recrystallized from acetonitrile and diethyl ether under cold
conditions. The pure product was obtained as a colorless oil
(76%). The final product was well characterized by 1H, 13C NMR
and MS. 1H NMR (CDCl3, 400 MHz) dH ppm: 1.99 (s, 3H, -CH3),
2.02–2.12 (m, 8H, -CH2), 2.73 (s, 6H, -CH3); 13C NMR (CDCl3,
100 MHz) dC ppm: 20.93, 43.65, 57.95, 58.94, 170.59; MS(ESI+)
m/z calcd. for C18H17BrNO2+ 239.13, found [M + H]+: [M + 2H]+
237 : 239
-CH2), 5.50 (brs, -OH), 6.15 (brs, -OH); 13C NMR (D2O, 100
MHz) dC ppm: 44.53, 49.94, 51.55, 54.58, 55.31, 56.15, 56.64,
57.97, 60.60, 176.61; MS(ESI+) m/z calcd. for C20H40N5O7
+
462.56, found [M - 2H]- 460.2
Synthesis of Ga(III) complex of (2-hydroxy-ethyl)-dimethyl-
[2-(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-
ethyl]-ammonium 8. Radiolabeling of the ligand 7 with 67Ga was
carried out in double distilled water under nitrogen atmosphere.
Gallium citrate (67Ga, 74 MBq) was added to the vial containing
the ligand and the temperature was raised to 80 ◦C. The pH of
the reaction mixture was adjusted to 6.5 with ammonium acetate
solution (0.5 M) to get optimum radiolabeling yield. After 4 h, the
reaction mixture was analysed using thin layer chromatography
in 10% ammonium acetate : methanol (1 : 1) solvent system. TLC
analysis demonstrated high radiochemical purity (above 96%) for
67Ga–DO3A-EA-choline.
Synthesis of (4,7-bis-t-butoxycarbonylmethyl-1,4,7,10-tetraaza-
cyclododec-1-yl)-acetic acid t-butyl ester 5. To a solution of
1,4,7,10-tetraazacyclododecane (0.5 g, 2.90 mmol) in acetonitrile
(50 mL) was added sodium bicarbonate (0.73 g, 8.71 mmol) at
0 ◦C under nitrogen. This was followed by the dropwise addition
of t-butylbromoacetate (1.7 g, 8.71 mmol). The reaction was
allowed to warm to room temperature and stirred for 12 h. The
reaction mixture was filtered and the filtrate was evaporated under
reduced pressure to obtain the crude product. It was purified by
column chromatography on a silica gel (eluent: dichloromethane–
methanol: 9.5/0.5) to afford the desired product as a white solid
(80%). The final product was well characterized by 1H, 13C NMR
and MS. 1H NMR (CDCl3, 400 MHz) dH ppm: 1.29 (s, 27H, -CH3),
2.77–2.99 (m, 16H, -CH2), 3.18–3.28 (m, 6H, -CH2); 13C NMR
(CDCl3, 100 MHz) dC ppm: 28.09, 47.40, 48.73, 49.05, 51.19,
58.00, 81.51, 169.54, 170.43; MS(ESI+) m/z calcd. for C26H50N4O6
514.70, found [M + H]+ 515.4
Cytotoxicity of DO3A-EA-choline
Cytotoxicity was determined using the MTT [3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide]
assay. Exponentially growing cells were plated in a 96 well
microtitre plate at a uniform cell density of 4,000 cells/well 24 h
before treatment. Cells were treated with varying concentrations
of the compound for various time intervals, 24 h, 48 h, 72 h
and MTT assays were performed. At the end of treatment,
negative control and treated cells were incubated with MTT at
a final concentration of 0.05 mg mL-1 for 2 h at 37 ◦C and the
medium was removed. The cells were lysed and the formazan
crystals were dissolved using 150 mL of dimethylsulfoxide.
