Synthesis and evaluation of amphiphilic cationic quinine-derived for antibacterial activity against methicillin-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2007.05.065

Several representative amphiphilic cationic quinine-derived have been synthesized and evaluated against methicillin- resistant Staphylococcus aureus. This is the first reported antibacterial activity of this class of compounds. In vitro the minimal inhibitory concentration values of the best compound Q7 ranged from 0.4 to 1.6 μg/mL (MBC < 3.2 μg/mL).

Full text of DOI:10.1016/j.bmcl.2007.05.065

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4102–4106
Synthesis and evaluation of amphiphilic cationic quinine-derived
for antibacterial activity against methicillin-resistant
Staphylococcus aureus
a
a
b
a,
*
Jian Lv, Yunbo Qian, Tingting Liu and Yongmei Wang
a
Department of Chemistry, The Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China
b
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, 110016 Liao Ning, China
Received 7 March 2007; revised 16 April 2007; accepted 18 May 2007
Available online 24 May 2007
Abstract—Several representative amphiphilic cationic quinine-derived have been synthesized and evaluated against methicillin-
resistant Staphylococcus aureus. This is the first reported antibacterial activity of this class of compounds. In vitro the minimal inhib-
itory concentration values of the best compound Q7 ranged from 0.4 to 1.6 lg/mL (MBC < 3.2 lg/mL).
ꢀ
2007 Elsevier Ltd. All rights reserved.
Drugs from a wide variety of pharmacological groups
are cationic amphiphilic in nature, such as antiarrhyth-
mics, local anesthetics, antimalarials, b-blockers, and
tissue damage. The number of effective antibiotics has
been reduced by the emergence of resistance to penicil-
8
,9
lin, methicillin, and, more recently, vancomycin,
problem that has been compounded by the recent emer-
a
1
tricyclic antidepressants. The drugs are prone to inter-
act with membrane phospholipids: the cationic nitrogen
is attracted to the negatively charged phosphate of the
phospholipid head-group, and the aromatic ring system
is directed toward the hydrophobic interior of the
phospholipid layer. The cationic amphiphilic nature
may have impact on drug pharmacokinetics and phar-
gence of methicillin-resistant S. aureus (MRSA) carriage
0,11
and disease in the community.
1
Antimalarial drugs are currently widely used to treat
patients with autoimmune dermatologic and rheumato-
logic diseases, and have also recently been proposed as
additional therapy for patients with human immunodefi-
2
macodynamics. In the past two decades, amphiphilic
cationic compounds have also been known to exhibit
1
2
ciency virus (HIV) infection. Indeed, the antibacterial
effect of these drugs may be especially important to these
often immunocompromised patients. Since many pa-
tients who receive antimalarials for the treatment of
non-infective inflammatory diseases are also immuno-
suppressed because of their disease or treatments, and
may have concomitant bacterial infections. Another
advantage of antimalarials is that they do not act directly
on the invading pathogens, but rather on the host cells,
so that there are few, if any, chances for the microorgan-
isms to become resistant to their effects. Quinine has been
used as an important natural antimalarial medicine.
3
strong antimicrobial activity. They exhibit rapid
activity against a broad range of microorganisms such
4
as bacteria (both Gram-positive and Gram-negative),
5
fungi, and certain viruses.
6,7
The impact on human health of Staphylococcus aureus
infection in community and hospital settings has led to
intensive investigation of this organism over recent
years. It is the causative agent of a wide range of dis-
eases, from carbuncles and food poisoning, through
more serious device and wound-related infections, to
life-threatening conditions. S. aureus produces a pleth-
ora of virulence factors that facilitate attachment, colo-
nization, cell–cell interactions, immune evasion, and
1
3
Wolf et al. investigated the effect of the antimalarial
drug on the growth and invasion of several bacteria, they
found that the invasive ability of E. agglomerans
(
Enterobacter agglomerans) and S. aureus was signifi-
cantly inhibited by 50 and 100 lM quinine sulfate (QS).
Keywords: Amphiphilic cationic; Quinine-derived; Antibacterial acti-
vity; MRSA.
