Cationic chalcone antibiotics. Design, synthesis, and mechanism of action

Article abstract of DOI:10.1021/jm049424k

This paper describes how the introduction of "cationic" aliphatic amino groups in the chalcone scaffold results in potent antibacterial compounds. It is shown that the most favorable position for the aliphatic amino group is the 2-position of the B-ring, in particular in combination with a lipophilic substituent in the 5-position of the B-ring. We demonstrate that the compounds act by unselective disruption of cell membranes. Introduction of an additional aliphatic amino group in the á-ring results in compounds that are selective for bacterial membranes combined with a high antibacterial activity against both Gram-positive and -negative pathogens. The most potent compound in this study (78) has an MIC value of 2 μM against methicillin resistant Staphylococus aureus.

Full text of DOI:10.1021/jm049424k

J. Med. Chem. 2005, 48, 2667-2677
2667
Cationic Chalcone Antibiotics. Design, Synthesis, and Mechanism of Action
Simon F. Nielsen,† Mogens Larsen,‡ Thomas Boesen,§ Kristian Schønning, and Hasse Kromann*,§
Lica Pharmaceuticals A/S, Symbion Science Park, Fruebjergvej 3, DK-2100 Copenhagen, Denmark
Received July 20, 2004
This paper describes how the introduction of “cationic” aliphatic amino groups in the chalcone
scaffold results in potent antibacterial compounds. It is shown that the most favorable position
for the aliphatic amino group is the 2-position of the B-ring, in particular in combination with
a lipophilic substituent in the 5-position of the B-ring. We demonstrate that the compounds
act by unselective disruption of cell membranes. Introduction of an additional aliphatic amino
group in the A-ring results in compounds that are selective for bacterial membranes combined
with a high antibacterial activity against both Gram-positive and -negative pathogens. The
most potent compound in this study (78) has an MIC value of 2 µM against methicillin resistant
Staphylococus aureus.
Chart 1. Linezolide (I) and Licochalcone A (II)
Introduction
The rapid development of resistance in clinically
important Gram-positive pathogens represents a serious
public health threat. The proportion of Staphylococus
aureus resistant to methicillin, oxacillin, or nafcillin
(MRSA) continues to rise and is now more than 50% in
intensive care units in the United States.1 Even more
critical is the development of vancomycin-resistant
enterococci (VRE). Vancomycin belongs to the glycopep-
tide drug family and has for long been regarded as the
last line of defense against drug-resistant strains, but
now about 25% of all enterococcal infections in ICUs in
the U.S. are resistant to vancomycin, and recently, the
first case of fully vancomycin-resistant S. aureus has
been reported.1-3
The last 10 years have seen only one significant novel
class of non-â-lactams emerge from basic research, the
oxazolidinones, of which the first drug, linezolid (I,
Chart 1), was approved in year 2000.4-6 However, the
speed of resistance development in bacteria creates a
continuous need for novel antibiotics; oxazolidinone
resistant bacteria have already appeared7,8
One of the most promising groups of compounds for
the future treatment of bacterial infections appears to
be the membrane active cationic peptide antibiotics.9-13
The main advantages are (a) the low likelihood of
resistance development, as resistance requires alter-
ation to the bulk membrane structure of the organism,
(b) activity against multiresistant pathogens, as the
compounds do not affect the classical antibiotic targets,
(c) rapid bacterial killing, and (d) a broad spectrum of
antibacterial activity.14,15
are not orally bioavailable; (c) stability in formulation
and stability in vivo; and (d) toxicity of the peptide or
degradation products.16
The oxygenated chalcone, licochalcone A (II, Chart
1), has previously been described as a moderate potent
antibacterial compound with activity against Gram-
positive bacteria.17,18 In this paper, we describe the
synthesis of a new group of compounds, the cationic
chalcone antibiotics, which combine the advantages of
cationic peptide antibiotics with the advantages of small
molecules, potentially eliminating the peptide-related
problems listed above.
A number of novel cationic antibacterial chalcones
have been prepared by systematic introduction of ali-
phatic amino substituents in different positions of the
chalcone rings. Due to the alkaline nature of the
aliphatic amines, the compounds are expected to be
protonated under physiological conditions.
On the other hand, cationic peptide antibiotics face
the problems related with the use of peptides as drugs:
(a) large therapeutic doses as a consequence of high
molecular weight and medium antibacterial activity; (b)
complicated route of administration, as the compounds
Chemistry
The chalcones described were all prepared by base-
catalyzed Claisen-Schmidt condensation22 of acetophe-
nones with appropriate benzaldehydes (e.g. Scheme 3)
in a yield of 2-73%.
Acetophenones having aminoalkylamino substituents
in the 2′- and 4′-positions (1, 2) were easily prepared
by noncatalyzed nucleophilic aromatic substitution us-
* Corresponding author. Current address: MedChem Aps, Frue-
bjergvej 3, Copenhagen, DK-2100, Denmark. Tel.: +45 3917 8301.
Fax: 3917 9901. E-mail: hk@medchem.dk
† Current address: LEO Pharma A/S, Ballerup, DK-2750, Denmark.
‡ Current address: H. Lundbeck A/S, Valby, DK-2500, Denmark.
§ Current address: MedChem Aps, Copenhagen, DK-2100, Denmark.
10.1021/jm049424k CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/04/2005

2668 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Nielsen et al.
Scheme 1. Preparation of
Scheme 3. Preparation of 5-Aryl Substituted
Aminoalkylamino-Substituted Acetophenonesa
Chalconesa
a Reagents and conditions:(a) 2′-F,4′-OMe-acetophenone, NaOH,
ethanol; (b) (hetero)arylboronic acid, Na2CO3, Pd(PPh3)2Cl2, DME/
water, reflux; Ar: cf. Table 2.
a Reagents and conditions: (a) K2CO3, N,N-dimethylethylene-
diamine, DMF, ∆; (b) (i) N,N-dimethylethylenediamine or
1-methylpiperazine, Pd2(dba)3, rac-BINAP, NaOBut, toluene ∆, (ii)
1 M HCl (aq).
of 17 using NBS in methylene chloride gave the desired
6-bromo isomer 18, which was reacted with phenyl
boronic acid to give the benzaldehyde 19 (Scheme 4).
Likewise palladium-catalyzed amination of 2-(3,5-
dibromophenyl)-[1,3]dioxane with 1-methylpiperazine,
deprotection, and subsequent Suzuki coupling with
phenylboronic acid gave the 3-(4-methylpiperazin-1-yl)-
5-phenylbenzaldehyde 21 (Scheme 4).
6-(4-Methylpiperazin-1-yl)biphenyl-3-carbaldehyde 23
and 3-(4-methylpiperazin-1-yl)biphenyl-4-carbaldehyde
25 were prepared by noncatalyzed nucleophilic aromatic
substitution of the appropriate fluorobenzaldehyde,
giving 22 and 24, respectively, followed by Suzuki
coupling using phenylboronic acid (Scheme 4).
The acetophenone 29 was prepared in a four-step
synthesis starting with the reduction of 5-bromo-2-
methoxybenzaldehyde using NaBH4 to give the benzyl
alcohol 26 (Scheme 5). The benzyl alcohol was converted
to the benzyl chloride 27 by thionyl chloride, and
subsequent reaction with aqueous dimethylamine gave
the amine 28. Conversion of 28 to the desired acetophe-
none 29 was performed under Heck conditions24,25 using
butoxyethene followed by hydrolysis.
The amide 30 was synthesized from the corresponding
carboxylic acid using thionyl chloride and dimethyl-
amine. Borane reduction of the amide gave the amine
31, which was converted to the acetopheneone 32 in a
similar manner as described for 30 (Scheme 5).
