N (8/1.5/0.5) to obtain
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
400 mg of a white solid (76% yield).
Ethyl
5-(3-(4-(3-([1,1’-biphenyl]-2-yloxy)propyl)piperazin-1-
yl)propoxy)-1-benzyl-2-methylindole-3-carboxylate (7) was ob-
tained from the monoalkylated piperazine 19b and the bromoether
2
0b according to the general procedure B. Trituration of the crude
residue into a 90:10 Et O/PE mixture gave a white solid (64% yield).
Ethyl 5-(3-(4-(3-([1,1’-biphenyl]-2-yloxy)ethyl)piperazin-1-
1
2
2
yl)ethoxy)-1-benzyl-2-methylindole-3-carboxylate (8) was obtained
from compounds 19a and 20a. The crude was purified by column
chromatography eluting with a mixture CH
200 mg of a white solid (46% yield).
2 2
Cl /MeOH 95:5 affording
Ethyl
5-(3-(4-(3-([1,1’-biphenyl]-2-yloxy)propyl)piperazin-1-
yl)ethoxy)-1-benzyl-2-methylindole-3-carboxylate (9) was obtained
from compounds 19b and 20a according to the general procedure B.
Trituration of the crude residue into a 90:10 Et
white solid (80% yield).
2
O/PE mixture gave a
Ethyl
5-(3-(4-(3-([1,1’-biphenyl]-2-yloxy)ethyl)piperazin-1-
yl)propoxy)-1-benzyl-2-methylindole-3-carboxylate (10) was ob-
tained from compounds 19a and 20b according to the general proce-
dure B. After trituration of the crude into a 90:10 Et
ture, a white solid (75% yield) was obtained.
2
O/hexane mix-
Ethyl 1-benzyl-5-(3-(4-(3-(4-fluorophenoxy)propyl)piperazin-1-
yl)propoxy)-2-methyl-1H-indole-3-carboxylate (11) was obtained
from chloro derivative 18 and amine 19 according to the general pro-
cedure B. Chromatography of the crude eluting with a mixture
EXPERIMENTAL SECTION
Chemistry. Starting materials were purchased from Aldrich-
Fluka and Apollo Scientific Ltd and used without further purification.
Chloroacetanilide was prepared in 95% yield according to literature
CH
id (74% yield).
Ethyl 1-benzyl-5-(2-((3-methoxy-4-((1-phenyl-1H-tetrazol-5-
yl)oxy)benzyl)amino)ethoxy)-2-methyl-1H-indole-3-carboxylate
12) was obtained according to general procedure C from amino de-
rivative 6d as a pale yellow solid (69% yield).
2 2 3
Cl /MeOH/Et N 95:4:1 allowed to recover 102 mg of a white sol-
35
1
13
19
procedure.
Bruker Avance III 400 MHz. Solubility was determined by H NMR
experiments in solution at 25 °C using 3-
trimethylsilyl)propionic-2,2,3,3-d acid sodium salt (TSP) as the in-
H, C and F NMR spectra were recorded with a
1
2
D O
(
4
(
33
ternal standard. HRMS spectra were registered on Agilent Technol-
ogies 6540 UHD Accurate Mass Q-TOF LC/MS, HPLC 1290 Infini-
ty. Purities of the final compounds were ≥98% as determined by
UHPLC.
Ethyl 1-benzyl-5-(3-((3-methoxy-4-((1-phenyl-1H-tetrazol-5-
yl)oxy)benzyl)amino)propoxy)-2-methyl-1H-indole-3-carboxylate
(13) was obtained according to general procedure C from amino de-
rivative 6c as a pale yellow solid (68% yield).
General procedure A. The suitable halo intermediate (176 mmol)
was added to a stirred mixture of phenol derivative (59 mmol) and
(8.1 g, 59 mmol) in ethanol (100 mL) and refluxed for 6 h.
After cooling, precipitated salt was filtered off and the filtrate was
concentrated under reduced pressure. The residue was diluted with
O and extracted with AcOEt. The combined AcOEt layers were
washed with brine, dried over anhydrous Na SO , concentrated under
vacuum and purified by column chromatography on silica gel (eluent
PE/AcOEt 95:5).