Acetophenone is used in consumer fragrances and as an
industrial solvent. Recently, the interest in the study of
*
Corresponding author. Tel./fax: +86 22 23502654; e-mail: ymw@
nankai.edu.cn
0
960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.065

J. Lv et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4102–4106
4103
some acetophenone derivatives arises from their signifi-
cant antimicrobial activity against Gram-positive bacte-
molecular structures. The IR spectra of pure products
Q2–7 indicated the presence of strong C@O bands at
1
4,15
16
ꢀ1
ria and fungi,
and antimutagenic activity. In the
1700–1710 cm . The signals from aromatic C@C bonds
ꢀ
1
1
present letter, based on the biological properties as well
as structural features of quinine and acetophenone, we
synthesized a series of amphiphilic cationic quinine-de-
rived compounds against S. aureus and methicillin-resis-
tant S. aureus as a step in studying potential
antibacterial activity of these chemical compounds.
at 1640–1579 cm were expressed. H NMR spectra of
compounds Q2–7 showed characteristic signals from
protons of quinoline heterocycle and aromatic ring in
the range of d 8.91–7.89 and 7.54–7.20 ppm, respec-
1
3
tively. The C NMR spectra of compounds Q2–7 also
showed the presence of C@O bands at 195.1–192.3 ppm.
The amphiphilic cationic quinine-derived compounds
were prepared from the quinine and the corresponding
a-bromo acetophenone derivatives (Scheme 1), which
can be easily prepared by the bromination of acetophe-
none derivatives with cupric bromide. Quinine and
a-bromo acetophenone derivatives were stirred in reflux-
ing acetone for 2 h, to give the corresponding amphi-
Amphiphilic cationic quinine-derived compounds and
quinine were tested against a panel of representative
pathogenic bacteria, together with ampicillin and kana-
mycin as references. Various methicillin-susceptible and
methicillin-resistant S. aureus were included in order to
evaluate the activities of this novel series of amphiphilic
cationic quinine-derived compounds to overcome bacte-
rial resistance. S. aureus ATCC 25923 is a methicillin-
susceptible strain. S. aureus CMAH 0430, S. aureus
CMAH 0504, and S. aureus CMAH 0515 are methicil-
lin-resistant clinical strains. Besides, all the quinine-de-
rived and quinine were also tested against other
Gram-positive and Gram-negative strains of bacteria,
such as Bacillus subtilis ATCC 6633, Micrococcus luteus
CMCC(B) 28001, Escherichia coli ATCC 25922, and
Pseudomonas aeruginosa ATCC 27853. The in vitro anti-
bacterial activities were reported as minimum inhibitory
concentrations (MICs) and minimum bactericidal con-
centrations (MBCs) that were determined by the broth
microdilution method as recommended by the NCCLS
philic cationic quinine-derived Q2–7 in 70–87% yields
(
1
Scheme 2). Both elemental and spectral (IR, H NMR
1
3
17
and C NMR) analysis data of all the synthesized
compounds are in full agreement with the suggested
OMe
OMe
Br-
O
R
N
O
N
Br
R
OH
OH
N
Acetone, reflux
N
Quinine(QN)
Quinine-derivatives
(National Committee for Clinical Laboratory
Standards).
1
8
Scheme 1.
The results in Table 1 show the antibacterial activity of
amphiphilic cationic quinine-derived compounds with
the reference compounds (ampicillin and kanamycin).
Ampicillin and kanamycin have good activity against
methicillin-susceptible S. aureus but not active against
the methicillin-resistant strains.
O
O
O
QN+
QN+
QN+
Br-
Br-
Br-
Br
Cl
Q2 (80%)
Q3 (70%)
Q4(72%)
Quinine was not active against S. aureus. However, the
amphiphilic cationic compound Q2 exhibited improved
activity compared to quinine against S. aureus and
methicillin-resistant S. aureus tested, which were sensi-
tive to the same extent (MICs = 12.5 lg/mL) (Table 1).
Thus, 4 -substituted bromine amphiphilic cationic com-
pound Q3 had better activity against methicillin-suscep-
tible and methicillin-resistant S. aureus (MICs 3.13–
O
O
QN+
Br-
QN+
O
Br-
QN+
Br-
0
NO2
Q5 (87%)
OMe
Q6 (80%)
Q7(75%)
6.25 lg/mL). However, the presence of the other groups
at the 4-position of the aromatic ring (such as –NO2
Scheme 2.
