Copolyanhydrides Based on OSA-SA
763
phosphorus were purchased from Shanghai Chemical Reagents
Factory (Shanghai, China). Analytically pure SA was also pur-
chased from Shanghai Chemical Reagents Factory, and recrys-
tallized twice from ethanol. Acetic anhydride was refluxed over
2-bromodecanoate (35 g, 0.1253 mol) was added over 10 min.
The reaction mixture was then refluxed for 18 h. The excess
base was neutralized with acetic acid, and ethanol was evapo-
rated under reduced pressure; water was added to dissolve the
inorganic salts, and the layers were separated. The aqueous layer
was extracted with diethyl ether (100 mL) and combined with
the above crude ester. The solution was dried with anhydrous
Na2SO4, filtered, and the solvent was removed at normal pres-
sure.The viscous oil was distilled under reduced pressure to pro-
duce decane-1,1,2-tricarboxylic acid triethyl ester (39 g, 87%).
The ester produced was hydrolyzed by heating under reflux
with 30% sodium hydroxide. After acidification with 6 M sul-
furic acid, the acidic solution was continuously extracted with
diethyl ether. The solvent was removed by distillation to afford
crude tricarboxylic acid, which was decarboxylated by heating
◦
magnesium strips and distilled, bp l39–142 C, before use.All the
solvents were dried over 4 Å molecular sieves and then distilled.
HeLa cells (human epithelial carcinoma cell line), purchased
from ATCC, was used for the cell growth inhibition assay. The
cells were cultivated in Dulbecco’s minimal essential medium
−
1
(
Gibco, BRL, USA) supplemented with 100 mL L foetal calf
−
1
serum (FCS; Gibco), penicillin (100 µg mL ), and strepto-
mycin (100 µg mL ) in a humidified atmosphere of 5% CO2
−
1
◦
at 37 C.
Instruments
◦
at 135–140 C. The crude OSA was dissolved in diethyl ether
IR spectra were obtained with a Bruker Equinox 55 Fourier trans-
form infrared (FT-IR) spectrometer. Ethyl 2-bromodecanoate
and polymer samples were prepared in dichloromethane and cast
onto NaCl plates for recording IR spectra. 2-Octylsuccinic acid
and treated with decolourizing charcoal. After evaporation of
the solvent, the product was recrystallized from ethanol to give
◦
OSA (22.8 g, 91%), mp 88–89 C.
1
δH (400.13 MHz, CDCl3) of OSA: 0.90 (t, 3H), 1.22–1.38
was pressed into a KBr pellet directly. H NMR spectra were
(
(
m, 10H), 1.40–1.56 (m, 4H), 2.58 (d, 2H), 2.66–2.76 (m, 1H)
Fig. 2).
obtained on a Bruker AV400 DQX400 NMR spectrometer, with
CDCl3 as solvent and tetramethylsilane as internal standard.
Thermal analyses were performed on a Perkin–Elmer sys-
tem consisting of a DSC 7 and TGA 7 analyzer with TAC7/DX
instrument controller. Data were processed using UNIX thermal
analysis system software on a DEC computer station. For differ-
ential scanning calorimetry (DSC), an average sample weight
Synthesis of Polymers
Copolyanhydrides were prepared from the reaction of a mix-
ture of acids (SA and OSA) with excess acetic anhydride under
reflux for 30 min in a flask with a side-arm equipped with a cap-
illary nitrogen inlet. The solution was allowed to cool to room
◦
−1
of 5–10 mg was heated at 10 C min under an argon atmo-
sphere. The melting point and the heat of fusion were measured
by DSC. For thermogravimetric analysis (TGA), an average sam-
◦
temperature, and excess solvent was removed at 70 C under
reduced pressure. Potassium acetate (1 mol-%) was added, then
◦
−1
the prepolymer underwent melt-polycondensation directly for
ple weight of 10 mg was heated at 10 C min under a flow of
argon. The decomposition temperature was detected by TGA.
The molecular weights of the polymers were determined on a
Waters gel permeation chromatography (GPC) system consist-
ing of a Waters 600 pump, and Waters 410 differential refractive
index detector. Samples were eluted in dichloromethane through
a Varian MicroPak G4000 and G3000 column installed in series
◦
9
0 min at 180 C; high vacuum (133.3 Pa) was applied. During
the polymerization, a strong nitrogen sweep with vigorous agita-
tion of the melt was performed for 30 s every 15 min. The crude
polymer was dissolved in dichloromethane and the catalyst was
removed from the polymer solution by filtration. The polymer
was precipitated in dry petroleum spirits from a dichloromethane
solution two times.The polymer was dried under vacuum at room
temperature over P2O5.
−
1
at a flow rate of 1.0 mL min . Molecular weights were deter-
mined relative to narrow dispersed polystyrene standards with a
molecular weight range of 1000–200000.
Cell Growth Inhibition Assay by MTT
The cell growth inhibition was evaluated by the modified MTT
Synthesis of Ethyl 2-Bromodecanoate
(
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide)
Decanoic acid (30 g, 0.174 mol) was heated with thionyl chloride
[16,17]
5
assay.
Briefly, thecellswereseededat1 × 10 cellsperwell
(
14 mL, 0.192 mol) for 90 min. The decanoyl chloride produced
in 96-well plates (Falcon), and incubated for 24 h in 200 µL cul-
ture media with 10% FCS to be allowed to attach overnight.
Then the media were replaced by serum-free medium. After
was allowed to cool and red phosphorus (0.05 g) was added.
The mixture was gently refluxed and dry bromine (10.5 mL,
0
.203 mol) was introduced over 6 h. Reflux was continued
until the evolution of hydrogen bromide ceased (12 h). The
-bromodecanoyl chloride produced was allowed to cool and
24 h, the media were placed in triplicate with graded concen-
trations of dissolved solutions of copolymers P(OSA-SA) 20:80
and P(OSA-SA) 30:70 in 0.1 M phosphate buffer at pH 7.4 and
2
absolute ethanol (50 mL) was added over 2 h. The reaction was
completed by heating for 4 h. Low-boiling fractions and excess
ethanol were removed by distillation at normal pressure. The
crude ethyl 2-bromodecanoate was dissolved in carbon tetra-
chloride and washed with water, sodium hydrogen carbonate
solution, and water. The solution was dried over calcium sulfate
◦
3
7 C, for 15 h. Then the cells were treated with MTT (Sigma,
−1
St Louis, MO, USA; 20 µL (5 g L )) for 4 h. After the removal
of the supernatant, the purple-blue sediment dissolved in 150 µL
per well of DMSO, and the optical densities were read on a multi-
well scanning spectrophotometer (Thermo Multiskan MK3) at
492 nm (A492). The growth inhibitory rate (GIR) of the treated
and distilled at normal pressure to give ethyl 2-bromodecanoate
cells was calculated by Eqn 1:
◦
(
37 g, 76.1%), bp 163–164 C.
GIR = (1 − [A492/A492(control)]) × 100%.
(1)
Synthesis of 2-Octylsuccinic Acid
The same amount of solvent 0.1 M phosphate buffered saline
(PBS) used as control for the solution of dissolved copolymers
is an explanation to the reference, which is also shown clearly
in Table 2.
To a solution of sodium (3 g, 0.13 mol) in 75 mL of abso-
lute ethanol, diethyl malonate (21 g, 0.131 mol) was added
◦
◦
dropwise at 70 C. After stirring for 30 min at 70 C, ethyl