I. Kubo et al. / Bioorg. Med. Chem. 11 (2003) 573–580
579
IR (KBr) 3360, 2960, 2890, 1640, 1590, 1552, 1480,
1395, 1250, 1170 cmꢂ1
Test strains
.
The microorganisms used for the assay, B. subtilis
ATCC 9372, B. ammoniagenes ATCC 6872, M. luteus
ATCC 4698, S. mutans ATCC 25175, P. acnes ATCC
11827, S. aureus ATCC 12598, S. aureus ATCC 33591
(MRSA), S. aureus ATCC 33592 (MRSA), E. coli
ATCC 9637, P. aeruginosa ATCC 10145, E. aerogenes
ATCC 13048, P. vulgaris ATCC 13315, S. choleraesuis
ATCC 35640, S. cerevisiae ATCC 7754, Z. bailii ATCC
60483, C. albicans ATCC 18804, and Aspergillus niger
ATCC 16404, used for this study were purchased from
the American Type Culture Collection (Manassas, VA).
P. aeruginosa IFO 3080 was available from our previous
works.7ꢂ9
Dodecyl protocatechuate (dodecyl 3,4-dihydroxybenzo-
ate) (8). This was obtained in 81% yield as a colorless
solid: 1H NMR (270 MHz, CDCl3, d): 0.89 (t,
J=6.6 Hz, 3H, –CH3), 4.24 (t, J=6.6 Hz, 2H, –OCH2),
6.66 (d, J=7.8 Hz, 1H, ArH), 7.40 (dd, J=2.3, 7.8 Hz,
1H, ArH), 7.53 (d, J=2.3 Hz, 1H, ArH); IR (KBr)
3530, 3360, 2960, 2890, 1700, 1625, 1550, 1480, 1395,
1290, 1180 cmꢂ1
.
Dodecyl 3,5-dihydroxybenzoate (9). This was obtained
1
in 74% yield as a colorless solid: H NMR (270 MHz,
CDCl3, d): 0.88 (t, J=6.6 Hz, 3H, –CH3), 4.21 (t,
J=6.6 Hz, 2H, –OCH2), 6.34 (t, J=2.3 Hz, 1H, ArH),
6.45 (d, J=2.3 Hz, 2H, ArH); IR (KBr) 3340, 2960,
2890, 1690, 1610, 1480, 1395, 1255, 1165 cmꢂ1. HRMS-
EI (m/z): [M]+ calcd for C19H30O4, 322.2140; found
322.2144.
Media
The culture media for the bacteria consisted of 0.9%
nutrient broth (BBL), 0.5% yeast extract (DIFCO), and
0.1% glucose (NYG ) except for the case of S.
mutans. For the culture of S. mutans, BHI consisting
of 3.7% brain heart infusion (DIFCO), and for the
culture of fungi, 2.5% malt extract (BBL) were used,
respectively.
Dodecyl 2,3-dihydroxybenzoate (10). This was obtained
1
in 74% yield as a colorless solid: H NMR (270 MHz,
CDCl3, d): 0.88 (m, 3H, –CH3), 3.86 (m, 2H, –CH2),
4.92 (t, J=6.8 Hz, 1H, –OCH2), 6.79 (d, J=7.8 Hz, 1H,
ArH), 7.03 (t, J=7.8 Hz, 1H, ArH), 7.53 (d, J=7.8 Hz,
1H, ArH); IR (KBr) 3100, 2960, 2860, 1716, 1660, 1545,
Antibacterial assay
1480, 1395, 1272 cmꢂ1
.
Broth macrodilution methods were used as previously
described4,5 with slight modifications. Briefly, serial
2-fold dilutions of the test compounds were prepared in
DMF, and 30 mL of each dilution was added to 3 mL of
NYG broth. These were inoculated with 30 mL of an
overnight culture of the test bacterium. After incubation
of the cultures at 37 ꢁC for 48ꢁh, the minimum inhibi-
tory concentration (MIC) was determined as the lowest
concentration of the test compound that demonstrated
no visible growth. The minimum bactericidal con-
centration (MBC) was determined as follows. After the
determination of the MIC, 100-fold dilutions with drug-
free NYG broth from each tube showing no turbidity
were incubated at 37 ꢁC for 48 h. The MBC was the
lowest concentration of the test compound that was not
visible in the drug-free cultivation.