Table 1. In vitro antibacterial activity of quinine-derived compounds (MIC/MBC, lg/mL)
Strain
Quinine Q2
Q3
Q4 Q5
Q6
Q7
Ampicillin Kanamycin
Staphylococcus aureus ATCC 25923
MRSA CMAH 0430
MRSA CMAH 0504
—
—
—
—
—
—
—
—
12.5/25
3.13/6.25
3.13/6.25
6.25/12.5
3.13/6.25
3.13/6.25
—
—
—
—
—
—
—
—
25/50 12.5/12.5 1.56/3.13 0.39/0.78
0.39/0.78
—
—
12.5/25
12.5/25
12.5/25
25/25
50/50 25/50
50/50 25/50
50/50 25/50
25/50 12.5/25
0.39/1.56
0.78/1.56
0.39/1.56
—
—
—
MRSA CMAH 0515
—
Bacillus subtilis ATCC 6633
Micrococcus luteus CMCC(B) 28001
Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853
3.13/6.25 0.39/0.39
6.25/6.25
0.39/0.39
0.39/0.39
—
6.3/12.5 6.25/6.25
25/50 12.5/12.5 3.13/3.13 0.39/0.78
—
—
100/—
100/—
—
—
—
—
50/100
100/—
50/100
—

4104
J. Lv et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4102–4106
or –OMe) gave similar or lowered activity against the S.
aureus, especially a chlorine substituent at the 4-position
of the aromatic ring Q4 was not active against S. aureus.
Interestingly, the amphiphilic cationic compound Q7,
by replacing the phenyl group of Q2 with the bulkier
b-naphthyl moiety, showed good activity against
S. aureus tested (MIC = 1.56lg/mL), and much better
against MRSA (MIC 0.39 to 0.78 lg/mL), while ampi-
cillin and kanamycin were inactive up to the concentra-
tion of 100 lg/mL. Furthermore, Q3 and Q7 had also
good activity against other Gram-positive bacteria
Table 2. Inhibition zone (diameter in mm) of quinine-derived com-
pounds and various antibiotics against S. aureus and three methicillin-
resistant S. aureus (MRSA) isolates by disk diffusion method
Antibiotics
30 lg/disk
Zone of inhibition (mm)
S. aureus
MRSA
CMAH
0430
MRSA
CMAH
0504
MRSA
CMAH
0515
Q3
Q7
24
26
34
30
14
28
27
32
32
23
23
26
—
2
22
23
—
—
17
—
—
—
26
22
22
25
—
—
17
—
—
—
27
24
Ampicillin
Cefazolin
Tetracycline
Kanamycin
Midecamycin
Levofloxacin
Cloramphenicol
Vncomycin
(
MIC 6 6.25 lg/mL) and weak activity against Gram-
15
—
—
—
27
24
negative bacteria, however, other amphiphilic cationic
compounds were not active against Gram-negative bac-
teria. The MBC data for the amphiphilic cationic
compounds except inactive compounds against Gram-
positive bacteria are shown in Table 1. The MBC value
was considered as the lowest amphiphile concentration
at which there was no viable cell present. In all cases,
the MBCs of the amphiphilic cationic compounds were
twofold higher than the corresponding MICs against
Gram-positive bacteria tested, suggesting a bactericidal
activity. Moreover, the MBC value of Q7 is only
2
0
Mueller–Hinton agar (Difco) with 2% NaCl, and other
kinds of antibiotics, such as ampicillin, cefazolin, tetra-
cycline, kanamycin, midecamycin, levofloxacin, chlor-
amphenicol, vancomycin, etc., were used as reference
antibacterial agents (Table 2). Although amphiphilic
cationic compounds Q3 and Q7 were a little weaker
against S. aureus than the other antibiotics, Q3 and
Q7 were more active against three isolates of MRSA.
As shown in Figure 2 (MRSA CMAH 0504), Q3, Q7,
1
.56 lg/mL against MRSA.
