Syntheses of dual-radioisotope-labeled CP-I, a GABAA receptor partial agonist

Article abstract of DOI:10.1002/jlcr.1889

CP-I is a potent subtype-selective GABA_A receptor partial agonist. Owing to its significant metabolic cleavage at C₈ observed in preliminary biotransformation studies with non-radiolabeled CP-I, the syntheses of CP-I labeled at the right or left hand side with ¹⁴C or labeled with 3H at the right hand side were required. The two compounds labeled with ¹⁴C at the left or right hand side were synthesized in 2 and 5 radio-synthetic steps using [¹⁴C]2-chloroacetyl chloride and [ ¹⁴C]NaCN as starting radiolabeled materials, respectively. CP-I was labeled with tritium at the right hand side by a tritium de-halogenation method. Batches of radiolabeled CP-I were mixed to give dual-radioisotope-labeled CP-I. An efficient approach to [¹⁴C]fluoropyridinyl imidazole was developed, and a short synthesis of iodo-substituted fluoropyridinyl imidazole was also achieved. The details of these syntheses are discussed. Copyright

Full text of DOI:10.1002/jlcr.1889

Research Article
Received 7 December 2010,
Revised 14 March 2011,
Accepted 15 March 2011
Published online 2 May 2011 in Wiley Online Library
(
wileyonlinelibrary.com) DOI: 10.1002/jlcr.1889
Syntheses of dual-radioisotope-labeled CP-I,
a GABA receptor partial agonist
A
a
Ã
b
Yinsheng Zhang, Laura Greenfield, and Yang Hong
c
CP-I is a potent subtype-selective GABA
in preliminary biotransformation studies with non-radiolabeled CP-I, the syntheses of CP-I labeled at the right or left hand
A 8
receptor partial agonist. Owing to its significant metabolic cleavage at C observed
1
side with C or labeled with 3H at the right hand side were required. The two compounds labeled with C at the left
4
14
1
4
14
or right hand side were synthesized in 2 and 5 radio-synthetic steps using [ C]2-chloroacetyl chloride and [ C]NaCN
as starting radiolabeled materials, respectively. CP-I was labeled with tritium at the right hand side by a tritium
de-halogenation method. Batches of radiolabeled CP-I were mixed to give dual-radioisotope-labeled CP-I. An efficient
1
4
approach to [ C]fluoropyridinyl imidazole was developed, and a short synthesis of iodo-substituted fluoropyridinyl
imidazole was also achieved. The details of these syntheses are discussed.
A
Keywords: C-14; tritium; dual-radioisotope labeling; halogen dance reaction; GABA receptor partial agonist
1
[ C]NaCN. On the other hand, tritium-labeled CP-I could be
4
Introduction
derived from iodo- or bromo-substituted analog H by tritium
CP-I, 2-f[2-(3-fluoropyrid-2-yl)-1H-imidazol-1-yl] methylg-1-pro- de-halogenation. The compound H should be disconnected to
pyl-5-cyano-1H-benzimidazole (compound 1), is a potent sub- give unlabeled A and I, while the intermediate I might be
type-selective partial agonist at the GABAA receptor complex.
This agonist significantly reduces in rate both spontaneous
synthesized from J (unlabeled B).
1
More specifically, [ C]chloroacetylchloride, a commercially
4
locomotor activity and the latency to assume a sleep posture available radioactive starting material, was utilized to synthesize
without the well-known side effects of commonly available
hypnotics. Significant metabolic scission of CP-I to benzimida-
the C-14-labeled CP-I at the left hand side via compound A by
1
following a known synthetic route (Scheme 1). [ C]NaCN was
4
zole alcohol 2 and fluoropyridine imidazole 3 in an NADPH- used to introduce a cyano group into the position 2 of pyridine.
dependent manner was observed from initial in vitro biotrans- A new synthetic approach to the imidazole ring was developed
1
formation studies using non-labeled CP-I (Figure 1).
to give compound B/14 (Scheme 2). The unknown 4-iodo-
The further metabolic stability studies of compounds 2 and 3 substituted fluoropyridine imidazole I/20 was synthesized by a
in the presence of NADPH showed that 2 was inert while 3 was halogen dancing approach and led to the preparation of iodo-
extensively metabolized. Therefore, both C-14- and H-3-labeled
substituted CP-I. The latter was converted to H-3-labeled CP-I by
CP-I were synthesized for use in in vivo biotransformation palladium catalyzed de-iodination with tritium gas (Scheme 3).
1
4
studies. For the C-labeled compounds, the label was placed on The dual-radiolabeled CP-I was prepared by combining two
1
4
3
single isotope-labeled compounds ([ C]CP-I, [ H]CP-I) in the
either side of C , the site of metabolic fragmentation, whereas
3
8
the H labeled was placed on the right-hand side. These labeled desired ratio of C-14 and H-3 (Scheme 4).
compounds were used to determine if all microsomal meta-
bolites formed after C
8
N-dealkylation (or its core-intact meta- Experimental
bolites) could be detected and quantified adequately (Figure 2).
