Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
9
J ¼ 6.4 Hz), 0.91 (d, 3H, J ¼ 6.4 Hz), 0.99 (s, 3H), 1.43 (s, 9H),
0.85e2.38 (remaining cholesterol & Lys protons), 3.12e3.21 (m,
3H), 3.63e3.66 (m, 8H), 3.70e3.75 (m, 4H), 4.14e4.15 (m, 1H),
4.31e4.43 (m, 2H), 5.34 (s, 1H), 8.69 (s, 2H). [ a ]D20: ꢀ7.6 (c 0.5,
CHCl3).
4.2.9. Synthesis of compound Fru-RGD-chol
Compound 11 (100 mg, 0.06 mmol) was added to a mixed sol-
vent of TFA (3 ml) and H2O (3 ml), and the reaction was stirred at r. t
for 24 h. Then TFA was removed, and the residue was diluted with
water and extracted with CH2Cl2 (30 mL ꢂ 3). The organic layer was
washed with saturated NaCl (30 mL ꢂ 2) and concentrated to give a
pale yellow semi-solid (86 mg). The pale yellow semi-solid was
further dissolved in of methanol (10 mL), and 10% Pd/C (10 mg) was
added. The reaction was stirred under a hydrogen pressure (0.4
Mpa) at 45 ꢁC for 48 h. Then, Pd/C was removed by filtration, and
the solvent in filtrate was removed. The residue was washed with
diethyl ether (5 mL ꢂ 5) to remove the small polar impurity
(10 mL ꢂ 5) to give a white solid (21.86 mg). 1H NMR (400 MHz,
4.2.6. Synthesis of compound 9
4-methylmorpholin (NMM, 0.127 mL, 1.15 mmol) and isobutyl
chloroformate (IBCF, 0.151 mL, 1.15 mmol) were added to a solution
of compound 8 (900 mg, 1.34 mmol) in CH2Cl2 (10 mL), and the
reaction was stirred at ꢀ5 ꢁC for 30 min. Then, a solution of com-
pound 21 (715 mg, 0.96 mmol) in CH2Cl2 (10 mL), was slowly added
dropwise. The reaction was stirred at r. t. overnight. The mixture
was washed with hydrochloric acid (1 N, 50 mL ꢂ 2) and saturated
NaCl (50 mL ꢂ 2). The organic layer was concentrated and purified
by silica gel column chromatography (CH2Cl2/CH3OH ¼ 50/1) to
give compound 9 as a pale yellow foamy solid (1.09 g, 81%). 1H NMR
CD3OD, ppm)
d
: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.8 Hz), 0.91 (d, 3H,
J ¼ 6.4 Hz), 0.98 (s, 3H), 0.86e2.37 (remaining cholesterol and Lys
protons), 2.50e2.79 (m, 10H), 2.90e3.05 (m, 2H), 3.17e3.20 (m,
2H), 3.62e3.76 (m, 12H), 3.77e3.94 (m, 5H), 4.18e4.35þ(m, 8H), 4.61
(br, 1H). HRMS (ESI) calculated for C66H110N8NaO21 [MþNa]þ-
1361.7678, found 1361.7674.
(400 MHz, CDCl3, ppm)
d: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91
(d, 3H, J ¼ 6.4 Hz), 0.98 (s, 3H), 1.41 (s, 9H), 0.85e2.37 (remaining
cholesterol & Lys protons & Arg protons), 2.54 (s, 4H), 2.78 (br, 2H),
3.05 (s, 2H), 3.13e3.21 (m, 1H), 3.29 (s, 2H), 3.61e3.63 (m, 8H), 3.67
(s, 2H), 3.93 (s, 2H), 4.19e4.28 (m, 2H), 4.44 (s, 2H), 4.91 (br, 1H),
5.04 (s, 2H), 5.09e5.10 (m, 2H), 5.32e5.33 (m, 1H), 7.25e7.33 (m,
10H). [ a ]D20: ꢀ6.2 (c 0.5, CHCl3).
4.2.10. Synthesis of compound 12
To a solution of compound 3 (1.04 g, 2.89 mmol) in CH2Cl2
(30 mL), were added DCC (795 mg, 3.86 mmol) and DMAP (47 mg,
0.39 mmol), and the reaction was stirred at ꢀ5 ꢁC for 30 min. A
solution of compound 6 (1.00 g, 1.93 mmol) in CH2Cl2 (20 mL) was
added dropwise and the reaction was stirred at r. t for 3 h. The
solvent was removed, and the residue was re-dissolved by ethyl
acetate (50 mL), and then filtrated. The filtrate was concentrated
and purified by silica gel column chromatography (petroleum
ether/ethyl acetate ¼ 5/1) to give compound 12 as a pale yellow
4.2.7. Synthesis of compound 10
Compound 9 (1.092 g, 0.78 mmol) was added to a mixture of
trifluoroacetic acid (TFA, 3 mL) and CH2Cl2 (9 mL), and the reaction
was stirred at r. t. for 20 min. The solvent was removed and the
residue was re-dissolved with CH2Cl2 (50 mL), followed by washed
with H2O (50 mL ꢂ 1), saturated NaHCO3 (50 mL ꢂ 1), and saturated
NaCl (50 mL ꢂ 2). The organic layer was concentrated to give
compound 10 as a light yellow solid (0.999 g, 99%). 1H NMR
viscous oil (1.78 g, 77%). 1H NMR (400 MHz, CDCl3, ppm)
d: 0.67 (s,
3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91 (d, 3H, J ¼ 6.4 Hz), 0.99 (s, 3H), 1.34
(s, 3H), 1.41 (s, 3H), 1.48 (s, 3H), 1.54 (s, 3H), 0.86e2.39 (remaining
cholesterol protons), 2.69 (s, 4H), 3.14e3.22 (m, 1H), 3.64e3.66 (m,
8H), 3.69e3.72 (m, 2H), 3.75e3.78 (m, 1H), 3.89e3.93 (m, 1H),
4.05e4.08 (m, 1H), 4.23e4.27 (m, 3H), 4.31 (d, 1H, J ¼ 2.6 Hz),
4.42e4.44 (m, 1H), 4.59e4.62 (m, 1H), 5.34 (s, 1H). HRMS (ESI)
calculated for C49H80O12Naþ [MþNa]þ-883.5542, found 883.5550. [
a ]D20: ꢀ17.6 (c 0.5, CHCl3).