Figure 1 shows the log(survivors) versus exposure time
plots for the different concentrations of amphiphilic cat-
ionic compound Q7 against S. aureus ATCC 25923 and
1
9
methicillin-resistant S. aureus CMAH 0504. Exposure
of the Q7 to bacterial cells was carried out in Mueller–
Hinton broth (Difco) medium. The concentration of
Q7 was kept at 5, 10, and 15 lg/mL against S. aureus
and at 2.5, 5, and 10 lg/mL against MRSA. When
exposed to Q7, all bacterial cells were killed within
2
0–30 min for MRSA (CMAH 0504) at 10 lg/mL,
whereas not until 40 min for S. aureus (ATCC 25923)
at 15 lg/mL. It is evident that with the compound Q7
the time required to kill MRSA is less than that for
the common S. aureus.
Next, amphiphilic cationic compounds Q3 and Q7,
which proved to have good activity against S. aureus
and MRSA, were tested for the efficacy of detecting S.
aureus and three MRSA isolates’ growth inhibition by
disk-diffusion method outlined by the NCCLS with
Figure 2. Comparison of activities of various antibacterial compounds
with Q3 and Q7 against MRSA (CMAH 0504) isolate by disk-
diffusion method.
Figure 1. log(survivors) versus exposure time plots for compound Q7 against (a) S. aureus (ATCC 25923): 1, control (without Q7); 2, 5 lg/mL; 3,
0 lg/mL; 4, 15 lg/mL; (b) MRSA (CMAH 0504): 1, control (without Q7); 2, 2.5 lg/mL; 3, 5 lg/mL; 4, 10 lg/mL.
1

J. Lv et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4102–4106
Table 3. Hemolytic activity of quinine-derived compounds
4105
Acknowledgments
Compound
Percent lysis of human red blood cells as a
function of compound concentration
This work was financially supported by the National
Natural Science Foundation of China (Grant Nos.
2
000
1000 lg/mL
500 lg/mL
250 lg/mL
20672061 and 20472032, China). We also thank Nankai
lg/mL
University State Key Laboratory of Elemento-organic
Chemistry for support.
Quinine
Q2
Q3
0
5
0
0
0
0
0
0
0
9
0
0
0
0
0
0
25
0
7
Q5
Q6
0
References and notes
0
55
0
17
Q7
1
2
. Grike, S.; Mohr, K.; Schrape, S. Biochem. Pharmacol.
989, 38, 2487.
1
. L u¨ llmann, H.; Mohr, K. Drug Phospholipid Interactions.
In Metabolism of Xenobiotics; Gorrod, J. W., Oelschl a¨ ger,
H., Caldwell, J., Eds.; Taylor & Francis: London, 1988.
. Hugo, W. B.; Russel, A. D. In Types of Antimicrobial
Agents, Principles and Practice of Disinfection, Preserva-
tion; Russel, A. D., Hugo, W. B., Ayliffe, G. A. J., Eds.,
and other antibiotics at 30 lg/mL diffused outward and
inhibit the growth of MRSA (CMAH 0504). The bacte-
ria on the left plate are susceptible to all but two drugs
3
(
ampicillin and kanamycin), whereas on the right is
resistant to three drug (cefazolin, midecamycin, levo-
floxacin). The compound Q7 was less active against
MRSA (CMAH 0504) than chloramphenicol, better
than vancomycin and tetracycline. Q3 also has the same
activity as vancomycin.
2
1
nd ed.; Blackwell Scientific Publications Oxford: Oxford,
992; pp 7–68.
4. Merianos, J. J. Quaternary Ammonium Antimicrobial
Compounds. In Disinfection, Sterilization, and Preserva-
tion; Block, S. S., Ed., 4th ed.; Lea and Febiger:
Philadelphia, 1991; pp 225–255.
5
. Ahlstrom, B.; Chelminska-Bertilsson, M.; Thompson, R.
A.; Edebo, L. Antimicrob. Agents Chemother. 1997, 41,
Since a number of the amphiphilic cationic compounds
exhibited strong antibacterial activity against MRSA,
they had potential to be developed as antibacterial ther-
apeutics. But because of the high affinity of the amphi-
5
44.
. Allenmark, S.; Lindstedt, M.; Edebo, L. U.S. Patent
080902, 1992.
