Asymmetric syntheses of radioactive and stable isotope-labeled β-amino acids

Article abstract of DOI:10.1002/jlcr.2916

β-amino acids 1 and 2 are α2δ agonists, which were developed for the treatment of generalized anxiety disorder and insomnia. The stable and radioactive isotope-labeled β-amino acids were required to support pre-clinical and clinical studies. Asymmetric sy

Full text of DOI:10.1002/jlcr.2916

Research Article
Received 12 December 2011,
Revised 27 January 2012,
Accepted 06 March 2012
Published online 28 March 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2916
Asymmetric syntheses of radioactive and
stable isotope-labeled b-amino acids
a
b
c
*
Yinsheng Zhang, Srirajan Vaidyanathan, Keith Garnes, and
Klaas Schildknegta
b-amino acids 1 and 2 are a2d agonists, which were developed for the treatment of generalized anxiety disorder and insomnia.
The stable and radioactive isotope-labeled b-amino acids were required to support pre-clinical and clinical studies. Asymmetric
syntheses of deuterium and carbon-14 labeled b-amino acids were achieved via chiral auxiliary and diastereoselective
hydrogenation methodologies, respectively. The details of these syntheses are reported.
Keywords: isotope-labeling; C-14; C-13; H-2; b-amino acids; asymmetric synthesis; anxiety disorder and insomnia
asymmetric Aldol reaction of chiral imines 11 and esters 10,
Introduction
followed by strong acid-promoted hydrolysis of sulfonamide
and ester groups.14 Method IV is relatively new and uses
a2d-Ligands are biologically active compounds that selectively
displace 3H-gabapentin from brain membranes, indicating a high
b-ketone acid 12 as a starting material. A two-step sequence of
amination and asymmetric hydrogenation offers the target 13.4
affinity interaction with the a2d subunit of voltage-gated calcium
channels.1 a2d-Ligands have been developed for the treatment
None of those approaches have been applied to synthesis
isotope labeled b-amino acids before.
Our syntheses of stable-isotope labeled compounds 3 and 4
of several conditions such as generalized anxiety disorder,
insomnia, fibromyalgia, epilepsy, neuropathic pain, anxiety,
depression, and attention deficient hyperactivity disorder.2
were accomplished in seven steps (Scheme 1) from commer-
Compounds 1 and 2 (Figure 1) were selected for detailed study
cially available chiral compound 14 and isotope labeled methyl
iodide (13CD3I] and ethyl bromide (CD3CD2Br), respectively,
of their pharmacology. Therefore, their stable-isotope labeled
based on the reported synthesis.3 The labeled halides were first
analogs 3 and 4 were required for use as internal standards for
phase I sample assays, and radiolabeled analog 5 was needed for
animal and human absorption, distribution, metabolism, and
elimination (ADME) and whole body autoradiography studies.
This article describes asymmetric syntheses of radioactive and
converted to Grignard reagents in situ then coupled with chiral
bromide 14 in the presence of LiCl and CuCl to furnish the
labeled chiral alkenes 15a and 15b in 78% and 95% yield,
respectively. The labeled alkenes 15a and 15b were oxidized
stable isotope-labeled compounds 3, 4, and 5. The synthesis
with Jones reagent to give chiral acids 16a and 16b. The rest
of unlabeled b-amino acids 1 and 2 has been previously
of the synthesis is illustrated through the use of acid 16a as a
reported.3,4 We have adapted the reported synthetic approaches
representative example. The purified unsaturated acid 16a was
to complete the syntheses of several isotope labeled b-amino acids
first activated by forming a mixed anhydride with trimethylacetyl
such as 3, 4, and 5 through chiral auxiliary and diastereoselective
chloride in situ and then combined with the Evans’ chiral
hydrogenation methodologies. Both final stable isotope-labeled
auxiliary 17 to generate chiral oxazolidinone 18a in 85% yield.
compounds 3 and 4 were prepared with a chemical purity of
An sodium bistrimethylsilylamide mediated alkylation of 18a
>99% and an isotopic enrichment of >98%, and carbon-14 labeled
with t-butylbromoacetate afforded a-substituted oxazolidinone
compound 5 was also synthesized with both radiochemical and
chemical purity of >99%.