(400 MHz, CDCl3, ppm)
d
: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91
(d, 3H, J ¼ 6.4 Hz), 0.98 (s, 3H), 0.86e2.36 (remaining cholesterol &
Lys protons & Arg protons), 2.56 (br, 4H), 2.94e3.20 (m, 5H), 3.27 (s,
2H), 3.62e3.65 (m, 10H), 3.92 (br, 2H), 4.25 (s, 2H), 4.42e4.55 (m,
2H), 4.90 (br, 1H), 5.04e5.09 (m, 4H), 5.33 (s, 1H), 7.28e7.32 (m,
10H). [ a ]D20: ꢀ6.2 (c 0.5, CH3OH).
4.2.11. Synthesis of Fru-chol
Compound 12 (3.49 g, 4.00 mmol) was added to a mixed solvent
of TFA (20 mL) and H2O (20 mL), and the reaction was stirred at r. t.
for 24 h. The TFA was removed, then the mixture was neutralized
with Na2CO3, and extracted by Ethyl acetate (50 mL ꢂ 3). The
organic layer was concentrated and purified by silica gel column
chromatography (petroleum ether/ethyl acetate ¼ 3/1) to give a
pale yellow viscous oil (1.197 g, 38%).1H NMR (400 MHz, CDCl3,
4.2.8. Synthesis of compound 11
To a solution of compound 3 (415 mg, 1.15 mmol) in CH2Cl2
(10 mL), were added N,N-diisopropylethylamine (DIPEA, 0.38 mL,
2.30 mmol), 1-(3-dimethylaminopropyl) 3-ethylcarbodiimide hy-
drochloride (EDCI, 294 mg, 1.54 mmol) and DMAP (188 mg,
1.54 mmol), and the reaction was stirred at ꢀ5 ꢁC for 30 min. A
solution of compound 10 (999 mg, 0.77 mmol) in CH2Cl2 (10 mL)
was added dropwise, and the mixture was stirred at r. t. overnight.
The mixture was washed with HCl solution (1 N, 30 mL ꢂ 2) and
saturated NaCl (30 mL ꢂ 2). The organic layer was concentrated,
and the residue was purified by silica gel column chromatography
CH2Cl2/CH3OH ¼ 8/1) to give compound 11 as a white foamy solid
ppm)
d
: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91 (d, 3H, J ¼ 6.4 Hz),
1.00 (s, 3H), 0.86e2.38 (remaining cholesterol protons), 2.68 (s, 4H),
3.16e3.21 (m, 1H), 3.42e3.50 (m, 7H), 3.62e3.69 (m, 10H),
4.25e4.36 þ(m, 4H), 5.34 (s, 1H). HRMS (ESI) calculated for
C
43H72NaO12[MþNa]þ- 803.4916, found 803.4912.
(977 mg, 77%). 1H NMR (400 MHz, CDCl [3], ppm)
d
: 0.67 (s, 3H),
4.3. Preparation and characterization of liposomes
0.86 (d, 6H, J ¼ 6.6 Hz), 0.91 (d, 3H, J ¼ 6.2 Hz), 0.98 (s, 3H), 1.33 (s,
3H), 1.39 (s, 3H), 1.46 (s, 3H), 1.52 (s, 3H), 0.86e2.36 (remaining
cholesterol & Lys protons & Arg protons), 2.51e2.75 (m, 8H),
2.90e3.06 (m, 2H), 3.13e3.33 (m, 5H), 3.62e3.64 (m, 8H),
3.66e3.69 (m, 2H), 3.72e3.76 (m, 1H), 3.87e3.93 (m, 3H),
3.99e4.02 (m, 1H), 4.22e4.26 (m, 3H), 4.30e4.31 (m, 1H),
4.42e4.47 (m, 3H), 4.59e4.61 (m, 1H), 4.88e4.91 (m, 1H), 5.02e5.14
(m, 4H), 5.33 (s, 1H), 7.28e7.34 (m, 10H). HRMS (ESI) calculated for
We selected the optimal prescription: cholesterol: soybean
phospholipid (SPC): ligand ¼ 33:64:3 (molar ratio), and lipid:
paclitaxel ¼ 30:1 (quality ratio), hydration solution was PBS
(pH ¼ 7.4). Briefly, the prescribed amount of lipid material and PTX
were accurately weighed to be dissolved with chloroform-
methanol mixed solution (V/V ¼ 2/1). And then the organic sol-
vent was removed by rotary evaporation at 37 ꢁC to give a complete
thin film. The thin film was hydrated in PBS for 0.5 h at 20 ꢁC.
Finally, it was further sonicated intermittently (80 W, 5S, 5S) for
C
85H127N9NaOþ23 [MþNa]þ-1664.8937, found 1664.8939. Mp:
46e48 ꢁC.[ a ]D20: ꢀ6.0 (c 0.5, CHCl3).