6
7
5
philic
cationic
compounds
toward
biological
. Wong, Y.-L.; Hubieki, M. P.; Curfaman, C. L.; Doncel,
G. F.; Dudding, T. C.; Savle, P. S.; Gandour, R. D.
Bioorg. Med. Chem. 2002, 10, 3599.
membranes, it was thought that they might have a sim-
ple lytic mode of action and be broadly cytotoxic against
human cells. We therefore determined the hemolytic
activity of these compounds as a measure of their cyto-
toxicity and an initial test of their suitability for further
development.
8. Centers for Disease Control and Prevention. Staphylococ-
cus aureus resistant to vancomycin—United States,
MMWR. Moribid. Morial. Wkly. Rep. 2002, 51, 565.
9
. Centers for Disease Control and Prevention. Public heath
dispatch: vancomycin-resistant Staphylococcus aureus—
Pennsylvania, MMWR. Moribid. Morial. Wkly. Rep.
Compounds Q5 and Q6 were the only compounds
other than quinine itself that did not lyse the eryth-
rocytes at any of the concentrations studied (Table
2
002, 51, 902.
1
0. Centers for Disease Control and Prevention. Four pedi-
atric deaths. from community-acquired methicillin-resis-
tant Staphylococcus aureus—Minnesota and North
Dakota, 1997–1999. MMWR. Morbid. Mortal. Wkly.
Rep. 1999, 48, 707.
2
1
3
). For the strongly antibacterial Q3 at concentra-
tions well above the MICs, for example, 1000 lg/
mL, hemolysis is only 7%, while for Q7, hemolysis
is 17% at this concentration, but drops to 9% at
11. Okuma, K.; Iwakawa, K.; Turnidge, J. D.; Grubb, W. B.;
Bell, J. M.; O’Brien, F. G.; Coombs, G. W.; Pearman, J.
W.; Tenover, F. C.; Kapi, M.; Tiensasitorn, C.; Ito, T.;
Hiramatsu, K. J. Clin. Microbiol. 2002, 40, 4289.
5
00 lg/mL, which is still well above the MICs. It is
clear that these quinine-derived ammonium salts have
surprisingly low lytic properties, although they are
also amphiphilic cationic compounds. Compounds
Q3 and Q7 show no hemolytic activity at concentra-
tions 100-fold greater than the MICs.
1
1
2. Boelaert, J. R.; Piette, J. J. Clin. Virol. 2001, 20, 137.
3. Wolf, R.; Tufano, M. A.; Ruocco, V.; Grimaldi, E.;
Ruocco, E.; Donna-rumma, G.; Baroni, A. Int.
J. Dermatol. 2006, 45, 661.
1
4. Gul, H. L.; Denizci, A. A.; Erciyas, E. Arzneim.-Forsch.
2002, 52, 773.
15. Rajabi, L.; Courreges, C.; Montoya, J.; Aguilera, R. J.;
In conclusion, a series of new amphiphilic cationic
quinine-derived compounds were synthesized and
characterized. It appears that Q3 and Q7 may be
able to maintain good antibacterial activity against
most pathogenic bacteria. Interestingly, the two
amphiphilic cationic quinine-derived compounds have
good activity against methicillin-susceptible and meth-
icillin-resistant S. aureus (from clinical isolates), which
may be to hold promise against drug-resistance bacte-
ria. Though Q3 and Q7 are amphiphilic cationic
compounds, they have surprisingly low lytic proper-
ties, and thus may be suitable lead candidates for
drug development.
Primm, T. P. Lett. Appl. Microbiol. 2005, 40, 212.
6. Miyazawa, M.; Shimamura, H.; Nakamura, S. J. Agric.
Food Chem. 2000, 48, 4377.
1
1
7. All new compounds gave satisfactory analytical and
spectral data.