Reaction of 4-bromobenzyl bromide with aqueous
dimethylamine afforded the amine 33, which was
converted into the phenyl analogue 34 under Suzuki
conditions (Scheme 6). Directed ortho-metalation of 34
using n-butyllithium in refluxing diethyl ether and
subsequent quenching with N,N-dimethylformamide
gave the desired benzaldehyde 35.
ing 2′- or 4′-fluoroacetophenone (Scheme 1). Acetophe-
nones with aminoalkylamino groups in the 3′-position
(3, 4) were synthesized by palladium-catalyzed reaction
of 2-(3-bromophenyl)-2-methyl-[1,3]dioxane with the
appropriate substituted aminoalkylamines followed by
acidic workup as described for similar compounds by
Buchwald et al.21 (Scheme 1). The use of the unprotected
3′-bromoacetophenone was not feasible, as the yield was
very low.
The nucleophilic aromatic substitution of 5-bromo-2-
fluorobenzaldehyde with various aminoalkylamines af-
forded the benzaldehydes 5-9 (Scheme 2). Direct syn-
thesis of the 5-bromo-2-(piperazin-1-yl)benzaldehyde (R
) H, Scheme 2) using an excess of piperazine resulted
in large quantities of polymeric material. An alternative
route was successfully explored in which the Boc-
protected benzaldehyde 7 was synthesized, coupled with
the selected acetophenone, and deprotected to give the
desired product (78, Table 4) by treatment with TFA.
Suzuki coupling23 of the benzaldehydes 5-9 with
various aryl boronic acids gave 5-aryl-substituted benz-
aldehydes (10-15).
In an attempt to explore the specific effect of the
aromatic substituent in the 5-position of the B-ring, a
number of chalcones with different aromatic rings in
the 5-position were prepared from the corresponding
5-bromochalcone (16) under Suzuki conditions (Scheme
3, Table 2).
The 3-methylpiperazine benzaldehyde 17 was pre-
pared by palladium-catalyzed amination using the 1,3-
dioxane-protected 3-bromobenzaldehyde. Bromination
Scheme 2. Preparation of 2,5-Disubstituted Benzaldehydesa
a Reagents and conditions: (a) K2CO3, aminoalkylamine, DMF; (b) arylboronic acid, Na2CO3, Pd(PPh3)2Cl2, DME/water, reflux.

Cationic Chalcone Antibiotics
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2669
Scheme 4. Preparation of 2,4-, 3,5-, 3,6-, or 4,5-Disubstituted Benzaldehydes To Be Used in the Chalcone Synthesis
Presented in Table 5a
a Reagents and conditions: (a) 1,3-propandiol, p-TsOH, toluene; (b) (i) 1-methylpiperazine, Pd2(dba)3, rac-BINAP, NaOBut, toluene, ∆,
(ii) HClaq; (c) NBS, CH2Cl2; (d) phenylboronic acid, Na2CO3, Pd(PPh3)2Cl2, DME/water, reflux; (e) K2CO3, 1-methylpiperazine, DMF.
The quaternary amines (84-86) (Chart 2) were
prepared by the treatment of the corresponding tertiary
amines with iodomethane in a suitable solvent.
ents in the A-ring and diverse substituents in the B-ring
have been prepared (Table 1). The position of the
aliphatic amine in the A-ring only has a marginal effect
on the activity, as compounds having the same amino
substituents in the 2′-, 3′-, or 4′-position are equipotent
(36-38). The distance between the aliphatic amino
group and the aromatic ring is not important for the
activity, as compounds having a dimethylethylendi-
amine, dimethylaminoethoxy, or dimethylaminomethyl
group have comparable activity (38-40). On the other
hand, the lipophilicity of the B-ring substituents ap-
pears to be the most important property with regard to
modulating the activity (41-43, 45), whereas the posi-
tion of the substituents has a minor effect on the activity
(43, 44). Generally, the compounds exhibit a broad
Gram-positive antibacterial spectrum (Table 1) but do
not affect Gram-negative pathogens (MIC > 300 µM).
All compounds in this series showed high protein
binding (>97%), low aqueous solubility, and a high
degree of hemolysis (80-100%) at 4 times the MIC.
Compared to licochalcone A (100% haemolysis) only a
marginally improvement has been achieved. These
results prompted us to investigate compounds having
aliphatic amines in the B-ring.
Results and Discussion
The oxygenated chalcone licochalcone A acts nonse-
lectively at both prokaryotic and eukaryotic cell mem-
branes (Table 6), making the compound unfit for the
treatment of bacterial infections. Licochalcone A is, due
to high lipophilicity, expected to interact with the
central lipophilic part of the membrane. The surface of
eukaryotic cell membranes is exclusively composed of
neutral zwittterionic phospholipids, whereas bacterial
membranes contain large amounts of negatively charged
phospholipids.11,20 Introduction of a positive charge in
the chalcone would make the basis for a strong electro-
static interaction with the surface of the bacterial
membrane, while the lipophilic interaction between the
chalcone and the interior of the membrane would be
retained. This would lead to a more potent compound
with reduced affinity for eukaryotic cells.
The major parameter used in the evaluation of the
compounds prepared is the in vitro antibacterial activity
against a panel of Gram-positive and -negative patho-
gens. In addition, the bacterial selectivity of the com-
pounds was examined. The selectivity is expressed as
the degree of hemolysis of human erythrocytes at 4
times the minimum inhibitory concentration value (MIC
value), which is the concentration level needed to obtain
a secure antibacterial efficacy in vivo.
Series B. Aliphatic Amines in the B-Ring. Intro-
duction of an aliphatic amine in the B-ring and a
lipophilic group in the A-ring results in compounds that
show the same selectivity profile and antibacterial
activity as seen for the compounds in series A (com-
pounds 43 and 46, Table 1).
Series A. Aliphatic Amines in the A-Ring. A
number of chalcones having aliphatic amino substitu-
It was envisioned that having a bulky group and an
aliphatic amine in the same ring would result in a

2670 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Nielsen et al.
Scheme 5. Preparation of 2,4- and 3,4-Subsituted
Scheme 6. Preparation of
Acetophenonesa
2-Dimethylaminomethyl-5-phenylbenzaldehydea
a Reagents and conditions: (a) Me2NH (aq); (b) phenylboronic
acid, Na2CO3, Pd(PPh3)2Cl2, DME/H2O; (c) (i) n-BuLi, diethyl ether,
reflux (6 h), (ii) DMF.
Table 1. In Vitro Antibacterial Activity of Chalcones with
Aliphatic Amines in Ring A against a Panel of Representative
Gram-Positive Bacteriaa
substituents
A-ring B-ring
MIC (µM)
compd
Ab
Bc
Cd
75
log Pe
LicA
36
37
38
39
40
41
42
43
44
45
46
40
40
40
40
40
40
300
75
20
20
10
20
40
40
40
40
40
40
NA
75
20
20
10
20
2-C
3-C
4-C
4-A
4-B
4-C
4-C
4-C
4-C
4-C
4-OPh
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
H
75
40
40
40
75
1.4
1.4
1.4
1.4
1.4
0
0.7
2.1
2.1
3.4
2.1
a Reagents and conditions: (a) NaBH4, ethanol; (b) SOCl2,
CH2Cl2; (c) Me2NH (aq); (d) (i) butoxyethene, palladium acetate,
1,3-bis(diphenylphosphino)propane, K2CO3, DMF/H2O, 80 °C; (ii)
HClaq; (e) (i) SOCl2, (ii) Me2NH (aq); (f) BH3·THF.
NA
4-Cl
75
5
5
10
10
4-OPh
3-OPh
4-N(Bu)2
4-C
change in the way the compounds interact with the
membrane, making them more selective.
