24
R.-j. Jiang et al. / Carbohydrate Research 400 (2014) 19–25
(2.4 mmol) was dissolved in 25 mL anhydrous ethylenediamino
solution, the mixture was stirred at 75 °C for 10 h under N2 protec-
tion, the reaction was monitored by thin-layer chromatography.
After the reaction was completed, the solution was concentrated
on a rotary evaporator, and followed by the addition of acetone.
The precipitate was collected by filtration and purified by
Sephadex G-25 with aqueous solution (yield = 84.9%). Yellow
amorphous solid, Rf = 0.35 (Ac2O/i-PrOH/NH3ÁH2O/H2O = 7:
7:5:4); 1H NMR (400 MHz, D2O, d): 4.93 (s, 7H, H-1 of CD), 3.81–
3.33 (m, H-2, 3, 4, 5, 6 of CD), 2.92–2.61 (m, 4H, CH2CH2 of
ethylenediamine).
4.2.6.2.
cyclodextrin (ATS-2NbCD).
Mono(6-artesunate-ethylenedi-amino-6-deoxy)-b-
ATS-2NbCD was finally obtained
from Sephadex LH-20 column chromatography and the yield was
50% (yellow amorphous solid). 1H NMR (400 MHz, D2O, d): 5.03
(s, 7H, H-1 of 2N-bCD), 3.95–3.24 (m, H-2, 3, 4, 5, 6, 7 of
2N-bCD), 2.70–2.39 (m, 8H, CH2CH2 of ATS, CH2CH2 of ethylenedi-
amine), 1.50–1.40 (s, 3H, 3-CH3 of ATS), 0.97–0.80 (m, 6H, 6, 9-CH3
of ATS). HRMS (ESI, m/z): [M+H]+ calcd for C63H103N2O41
,
1543.6038; found, 1543.6023.
4.2.6.3.
Mono(6-artesunate-diethylenetriamino-6-deoxy)-b-
cyclodextrin (ATS-3NbCD).
ATS-3NbCD was finally obtained
4.2.4. Synthesis of mono(6-diethylenetriamino-6-deoxy)b-
cyclodextrin (3N-bCD)
from Sephadex LH-20 column chromatography and the yield was
43% (yellow amorphous solid). 1H NMR (400 MHz, D2O, d): 5.00
(s, 7H, H-1 of 3N-bCD), 3.95–3.20 (m, H-2, 3, 4, 5, 6, 7 of 3N-
bCD), 2.70–2.40 (m, 12H, CH2CH2 of ATS, CH2CH2 of diethylenetri-
amine), 1.45–1.35 (s, 3H, 3-CH3 of ATS), 0.97–0.80 (m, 6H, 6,9-CH3
of ATS). HRMS (ESI, m/z): [M+Na]+ calcd for C65H107N3O41Na,
1608.6460; found, 1608.6407.
Mono(6-diethylenetriamino-6-deoxy)b-cyclodextrin (3N-bCD)
was synthesized by the method described below. 3.0 g OTS-bCD
(2.4 mmol) was dissolved in 30 mL anhydrous diethylenetriamino
solution, the mixture was stirred at 80 °C for 12 h under N2 protec-
tion, the reaction was monitored by thin-layer chromatography.
After the reaction was completed, the mixture was cooled to room
temperature and precipitated in acetone. The precipitate was col-
lected by filtration and purified by Sephadex G-25 with aqueous
solution (yield = 64.5%). Yellow amorphous solid, Rf = 0.25 (Ac2O/
i-PrOH/NH3ÁH2O/H2O = 7:7:5:4); 1H NMR (400 MHz, D2O, d): 5.03
(s, 7H, H-1 of CD), 3.90–3.39 (m, H-2, 3, 4, 5, 6 of CD), 2.85–2.65
(m, 8H, CH2CH2 of diethylenetriamine).
4.2.6.4. Mono(6-artesunate-triethylenetetraamino-6-deoxy)-b-
cyclodextrin (ATS-4NbCD).
ATS-4NbCD was finally obtained
from Sephadex LH-20 column chromatography and the yield was
40% (yellow amorphous solid). 1H NMR (400 MHz, D2O, d): 5.02
(s, 7H, H-1 of 4N-bCD), 3.95–3.34 (m, H-2, 3, 4, 5, 6, 7 of
4N-bCD), 2.70–2.40 (m, 16H, CH2CH2 of ATS, CH2CH2 of triethyl-
enetetraamine), 1.40–1.30 (s, 3H, 3-CH3 of ATS), 0.97–0.75 (m,
6H, 6,9-CH3 of ATS). HRMS (ESI, m/z): [M+Na]+ calcd for C67H112N4-
O41Na, 1651.6882; found, 1651.6720.
