Chalcone inhibitors of the NorA efflux pump in Staphylococcus aureus whole cells and enriched everted membrane vesicles

Article abstract of DOI:10.1016/j.bmc.2012.05.025

A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC₅₀-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms.

Full text of DOI:10.1016/j.bmc.2012.05.025

Bioorganic & Medicinal Chemistry 20 (2012) 4514–4521
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chalcone inhibitors of the NorA efflux pump in Staphylococcus aureus
whole cells and enriched everted membrane vesicles
Jes Gitz Holler a, , Hans-Christian Slotved b, Per Mølgaard a, Carl Erik Olsen c, Søren Brøgger Christensen a
⇑
a University of Copenhagen, Faculty of Pharmaceutical Sciences, Department of Medicinal Chemistry, Denmark
b Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen, Denmark
c University of Copenhagen, Faculty of Life Sciences, Department of Basic Sciences and Environment, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated
Received 5 March 2012
Revised 1 May 2012
Accepted 11 May 2012
Available online 18 May 2012
ethidium bromide efflux. Five of the chalcones (5–7, 9, and 10) were active and two chalcones (9 and 10)
were equipotent to reserpine with IC50-values of 9.0 and 7.7 lM, respectively. Twenty chalcones were
subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds
5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results sug-
gest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux
transporters of microorganisms.
Keywords:
Chalcones
Staphylococcus aureus
MRSA
Ó 2012 Elsevier Ltd. All rights reserved.
Antibiotic resistance
Antibiotics
Natural compounds
1. Introduction
efflux pump inhibitor (EPI) with an antibiotic is a possibility for
overcoming MDR.14 Recently we found a p-coumaroyl ester of
The emergence of multidrug resistance among pathogenic
microorganisms makes presently used antibiotics inferior in the
combat of infections.1 Frequently the resistance is mediated by
multidrug resistance (MDR) pumps, which are members of the
large classes of transporters, known to be involved in uptake and
exchange of key nutrients, ions, metabolic vaste, chemotactics,
communication and hazardous substances.2 The Staphylococcus
aureus native pump NorA of the Major Facilitator Superfamily
(MFS) belongs to a class of proton motive force (PMF) dependant
pumps that include symporters and antiporters. These transporters
are involved in the utilization of sugars, anions, metabolites and
drugs.3,4 Among the drugs identified as substrates for this pump
are fluoroquinolones,5,6 phenothiazines, thioxanthenes,7 quarter-
nary ammonium compounds and anti-septics,8,9 verapamil,10 ome-
prazole,11 reserpine, totarol, ferruginol, carnosic acid, and dyes like
ethidium bromide, rhodamine, and acridines.12 Overexpression of
the transporters mediates drug resistance.13 A survey conducted
in the USA revealed that about half of bloodstream isolates of S.
aureus (n = 232) possess efflux pumps that contributed strongly
to resistance and 22.8% of these were identified as NorA-overex-
pressors.13 Recent clinical results indicate that combination of an
rhamnose that act as a potent inhibitor of the NorA pump.15
Encouraged by this finding we screened a library of 117 chalcones
assuming that the cinnamoyl moiety was crucial for the pump
inhibitory activity.
The present study reports the NorA inhibitory activities of the
chalcones and their ability to potentiate the activity of ciprofloxa-
cin against resistant S. aureus.
2. Material and methods
2.1. Preparation of compounds
Compound 1–7, 12–16 and 20 were synthesized as described
previously.16–20 Compounds 8–11, 17–19 are new compounds.
Their preparation is reported below. The nuclear magnetic reso-
nance (NMR) spectra were recorded on a Varian Mercury 300 MHz
instrument and high-resolution mass-spectrometry (HRMS) on a
Bruker micrOTOF-Q mass spectrometer.
R2'
R4
R3'
R4'
R1' R5
R3
R2
⇑
Corresponding author. Address: University of Copenhagen, Faculty of Pharma-
ceutical Sciences, Department of Drug Design and Pharmacology, Universitetspar-
ken 2, 2100 Copenhagen, Denmark. Tel.: +45 35 33 62 93; fax: +45 35 33 60 41.
E-mail address: jgh@farma.ku.dk (J.G. Holler).
R5'
O
R1
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.025

J. G. Holler et al. / Bioorg. Med. Chem. 20 (2012) 4514–4521
4515
and C5), 161.38 (C4), 190.66 (CO), 125.47 (CO-C@), 144.97 (C-
C@), 132.20 (C20 and C60), 115.66 (C30 and C50), 164.42 (C40). HRMS:
Calcd for [C25H26NO3]+: 388.1907. Found 388.1896.
0
0
0
0
0
1. R1,2,3,4,5 = H, R1 ,2 ,4 ,5 = H, R3 = –OCH2–CH2–N(CH3)2.
0
0
0
0
0
2. R1,2 = OCH3, R3,4,5 = H, R1 ,2 ,4 ,5 = H, R3 = –OH.
3. R1,4,5 = H, R2,3 = OCH3, R1 ,2 ,4 ,5 = H, R3 = –OH.
0
0
0
0
0
0
0
0
0
0
4. R1,3 = OCH3, R2,4,5 = H, R1 ,2 ,4 ,5 = H, R3 = –OH.
2.1.3. Preparation of 4-dimethylamino-40-
0
0
0
0
5. R1 = OCH3, R2,4 = H, R3 = –OH, R5 = –CH3, R1 ,2 ,4 ,5 = H,
dimethylaminoethoxychalcone (10)
0
R3 = –OH.
The title compounds was prepared as described above for
(8) using 40-(2-dimethylaminoethoxy)acetophenone (99 mg, 0.48
mmol) and 4-dimethylaminobenzaldehyde (72 mg, 0.48 mmol) in
MeOH (2 mL) as starting materials, mp 124–125 °C.
0
0
0
0
0
6. R1,4 = OCH3, R2,3,5 = H, R1 ,2 ,4 ,5 = H, R3 = –OH.
0
0
0
0
7. R1,2,4,5 = H, R3 = –OCH2–CO–OH, R1 ,2 ,3 ,4 = H,
0
R5 = –OCH2–CH–CH2.
0
0
0
0
0
8. R1 = F, R2,3,4,5 = H, R1 ,2 ,4 ,5 = H, R3 = –OCH2–CH2–N(CH3)2.
9. R1,2,4,5 = H, R3 = OC6H5, R1 ,2 ,4 ,5 = H,
R3 = –OCH2–CH2–N(CH3)2.
10. R1,2,4,5 = H, R3 = –N(CH3)2, R1 ,2 ,4 ,5 = H,
R3 = –OCH2–CH2–N(CH3)2.
