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4H), 4.85 (d, J=7.2 Hz, 2H), 5.12 (m, 1H), 7.09 (m, 1H), 7.13 (m,
1H), 7.16 ppm (m, 1H); 13C NMR (75 MHz, CDCl3): d=19.4, 20.1,
23.1, 24.8, 25.2, 27.5, 28.4, 33.1, 33.2, 37.4, 37.7, 37.8, 39.8, 62.5,
115.8, 128.3, 131.3, 132.2, 132.3, 134.2, 139.5, 142.1, 165.5 ppm; ESI-
MS: m/z: 427 [M+H]+.
(CCl4): n˜max =1654, 1686, 1719, 2858, 2959 cmꢀ1; H NMR (300 MHz,
CDCl3): d=0.84–0.98 (m, 12H), 1.18 (m, 12H), 1.37 (m, 2H), 1.52 (m,
1H), 1.62 (m, 2H), 1.77 (brs, 3H), 2.02 (m, 4H), 5.96 (d, J=10.6 Hz,
1H), 7.20 (d, J=14 Hz, 1H), 7.37 (m, 2H), 7.39 (m, 1H), 7.55 (m, 2H,
m), 7.59 ppm (m, 1H); 13C NMR (75 MHz, CDCl3): d=16.2, 20.2, 23.1,
24.1, 25.3, 25.6, 28.4, 32.3, 35.7, 37.9, 39.1, 39.8, 122.4, 125.5, 129.3,
131.3, 134.3, 135.2, 143.3, 146.6, 187.0 ppm; ESI-MS: m/z: 397 [M+
H]+.
1-O-Cinnamoylphytol (11): Viscous liquid; yield: 826 mg, 88%; IR
(CCl4): n˜max =1654, 1668, 1724, 2867 cmꢀ1
;
1H NMR (300 MHz,
CDCl3): d=0.87 (m, 12H), 1.15 (m, 12H), 1.34 (m, 2H), 1.38 (m, 2H),
1.53 (m, 1H), 1.75 (brs, 3H), 2.04 (m, 4H), 4.74 (d, J=7.5 Hz, 2H),
5.43 (t, J=6.0 Hz, 1H), 6.44 (d, J=15.0 Hz, 1H), 7.40 (m, 3H), 7.54
(m, 2H), 7.68 ppm (d, J=16.2 Hz, 1H); 13C NMR (75 MHz, CDCl3):
d=16.4, 19.6, 22.6, 24.4, 24.8, 25.0, 27.9, 32.6, 32.7, 36.7, 36.9, 37.8,
39.3, 39.8, 61.5, 118.2, 128.0, 128.8, 130.4, 130.6, 134.4, 142.8, 144.6,
167.0 ppm; ESI-MS: m/z: 470 [M+H]+.
Biology
In vitro antimicrobial and drug resistance reversal (DRR) assay: This
assay used E. coli cultures, namely the NAL-resistant DH5a strain
(MTCC 1652), procured from IMTEC (Chandigarh, India), and the
NAL-sensitive strain, CA8000, which was a kind gift to CSIR-CIMAP
by Dr. Sushil Kumar (Former Director, CIMAP, Lucknow, India). Mul-
tidrug-resistant clinical isolate KG4 (MDREC) was a kind gift from
Dr. Mastan Singh and Dr. M. K. Gupta of K.G.M. University, Lucknow
and was characterized previously.[25] The MIC values for antibacteri-
al and DRR activities were determined by a twofold serial dilution
broth assay and detected from the observatory data as per CLSI
guidelines[37] using NAL and TET (Sigma–Aldrich) as positive con-
trols. The MIC values reported were consistent across three inde-
pendent experiments performed in duplicate. The DRR potential of
various phytol derivatives was assessed by measuring the MIC
value of NAL in combination with 10 mgmLꢀ1 of the derivatives
against CA8000 and DH5a strains of E. coli, following procedures
described in our earlier publications.[24,25]
Diphytyloxalate (12): Colorless oil; yield: 413.5 mg, 64%; IR (CCl4):
1
n˜max =1654, 1742, 2862, 2926 cmꢀ1; H NMR (300 MHz, CDCl3): d=
0.85–0.96 (m, 24H), 1.26 (m, 24H), 1.48 (m, 4H), 1.55 (m, 4H), 1.67
(m, 2H), 1.74 (brs, 6H), 2.37 (m, 8H), 4.92 (d, J=7.2 Hz, 4H),
5.83 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d=16.3, 19.6, 22.6,
24.8, 25.0, 27.9, 33.2, 34.0, 37.1, 37.9, 39.8, 63.3, 121.9, 145.6,
164.2 ppm; ESI-MS: m/z: 647 [M+H]+.