4.2.5. Synthesis of mono(6-triethylenetetraamino-6-deoxy)-b-
cyclodextrin (4N-bCD)
The synthetic procedures for mono(6-triethylenetetraamino-6-
deoxy)-b-cyclodextrin (4N-bCD) were similar to the 3N-bCD
described above. (yield = 54.5%). Yellow amorphous solid, Rf =
0.19 (Ac2O/i-PrOH/NH3ÁH2O/H2O = 7:7:5:4); 1H NMR (400 MHz,
D2O, d): 5.08 (s, 7H, H-1 of CD), 3.89–3.38 (m, H-2, 3, 4, 5, 6 of
CD), 2.85–2.61 (m, 12H, CH2CH2 of triethylenetetraamine).
4.3. Characterization
1H NMR and 2D ROESY NMR spectra were recorded on a
Bruker Avance DRX 400 MHz spectrometer at 298 k. Chemical
shifts (ppm) are given with respect to residual nondeuterated sol-
vent protons (d 2.50 ppm for DMSO and d 4.80 ppm for HDO).
Electrospray ionization mass spectra (ESI MS) were recorded on
an Agilent LC/MSD TOF mass spectrometer in D2O. Powder
X-ray diffraction (XRD) was measured in a D/max-3B diffractom-
4.2.6. General procedure for synthesis of ATS-b-CD conjugates
Mono(6-artesunate-amino-6-deoxy)-b-cyclodextrin
(ATS-
1NbCD) was synthesized by the method described below. A solu-
tion of 0.5 g ATS (1.2 mmol), 0.4 g EDCI (2.0 mmol), and 0.3 g NHS
(2.3 mmol) in DMF (20 mL) was stirred for 45 min in an ice bath,
then 1.1 g 1N-bCD (1.0 mmol) was added to the reaction. The
mixture was allowed to react for additional 2 h in an ice bath
and subsequently for 15 h at room temperature. After the reac-
tion was completed, the solution was removed by a rotary evap-
orator, and followed by the addition of double-distilled water.
The clear solution was collected by filtration, and then was
poured into acetone. The precipitate was collected and purified
on a Sephadex LH-20 column with aqueous solution as eluent.
The synthetic procedures for mono(6-artesunate-ethylenedi-
amino-6-deoxy)-b-cyclodextrin (ATS-2NbCD), mono(6-artesu-
nate-diethylenetriamino-6-deoxy)-b-cyclodextrin (ATS-3NbCD),
and mono(6-artesunate-triethylenetetraamino-6-deoxy)-b-cyclo-
dextrin (ATS-4NbCD) were similar to the ATS-1NbCD described
above.
eter using Cu-ja (j = 15460 Å) with 40 kV, and a scanning rate of
5/min. Powder samples were mounted on a sample holder and
scanned with a step size of 2h = 0.02° between 2h = 3° and 70°.
Thermogravimetric analysis (TGA) was performed under constant
nitrogen flow (50 mL/min), with a heating rate of 10 °C/min using
a NETZSCH STA449F3 instrument. The heating scans were per-
formed on 8.0–12.0 mg of the samples in the temperature range
of 25–500 °C.
4.4. Measurement of aqueous solubility
The aqueous solubility of the conjugates was assessed by
preparation of their saturated aqueous solutions.38 An excess
amount of conjugate was put in 5 mL distilled water (pH ca.
7), and the mixture was stirred for 1 h. After removing the
insoluble material by filtration, the filtrate was evaporated under
reduced pressure to dryness and the residue was dosed by
weighing method.
4.2.6.1.
(ATS-1NbCD).
Mono(6-artesunate-amino-6-deoxy)-b-cyclodextrin
ATS-1NbCD was finally obtained from Sepha-
4.5. Cells and medium
dex LH-20 column chromatography and the yield was 57% (white
amorphous solid). 1H NMR (400 MHz, D2O, d): 4.97 (s, 7H, H-1 of
1N-bCD), 3.85–3.44 (m, H-2, 3, 4, 5, 6, 7 of 1N-bCD), 2.73–2.49
(m, 4H, CH2CH2 of ATS), 1.40–1.31 (s, 3H, 3-CH3 of ATS), 0.95–
0.75 (m, 6H, 6,9-CH3 of ATS). HRMS (ESI, m/z): [M+H]+ calcd for
Human colon cancer lines HCT116, LOVO, SW480, HT-29, and
glioma cells C6 were cultured at 5 Â 105/mL in RPMI 1640 supple-
mented with 10% heat inactivated fetal bovine serum (FBS) at 37 °C
in a humidified atmosphere of 5% CO2 in air.
C61H98NO41, 1500.5616; found, 1500.5632.