A. Pekošak et al.
1
(
Valkenswaard, the Netherlands) and used as received unless stated
otherwise. Reactions were performed at ambient temperature unless CDCl
stated otherwise. Reactions were monitored by thin-layer chromatography (s, 1H, CHbenzamide), 3.95 (m, 2H, CH2piperidine), 3.82 (s, 3H, CH
3 3
on pre-coated silica 60 F254 aluminum plates (Merck, Darmstadt, (m 2H, CH2piperidine), 1.92 (m, 2H, CH2piperidine), 1.40 (s, 9H, (CH )
R
f
value, 0.45 (ethyl acetate/hexane = 50/50); H-NMR (250.13 MHz,
) δ 8.02 (s, 1H, CHbenzamide), 7.58 (d, 1H, J = 7.5 Hz –NH–CO–), 6.29
), 2.93
),
163.69
), 133.10
using a rotary evaporator (Rotavapor® R II, Flawil, Switzerland). Flash (CHAr), 112.43 (CArCl), 111.68 (CArC¼O), 97.82 (CHAr), 79.61 (–C(CH ),
column chromatography was performed manually on silica gel 60 Å 56.14 (O–CH ), 52.15 (CHpiperidineNH), 46.40 (CH2piperidine), 31.98
Merck, Darmstadt, Germany) or on a Büchi (Flawil, Switzerland) Sepacore (CH2piperidine), 28.46 ((CH )+; ESI-HRMS: calculated for C18 26ClN
3
3
1
3
Germany). Spots were visualized by ultraviolet (UV) light, bromocresol 1.18 (m 2H, CH2piperidine);
3
C-NMR (125.78 MHz, CDCl )
green, and ninhydrine. Solvents were evaporated under reduced pressure (C¼O), 157.39 (CArOCH ), 154.77 (C¼O–O–), 146.66 (CArNH
3
2
3 3
)
3
(
3
)
3
H
3 4
O :
system (comprising a C-620 control unit, a C-660 fraction collector, two 383.87, found 384.17 [M + H] .
C-601 gradient pumps, and a C-640 UV detector) equipped with Büchi
Sepacore pre-packed flash columns. NMR spectroscopy was performed
4
-(4-amino-5-chloro-2-methoxybenzamido)piperidin-1-ium
1
chloride (10)
on a Bruker (Billerica, MA, USA) Avance 250 (250.13 MHz for H) or a Bruker
1
13
Avance 500 (500.23 MHz for H and 125.78 MHz for C) with chemical
shifts (δ) reported in parts per million (ppm) relative to the solvent (CDCl
Tert-butyl 4-(4-amino-5-chloro-2-methoxybenzamido)piperidine-1-carboxylate
(1 eq, 95 mg, 0.25 mmol) was dissolved in 1,4-dioxane (5.5 mL), and
HCl (4 M in 1,4-dioxane, 3.5 mL) was added at 0 °C. The mixture was
allowed to stir at room temperature for 30 min followed by drying with
MgSO and concentration in vacuo. The residue was washed two times
4
with diethyl ether (20 mL) and collected by filtration. The precipitate
was dried in a vacuum oven for 24 h at 40 °C to obtain 10 in 99% yield
3
,
9
1
13
1
13
2 3 2
H 7.26 ppm, C 77.16 ppm; D O, H 4.79 ppm; (CD ) SO, C 39.52 ppm).
Electrospray ionization-high resolution mass spectrometry (ESI-HRMS)
was carried out using Bruker microTOF-Q instrument in a positive ion
mode (capillary potential of 4500 V). Analytical HPLC was performed on a
Jasco (Easton, MD, USA) PU-2089 Plus station with a Alltima C18 5 μm
(250 × 4.6 mm) column (Grace, Breda, the Netherlands) (A) or Fermentation
(
78 mg, 0.24 mmol) and >98% purity, determined by HPLC (column A,
solvents D/H, 55/45, v/v, wavelength 254 nm, column temperature –
Monitoring Column 9 μm (150 × 7.8 mm) (Bio-Rad, Veenendaal, the
Netherlands) (B) with Jasco UV-2075 Plus UV detector (254 nm) and NaI
radioactivity detector (Raytest, Straubenhardt, Germany) at ambient
ꢀ1
ambient, flow 1 mL · min , Rt 4.5 min).