General procedure B. A mixture of the suitable halo intermediate
Ethyl
1-benzyl-5-(4-((3-methoxy-4-((1-phenyl-1H-tetrazol-5-
K
2
CO
3
yl)oxy)benzyl)amino)butoxy)-2-methyl-1H-indole-3-carboxylate
(14) was obtained from the amino indole 6e according to the general
procedure C as a pale yellow solid (74% yield).
H
2
Ethyl
1-benzyl-2-methyl-5-(3-(((1-phenyl-1H-tetrazol-5-
2
4
yl)methyl)amino)propoxy)-1H-indole-3-carboxylate (15) was ob-
tained from amine 6c and 5-(chloromethyl)-1-phenyl-1H-tetrazole
according to the general procedure B. After chromatography using as
eluent CH
collected (46% yield).
Ethyl 1-benzyl-5-(3-((4-fluoro-3-methoxybenzyl)amino)propoxy)-
2 2 3
Cl /AcOEt/Et N 70:29:1, a pale yellow solid (123 mg) was
(11 mmol) and NaI (3.3 g, 22 mmol) in CH
0 min. After cooling to room temperature, piperazine or the suitable
amino derivative (33 mmol) and anhydrous K CO (44 mmol) were
added and the resulting mixture was refluxed again for 6-12 h. The
reaction mixture was diluted with H O and extracted with CH Cl
The combined organic layers were dried over anhydrous Na SO and
3
CN (30 mL) refluxed for
3
3
0
2
3
2-methyl-1H-indole-3-carboxylate (16). A mixture of 4-fluoro-3-
methoxybenzaldehyde (43 mg, 0.28 mmol), Ti(Oi-Pr) (166 ꢀl, 0.59
mmol) and amine 6c (50 mg, 0.14 mmol) in EtOH (1 mL) was stirred
under nitrogen atmosphere at r.t. overnight. NaBH (8 mg, 0.21
4
2
2
2
.
2
4
4
concentrated under vacuum. If not stated otherwise, the crude was
purified by chromatography on silica gel.
General procedure C. Aldehyde 21 (243 mg, 0.82 mmol) was
added to a MeOH (10 mL) solution of the suitable amine 6c-e (0.82
mmol). The resulting suspension was stirred at room temperature
mmol) was then added and the resulting mixture was stirred at room
temperature for additional 5 h. The reaction was quenched with 2 M
3
6,37
NH
ic layers were dried over anhydrous Na
vacuum. Chromatography of the crude on silica gel (eluent
4
OH aq. solution and extracted with AcOEt. The combined organ-
2
SO and concentrated under
4
overnight. NaBH
mixture was heated for 4 h at 50 °C. After cooling, the mixture was
diluted with H O and extracted with CH Cl . The organic layer was
dried over anhydrous Na SO and concentrated under reduced pres-
4
(187 mg, 4.92 mmol) was added and the reaction
CH Cl
Ethyl
2
2
/MeOH 95:5) afforded 50 mg of a colourless oil (71% yield).
1-benzyl-5-(2-hydroxy-3-((3-methoxy-4-((1-phenyl-1H-
2
2
2
tetrazol-5-yl)oxy)benzyl)amino)propoxy)-2-methyl-1H-indole-3-
carboxylate (17). LiClO (44 mg, 0.41 mmol) was added to a solu-
2
4
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 7
3
tion of epoxide 1 (150 mg, 0.41 mmol) in dry CH CN (2 mL) under
ASSOCIATED CONTENT
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nitrogen atmosphere and the mixture was stirred at r.t. for 1 h. Then
the amine 22 (122 mg, 0.41 mmol) was added and the reaction mix-
Supporting Information
ture was stirred overnight. The mixture was diluted with H
tracted with AcOEt. The combined organic layers were dried over
anhydrous Na SO . After solvent evaporation under reduced pressure,
the crude was purified by column chromatography on Silica gel (elu-
2
O and ex-
More experimental details (e.g. all compounds characterization,
metabolism assays procedure and computational methods) are
given in the Supporting Information.
2
4
2 2 3
ent CH Cl /MeOH/Et N 97:2:1) affording 60 mg (22%) of a white
solid.