Compound Q2: yield: 80%. Mp 176–178 ꢁC. IR (KBr) m
ꢀ1
(
1
1
cm ): 3445, 2953, 2883, 1702, 1640, 1601, 1559, 1450,
1
024, 917, 832. H NMR (300 MHz, DMSO-d
.21–0.96 (m, 2H), 1.59–1.56 (m, 1H), 1.80–1.78 (m, 1H),
6
) d (ppm): d
2.01–1.99 (m, 2H), 2.86–2.79 (m, 2H), 3.83–3.71 (m, 2H),
3.98 (s, 3H), 4.27–4.23 (m, 1H), 4.43–4.38 (m, 1H), 4.60–
4.58 (m, 1H), 5.17 (d, J = 10.5 Hz, 1H), 5.35 (d,

4106
J. Lv et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4102–4106
1
3
J = 17.4 Hz, 1H), 5.91–5.80 (m, 1H), 6.38 (d, J = 7.5 Hz,
J = 8.4 Hz, 1H), 8.89 (d, J = 4.0 Hz, 1H). C NMR (75
MHz, DMSO-d ): 193.2, 159.0, 148.4, 148.2, 144.1, 140.9,
1
2
H), 7.20 (s, 1H), 7.54–7.46 (m, 5H), 7.89 (d, J = 15.6 Hz,
6
1
3
H), 8.04–8.00 (m, 2H), 8.91 (d, J = 4.5 Hz, 1H).
): 194.1, 158.5, 148.1, 144.4,
44.0, 139.0, 135.8, 134.8, 132.3, 129.7, 129.3, 126.0, 125.6,
C
138.8, 132.3, 130.9, 128.5, 126.3, 124.2, 123.0, 119.9,
116.8, 101.5, 65.8, 62.8, 61.0, 57.9, 57.2, 56.8, 53.8, 37.6,
NMR (75 MHz, DMSO-d
6
+
1
1
2
26.2, 24.9, 22.3. MS (ESI): 473 [MꢀBr] . Calcd for
21.1, 116.3, 101.5, 66.0, 63.6, 60.8, 57.8, 56.7, 37.3, 26.0,
5.5, 22.1. MS (ESI): 443 [MꢀBr] . Calcd for
28 2 4
C H33BrN O : C, 62.93; H, 6.01; N, 5.06%. Found: C,
+
62.90; H, 6.04; N, 5.03%.
Compound Q7: yield: 75%. Mp 170–172 ꢁC. IR (KBr) m
(cm ): 3345, 3113, 2946, 2888, 1710, 1678, 1620, 1588,
1508, 1473, 1433, 1357, 1262, 1239, 1120, 1042, 1228,
1085, 1024, 917, 832. MS (ESI): 488.1. H NMR
C
28
H31BrN
2
O
3
: C, 75.82; H, 7.04; N, 6.32%. Found: C,
ꢀ
1
75.80; H, 7.01; N, 6.35%.
Compound Q3: yield: 70%. Mp 180–182 ꢁC. IR (KBr) m
ꢀ
1
1
(
1
cm ): 3414, 3147, 2939, 1701, 1637, 1619, 1586, 1507,
NMR
1
H
469, 1397, 1239, 1024, 997, 860, 837.
6
(400 MHz, DMSO-d ) d (ppm): 1.09–0.99 (s, 1H), 1.91–
(
2
3
300 MHz, DMSO-d ) d (ppm): d 1.18–0.96 (m, 1H),
.11–1.92 (m, 4H), 2.87 (s, 1H), 3.32 (d, J = 8.8 Hz, 2H),
.85–3.72 (m, 2H), 4.07 (s, 3H), 4.21–4.18 (m, 1H), 4.59
6
2.12 (m, 3H), 2.46 (s, 2H), 2.88 (s, br, 1H), 3.35 (s, 1H),
3.81–3.89 (m, 2H), 4.11 (s, 3H), 4.32–4.30 (m, 1H), 4.40–
4.65 (m, 2H), 5.02 (d, J = 10.4 Hz, 1H), 5.24 (d,
J = 17.2 Hz, 1H), 5.83–5.73 (m, 3H), 7.14 (s, 1H), 7.41
(d, J = 8.4 Hz, 1H), 7.40–7.43 (m, 3H), 7.94 (d.