11. R1,3,5 = H, R2,4 = OCH3, R1 ,3 ,4 ,5 = H, R2 = –O–OC–C6H5.
12. R1,3,4,5 = H, R2 = N(CH3)2, R1 ,2 ,3 = OCH3, R4 ,5 = H.
13. R1,3,5 = H, R2,4 = OCH3, R1 ,2 ,3 = OCH3, R4 ,5 = H.
1H NMR (300 MHz, CDCl3): d 2.36 (s, 6H, (CH3)2N-CH2) 2.77 (t,
J = 5.71 Hz, 2H, CH2-N) 3.05 (s, 6H, (CH3)2N-C4) 4.15 (t,
J = 5.71 Hz, 2H, CH2-O) 6.70 (d, J = 8.79 Hz, 2H, H30 and H50) 7.00
(d, J = 8.79 Hz, 2H, H2 and H6) 7.36 (d, J = 15.24 Hz, 1H, CO-CH)
7.56 (d, J = 8.79 Hz, 2H, H2 and H6) 7.80 (d, J = 15.24 Hz, 1H, CO-
C@CH) 8.03 (d, J = 9.08 Hz, 2H, H20 and H60).
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
13C NMR (75 MHz, CDCl3):
d 40.41 [(CH3)2N-CH2], 45.97
0
0
0
0
0
[(CH3)2N-C4], 59.07 (N-CH2), 67.00 (O-CH2), 123.99 (C1), 131.85
(C2 and C6), 113.13 (C3 and C5), 153.99 (C4), 132.94 (C10),
131.92 (C20 and C60), 115.55 (C30 and C50), 164.12 (C40), 191.11
(CO), 116.93 (CO-C), 147.27 (CO-C@C). HRMS: Calcd for
[C21H27N2O2]+: 339.2067. Found 339.2062.
0
0
0
0
0
14. R,1,2 = OCH3, R3,4,5 = H, R1 ,2 ,4 ,5 = H, R3 = O–CH2–CH–CH2.
0
0
0
0
0
15. R1.3 = OCH3, R2,4,5 = H, R1 = OCH3, R2 ,3 ,4 ,5 = H.
16. R1,3,5 = OCH3, R2,4 = H, R1 ,2 ,4 ,5 = H, R3 = OH.
17. R1,2,4,5 = H, R3 = –CN, R1 ,2 ,4 ,5 = H, R3 = –OCH2–CH2–N(CH3)2.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
18. R1,3,4,5 = H, R2 = –F, R1 ,2 ,4 ,5 = H, R3 = –OCH2–CH2–N(CH3)2.
0
0
0
0
0
19. R1,2,4,5 = H, R3 = –F, R1 ,2 ,4 ,5 = H, R3 = –OCH2–CH2–N(CH3)2.
2.1.4. Preparation of 3,5-dimethoxy-30-benzoyloxychalcone (11)
A solution of sodium hydroxide (6 M, 1 mL) was added to a
solution of 3-hydroxyacetophone (113 mg, 0.83 mmol) and 3,5-
dimethoxybenzaldehyde (138 mg, 0.83 mmol) in MeOH (1 mL).
The reaction mixture was stirred at 50 °C for 18 h and at 4 °C over-
night. The precipitate was washed with MeOH and water to give
3,5-dimethoxy-3-hydroxychalcone (100 mg, 43%). To a solution
of the crude chalcone (90 mg, 0.3 mmol) in dichloromethane
(3 mL) was added pyridine (0.7 mL) and benzoyl chloride (53 mg,
0.38 mmol). The reaction mixture was stirred at room temperature
for 4 h.
To the reaction mixture was added a saturated aqueous solution
of NaHCO3 (30 mL), and the mixture was extracted with ethyl ace-
tate (3 ꢀ 20 mL). The combined organic layers were sequentially
washed with an aqueous solution of NaOH (2 M, 10 mL), HCl
(2 M, 10 mL), and brine (20 mL), dried over magnesium sulfate,
and concentrated in vacuo. Recrystallization of the residue (MeOH
and water) afforded the title compound 66 mg (53.6%), mp 110–
111 °C.
20. R1,3,4,5 = H, R2 = –OCH2–CH2–N(CH3)2,
0
0
0
0
0
R1 ,2 ,3 = OCH3, R4 ,5 = H.
2.1.1. Preparation of 2-fluoro-40-dimethylaminethoxychalcone
(8)
To an aqueous solution of sodium hydroxide (6 M, 0.5 mL) was
added
a
solution of 40-(2-dimethylaminoethoxy)acetophenone
(99 mg, 0.48 mmol) and 2-fluorobenzaldehyde (60 mg, 0.48 mmol)
in MeOH (2 mL). The reaction mixture was stirred at room temper-
ature for 2 h and left at 4 °C overnight to give a precipitate. Recrys-
tallization (MeOH–water) afforded the title compound (107 mg,
70%), mp 60–61 °C.
1H NMR (300 MHz, methanol-d4): d 2.34 (s, 6H, [(CH3)2N-CH2])
2.79 (t, J = 5.42 Hz, 2H, N-CH2) 4.19 (t, J = 5.57 Hz, 2H, O-CH2) 7.07
(d, J = 9.08 Hz, 2H, H30 and H50) 7.18 (ddd, J = 11.0, 8.0, 1.0 Hz, 1H,
H3) 7.25 (td, J = 7.00, 1.00 Hz, 1H, H5) 7.38–7.50 (m, 1H) 7.76–7.94
(m, 1H, H6) 7.89 (d, J = 16.00 Hz, 1H, COCH@C) 7.81 (d, J = 16.00 Hz,
1H, COC@CH) 8.08 (d, J = 8.79 Hz, 2H, H2 and H6).
13C NMR (75 MHz, CDCl3): d 45.96 [(CH3)2N-CH2], 59.02 (N-
CH2), 67.04 (O-CH2), 124.33 (C1), 163.06 (d, J = 250 Hz, C2),
117.19 (d, J = 22 Hz, C3), 133.46 (d, J = 9 Hz, C4), 126.05 (C5),
125.09 (d, J = 4.5 Hz, C6), 130.35 (C10), 132.29 (C20 and C60),
115.73 (C30 and C50), 164.57 (C40), 190.30 (CO), 126.01 (CO-C),
137.17 (d, J = 3.7 Hz, CO-C@C). HRMS: Calcd for [C19H21FNO2]+:
314.1551. Found 314.1540.
1H NMR (300 MHz, acetonitrile-d3): d 3.81 (s, 6H, [CH3O]2) 6.57
(t, J = 2.34 Hz, 1H, H4) 6.92 (d, J = 2.34 Hz, 2H, H2 and H6) 7.52
(ddd, J = 8.00, 2.50, 1.00 Hz, 1H, H40) 7.59 (t, J = 8.00 Hz, 2H, H300
and H500) 7.63 (t, J = 8.00 Hz, 1H, H50) 7.74 (dt, J = 7.50, 1.50 Hz,
1H, H400) 7.67 (d, J = 15.50 Hz, 1H, COCH@C) 7.73 (d, J = 15.50 Hz,
1H, COC@CH) 7.96 (t, J = 1.90 Hz, 1H, H20) 8.03 (dt, J = 7.50,
1.50 Hz, 1H, H60) 8.20 (dt, J = 7.00, 2.00 Hz, 2H, H200 and H600).
13C NMR (75 MHz, acetonitrile-d3): d 56.24 (CH3O), 130.33 (C1),
107.47 (C2), 162.14 (C3), 103.77 (C4), 162.14 (C5), 107.47 (C6),
137.79 (C10), 122.97 (C20), 152.37 (C30), 127.07 (C40), 127.56 (C50),
130.92 (C60), 189.74 (CO), 123.30 (CO-C), 145.50 (CO-C@C),
166.05 (OCO), 140.50 (C100), 130.92 (C200 and C600), 129.85 (C300
and C500), 134.93 (C400). HRMS: Calcd for [C24H20NaO5]+: 411.1203.