By labeling CP-I with different isotopes, it was possible to
monitor simultaneously the two radio-isoforms without the
confusion of changes in specific activity of mono-radiolabeled
General methods
All reactions were carried out under an atmosphere of nitrogen
unless otherwise stated. LC-MS data were obtained on a Water
1
,2
metabolites.
The retro-synthetic analysis for the incorporation of a single
C label at both sides of CP-I at position 2 on the substituted
1
4
a
Radiochemical Synthesis, Research API, Groton Laboratories, Pfizer Inc., Groton,
CT 06340, USA
imidazole rings is shown in Figure 3. The disconnection of the
-N bond gives the retro-targets A and B. Further disconnec-
C
8
1
b
Supply Chain, Pharmaceutical Sciences, Pfizer Inc., Groton, CT 06340, USA
tion of A provides labeled building block D and unlabeled
intermediate C, which can be synthesized from commercially
available compound E. The further disconnection of the labeled
B gives the target cyanopyridine synthon F, which can be
prepared from building block G. The carbon-14 radiolabel can
c
Radiochemistry-Discovery Chemical Synthesis, Bristol-Myers Squibb, Princeton,
NJ 08543, USA
*Correspondence to: Yinsheng Zhang, Radiochemical Synthesis, Research API,
Groton Laboratories, Pfizer Inc., Groton, CT 06340, USA.
1
4
be incorporated into F by reacting synthon G with [ C]KCN or E-mail: yinsheng.zhang@pfizer.com
J. Label Compd. Radiopharm 2011, 54 411–417
Copyright r 2011 John Wiley & Sons, Ltd.

Y. Zhang et al.
NC
N
N
N
N
OH
NC
N
microsomes, O2
NADPH
+
N
N
N
N
H
F
+
C8
N
F
3
2
CP-I
1
NADPH
inert
extensively metabolized
Figure 1. The NADPH-dependent microsomal-mediated metabolic scission of 1 into 2 and 3.
14C
14C
14C
3H
NC
N
NC
N
NC
N
N
N
N
N
N
N
N
N
*
*
C8
*
C8
C8
N
N
N
N
T
F
F
F
[14
C] CP-I-L
[14C/ H] CP-I
3
[14
C] CP-I-R
Figure 2. Positions of C-14 and H-3 labels.
14C
NC
N
3H
N
N
N
C8
N
T
F
[14
C/ H] CP-I
3
H
N
N
NC
NC
N
N
N
Cl
N
N
*
N
+
*
N
F
N
X (Br, I)
H
F
A
H
B
O
NC
NH2
X (I, Br)
Cl
N
N
N
Cl
+
NH
*
*
N
N
F
F
H
F
C
I
D
[14
C]NaCN or [ C]KCN
+
14
N
NC
NO2
Cl
F
N
H
N
N
F
F
E
J
G
14
Figure 3. Retro-synthetic pathway of [ C]CP-I and [3H]CP-I.
www.jlcr.org
Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 411–417

Y. Zhang et al.
NC
NO2
NC
NH2
NH
NC
NO2 CH CH CH NH HCl
5% Pd/C/50psi
3
2
2
2
K2CO3/DMF/RT/5h
H2/EtOAc/RT/2h
NH
Cl
8
5%
90%
4
5
6
N
N
O
N
NC
NC
N
N
N
Cl
N
F
Cl
F
*
Cl
*
*
H
3
7
N
N
N
EtOAc/TEA
reflux/20h
NaOH/THF
H O
2
6
0%
7
0%
8
[14C] CP-I-L
9
14
Scheme 1. Synthesis of [ C]CP-I-L.
[14
DMSO
C]NaCN
EtOH/HCl
HCl
ethylenediamine
o
0
C/5h
NH
N
*
*
F
N
*
HN
F
N
F
30oC/24h
55%
F
N
CN
F
N
77%
OEt
1
0
11
1
2
13
N
NC
N
N
Cl
NC
N
N
BaMnO4/CH2Cl2
reflux/10h
N
*
N
*
F
N
15
N
95%
HN
NaOH
14
THF/H2O
F
73%
16 [14C] CP-I-R
14
Scheme 2. Synthesis of [ C]CP-I-R.
NC
I
N
Cl
I
1
) LDA/THF
1) LDA/THF
o
o
N
-
78 C
-78 C
o
o
N
2) I2/-78 C
N
2) H2O/-78 C
51%
F
N
F
N
15
N
F
N
48%
HN
HN
NaOH
THF/H2O
HN
3
19
2
0
8
6%
N
N
T2 /5% Pd/C
DMF/RT 1hr
NC
N
N
NC
N
N
N
N
N
3H
N
I
F
21
F
3
2
2 [ H]CP-I-R
3
Scheme 3. Synthesis of [ H]CP-I-R.