1-Bromo-3,7,11,15-tetramethyl-2-hexadecene (13): PBr3 (3 mmol)
was added to solution of phytol (2 mmol) in dry benzene. The re-
action mixture was left at 48C for 30 min, followed by stirring at
room temperature (30–358C) for 3 h. After completion, the solvent
was removed by evaporation, and the residue was extracted with
chloroform. The combined organic extracts were dried and evapo-
rated to give crude products, which on purification by column
chromatography on silica gel, afforded pure product 13 as a viscous
dirty-white liquid in 86% yield (619.5 mg) (purity >95%): IR (CCl4):
In addition, synergy studies of most active combinations were also
performed using the broth checkerboard method with TET against
MDREC-KG4.[38] Cation-adjusted Mueller-Hinton broth (150 mL) was
added to each well of a 96-well plate. The last four columns of
wells served as controls for E. coli growth and plate sterility. The
final concentrations ranged from 6.25 to 800 mgmLꢀ1 for tetracy-
cline and from 0.78 to 100 mgmLꢀ1 for test compounds. Thus, each
of the 64 wells had a unique combination of antibiotics and test
compounds. The final bacterial inoculum in each well was 5ꢂ
105 CFUmLꢀ1, except for the negative control. The plates were in-
cubated at 378C for 24 h. The MIC was recorded as the last dilution
without any turbidity, as per CLSI guidelines. Later, the MIC values
were converted from mgmLꢀ1 to micromolar concentrations.
n˜max =1664, 2928, 1460 cmꢀ1 1H NMR (300 MHz, CDCl3): d=0.83–
;
0.88 (m, 12H), 1.26 (m, 12H), 1.38 (m, 2H), 1.58 (m, 1H), 1.62 (m,
2H), 1.76 (brs, 3H), 2.30 (m, 4H), 3.57 (d, J=6.8 Hz, 2H), 5.25 ppm
(m, 1H); 13C NMR (75 MHz, CDCl3): d=16.2, 19.7, 22.6, 24.4, 24.8,
25.2, 27.9, 29.1, 32.7, 34.0, 36.8, 37.2, 37.8, 39.3, 39.8, 118.0,
141.1 ppm; ESI-MS: m/z: 359 and 361 [M+H]+ 381 [M+Na]+.
,
3,7,11,15-Tetramethyl-2-hexadecenal (14): PCC (3 mmol) was
added to a solution of phytol (2 mmol) in dry CH2Cl2. The reaction
mixture was heated at reflux for 6 h, then the solvent was removed
by evaporation. The residue was extracted with chloroform in
excess water. The combined organic extracts were dried and
evaporated to give crude products which, after purification by
column chromatography on silica gel, afforded pure product 14 as
a viscous colorless liquid in 74% yield (435.5 mg) (purity >95%): IR
Time–kill studies: Time–kill studies of TET alone and in combination
with compounds 4, 9, 10, 11, and 14 against MDREC-KG4 strains
was conducted at MIC, 2xMIC, and 4xMIC concentrations using
a method described previously by Eliopoulos and Moellering.[39]
Each analysis was done in triplicate with a control without test
sample. Time–kill curves were derived by plotting log10 CFUmLꢀ1
against time (h). Time–kill kinetics were also studied in combina-
tions of antibiotics and test compounds at the reduced concentra-
tions at which maximum synergy was observed.
1
(CCl4): n˜max =1639, 1690, 2867, 2926, 2953 cmꢀ1; H NMR (300 MHz,
CDCl3): d=0.85 (m, 12H), 1.14 (m, 12H), 1.35 (m, 2H), 1.37 (m, 2H),
1.52 (m, 1H), 1.67 (brs, 3H), 1.97 (m, 4H), 5.89 (d, J=7.8 Hz, 1H),
9.98 ppm (d, J=7.8 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=16.4,
19.6, 22.6, 24.4, 24.8, 25.0, 27.9, 32.6, 32.7, 36.4, 36.6, 37.8, 39.3,
122.4, 142.8, 209.0 ppm; ESI-MS: m/z: 295 [M+H]+.
Ethidium bromide efflux studies: Fluorometric determination of
ethidium bromide (EB) efflux was performed as per reported meth-
ods.[40] To obtain metabolically active cells, MDREC-KG4 cultures
were grown in 10 mL MHB (pH 7.3ꢁ0.2) with an optical density
(OD) of 0.6 at 600 nm. The cells were collected by centrifugation at
16060ꢂg for 3 min and washed with phosphate-buffered saline
(PBS). EB (25 mgmLꢀ1) was added to the bacterial suspension,
which was then incubated for 60 min at 258C in the absence/pres-
ence of test compounds (4, 9, 10, 11, and 14) at their MECs. The
EB-loaded bacterial suspensions were centrifuged at 16060ꢂg for
3 min, the supernatant was discarded, and the pellet was resus-
pended in cold PBS (1ꢂ). The tubes were then placed on ice. Ali-
3,7,11,15-Tetramethyl-2’-phenyloctadec-1(1’),2(3)-diene-2’-one
(15): BF3·Et2O (5 mmol) was gradually added to a stirred solution of
14 (2 mmol) and acetophenone (2 mmol). The reaction mixture
was heated at reflux for 1 h and then left at room temperature
overnight (14 h). After completion of the reaction, the mixture was
diluted with diethyl ether (50 mL), followed by washing with dis-
tilled water (5ꢂ100 mL) to discharge the BF3·Et2O complex. The
combined organic extract was dried over anhydrous Na2SO4 and
evaporated to give crude products which, after purification by
column chromatography on silica gel, afforded pure product 15 as
a reddish viscous liquid in 62% yield (491 mg) (purity >95%): IR
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ChemMedChem 2014, 9, 1860 – 1868 1866