R
f
value, 0.0 (ethyl acetate/hexane = 70/30 + 0.1% triethylamine);
H-NMR (250.13 MHz, D O) δ 7.53 (s, 1H, CHbenzamide), 6.47 (s, 1H,
CHbenzamide), 4.00 (m, 1H, CHpiperidine), 3.73 (s, 3H, CH
= 17.5 Hz, 2H, CH2piperidine), 3.04 (m 2H, CH2piperidine), 2.09 (q, J
= 15 Hz, 2H, CH2piperidine), 1.69 (m 2H, CH2piperidine);
temperature. Acetonitrile (CH
buffer 1 (4 mM sodium formate and 4% dimethylformamide) (F), buffer 2
0.1 M ammonium formate and 1% dimethylformamide, pH = 4.1) (G),
3
CN) (C), methanol (MeOH) (D), water (E),
1
2
3
1
), 3.37 (q, J = 5 Hz,
(
J
J
2
1
= 2.5 Hz,
solution 1 (0.1 M ammonium formate) (H), and solution 2 (0.005 M sulfuric
acid) (I) were used as mobile phases. Chromatograms were acquired with
Raytest GINA Star software (version 5.8). Semi-preparative isocratic HPLC
was performed on Jasco UV-2087 Plus station with an Alltima C18 5 μm
13
2
C-NMR
(
1
4
125.78 MHz, (CD
3
)
2
SO) δ 163.82 (C¼O), 157.72 (CArOCH
3
), 148.95 (CArNH
2
),
),
31.74 (CHAr), 111.06 (CArCl), 109.27 (CArC¼O), 97.98 (CHAr), 56.42 (CH
3
4.45 (CHpiperidineNH), 42.28 (CH2piperidine), 28.66 (CH2piperidine); ESI-HRMS:
(
250 × 10 mm) column (Grace, Breda, the Netherlands) 1(J) using
+
calculated for C13
for C13H N O : 250.16, found 251.15 [M + H] .
20 3 2
H
18ClN
3 2
O : 283.12, found 284.11 [M + H] and calculated
ꢀ
MeOH/buffer 2 (65/35, v/v) as a eluent, flow rate 4 mL · min (method
A), a Jasco UV-2075 Plus UV detector (254 nm), a homemade radioactivity
detector, and Jasco ChromNAV CFR software (version 1.14.01).
+
+
Radiosynthesis
11
[
C]Benzyl iodide (4)
Synthesis
1
1
14
11
[
C]CO
2
was produced by the N(p,α) C nuclear reaction performed in
/N gas mixture using IBA Cyclone 18/9 (IBA, Louvain-la Neuve,
Belgium). After trapping of [ C]CO
Clebopride (4-amino-N-(1-benzylpiperidin-4-yl)-5-chloro-2-
methoxybenzamide (8)
a 0.5% O
2
2
1
3
1
1
2
from the target in a stainless trap
2 2
dispensed in liquid N , [ C]CO was transferred with a He flow of
1
1
Clebopride was synthesized according to the procedure published by
R.H. Mach et al. in 1993.
