30
R. Zhao et al. / Nuclear Medicine and Biology 72–73 (2019) 26–35
(m, 6H). 13C NMR (100 MHz, CDCl3) δ 147.70, 146.71, 130.02, 126.49,
113.59, 108.29, 74.55, 60.93, 60.06, 55.97, 51.83, 41.57, 40.52, 39.69,
29.07, 25.37, 24.11, 21.77. HRMS calcd. for C21H27D6NO4 [M + H]+
370.2786 found 370.3505.
The samples were analyzed by LC/MS with an Agilent LC/MSD TOF
instrument in positive ESI mode and an Agilent HPLC 1100 series sys-
tem. The HPLC conditions were as follows: stationary phase: Agilent
Poroshell 120 EC-C18 2.7 μ 3.0 × 50 mm; mobile phase: 0.4 mL/min:
UV detection: 280 nm; and ESI positive mode. The following gradient
was used: from 0 to 15 min, gradient 0.1% formic acid 100–0%, ACN
0–100%; and from 15 to 20 min, 100% ACN.
2.2.7. 3-(((2R,3R,11bR)-2-Hydroxy-3-isobutyl-10-methoxy-1,3,4,6,7,11b-
hexahydro-2H-pyrido[2,1-a]isoquinolin-9-yl)oxy)propyl-1,1,2,2,3,3-d6 4-
methylbenzenesulfonate, (7)
2.5. Solid-phase extraction (SPE) purification
Compound 7 was prepared from compound 1 (100 mg, 0.33 mmol),
compound 5 (140 mg, 0.35 mmol), cesium carbonate (Cs2CO3, 427 mg,
1.3 mmol) and acetone (30 mL) following the same procedure de-
scribed for compound 3. The concentrated residue was purified by FC
(DCM/MeOH/NH3·H2O = 95/5/0.5) to give 100 mg of compound 7
(yield 58%) as a pale-yellow solid. 1H NMR (400 MHz, CDCl3) δ
7.78–7.75 (m, 2H), 7.25–7.27 (m, 2H), 6.66 (s, 1H), 6.51 (s, 1H), 3.77
(s, 3H), 3.39–3.38 (m, 1H), 3.13–2.99 (m, 4H), 2.63–2.56 (m, 2H),
2.47–2.40 (m, 4H), 2.00–1.95 (m, 1H), 1.70–1.72 (m, 2H), 1.53–1.48
(m, 2H), 1.13–1.15 (m, 1H), 0.96–0.94 (m, 6H). 13C NMR (100 MHz,
CDCl3) δ 147.80, 146.80, 130.00, 126.61, 113.79, 108.76, 74.58, 60.91,
60.08, 56.15, 51.88, 41.62, 40.55, 39.70, 29.10, 25.36, 24.12, 21.78.
HRMS calcd. for C39H52D6N2O6 [M + H]+ 524.2875 found 524.2927.
The reaction mixture was cooled to room temperature, and 7 mL of
water was added. The mixture was loaded onto different cartridges
(eight different cartridges were tested: Sep-Pak Oasis, tC2, C8, tC18,
CN, Chromafix C18 Hydrox, Chromafix C18, and Chromfix C4). The
loaded cartridges were first washed with 10 mL of water and then
eluted with 2 mL of gradient concentrations of ethanol-water or
acetonitrile-water (10%, 20%, 30%, 40%, 50% and 60%). The resulting elu-
ates were analyzed by HPLC. To optimize the elution volume, the se-
lected cartridges were washed with water, followed by different
concentrations (25%, 30% or 35%) of ethanol-water in 2 mL portions.
The flow rate was approximately 10 mL/min.
2.6. Semipreparative HPLC purification method
2.2.8. (2R,2′R,3R,3′R,11bR,11b'R)-9,9′-((Propane-1,3-diyl-d6)bis(oxy))bis
(3-isobutyl-10-methoxy-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]
isoquinolin-2-ol), (8)
The reaction mixture was cooled to room temperature, and 7 mL of
water was added to this mixture. The mixture was loaded onto an
Oasis HLB (3 cc) cartridge and then washed with 10 mL of water. [18F]
9 was eluted with 1 mL of acetonitrile. This solution was diluted with
1 mL of 10 mM AFB and injected into the HPLC system (Gemini 250
× 10 mm, ACN/10 mM AFB = 45/55, 3 mL/min). The eluate from 14.0
to 15.5 min was collected and diluted with 25 mL of water. This mixture
was loaded onto a tC18 cartridge, from which the desired product, [18F]
9, was eluted with 1 mL of ethanol.
