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phy on silica (1% MeOH/CH2Cl2) to furnish lamellarin acetate with
an unsaturated D-ring.
For suspended cells, 75 mL of the XTT reagent (prepared from 5 mL
of 1 mgmLÀ1 XTT sodium in water and 100 mL of 0.383 mgmLÀ1
PMS in water) was added to each well, and the cells were further
incubated for 4 h at 378C with 95% humidity and 5% CO2. After-
wards, the absorbance of orange formazan at 492 nm was mea-
sured with a reference wavelength of 690 nm using a SpectraMax
Plus 384 microplate reader.
General Procedure for the KOH/EtOH Deacetylation
To the lamellarin acetate (1.0 equiv) was added 5% KOH in EtOH
(10 mLmmolÀ1) at room temperature. The mixture was stirred at
room temperature until a clear solution was obtained (5 min). At that
time, the reaction was immediately acidified with 2 N HCl. The mate-
rial was extracted with EtOAc (3 times), and the combined organic
layers were dried over Na2SO4, filtered, and concentrated under re-
duced pressure to provide crude product, which was purified by re-
crystallization (MeOH/CH2Cl2) to furnish the desired lamellarin.
For each well, the background absorbance (averaged from the
wells containing the same volume of complete culture medium)
was subtracted from either A550 or A492 to get the absolute absorb-
ance. The average value from the duplicate wells, which had been
treated with each concentration of the test compounds, was then
compared with that of the untreated wells to yield the percentage
of surviving cells. The IC50 value was finally calculated from the
dose-response curve as the concentration that inhibits the cell
growth by 50% in comparison with the negative control following
48 h of exposure to each test compound. The results shown in
Table 5 are expressed as the mean IC50 value from at least two in-
dependent experiments, excluding those with a variation greater
than 10%. The standard deviations are omitted for visual clarity.
Cytotoxicity Assays
The cell lines used in this study were either purchased from the
American Type Culture Collection (ATCC, Manassas, VA, USA) or re-
ceived as gifts from other sources (Supporting Information Table S1).
Dulbecco’s Modified Eagle Medium (DMEM), as well as Ham’s F12
and RPMI 1640 media, were supplied in powder form by HyClone
Laboratories (Logan, UT, USA), while fetal bovine serum (FBS) and
0.25% trypsin-EDTA were obtained from J R Scientific, Inc. (Wood-
land, CA, USA) and Gibco (Grand Island, NY, USA), respectively. In ad-
dition, bovine insulin, DMSO, doxorubicin, etoposide, glucose, l-glu-
tamine, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide), penicillin-streptomycin, phenazine methosulfate (PMS),
and sodium pyruvate were supplied by Sigma–Aldrich (St. Louis,
MO, USA), whereas XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide) was from Fluka Chemie (St. Louis,
MO, USA). All materials were used as received.
Lipophilicity Determinations
Formamide and 7 reference compounds, including benzyl alcohol,
benzonitrile, methyl benzoate, benzophenone, naphthalene,
benzyl benzoate, and diphenyl ether, were obtained from commer-
cial sources with at least 98% purity and used as received without
further purification. Methanol (HPLC grade) and deionized water
with a resistivity of 18.2 MW·cm (freshly obtained from a Barnstead
MicroPure water purification system, Thermo Scientific, Waltham,
MA, USA) were employed in the preparation of all samples and
mobile phase for HPLC analyses.
Among the 11 cancerous and 1 normal cell lines used for cytotoxi-
city screening of lamellarins and analogs, 10 cell lines were adher-
ent to the culture wells, whereas only HL-60 and P388 grew in sus-
pension. Each cell line was maintained in an appropriate culture
medium supplemented with essential nutrients (Supporting Infor-
mation Table S1) and maintained using standard procedures at
378C with 95% humidity and 5% CO2. All the test compounds and
positive controls, including doxorubicin and etoposide, were pre-
pared as 10 mgmLÀ1 stock solutions in DMSO and freshly diluted
with the corresponding cell culture medium for each cell line on
the day of analysis.
All lamellarins and analogs were predissolved in either THF or
DMSO and further diluted to achieve the concentration of
0.1 mgmLÀ1 in MeOH and water (75:25). Similarly, all the 7 refer-
ence compounds were also prepared as a reference mixture in
MeOH and water (75:25). The lamellarin samples were then subject
to chromatographic log P determinations previously established
for these compounds.[2]
As suggested by the OECD guidelines,[19] all HPLC analyses were
performed using Isocratic elution with methanol:water (75:25) for
15–75 min. On a daily basis, an Agilent 1200 series LC system in-
stalled with a ZORBAX Eclipse Plus C18 column (Agilent Technolo-
gies, Santa Clara, CA, USA) was calibrated using the reference mix-
ture. The retention time (tR) of each reference compound was de-
termined and subsequently converted to the retention factor (k)
using the equation k=(tRÀt0)/t0, where t0 is the column dead time
determined using the unretained formamide. Next, the resulting
log k values of 7 reference compounds were regressed against
their corresponding literature log P data to generate a calibration
curve. Afterwards, the same chromatographic conditions were
then applied to each test compound to determine the retention
factor, which was finally used to calculate the log P value from the
calibration curve constructed on the same day. The results report-
ed in Table 5 are the mean values from at least triplicate determi-
nations with a standard deviation of less than Æ0.1 log unit ac-
cording to the OECD guidelines.
Prior to the assay, the cells were inoculated as a suspension in the
corresponding cell culture medium (100 mL for adherent cells and
75 mL for suspended cells) into 96-well microtiter plates (Costar No.
3599, Corning Incorporated, Corning, NY, USA) at a density of
5000–20000 cells per well, depending on their growth rates. Ad-
herent and suspended cells were then allowed to grow at 378C
with 95% humidity and 5% CO2 for 24 h and 30 min, respectively.
The cytotoxicity assay was initiated by adding an equal volume of
cell culture medium containing either each test compound, posi-
tive control, or DMSO, at predetermined concentrations. Following
48 h of exposure to various treatments, cell viability was deter-
mined using MTT assay for adherent cells or XTT assay for suspend-
ed cells, as described below.
For adherent cells, 100 mL of the MTT reagent (0.5 mgmLÀ1 in
serum-free cell culture medium) was added to each well, and the
microtiter plates were further incubated for 2.5–4 h at 378C with
95% humidity and 5% CO2. The medium was subsequently re-
placed with 100 mL of DMSO to dissolve the purple formazan
before the absorbance at 550 nm was measured using a Spectra-
Max Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA,
USA) with a reference wavelength of 650 nm.
Structure–Activity and Structure–Lipophilicity Relationships
Whenever possible, matched molecular pairs analysis was carried
out to systematically determine both the structure–cytotoxicity
and structure–lipophilicity relationships of lamellarins and analogs.
Chem. Asian J. 2015, 10, 925 – 937
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