Dodecyl 3-hydroxy-4-methoxybenzoate (11). This was
1
obtained in 84% yield as a colorless solid: H NMR
(270 MHz, CDCl3, d): 0.88 (t, J=6.6 Hz, 3H, –CH3),
4.20 (s, 3H, –OCH3), 4.26 (t, J=6.6 Hz, 2H, –OCH2),
6.73 (d, J=8.7 Hz, 1H, ArH), 7.43 (dd, J=2.2, 8.7 Hz, 1H,
ArH), 7.65 (d, J=2.2Hz, 1H, ArH); IR (KBr) 3290, 2960,
2890, 1690, 1630, 1600, 1480, 1395, 1251, 1180 cmꢂ1
.
Dodecyl 4-hydroxy-3-methoxybenzoate (12). This was
1
obtained in 66% yield as a colorless solid: H NMR
(270 MHz, CDCl3, d): 0.88 (t, J=6.6 Hz, 3H, –CH3),
3.89 (s, 3H, –OCH3), 4.20 (t, J=6.6 Hz, 2H, –OCH2),
6.78 (d, J=8.7 Hz, 1H, ArH), 7.47 (dd, J=2.2, 8.7 Hz,
1H, ArH), 7.53 (d, J=2.2 Hz, 1H, ArH); IR (KBr)
3360, 2960, 2890, 1645, 1600, 1530, 1480, 1395, 1252,
1170 cmꢂ1
.
Time–kill study
3,4-Dihydroxyphenyl decanoate (13). This was obtained
1
in 84% yield as a colorless solid: H NMR (270 MHz,
The cultivation with dodecyl gallate was performed the
same as the above MIC assay. Samples were withdrawn at
selected time points, and serial dilutions were performed
in sterile saline before the samples were plated onto NYG
agar plates. After the plates were incubated at 37 ꢁC for
24 h, colony forming units (CFU) were estimated.
CDCl3, d): 0.89 (t, J=6.6 Hz, 3H, –CH3), 2.60 (t,
J=6.8 Hz, 2H, –CH2), 6.18 (s, 1H), 6.31 (d, J=7.8 Hz,
1H, ArH), 6.57 (d, J=7.8 Hz, 1H, ArH). IR (nujol)
3480, 3300, 2780, 2720, 1700, 1750, 1650, 1620, 1540,
1250, 1190, 1160 cmꢂ1
.
Heptyl coumarate [heptyl 3-(4-hydroxyphenyl)-2-pro-
penoate] (14). Obtained in 71% yield as a colorless
solid: 1H NMR (270 MHz, CDCl3, d): 0.89 (t,
J=6.6 Hz, 3H, –CH3), 4.18 (t, J=6.6 Hz, 2H, –OCH2),
6.25 (d, J=16.2 Hz, 1H, –CH), 6.88 (d, J=7.8 Hz, 1H,
ArH), 7.0 (d, J=7.8 Hz, 1H, ArH), 7.10 (s, 1H, ArOH),
7.57 (d, J=16.2 Hz, 1H, –CH). IR (nujol) 3520, 3370,
2760, 2720, 1700, 1480, 1660, 1620, 1550, 1320, 1290,
Adsorption test
The test strain was cultured with shaking in YPD broth
overnight at 30 ꢁC and washed twice with 50 mM MOPS
buffer (pH 6.0). After each gallate ester was mixed with
or without yeast cells (108 cells/mL) in the above buffer
at 30 ꢁC, the suspension was vortexed for five seconds.
Absorbance of the supernatants obtained by cen-
trifugation for 2 min was measured at 272 nm.
1200 cmꢂ1
.