J = 8.8 Hz, 1H), 8.19–8.04 (m, 4H), 8.76 (s, 1H), 9.02
(
d, J = 10.0 Hz, 2H), 5.50–5.41 (dd,
J
1
= 10.0,
= 10.4 Hz, 1H), 5.14–5.36 (dd, J = 17.6, J = 10 Hz,
J
1
1
2
1
2
H), 5.81–5.67 (m, 3H), 7.11 (s, 1H), 7.42 (d, J = 9.2 Hz,
H), 7.71 (d, J = 4.0 Hz, 1H), 7.96–7.87 (m, 3H), 8.10 (d,
1
3
6
(s, 1H). C NMR (100 MHz, DMSO-d ) d (ppm): 193.2,
1
3
J = 8.4 Hz, 2H), 8.76 (d, J = 4 Hz, 1H). C NMR (75
MHz, DMSO-d ): 193.2, 158.4, 148.1, 144.4, 144.1, 140.8,
1
1
2
159.0, 148.4, 148.2, 144.1, 140.9, 138.8, 132.3, 130.9,
128.5, 126.3, 124.2, 123.0, 119.9, 116.8, 101.5, 65.8, 62.8,
61.0, 57.9, 57.2, 56.8, 53.8, 37.6, 26.2, 24.9, 22.3. MS-ESI:
6
39.0, 133.6, 131.3, 129.8, 126.0, 122.7, 121.1, 116.2,
01.6, 65.9, 63.5, 60.7, 57.8, 56.8, 37.3, 31.4, 26.0, 25.5,
+
m/z: Positive polarity: 493 [MꢀBr] . Calcd for
+
2.1. MS (ESI): 478 [MꢀBr] . Calcd for C28
H30Br
N
2 2
O
3
:
32 2 3
C H33BrN O : C, 67.01; H, 5.80; N, 4.88%. Found: C,
C, 55.83; H, 5.02; N, 4.65%. Found: C, 55.85; H, 5.01; N,
.60%.
Compound Q4: yield: 72%. Mp 174–176 ꢁC. IR (KBr) m
66.99; H, 5.78; N, 4.84%.
4
18. National Committee for Clinical Laboratory Standards.
Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically. Fifth Edition; Approved
standard M7-A5. Wayne, PA: NCCLS; 2001.
ꢀ
1
(
cm ): 3414, 3125, 2960, 1703, 1638, 1584, 1463, 1400,
1
1
262, 1084, 871, 800. H NMR (300 MHz, DMSO-d ) d
6
(
ppm): d 1.08–1.06 (m, 1H), 2.12–2.05 (m, 4H), 2.87 (s,
19. The killing time studies were performed with compound Q7
at the final concentrations higher than MBC values against
MRSA CMAH 0504 and S. aureus ATCC 25923, respec-
tively. The mid-logarithmic phase cultures were appropri-
ately diluted with Mueller–Hinton broth medium to achieve
1
4
1
1
H), 3.11 (s, 2H), 3.85–3.74 (m, 2H), 4.07 (s, 3H), 4.22–
.19 (m, 1H), 4.63–4.58 (m, 2H), 5.01 (d, J = 10.4 Hz,
H), 5.21 (d, J = 17.2 Hz,1H), 5.81–5.64 (m, 3H), 7.12 (s,
H), 7.44–7.41 (m, 1H), 7.75–7.71 (m, 3H), 7.95 (d,
5
J = 9.2 Hz, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.76 (d,
J = 4.4 Hz, 1H). C NMR (75 MHz, DMSO-d ): 193.2,
a final inoculum of 3–5 · 10 cell/mL. The same inoculum
1
3
was added to drug-free medium as a growth control. The
test tubes were incubated in a water bath at 30 ꢁC. Next,
100 lL of samples was taken from each tube at known time
intervals, centrifuged, and washed three times with fresh
Mueller–Hinton broth. After vortexed vigorously for 10 s,
the suspension was suitably diluted and spread onto
Mueller–Hinton agar plates. The plates were incubated at
30 ꢁC for 24 h and colonies were counted. Each number of
viable cells was determined from three-independent exper-
iments performed in triplicate.
6
1
1
6
5
5
58.4, 148.1, 144.4, 144.1, 140.8, 139.0, 133.6, 132.2,
31.3, 129.8, 126.0, 122.7, 121.1, 116.2, 101.6, 65.9, 63.5,
0.7, 57.8, 56.8, 37.3, 31.4, 26.0, 25.5, 22.1. MS (ESI):
+
21 [MꢀBr] . Calcd for C H BrN O : C, 60.28; H,
2
8
30
2
3
.42; N, 5.02%. Found: C, 60.24; H, 5.40; N, 5.00%.