Previous papers on bioactive chalcones highlight the
importance of bulky substituents in the 5- position.17,26,27
We have prepared a number of analogues with increas-
ingly bulky substituents in the 5-position, while the
aliphatic amine in the 2-position (47-52, Table 2) was
kept. An increase of the bulk in the 5-position gradually
increases the activity of the compounds (47-52). Ana-
logues with a phenyl group (50) or a 3,5-dimethylphenyl
group (51) in the 5-position are more than 60 times as
potent as the nonsubstituted analogue 47.
In addition, the substitution pattern of the phenyl
ring in the 5-position have been evaluated (Table 2,
compounds 51-61). In general, nonpolar substituents
are tolerated in the 2′′- or 3′′-position (53, 57, 58), while
introduction of polar substituents dramatically reduces
potency (60, 61). Substituents in the 4′′-positon appear
to be detrimental to the activity (59). The preferred
aromatic substituent in the 5-position in this study is
3,5-dimethylphenyl (51).
a Each value represents the mean of three experiments. b S.
aureus ATCC33591 (resistant to methicillin). c Enterococcus faeci-
um #17501 (vancomycin-resistant clinical isolate). d E. faecium
ATCC292121. e log P is calculated as Σπ of substituents on the
B-ring.
nitrogen atom spaced to the B-ring by a single methyl-
ene group (56) reduces the activity, whereas an aliphatic
nitrogen atom in a more distant position results in
potent compounds independent of the linker (52-54).
As expected, a complete masking of the aliphatic amino
group as an amide (55) totally eliminates the activity.
Having identified the 2-amino-5-phenyl combination
as the preferred substitution pattern, a further inves-
tigation of the structure-activity relationships was
initiated. A library of analogues with different polar and
nonpolar substituents (Br, OBu, NH2, F) in the A-ring
was synthesized according to a statistical design (data
The distance between the aliphatic amino group and
the B-ring is of importance for the activity. An aliphatic

Cationic Chalcone Antibiotics
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2671
Table 2. In Vitro Antibacterial Activity of Chalcones with
Aliphatic Amines in Ring B against a Panel of Representative
Gram-Positive Bacteriaa
Table 3. In Vitro Antibacterial Activity of Chalcones with
Aliphatic Amines in Rings A and B against a Panel of
Representative Gram-Positive and -Negative Bacteriaa
substituents
Y
MIC (µM)
compd
X
Ab
Bc
Cd
MIC (µM)
substituents:
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
2-A
2-A
2-A
2-A
2-A
2-A
2-D
2-E
2-F
2-B
2-D
2-D
2-D
2-D
2-D
H
300
75
10
5
300
150
20
300
150
20
10
5
10
20
20
compd
A-ring
Ab
Bc
Cd
De
Me
t-Bu
62
63
64
65
66
67
68
69
70
71
72
73
74
75
2-NH2
5
10
10
10
10
10
5
5
5
10
20
10
10
5
5
10
10
10
10
10
5
10
10
10
20
10
10
10
10
10
20
10
10
5
NA
NA
NA
10
40
75
75
20
20
40
40
20
40
20
Ph
10
3-NH2
4-NH2
2-C
3,5-(Me)2-Ph
2-OMe-Ph
2-OMe-Ph
2-OMe-Ph
2-OMe-Ph
Ph
5
5
10
10
10
10
10
2-A
2-B
2-B, 4-OCH3
3-C
3-A
3-B
3-B, 4-OCH3
4-C
10
NA
NA
150
10
NA
5
150
5
5
150
20
10
10
10
10
20
20
20
20
2-CF3-Ph
3-CF3-Ph
4-CF3-Ph
3-pyridyl
3-(CH2OH)-Ph
5
NA
75
NA
NA
75
NA
75
NA
NA
4-A
4-B
a Each value represents the mean of three experiments. b S.
aureus ATCC33591 (resistant to methicillin). c E. faecium #17501
(vancomycin-resistant clinical isolate). d E. faecium ATCC29212.
e E. coli ATCC25922.
a Each value represents the mean of three experiments. b S.
aureus ATCC33591 (resistant to methicillin). c E. faecium #17501
(vancomycin-resistant clinical isolate). d E. faecium ATCC29212.
position favors a three-atom linker to obtain the most
potent compounds (69, 70), while a one-atom linker,
especially in combination with a 4′-methoxy group is
unfavorable (71, 72). A one-atom linker appears to be
the preferred in the 4′-position (75). A majority of the
compounds prepared for this series are active against
both Gram-positive and -negative organisms (Escheri-
chia coli).
(b) Effect of Changing the Amine in the B-Ring.
To investigate the effect of introduction of different
amines in the 2-postion of the B-ring, a number of
analogues were prepared retaining the most favorable
A-ring (Table 4). Chalcones having the N-methylpi-
perazine (76) and N,N-dimethylaminoethoxy (69) groups
in the 2-position are equipotent, whereas the introduc-
tion of the N,N′,N′-trimethylethylendiamine group (77)
results in a less potent compound. The most potent class
of compounds has a piperazine in the 2-position (78).
The high activity of this secondary amine may reflect
the fact that the latter compound is more basic and
consequently has a higher degree of protonation in the
assay.28 Given the mechanism of action described below,
this fact should be favorable for the binding in the
membrane.
(c) Position of the Amine and the Phenyl Group
in Ring B. The effect of moving the amine and/or the
phenyl group was investigated (79-83, Table 5). Moving
the phenyl group to the 4-position of the B-ring (80) only
marginally changes the activity. The same pattern is
seen when moving the amine to the 3-position and
keeping the phenyl in the 5-position (81). However,
having the amine in the 3-position and a phenyl in
6-position (82) or an amine in the 4-position and phenyl
in the 5-position (83) significantly reduce the activity.
not shown).9 Surprisingly, the substituents on the
A-ring have little or no influence on the activity,
indicating that the A-ring is less important for binding.
Compounds having aniline as the A-ring (62-64) appear
to be more selective for the bacterial membrane (20-
30% hemolysis at 4 times the MIC as compared to 50-
100% for the rest). This may be due to a different
orientation of the compounds in the eukaryotic cell
membrane as a consequence of the increased polarity
of the A-ring. In addition, the protein binding is lower
than for the rest of the compounds (75-88% as com-
pared to 94-97%), but the solubility is relatively low
(approximately 0.2 mg/mL).
Series C. Aliphatic Amines in the A -and B-Ring.
On the basis of the observations described above and
in an attempt to take full advantage of the negatively
charged bacterial membrane, a number of compounds
with aliphatic amines in both the A- and B-ring were
prepared. It was the hope that this would lead to an
improvement of the activity, selectivity, protein binding,
and solubility of the compounds.
(a) Effect of Changing the Aliphatic Amine in
the A-Ring. A series of chalcones with diverse aliphatic
amines in the A-ring and the (2-dimethylaminoethoxy-
5-(3,5-dimethylphenyl) B-ring was prepared (Table 3,
65-75). The data shows that the position and type of
linker between the aliphatic nitrogen and the A-ring
have relatively small effect on the antibacterial activity.
In the 2′-position, a one-atom linker to the aliphatic
nitrogen in combination with a methoxy group in the
4′-position is the optimal combination (68), showing
slightly better activity than the compounds having
longer linkers or no 4′-methoxy group (65-67). The 3′-

2672 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Nielsen et al.
