W. Wei et al.
Journal of Controlled Release 285 (2018) 187–199
(
NMM), Maleic anhydride, 6-aminocaproic acid, Dithiodipropionic acid
then the filtrate was dried to produce a crude product, which was
purified by silica gel column chromatography (dichloromethane/me-
and thiodipropionic acid were purchased from Aladdin Industrial
Corporation (Shanghai,China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe-
nyltetrazoliumbromide (MTT), trypsin, Dulbecco's modified eagle medium
thanol (100:2) to obtain 270 mg of DSSM (Compound 7) as white solid
1
(40% yield). H NMR (400 MHz, DMSO‑d
6
, ppm) of DSSM: δ 7.97–8.01
(DMEM, high glucose) and Roswell Park Memorial Institute (RPMI-1640)
(2H,d), 7.85–7.88(1H,d), 7.7–7.73 (1H,t), 7.64–7.69 (2H,t), 7.37–7.42
(4H,m), 7.14–7.18 (1H,t), 7.03 (2H,S), 5.75–5.82 (1H,t), 5.38–5.41
(1H,d), 5.08–5.1 (3H,m), 5.0–5.02 (1H,d), 4.9–4.94 (2H,t), 4.42–4.45
were bought from Gibco (Beijing, China). All other reagents and solvents
were of analytical or HPLC grade.
(
1H,S), 4.15–4.17 (2H,t), 4.01–4.03 (3H,S), 3.65–3.68 (1H,d), 3.6–3.65
2.2. Synthesis of DTX-Maleimide prodrugs
(2H,t), 2.24–2.91 (12H,m), +0.97–1.90 (24H,m). ESI-MS (m/z):
[
M + Na]+ = 1145.5, [M + K] = 1161.4.
Synthesis of DTX-Maleimide (DM): The prodrug DM was established
Synthesis of DTX-S-Maleimide (DSM): Firstly, thiodipropionic acid
by a two-step reaction. Firstly, 6-maleimidocaproic acid was synthe-
sized as described previously [18]. Then DM was prepared through
conjugating EMC to the C2’-hydroxyl position of DTX via a simple ester
bond. Briefly, DTX (403.9 mg, 0.5 mmol) and EMC (116.2 mg,
(356 mg, 2 mmol) was dissolved in 20 ml acetic anhydride and stirred at
30 °C for 3 h. Excess solvent was removed with anhydrous toluene in
high vacuo three times at 30 °C to prepare compound 8. Then this
product (Compound 8) was dissolved in 15 ml anhydrous di-
chloromethane, Mal-OH (310.2 mg, 2.2 mmol) and DAMP (48.89 mg,
0.4 mmol) were added into the solution. The reaction proceeded over-
night at 25 °C under nitrogen. TLC was utilized to monitor the reaction
process. At the end of reaction, solvent was evaporated and the crude
product was purified by silica gel column chromatography (di-
chloromethane/methanol (100:0.75) to give about 380 mg compound 9
as a yellow oily solid (60% yield).
0.55 mmol) were dissolved in 10 ml anhydrous dichloromethane, then
DCC (206.2 mg, 1 mmol) and DMAP (12.2 mg, 0.1 mmol) were added
and stirred under nitrogen for 12 h at room temperature. Upon com-
pletion, filtered to remove the insoluble solids N, N-dicyclohexylurea
(
DCU). Then the filtrate was dried under vacuum. The crude product
was further purified by a silica gel column (dichloromethane/methanol
100:1.5)), and obtain 313.4 mg white powder (mass, 62% yield). 1
NMR (400 MHz, DMSO-d ppm) of DM: 7.97–7.99 (2H,d),
.83–7.87(1H,d), 7.67–7.73 (1H,t), 7.64–7.67 (2H,t), 7.34–7.41
(
H
6
,
δ
Next, a solution of compound 9 (198.7 mg, 0.66 mmol) in 15 ml
anhydrous dichloromethane was activated by HATU (456.3 mg,
1.2 mmol) at 0 °C for 30 min. Then 4-methylmorpholine (NMM)
(0.13 mL, 1.2 mmol) and DTX (484.7 mg, 0.6 mmol) in 10 ml anhydrous
dichloromethane was added to the mixture, and further stirred under
nitrogen at 25 °C for 12 h. TLC was utilized to monitor the reaction
process. At the end of reaction, filtered to remove the insoluble solids,
then the filtrate was dried to produce a crude product, which was
purified by silica gel column chromatography (dichloromethane/me-
thanol (100:2) to obtain about 320 mg of DSM (Compound 10) as white
7
(
(
4H,m), 7.13–7.18 (1H,t), 7.0 (2H,S), 5.75–5.78 (1H,S), 5.38–5.40
1H,d), 4.89–5.08 (6H,m), 4.39–4.42 (1H,S), 4.01–3.62 (6H,m),
2
.36–2.39 (2H,t), 2.21–2.23 (4H,m), 0.98–1.78 (30H,m). ESI-MS (m/z):
+
[
M + Na] = 1023.5.