19a with excellent diastereoselectivity (>98% i.e., based on
HPLC assay) and good yield. Compound 19a was treated with
LiOH-H2O2 in a mixture of THF and water resulting in the formation
of sufficiently pure chiral acid 20a. The key Curtius re-arrangement
Results and discussion
Available methodology for asymmetric synthesis of b-amino
acids with one chiral center is shown in Figure 2.5–7 Method I
starts with unsaturated acid 6. Asymmetric Michael addition with
aRadiochemistry Group, Chemical R&D, Groton Lab. Pfizer Inc, North Chicago, IL, USA
bChemical Process R &D, Abbott Laboratories, North Chicago, IL, USA
chiral benzylamine 7 followed by catalytic de-benzylation gives
the amino acid 13.8–11 Both methods II and III are based on the
chiral auxiliary approach. Method II utilizes Evans’ auxiliary to
generate a chiral center through asymmetric alkylation of imide
9. A carboxylic acid group from 9 is then converted to a b-amino
group by Curtis re-arrangement.12,13,3 Method III involves
cSynthesis Services, Radiosynthesis, ABC Laboratories Inc, Columbia, MO, USA
*Correspondence to: Yinsheng Zhang, Radiochemical Synthesis, Chemical R&D,
Pfizer Inc., Groton, CT, USA.
E-mail: yinsheng.zhang@yahoo.com
J. Label Compd. Radiopharm 2012, 55 166–170
Copyright © 2012 John Wiley & Sons, Ltd.

Y. Zhang et al.
O
NH2
S
R
HO
R
*
3) R=CH2CH213CD3, * = 12
4) R=CH2CH2CD2CD3, * = 12
C
5) R=CH2CH2CH3, * = 14
C
1) R=CH2CH2CH3 ,* = 12
2) R=CH2CH2CH2CH3, * = 12
C
C
C
Figure 1. Structures of unlabeled and labeled b-amino acids 1, 2, 3, 4, and 5.
O
O
O
R
O
R
N
HO
O
6
Br
Ph
N
H
7
Ph
Bn
9
OtBu
1) Michael addition
2) De-benzylation by Pd
1) asymmetric alkylation
2) Curtius rearrangement
8
I
II
O
NH2
R
HO
1) amination
2) asymmetric hydrogenation
of enamide
13
>95% e.e
1) asymmetric Aldol-type
2) de-t-butanesulfinyl group
III
IV
O
NH3
O
O
O
OtBu
10
R
S
R
HO
N
12
11
Figure 2. Available methodology for b-amino acids.
[
13C]ICD3 /Mg/ether
LiCl/CuCl/THF
0oC to RT/16hr 78%
R
CrO3/H2SO4
Br
O
0oC to RT/15hr
or
R
BrCD2CD3/Mg/ether
LiCl/CuCl/THF
OH
15 a) R=13CD3
b) R=CD3CD2
61-80%
14
16a, 16b
0oC to RT/16hr 95%
Me3CCOCl
Et3N/LiCl/THF
RT/18hr
NaHMDS
LiOH
O
O
O
O
BrCH2CO2tBu
30% H2O2
THF/H2O
RT/5hr
-75oC/4hr
13CD3
13CD3
O
O
N
85%
O
N
RT/16 hr
13CD3
O
OH
83%
Ph
Me
OtBu
Ph
Me
O
NH
Me
99%
O
16a
Ph
18a
19a
17
O
O
DPPA
Et3N
C
N
13CD3
H2N
HCl
13CD3
HO
13CD3
3M HCl
MTBE
reflux/18h
80oC/15h
OtBu
OH
3
O
60%
O
OtBu
O
20a
21a
Scheme 1. Synthesis of stable-isotope labeled compounds 3 and 4.
was carried out by heating acid 20a with diphenyl-phosphoryl for preliminary studies. However, several issues were identified
azide (DPPA) in the presence of triethylamine to afford crude that did not make it amenable for scale-up4 and current Good
isocyanate 21a. Without further purification, crude 21a was Manufacturing Practices (cGMP) radiosynthesis, such as the highly
hydrolyzed with 3M HCl to form labeled compound 3 as an HCl salt toxic waste produced by the Jones oxidation and the use of
in 60% yield after purification. The overall yield of 3 from hazardous reagents, hydrogen peroxide, and DPPA. Therefore, a
the labeled methyl iodide was 16.7% with >98% of isotope more efficient route (Scheme 2) was developed for the synthesis of
enrichment. The labeled compound 4 was prepared in a similar compound 1 on a large scale4 and adapted to prepare carbon-14
fashion from chiral acid 16b (R = CD2CD3).
labeled cGMP material for supporting human ADME studies.