Table 4. In Vitro Antibacterial Activity of Chalcones with
Aliphatic Amines in Rings A and B against a Panel of
Representative Gram-Positive and -Negative Bacteriaa
Table 6. Mechanism of Action: In Vitro Activity and Effect on
Bacterial Membrane Integrity (BacLight) and Hemolysis of
Selected Analoguesa
MIC
S. aureus
(µM)
%
%
membrane
integrity
hemolysis
(4 × MIC)
compound
control
licochalcone A
39 (series A)
53 (series B)
68 (series C)
78 (series C)
ciprofloxacin
100
0
40
40
10
5
2
2
100
100
80
<1
0
0
0
2
MIC (µM)
3
102
0
substituents:
a Each value represents the mean of three experiments.
compd
X
Ab
Bc
Cd
De
69
76
77
78
2-A
2-D
2-G
2-H
5
5
10
2
10
5
10
5
10
5
10
5
20
10
20
5
and a very favorable aqueous solubility at physiological
pH (>1 mg/mL). Due to the promising results, this group
of compounds is currently under further investigation
Mechanism of Action
The mechanism of action has been investigated and
validated by four different methods.
(a) Hemolysis. Human erythrocytes were exposed to
different concentration of the test compound and the
proportion of lysed cell was measured.29 The amino
chalcones from series A and B like licochalcone A have
pronounced hemolytic properties, which support the
underlying thesis of this work that licochalcone A and
the novel amino chalcones share the same mechanism
of action (Table 6).
a Each value represents the mean of three experiments. b S.
aureus ATCC33591 (resistant to methicillin). c E. faecium #17501
(vancomycin-resistant clinical isolate). d E. faecium ATCC29212.
e E. coli ATCC25922.
Table 5. In Vitro Antibacterial Activity of Chalcones with
Aliphatic Amines in Rings A and B against a Panel of
Representative Gram-Positive and -Negative Bacteriaa
(b) Kinetics of Bacterial Killing. Time-kill experi-
ments were carried out in order to investigate the
kinetics of the killing of the bacteria (Figure 1). The
extremely fast killing kinetics seen for licochalcone A
(Figure 1A) indicates an immediate bacteriocidal effect
on the membrane structure of the cell. Furthermore, the
three amino chalcones (40, 53, 76) were shown to have
bactericidal profile even at concentrations corresponding
to the MIC value (Figure 1B-D). For the compounds
belonging to series A (e.g. 40) and series C (e.g. 76), a
concentration-dependent bacterial killing was observed.
The compound from series B (e.g. 53) shows a slower
bacterial killing that appears to be time-dependent. This
observation may reflect that the compounds due to
different orientation in the membrane induce slightly
different mechanisms of membrane damage, as also
seen for cationic peptides.16
substituents
MIC (µM)
compd
X
Y
Ab
Bc
Cd
De
20
40
75
NA
NA
79
80
81
82
83
2-D
2-D
3-D
3-D
4-D
5-Ph
4-Ph
5-Ph
6-Ph
5-Ph
20
20
20
75
75
20
40
40
75
75
40
40
40
75
150
a Each value represents the mean of three experiments. b S.
aureus ATCC33591 (resistant to methicillin). c E. faecium #17501
(vancomycin-resistant clinical isolate). d E. faecium ATCC29212.
e E. coli ATCC25922.
(c) BacLight assay.29 The integrity of the bacterial
membranes was investigated by the use of the BacLight
fluorescence assay. The bacteria are labeled with a
mixture of SYTO9 green-fluorescent nucleic acid stain
and the red-fluorescent nucleic acid stain propidium
iodide. The SYTO9 stain generally labels all bacteria
in a population, whereas propidium iodide only pen-
etrates damaged membranes, labeling solely the bac-
teria with damaged membranes. The difference in
spectral characteristics and ability to penetrate healthy
bacterial cells can be used to investigate the degree of
membrane damage. We have investigated the membrane-
damaging effect of licochalcone A and selected com-
pounds representing the three series of compounds
presented in this paper (39, 53, 68, 78). As seen in Table
6, all compounds tested produce almost complete mem-
brane damage within 15 min (g97% permeability
compared 0% for the nondrug control), strongly sup-
porting the hypothesis that damage of the membrane
The reason for the lower activity of these compounds
may be an unfavorable orientation of the chalcone in
the cell membrane (82) or a result of bringing the
lipophilic and hydrophilic parts of the molecule close
together, making the incorporation of the molecule in
cell membrane difficult (83).
The idea of introducing two aliphatic amines in the
molecule in order to improve the in vitro performance
has proven successful. The most noteworthy feature of
the compounds in series C is that, in addition to
retaining the potency seen in the best compounds of
series A and B, they are, in general, practically non-
hemolytic (0-10%) at 4 times the MIC. One of the most
active compounds (78) is as potent in our in vitro assay
as linezolide (MIC ) 2 µM against S. aureus) and the
degree of hemolysis is negligible (<4%) even at a
concentration of 150 µM. Furthermore, the compounds
in series C have acceptable protein binding (50-87%)

Cationic Chalcone Antibiotics
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2673
Figure 1. Time-kill kinetics and dose-response effect of licochalcone A and selected analogues representing the three series
against S. aureus (ATTC 29213): (A) licochalcone A, (B) 40, (C) 53, (D) 76. Compound concentrations: 8 × MIC (9), 4 × MIC (2),
2 × MIC (×), MIC (/), 0.5 × MIC (b), and control ([).
Chart 2. In Vitro Activity of the Quaternary Chalcones
Analogoues against S. aureusa
(c) Synthesis of Quaternary Amine Analogues.
To conclusively prove the suggested mechanism of
action, a number of quaternary amine analogues were
synthesized and their activities were compared with
those of the corresponding tertiary amine analogues
(Chart 2). It is highly unlikely that the quaternary
compounds, being charged at any time, can penetrate
the bacterial membrane. As seen in Chart 2, the
compounds are equipotent to the parent compounds,
fully supporting the suggested mechanism of action.
Conclusion
In this report, we describe the synthesis and antibac-
terial effect of a novel class of chalcones having aliphatic
amino substituents. The mechanism of action has been
investigated and it is shown that the compounds act by
disruption of the cell membrane. We describe how
generally membrane damaging chalcones (e.g. licochal-
cone A) were converted into a class of chalcones that
selectively disrupt bacterial membranes without affect-
ing eukaryotic cell membranes via the introduction of
aliphatic amino groups in both rings of the chalcone,
resulting in compounds being cationic at physiological
pH. The antibacterial spectrum has been expanded, as
many of the compounds are equally potent against
Gram-positive and -negative pathogens. Due to the
novel mechanism of action, the compounds are active
against drug resistant pathogens such as MRSA and
VRE, making them very attractive for further develop-
ment.
a The activity of the corresponding tertiary analogues is written
in italics. Each value represents the mean of three experiments.
is the mechanism of action. On the other hand, the
degree of hemolysis is related to the series of compounds
as described previously. Series A, exemplified by com-
pound 39, and series B, exemplified by compound 53,
causes >80% hemolysis at 4 times the MIC, whereas
compounds belonging to series C (68, 78) show less than
10% hemolysis, even though the bacterial membrane is
completely disrupted, indicating a significant degree of
selectivity. The data demonstrate that the compounds
from the three series have the same mechanism of
action on bacterial cells, but at the same time they have
dramatically different affinity for the eukaryotic mem-
branes. Ciprofloxazine, which was included as a nega-
tive control, does not affect the bacterial membrane or
cause hemolysis in our assays.
In addition, the protein binding has been reduced to
an acceptable level and the aqueous solubility has been
improved more than 500 times, making administration
and absorption feasible.

2674 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Nielsen et al.
°C. The solution was cooled to 0 °C and pH was adjusted to 13
using 6 M NaOH (aq) and extracted with Et2O (4 × 100 mL).