N
NC
N
N
N
N
NC
NC
N
N
N
*
N
*
N
MeOH
N
+
N
N
N
3H
N
3H
F
F
F
[14C]CP-I-L
3
23 [ H/14C]CP-I
3
9
2
2 [ H]CP-I-R
3 14
Scheme 4. Preparation of [ H/ C]CP-I.
J. Label Compd. Radiopharm 2011, 54 411–417
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org

Y. Zhang et al.
Micromass LCZ mass spectrometer with flow injection analysis 11 mmol). The resulting mixture was stirred at 301C for 24 h.
and electrospray ionization (ESI). H/ H and C NMR spectra After cooling to room temperature, the solid was filtered off and
1
3
13
were recorded on a Varian Gemini 400 MHz instrument. washed with CH Cl (3 ꢁ 3 ml). The filtrate was washed with
2
2
Chemical purity of all labeled compounds was determined by brine (3 ꢁ 2 ml) and dried over MgSO4 and concentrated in
HPLC and LC-MS. Purifications were done by flash column vacuo to give the crude product. The pure product was obtained
chromatography on Biotage Flash 40 system. Quantitation of by flash chromatography (THF/hexane = 1/4) (73.8 mg, 33 mCi,
1
3
radioactivity of C-14- and H-3-labeled compounds was per- 54 mCi/mmol, 55%). H NMR (CDCl ): d 7.97 (dd, J = 7.6 Hz,
3
H-H
4
3
4
formed using a Packard 2200CA liquid scintillation analyzer, with
Scintiverse BD cocktail used throughout. Commercial reagents (dd,
and solvents were purchased from Aldrich and used as-received and identified as 2,6-[ C ]dicyanopyridine (8.2 mg). H NMR
J
H-F = 5.8 Hz), 7.62 (dd,
J
H-H = 7.3 Hz,
J
H-F = 1.7 Hz), 7.21
3
3
J
H-H = 8.5 Hz, JH-F = 2.6 Hz); The by-product was isolated
1
4
1
2
1
4
1
unless otherwise noted. [ C]Chloroacetyl chloride (50 mCi, (CDCl ): d 8.06 (t, 1H), 7.91 (d, 2H). H NMRs of both C-14-labeled
3
1
0 mCi/mmol) and [ C]NaCN (60 mCi, 54 mCi/mmol) were product and by-product were consistent with the unlabeled
4
5
purchased from American Radiolabeled Chemicals, Inc. Inter- authentic compounds.
mediates 3 and 15 and compound 1 were provided by Chemical
1
4
R&D, Groton Lab, Pfizer Inc. 3-Amino-4-n-propylaminobenzoni- 6-Fluoro-2-(4,5-dihydro-1H-½2- Cꢀimidazol-2-yl)pyridine 13
trile 6 was synthesized according to the procedures described
1
4
solution of 6-fluoro[2- C]picolinonitrile 11 (73 mg,
3
by Li. All known compounds were identified by comparison of
NMR spectra to those reported in the literature.
To
3
a
2.4 mCi, 0.6 mmol) in EtOH (1.5 ml) and CH
2
Cl (1.5 ml) at 01C
2
was added dry HCl gas during 15 min. The reaction solution was
stirred at room temperature for 4 h and then bubbled with dry
nitrogen gas to remove the excess HCl gas and some solvent.
A solution of EtOH/THF (1/1.5 ml) was added and the resulting
1
4
2
carbonitrile 8
-Chloromethyl-1-propyl-1H-benzol[d]½2- Cꢀimidazole-5-
To a solution of 3-amino-4-n-propylaminobenzonitrile 6 (0.515g, mixture was cooled to 01C, then ethylenediamine (200 mg,
.94mmol) and triethylamine (0.45 ml) in ethyl acetate (6ml) was 3.33 mmol) was added. The resulting suspension was stirred at
2
1
4
slowly added [ C]chloroacetyl chloride (50 mCi, 50mCi/mmol, 51C for 1 h and at room temperature for 15 h. The solvent was
.0 mmol) at room temperature. The reaction mixture was stirred evaporated in vacuo. The oily residue was diluted with CH Cl
at room temperature for 30min and then at 601C for 20h. After (15 ml) and washed with brine (2 ꢁ 4 ml). The organic layer was
cooling to room temperature, the mixture was diluted with water dried over MgSO and concentrated in vacuo to give the titled
5ml). The organic solution was washed with 1.0 M NaOH product as an off-white solid (76.6 mg, 24.9 mCi, 54 mCi/mmol,
1
2
2
4
(
(
(
2ꢁ 5 ml), then with 0.25 M KH
2
5ml). The organic layer was dried over Na SO
2
PO
4
(5 ml) followed by brine 77%). The pure product was obtained by flash chromatography
3
1
). H NMR (CDCl
4
and concentrated (5% MeOH in CH
2
Cl
2
3
): d 7.98 (dd, JH-H = 7.98 Hz,
4
3
4
JH-F = 2.2 Hz), 7.80 (dd, J = 7.90 Hz, J = 15.6 Hz), 6.94 (dd,
in vacuo to give the crude product, which was recrystallized from
EtOAc/hexane (1/2) to afford the titled compound as brown
crystals (151.7mg, 32.5mCi, 50mCi/mmol, 60%). H NMR of [ C]8 164.5 (d, JC-F = 4.1 Hz), 161.5 (d, JC-F = 239.5 Hz ), 144.3 (d, JC-F =
was consistent with the unlabeled compound reported in the 12.2Hz), 142.1 (d, J = 7.5 Hz), 118.6 (s), 109.5 (d, J = 36.2Hz),
H-H
H-F
13
3
3
3
JH-H = 8.0 Hz, JH-F = 2.6 Hz), 3.77 (b, 4H); C NMR (CD OD): d
3
1
14
4
1
3
2
C-F
C-F
2
literature.