For our cGMP radiosynthesis, we used commercially available
This route proved satisfactory to provide small amounts of
compounds 1 and 2 and their stable isotope-labeled analogs potassium ethyl [1-14C]malonate 24 as our C-14 source and
J. Label Compd. Radiopharm 2012, 55 166–170
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

Y. Zhang et al.
O
*
O
EtO
OK
O
CH3
CDI
EtOAc
RT/4hr
O
CH3
24
O
O
CH3
MgCl2, Et3N, 45oC/16hr
70%
N
HO
N
*
*
EtO
23
22
25
Ac2O
Ac
NH4OAc
EtOH,
(R)-mTCFP-Rh(COD)BF4
H2, MeOH, 50oC, 10psi
Py/Isooctane
O
*
NH CH3
O
*
NH2 CH3
100oC/15hr
60oC/5hr
EtO
EtO
95%, 95% de
85%
27
26
Cl
Ac
O
1) HCl, 100oC/20hr
2) Recryst. (IPA/toluene)
O
*
NH3 CH3
NH CH3
P
P
HO
EtO
*
76%, 99.8% e.e.
*C=14
C
28
5
(R)-mTCFP
Scheme 2. cGMP Synthesis of radioactive isotope-labeled compound 5.
chromatography on Biotage Flash 40 system. Quantitation of
radioactivity of C-14 labeled compounds was performed using
a Packard 2200CA liquid scintillation analyzer, with Scintiverse
BD cocktail used throughout. Commercial reagents and solvents
were purchased from Aldrich and used as received unless
otherwise noted. Potassium ethyl [1-14C2]malonate 24 (50 mCi,
54.7 mCi/mmol) was purchased from PerkinElmer. [13C,D3]methyl
iodide and [D5]ethylbromide were purchased from CDN Isotopes,
Inc. Intermediate 22 and ligand (R)-mTCFP were provided by
Chemical R&D, Sandwich Lab, Pfizer Inc. All known compounds
diluted the labeled material with its unlabeled analog to reduce
its specific activity. A 6-step cGMP radiosynthesis is shown in
Scheme 2. The synthesis started with activation of (R)3-methyl-
hexanoic acid 22 with 1,1’-carbonyldiimidazole to generate
imidazolide 23 in situ. Imidazolide 23 was reacted with 24 (pre-
15
diluted with unlabeled material) in the presence of MgCl2 to
give the labeled b-ketoester 25 in 70% yield. Treatment of
ketoester 25 with ammonium acetate in ethanol at 60ꢀC
afforded enamine 26, which was immediately reacted with
acetic anhydride in isooctane at 100ꢀC to form enamide 27. were identified by comparison of NMR spectra to those reported
in the literature or authentic samples.
The key asymmetric hydrogenation of enamide 27 in methanol
and isooctane was accomplished using (R)-mTCFP as a ligand
and Rh(COD)BF4 as a catalyst to produce the desired acetamide
28 with excellent diasteroselectivity (>95% de) in 95% yield.16
Acidic hydrolysis of acetamide 28 with aqueous HCl solution
was carried out to give the desired C-14 labeled cGMP material
5 as an HCl salt after recrystallization from IPA/toluene. The
overall radiochemical yield from potassium ethyl [1-14C]malonate
9-([13C]-Trideuteriomethyl)-(R)-2,6-dimethylnon-2-ene (15a)
A flame-dried three-necked flask was charged with magnesium
(4.5 g, 186 mmol) and diethylether (60 mL). A solution of 13CD3I
(Aldrich, 15.0 g, 104 mmol) in diethylether (15 mL) was added
slowly. Upon addition of all the iodide, the mixture was heated
at 30ꢀC for 1 hour and then cooled at room temperature. A
24 was 43% with a radiochemical purity of 99.2% and a specific second flame-dried three-necked flask was charged with lithium
chloride (0.9 g, 21 mmol), cuprous chloride (1.04 g, 10.5 mmol)
and tetrahydrofuran (distilled, 75 mL). (S)-(+)-citronellylbromide
14 ( 11.5 g, 52 mmol) was added slowly at room temperature.