The organic phase was dried (K2CO3) and the solvent was
removed in vacuo. The resulting crude oil was purified by CC
using EtOAc-Et3N 20:1. General procedure B gave the title
compound as a yellow oil in 72% yield (11.2 g). 1H NMR
(CDCl3): δ 7.21 (t, J ) 7.9 Hz, 1H), 7.15-7.12 (m, 2H), 6.85
(bd, J ≈ 8 Hz, 1H), 5.66 (t, J ) 5.3 Hz, 1H), 3.13 (q, J ) 6.6
Hz, 2H), 2.50 (s, 3H), 2.44 (t, J ) 6.6 Hz, 2H), 2.25 (s, 6H).
GC-MS: 206 (M).
General Procedure C for the Aromatic Nuclephilic
Substitution of Fluorobenzaldehydes (5-9, 22, 24). Ex-
ample: 5-Bromo-2-(4-methylpiperazin-1-yl)benzalde-
hyde (5). To a stirred solution of 5-bromo-2-fluorobenzalde-
hyde (40 g, 197 mmol) in dry DMF (200 mL) were added K2CO3
(81.7 g, 590 mmol) and 1-methylpiperazine (30 g, 290 mmol),
and the mixture was left overnight at 100 °C. The reaction
mixture was cooled to room temperature, quenched with H2O
(200 mL), and extracted with Et2O (3 × 100 mL). The combined
organic phases were washed with H2O, dried (Na2SO4), and
evaporated in vacuo. The resulting crystals were recrystallized
(heptane) to give the desired product.
Experimental Section
Chemistry. Thin-layer chromatography (TLC) was per-
formed on silica gel F254 plates (Merck). All compounds were
detected using UV light. 1H and 13C NMR spectra were
recorded on a Bruker AC-300 F spectrometer, using CDCl3 or
DMSO-d6 as solvent. Chemical shifts are given in ppm (δ)
using TMS as the internal standard, and coupling constants
(J) are given in hertz. Mass spectra (GC-MS) were recorded
using an Agilent Technologies 6890N and were all >95% pure.
Mass spectra (LC-MS) were recorded using a Waters Alliance
HPLC-system coupled to a Quatro Micro triple quadropol mass
spectrometer (Micromass) operating in positive (ESI) mode.
Separation was performed on a XTerra MS C18 column (150
× 2.1 mm i.d., 3.5 µm particle size). All intermediate com-
pounds were characterized by either GC-MS or LC-MS and
all final compounds were confirmed by LC-MS. The purity of
the compounds (>95%) was determined using a Waters Alli-
ance 2690 separation module and Waters 996 PDA-detector
(Waters, Milford, MA). Separation was performed on a XTerra
MS C18 column (150 × 2.1 mm i.d., 3.5 µm particle size)
(Waters Milford, MA).
For system I, initial conditions were 40% mobile phase A
(acetonitrile) and 60% mobile phase B (10 mM ammonium
acetate pH 9.5). During the first 20 min, the mobile phase was
changed via a linear gradient to 90% A and 10% B.
For system II, initial conditions were 70% mobile phase A
(0.1% (v/v) formic acid in MilliQ-water) and 30% mobile phase
B (methanol). During the first 10 min, the mobile phase was
changed via a linear gradient to 10% A and 90% B, and this
gradient was maintained for 10 min.
The accurate mass measurements (HRMS) were obtained
on a VG AutoSpec mass spectrometer (Micromass, Manchester,
UK) equipped with an EI source. The instrument was operated
at a resolution of 10000 fwhm.
Column chromatography (CC) was performed on Merck
silica gel 60 (0.063-0.200 mm). All solvents and reagents were
obtained from Fluka or Aldrich and used without further
purification except DMF and THF, which were stored over 3
Å molecular sieves.
General Procedure A for the Aromatic Nuclephilic
Substitution of Fluoroacetophenones (1, 2). Example:
1-[2-(2-Dimethylaminoethylamino)phenyl]ethanone (1).
A solution of 2′-fluoroacetophenone (5.1 mL, 40 mmol), N,N-
dimethylethylenamine (11.1 g, 50 mmol), and K2CO3 (11.1 g,
50 mmol) in dry DMF (20 mL) was refluxed under an
atmosphere of argon for 18 h. The solvent was removed in
vacuo and H2O (50 mL) was added to the resulting residue.
The aqueous phase was extracted with Et2O (2 × 100 mL) and
the organic phase was dried (K2CO3) and evaporated in vacuo
to yield the desired product, which was used without further
purification.
General procedure A gave the title compound as a yellow
oil in 58% yield (5.0 g). 1H NMR (CDCl3): δ 8.95 (bs, 1H), 7.73
(dd, J ) 8.0 Hz, J ) 1.6 Hz, 1H), 7.35 (td, J ) 8.0 Hz, J ) 1.6
Hz, 1H), 6.69 (bd, J ) 8.0 Hz, 1H), 6.58 (td, J ) 7.1 Hz, J )
1.6 Hz, 1H), 3.30 (q, J ) 6.5 Hz, 2H), 2.58 (t, J ) 6.5 Hz, 2H),
2.56 (s, 3H), 2.30 (s, 6H). GC-MS: 206 (M).
General procedure C gave the title compound as yellow
crystals in 54% yield (30 g). 1H NMR (CDCl3) δ 10.24 (s, 1H),
7.91 (d, J ) 2.4 Hz, 1H), 7.61 (dd, J ) 8.7 Hz, J ) 2.4 Hz, 1H),
7.02 (d, J ) 8.7 Hz, 1H), 3.11 (m, 4H), 2.63 (m, 4H), 2.39 (s,
3H). GC-MS: 282 (M).
General Procedure D for the Suzuki Coupling of
Substituted Benzaldehydes (10-15, 19, 21, 23, 25, 34).
Example: 3′,5′-Dimethyl-4-(4-methylpiperazin-1-yl)biphenyl-
3-carbaldehyde (10). To a solution of 5-bromo-2-(4-methylpi-
perazin-1-yl)benzaldehyde 5 (13.5 g, 47.6 mmol) and 3,5-
dimethylphenylboronic acid (8.6 g, 57.2 mmol) in DME (130
mL) was added a solution of Na2CO3 (5.2 g, 143 mmol) in H2O
(70 mL). The mixture was flushed with argon for 2 min
followed by addition of Pd(PPh3)2Cl2 (1.0 g, 3 mol %). The
reaction was heated to reflux and left overnight under an
atmosphere of argon. The solution was cooled, washed with 2
M Na2CO3 (aq), extracted with Et2O (3 × 40 mL), and purified
by CC.
General procedure D gave the title compound as yellow
1
crystals in 74% yield (10.9 g). H NMR (DMSO-d6): δ 10.22
(s, 1H), 7.91 (d, J ) 2.4 Hz, 1H), 7.87 (dd, J ) 8.5 Hz, J ) 2.4
Hz, 1H), 7.28-7.24 (m, 3H), 6.98 (bs, 1H), 3.12 (m, 4H), 2.56
(m, 4H), 2.33 (s, 6H), 2.25 (s, 3H). GC-MS: 280 (M).
General Procedure E for the Buchwald-type Coupling
of Bromobenzaldehydes (17, 20). Example: 3-(4-Meth-
ylpiperazin-1-yl)benzaldehyde (17). A solution of 3-bro-
mobenzaldehyde (18.5 g, 100 mmol), 1,3-propandiol (9.1 g, 120
mmol), and p-TsOH (0.1 g) in toluene (200 mL) was refluxed,
azeotropically removing H2O using a Dean-Stark water
separator. After 18 h, the solution was washed with 5% Na2-
CO3 (100 mL) and dried (K2CO3) and the solvent removed in
vacuo. The resulting 1,3-dioxane was used without further
purification.