The residue from the synthesis of compound 7 was purified by FC
(DCM/MeOH/NH3·H2O = 90/9/1) to give 5.8 mg of compound 8
(yield 5%) as a pale-yellow solid. 1H NMR (400 MHz, CDCl3) δ
6.67–6.34 (m, 2H), 3.81 (s,6H), 3.39–3.43 (m, 2H), 3.15–2.99 (m, 8H),
2.62–2.59 (m, 4H), 2.48–2.43 (m, 2H), 1.99 (t, J = 10.8, 2H), 1.73–1.63
(m, 4H), 1.63–1.46 (m, 6H), 1.46–1.03 (m, 2H), 0.97–0.93 (m, 12H).
13C NMR (100 MHz, CDCl3) δ 147.88, 147.14, 129.81, 126.67, 113.84,
108.67, 74.85, 61.10, 60.24, 56.29, 52.05, 41.78, 40.74, 39.90, 29.28,
25.55, 24.37, 21.98. HRMS calcd. for C39H52D6N2O6 [M + H]+
657.4671 found 657.4100.
2.7. In vitro binding assay for IC50 determination
Tissue homogenates of striatum (dissected from mouse brain) were
prepared in buffer 1 (50 mM of HEPES, 0.32 M sucrose, pH = 7.4). Com-
pounds were examined for their ability to compete with [18F]9 for bind-
ing at concentrations ranging from 10−6 to 10−11 M. The binding assays
were performed in glass tubes (12 × 75 mm) with final volumes of
0.25 mL. In the nonspecific binding assay, 50 μM ( )-tetrabenazine
(TBZ), was used as the positive control. After incubation for 1 h at
room temperature, the bound ligand was separated from the free ligand
by filtration through glass fiber filters. The filters were washed three
times with 4 mL of ice-cold PBS buffer at pH 7.4, and the radioactivity
of the material remaining on the filters was counted with a gamma
counter (WIZARD2, Perkin-Elmer). Data were analyzed using the non-
linear least-square curve fitting program LIGAND to determine IC50
values.
2.3. Nucleophilic [18F]fluorination of 9 under different conditions
A solution containing [18F]fluoride (37–74 MBq) was loaded onto an
activated QMA light cartridge and eluted with 1 mL of K222/K2CO3 solu-
tion (2 mg K2CO3, 10 mg K222 in 0.15 mL of water and 0.85 mL of aceto-
nitrile) into a glass test tube. The solution was dried under a flow of
argon at 90 °C and azeotropically dried with 2 mL of acetonitrile. The
precursor (1 mg) was dissolved in 1 mL of solvent (DMSO, DMF, DMA
or ACN) and added to the dried [18F]F−/K222/K2CO3 complex. The reac-
tion mixture was heated to different temperatures (70 °C, 75 °C, 90 °C,
110 °C or 130 °C). Aliquots (20 μL) were removed at different time
points (5, 10 and 15 min), and the aliquots were quenched in ice
water prior to analysis by HPLC.
2.4. Analysis of the radiochemical yield, radiochemical purity and amount
of chemicals by HPLC
2.8. In vivo biodistribution in mice
Five normal CD-1 mice per group were used for each biodistribution
study. The assay used 0.15 mL of solutions containing different concen-
trations of 3, 6, and 8. HPLC-purified [18F]9 in 0.1% bovine serum albu-
min was injected into the tail vein of CD-1 mice (33–37 g, male) while
they were under isoflurane anesthesia (1.11 MBq per mouse). The
mice (n = 5) were sacrificed by cervical dislocation at 30 min postinjec-
tion. The organs or tissues of interest were removed and weighed, and
radioactivity was counted with an automatic gamma counter. The per-
centage of the dose per organ was calculated by comparing the tissue
counts with suitable diluted aliquots of the injection material. Different
regions of the brain corresponding to the cerebellum (CB), striatum
(ST), hippocampus and cortex were dissected from the brains and
counted separately.
The radiochemical yield (RCY) and radiochemical purity (RCP) were
analyzed by HPLC with a gamma ray radio detector and a UV/Vis detec-
tor (Agilent 1200 series, Ascentis C18 column, 150 × 4.6 mm; 280 nm)
with the following mobile phase (1 mL/min) gradient. From 0 to 2 min,
isocratic 10 mM ammonium formate buffer (AFB) 95%, ACN 5%; from 2
to 5 min, gradient AFB 95–30%, ACN 5–70%; from 5 to 10 min, gradient
AFB 30–0%, ACN 70–100%; from 10 to 15 min, gradient AFB 0–95%, ACN
100–5%; and from 15 to 20 min, 95% AFB (retention times: [18F]fluo-
ride = 1.4 min, [18F]9 = 7.8 min). The amounts of compounds 3, 6, 7,
and 8 were quantified using this HPLC system; standard calibration
curves were plotted using authentic sample solutions at least 6 different
concentrations from 0.01 μg to 1 μg (Fig. S1B).