The resulting mixture was stirred at 25ꢀC for 10 minutes and at
0ꢀC for an additional 30 minutes. The solution of the Grignard
reagent was slowly added to the solution of the cuprate mixture
at 0ꢀC. The resulting mixture was stirred for 16 hours at room
temperature. After the mixture was quenched with ammonium
chloride (25 mL, saturated), it was poured into a mixture of
diethyl ether (500 mL) and ammonium chloride (500 mL, 2N).
The organic phase was separated, washed with brine, dried
(Na2SO4), and concentrated under vacuum to give 15a (6.4 g,
78%) as a colorless oil. 1HNMR (CDCl3): d 5.03 (t sept, 3J = 7.1
Hz, 4J = 1.5 Hz, 1H), 1.89 (m, 2H), 1.61 (d, 4J = 1.2 Hz, 3H), 1.53
(s, 3H), 1.41–1.12 (m, 5H), 1.11–0.95 (m, 2H), 0.79 (d, J = 6.2 Hz, 3H).
activity of 8.4 mCi/mmol. This labeled cGMP material with a high
specific activity was further diluted with GMP unlabeled material
to furnish the final dose (105.29 mCi/mmol or 0.502 mCi/mg).
In summary, we have utilized a chiral auxiliary methodology to
synthesize two stable isotope-labeled b-amino acids from a
readily available stable isotope sources. Also, we have demon-
strated that cGMP radioactive materials were prepared in four
steps using a more efficient approach with a key asymmetric
reduction step.
Acknowledgements
The authors thank Che Huang, Laura Greenfield, Wayne Stolle,
and Anil Rane for the helpful discussions during the imple-
mentation of this project.
7-(13C-Trideuteriomethyl)-(R)-4-methylheptanoic acid (16a)
Experimental section
A round-bottomed flask was charged with chromium trioxide
(21.2 g, 212 mmol), H2O (60 mL) and cooled to 0ꢀC. Sulfuric acid
General methods: All reactions were carried out under an (18.0 mL, 335 mmol) was slowly added to this cold solution. The
atmosphere of nitrogen unless otherwise stated. Liquid chroma- resulting mixture was stirred at 0ꢀC for 15 minutes. Another flask
tography-mass spectrometry (LC-MS) data were obtained on a was charged with 15a (6.4 g, 40.5 mmol) and acetone (50 mL)
Waters Micromass LCT mass spectrometer with flow injection and cooled to 0ꢀC. The solution of the oxidant via a dropping
analysis and electrospray ionization. 1H and 13C NMR spectra funnel at 5ꢀC over a period of 4 hours was slowly added to this
were recorded on a Varian Gemini 300 MHz instrument. Chemi- cold solution. After the addition, the mixture was stirred for
cal purity of all compounds was determined by HPLC and 16 hours at room temperature, quenched with brine (20 mL),
LC-MS. Purifications were performed using flash column concentrated under vacuum, and further diluted with brine
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 166–170

Y. Zhang et al.
(20 mL). The aqueous layer was extracted with tert-butyl methyl (CDCl3): d 2.81 (m, 1H), 2.41 (ddd, 2J = 77.6 Hz, 3J = 9.7 Hz, 5.1 Hz,
ether (4 Â 60 mL). The combined organic layers were washed 2H), 1.62 (m, 1H), 1.49–1.32 (m, 2H), 1.36 (s, 9H), 1.29–0.98
with brine (200 mL), dried (Na2SO4), and concentrated under (m, 5H), 0.85 (d, J = 6.4 Hz, 3H).
vacuum to give a viscous brown tar. The crude material was
8-(13C-Trideuteriomethyl)-(3S, 5R)-3-amino-5-methyloctanoic acid (3)
purified by flash column chromatography (heptane/ethyl acetate,
gradient 6:1 to 2:1) to afford 16a (4.4g, 73.3%) as a colorless oil.
1HNMR (CDCl3): d 2.30 (m, 2H), 1.60 (m, 1H), 1.46–1.32 (m, 2H),
Triethylamine (3.5 mL, 25 mmol) and diphenylphosphoryl azide
(DPPA, 4.7 mL, 22.6 mmol) was added to a mixture of 20a
(5.2 g, 20 mmol) in anhydrous toluene (120 mL). The resulting
mixture was stirred for 30 minutes at room temperature, then
1.30–1.14 (m, 3H), 1.10–0.99 (m, 1H), 0.81 (d, J = 6.7 Hz, 3H).