50.2 (b); HRMS (ESI) m/z Found 165.0708, calcd for C
1
8 8 3
H FN :
65.0702.
1
-Propyl-2-2-(2-fluoropyrid-6-yl)-1H-imidazol-1-yl-methyl-5-
1
4
14
14
cyano-1H-½2- Cꢀbenzimidazole 9 (½ CꢀCP-I-L)
6-Fluoro-2-(1H-½2- Cꢀimidazol-2-yl)pyridine 14
To a solution of compound 8 (142.6 mg, 30.5 mCi, 0.61 mmol) To a solution of compound 13 (76 mg, 0.46 mmol, 24.7 mCi) in
and compound 3 (106.1 mg, 0.65 mmol) in THF (30 ml) was CH Cl (12 ml) was added BaMnO (868.8 mg, 3.39 mmol). The
2
2
4
added a solution of NaOH (81 mg) in water (31 ml) slowly at suspension was stirred at 551C for 10 h. After cooling to room
room temperature. The resulting mixture was stirred at room temperature, the precipitate was filtered off and washed with
temperature overnight and at 451C for 1 h. After cooling the CH Cl (3 ꢁ 10 ml). The filtrate was concentrated in vacuo to give
2 2
reaction mixture at 01C for 30 min, the precipitate was collected the crude product. The pure product was obtained by flash
by filtration and dried under reduced pressure to give the crude chromatography (5% MeOH in CH Cl ) (71.2 mg, 23.5 mCi,
3
2
2
1
3
product. The pure product was obtained by flash chromato- 54 mCi/mmol, 95%). H NMR (CD OD): d 7.98 (q, JH-H = 8.0 Hz,
4
5
3
graphy (EtOAc/hexane = 1/4) as a off-white solid (160 mg, 1H), 7.89 (ddd, JH-F = 2.4, JH-H = 8.0 Hz, JH-H = 0.66 Hz, 1H), 7.19
1
3
3
4
2
2 mCi, 50 mCi/mmol, 70%). H NMR (CD OD): d 8.17 (q), 8.03 (b, 2H), 7.0 (ddd, J = 2.4, J = 8.13 Hz, J = 0.66 Hz 1H);
3 H-F H-H H-H
13 1
(
q), 7.93 (q), 7.83 (s), 7.62 (q), 7.21(q), 6.36 (s), 4.43(t), 1.97(q),
3
OD): d 162.4 (d), 152.5 (s), 148.3, 138.3, 142.6 (d, JC-F = 7.6 Hz), 116.6 (d, 2C), 108.5 (d, JC-F = 38 Hz).
30.1, 126.6, 125.3, 124.1, 120.4, 120.0, 111.2, 108.5, 105.6, 46.1, HRMS (ESI) m/z Found 163.0542, calcd for C H FN : 163.0546.
3
3
C NMR (CD OD): d 163.5 (d, JC-F = 240.5 Hz), 147.0 (S), 144.9 (s),
2
1
.04(t); C NMR (CD
3
3
1
1
4
2
0
8
6
4.6, 23.4, 11.3; HPLC condition for purity: YMC ODS-AQ, 5 mm,
50 ꢁ 4.6 mm, column temperature: 301C, Mobile Phase A: 1-Propyl-2-2-(2-fluoropyrid-6-yl)-1H-½2- Cꢀimidazol-1-yl-
.01 M TEA pH 2.5 w/HClO ; Mobile Phase B: CH CN, 15% B methyl-5-cyano-1H-benzimidazole, 16 (½ CꢀCP-I-R)
14
1
4
4
3
linear gradient to 60% over 20 min, hold A:B 40:60 to 45 min.
Flow rate = 1.0 ml/min, UV detection: 220 nm.