Found 411.1191.
2.1.2. Preparation of 4-phenoxy-40-
dimethylaminoethoxychalcone (9)
Compound (9) was prepared as described above for 8 using 40-
(2-dimethylaminoethoxy)acetophenone (99 mg, 0.48 mmol) and
4-phenoxybenzaldehyde (72 mg, 0.48 mmol) in MeOH (2 mL) as
starting materials, 101–102 °C.
1H NMR (300 MHz, methanol-d4) d: 2.35 (s, 6H, (CH3)2N) 2.79 (t,
J = 6.00 Hz, 2H, N-CH2) 4.19 (t, J = 6.00 Hz, 2H, CH2-O) 7.07 (d,
J = 15.00 Hz, 1H, HC@C) 7.01 (d, J = 15.00 Hz, 1H, HC@C) 6.99 (d,
J = 7.50 Hz, 2H, H200 and H600) 7.07 (d, J = 7.33 Hz, 2H, H30 and
H50) 7.18 (t, J = 8.00 Hz, 1H, H400) 7.40 (t, J = 8.00 Hz, 2H, H300 and
H500) 7.71 (d, J = 7.00 Hz, 2H, H2 and H6) 7.73 (d, J = 8.50 Hz, 2H,
H3 and H5) 8.09 (d, J = 8.50 Hz, 2H, H20 and H60).
2.1.5. Preparation of 4-cyano-40-dimethylaminethoxychalcone
(17)
To an aqueous solution of sodium hydroxide (6 M, 0.5 mL) was
added
a
solution of 40-(2-dimethylaminoethoxy)acetophenone
(99 mg, 0.48 mmol) and 4-cyanobenzaldehyde (63 mg, 0.48 mmol)
in MeOH (2 mL). The reaction mixture was stirred at room temper-
ature for 2 h and left at 4 °C overnight to give a precipitate. Recrys-
tallization (MeOH–water) afforded the title compound (107 mg,
70%), mp 120–121 °C.
13C NMR (75 MHz, methanol-d4) d: 45.98 [(CH3)2N], 59.04 (C-N),
67.05 (C-O), 157.59 (C100), 119.43 (C200 and C600), 131.24 (C300 and
C500), 121.60 (C400), 132.37 (C1), 131.73 (C2 and C6), 120.94 (C3

4516
J. G. Holler et al. / Bioorg. Med. Chem. 20 (2012) 4514–4521
1H NMR (300 MHz, methanol-d4) d 2.80 (t, J = 5.42 Hz, 2H,
(>99%, Merck, Germany) due to lower solubility of chalcones in
aqueous media. Then diluted in MHB II (MHB II, Oxoid, Hamp-
NCH2) 4.21 (t, J = 5.42 Hz, 2H, OCH2) 7.06–7.12 (m, 2H, H30 and
H50) 7.77 (d, J = 7.03 Hz, 2H, H2 and H6) 7.90 (m, 4H, COC@CH,
COCH@C, and H3 and H5) 8.07–8.16 (m, 2H, H20 and H60). 13C
NMR (75 MHz, CDCl3): d 45.98 [(CH3)2N-CH2], 59.03 (N-CH2),
67.09 (O-CH2), 141.06 (C1), 130.31 (C2 and C6), 133.91 (C3 and
C5), 114.36 (C4), 131.93 (C10), 132.41 (C20 and C60), 115.76 (C30
and C50), 164.70 (C40), 189.91 (CO), 126.26 (CO-C), 142.75 (CO-
C@C), 119.56 CꢁN. HRMS: Calcd for [C20H21N2O2]+: 321.1598.
Found 321.1581.
shire, UK) to
a
final concentration of 20 lg/mL (final
[DMSO] 6 1%). This preparation allowed clear solutions with no
precipitation. Chemicals used were CCCP (carbonyl cyanide 3-
chlorophenylhydrazone, P98.0% (HPLC), Fluka) and EtBr (ethi-
dium bromide solution (BioReagent, for molecular biology,
10 mg/mL in H2O, Sigma–Aldrich). The positive control was
reserpine (P99.0%, HPLC, Fluka, Switzerland) at 20 lg/ml, and
the negative control was 1% DMSO in MHB II. Background was
measured on a resuspended pellet in clean MHB II. Measure-
ments were carried out in duplicate on a spectrofluorometer
(Perkin Elmer, LS-50B luminescence spectrometer, England)
using excitation kex = 530 nm, emission: kem = 600 nm. Slit
5 mm excitation and 10 mm emission. Mean results were ex-
pressed as percentage reduction of efflux compared to control.
2.1.6. Preparation of 3-fluoro-40-dimethylaminethoxychalcone
(18)
The title compounds was prepared as described above for
8 using 40-(2-dimethylaminoethoxy)acetophenone (99 mg, 0.48
mmol) and 3-fluorobenzaldehyde (60 mg, 0.48 mmol) in MeOH
(2 mL) as starting materials, mp 59–60 °C.
1H NMR (300 MHz, methanol-d4) d: 2.28 (s, 6H, [(CH3)2N-CH2])
2.72 (t, J = 5.57 Hz, 2H, N-CH2) 4.09 (t, J = 5.57 Hz, 2H, O-CH2) 6.94
(d, J = 8.79 Hz, 2H, H30 and H50) 6.98–7.07 (m, 1H, H2) 7.24–7.36
(m, 3H, H4, H5, and H6) 7.48 (d, J = 16.00 Hz, 1H, CO-CH@C) 7.65
(d, J = 16.00 Hz, 1H, CO-C@CH) 7.95 (d, J = 8.79 Hz, 2H, H20 and H60).
13C NMR (75 MHz, CDCl3): d 45.75 [(CH3)2N-CH2], 58.01 (N-
CH2), 65.97 (O-CH2), 137.27 (d, J = 7.5 Hz, C1), 132.00 (d,
J = 22 Hz, C2), 163.04 (d, J = 244 Hz, C3), 117.48 (d, J = 22 Hz, C4),
130.62 (d, J = 22 Hz, C5), 124.67 (d, J = 2.2 Hz, C6), 131.62 (C10),
131.09 (C20 and C60), 114.56 (C30 and C50), 162.89 (C40), 189.03
(CO), 123.02 (CO-C), 142.95 (CO-C@C). HRMS: Calcd for
[C19H21FNO2]+: 314.1551. Found 314.1537.
2.4. Preparation ofeverted membrane vesicles. Measurement of
the proton gradient using acridine orange and inhibition of
Hoechst 33342 efflux
To investigate NorA inhibitory activity of the inhibitors in ab-
sence of cytosolic machinery a subcellular model of everted mem-
brane vesicles with the NorA efflux pump, was constructed. The
method is based on the cloning of the norA gene into an E. coli
expression vector and the subsequent transformation of the vector
into E. coli and expression of NorA. The advantage is that NorA can
be expressed in absence of other S. aureus native efflux pumps.