7-(13C-Trideuteriomethyl)-(4R,5S)-4-methyl-3-[(R)-4-methylhepta-
noyl]-5-phenyloxazolidin-2-one (18a)
for 18 hours at reflux. After the mixture was cooled at room
temperature, hexane (7 mL) and water (8 mL) were added. The
organic layer was separated and washed with water (4 mL). The
solvent was evaporated under vacuum to give crude 21a.
The crude 21a was then dissolved in 3M HCl (10 mL) and heated
at 80ꢀC for 15 hours. After cooling at room temperature, the
reaction mixture was diluted with diethyl ether (8 mL). The
aqueous layer was separated and extracted with diethyl ether
(5 mL) and treated with charcoal at 60ꢀC for 30 minutes. After
removal of charcoal, the aqueous layer was evaporated under
vacuum to dryness. The crude product was triturated in
acetonitrile at 23ꢀC for 1 hour to afford 3 as an HCl salt (2.56 g,
Triethylamine (4.4 mL, 60 mmol) and trimethylacetyl chloride
(4.34 g, 36.0 mmol) at 0ꢀC with stirring was added to a solution
of 16a (4.4 g, 29.7 mmol) in THF (200 mL). The resulting mixture
was stirred at 0ꢀC for 1 hour. A mixture of (4R,5S)-(+)-4-methyl-
5-phenyl-2-oxazolidinone (17, 6.0 g, 34 mmol) and LiCl (1.27 g,
30 mmol) were added. The resulting mixture was stirred for
18 hours at room temperature. The precipitate formed in the
reaction mixture was removed by filtration, washed with diethyl
ether (2 Â 10 mL). The filtrate was washed with brine (2 Â 30 mL),
dried over MgSO4, and concentrated under vacuum. The crude
product (11.2 g) was purified by flash column chromatography
(heptane-ethyl acetate, 10:1) to afford 18a (7.5 g, 82%) as a
1
60%). Mp.133–134ꢀC. HNMR (CD3OD): d 3.63(m, 1H), 2.68 (ddd,
3
2J = 38.9 Hz, J = 7.2 Hz, 4.9 Hz, 2H), 1.73–1.53 (m, 2H), 1.52–1.12
(m, 5H), 0.98 (d, J = 5.9 Hz, 3H). 13CNMR (CD3OD): d 172.4, 46.7,
40.1, 39.1, 36.6, 28.7, 19.7, 18.4, 13.5 (septet, intense). MS(APCl+):
m/z 178 (M + H).
1
colorless oil. HNMR (CDCl3): d 7.48–7.30 (m, 5H), 5.68 (d, J = 5.8
Hz, 1H), 4.78 (qn, J = 7.3 Hz, 1H), 3.08–2.85 (m, 2H), 1.78–1.63
(m, 1H), 1.61–1.45 (m, 2H), 1.41–1.25 (m, 3H), 1.23–1.10 (m, 1H),
0.93 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H).
8-(13C-Trideuteriomethyl)-(3S,5R)-ter-butyl-5-methyl-3-((4R,5S)-4-
Analytical data for (3S,5R)-3-amino-5-methyl[D5]nonanoic acid
1
(4): HNMR (CD3OD); d 5.13 (bs, 3H), 3.38–3.58(m, 1H), 2.42–2.57
(m, 1H), 2.23–2.42 (m, 1H), 1.56–1.72 (m, 2H), 1.17–1.46 (m, 5H);
0.92 (d, 3H).13CNMR (CD3OD): d 176.4, 47.5, 40.5, 38.9, 38.6, 36.4,
29.0, 28.9, 22.8 (m), 18.4, 13.2 (m). MS (APCl+): m/z 193 (M+ H).