Compound Q5: yield: 87%. Mp 199–201 ꢁC. IR (KBr) m
(
1
ꢀ1
cm ): 3345, 3171, 2953, 2883, 1705, 1620, 1687, 1509,
1
451, 1342, 1228, 1185, 1024, 917, 832. H NMR
(
300 MHz, DMSO-d (ppm): 1.00–0.97 (m,
6
)
d
d
1
1
4
H),1.23–1.19 (m, 1H), 1.60–1.57 (m, 1H), 1.83 (br,
H), 2.88–2.82 (m, 2H), 3.35 (s, 4H), 3.84–3.81 (m, 2H),
.00 (s, 3H), 4.36 (d, J = 13.5 Hz, 1H), 4.56 (d,
20. National Committee for Clinical Laboratory Standards.
Performance Standards for Antimicrobial Disk Suscepti-
bility Tests. Eighth Edition; Approved Standard M2-A8.
Wayne, PA: NCCLS; 2003.
J = 13.5 Hz, 1H), 4.69–4.65 (m, 1H), 5.18 (d,
J = 10.8 Hz, 1H), 5.93–5.57 (m, 1H), 6.42 (d,
J = 7.5 Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 7.51–7.47 (dd,
21. Human blood from healthy volunteers was collected in
10 mL Vacationer tubes containing sodium heparin as
anticoagulant. The cells were washed three times with
calcium- and magnesium-free phosphate-buffered saline
(PBS) and centrifuged at 2000g for 10 min. The third
supernatant liquid was clear and colorless. Then 0.1 mL
erythrocyte suspension diluted with PBS (erythrocyte
J
1
2
= 2.4, J = 1.2 Hz, 1H), 8.06–8.02 (m, 2H), 8.19 (d,
J = 8.7 Hz, 2H), 8.37 (d, J = 8.7 Hz, 2H), 8.93 (d,
J = 4.8 Hz, 1H). C NMR (75 MHz, DMSO-d ): 192.7,
1
3
6
1
1
6
4
5
51.1, 150.1, 148.2, 145.7, 142.0, 138.8, 130.9, 130.7,
30.5, 128.5, 125.2, 124.2, 123.7, 119.8, 117.2, 65.6, 63.2,
1.2, 56.9, 53.7, 37.9, 37.5, 26.7, 25.0, 22.3. MS (ESI):
9
concentration around 1.0 · 10 cells/mL) was mixed with
+
88 [MꢀBr] . Calcd for C H BrlN O : C, 59.16; H,
0.1 mL of test substances at a series of concentrations (1–
2000 lg/mL). The mixtures were incubated at 37 ꢁC for
1 h. After incubation, tubes were centrifuged at 2000g for
10 min. The supernants were transferred into 96-well
polystyrene plates (Costar 3590, incorporated) and the
optical density was measured at 540 nm using MTP120
microplate reader (Colona Electric, Japan). The values for
0% and 100% lysis were determined by incubating
erythrocytes with PBS, and 0.1% (v/v) Triton X-100
(Amresco 0694), respectively. Assays were carried out in
triplicate and the results were confirmed in three-indepen-
dent experiments.
2
9
30
3
5
.32; N, 7.39%. Found: C, 59.13; H, 5.33; N, 7.38%.
Compound Q6: yield: 80%. Mp 195–197 ꢁC. IR (KBr) m
ꢀ1
(
1
cm ): 3324, 3171, 2975, 2888, 1700, 1621, 1579, 1520,
H
1
509, 1475, 1459, 1343, 1231, 1085, 992, 931, 855.
NMR (300 MHz, DMSO-d ) d (ppm): d 1.23–0.93 (m,
6
2H), 1.56 (d, J = 9.2 Hz, 1H), 2.11–1.79 (m, 3H), 2.89–
2.78 (m, 2H), 3.35 (s, 3H), 3.84–3.81 (m, 3H), 3.97 (s,
3H), 4.18–4.07 (m. 1H), 4.37 (d, J = 13.2 Hz, 1H), 4.74–
4.58 (m, 2H), 5.13 (d, J = 10.8 Hz, 1H), 5.34 (d, J = 17.2,
1H), 5.91–5.83 (m, 1H), 6.42 (d, J = 7.2 Hz, 1H), 7.43–
8.18 (m, 5H), 8.40 (d, J = 9.2 Hz, 1H), 8.44 (d,