Preparation of everted membrane vesicles was carried out from
an overnight culture of E. coli DH10B bearing the plasmid pTr-
cHis2C-norA or pTrc99A according to Yu et al., 2002.23 The method
was modified by adding protease inhibitor PMSF (phen-
ylmethanesulfonyl fluoride, P98.5%, Sigma, USA) (0.1 mM final) be-
fore disruption through French Press and Pancreatic DNAse (Sigma,
USA) (0.1 mg/mL final) to the cell lysate before centrifugation. Ves-
icles were adjusted to 20 mg/mL in 50 mM potassium phosphate
buffer (pH 7.2) supplemented with 10% glycerol using BCA™ Pro-
tein Assay Kit (Thermo Scientific, USA) and stored in aliquots of
0.2 mL at ꢂ70 °C until use. To ensure viable function a measure-
ment of the proton gradient across the membrane was performed.
2.1.7. Preparation of 4-fluoro-40-dimethylaminethoxychalcone
(19)
The title compounds was prepared as described above for
(8) using 40-(2-dimethylaminoethoxy)acetophenone (99 mg, 0.48
mmol) and 4-fluorobenzaldehyde (60 mg, 0.48 mmol) in MeOH
(2 mL) as starting materials, 109–110 °C.
1H NMR (300 MHz, methanol-d4): d 2.35 (s, 6H, [(CH3)2N-CH2])
2.80 (t, J = 5.42 Hz, 2H, N-CH2) 4.20 (t, J = 5.42 Hz, 2H, O-CH2) 7.08
(d, J = 8.79 Hz, 2H, H30 and H50) 7.17 (t, J = 8.79 Hz, 2H, H3 and H5)
7.74 (s, 2H, HC@CH) 7.80 (dd, J = 8.79, 5.27 Hz, 2H, H2 and H6) 8.11
(d, J = 9.08 Hz, 2H, H20 and H60).
Vesicles were diluted to a concentration of 80 lg protein/mL in
13C NMR (75 MHz, CDCl3): d 45.98 [(CH3)2N-CH2], 59.05 (N-
CH2), 67.06 (O-CH2), 133.00 (C1), 132.00 (d, J = 16 Hz, C2 and C6),
117.15 (d, J = 22 Hz, C3 and C5), 164.9 (d, J = 248 Hz, C4), 132.71
(C10), 132.27 (C20 and C60), 115.69 (C30 and C50), 164.51 (C40),
190.54 (CO), 122.78 (CO-C), 144.20 (CO-C@C). HRMS: Calcd for
[C19H21FNO2]+: 314.1551. Found 314.1549.
2 mL 50 mM potassium HEPES (HEPES Enzyme Grade, 99% mini-
mum (Fischer Scientific, USA), 8.5 mM sodium chloride (sodium
chloride, Certified ACS Crystalline (Fischer Scientific, USA) 2 mM
magnesium (magnesium chloride hexahydrate, GR, ACS Crystals
(EMD Chemicals Inc., USA) (pH 7.2) buffer. Acridine orange was
added to a final concentration of 500 nM. Addition of Mg2+-ATP
(adenosine 50-triphosphate magnesium salt P95%, bacterial. Sigma,
2.2. Bacterial strains and growth conditions
USA) to a final concentration of 50
force. The ionophores valinomycin (P98% Sigma, USA) and nigeri-
cin (sodium salt >98%, Sigma, USA) were added (2 and 5 M, respec-
tively) to dissipate the electrical and proton gradients, respectively.
Norfloxacin (P98%, Sigma, USA) as a positive control, was added to
lM generated a proton motive
S. aureus ATCC 29213, SA1199B and K1758 was used for poten-
tiation and MIC determinations. K1758 is a norA knock-out and
SA1199B is a norA overexpressor derived from a methicillin sus-
ceptible S. aureus bloodstream isolate.21,22 These were provided
by Dr. Glenn W. Kaatz, John D. Dingell VA Medical Center, Detroit
MI, USA. Strains were cultivated in Mueller Hinton growth medium
II (Oxoid, Hampshire, UK).
l
a final concentration of 10 lM. Fluorescence was determined using
a Shimadzu RF-5301PC spectrofluorometer and wavelengths of
494 nm (excitation) and 530 nm (emission). Measurement of inhi-
bition of Hoechst 33342 efflux was carried out by diluting vesicles
to a concentration of 80 lg protein/mL in 2 ml 50 mM potassium
HEPES, 8.5 mM sodium chloride, 2 mM magnesium chloride (pH
Escherichia coli DH10B background with pTrcHis2C-norA23 was
kindly provided by Dr. David C. Hooper. Control strain DH10B con-
taining pTrc99A was constructed by transformation using CaCl2 as
stated by.24
7.2) buffer. Within 10 s of running time Hoechst 33342 (P98%, Sig-
ma, USA) was added to a final concentration of 50
rescence became stable (ꢃ30 s), NorA was activated by addition of
Mg2+-ATP (50
M final concentration) to generate proton motive
lM. When fluo-
2.3. Ethidium bromide efflux inhibition assay
l
force. At 150–160 s of assay time the inhibitor was added. Fluores-
cence was determined using wavelengths of excitation
kex = 355 nm, emission: kem = 457 nm over a 300 s time course.
The assay was performed according to.25 Test solutions was
prepared by dissolving compound in dimethyl sulfoxide (DMSO)

J. G. Holler et al. / Bioorg. Med. Chem. 20 (2012) 4514–4521
4517
Measurements were carried out in triplicate on independent vesicle
3.2. Inhibition of NorA mediated ethidiumbromide efflux
batches and expressed as mean of three with standard deviations.
Among the 117 chalcones previously screened for antiplasmod-
ic activity 10 compounds inhibited the NorA transporter just as
efficient as or better than the positive control reserpine at a con-
2.5. Susceptibility and potentiation studies
centration of 20 lg/mL. The IC50-values for the 10 most potent
chalcones as inhibitors of ethidium bromide efflux are presented
in Table 1. Chalcones 9 and 10 are equipotent to the positive con-
trol reserpine with IC50-values of 9.0 and 7.7 lM, respectively.
Compounds 5–8 are approximately half as potent. Examples of
concentration–response relationships for 5, 7, 9, and reserpine
are depicted in Figure 1. The high MIC-value (25 lg/mL) showed
that the inhibition of EtBr efflux by 9 was not caused by a killing
Minimum inhibitory concentration (MIC) determination was by
the microdilution method. Final volume in each well was 200 lL
with an inoculum size of 5 ꢀ 105 cfu/mL. The MIC was the lowest
concentration at which no visible growth was observed after 20 h
of incubation at 35 °C as inspected on a Micur-Viewer. Positive
controls were a bacterial suspension in MHB II and a bacterial sus-
pension in MHB II with DMSO in concentrations corresponding to
the highest quantity present (ꢄ1%). Negative controls were wells
with only MHB II and MHB II with test compound. A MIC-determi-
nation control for solvent DMSO and ciprofloxacin (Ratiopharm
ApS, Denmark) were used as positive controls. All MIC-determina-
tions were performed in accordance with CLSI/EUCAST guidelines
in duplicate or more.
of the S. aureus. The remaining compounds did not exhibit antibac-
terial activity within the range 0.2–25 lg/mL.