2-(3-Bromophenyl)-[1,3]dioxane (14.0 g, 54 mmol), 1-meth-
ylpiperazine (6.6 g, 64.8 mmol), Pd2(dba)3 (0.25 g, 0.3 mmol, 1
mol % Pd), rac-BINAP (0.52 g, 0.8 mmol), and NaOBut (9.1 g,
91.8 mmol) were stirred under argon in toluene (100 mL) at
100 °C for 18 h. The dark brown mixture was poured into an
ice cold solution of HCl (aq, 1 M, 200 mL) and stirred
vigorously for 2 h at 25 °C. The solution was cooled to 0 °C
and pH was adjusted to 13 using 6 M NaOH (aq) and extracted
with Et2O (4 × 100 mL). The organic phase was dried (K2-
CO3) and the solvent was removed in vacuo. The resulting
crude oil was purified by flash chromatography using EtOAc-
Et3N 20:1.
General Procedure B for the Buchwald-type Coupling
of 3′-Bromoacetophenones (3, 4). Example: 1-[3-(2-Di-
methylaminoethylamino)phenyl]ethanone (3). A solution
of 3′-bromoacetophenone (19.9 g, 100 mmol), 1,3-propandiol
(9.1 g, 120 mmol), and p-TsOH (0.1 g) in toluene (200 mL) was
refluxed, azeotropically removing H2O using a Dean-Stark
water separator. After 18 h, the solution was washed with 5%
Na2CO3 (100 mL) and dried (K2CO3) and the solvent removed
in vacuo. The resulting 1,3-dioxane was used without further
purification.
General procedure E gave the title compound as brown oil
in 77% yield (17 g). 1H NMR (DMSO-d6): δ 9.94 (s, 1H), 7.45-
7.40 (m, 2H), 7.31-7.27 (m, 2H), 3.21 (bt, J ) 4.9 Hz, 4H),
2.46 (bt, J ) 4.9 Hz, 4H), 2.22 (s, 3H). GC-MS: 204 (M).
2-Bromo-5-(4-methylpiperazin-1-yl)benzaldehyde (18).
A solution of compound 17 (10 g, 49 mmol) in CH2Cl2 (150 mL)
was slowly added a solution of NBS (22 g, 98 mmol) in CH2-
2-(3-Bromophenyl)-2-methyl-[1,3]dioxane (20.4 g, 79 mmol),
dimethylethylenediamine (11.0 mL, 95 mmol), Pd2(dba)3 (365
mg, 0.4 mmol, 1 mol % Pd), rac-BINAP (500 mg, 0.8 mmol),
and NaOBut (10.7 g, 111 mmol) were stirred under an
atmosphere of argon in toluene (100 mL) at 80 °C for 18 h.
The dark brown mixture was poured into an ice cold solution
of HCl (aq, 1 M, 200 mL) and stirred vigorously for 2 h at 25

Cationic Chalcone Antibiotics
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2675
Cl2 (450 mL). The mixture was stirred for 10 min at rt and
washed with 2 M NaOH (aq), and the organic phase was
removed in vacuo. The resulting oil was purified by CC, giving
the title compound as black oil (90% pure) in 95% yield. The
product was used without further purification. 1H NMR
(CDCl3): δ 10.29 (s, 1H), 7.47 (d, J ) 8.7 Hz, 1H), 7.40 (d, J )
3.4 Hz, 1H), 7.00 (dd, J ) 8.7 Hz, J ) 3.4 Hz, 1H), 3.26 (bt, J
≈ 6 Hz, 4H), 2.58 (bt, J ≈ 6 Hz, 4H), 2.36 (s, 3H). GC-MS:
282 (M).
(5-Bromo-2-methoxyphenyl)methanol (26). To a solu-
tion of 5-bromo-o-anisaldehyde (21.5 g, 0.10 mol) in 96% EtOH
(100 mL) was added NaBH4 (3.8 g, 0.10 mol). The mixture was
stirred overnight, and the solvent was removed in vacuo. The
residue was dissolved in EtOAc and washed with H2O, and
the organic phase was evaporated in vacuo. The resulting oil
was purified by CC giving the title compound as white crystals
(13.9 g, 64%). 1H NMR (DMSO-d6): δ 7.47 (bd, J ) 2.7 Hz,
1H), 7.37 (dd, J ) 8.7 Hz, J ) 2.7 Hz, 1H), 6.90 (d, J ) 8.7 Hz,
1H), 5.18 (t, J ) 5.6 Hz, 1H), 4.46 (d, J ) 5.6 Hz, 2H), 3.77 (s,
3H). GC-MS: 216 (M).
4-Bromo-2-chloromethyl-1-methoxybenzene (27). A so-
lution of compound 26 (13.9 g, 64 mmol) in CH2Cl2 (100 mL)
was slowly added SOCl2 (5.1 mL, 70 mmol) and the mixture
was stirred for 2 h at rt. The solvent and excess of SOCl2 were
removed in vacuo, giving 27 as red oil (15 g, 100%), which was
used without further purification. 1H NMR (DMSO-d6): δ 7.60
(d, J ) 2.5 Hz, 1H), 7.51 (dd, J ) 8.7 Hz, J ) 2.5 Hz, 1H), 7.01
(d, J ) 8.7 Hz, 1H), 4.68 (s, 2H), 3.84 (s, 3H). GC-MS: 234
(M).
1H), 7.00 (d, J ) 2.9 Hz, 1H), 6.80 (dd, J ) 8.6 Hz, J ) 2.9 Hz,
1H), 3.75 (s, 3H), 3.41 (s, 2H), 2.20 (s, 6H). GC-MS: 243 (M).
1-(2-Dimethylaminomethyl-4-methoxyphenyl)etha-
none (32). Same procedure as described for compound 29 (36
mmol) gave the title compound as a yellow oil in 80% yield.
1H NMR (DMSO-d6): δ 7.46 (d, J ) 8.6 Hz, 1H), 7.05 (d, J )
2.9 Hz, 1H), 6.88 (dd, J ) 8.6 Hz, J ) 2.9 Hz, 1H), 3.81 (s,
3H), 3.59 (s, 2H), 2.46 (s, 3H), 2.11 (s, 6H). GC-MS: 207 (M).
(4-Bromobenzyl)dimethylamine (33). To a solution of
4-bromobenzyl bromide (50 g, 200 mmol) in dioxane (100 mL)
was added dimethylamine (8 M solution in H2O, 100 mL, 800
mmol), and the mixture was stirred for 3 h at rt. The mixture
was extracted with Et2O to give 37 as yellow oil (38.4 g, 90%).
Bp: 68-70 °C/2 mbar. 1H NMR (DMSO-d6): δ 7.49 (d, J ) 7.5
Hz, 2H), 7.24 (d, J ) 7.5 Hz, 2H), 3.33 (s, 2H), 2.12 (s, 6H).
GC-MS: 213 (M).
4-Dimethylaminomethylbiphenyl-3-carbaldehyde (35).
To a solution of compound 34 (11.6 g, 55 mmol) in Et2O was
added n-BuLi (2.5 M in THF, 65 mmol), and the mixture
refluxed for 6 h under an atmosphere of argon. The solution
was cooled on ice and DMF (60 mmol) was added. The reaction
was stirred overnight, quenched with H2O, and extracted with
EtOAc. The desired product was isolated as yellow oil after
distillation (5.3 g, 40%). Bp: 130-145 °C/0.015 mbar. 1H NMR
(DMSO-d6): δ 10.38 (s, 1H), 8.03 (d, J ) 2.0 Hz, 1H), 7.89 (dd,
J ) 7.9 Hz, J ) 2.0 Hz, 1H), 7.73-7.69 (m, 2H), 7.53-7.39
(m, 4H), 3.76 (s, 2H), 2.17 (s, 6H). GC-MS: 239 (M).