methyl-2-oxo-5-phenyloxazolidine-3-carbonyl)octanoate (19a)
A flame-dried three-necked flask was charged with 18a (7.5 g,
24.3 mmol) and anhydrous THF (100 mL). Sodium bistrimethylsi-
lylamide (1M in THF, 30 mL) was added dropwise at À70ꢀC over
10 minutes. The mixture was stirred at À70ꢀC for 1 hour. A
solution of tert-butyl bromoacetate (15.0g, 77 mmol) in anhydrous
THF (10 mL) was added via syringe over 30 minutes. The mixture
was stirred for 4 hours at À70ꢀC and then was warmed at room
temperature and stirred for 16 hours. The mixture was quenched
by addition of H2O (10 mL) and then ammonium chloride (2N,
100mL). The mixture was extracted with tert-butyl methyl ether
(3Â 100 mL). The combined organic phases were washed with
brine (100 mL), dried over Na2SO4, and concentrated under
vacuum to give the crude product as a colorless oil, which was
further purified by flash column chromatography (heptane-ethyl
acetate, 9:1) to give 19a (8.5g, 83%) as a white solid. 1HNMR
(CDCl3): d 7.40–7.22 (m, 5H), 5.58 (d, J = 7.4 Hz, 1H), 4.69 (qn,
J = 5.9 Hz, 1H), 4.25 (m, 1H), 2.50 (ddd, 2J = 83.1 Hz, 3J = 9.7 Hz, 5.1
Hz, 2H), 1.65 (qn, J = 6.8 Hz, 1H), 1.45–0.98 (m, 7H), 1.34 (s, 9H),
0.85 (d, J = 6.3 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H).
(R)-Ethyl 5-methyl-3-[1-14C]oxooctanoate (25)
A 100 mL flask was charged with 1,1’-carbonyldiimidazole
(0.7293 g, 4.50 mmol) and ethyl acetate (4.20 mL) to form a
suspension. A solution of compound 22 (0.5855 g, 4.5 mmol) in
ethyl acetate (4.2 mL) at room temperature was added slowly
to the suspension. The resulting mixture was stirred at room
temperature for 4 hours and then evaporated under vacuum to
remove CO2 to give a solution A. Another flask was charged
with [1-14C]potassium ethyl malonate 24 (50 mCi, 0.914 mmol,
54.7 mCi/mmol), potassium ethyl malonate (0.603 g, 3.54 mmol),
MgCl2 (0.381 g, 4.00 mmol) and ethyl acetate (5.0 mL). The result-
ing mixture was stirred at room temperature for 10 minutes.
Triethylamine (0.4624 g, 4.57 mmol) dropwise was added to this
mixture at such a rate as to maintain the internal temperature
below 30ꢀC to form a suspension. The suspension was stirred
at room temperature for 1 hour. The solution A was added to
the suspension at room temperature. The resulting mixture was
stirred at 45ꢀC for 16 hours and then cooled to room tempera-
ture. 4M HCl (5.7 mL) was added to this mixture at such a rate
to keep the internal temperature below 25ꢀC. The mixture
was allowed to stand and to form a clear bi-phase mixture.
The organic layer was separated and washed with water
(2 Â 3.0 mL), 6% NaHCO3 (4.6 mL) and water (2 Â 3.0 mL) and
dried over Na2SO4 (5.0 g). The solvent was evaporated under
vacuum to give compound 25 (834.5 mg, 70%) with a total
activity of 35.0 mCi and a specific activity of 8.40 mCi/mmol.
1HNMR (CDCl3): d 4.20 (q, 2H), 3.42 (s, 2H), 2.52 (dd, 1H), 2.33
(dd, 1H), 2.03 (m, 1H), 1.30–1.10 (m, 6H), 1.28 (t, 3H), 0.90 (m, 6H).
7-(13C-Trideuteriomethyl)-(2S,4R)-2-(2-tert-butoxy-2-oxoethyl)-4-
methylheptanoic acid (20a)
A solution of lithium hydroxide hydrate (1.68 g, 40 mmol) in
hydrogen peroxide (30%, 14.4 mL) and water (32 mL) at 0ꢀC
was slowly added to a solution of 19a (8.4 g, 19.9 mmol) in THF
(180 mL) and water (60 mL). Then, the resulting mixture was
warmed at room temperature and stirred for 5 hours. A solution
of sodium sulfite (6.0 g) and sodium bisulfite (6.0 g) in water
(60 mL) was added. The mixture was extracted with a mixture
of tert-butyl methyl ether and heptane (50/50 v/v, 250 mL). The
organic phase was dried over Na2SO4 and concentrated in vacuo.