ꢀ1
1
0 mL · min into 100 μL of 1 M phenylmagnesium bromide in THF at
3
5 °C. After obtaining the maximum radioactivity in the vessel, the
R
f
value, 0.30 (ethyl acetate/hexane = 70/30 + 0.1% triethylamine);
H-NMR (250.13 MHz, CDCl ) δ 7.99 (s, 1H, CHbenzamide), 7.58 (d, 1H,
J = 7.5 Hz, –NH–CO–), 7.21 (m, 5H, CHAr), 6.20 (s, 1H, CHbenzamide), 4.32
s, 2H, –CH –), 3.77 (s, 3H, CH ), 2.77 (m, 2H, CH2piperidine), 2.21 (m,
H, CH2piperidine), 1.90 (m, 2H, CH2piperidine), 1.55 (m, 2H, CH2piperidine);
ꢀ
1
1
reaction mixture was stirred using He flow (10 mL · min ) for 1 min
and additional 1 min at a He flow of 50 mL · min . 100 μL 1 M LiAlH
THF was added and immediately evaporated to dryness by heating the
3
ꢀ
1
4
in
(
2
2
3
ꢀ
1
reaction vial to 130 °C under a He flow (50 mL · min ) for 90 s.
Subsequently, 100 μL of 57% hydriodic acid (HI) in water was added
at 0 °C, and the mixture was left to react for 2 min at 120 °C to yield
1
3
C-NMR (125.78 MHz, CDCl
), 133.09 (CHAr), 129.51 (CHAr), 128.37 (CHAr), 127.43 (CHAr), 112.59
ArCl), 111.65 (CArC¼O), 97.85 (CHAr), 62.99 (–CH –), 56.10 (O–CH ), 52.06
CHpiperidineNH), 45.92 (CH2piperidine), 29.72 (CH2piperidine); ESI-HRMS:
3
) 163.70 (C¼O), 157.41 (CArOCH
3
), 146.57
(CArNH
2
11
[
C]benzyl iodide. The product was dissolved in 3 mL dichloromethane
(C
2
3
and passed through 5 × 0.5 cm column filled with potassium carbonate
and magnesium sulfate (approx. 8/2 by volume) into a second reaction vial,
containing one potassium hydroxide (KOH) pellet (96 ± 9 mg (n = 20)). The
identity of the purified product was confirmed with analytical HPLC by
co-injection of the product and authentic reference of benzyl iodide (column
A, solvents C/E, 60/40, v/v, wavelength 254 nm, column temperature –
(
+
3 2
calculated for C20H24ClN O : 373.88, found 374.16 [M + H] .
Tert-butyl 4-(4-amino-5-chloro-2-methoxybenzamido)piperidine-
-carboxylate (9)
1
ꢀ1
ambient, flow 1 mL · min , Rt 14.5 min). In addition, synthetic intermediates
were identified with analytical HPLC by co-injection of the corresponding
intermediate and authentic reference of benzoic acid or benzyl alcohol
Triethylamine (1 eq, 690 μL, 496 μmol) was added to 4-amino-5-chloro-2-
methoxybenzoic acid 7 (1 eq, 100 mg, 496 μmol) and dichloromethane
(10 mL) to give a white suspension. The mixture was cooled to 0 °C,
1
1
(
[ C]benzoic acid (2): (column B, solvents I/C, 80/20, v/v, wavelength
and ethylchloroformate (1 eq, 480 μL, 496 μmol) was added and stirred
at 0 °C for 10 h. A solution of tert-butyl 4-aminopiperidine-1-carboxylate
ꢀ
1
2
54 nm, column temperature – ambient, flow 0.65 mL · min , Rt 8.5 min));
11
([ C]benzyl alcohol (3): (column A, solvents C/E, 50/50, v/v, wavelength
(
(
1 eq, 970 mg, 496 μmol) in dichloromethane (10 mL) and triethylamine
1 eq, 690 μL, 496 μmol) was added at ambient temperature, and the
ꢀ1
254 nm, column temperature – ambient, flow 1 mL · min , Rt 5.2 min)).
mixture was stirred for 6 h. The solvent was dried with MgSO
concentrated in vacuo. The crude compound was purified using flash
column chromatography (ethyl acetate/hexane = 50/50) to obtain 9 in
4
and
11
[
C]tribenzylamine (6)
A mixture of dibenzylamine 5 (1 eq, 1.9 mg, 9.6 μmol) and potassium
52.6% yield (100 mg, 261 μmol).
carbonate (1.5 eq, 2.0 mg, 14 mmol) or cesium carbonate (1.5 eq,
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 342–348