General Procedure F for the Synthesis of Substituted
Chalones by Claisen Condensation (16, 36-52-56, 60,
62-83). To a solution of acetophenone (2 mmol) and benzal-
dehyde (2 mmol) in 96% EtOH (10 mL) was added 8 M NaOH
(aq, 0.3 mL), and the reaction was stirred for 18 h at 25 °C.
The mixture was evaporated on Celite and the product was
isolated by CC. When indicated, the compounds were used as
the free base, but in some cases the compounds were isolated
as the fumarate salt or the oxalate salt. The aminochalcone
was dissolved in MeOH:Et2O (1:9 v/v, 10 mL), and a solution
of fumaric acid or oxalic acid in MeOH:Et2O (1:9 v/v) was
added. Alternatively, the aminochalcones were isolated as their
HCl salt. The aminochalcone was dissolved in a small amount
of 96% EtOH, and a 3 M solution of concentrated HCl in EtOH
was added. The desired compound was precipitated by addition
of Et2O. The resulting crystals were filtered off and, when
indicated, recrystallized from CH3CN.
Example: (E)-3-(2,4-Dichlorophenyl)-1-[2-(2-dimethyl-
aminoethylamino)phenyl]propenone (36). General pro-
cedure F (3 mmol) gave the oxalate salt of title compound as
yellow crystals in 69% yield. 1H NMR (DMSO-d6): δ 9.13 (t, J
) 5.0 Hz, 1H), 8.24 (t, J ) 6.9 Hz, 2H), 8.08 (d, J ) 13.9 Hz,
1H), 7.90 (d, J ) 16.0 Hz, 1H), 7.72 (d, J ) 7.2 Hz, 1H), 7.56-
7.40 (m, 2H), 6.93 (d, J ) 7.0 Hz, 1H), 6.70 (t, J ) 5.2 Hz,
1H), 3.70-3.58 (m, 2H), 3.20 (t, J ) 6.0 Hz, 2H), 2.75 (s, 6H).
13C NMR (DMSO-d6): δ 190.1, 164.1, 150.7, 135.7, 135.1, 134.8,
132.4, 131.5, 129.7, 129.3, 127.8, 126.6, 117.9, 114.7, 111.7,
54.9, 42.6, 37.2. LC-MS: 363.1 (M+1). HR-MS (C19H20Cl2N2O)
Calcd.: 362.0953. Found: 362.0972.
(5-Bromo-2-methoxybenzyl)dimethylamine (28). A so-
lution of compound 27 and dimethylamine (2 M solution in
THF, 23 mL, 45 mmol) was stirred for 18 h at rt. The organic
solvent was removed in vacuo and the residue was extracted
with Et2O and washed with 2 M NaOH (aq), giving the product
1
as orange oil (4.0 g, 82%). H NMR (DMSO-d6): 7.42 (d, J )
2.5 Hz, 1H), 7.38 (dd, J ) 8.6 Hz, J ) 2.5 Hz, 1H), 6.94 (d, J
) 8.6 Hz, 1H), 3.76 (s, 3H), 3.36 (s, 2H), 2.15 (s, 6H). GC-MS:
243 (M).
1-(3-Dimethylaminomethyl-4-methoxyphenyl)etha-
none (29). A solution of compound 28 (7.0 g, 29 mmol),
butoxyethene (10 g, 100 mmol), Pd(OAc)2 (200 mg, 0.9 mmol),
1,3-bis(diphenylphosphino)propane (1.8 mmol), and K2CO3 (30
mmol) in DMF (50 mL) and H2O (3 mL) under an atmosphere
of argon was heated at 80 °C overnight. The mixture was
poured into a solution of 2 M HCl (aq) and stirred for 1 h. The
solution was adjusted to basic pH using a solution of 2 M
NaOH (aq) and extracted with CH2Cl2. The organic phase was
evaporated on Celite and the residue was purified by CC to
give the title product as orange oil in 42% yield (2.5 g). 1H
NMR (CDCl3): δ 7.90 (s, 1H), 7.88 (dd, J ) 8.9 Hz, J ) 2.3
Hz, 1H), 6.89 (d, J ) 8.9 Hz, 1H), 3.88 (s, 3H), 3.44 (s, 2H),
2.55 (s, 3H), 2.25 (s, 6H). GC-MS: 207 (M).
2-Bromo-5-methoxy-N,N-dimethylbenzamide (30). A
mixture of 2-bromo-5-methoxybenzoic acid (30 g, 130 mmol)
and SOCl2 (15 mL, 200 mmol) was refluxed for 1 h. Excess
SOCl2 was removed in vacuo, and the product was dissolved
in Et2O (100 mL) and added to dimethylamine (aq, 8 M, 400
mL, 3.2 mol). The reaction was stirred vigorously for 1 h and
extracted with Et2O. Evaporation in vacuo gave the desired
product as a dark oil (33 g, 99%) that was used without further
purification. 1H NMR (CDCl3): δ 7.43 (dt, J ) 9.0 Hz, J ) 1.5
Hz, 1H), 6.81-6.75 (m, 2H), 3.79 (s, 3H), 3.13 (s, 3H), 2.87 (s,
3H). GC-MS: 257 (M).
General Procedure G for the Synthesis of Substituted
Chalones by Suzuki Couplings (57-59, 61). Example:
(E)-1-(2-Fluoro-4-methoxyphenyl)-3-[4-(4-methylpiperazin-
1-yl)-2′-trifluoromethylbiphenyl-3-yl]propenone (57). A
solution of compound 16 (0.40 g, 0.92 mmol) and 2-(trifluo-
romethyl)phenylboronic acid (1.11 mmol) in DME (10 mL) was
added a solution of Na2CO3 (290 mg, 2.8 mmol) in H2O (5 mL).
The mixture was flushed with argon for 2 min followed by
addition of Pd(PPh3)2Cl2 (19 mg, 3 mol %). The reaction was
heated at reflux overnight under an atmosphere of argon. The
solution was cooled, washed with 2 M Na2CO3 (aq), extracted
with Et2O (3 × 20 mL), and purified by CC.
(2-Bromo-5-methoxybenzyl)dimethylamine (31). Bo-
rane (1 M solution in THF, 300 mL, 0.30 mol) in THF (150
mL) was slowly added compound 30 (33 g, 0.13 mol). The
reaction was stirred for 5 h under an atmosphere of argon
followed by careful addition of MeOH. Evaporation in vacuo
followed by addition of 6 M HCl (aq) resulted in a suspension
that was stirred for 18 h. The mixture was made alkaline using
2 M NaOH (aq) and extracted with Et2O to give the desired
product as a clear oil after distillation (16.1 g, 51%). Bp: 100-
General procedure G gave the title compound as yellow oil
1
in 20% yield. H NMR (CDCl3): δ 8.14 (dd, J ) 16.1 Hz, J )
2.1 Hz, 1H), 7.86 (t, J ) 8.7 Hz, 1H), 7.74 (d, J ) 7.4 Hz, 1H),
7.63-7.30 (m, 6H), 7.07 (d, J ) 8.7 Hz, 1H), 6.77 (dd, J ) 8.7
Hz, J ) 2.4 Hz, 1H), 6.61 (dd, J ) 16.1 Hz, J ) 2.4 Hz, 1H),
3.85 (s, 3H), 3.08 (m, 4H), 2.65 (bs, 4H), 2.38 (s, 3H). 13C NMR
1
103 °C/0.05 bar. H NMR (DMSO-d6): δ 7.46 (d, J ) 8.6 Hz,

2676 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Nielsen et al.