3.3. Inhibition of the NorA efflux pump in enriched membrane
vesicles
Potentiation studies was carried out as a checkerboard assay
determining ciprofloxacin MIC in the presence of increasing
amounts EPI against S. aureus SA1199B, ATCC 29213 and the norA
knock-out strain K1758. Assays were carried out in microtitre
plates using the checkerboard synergy method as adapted from
Lomovskaya et al. 2001 and Smith et al. 2005.26,27 Twofold serial
dilutions in MHB were carried out, yielding end concentrations of
The functionality of the NorA pump enriched vesicles and con-
trol vesicles upon addition of Mg2+-ATP were verified by measuring
fluorescence changes caused by the activation of F0F1 H+-ATPase
(Data not shown) using the optical probe acridine orange.
NorA-enriched vesicles and control vesicles showed similar
magnitude of change in fluorescence. Valinomycin dissipated the
electrical gradient and nigericin the proton gradient. When nor-
0.05–12.5
l
g/mL and 0.2–25
l
g/mL for ciprofloxacin and EPI,
L with an inocu-
floxacin was added at 10 lM, the fluorescence changed dramati-
respectively. Final volume in each well was 200
l
lum size of 5 ꢀ 105 cfu/mL. Plates were incubated at 35 °C for 20 h
and read as above. Ciprofloxacin-EPI interactions were classified
using Fractional Inhibitory Concentration (FIC) index. The FIC index
was calculated as the sum of Fractional Inhibitory Concentrations
(FICs) of each of the drug.
cally for the NorA-enriched vesicles indicating loss of proton
gradient due to the operation of the NorA efflux pump. Test com-
pounds and the positive control verapamil provoked a time-depen-
dent increase in fluorescence as expected for NorA inhibitors.
Analogously the 20 most active chalcones that inhibited the ef-
flux of Hoechst 33342 dye is depicted in Figure 2. No decrease in
fluorescence was observed from control vesicles lacking NorA, con-
sistent with Hoechst 33342 being a substrate for this efflux
pump.23 Chalcones 2, 3, and 11–15 exerted activity similar to or
less than verapamil, whereas the remaining 13 compounds were
better at inhibiting Hoechst 33342 efflux.
FICs = (MIC of drug A or B in combination)/(MIC of drug A or B
alone). FIC indices are 60.5 = synergy. 1.0 = Additivity. 2.0 = Indif-
ferent and P4.0 = antagonism.7
3. Results
When adding the chalcones or verapamil to the vesicles a time-
dependent increase in fluorescence occurred, consistent with
activity through inhibition of NorA (Fig. 3). Some compounds also
exert quenching of the Hoechst 33342 dye when testing them
without first adding the Mg2+-ATP, for example, 20 (Fig. 3). The
quenching appeared instantly, and the magnitude was increased
with increasing concentrations of the inhibitor. In the control ves-
icles pTrc99A, lacking NorA a similar, but smaller quenching was
evident. The lack of NorA in these proves that the pump is not in-
volved in the observed changes (Fig. 3).
3.1. Origin of chalcones
The majority of the chalcones initially screened were previously
prepared in an attempt to find new drugs against malaria.17 The
new chalcones (8–11, and 17–19) have been prepared by a Clais-
en–Schmidt condensation of the appropriate benzaldehyde with
the appropriate acetophenone. Structures of new compounds were
substantiated by 1H, 13C NMR, and HRMS.
Table 1
Inhibition of EtBr efflux in SA1199B by the 10 most potent inhibitors
Compound
Concentration of EPI (
l
g/mL)
20
IC50
(lg)
IC50 (lM)
2.5
5
10
1
2
3
4
5
6
7
8
21.8
11.7
14.0
17.3
36.6
23.3
14.7
33.7
43.4
48.0
30.0
36.1
38.0
38.3
24.1
63.7
56.6
46.2
48.5
68.8
96.5
48.1
56.9
73.3
64.3
62.7
84.3
82.7
79.9
70.3
97.5
97.4
71.8
83.6
8.4
6.9
7.6
8.3
3.8
4.3
5.7
5.7
3.5
2.6
5.1
28.4
24.3
26.5
29.1
13.4
15.0
16.9
18.1
9.0
90.8
87.9
89.6
91.7
91.0
87.1
97.0
99.1
98.7
85.1
9
10
Reserpine
7.7
8.2
Data are presented are the mean of two measurements and expresses percent
reduction of ethidium bromide efflux at a given concentration.
Figure 1. Dose–response relationships of compounds 5, 7, 9 and positive control
reserpine at inhibiting EtBr-efflux in the NorA-overexpressor SA1199B.

4518
J. G. Holler et al. / Bioorg. Med. Chem. 20 (2012) 4514–4521
Figure 2. Inhibition of NorA-mediated Hoechst 33342 efflux by the chalcones. NP is the natural product kaempferol-3-O-
a-L-(2,4-bis-E-p-coumaroyl)rhamnoside isolated
from Persea lingue Nees.15 Black bars represents EPI activity at 2
l
g/mL and grey bars the activity when normalized to verapamil equivalents. EPI levels presented are the
mean of three measurements with standard deviation depicted as error bars.
In the control vesicles with no NorA present (pTrc99A) and
using acridine orange instead of Hoechst 33342, compound 1–10
and 17–20 and verapamil exerted an activity similar to that of
nigericin (e.g., 20 in Fig. 4) indicating a disruption of the proton
gradient. Compound 2–7 showed no changes of acridine orange
fluorescence (e.g., chalcone 5 Fig. 4). Norfloxacin did not exert
activity, nor did DMSO.
3.4. Susceptibility testing and potentiation studies
Compounds were studied for antimicrobial activity by means of
a MIC determination and potentiating studies were carried out by
checkerboard combinations of inhibitors with known NorA sub-
strate ciprofloxacin. The ability of the 10 most potent chalcones
to enhance the antibacterial activity of ciprofloxacin was evaluated
against three strains of S. aureus (SA1199B, ATCC 29213 and
K1758) and is comprised in Table 2. A FIC-index 60.5 represents
synergic activity.
Figure 3. Hoechst 33342 efflux inhibition in vesicles pTrcHis2C-NorA with and
without addition of ATP and control vesicles pTrc99A. (A) 100 nM Hoechst 33342,
(B) 50 l lg/mL chalcone 20.