To a solution of compound 14 (71.2 mg, 23.5 mCi, 0.435 mmol)
and compound 15 (102.8 mg, 0.44 mmol) in THF (20 ml) was
added a solution of NaOH (60 mg) in water (22 ml) slowly at
1
4
6
-Fluoro½2- Cꢀpicolinonitrile 11
room temperature. The resulting mixture was stirred at room
1
To a solution of [ C]NaCN (1.1 mmol, 60 mCi, 54 mCi/mmol) in temperature overnight and at 45 C for 1 h. After cooling the
4
o
dry DMSO (1 ml) at 351C was added 2,6-difluoropyridine (1.266 g, reaction mixture at 01C for 30 min, the precipitate was collected
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Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 411–417

Y. Zhang et al.
by filtration, dried under reduced pressure to give the crude reaction mixture at 01C for 30 min, the precipitate was collected
product. The pure product 16 was obtained by flash chromato- by filtration, dried under reduced pressure to give the crude
graphy (EtOAc/hexane = 1/4) as a off-white solid (115 mg, product. The pure product was obtained by flash chromato-
1
1
8
1
1
4
5
0
7.2 mCi, 53.8 mCi/mmol, 73%). H NMR (CD OD): d 8.17 (q), graphy (EtOAc/hexane = 1/4) as a off-white solid (183.8 mg,
3
1
.03 (q), 7.93 (q), 7.83 (S), 7.62 (q), 7.21(q), 6.36 (S), 4.43(t), 86%). H NMR (DMSO-d ): d 8.32, 7.91. 7.75 (d), 7.57 (d), 7.49,
.97(q), 1.04(t); C NMR (CD OD): d 162.4 (d), 152.5 (s), 148.3, 7.45, 7.16 (s,1H), 6.04 (s), 4.30 (t), 2.48 (q), 0.85 (t), C NMR
3
38.3, 130.1, 126.6, 125.3, 124.1, 120.4, 120.0, 111.2, 108.5, 105.6, (DMSO-d ): d 162.4 (d), 152.5 (s), 148.3, 138.3, 130.1, 126.6, 125.3,
6.1, 44.6, 23.4, 11.3; HPLC condition for purity: YMC ODS-AQ, 124.1, 120.4, 120.0, 111.2, 108.5, 105.6, 46.1, 44.6, 23.4, 11.3;
mm, 250 ꢁ 4.6 mm, column temperature: 301C, Mobile Phase A: HRMS (ESI) m/z Found 486.0462, calcd for C20H16FIN : 486.0465.
6
.01 M TEA pH 2.5 w/HClO ; Mobile Phase B: CH CN, 15% B linear
gradient to 60% over 20 min, hold A:B 40:60 to 45 min. Flow 2-[2-(6-Fluoro½4- Hꢀpyrid-2-yl)1H-imidazol-1-yl]methyl-1-
rate = 1.0 ml/min, UV detection: 220 nm.
-fluoro-2-(1H-imidazol-2-yl)-3-iodopyridine 19
6
1
3
13
6
4
3
3
3
propyl-5-cyano-1H-benzoimidazole, 22 (½ HꢀCP-I-R)
A trisorber flask was charged with compound 21 (1.0 mg) and
% Pd/C (1 mg) in MeOH (0.5 ml). The reaction mixture was
6
5
reduced with 800 mCi carrier free tritium gas on a commercial
tritiation manifold (TRI-SORBER R Tritiation Manifold, IU/US
System, Inc.) for 2 h. After completion, the reaction mixture was
diluted with MeOH (1 ml) and the Pd/C catalyst was removed by
filtration. Labile products were removed by co-evaporation with
ethanol and the crude product was subjected to reverse phase
HPLC purification. The fractions containing the desired pure
product were pooled, concentrated, and reconstituted in
ethanol to give 74.7 mCi of the final product 22 with a specific
activity of 23.7 Ci/mmol and radiochemical purity of 99.52%.
HPLC condition for purity: YMC ODS-AQ, 5 um, 250 ꢁ 4.6 mm,
column temperature: 301C, Mobile Phase A: 0.01 M TEA pH 2.5
To a solution of diisopropylamine (2.9 ml) in dry THF (10 ml) was
added a solution of n-BuLi (2.5 M, 12 ml) at 01C. The solution
was stirred at 01C for 30 min and further cooled toꢂ781C. To this
cold solution was added a solution of compound 3 (2.0 g,
1
2.3 mmol) in THF ( 5 ml) at ꢂ781C to give a yellow suspension.
The suspension was stirred at ꢂ781C for 4 h and then to this
cold suspension was added a solution of I (3.68 g, 14.6 mmol) in
2
THF (5 ml). The resulting mixture was stirred atꢂ781C for 10 h
and slowly warmed to room temperature over 10 h. To the
reaction mixture was added water (5 ml) and aqueous saturated
NaHCO (5 ml). The mixture was extracted with ether (4 ꢁ 25 ml).