Synthesis of N-(2-Hydroxyethyl) Maleimide (Mal-OH): N-(2-hydro-
xyethyl) maleimide (Mal-OH) was prepared according to a previously
reported method [3, 34]. Firstly, 30.0 g maleic anhydride (306 mmol)
was dissolved in toluene and 22.4 ml furan (309 mmol) was slowly in-
jected into the solution via syringe. After the mixture stirred for 24 h at
room temperature, the reaction was stopped and the white crystals were
precipitated from the mixture, collected and washed with toluene. The
white powder was dried in a vacuum oven to prepare furan-protected
maleic anhydride. Secondly, the furan-protected maleic anhydride
solid (50% yield). 1H NMR (400 MHz, DMSO‑d
, ppm) of DSM: δ
6
7.97–7.99 (2H,d), 7.87 (1H,d), 7.67–7.72 (1H,t), 7.61–7.65 (2H,t),
7.37–7.41 (4H,m), 7.13–7.19 (1H,t), 7.03 (2H,S), 5.75–5.82 (1H,t),
5.37–5.41 (1H,d), 5.03–5.1 (3H,m), 5.0–5.03 (1H,d), 4.87–4.93 (2H,t),
4.41–4.45 (1H,S), 4.12–4.17 (2H,t), 4.01–4.07 (3H,S), 3.65–3.68
(1H,d), 3.6–3.65 (2H,t), 2.24–2.72 (12H,m), 0.97–1.88 (24H,m). ESI-
MS (m/z): [M + Na]+ = 1113.7, [M + K]+ = 1129.6.
(
1
4.98 g, 30 mmol) was suspended in methanol and stirred at 0 °C. Then
.8 ml of ethanolamine (30 mmol) was added dropwise to the solution,
stirred and refluxed at 80 °C for 24 h. Then the reaction mixture was
cooled to 0 °C, the product was crystallized from the mixture and col-
lected to obtain furan-protected N-(2-hydroxyethyl)-maleimide. Finally,
furan-protected N-(2-hydroxyethyl)-maleimide (2.1 g, 10 mmol) in 10 ml
toluene was stirred and refluxed under nitrogen for 12 h. The reaction
mixture was hot filtered, and the filtrate was stored at 4 °C overnight.
The product crystallized from the mixture, then collected, dried under
vacuum for 24 h to obtain N-(2-hydroxyethyl)-maleimide (Mal-OH).
Synthesis of DTX-SS-Maleimide (DSSM): Firstly, dithiodipropionic
acid (420 mg, 2 mmol) was dissolved in 20 ml acetic anhydride and
stirred at 30 °C for 3 h. Excess solvent was removed with anhydrous
toluene in high vacuo three times at 30 °C to prepare compound 5. Then
this product (Compound 5) was dissolved in 15 ml anhydrous di-
chloromethane, Mal-OH (310.2 mg, 2.2 mmol) and DAMP (48.89 mg,
2.3. Incubation with bovine serum albumin and plasma serum
Compound DM, DSM and DSSM (300 μM) were added to a solution
of BSA (700 μM in PBS, pH 7.4) and incubated at 37 °C for albumin
binding studies. Then 50 μL samples were directly analyzed using HPLC
after an incubation time of 5 min, 30 min and 90 min. Similarly, blood
plasma (EDTA-stabilized) was taken from healthy Sprague-Dawley rats.
Then, DM, DSM and DSSM were added to plasma serum at 37 °C and
were incubated for the same time. A 50 μL sample was analyzed by
high-performance liquid chromatography (HPLC). In addition, for
blocking study, plasma serum was preincubated with an excess amount
of EMC for an hour before adding compound DM, DSM and DSSM. At
last, compound DTX (300 μM), which is not functionalized with the
maleimide group as a negative control, was also added to a solution of
BSA (700 μM in PBS, pH 7.4) and incubated at 37 °C for albumin
binding studies. All the samples were analyzed by an analytical HPLC
using a Waters e2695 pump, a Waters 2489 UV detector (λ = 254 nm)
and the Empower 3 software (Waters Corp.). Column: Waters Symmetry
300™ C18 (250 mm × 4.6 mm, 5 μm); column temperature: 35 °C; flow
rate: 1.0 mL/min; mobile phase A: acetonitrile containing 0.1% tri-
fluoroacetic acid; mobile phase B: water containing 0.1% trifluoroacetic
acid; gradient: 0–5 min, 30% mobile phase A; 5–10 min, increase to
40% mobile phase A; 10–15 min, 40% mobile phase A; 15–35 min, in-
crease to 80% mobile phase A; 35-38 min, 80% mobile phase A; 38-
40 min, decrease to 30% mobile phase A; 40–45 min, 30% mobile phase
A; injection volume: 10 μL.
0.4 mmol) were added into the solution. The reaction proceeded over-
night at 25 °C under nitrogen. TLC was utilized to monitor the reaction
process. At the end of reaction, solvent was evaporated and the crude
product was purified by silica gel column chromatography (di-
chloromethane/methanol (100:0.75) to give about 430 mg compound 6
as a yellow oily solid (65% yield).
Next, a solution of compound 6 (219.8 mg, 0.66 mmol) in 15 ml
anhydrous dichloromethane was activated by HATU (456.3 mg,
1.2 mmol) at 0 °C for 30 min. Then 4-methylmorpholine (NMM)
(
0.13 mL, 1.2 mmol) and DTX (484.7 mg, 0.6 mmol) in 10 ml anhydrous
dichloromethane was added to the mixture, and further stirred under
nitrogen at 25 °C for 12 h. TLC was utilized to monitor the reaction
process. At the end of reaction, filtered to remove the insoluble solids,
189