M Mg2+-ATP; (C) 2
All compounds, except 1, 8, 10 and verapamil exerted fourfold
reductions of ciprofloxacin MIC against both strains expressing
NorA. Positive control reserpine showed same degree of potentia-
tion at 6.25
pound 9 showed the highest degree of potentiation (fourfold at
3.13 g/mL) and was approximately twice as potent as reserpine
lg/mL and compound 5, 7 were equally potent. Com-
l
and chalcones 5 and 7. FIC-indices calculated for the combinations
exerting a minimum of fourfold reduction in MICciprofloxacin were
<0.5 for all but 1, 8, 10 and verapamil against both strains. These
combinations are therefore synergic in their activity. The potentia-
tion was concentration dependent and compounds 5 and 7 both
showed an 8-fold and 16-fold reduction of ciprofloxacin MIC at
12,5 and 25
16-fold reduction of ciprofloxacin MIC at 6.25 and 12.5
respectively. However at 25 g/mL 9 was antibacterial alone. None
of the other compounds showed antibacterial activity at 0.2–
25 g/mL. The concentration of compound 9 needed for exhibiting
l
g/mL, respectively. Compound 9 exerted 8-fold and
lg/mL,
l
l
a synergistic activity was approximately eight times less than the
MIC value, making it unlikely that the antibacterial activity is con-
tributing essentially to more than the efflux activity. Chalcone 10
exerted surprisingly low potentiating activity, which is contradic-
tory to the potent EtBr efflux inhibitory activity. None of the
Figure 4. Measurement of fluorescence changes of acridine orange in control
vesicles pTrc99A. (A) 500 nM acridine orange, (B) 50
chalcone 5 or 20.
l lg/ml
M Mg2+-ATP; (C) 2

J. G. Holler et al. / Bioorg. Med. Chem. 20 (2012) 4514–4521
4519
Table 2
Results of potentiation studies of chalcones with ciprofloxacin against S. aureus strains with different expression levels of norA
Compound(s)
D
norA K1758
ATCC29213
CIP MIC
SA1199B
CIP MIC
FIC-index (ATCC 29213)
FIC-index (SA1199B)
CIP MIC
[EPI]
[EPI]
[EPI]
Ciprofloxacin
+1
+2
+3
+4
+5
+6
+7
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.2
0.2
0.4
0
25
12.5
25
12.5
6.25
12.5
6.25
25
3.13
25
6.25
25
0.4
0.2⁄
0.1
0.1
0.1
0.1
0.1
0.1
NAP
0.1
0.2
0.1
0.4
0
25
6.25
25
6.25
12.5
12.5
3.13
25
6.25
12.5
6.25
25
6.25
3.13
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
NAP
1.56
6.25
0
25
12.5
25
12.5
6.25
12.5
6.25
25
3.13
25
6.25
25
NAP
>0.5
0.25
0.25
0.25
0.25
0.25
0.25
>1
0.26
0.5
0.25
>1
NAP
>0.5
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.38
NAP
0.25
>1
+8
+9
+10
+Reserpine
+Verapamil
Reserpine and verapamil was included as positive controls.
NAP = no antimicrobial potentiation. Presented results are the combinations that produce at least fourfold decrease in ciprofloxacin MIC except marked ꢅ producing only
twofold decrease. For each strain two columns are shown representing the MIC of ciprofloxacin (CIP MIC) and the corresponding EPI concentration ([EPI]) to produce this MIC.
MIC and EPI are lg/mL.
compounds potentiated the activity of ciprofloxacin against the
NorA deletion strain, confirming that the activity was directed at
the NorA function.
Some compounds instantly exerted quenching of the Hoechst
33342 fluorescence when added to unenergized vesicles as well
as the energized pTrc99A control vesicles. This quenching was in
both cases increased with higher concentrations of the test com-
pound. A difference in the magnitude of this interference may be
explained by the differences of the two types of vesicles, one being
enriched with NorA and containing up to 5–10% of this protein in
the membrane and the other lacking NorA thereby being different
matrices for Hoechst 33342 accumulation and equilibrium in the
membrane.23
4. Discussion and conclusion
A kaempferol rhamnoside has been shown to be a potent NorA
inhibitor.15 Inspired by the structural similarities of the coumaroyl
substituents and chalcones a series of 117 chalcones were tested as
EPI inhibitors. At 20 lg/mL 10 of the chalcones inhibited NorA as
Inoue et al. (1982) has shown that quenching of fluorescence for
some chalcones could be the result of interference with PMF. This
possibility was precluded by measuring fluorescence changes in
the control vesicles using the proton gradient probe acridine or-
ange in presence of the EPIs.33 Chalcone 1, 8–10, 17–20 and verap-
amil exerted activity similar to nigericin, which interferes with the
proton gradient. The remainder did not show changes of acridine
orange fluorescence (e.g., chalcone 5, Fig. 4). Neither norfloxacin
nor the solvent DMSO interferes with the fluorescence observed.
Verapamil is known to inhibit NorA induced norfloxacin ef-
flux,10 but also for being a calcium channel blocker. However,
verapamil does also inhibit H+-ATPase generated trans-membrane
proton electrochemical gradient.34 These properties of verapamil
could explain the observed PMF interruption. Interestingly all the
more potent chalcones (1, 7–10, and 17–19) carry a dimethylami-
noethoxy substituent. This structural feature seems to interfere
with the PMF or acridine orange in some way similar to the action
of verapamil and nigericin. The observed NorA effect of the
dimethylaminoethoxy chalcones may therefore be the result of a
proton gradient disruption as well as a specific inhibition of NorA.
A mechanism could be inhibition of the H+-ATPase and thereby
indirectly the NorA pump through elimination of the energy
source. Of interest is the finding that phenothiazines and thiox-
anthenes in addition to affect MDR efflux pumps pertubates the
membrane energetics of the staphylococcal membrane.7 These
findings are interesting as the phenothiazines and thioxanthenes
contain amino-sidechains and especially chlorpromazine has a
dimethylamino-propyl sidechain which is similar to the dimethyl-
aminoethoxy side chain of the potent chalcones.
efficient as did reserpine (Table 1). The IC50 values of standard
compounds like reserpine, thioridazine and prochlorperazine are
reported to be 10
lM and a tryptamine derivative and SSRI paroxe-
tine 18 and 25
l
M, respectively.7,29,30 A synthetic compound of a
bisaryl urea EPI attached to ofloxacin (one of the most potent NorA
EPI found) have been shown to produce an inhibition response
over 84% at 10
rol,22 orizabin IX, XV32 has EtBr-efflux inhibitory activity 10–
50 M, respectively. Consequently 1–10 can be concluded to be
l
M,31 and natural products carnosic acid,12 tota-
l
medium potent EPI inhibitors.
To verify that the effect was caused by NorA inhibitory activity a
simpler system consisting of everted membrane vesicles enriched
with NorA were constructed. The purpose was to show the activity
of the EPIs against NorA in an isolated manner. The system was
prepared by overexpression of norA in E. coli and the construction
of everted membrane vesicles. The absence of native S. aureus
membrane transporter proteins as well as cytosolic machinery in
these vesicles constituted a strong tool for the evaluation of NorA
inhibitory activity. Showing that the EPIs exerts inhibition of this
system provided further confirmation of the activity against NorA
and also a separation of the activity from e.g. cellular targets and
the possible interference of other efflux pumps of the S. aureus
membrane.