3
The combined ether layers were dried with MgSO
concentrated in vacuo to give the crude product. The pure
4
and
4 3
w/HClO ; Mobile Phase B: CH CN, 15% B linear gradient to 60%
over 20 min, hold A:B 40:60 to 45 min. Flow rate = 1.0 ml/min, UV
detection: 220 nm; HPLC condition for purification: YMC ODS-
AQ, 5 mm, 250 ꢁ 6 mm, column temperature:251C, Mobile Phase
product was obtained by flash chromatography (EtOAc/hexane =
1
/4) as a white solid (1.71 g, 48%). H NMR (CD
1
7
2
3
OD): d 8.38 (t, 1H),
.66 (q, 1H), 7.20 (b, 2H); C NMR (CD OD): d 163.5 (d, JC-F =
3
3
40.5 Hz), 153.0 (d, JC-F = 7.6 Hz), 148.4 (d, JC-F = 15.9 Hz), 144.9
1
3
1
3
A: 0.1% TFA in water, Mobile Phase B: 0.1% TFA in CH CN, 15% B
linear gradient to 30% over 5 min, hold A:B 70:30 to 22 min, hold
A:B 30:70 to 30 min Flow rate = 2.0 ml/min, UV detection:
3
4
2
(s, 2C), 120.2 (d, J = 3.8Hz), 76.3 (d, J = 45.4 Hz); HRMS (ESI)
C-F C-F
8 3
m/z Found 288.9514, calcd for C H10FIN : 288.9512.
3
20 nm. H NMR (CD
1
2
3 3
OD): d 8.10; H NMR (CD OD): d 8.08 (q),
8
(
.02 (d), 7.85 (s), 7.76 (d), 7.70 (b), 7.61 (d), 7.258 (b), 7.07 (d), 6.28
s), 4.43(t), 1.93(q), 1.01(t).
6
-fluoro-2-(1H-imidazol-2-yl)-4-iodopyridine 20
To a solution of diisopropylamine (0.725 ml) in dry THF (5 ml)
was added a solution of n-BuLi (2.5 M, 3 ml) at 01C. The solution 2-[2-(6-Fluoro½4- Hꢀpyrid-2-yl)1H-imidazol-1-yl]methyl-1-
was stirred at 01C for 30 min and then cooled to ꢂ781C. To this propyl-5-cyano-1H-½2- Cꢀbenzoimidazole, 23 (½ H= CꢀCP-I)
cold solution was added a solution of compound 19 (1.0 g,
3
1
4
3
14
1
4
A solution of [ C]CP-I-L (9, 250 mCi, 14 mCi/mg) in MeOH (2.5 ml)
was combined with a solution of [ H]CP-I-R (22, 2500 mCi) in
MeOH (7.5 ml) to form a solution of [ H/ C]CP-I (250 mCi of C-14
and 2500 mCi of H-3 in 10 ml of MeOH) with H-radiochemical
purity of 99.4% and C radiochemical purity of 99.6%. The final
3.46 mmol) in THF (2 ml) at ꢂ781C to give a yellow suspension.
3
The suspension was stirred at ꢂ781C for 4 h and then to this
cold suspension was added water (10 ml). The resulting mixture
was stirred at room temperature for 2 h and then extracted with
ether (4 ꢁ 20 ml). The combined ether layers were dried with
3
14
3
1
4
1
4
specific activity was determined to be 14.0 mCi/mg for C and
MgSO and concentrated in vacuo to give the crude product.
The pure product was obtained by flash chromatography
4
3
1
38 mCi/mg for H. HPLC condition for purity: YMC ODS-AQ,
5 mm, 250 ꢁ 4.6 mm, column temperature: 301C, Mobile Phase A:
0.01 M TEA pH 2.5 w/HClO ; Mobile Phase B: CH CN, 15% B linear
1
EtOAc/hexane = 1/4) as a white solid (0.55 g, 51%). HNMR
(
(
5
4
3
4
3
CD
3
OD): d 8.29 (t, JH-F = 1.0 Hz, JH-H = 1.1 Hz. 1H), 7.46 (q, JH-F
=
4
13
gradient to 60% over 20 min, hold A:B 40:60 to 45 min. Flow
rate = 1.0 ml/min, UV detection: 220 nm.
2.9 Hz, J = 1.1Hz, 1H), 7.21(b, 2H); CNMR (CD OD): d 163.4
H-H 3
1 3
(
d, JC-F = 253.1 Hz), 147.1 (d, JC-F = 15.9Hz), 143.5 (s), 126.0 (s),
3
125.9 (s), 117.9 (s), 117.5(s), 109.0 (d, JC-F = 8.0 Hz); HRMS (ESI) m/z
Found 288.9514, calcd for C H FIN : 288.9512.
3
8
10
Results and discussion
1
4
1-Propyl-2-2-(2-fluoro-4-iodo-pyrid-6-yl)-1H-imidazol-1-yl-
methyl-5-cyano-1H-benzimidazole 21
Synthesis of ½ CꢀCP-I labeled at the left hand side
Our first goal was to introduce a C-14 label into the
To a solution of compound 15 (102.8 mg, 0.44 mmol) and benzimidazole ring system at the left hand side of CP-I.