The residue was dissolved in pentane (50 mL), and a brief
(R,Z)-Ethyl 3-acetamido-5-methyl[1-14C]oct-2-enoate (27)
agitation caused the chiral auxiliary to precipitate. The mixture A 50 ml flask was charged with AcONH4 (0.6985 g, 9.06 mmol), 25
was left to stand for 16 hours at room temperature. The solids (0.7943 g, 3.96 mmol, 33.3 mCi) and anhydrous ethanol (6.0 mL)
were removed by filtration, and the filtrate was concentrated to form a suspension. The suspension was stirred at 60ꢀC for a
under vacuum to afford 20a (5.2 g, 99%) as a colorless oil. 1HNMR minimum of 5 hours and then cooled to 35ꢀC and diluted with
J. Label Compd. Radiopharm 2012, 55 166–170
Copyright © 2012 John Wiley & Sons, Ltd.
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Y. Zhang et al.
isooctane (4.0 mL). The mixture was concentrated in vacuo to to facilitate precipitation. The solid was collected by filtration
form a semi-solid. Isooctane (4 mL) was added to the semi-solid, with Buchner funnel fitted with a filter paper while it was cold,
and the resulting mixture was stirred for 5 minutes. The solid was and washed with cold toluene pre-cooled at 0ꢀC (4.0 mL Â 4).
removed by filtration and washed with isooctane (3 Â 2.0 mL). The resultant solid was dried under high vacuum overnight
The filtrate was concentrated to the volume of about 1 mL. and further recrystallized from a mixture of 2-propanol and
Isooctane (4.5 mL), Ac2O (0.90 mL), and pyridine (0.90 mL) were toluene (1/2, 4.5 mL) to give 5 (0.592, 76%, 8.4 mCi/mmol). After
added to the residue. The resulting mixture was heated at radio-dilution, the final cGMP material (6.0284 g) was obtained
100ꢀC for 15 hours with stirring and then cooled to 5ꢀC. Water with a specific activity of 105.29 mCi/mmol (0.502 mCi/mg) and
(3.0 mL) was added, and the resulting mixture was stirred for radiochemical and chemical purity of >99.0%. 1HNMR (DMSO-d6):
2.5 hours at room temperature. The organic layer was separated, d 12.7 (bs, 1H), 8.17 (bs, 1H), 3.38 (m, 1H), 2.69 (dd, J = 6, 17 Hz,
washed with water (2 Â 2.0 mL), H2SO4 solution (2 Â 2.0 mL, 1H), 2.53 (dd, J= 7, 17 Hz, 1H), 1.61 (m, 2H), 1.2 (m, 4H), 1.10
1.0 mL conc. H2SO4, and 10.0 mL water), then water (2 Â 2.0 mL) (m, 1H), 0.80 (m, 6h); 13CNMR (DMSO-d6): d 172.3 (1C), 46.5(1C),
and dried over anhydrous Na2SO4. The Na2SO4 was filtered 40.2(1C), 39.2(1C), 36.5(1C), 28.8(1C), 19.5(1C), 18.6(1C), 13.4 (1C);
off and washed with isooctane (3 Â 2.0 mL). The filtrate was HPLC method: Column Phenomenex, Synergi Polar-RP, 4 mm,
concentrated under vacuum to give 27 (0.8165 g, 8.4 mCi/mmol, 150x4.6; Mobile phase A = Buffer (25 mM potassium phosphate
28.4 mCi, 85.3%). 1HNMR (CDCl3): d 11.20 (s, 1H), 4.90 (s, 1H), 4.17 pH6.3):MeOH 90:10, B = Buffer:MeOH 10:90; from 0%B to 100%
(q, 2H), 2.88 (dd, 1H), 2.34 (dd, 1H), 2.14 (s, 3H), 1.79 (m, 1H), 1.30 in 60 minutes; Flow rate = 1 mL/minute, Temperature= 30ꢀC; UV
(m, 7H), 0.89 (m, 6H); 13CNMR (CDCl3): d 169.1 (1C), 168.3 (1C), @210 nm.
157.8 (1C), 97.2 (1C), 59.8 (1C), 42.7 (1C), 36.3 (1C), 31.4 (1C),
29.0 (1C), 28.8 (1C), 22.9 (1C), 19.0 (1C), 14.2 (1C).
(3S,5R)-Ethyl 3-acetamido-5-methyl[1-14C]octanoate (28)
Conflict of Interest
The authors did not report any conflict of interest.