AUTHOR INFORMATION
2-(2-Bromoethoxy)-1,1’-biphenyl (18a) was prepared as a white
solid (6.3 g, 38% yield) according to the general procedure A from
Corresponding Authors
*
1
2
,2-dibromoethane and 2-phenylphenol.
-(3-Bromopropoxy)-1,1’-biphenyl (18b) was prepared as a white
To whom correspondence should be addressed. For G.C.:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
phone, +39 075 585 5629; fax, +39 075 45646; e-mail, gabrie-
le.cruciani@unipg.it. For G.W.K.: phone, +1 313 576-4491; e-
mail, gkaatz@juno.com.
solid according to the general procedure A from 1,3-dibromopropane
and 2-phenylphenol (15.7 g, 90% yield).
1-(2-[(1,1’-Biphenyl)-2-yloxy)ethyl]piperazine (19a). The title in-
termediate was obtained from 18a and piperazine according to the
general procedure B. The oily crude residue was acidified with 5 N
HCl. The resulting precipitate was filtered off, washed with Et O and
Author Contributions
‡
These authors contributed equally.
2
then dissolved in 5 N NaOH. The aqueous phase was extracted with
AcOEt and the combined organic layers were dried over anhydrous
Funding Sources
Work performed in the Kaatz laboratory was supported in part by
a VA Biomedical Laboratory Research and Development grant
Na
yield) used without further purification.
-(3-[(1,1’-Biphenyl)-2-yloxy)propyl]piperazine (19b) was obtained
2 4
SO and concentrated under vacuum affording a white solid (58%
1
(
grant number BX000465-01A2). Work performed by S.L. was
from 18b and piperazine according to the general procedure B. Puri-
fied as described for compound 19a, a white solid (62% yield) was
obtained.
supported by a Molecular Discovery (London, UK) grant (grant
number MD011-2014).
3
-Methoxy-4-((1-phenyl-1H-tetrazol-5-yl)oxy)benzaldehyde (21).
t-BuOK (3.0 g, 26.6 mmol) was added to a solution of vanillin (3.40
g, 22.1 mmol) in dry DMF (50 mL) under nitrogen atmosphere. Upon
complete dissolution of the base, 5-chloro-1-phenyltetrazole (4.00 g,
22.1 mmol) was added and the mixture was stirred at r.t. for 18 h. The
reaction mixture was poured into ice water and the formed precipitate
ABBREVIATIONS
MRSA, methicillin-resistant Staphylococcus aureus; EPI(s), ef-
flux pump inhibitor(s); MDR, multiple drug resistance; EtBr, eth-
idium bromide; MIC, minimum inhibitory concentration; SAR,
structure-activity relationship; FLAP, fingerprints for ligands and
proteins; MIFs, Molecular Interaction Fields; HBD, hydrogen
Bond donor; HBA, hydrogen Bond acceptor; HLM, human liver
was filtered off under vacuum, washed with H
vacuum obtaining 4.8 g of a white solid (73% yield).
3-Methoxy-4-((1-phenyl-1H-tetrazol-5-
yl)oxy)phenyl)methanamine (22). A solution of Na
1.95 mmol) in H O (1.2 mL) was added to a mixture of aldehyde 21
(500 mg, 1.86 mmol) in EtOH (4 mL). After a solution of NH
OH ⋅
HCl (181 mg, 2.60 mmol) in H O (1.8 mL) was added slowly to the
mixture and the resulting solution was stirred at r.t. for 24 h. H O was
added and the mixture was extracted with AcOEt; the organic layer
was dried over anhydrous Na SO and concentrated under reduced
pressure. Flash chromatography of the crude on silica gel (eluent
CH Cl /MeOH 95:5) afforded 3-methoxy-4-((1-phenyl-1H-tetrazol-5-
2
O and dried under
(
2
3
CO (207 mg,
2
2
microsomes;
DMF,
N,N-dimethylformamide;
TSP,
3-
2
(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt; LBVS, lig-
and-based virtual screening.
2
2
4
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2
2
yl)oxy)benzaldehyde oxime as a white solid (420 mg, 72% yield).
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.
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2
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.
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4
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.
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4
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.
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.
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