1
compound 20 (132.9 mg, 0.46 mmol) in THF (20 ml) was added a Scheme 1 illustrates a route to [ C]CP-I-L (9) via coupling of
solution of NaOH (60 mg) in water (22 ml) slowly at room [ C]chloromethyl compound 8 and aryl imidazole 3. Thus, aryl
temperature. The resulting mixture was stirred at room chloride 4 was reacted with n-propylamine in the presence of
4
1
4
3
temperature overnight and at 451C for 1 h. After cooling the
2 3
K CO to give amino adduct 5 in 85% yield. The selective
J. Label Compd. Radiopharm 2011, 54 411–417
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org

Y. Zhang et al.
reduction of the nitro group in amine 5 with 5% Pd/C in EtOAc midazolam, we developed an efficient approach to imidazole
yielded diamine 6 in 90% yield. The diamine 6 can be reacted derivatives via diamine coupling with acetimidic acid ethyl ester
5
with 2-chloro-acetimidic acid methyl ester, or a similar electro- hydrochloride (Figure 4, route II). The later can be easily
phile such as 2-chloro-1,1,1-trimethoxy-ethane, chloroacetic acid prepared in situ from a nitrile. Therefore, we were focused on
anhydride or chloroacetyl chloride to form the chloromethyl the new route II and explored a way to introduce a cyano group
3
14
compound 8. However, [ C]chloroacetyl chloride was selected into position 2 of the pyridine.
because it was commercially available and easily prepared. The The literature showed that 2-fluoro-5,6-dichloro pyridine
cyclization of the diamine 6 with [ C]chloroacetyl chloride could be selectively converted to 2-cyano-5,6-dichloropyridine
1
4
1
4
formed the desired [ C]8 in 60% isolated yield.
using excess NaCN as a cyanation reagent and DMSO as
6
In the original synthesis of unlabelled CP-I (1), the final solvent. These reaction conditions were adopted to make the
N-alkylation used NaH as base and DMF as solvent. In our desired 2-cyano-6-fluropyridine. Unfortunately, the undesired
radiosynthesis, the N-alkylation was carried out using an di-cyano substituted pyridine 18 was isolated as a major
aqueous NaOH solution in THF as a more environmentally product. However, after changing the molar ratio of 2,6-
1
4
friendly alternative, and this afforded 70% of the final [ C]CP-I-L. difluoropyridine 10 and NaCN from 1/3 to 10/1 and the
This route only involved two radiosynthetic steps and gave a temperature from 60 to 301C, we were able to synthesize the
42% overall radiochemical yield with a radiochemical purity of 2-cyano-6-fluoropyridine 17 in 65% yield and its C-14-labeled
9
9.7% and a specific activity of 49.9 mCi/mmol.
analog 11 in 55% yield (see entries 5 & 6 in Table 1). Using CuCN
as a cyanating reagent (entry 3 in Table 1) gave a very poor yield
of 17 after 24 h of heating at 1001C.
1
4
Synthesis of ½ CꢀCP-I labeled at the right hand side
1
During drug development of CP-I, [ C]CP-I labeled at right hand
side was also required to support animal and human studies. converted to the [ C]acetimidic acid ethyl ester hydrochloride 12
Scheme 2 shows a 4-step radiosynthesis of [ C]CP-I-R via by treating 11 with dry HCl gas in EtOH at 01C for 5 h. Compound
N-alkylation of a key C-14-labeled imidazole derivative 14 with 12 was not isolated but reacted directly with ethylenediamine to
the unlabeled chloromethyl compound 15.
4
4
14
The desired [ C]2-cyano-6-fluoropyridine 11 was then easily
1
4
1
4
1
4
give [ C]dihydroimidazole 13 in 77% yield after flash chromato-
According to literature, the unlabeled imidazole derivative 3 graphy. The compound 13 was then oxidized by BaMnO to
4
can be prepared by the reaction of 6-fluoro-pyridine-2- desired imidazole 14 in 95% yield. The final N-alkylation of
carbaldehyde with glyoxal and ammonium hydroxide (route I compound 14 with compound 15 in an aqueous NaOH/THF
1
4
in Figure 4). However, none of the C-14-labeled compounds solution furnished the target, [ C]CP-I-R, in 73% yield. Overall
1
4
could be easily synthesized because both [ C]aldehydes are radiochemical yield for 5 steps was 33% with a radiochemical
unstable. In our lab, during the preparation of isotope-labeled purity of 99.2% and a specific activity of 54.0mCi/mmol.
NH OH/MeOH
4
o
+
CHO
OHC CHO
18hr/0 C-RT
I
F
N
4
0%
N
F
N
1
) diamine
HN
HCl/EtOH
NH
HCl
3
II R-CN
R
2) MnO or BaMnO4
2
OEt
CH Cl2
2
6
0-95%
Figure 4. Possible approaches to 2-fluoro-6-(1H-imidazol-2-yl)pyridine 3.