A pressure reactor was charged with compound 27 (0.8165,
8.4 mCi/mmol, 28.4 mCi) and isooctane (3.0 mL, pre-treated with
dry N2 gas for 10 minutes). A solution of (R)-mTCFPRhCODBF4 References
(11.94 mg, 0.0213 mmol) in MeOH (3.0 mL, pre-treated with dry
[1] K. Ohashi, M. Kawai, N. Ninomiya, C. Taylor, Y. Kurebayashi, Pharmacology
N2 gas for 10 min) under N2 gas was added to the reaction
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mixture. The resulting mixture was purged with N2 (three times)
[2] D. J. Rawso, J. B. Schwarz, WO 2007/052134 A1 20070510. CAN
and then with H2 gas (four times). A H2 gas pressure of 10 psi was
146:482239.
applied to the pressure reactor. The reactor was heated at 50ꢀC
[3] J. Magano, D. Bowles, B. Conway, T. N. Nanninga, D. Winkle. Tetrahedron
for 15 hours. After completion of the reaction determinate by
GC-MS, the mixture was evaporated under vacuum to give the
crude product. The crude material was dissolved in isooctane
(15.0 mL) and washed with water (2.0 mL Â 2). The organic layer
was filtered and concentrated under vacuum to give the desired
compound 28 (0.7796 g, 94.7%, 26.88 mCi, 8.4 mCi/mmol).
1HNMR (CDCl3): d 5.98 (d, 1H), 4.35 (m, 1H), 4.15 (q, 2H0, 2.47
(m, 2H), 1.97 (s, 3H), 1.57 (m 1H), 1.00–1.40 (m, 8H), 0.90 (m, 6H).
Lett. 2009, 50, 6325–6328, and references sited therein.
[4] J. Magano, B. Conway, D. Bowles, J. Nelson, T. N. Nanninga, D. Winkle,
Tetrahedron Lett. 2009, 50, 6329–6331.
[5] C. S. Dexter, F. W. Jacson, J. Org. Chem. 1999, 64, 7579–7585.
[6] T. K. Chakraborty, A. Ghosh. Synlett 2002, 12, 2039–2040, and references
sited therein.
[7] E. Juaristi, Enantioselective synthesis of b-aminoacids, Wiley-VCH:
Nork York, 1997.
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[9] M. E. Bunnage, S. G. Davies, P. M. Roberts, A. D. Smith, J. M. Withey,
Org. Biomol. Chem. 2004, 2(19), 2763–2776.
(3S,5R)-3-Amino-5-methyl[1-14C]octanoic acid hydrochloride (5)
[10] J. Monfray, Y. Gelas-Mialhe, J. C. Gramain, R. Remuson, Tetrahedron:
Asymmetry 2005, 16(5), 1025–1034.
Compound 28 (0.7796 g, 3.20 mmol, 26.88 mCi, 8.40 mCi/mmol)
was stirred in MeOH (1.2 mL) and water (2.0 mL) at 35ꢀC. A total [11] F. Liu, W. Yu, W. Ou, X. Wang, L. Ruan, Y. Li, X. Peng, X. Tao, X. Pan,
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Tedrow, J. Org. Chem. 1999, 64, 6411–6417.
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[14] T. P. Tang, J. A. Ellman, J. Org. Chem. 1999, 64, 12–13.
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[16] G. Hoge, H. P. Wu, W. S. Kissel, D. A. Pflum, D. J. Greene, J. Bao, J. Am.
Chem. Soc. 2004, 126, 5966; (R)-mTCFP ligand refers to “methyltri-
chickenfootphos”, as it is known in the Pfizer lab.
of 37% HCl (2.2 mL) in one portion was added to this mixture.
The resulting mixture was heated at 100ꢀC for not less than
20 hours and then cooled to 55ꢀC. Toluene (3.0 mL) was added,
and the resulting mixture was heated at 55ꢀC for 10 minutes with
stirring. After standing for 10 minutes, the top toluene layer was
removed, and the aqueous layer was extracted with toluene
(3.0 mL Â 2) at 60ꢀC. Toluene (2.0 mL) was added to the aqueous
layer at 60ꢀC. The final mixture was cooled slowly from 60ꢀC to
À10ꢀC over a period of 3 hours and kept at À10ꢀC for 5 hours
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