The chalcones and the control verapamil produced a time-
dependent increase in fluorescence, consistent with an inhibition
of the Hoechst 33342 efflux. Thirteen of the chalcones (1, 4–10,
and 16–20) were more potent inhibitors than verapamil. The more
potent inhibitor reserpine could not be used as a positive control
because of interference with the fluorescence. Also the use of EtBr
was found unsuited to study NorA activity in the everted mem-
brane vesicles due to the lack of intracellular components, which
afforded only a poor difference in fluorescence of intra- and ex-
tra-vesicular EtBr. Similar other dyes including Pyronin Y, Acrifla-
vine, TMA-DPH, Nile Red, NPN, DMP and NBD-X generated poor
results (data not shown).
Chalcone inhibitors bearing the dimethylamino-ethoxy side-
chain might therefore also act by interfering with membrane ener-
getics and consequently indirectly the NorA function, but this
matter warrants further investigation.
Six chalcones (2–7) exerted good efflux inhibitory activity, but
did not show any interference with the PMF according to the

4520
J. G. Holler et al. / Bioorg. Med. Chem. 20 (2012) 4514–4521
investigations with acridine orange. These six compounds probably
are specific inhibitors of NorA-activity.
floxacin. Compound 4 was further investigated in mice and was
found to inhibit a parasitic infection with Plasmodium yoelii at a
dose of 60 mg/day without killing the host.44 The very small struc-
tural difference in between 2, 3, 4 and 6 (substitution pattern of the
methoxy group) reveals that it is difficult to predict selectivity of
the compounds from their structures. However, the results of the
present study reveal that appropriate substitution of the chalcone
skeleton might afford drugs for combating infection with multi
resistant microorganisms.
Five of these chalcones are all hydroxylated at the 40-position.
This feature is also found in flavones and isoflavones previously
studied.35,36 In addition 40-position hydroxylated chalcones iso-
lated from Dalea versicolor were able to potentiate synergistically
the activity of berberine, against S. aureus and Bacillus cereus.37
The present study suggests that in addition to the dimethylami-
noethoxy group, a hydroxy at 40-position of the B-ring and a meth-
oxy group in the 2-position of the A-ring is important for the
synergistic activity. The substitution pattern with a methoxy at
2-position in the A-ring and an additional methoxy at either posi-
tion 3, 4 or 5 does not interfere with activity, while compounds
with no substituents or a methoxy group at different positions,
potentiate less. A chalcone tested with a hydroxy at 40-position
in the B-ring and a methyl group in 2 and 4-position in the A-ring
showed no NorA inhibition determined in the EtBr-efflux assay
(data not shown). This is comparable to the findings of Guz et al.
(2001) who have investigated structure activity relationships of
several flavonolignans and flavones with affinity to NorA and found
importance of having a methoxy group instead of hydroxy at 40-po-
sition of flavones.38
Acknowledgements
We kindfully thank Dr. Glenn W. Kaatz at The John D. Dingell
Department of Veteran’s Affairs Medical Center, Detroit, Michigan,
USA and Dr. David C. Hooper, Division of Infectious Diseases, Mas-
sachusetts General Hospital, Harvard Medical School, Boston, USA
for providing strains.
A special thanks is addressed to Dr. Hiroshi Nikaido at the
Department of Molecular and Cell Biology, University of California,
Berkeley for access to his laboratory and vast knowledge on the
subject.
We investigated the 10 most potent EPIs for intrinsic antimicro-
bial activity in a MIC determination against assay strains as well a
checkerboard combinations with the known NorA substrate cipro-
floxacin. The majority of the compounds exhibited fourfold reduc-
tions of ciprofloxacin MIC against both strains expressing NorA and
no potentiating effect against the NorA-knock out strain. These
findings in conjunction with the efflux inhibitory assays are strong
evidence that the chalcones inhibit NorA-activity. The degree of
potentiating was similar to reserpine for several compounds (5, 7
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.025.
References and notes
1. Tegos, G. P.; Haynes, M.; Strouse, J. J.; Khan, M. M.; Bologa, C. G.; Oprea, T. I.;
Sklar, L. A. Curr. Pharm. Des. 2011, 17, 1291.
2. Li, X. Z.; Nikaido, H. Drugs 2004, 64, 159.
3. Yoshida, H.; Bogaki, M.; Nakamura, S.; Ubukata, K.; Konno, M. J. Bacteriol. 1990,
172, 6942.
4. Saier, M. H., Jr.; Beatty, J. T.; Goffeau, A.; Harley, K. T.; Heijne, W. H.; Huang, S.
C.; Jack, D. L.; Jahn, P. S.; Lew, K.; Liu, J.; Pao, S. S.; Paulsen, I. T.; Tseng, T. T.; Virk,
P. S. J. Mol. Microbiol. Biotechnol. 1999, 1, 257.
5. Kaatz, G. W.; Seo, S. M.; Ruble, C. A. Antimicrob. Agents Chemother. 1993, 37,
1086.
6. Neyfakh, A. A.; Borsch, C. M.; Kaatz, G. W. Antimicrob. Agents Chemother. 1993,
37, 128.
and 9) at 6.25
at 3.13 g/ml, being twice as potent as reserpine, and compounds 5
and 7. The activity was concentration dependent in that a concen-
tration of 12.5 g/ml EPI compounds 5 and 7 showed an eightfold
decrease, and at 25 g/ml a 16-fold reduction was observed with-
lg/ml. Compound 9 showed a fourfold potentiation
l
l
l
out having intrinsic antibacterial activity. Compound 9 exhibited
8-fold and 16-fold reduction of ciprofloxacin MIC at 6.25 and
12.5
rial at 25
l
g/ml, but MIC-determination showed that 9 was antibacte-
7. Kaatz, G. W.; Moudgal, V. V.; Seo, S. M.; Kristiansen, J. E. Antimicrob. Agents
Chemother. 2003, 47, 719.
8. Lewis, K.; Klibanov, A. M. Trends Biotechnol. 2005, 23, 343.
9. Noguchi, N.; Tamura, M.; Narui, K.; Wakasugi, K.; Sasatsu, M. Biol. Pharm. Bull.
2002, 25, 1129.
10. Ng, E. Y.; Trucksis, M.; Hooper, D. C. Antimicrob. Agents Chemother. 1994, 38,
1345.
11. Vidaillac, C.; Guillon, J.; Arpin, C.; Forfar-Bares, I.; Ba, B. B.; Grellet, J.; Moreau,
S.; Caignard, D. H.; Jarry, C.; Quentin, C. Antimicrob. Agents Chemother. 2007, 51,
831.
12. Stavri, M.; Piddock, L. J.; Gibbons, S. J. Antimicrob. Chemother. 2007, 59, 1247.
13. DeMarco, C. E.; Cushing, L. A.; Frempong-Manso, E.; Seo, S. M.; Jaravaza, T. A.;
Kaatz, G. W. Antimicrob. Agents Chemother. 2007, 51, 3235.