Table 1. Study on cyanation of 2,6-difluoropyridine with metal cyanides
Entry
Mole ] MCN
1 eq NaCN
Temperature (1C)
Time (h)
Ratio 17/18
Yields (major)
1
2
3
4
5
6
60
40
100
40
30
30
4
15
24
15
24
24
1/8
1/5
1/1
6/1
9/1
9/1
45% (18)
38% (18)
10% (17)
32% (17)
65% (17)
55% (17)
1 eq NaCN
1 eq CuCN
0.1 eq NaCN
0.1 eq NaCN
1
4
0.1 eq [ C]NaCN
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Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 411–417

Y. Zhang et al.
3
is used more often and is more convenient for obtaining
the desired ratio of two isotopomers. In addition, the single-
Synthesis of ½ HꢀP-I labeled at the right hand side
The preliminary metabolic mechanism study suggested that isotope-labeled compounds were required for in vitro and
only position 4 in the pyridine ring system was metabolically in vivo studies.
stable. Therefore, the tritium label has to be introduced into
In our case, C-14- and H-3-labeled CP-I were synthesized as
position 4 to avoid the loss of tritium in vivo. It was decided that described, and combined together as a 1/10 ratio of the total
a dehalogenation approach would be the easiest way to activity of C-14 to H-3 to support the dual-radiolabeled studies.
incorporate a tritium into the desired position. To synthesize
the 4-iodo or bromo substituted CP-I (21), the 4-iodo- or bromo-
substituted derivative of compound 3 was desired.
The final dual-radioisotope-labeled CP-I was prepared as a
3
methanol solution with H-radiochemical purity of 99.4% and
1
4
C radiochemical purity of 99.6%. The final specific activity was
1
4
3
Normal halogenation reactions, such as treatments with Br
and NBS, failed to introduce a halogen (I or Br) into the desired
2
determined to be 14.0 mCi/mg for C and 138 mCi/mg for H.
position 4 in the compound 3. However, a ‘halogen-dance’ Conclusion
phenomenon reported by several chemists in the literature led
7
us to the desired 6-fluoro-2-(1H-imidazol-2-yl)-4-iodopyridine 20. Having established valid routes for the key labeled inter-
Treatment of compound 3 with 2.2 eq of LDA in THF at ꢂ781C mediates, two C-14-labeled CP-I isotopomers and one H-3-
followed by quenching with I
2
at ꢂ781C gave 6-fluoro-2-(1H- labeled CP-I isotopomer were synthesized in good overall
imidazol-2-yl)-3-iodopyridine 19 in a 48% isolated yield. The yields and 499% radiochemical purity. An efficient route to
isolated 3-iodo substituted pyridine 19 was then treated with the C-14-labeled fluoropyridine imidazole was developed via
2
.2 eq of LDA in THF again at ꢂ781C followed by quenching with the acetimidic acid ester approach. This new approach to the
water to afford the desired 4-iodo substituted pyridine 20 in imidazole ring system can be easily expanded to prepare other
a 51% isolated yield. Nuclear Overhauser Effect analysis of isotopically labeled drugs. A short synthesis of iodo substituted
regioisomers 19 and 20 further proved the structures.
fluoropyridine imidazole was also achieved via a ‘halogen-
Having the desired 4-iodo-substituted pyridine 20 in hand, dancing’ approach.
the synthesis continued with N-alkylation of 20 using chloro-
methyl compound 15 under the same condition as the
1
4
preparation of [ C]CP-I to furnish 4-iodo-substituted CP-I (21).
The de-iodination of 21 was carried out with 800 mCi of tritium
gas and 5% Pd/C as a catalyst. A total of 74 mCi of pure [3 H]CP-I
was isolated at a specific activity of 23.7 Ci/mmol and a
radiochemical purity of 99.5% after preparative HPLC. H NMR
showed tritium was located at the expected position.
References
[
[
[
[
1] C. L. Shaffer, C. S. Langer, J. Pharm. Biomed. Anal. 2007, 43,
1195–1205.
2] C. J. Nonckreman, P. G. Rouxhet, C. C. Dupont-Gillain, J Biomed.
Mater. Res. A 2007, 81A(4), 791–802.
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2002050062, 2002.
4] Y. Xu, B. Han, L., Xie, G. Maynard, WO 2005080355, 2005.
3
3
14
Preparation of ½ H= CꢀCP-I
Generally speaking, there are two ways to prepare dual
radiolabeled compounds, i.e. (1) synthesize two radiolabeled
intermediates and then couple them to make a dual-labeled
target; (2) prepare single-labeled final compounds separately
and then combine them in the desired ratio. Of these, method 2
[5] Y. Zhang, P. W., Woo, J. Hartman, Tetrahedron Lett. 2005, 46,
087–2091.
2
[
6] J. A. Orvik, A. P., Fung, J., Love, T. J. Dietsche, US 4766219, 1988.
7] T. J. M. Schnuerch, M. Spina, A. F. Khan, M. D. Mihovilovic,
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[
J. Label Compd. Radiopharm 2011, 54 411–417
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org