(DMSO-d6) δ 190.2, 164.8 (J ) 12.5 Hz), 163.4 (J ) 151.3 Hz),
153.5, 143.4 (J ) 25.4), 133.3, 132.5 (J ) 5.6 Hz), 130.5, 130.1
(J ) 186 Hz), 128.7, 127.5, 126.7, 126.4, 124.7, 124.0, 123.6,
123.2, 120.4, 119.3 (J ) 13.1 Hz), 111.6, 100.1 (J ) 23.3 Hz),
55.4, 55.0, 51.5, 43.9. HR-MS (C28H26F4N2O) Calcd.: 498.1930.
Found: 498.1941.
An overnight culture of S.aureus ATCC29213 was diluted
1:20 in Mueller-Hinton broth and grown under aeration until
reaching an OD450 of 0.6-0.7. The bacterial suspension was
washed once in sterile H2O and then resuspended in one-fifth
of its original volume. Then, the resulting bacterial suspension
was diluted 1:20 in either test compound at a concentration
of 4 × MIC or in DMSO diluted in H2O at the same
concentration as used to dissolve the test compound (usually
1%). Ciprofloxacin at 2 µg/mL (corresponding to 4 × MIC) was
included as a control. The suspensions were incubated at 37
°C for 15 min under gentle agitation. Then a 10 µL sample
was removed and immediately diluted 1:100 in Mueller-
Hinton broth for CFU and cell viability determination, and
the remaining suspensions was washed and resuspended in
distilled H2O at an OD600 of approximately 0.4. A 100 µL
volume of the resulting bacterial suspension was added in
triplicate to a microtiter plate and an equal volume of BacLight
reagent was added. Plates were then incubated in the dark
for 15 min and then green fluorescence (SYTO-9) was read at
530 nm and red fluorescence was read at 630 nm using a Bio-
Tek FL600 fluoremeter with an excitation wavelength of 485
nm. The ratio of green to red fluorescence was normalized to
the untreated control and expressed as percentage of the
control.
Determination of Hemolysis Activity. Hemolytic activity
was determined using horse erythrocytes (supplied by State
Serum Institut, Copenhagen) in an assay modified after
Hilliard et al.29 Briefly, horse erythrocytes were washed three
times in Dulbeccos phosphate-buffered saline (D-PBS) and
resuspended at a final concentration of 5 × 108 RBC/mL in
D-PBS. To aliquots of the cell suspension were added 4
volumes of either test compound at a concentration 4 × MIC
in D-PBS/1% DMSO or D-PBS/1% DMSO (negative control
yielding 0% hemolysis) or 0.1% NH4OH or sterile H2O (positive
controls). Cell suspensions were then incubated at 37 °C for
90 min under gentle agitation. At the end of incubation,
samples were centrifuged at 1300g for 5 min, and hemoglobin
release was determined by measuring A540 of the supernate
using A670 as reference. As sterile H2O consistently gave higher
A540-determinations than the 0.1% NH4OH control, the sterile
H2O positive control was chosen as positive control yielding
100% hemolysis.
General Procedure H for the Synthesis of Chalcones
with one Quartinary Amine (84, 85). Example: (E)-4-{3-
[3-(2-Fluoro-4-methoxyphenyl)-3-oxopropenyl]-2′-meth-
oxybiphenyl-4-yl}-1,1-dimethylpiperazin-1-ium Iodide
(85). Iodomethane (25 mg, 0.17 mmol) was added to a solution
of compound 53 (23 mg, 0.05 mmol) in THF (10 mL) and
stirring was continued for 16 h. The precipitate was collected
by filtration and was washed with THF to give the product as
1
yellow crystals in 56% yield. H NMR (DMSO-d6): δ 7.81 (d,
J ) 16.8 Hz, 1H), 7.71-7.60 (m, 2H), 7.48 (dd, J ) 8.4 Hz, J
) 1.8 Hz, 1H), 7.32-7.17 (m, 4H), 7.04 (d, J ) 7.5 Hz, 1H),
6.96 (t, J ) 7.5 Hz, 1H), 6.79 (dd, J ) 9.0 Hz, J ) 1.9 Hz, 1H),
6.72 (dd, J ) 13.0 Hz, J ) 2.2 Hz, 1H), 3.73 (s, 3H), 3.64 (s,
3H), 3.48 (m, 4H), 3.18 (m, 4H), 3.10 (s, 6H). 13C NMR (DMSO-
d6) δ 188.5, 164.1 (J ) 11.5 Hz), 161.0 (J ) 250.2 Hz), 154.7,
152.6, 140.4, 132.9, 132.8, 132.5, 131.8 (J ) 4.4 Hz), 130.7,
127.9 (J ) 7.5 Hz), 127.5, 127.4, 125.8 (J ) 5.1 Hz), 120.3,
118.6 (J ) 13.7 Hz), 118.3, 112.7, 111.9, 101.0 (J ) 25.6 Hz),
58.1, 56.7, 52.7, 50.0, 45.7. LC-MS: 475.2 (M).
(E)-3-[4-(2-Trimethylammoniumethoxy)-3′,5′-dimethyl-
biphenyl-3-yl]-1-(2-trimethylammonium-4-methoxy-
phenyl)propenone Diiodide (86). A solution of 68 (69 mg,
0.16 mmol) in iodomethane (2.3 g, 16 mmol) was stirred for
16 h. Iodomethane was removed under reduced pressure and
the residue was washed with THF to give the product as yellow
crystals in 66% yield (70 mg). 1H NMR (DMSO-d6): δ 8.17 (d,
J ) 2.3 Hz, 1H), 8.13 (d, J ) 8.7 Hz, 1H), 7.97 (d, J ) 15.7 Hz,
1H), 7.82-7.70 (m, 2H), 7.36-7.26 (m, 5H), 7.00 (bs, 1H), 4.90
(s, 2H), 4.63 (bs, 2H), 3.93 (s, 3H), 3.91 (m, 2H), 3.22 (s, 9H),
3.04 (s, 9H), 2.35 (s, 6H). 13C NMR (DMSO-d6): δ 192.6, 161.2,
155.7, 138.8, 137.8, 133.7, 133.1, 132.5, 129.8, 129.2, 128.6,
126.0, 124.2, 123.7, 123.1, 121.8, 114.9, 113.3, 63.9, 63.8, 62.1,
55.9, 53.1, 52.5, 20.9. LC-MS: 516.3 (M).
Determination of MIC. MIC was determined in triplicate
in a microdilution assay using Mueller-Hinton agar as
described by the National Committee for Clinical Laboratory
Standards (NCLLS).31 This method was modified to include
an uninoculated dilution series of test compounds to facilitate
MIC determination if the test compound should precipitate.
MIC was determined as the lowest concentration of test
compound able to inhibit visible growth of bacteria.
Kinetics of Bacterial Killing. For the determination of
the killing curve of a test compound, a dilution series of test
compound was made using 2 mL of MH-broth cultures in
microtiter plates and an inoculum of approximately 5 × 105
CFU/mL as described by Amsterdam.32 At the time points
indicated, 100 µL samples were withdrawn from the test tubes,
serially diluted, and spotted in duplicate on unselective agar
plates to determine CFU. Test compounds with bactericidal
activity were capable of decreasing surviving colony counts
(CFU/mL) when incubated with bacteria.
Determination of Solubility. Solubility of the compounds
was determined by preparing a saturated solution of compound
in 0.3 M phosphate buffer (pH 7.4 ( 0.3) in a brown glass tube.
The suspensions were rotated slowly for 24 h. Aliquots were
centrifuged for 10 min at 14 000 rpm, and supernatants were
diluted in 40% (v/v) acetonitrile in water prior to HPLC
analysis (system I). Concentrations of analytes were quantified
against a standard curve and used as a term of solubility.
Supporting Information Available: Experimental data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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