Optical density was measured on 150 mL of extracts at 570 nm
(reference filter: 630 nm). Mitochondrial activity was expressed
as percentage of viability compared to negative control (mean
SD of triplicate cultures). Percentage of viability = [OD (570 nm
- 630 nm) test product/OD (570 nm - 630 nm) negative control]
¥ 100%. Percentage viability of HEK cells was plotted against
(0.001–10 mM) concentration range for DO3A-EA-choline.
Synthesis
of
(2-acetoxy-ethyl)-dimethyl-[2-(4,7,10-tris-t-
butoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-ethyl]-
ammonium 6. To a solution of 1,4,7,10-tetraazacyclododecane-
1,4,7-tris(t-butyl acetate) (0.5 g, 0.97 mmol) in acetonitrile
(50 mL) was added potassium carbonate (1.34 g, 9.72 mmol)
under nitrogen. The reaction temperature was raised to 35 ◦C
followed by the dropwise addition of 2 (0.45 g, 1.94 mmol). The
reaction was stirred at 35 ◦C for 12 h. The reaction mixture
was cooled to room temperature, filtered and the filtrate was
evaporated under reduced pressure to obtain the crude product. It
was recrystallized from dichloromethane and diethyl ether under
cold conditions to afford the pure compound as a brown oily
1
product (84%). The final product was well characterized by H,
13C NMR and MS. 1H NMR (CDCl3, 400 MHz) dH ppm: 1.46 (s,
30H, -CH3), 2.15 (m, 6H, -CH2), 2.75–2.95 (m, 12H, -CH2), 3.10
(m, 4H, -CH2), 3.47 (m, 6H, -CH3), 3.61 (m, 8H, -CH2), 3.81 (s,
2H, -CH2); 13C NMR (CDCl3, 100 MHz) dC ppm: 28.18, 49.61,
50.49, 51.47, 52.91, 57.84, 60.42, 66.09, 81.58, 170.14, 171.28;
Cell uptake assay
Cell uptake studies were performed using radiolabeled 67Ga-
DO3A-EA-choline. The specificity of choline conjugates to bind
to cell surface receptors on tumor cells was examined by receptor
binding assays on U-87 MG cell line grown in normal DMEM
(10% serum). Monolayer cultures of the cell lines were washed with
HBSS and were then incubated for 2 h in HBSS at 37 ◦C prior to
the experiment. Binding experiments were conducted at 37 ◦C. The
cell line cultures were then incubated for 40 min with 67Ga-DO3A-
EA-choline at 37 ◦C in HBSS containing various concentrations
(0.00001 mM–1 mM) in the absence and presence of 100 fold
excess unlabeled choline for estimation of total binding and non
specific binding respectively. Specific binding was obtained by
subtracting non specific binding from total binding. At the end
of each experiment, the cells were washed with cold PBS and 0.9%
saline four times. The cell associated radioactivity was determined
by gamma scintillation counting. Scatchard plot analysis was done
using EQUILIBRATE software from graph pad.
MS(ESI+) m/z calcd. for C34H66N5O8 674.50, found [M + 3H]+
+
677.5
Synthesis
of
(2-hydroxy-ethyl)-dimethyl-[2-(4,7,10-tris-
carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-ethyl]-ammo-
nium 7. To a solution of 6 in anhydrous dichloromethane was
added trifluoroacetic acid (4 mL) and the reaction was stirred for
24 h at room temperature. The reaction progress was monitored
by TLC. The solvent was evaporated to dryness under reduced
pressure to obtain the crude product. The product was washed
well with chloroform (3 ¥ 50 mL). Addition of cold diethyl ether
led to precipitation of pure product as a pale white solid (65%).
1
The final product was well characterized by H, 13C NMR and
MS. 1H NMR (D2O, 400 MHz) dH ppm: 1.93 (s, 6H, -CH3),
2.61–3.05 (m, 16H, -CH2), 3.28–3.70 (m, 8H, -CH2), 3.97 (s, 6H,
1598 | Org. Biomol. Chem., 2011, 9, 1591–1599
This journal is
The Royal Society of Chemistry 2011
©