14. Amaral, L.; Martins, A.; Molnar, J.; Kristiansen, J. E.; Martins, M.; Viveiros, M.;
Rodrigues, L.; Spengler, G.; Couto, I.; Ramos, J.; Dastidar, S.; Fanning, S.;
McCusker, M.; Pages, J. M. In Vivo 2010, 24, 409.
15. Holler, J. G.; Christensen, S. B.; Slotved, H. C.; Rasmussen, H. B.; Guzman, A.;
Olsen, C. E.; Petersen, B.; Molgaard, P. J. Antimicrob. Chemother. 2012, 67, 1138.
16. Nielsen, S. F.; Christensen, S. B.; Cruciani, G.; Kharazmi, A.; Liljefors, T. J. Med.
Chem. 1998, 41, 4819.
lg/ml. This is approximately eight times the concentra-
tion needed for the fourfold reduction of ciprofloxacin MIC
towards SA1199B and an IC50 to MIC ratio of 0.14 which makes it
unlikely that the antibacterial activity is contributing to the ef-
flux-inhibition. The activity of these EPIs is comparable to e.g. p-
cyanophenyl amine,39 totarol22 and stolineferin I.40 It is puzzling
why compound 10 does not potentiate ciprofloxacin when inhibit-
ing well in both efflux assays. An explanation could be that 10 as a
dicationic compound is very much like pentamidine, which is a
splendid transporter substrate.41 Pentamidine has a strong affinity
to the NorA pump as well as QacA and MepA MDR-systems and
therefore is readily pumped out and not saturating the efflux
systems.42,43
It has been shown that compounds 2, 3, 4, and 6 inhibit the
growth of Leishmania major promastigotes, but at higher concen-
trations they interfere with proliferation of human lymphocytes
provoked by PHA. Compounds 2 and 6 almost completely inhibit
this response at 10
tion of ciprofloxacin MIC was exerted at 12.5
17. Nielsen, S. F.; Boesen, T.; Larsen, M.; Nielsen, M. B.; Kromann, H. USA Patent,
2008. http://ip.com/patent/US7423181.
18. Jasinski, J. P.; Butcher, R. J.; Khaleel, V. M.; Sarojini, B. K.; Narayana, B. Acta
Crystallogr., Sect. E 2011, 67, O813.
19. Zhai, L.; Chen, M.; Blom, J.; Theander, T. G.; Christensen, S. B.; Kharazmi, A. J.
Antimicrob. Chemother. 1999, 43, 793.
lg/mL. As potentiating agents a fourfold reduc-
lg/mL, and this small
20. Packman, A. M.; Rubin, N. Am. J. Pharm. (1835–1936) 1962, 134, 35.
21. Kaatz, G. W.; Seo, S. M.; Ruble, C. A. J. Infect. Dis. 1991, 163, 1080.
22. Smith, E. C.; Kaatz, G. W.; Seo, S. M.; Wareham, N.; Williamson, E. M.; Gibbons,
S. Antimicrob. Agents Chemother. 2007, 51, 4480.
difference makes it unlikely that 2 and 6 are safe to use for sys-
temic application. Compound 3 showed slight toxic levels at
10 lg/mL. This chalcone only reveal potentiating effect at 25 lg/
23. Yu, J. L.; Grinius, L.; Hooper, D. C. J. Bacteriol. 2002, 184, 1370.
24. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual,
2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989.
25. Kaatz, G. W.; Seo, S. M.; O’Brien, L.; Wahiduzzaman, M.; Foster, T. J. Antimicrob.
Agents Chemother. 2000, 44, 1404.
mL, making it unlikely that this compound possess a potential
for treatment of infections with multi resistant microorganisms.
In contrast, compound 4 did not affect the proliferation of human
lymphocytes in the concentrations in which it potentiated cipro-

J. G. Holler et al. / Bioorg. Med. Chem. 20 (2012) 4514–4521
4521
26. Lomovskaya, O.; Warren, M. S.; Lee, A.; Galazzo, J.; Fronko, R.; Lee, M.; Blais, J.;
Cho, D.; Chamberland, S.; Renau, T.; Leger, R.; Hecker, S.; Watkins, W.; Hoshino,
K.; Ishida, H.; Lee, V. J. Antimicrob. Agents Chemother. 2001, 45, 105.
27. Smith, E.; Williamson, E.; Zloh, M.; Gibbons, S. Phytother. Res. 2005, 19, 538.
29. Michalet, S.; Cartier, G.; David, B.; Mariotte, A. M.; Dijoux-franca, M. G.; Kaatz,
G. W.; Stavri, M.; Gibbons, S. Bioorg. Med. Chem. Lett. 2007, 17, 1755.
30. Kaatz, G. W.; Moudgal, V. V.; Seo, S. M.; Hansen, J. B.; Kristiansen, J. E. Int. J.
Antimicrob. Agents 2003, 22, 254.
37. Belofsky, G.; Percivill, D.; Lewis, K.; Tegos, G. P.; Ekart, J. J. Nat. Prod. 2004, 67,
481.
38. Guz, N. R.; Stermitz, F. R.; Johnson, J. B.; Beeson, T. D.; Willen, S.; Hsiang, J.;
Lewis, K. J. Med. Chem. 2001, 44, 261.
39. Sangwan, P. L.; Koul, J. L.; Koul, S.; Reddy, M. V.; Thota, N.; Khan, I. A.; Kumar,
A.; Kalia, N. P.; Qazi, G. N. Bioorg. Med. Chem. 2008, 16, 9847.
40. Cherigo, L.; Pereda-Miranda, R.; Fragoso-Serrano, M.; Jacobo-Herrera, N.; Kaatz,
G. W.; Gibbons, S. J. Nat. Prod. 2008, 71, 1037.
31. German, N.; Wei, P.; Kaatz, G. W.; Kerns, R. J. Eur. J. Med. Chem. 2008, 43, 2453.
32. Pereda-Miranda, R.; Kaatz, G. W.; Gibbons, S. J. Nat. Prod. 2006, 69, 406.
33. Inoue, B.; Inaba, K.; Mori, T.; Izushi, F.; Eto, K.; Sakai, R.; Ogata, M.; Utsumi, K. J.
Toxicol. Sci. 1982, 7, 245.
34. Terland, O.; Gronberg, M.; Flatmark, T. Eur. J. Pharmacol. 1991, 207, 37.
35. Stermitz, F. R.; Scriven, L. N.; Tegos, G.; Lewis, K. Planta Med. 2002, 68, 1140.
36. Morel, C.; Stermitz, F. R.; Tegos, G.; Lewis, K. J. Agric. Food Chem. 2003, 51, 5677.
41. Markham, P. N.; Westhaus, E.; Klyachko, K.; Johnson, M. E.; Neyfakh, A. A.
Antimicrob. Agents Chemother. 1999, 43, 2404.
42. Nikaido, H. Annu. Rev. Biochem. 2009, 78, 119.
43. Hassan, K. A.; Skurray, R. A.; Brown, M. H. J. Mol. Microbiol. Biotechnol. 2007, 12,
180.
44. Kharazmi, A.; Christensen, S. B.; Ming, C.; Theander, T. G. USA Patent, 2003.
http://www.freepatentsonline.com/6603046.html.